Upstream proteolysis by Ste24 does not require a C-terminal methyl ester as revealed using 33-residue a-factor precursor peptide substrates synthesized via epimerization-free methods

Upstream proteolysis by Ste24 does not require a C-terminal methyl ester as revealed using 33-residue a-factor precursor peptide substrates synthesized via epimerization-free methods | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Upstream proteolysis by Ste24 does not require a C-terminal methyl ester as revealed using 33-residue a-factor precursor peptide substrates synthesized via epimerization-free methods Taysir K. Bader, Shanica M. Brown, Christine A. Hrycyna, Mark D. Distefano This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5094096/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2025 Read the published version in International Journal of Peptide Research and Therapeutics → Version 1 posted 16 You are reading this latest preprint version Abstract Protein prenylation is a post-translational modification that links a specific cytosolic protein with a hydrophobic isoprenoid lipid resulting in membrane localization of the target protein. Following prenylation, some proteins undergo proteolytic removal of a C-terminal tripeptide. This step results in a protein terminating in a prenylcysteine residue that is subsequently methylated by the membrane-associated methyltransferase ICMT. In some cases, this proteolytic reaction is catalyzed by ZMPSTE24 in humans or Ste24 in yeast. The molecular recognition demonstrated by this family of proteases is intriguing as they also cleave at an unrelated additional site upstream from the C-terminus that has no obvious structural similarity. From a medical perspective, these events are particularly important for the posttranslational processing of protein lamin A, as mutations in ZMPSTE24 that impair its activity lead to accelerated premature aging progeroid diseases. A central question in the field regards the structure of the C-terminus of processed lamin A and whether methylation is essential for subsequent upstream cleavage. Herein, a series of 33-residue peptides based on the structure of the precursor for the peptide pheromone a -factor from yeast, a substrate for both Ste24 and ZMPSTE24. These peptides were synthesized with a fluorescent donor-quencher pair and incorporated either a C-terminal methyl ester to mimic the native substrate, a free acid to mimic an unmethylated peptide or an amide to mimic differently modified C-termini. Their preparation presented several synthetic challenges due to the presence of a C-terminal methyl ester, an epimerization-prone C-terminal cysteine and an acid-sensitive farnesyl group. The synthesis of those peptides and an analysis of their cleavage at the upstream site catalyzed by Ste24 are reported here. In vitro fluorescence-based proteolytic cleavage assays showed that all of these peptides were processed at similar rates, suggesting that C-terminal methylation is not a prerequisite for subsequent upstream proteolysis. a-Factor farnesylation peptide epimerization progeria solid phase peptide synthesis Ste24 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Protein prenylation is a post-translational modification that links certain cytosolic proteins with a hydrophobic isoprenoid lipid. The primary purpose of this modification is to facilitate membrane localization and association, although there are examples of prenylated proteins that remain cytosolic (Casey 1992; Palsuledesai and Distefano 2015). Prenylated proteins are involved in numerous signal transduction pathways (Gelb et al. 2006; Berndt et al. 2011; Palsuledesai and Distefano 2015; Wang and Casey 2016), and often are initially synthesized with a C-terminal CaaX sequence, where C is cysteine, a is generally an aliphatic residue and X can be one of a number of amino acids. One of the most common forms of prenylation, farnesylation, occurs as the first of three enzymatic steps in the CaaX protein post-translational processing pathway (Fig. 1). The first step is the transfer of an isoprenoid moiety to the cysteine of the C-terminal CaaX sequence(Wang and Casey 2016). The isoprenoid can be either a farnesyl (3 isoprene repeats, 15 carbons) or a longer geranylgeranyl (4 isoprene repeats, 20 carbons) chain, which are added by either protein farnesyltransferase (FTase) or types 1, 2, or 3 geranylgeranyltransferase (GGTase I, II or III) respectively (Casey 1992; Palsuledesai and Distefano 2015). The second step in the pathway is the endoproteolytic removal of the aaX sequence by zinc metalloprotease sterile 24 (ZMPSTE24) or Ras converting CaaX endopeptidase 1 (Rce1) (Ma et al. 1992; Ashby et al. 1992; Trueblood et al. 2000). The final step is carboxyl methylesterification of the newly exposed C-terminal prenylcysteine by protein- S -isoprenylcysteine O-methyltransferase (ICMT)(Hrycyna and Clarke 1990; Stephenson and Clarke 1990; Marr et al. 1990; Hrycyna et al. 1991; Pillingers et al. 1994; Dai et al. 1998). The resulting proteins are then shuttled to various membranes or further processed to carry out their biological functions ZMPSTE24 is a unique membrane-bound zinc metalloprotease localized to the endoplasmic reticulum and the inner nuclear membrane (Schmidt et al. 1998; Barrowman et al. 2008). In addition to its function of endoproteolytic cleavage of aaX tripeptides from the C-termini of prenylated proteins that is similar to that of Rce1 (Schmidt et al. 1998; Porter et al. 2007), ZMPSTE24 can also catalyze a second site-specific cleavage upstream from the prenylated cysteine (Tam et al. 1998); interestingly, there is no obvious sequence similarity between the two different cleavage sites. The mechanism behind this bifunctional behavior is poorly understood, yet quite important in human health. The only bona fide human substrate of ZMPSTE24 is the prenylated protein prelamin A (Corrigan et al. 2005; Barrowman et al. 2012; Casasola et al. 2016; Babatz et al. 2021). ZMPSTE24-catalyzed cleavage at two different sites of that protein leads to the release of a 15-residue prenylated peptide from the C-terminus along with the mature nuclear scaffold protein lamin A (Fig. 2). While lamin A is essential for properly forming the nuclear lamina and providing mechanical stability, the function of the 15-residue prenylated peptide product is unknown (Casasola et al. 2016). Mutations in ZMPSTE24 that prevent the second upstream cleavage result in accelerated aging progeroid diseases (Young et al. 2005; Fong et al. 2009; Yang et al. 2010; Gordon et al. 2018). Thus, a better understanding of ZMPSTE24 function and its substrate recognition would be helpful for both the development of treatments for progeroid diseases and for providing more detailed molecular insights into the process of human aging. Intriguingly, in recent years, potentially new functions have been ascribed to ZMPSTE24 including that of a “translocon unclogger”, where misfolded proteins from the translocon are cleared during signal recognition-particle-independent protein translocation (Ast et al. 2016; Kayatekin et al. 2018). It was observed that ZMPSTE24 could clear misfolded human islet amyloid polypeptide, which is common in patients with type 2 diabetes (Kayatekin et al. 2018). This has led to the suggestion that ZMPSTE24 plays a critical role in the ER-associated degradation pathway (Avci and Lemberg 2015), and that its downstream substrates may be druggable targets (Goblirsch and Wiener 2020). Perhaps more surprisingly, some evidence suggests that ZMPSTE24 serves as an “intrinsic broad-spectrum antiviral protein” that is recruited to prevent the fusion of viral membranes and endosomal membranes (Guo et al. 2021), thus protecting against viral infections such as influenza and even SARS-CoV-2 (Fu et al. 2017; Shilagardi et al. 2022). These results cast doubt on the idea that its primary cellular function is to be CaaX protease (Goblirsch and Wiener 2020). Clearly, a better understanding of how ZMPSTE24 recognizes its substrates is critical for understanding its cellular function. ZMPSTE24 has several intriguing structural features. It consists of seven transmembrane α-helices that together form a novel “α-barrel,” which includes a large (> 12,000 Å 3 ) chamber where substrate binding and catalysis take place. Proteolytic activity requires an HExxH zinc metalloprotease consensus sequence, where the two histidines and the glutamate residue bind a catalytic zinc atom (Jongeneel et al. 1989; Clark et al. 2017). For CaaX processing, it has been proposed that the endoproteolytic cleavage of the -aaX motif and carboxylmethylation by ICMT may occur before the upstream cleavage step, which would first involve substrate entry into the ZMPSTE24 cavity, -aaX cleavage, product release, ICMT-catalyzed methylation and reentry into the enzyme reaction chamber for the upstream cleavage event (Michaelis et al. 1992; Fujimura-Kamada et al. 1997; Boyartchuk et al. 1997; Tam et al. 1998; Schmidt et al. 2000; Barrowman et al. 2008; Barrowman and Michaelis 2013). However, to date, there has been no conclusive evidence to confirm the validity of that model. If carboxylmethylation is an absolute prerequisite for the upstream cleavage step, then inhibition of ICMT would lead to laminopathy symptoms similar to progeroid diseases, since unmethylated lamin A would retain the C-terminal 15 residue prenylated peptide. ICMT inhibition is currently being considered as potential therapeutic target for some Ras-based cancers (Bergo et al. 2004; Bergman et al. 2011; Ahearn et al. 2021), and thus it is essential to clarify this question. There is a possible precedent for this effect with HIV aspartyl protease inhibitor drugs, which were found to cause lipodystrophy through off-target inhibition of ZMPSTE24, thus leading to the accumulation of farnesylated prelamin A (Coffinier et al. 2007; Clarke 2007; Hudon et al. 2008; Clark et al. 2017). Ste24 is the yeast homolog of ZMPSTE24 (Michaelis and Barrowman 2012; Barrowman and Michaelis 2013; Goblirsch and Wiener 2020) and the two enzymes share significant sequence and structural similarity (Pryor et al. 2013; Clark et al. 2017; Goblirsch et al. 2020). Importantly, those similarities extend to their function as well; both enzymes can process their homolog’s substrates, and ZMPSTE24 has been found to rescue the function of yeast mutants lacking Ste24 activity (Tam et al. 1998). However, Ste24 is more amenable to purification and functional assays, making it more attractive for biochemical experiments (Boivin et al. 1993; Hrycyna and Clarke 1993). To address the question as to whether a Ste24 substrate must first undergo processing at the C-terminus before upstream cleavage can occur, a series of 33-residue a -factor analog precursor peptides containing a fluorescent donor-quencher pair were synthesized. These peptides incorporated either a C-terminal methyl ester to mimic the native substrate, the free acid to mimic an unmethylated peptide or an amide to mimic a differently modified C-terminus. These peptides were used as substrates for a quantitative in vitro fluorescence-based proteolytic cleavage assay. The synthesis of these peptides presented several synthetic challenges due to the presence of a C-terminal methyl ester, an epimerization-prone C-terminal cysteine and an acid-sensitive farnesyl group. The synthesis of those peptides and an analysis of their cleavage at the upstream site catalyzed by Ste24 are reported here. Materials and Methods Materials HPLC grade H 2 O and CH 3 CN, and sequencing grade dimethyl formamide (DMF), were purchased from Fisher Scientific. Protected amino acids, resins, O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 6-Chloro-1-hydroxybenzotriazole (Cl-HOBt) were purchased from Chem-Impex International. C-18 reverse phase Sep-Paks Ò (820 mg, 55-105 µm particle size) were purchased from Waters corporation. Sequencing-grade Trypsin was purchased from Promega and reconstituted according to the manufacturer’s specifications. Bulk E. coli polar lipids were purchased from Avanti Polar Lipids. n-Dodecyl-B-D-maltopyranoside (DDM) was purchased from Anatrace. TALON Ò metal affinity resin was purchased from Clontech. Amicon® Ultra Centrifugal Filters (30,000 MWCO) were purchased from Millipore. All other reagents were purchased from Sigma-Aldrich and were used without further purification. Methods Chemical synthesis 2.2.1.1 | Synthesis of Fmoc- L -Cys-OMe 7a Fmoc- L -cysteine hydrate ( 7a , 2 g, 5.54 mmol) was dissolved in 15 mL CH 3 OH, and 6 drops of concentrated HCl were added to catalyze the reaction. The solution was stirred for 24 h, affording a white slurry that was dissolved with acetone. The solvent was then removed by rotary evaporation at 25 °C, and the material was dried under vacuum for 5 h yielding Fmoc- L -Cys-OMe ( 8a , 1.96 g, 99% yield) as a white solid. The 1 H NMR in CDCl 3, was consistent with previously reported data (Diaz-Rodriguez et al. 2015). Synthesis of Fmoc-D-Cys-OMe 7b Fmoc- D -cysteine hydrate ( 7b , 1 g, 2.77 mmol) was dissolved in 15 mL CH 3 OH, and 6 drops of concentrated HCl were added to catalyze the reaction. The solution was stirred for 24 h, affording a white slurry that was redissolved in acetone. The solvent was then removed by rotary evaporation at 25 °C, and the material was dried under vacuum for 5 h yielding Fmoc-D-Cys-OMe ( 8b , 0.97 g, 99% yield) as a white solid. The 1 H NMR in CDCl 3, was consistent with previously reported data (Diaz-Rodriguez et al. 2015). Resin loading for the production of peptides with L -Cys-OMe C-terminus 9a To load 8a onto the solid support, 0.82 g (1.44 mmol) of trityl-chloride resin (100-200 mesh, 1.75 mmol/g loading) was placed in a polypropylene filter syringe and washed with CH 2 Cl 2 for 1 min 3x. Compound 8a (1.96 g, 5.48 mmol) was then dissolved in 9 mL CH 2 Cl 2 (600 mM) along with 995 µL (5.74 mmol, 600 mM) DIPEA and added to the resin which was placed on a rotator for 24 h. Unreacted positions were then capped by adding 1 mL CH 3 OH to the solution and allowing it to rotate for 15 min. The resin was subsequently washed with CH 2 Cl 2 for 2 min 3x and dried under a vacuum. To quantify resin loading, three samples of 10 mg were weighed and then placed in a filter syringe. Each sample was subjected to Fmoc deprotection with 1 mL 20% piperidine in DMF for 30 min. These solutions were then transferred to 25 mL volumetric flasks, and EtOH was used to wash the resins in 5 mL batches and then transferred to the same flasks until the fill lines were reached. A standard curve was then constructed consisting of Fmoc-OSu in EtOH at 1 mM, 0.75 mM, 0.5 mM, 0.25 mM, 0.125 mM, and 0.0625 mM concentrations. The absorbance of the standard curve and samples were all read in triplicate at 301 nm. A resin loading of 0.12 mmol/g was obtained. Resin loading for the production of peptides with D-Cys-OMe C-terminal 9b To load 8b onto the solid support, 0.17 g (0.323 mmol) of trityl-chloride resin (100-200 mesh, 1.90 mmol/g loading) was placed in a polypropylene filter syringe and washed with CH 2 Cl 2 for 1 min 3x. Compound 8b (0.22g g, 0.616 mmol) was then dissolved in 2 mL CH 2 Cl 2 (final concentration 300 mM) along with 220 µL (1.26 mmol, final concentration 300 mM) DIPEA and added to resin which was placed on a rotator for 24 h. Unreacted positions were then capped by adding 0.5 mL CH 3 OH to the solution and allowing it to rotate for 15 min. The resin was subsequently washed with CH 2 Cl 2 for 2 min 3x and dried under a vacuum. The resin was quantified in the same manner as 9a . A resin loading of 1.49 mmol/g was obtained. Peptide synthesis, prenylation and characterization General Procedure for Peptide Synthesis on Gyros PS3 automated synthesizer Unprenylated precursors to peptides 1a and 2a were synthesized using a Gyros PS3 automated peptide synthesizer employing Fmoc/HCTU-based chemistry. Resins (0.15 mmol) for the appropriate peptide (Fmoc-Cys-OMe Trt resin for peptide 1a , or Fmoc-Cys(Trt)-OH Wang resin (100-200 mesh) for peptide 2a were placed in a reaction vessel, and swelled in DMF for 10 min 3x. The Fmoc group on the first amino acid was then removed using 20% piperidine in DMF for 5 min 2x. Four equiv of the subsequent amino acid were activated with an equimolar amount of HCTU in 2 mL DMF with 800 mM DIPEA and 300 mM Cl-HOBt for 3 min. This solution was then transferred to the resin, and 2 mL of DMF was used to wash the amino acid vial before being transferred to the reaction vessel, resulting in an amino acid/HCTU/Cl-HOBt concentration of 150 mM and a DIPEA concentration of 400 mM. The coupling was carried out for either 20 or 60 min with N 2 -mediated mixing for 1 s every 10 s. D, G, N, P, Y, K(Dnp), and Abz were coupled for 60 min. All other amino acids were coupled for 20 min. After all amino acids were coupled, a final Fmoc deprotection step was carried out and the resin was washed with CH 2 Cl 2 for 5 min (3x) and then dried in vacuo. General Procedure for Peptide Synthesis on Gyros Chorus automated synthesizer Unprenylated precursor to peptide 3a was prepared using a Gyros Chorus automated peptide synthesizer. Low-loading rink amide MBHA resin (0.2 mmol, 100-200 mesh) was placed in a polypropylene syringe with a stopcock and swelled in 5 mL DMF for 10 min 3x. The Fmoc group was removed from the resin by incubating with 5 mL 20% piperidine in DMF for 5 min 2x, then washed with 5 mL DMF for 2 min 3x. Fmoc-Cys(Trt)-OH (468 mg, 0.8 mmol, 4 equv) and 331 mg (0.8 mmol, 4 equiv) of HCTU were dissolved in 5 mL of 400 mM M DIPEA (150 mM final conc) and added to the resin, placed on a rotator and allowed to react for 1 h, after which the Ninhydrin test showed complete consumption of the amine (Kaiser et al. 1970; Vilaseca and Bardaji 1995). The resin was washed with DMF as above and placed in the instrument’s reaction vessel. The resin was then swelled in 10 mL DMF for 10 min 3x and the Fmoc group was removed using 10 mL 20% piperidine in DMF for 5 min, 2x. Subsequent amino acids (5 equiv) were activated with an equimolar amount of HCTU in 400 mM DIPEA at 150 mM and added to the resin. The coupling was carried out for 20 min, after which the resin was washed with 10 mL for 30 secs 3x, then the coupling was repeated, and the resin was washed again. After the coupling, any unreacted positions were capped using 50 % Ac 2 O in 400 mM DIPEA for 15 min before washing again with DMF. The Fmoc group was then removed using 10 mL of 20% piperidine for 5 min 2x before washing with DMF. K(Dnp) and Abz were also coupled manually using the same procedure as the first amino acid, and each required 1 h for reaction completion. After all amino acids were coupled, a final Fmoc deprotection step was carried out and the resin was washed with CH 2 Cl 2 for 1 min 6x and then dried in vacuo. General Procedure for Peptide Cleavage Peptide cleavage and global side chain deprotection were carried out by first placing an aliquot of 0.075 – 0.1 mmol of the peptide on-resin in a polypropylene filter syringe with a polypropylene Luer cap. Reagent K (10 mL, 82% TFA, 2.5% ethanedithiol, 5% thioanisole, 5% phenol, and 5% H 2 O ) was added to the syringe and rotated for 2 h. Next, the solution was drained into a 50 mL polypropylene centrifuge tube, and TFA (10 mL) was used to wash the resin in 2 mL batches. A gentle N 2 stream was used to evaporate excess TFA over an additional h until approximately 2 mL of solution remained. The peptide was then precipitated by adding Et 2 O to the 50 mL mark and cooling in a dry ice/ i -PrOH bath. The peptide was then pelleted by centrifugation at 3,000 x g for 5 min. This procedure was repeated twice, with resuspension of the solid peptide in fresh Et 2 O through vortexing for 2 min. After the third Et 2 O precipitation, the tube was placed uncovered in a fume hood for 1 h to dry. Next, 3 mL of HOAc and 2 mL of H 2 O were added to the peptide, and incubated at rt for 10 min to allow the solid to fully dissolve. The solution was then diluted to 10 mL with H 2 O, flash-frozen in liquid N 2 , and then lyophilized. This solubilization and lyophilization process was crucial for two reasons: First, it facilitated the complete deprotection of the tryptophan side chain Boc protecting groups, which we have observed to be sluggish and results in the observation of a +44 Da side product believed to be a carbamic acid intermediate. Second, this procedure improved the solubility of the peptides in DMF for the subsequent prenylation step. General Procedure for Peptide Prenylation All the solvents used in this procedure were sparged with N 2 for 3 h to deoxygenate them and prevent disulfide formation. DMF was added to the lyophilized peptide to dissolve it and then Ellman’s assay was used to quantify the amount of free thiol in the solution.(Riener et al. 2002) If the concentration was significantly higher than 1 mM, then it was adjusted to that concentration with more DMF. LC-MS analysis was used to confirm the presence of the peptide before proceeding with the reaction. Once confirmed, farnesyl bromide (5 equiv) was diluted 10-fold v/v in DMF and then added dropwise to the peptide solution. The centrifuge tube was then vortexed for 30 sec to fully dissolve the farnesyl bromide. Zn(OAc) 2 •H 2 O (5 equiv) were dissolved 2 M NaOAc, pH. 5.0. The buffer volume was determined based on the volume of DMF used so that the final solvent composition was 9:1 DMF/2M NaOAc buffer. Once the Zn(OAc) 2 •H 2 O was fully dissolved, it was added to the peptide solution, and the tube was vortexed for 30 sec before being placed on a rotator overnight. The next day, LC-MS was used to confirm the completion of the reaction (>90% conversion). Once complete, 5% HOAc was added to the solution to both quench the reaction and help maintain peptide solubility. The solution was then filtered through a 0.2 µm GHP syringe filter and purified by HPLC. It is essential to do this step promptly, or the peptide will precipitate out of the solution. General Method for LC-MS analysis LC-MS analysis was performed using an Agilent 1200 series system (Windows 10, ChemStation Software, G1322A Degasser, G1312A binary pump, G1329A autosampler, G1315B diode array detector, 6130 quadrupole) equipped with a C18 column (Agilent ZORBAX 300-SB-C18, 5 μM, 4.6 X 250 mm). Separations were performed at a flow rate of 1 mL/min. One H 2 O/CH 3 CN solvent system containing 0.1% TFA was used, consisting of solvent A (H 2 O with 0.1% TFA) and solvent B (CH 3 CN with 0.1% TFA). Samples were filtered through a 0.2 µm GHP filter before injecting into the instrument. The gradient used was sample dependent and is indicated in the figure legends. Note that in samples containing DMF, a 10 min hold at 1% B at the beginning of the method before starting the gradient significantly enhanced the resolution. General Method for Two-Stage HPLC purification HPLC purification was performed using an Agilent 1100 series system (Windows 7, ChemStation Software, G1312A binary pump, G1329A autosampler, G1315B diode array detector, Teledyne Foxy R1 fraction collector). Samples were filtered through a 0.2 µm GHP syringe filter before injecting into the instrument. Purification was performed first on a preparative scale (10 - 20 mg peptide per injection, Agilent Pursuit C18, 5 μm, 250 × 21.2 mm) with a 5 mL/min flow rate and using the same Solvent A/Solvent B system described above. The gradient was as follows: 1-10 min hold at 30% B, 10-70 min ramp to 100% B. Peptides 1 - 3 were then further purified on a semi-preparative scale (2 - 10 mg peptide per injection, Agilent ZORBAX 300SB-C18, 5 μm, 9.4 × 250 mm) with 4 mL/min flow rate using the same solvent A/Solvent B system described above. The gradient for peptide 1a was as follows: 1-5 min hold at 20% B, 10 min ramp to 45% B, 20 min ramp to 55% B, 1 min ramp to 100% B. The gradient for peptides 2a and 3a was as follows: 1-5 min hold at 20% B, 10 min ramp to 40% B, 20 min ramp to 50% B, 1 min ramp to 100% B. General Method for Sep-Pak Purification of 33mer Peptides Prenylated peptides were purified using a simple Sep-Pak solid phase extraction procedure. The cartridges were first conditioned using 10 mL of 100% solvent B, followed by equilibration with 10 mL of 100% solvent A. Prenylation reaction mixtures (5 mL) containing peptides 1 -3 were then diluted 5-fold with solvent A, and loaded onto the cartridges. The cartridges were then washed using 10 mL of 100% solvent A and 10 mL of 30% solvent B, before eluting the peptide using 10 mL of 80% solvent B. The organic solvent was then removed using a gentle stream of N 2 before lyophilizing the peptides and redissolving them in DMSO. Synthesis of Peptide 1a Peptide 1a was synthesized using a PS3 automated peptide synthesizer and cleaved from the resin, prenylated, and purified by HPLC as described above. Pooled HPLC fractions were lyophilized and then redissolved in DMSO. The concentration was measured by diluting the peptide in 6 M Gdm•HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 360 nm (ɛ 360 = 17,500 M -1 cm -1 ) (Hsu et al. 2019). ESI-MS: for C 190 H 289 N 44 O 56 S 2 3+ [M+3H] 3+ ; calcd 1383.0204, found 1383.0203. Synthesis of Peptide 2a Peptide 2a was synthesized using a PS3 automated peptide synthesizer and cleaved from the resin, prenylated, and purified by HPLC as described above. Pooled HPLC fractions were lyophilized and then redissolved in DMSO. The concentration was measured by diluting the peptide in 6 M Gdm•HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 360 nm (ɛ 360 = 17,500 M -1 cm -1 ) (Hsu et al. 2019). ESI-MS: for C 189 H 287 N 44 O 56 S 2 3+ [M+3H] 3+ ; calcd 1378.3485, found 1378.3499. Synthesis of Peptide 3a Peptide 3a was synthesized using a Chorus automated peptide synthesizer and cleaved from the resin, prenylated, and purified by HPLC as described above. Pooled HPLC fractions were lyophilized and then redissolved in DMSO. Concentration was measured by diluting the peptide in 6 M Gdm• HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 360 nm (ɛ 360 = 17,500 M -1 cm -1 ) (Hsu et al. 2019).ESI-MS: for C 189 H 289 N 45 O 55 S 2 3+ [M+3H] 3+ ; calcd 1378.0205, found 1378.0190. Synthesis of Peptide 4b Peptide 4b was synthesized using a PS3 automated peptide synthesizer and cleaved from the resin, prenylated, and purified by HPLC as described above. Pooled HPLC fractions were lyophilized and then redissolved in DMSO. Concentration was measured by diluting the peptide in 6 M Gdm• HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 280 nm (ɛ 280 = 5,810 M -1 cm -1 ).(Gill and von Hippel 1989) ESI-MS: for C 58 H 82 N 9 O 11 S + [M+H] + ; calcd 1112.5850, found 1112.5831. Synthesis of Peptide 5b Peptide 5b was synthesized by hydrolysis of the methyl ester of peptide 4b as described below. After hydrolysis and reaction quenching, the peptide was isolated using the same Sep-Pak procedure described above. After lyophilizing the peptide and redissolving in DMSO, the concentration was measured by diluting the peptide in 6 M Gdm• HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 280 nm (ɛ 280 = 5,810 M -1 cm -1 ).(Gill and von Hippel 1989) ESI-MS: for C 57 H 80 N 9 O 11 S + [M+H] + ; calcd 1098.2693, found 1098.2714. Synthesis of Peptide 6b Peptide 6b was synthesized using a Chorus automated peptide synthesizer and cleaved from the resin, prenylated, and purified by HPLC as described above. Pooled HPLC fractions were lyophilized and then redissolved in DMSO. Concentration was measured by diluting the peptide in 6 M Gdm• HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 280 nm (ɛ 280 = 5,810 M -1 cm -1 ).(Gill and von Hippel 1989) ESI-MS: for C 57 H 81 N 10 O 10 S + [M+H] + ; calcd 1097.5853, found 1097.5868. Synthesis of Peptide 11 Peptide 11 was synthesized using a PS3 automated peptide synthesizer and cleaved from the resin and purified by HPLC as described above. Pooled HPLC fractions were lyophilized and then redissolved in DMSO. Concentration was measured by diluting the peptide in 6 M Gdm• HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 310 nm (ɛ 310 = 2,400 M -1 cm -1 ).(Ito et al. 2001) ESI-MS: for C 36 H 55 N 9 O 13 S + [M+H] + ; calcd 853.3640, found 853.3642. MS-MS analysis of 33mer peptides In order to confirm correct peptide sequence MS-MS analysis was carried out using ThermoFisher Orbitrap Fusion Lumos Tribrid Mass Spectrometer. To prevent loss of the farnesyl group in the MS 2 fragmentation step, data-dependent Electron Transfer Dissociation (ETD) activation was used along with EThcD collision energy type. Chromatographic separation was performed using a nano-flow 300 Å pore size C3 column with a 1 µL/min flow rate. The gradient used was as follows: 1-5 min, hold at 30% B. 5-15 min, ramp to 90% B. 1 min, ramp to 100%. Parent ions (+3, +4, and +5 charge states) were fragmented via ETD to obtain daughter ions that were primarily z and c ions. Data is summarized in Tables S1-3. Methyl ester peptide hydrolysis Peptides 1a and 4a were each hydrolyzed to their corresponding C-terminal carboxylic acids through saponification reactions. The peptide (100 µM) in 0.5 M NaOH with 50% v/v CH 3 CN for 1 h at rt. The reaction was then quenched by adding 20% HOAc, which also neutralized the base and prevented any subsequent epimerization. Trypsin digestion Three aliquots of 140 µL of 50 mM NH 4 HCO 3 solutions and 40 µL of CH 3 CN were prepared in 1.5 low-adhesion microcentrifuge tubes. Peptides 1-3 were added from DMSO stocks to a final concentration of 0.1 mM. Trypsin (15 µL of a 20 µg/mL stock) was added to each tube to yield a final concentration of 1.5 µg/mL. The tubes were incubated at 37 ˚C overnight with rotation before subjecting to LC-MS analysis with and without the addition of the appropriate authentic standard peptides 4 - 6 . Enzymatic reactions Plasmids and yeast strains The pCH1283 plasmid used for this study contains a His 10 -HA 3 tag on the N-terminus of the Ste24 gene ( 2μ URA3 P PGK -His 10 -HA 3 -Ste24 ). This plasmid was transformed into the yeast strain SM3614 that has a double deletion for endogenous Ste24 and Rce1 ( MATa trp1 leu2 ura3 his4 can1 ste24Δ::LEU2 rce1Δ::TRP1 )(Tam et al. 1998). Crude membrane preparation To prepare crude membrane samples, a small culture of synthetic complete supplement mixture without uracil (SC-URA) was inoculated with pCH1283 WT strain and incubated overnight at 30 ⁰C. This starter culture was then used to inoculate a larger culture at a ratio of 15 mL to 1 L and grown to log phase (OD 600 0.3-0.5) after which time the cultures were harvested at 4000 ´ g and the pellets stored at -80 ⁰C until needed. Cell pellets were lysed using yeast sorbitol buffer (0.3 M sorbitol, 0.1 M NaCl, 12 mM MgCl 2 , 1% aprotinin, 3 mM AEBSF, 1 mM DTT, 10 mM Tris-HCl, pH 7.5). Lysis buffer was added at a ratio of 1 mL to 800 OD’s culture pellets, vortexed and left on ice for 15 min. The resulting suspension was frozen and thawed twice in liquid nitrogen and then further lysed by passing through a French press twice at 18,000 psi. The solution was then centrifuged twice at 500 ´ g for 10 min to remove cell debris and then once more at 100,000 ´ g for 1 h at 4⁰C. The supernatant was removed, and the pellet resuspended in 10 mM Tris-HCl pH 7.5 and then stored at -80 ⁰C. The protein concentration of the crude membrane samples was measured using a Coomassie blue protein assay employing BSA as a standard (Sedmak and Grossberg 1977). Protein Purification Crude membrane samples were purified by first solubilizing in buffer A (0.3 M sorbitol, 0.1 M NaCl, 6 mM MgCl 2 , 10 mM Tris, pH 7.5, 10% glycerol, 1% aprotinin, 2 mM AEBSF), 1% n-Dodecyl-b-D-maltopyranoside (DDM), and 20 mM imidazole. This solution was rocked at 4 ⁰C for 1 h and then centrifuged at 100,000 ´ g for 45 min. The supernatant was then added to Talon® metal affinity resin beads (Clontech, Inc.) and rocked at 4 ⁰C for 1 h (25 mg of protein to 1 mL of resin). The resulting resin mixture was then washed twice with buffer B (buffer A plus 40 mM imidazole and 1% DDM), once with buffer C (buffer A plus 40 mM imidazole, 1% DDM and 0.5 M KCl), and once with buffer D (buffer A plus 40 mM imidazole, 0.1% DDM and 0.5 M KCl). The protein was finally eluted using buffer E (buffer A plus 250 mM imidazole, 0.1% DDM) into a Amicon® Ultra Centrifugal Filter 30,000 MWCO (Millipore). The sample was then concentrated to desired volume by centrifuging at 4,000 ´ g for 20-30 min at 4 ⁰C and then stored at -80 ⁰C (Anderson et al. 2005). Protein concentration was calculated using an amido black protein assay using BSA as a standard (Schaffner and Weissmann 1973). SDS-PAGE and immunoblot analysis To assess protein purity, 1 mg of protein (diluted in 2X SDS loading buffer (0.5 M Tris-HCl, pH 6.8, 30% sucrose (w/v), 10% sodium dodecylsulfate (w/v), 3.5 M 2-mercaptoethanol and 0.1% bromophenol blue (w/v)) was loaded onto a 4–15% Mini-PROTEAN® TGX™ precast protein gel and run at 165 V for 45 min. The gel was then stained at rt overnight in Coomassie Blue (0.3 M Coomassie Brilliant Blue, 10% HOAc, 40% CH 3 OH) and then de-stained with 10% HOAc/30% CH 3 OH. For immunoblot analysis, 0.05 μg pure protein (also diluted in 2X SDS loading buffer) was loaded onto 4–15% Mini-PROTEAN® TGX™ a precast protein gel and run under similar conditions as described above. The resulting gel was then used to transfer the protein onto a nitrocellulose membrane (Cytiva Amersham™ Protran™ NC Nitrocellulose) at 100 V for 90 min. Membranes were blocked overnight with 20% milk in PBST (1x PBS buffer, 0.1% Tween-20) at 4 ⁰C, followed by treatment for 2 h with the primary antibody (mouse, anti-HA at 1:15,000) in 5% milk in PBST. After washing with PBST, the membrane was incubated for 1 h with the secondary antibody (goat-anti-mouse-HRP, 1:4,000) in 4% milk in PBST. The resulting protein bands were visualized with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and a GeneGnome XRQ (SynGene) instrument. Ste24 activity assay for LC-MS analysis A stock of bulk E. coli lipids in CHCl 3 was placed in a glass scintillation vial, and the chloroform was removed under vacuum in a rotary evaporator at 30 ˚C. Afterward, 150 mM Tris buffer, pH 7.5, was added to give a final lipid concentration of 10 mg/mL in order to hydrate the lipids. The water bath of the rotary evaporator was then heated to 70 ˚C with rotation but no vacuum for 30 min and then fully dissipated by sonication for 30 mins. This solution was stored at -20 ˚C until before usage, when it was further diluted to 0.625 mg/mL in Tris buffer (10 mM Tris•HCl, pH 7.5). A stock solution of Ste24 enzyme in DDM was diluted to 0.15 µg/µL in 10 mM Tris Buffer, pH 7.5. An aliquot (40 µL) of this solution was added to 80 µL of the aforementioned 0.625 mg/mL lipid suspension solution. Afterward, 520 µL of 150 mM Tris•HCl (pH 7.5) was added to break the detergent vesicles and translocate the enzyme into the lipid vesicles. This solution was incubated on ice for 10 min before aliquoting 160 µL into three low-adhesion microcentrifuge tubes and incubating at 30 ˚C for 5 min. Meanwhile, three peptide solutions containing peptides 1a - 3a at 0.15 mM in 150 mM Tris•HCl (pH 7.5) were prepared. A 40 µL aliquot of this solution was added to the enzyme solution, and the mixture was incubated at 30 ˚C for 10 min followed by the addition of HOAc (50 µL) quench the reaction, and 100 µL of CH 3 CN was added to fully solubilize the reaction mixture. Each solution was subjected to LC-MS analysis without filtration. Kinetic Analysis First, a standard curve was created by plotting relative fluorescence units (RFU) versus concentration of the unquenched peptide product (peptide 13 ). To do this, 0.75 μg of purified Ste24 was rapidly reconstituted into 6.25 μg of E.coli polar lipid (Avanti Polar Lipids) in 150 mM Tris-HCl, pH 7.5 and incubated on ice for 10 min. Varying concentrations of peptide 1a (0 to 50 μM) were incubated with the pure WT Ste24 and fluorescence readings were obtained at 30 ⁰C. Fluorescence readings were then plotted versus concentration to create the standard curve. A calibration curve was also created by incubating equimolar amounts of peptides 13 and 14 (0 to 50 μM) with pure WT Ste24 protein using the same conditions as for the standard curve. This calibration curve allowed for the correction of the inner filter effect by calculating a correction factor (C). This was calculated from the ratio of RFU between the two calibration curves at each concentration of each peptide in the assay. The calculated values were then used to multiply the raw RFU units to produce corrected fluorescence values for subsequent analyses. Protein samples were prepared using standard assay conditions as described above and incubated with increasing concentrations of peptides (0 to 50 μM). Fluorescence values were then collected at 30 sec intervals for 1 h using excitation and emission wavelengths of 320 nm and 420 nm respectively. From the fluorescence progress curves, the initial rates were calculated using the first linear region (typically the first 10 min) and converted to specific activities using the extinction coefficient. The specific activities were corrected by multiplying by the correction factor, C, and then plotted against substrate concentration. The kinetic parameters were established by fitting specific activity and substrate concentrations to the Michaelis-Menten equation model {V = V max [S]/(K M + [S])} using Kaleidagraph (v5.0.3). For the cooperative model the equation V = {V = V max [S] n /(K M n + [S] n )} was used. In these equations, [S] is the substrate (peptide) concentration and n is the Hill coefficient. Results Substrate design for testing Ste24 dependence on C-terminal structure To probe the role of the C-terminus in ordering processing by Ste24, three peptide analogs based on the 33 residue C-terminus from the precursor of the mating pheromone a -factor were designed and synthesized (Fig. 3). The first, 1a , contained a C-terminal methyl ester, modeling a substrate for Ste24 containing a fully processed C-terminus. The second peptide analog ( 2a ) had a free C-terminal carboxylate, representing the unmethylated precursor. The final peptide, 3a , contained an unnatural C-terminal amide; that substrate would mimic the neutral character of 1a while eliminating the synthetically more problematic methyl ester. If carboxylmethylation is required for Ste24 cleavage at the upstream site, the methyl ester-containing peptide ( 1a ) would be a significantly better Ste24 substrate than 2a . Given its neutral charge, 3a would also be expected to react at a rate comparable to that of 1a . In contrast, if carboxylmethylation is not a prerequisite, then 2a could have similar Ste24 reactivity. Peptide 1a was synthesized using the side-chain anchoring strategy outlined in Fig. 4 while peptides 2a and 3a were prepared using more routine methods. To measure the activity of these substrates using an assay that avoids the use of radioactivity, each peptide was designed to contain an Abz/Dnp donor-quencher FRET pair flanking the N-terminal cleavage site with the Abz group positioned on the N-terminus and a Lys(Dnp) group located 4 residues downstream of the cleavage site; that donor-quencher pair has been used extensively in related peptides designed to probe the first cleavage that occurs C-terminal to the prenylcysteine residue (Hollander et al. 2000; Kyro et al. 2011; Hildebrandt et al. 2016, 2024; Arachea and Wiener 2017a). After Ste24 N-terminal cleavage, two peptide fragments (Fig. 5) would be liberated, including one with an unquenched Abz fluorophore, thus causing an increase in fluorescence that could be used to measure enzymatic activity. Synthesis of a-Factor 33mer precursor 1a with a C-termin al methyl ester To synthesize the methyl ester-containing peptide analogue 1a , a side-chain-anchoring methodology first developed by Barany et al (Barany et al. 2003) and later optimized by Distefano and coworkers was utilized (Fig. 4) (Diaz-Rodriguez et al. 2012, 2015, 2018; Diaz-Rodriguez and Distefano 2017; Bader et al. 2019; Morstein et al. 2022). In brief, the carboxylic acid of Fmoc-L-cysteine ( 7a) was esterified in methanolic HCl to yield methyl ester 8a with a free side chain thiol. The thiol of 8a was alkylated with trityl chloride resin in the presence of DIPEA resulting in the formation of a side chain-anchored cysteine methyl ester ( 9a ) suitable for SPPS. Next, the full peptide chain was elongated via automated peptide synthesis using standard HCTU/Fmoc chemistry. Single couplings (20 min) were used for all positions except D, G, N, P, Y, K(Dnp), and Abz, which were coupled for 60 mins. To suppress epimerization, those coupling reactions were supplemented with Cl-HOBt (Han et al. 1997; Montalbetti and Falque 2005; Albericio and El-Faham 2018). It is important to note that repeated exposure to HCTU can cause the development of life-threatening anaphylaxis, and so it should be handled with either a respirator or in a well-ventilated fume hood (McKnelly et al. 2020). Additionally, Cl-HOBt can be explosive under certain conditions and as a result should also be handled with care. (Wehrstedt et al. 2005; Malow et al. 2007) Once the complete peptide chain was assembled, the peptide was cleaved and globally deprotected using Reagent K, yielding the crude C-terminal methyl ester-containing peptide 10a with a free thiol (Fig. S1). This peptide was then prenylated with farnesyl bromide under mildly acidic conditions in the presence of Zn(OAc) 2 (Fig. S2) (Xue et al. 1992; Wollack et al. 2009; Bader et al. 2019). A critical consideration for this step was to deoxygenate the solvents properly through N 2 sparging to prevent disulfide formation. Additionally, earlier reports used a solvent mixture containing 0.1% TFA (Diaz-Rodriguez et al. 2012, 2015; Vervacke et al. 2014; Bader et al. 2019); this was found to decrease the reaction yield, likely due to over-acidification of the reaction and an associated decrease in the nucleophilicity of the Zn-thiolate species thought to be involved in the reaction. The original description of this reaction used 0.025% TFA, not 0.1%.(Xue et al. 1992) Improved results were observed when the reaction was buffered to pH 5.0 with sodium acetate (Wollack et al. 2009; Morstein et al. 2022). Prenylated peptide 1a was then purified by HPLC using a two-stage process first involving preparative scale HPLC using a broad range gradient, which resulted in purity of approximately 70% based on 220 nm integration in analytical LC-MS. This peptide was further purified to >95% on a semi-preparative scale using a targeted gradient ranging from 45-55% solvent B over 20 min (Table 1, Fig. S3) to yield pure 1a (Fig. S4). The structure of the desired peptide was confirmed by LC-MS/MS using ETD to minimize the loss of the farnesyl group that is common with these types of peptides. Complete coverage of all z and c type ions was observed (Table S1). The position of the farnesyl group was confirmed from analysis of the C-terminal c and z ions. Table 1. Purification of 33mer peptides based on a -factor. Peptide Crude purity (%) Epimerization in crude product (%) Purity after initial purification (%) Purity after final purification (%) Epimerization after final purification (%) 1a 31 5 70 97 2 2a 20 36 73 95 4 3a 27 <1 75 99 <1 Synthesis of a-factor precursor analogues 2a and 3a Peptide 2a was synthesized using identical conditions to peptide 1a but starting with Fmoc-Cys on Wang resin. Cleavage from resin (Fig. S1) to yield crude 11a , prenylation (Fig. S5) and preparative HPLC purification (Fig. S6) provided pure peptide 2a (Fig. S7). Peptide 3a was synthesized using unloaded Rink MBHA amide resin using the same HCTU/Fmoc chemistry but without Cl-HOBt, as it was found to lower crude peptide purity due to decreased coupling efficiency (Vrettos et al. 2017; Albericio and El-Faham 2018). Double couplings (20 min) were used instead of single couplings for all positions except C, K(Dnp), and Abz, which were all coupled manually for 60 min to allow reaction monitoring using the Ninhydrin test (Kaiser et al. 1970; Vilaseca and Bardaji 1995). Also, an acetic anhydride capping step was added between each coupling cycle to prevent truncated side products that would further complicate the HPLC purification. Cleavage from resin (Fig. S1) to yield crude 12a , prenylation (Fig. S8) and preparative HPLC purification (Fig. S9) gave pure peptide 3a (Fig. S10). The semi-preparative HPLC purification of 2a and 3a was similar to that of 1a but the targeted gradient spanned 40-50% B due to the increased polarity of analogues 2a and 3a (Fig. S5, Fig. S6). The best crude purity obtained for the unprenylated peptides was for 10a synthesized through side chain anchoring methodology, followed by peptide 12a , and then peptide 11a (having the lowest crude purity (Table 1, Fig. S1 ). The structures of 2a and 3a were confirmed by LC-MS/MS using ETD (Tables S2 and S3). For 2a , good coverage including all z ions except z1, z3, z5, z6, z13, z15, z19, and z26 was observed; those missing ions were compensated for by the presence of complementary y ions in all cases except y19. Good coverage of c ions was also observed with this peptide. When missing, those positions were confirmed by overlapping a and b ions for all positions except c12. For 3a , excellent coverage including all z ions except z11 was obtained with y11 being observed instead. Good coverage of c ions was also observed with this peptide with missing ions being confirmed by overlapping a or b ions for all positions (b5, b6, b7, b10, b11, a13, b20, b22, b27, b33) except c12. For both 2a and 3a , the position of the farnesyl group was confirmed from analysis of the C-terminal c and z ions. Epimerization analysis of a-Factor 33mer analogues Before testing the peptides for activity with the Ste24 enzyme, it was essential to determine their enantiomeric purity. C-terminal cysteines are prone to epimerization (Han et al. 1997; Kondasinghe et al. 2017), and it was unknown whether a -Factor containing a D-Cys would have the same reactivity as a-Factor containing L-Cys in Ste24-catalyzed proteolysis. Due to the length of these peptides, it was unlikely that there would be a detectable retention time difference between the two epimeric 33mer peptides in LC-MS analysis. Thus, each of the three peptides was subjected to trypsin digestion, resulting in much shorter 8 residue fragments containing the prenylated cysteines that should be easily resolvable via LC-MS analysis. To confirm this, authentic D-cysteine-containing standards were synthesized. Peptide 4b (Fig. 3) was synthesized using the same procedure required to obtain peptide 1a but starting from Fmoc-D-cysteine hydrate. Peptide 5b (Fig. 3) was produced by hydrolyzing the methyl ester of peptide 4b through a simple saponification reaction with NaOH. Peptide 6b (Fig. 3) was synthesized using the same procedure as peptide 3a but also using Fmoc-D-cysteine hydrate. Analysis of tryptic digests of peptides 1a, 2a, and 3a each showed a major dominant peak in the LC-MS chromatogram for 4a , 5a and 6a , respectively), along with a minor isobaric peak that integrated to 2% ( 4b from 1a ), 4% ( 5b from 2a ) and 95% enantiomerically pure (Fig. 6). To determine whether the high enantiomeric purities of the purified peptides noted above accurately reflected the fidelity of the synthesis or were a consequence of the subsequent HPLC purification, the crude peptides were also subjected to the same trypsin digestion analysis following the prenylation reaction without the HPLC purification. Subjecting these crude peptides to tryptic digestion and subsequent LC-MS analysis showed that for the methyl ester-containing peptide 1a , there was <5% epimerization in the crude peptide (Fig. 7, Table 1). A similar result was observed in the analysis of crude amide-containing peptide 3a . In contrast, LC-MS analysis of the tryptic digest of crude carboxylic acid-containing peptide 2a displayed two peaks, one corresponding to 5a integrating to 64%, and a peak corresponding to the epimer 5b integrating to 36%. Thus, substantial Cys epimerization occurred in the peptide prepared using conventional anchoring through the C-terminal carboxylate. This highlights the advantage of the side-chain anchoring methodology over traditional Wang resin for such peptides. It should be noted that side chain anchoring can also yield C-terminal acids via saponification of the methyl ester (or acidolytic cleavage of C-terminal t-butyl esters) as was done to obtain peptide 5b . In fact, peptide 2a was also prepared in this manner from peptide 1a after only 1 h of incubation with NaOH at room temperature, followed by neutralization with glacial acetic acid (Fig. S11). For especially sensitive cases, bases have been identified that minimize epimerization in this hydrolytic reaction (Nicolaou et al. 2005). Evaluation of peptides as substrates for Ste24 Once the structures and enantiomeric purities of the purified peptides were confirmed, they were subjected to Ste24-catalyzed cleavage using two in vitro assays. First, to confirm that the peptides were cleaved by Ste24 at the correct site, each peptide was incubated with Ste24 for 10 min at 30 ˚C and then subjected to LC-MS analysis. The predicted cleavage products were observed for each peptide (Fig. 5, Fig. S12 ( 1a ), S13 ( 2a ) and S14 ( 3a )), demonstrating that Ste24 cleaved the a -factor analog peptides between the Thr and Ala residues upstream of the farnesyl-modified C-terminus. Although trace levels of non-specific cleavage was observed at high concentrations of peptide, the vast majority of cleavage occurred at the target site. Next, the kinetics of the enzymatic cleavage reactions were studied using an in vitro quantitative fluorescence enzymatic assay with purified and Ste24 reconstituted into membranes. In these assays, the peptide concentration was varied and the initial reaction rate was determined using the increase in Abz fluorescence as the Dnp quencher was proteolytically removed (Fig. 5). To determine specific activities for each peptide with Ste24, a standard curve was first generated to facilitate the conversion of raw fluorescence data from relative fluorescence units (RFU) to concentration. A calibration curve was also used to adjust for the inner filter effect resulting from the internal quenching properties of the Abz/Dnp FRET pair which occurs when using a high concentration of the fluorogenic peptide substrate (Hildebrandt et al. 2016; Arachea and Wiener 2017b). To obtain kinetic parameters for the three peptides ( 1a , 2a and 3a ), purified Ste24 was incubated with each peptide at varying concentrations. After conversion of the fluorescence data from RFU to concentration and adjustments due to inner filter effects, the rates were plotted versus peptide concentration (Fig. 8A). The data was then fit to the Michaelis-Menten equation using a non-linear least-squares algorithm to obtain the kinetic parameters, K M and V max (Table 2). The V max and K M values obtained for the methyl ester substrate, peptide 1a , were 3.3 ´ 10 3 pmol/mg/min and 7.3 μM, respectively. The K M value measured here was comparable to earlier reported values of 11 µM (Pryor et al. 2013), measured using a different assay, and 9.1 µM (Hsu 2018) obtained using the fluorescence assay described here. The V max value is lower, which may be due to variability in the purity and activity of the enzyme preparations. Moreover, the relative insolubility of the peptides resulted in significant scatter in the data at concentrations above 20 µM making determinations of V max challenging. Overall, these data confirm that Ste24 cleaved peptide 1a with similar efficiency as previously determined. For peptide 2a , the C-terminal acid, the K M and V max values obtained were 15 μM and 4.6 ´ 10 3 pmol/mg/min. Lastly, peptide 3a , which contains a neutral C-terminal amide, yielded kinetic values of 11 μM and 6.1 ´ 10 3 pmol/mg/min for K M and V max respectively. It is interesting to note that there appears to be some deviation from Michaelis-Menten behavior with all three peptides and a better fit was obtained by adding a cooperativity term in the equation (Fig. 8B). This type of behavior has been previously observed with other membrane-bound enzymes acting on insoluble substrates (Lister et al. 1988; Burke et al. 1995). While this analysis changed the kinetic constants, it did not reveal any major differences in the relative catalytic efficiencies detected with these different peptide substrates (Table 2). Collectively, the K M and V max values for these three different peptides vary by a factor of 2 or less indicating that the identity of the C-terminal group does not significantly impact the rate of cleavage at the upstream site. From a biological perspective, this is a key finding that suggests that ability of Ste24 to cleave the upstream site in susbtrates that undergo CAAX processing is independent of carboxylmethylation. An important consequence of this is that inhibitors of the methyltransferase ICMT, which may be useful as therapeutic agents for cancer, should not inhibit the processing of prelamin A. Table 2 . Kinetic parameters for Ste24 cleavage of peptides 1a , 2a and 3a . Peptide K M (µM) K M rel V max (nmol/min/mg) V max rel V max /K M V max /K M rel Hill coeff. n c (replicates) Ester ( 1a ) M.M. a Coop. b 7.3 ± 4.5 5.6 ± 1.0 1.0 1.0 3.3 ± 0.70 2.6 ± 0.24 1.0 1.0 0.45 0.46 1.0 1.0 2.2 ± 0.63 4 4 Acid ( 2a ) M.M. a Coop. b 15 ± 5.8 9.2 ± 0.68 2.1 1.6 4.6 ± 0.74 3.2 ± 0.25 1.4 1.2 0.31 0.35 0.68 0.76 4.9 ± 1.8 7 7 Amide( 3a ) M.M. a Coop. b 11 ± 4.8 8.2 ± 1.0 1.5 1.5 6.1 ± 1.0 4.7 ± 0.54 1.8 1.8 0.55 0.57 1.2 1.2 4.6 ± 2.7 6 6 a Values calculated with standard Michaelis-Menten kinetic model. b Values calculated with Michaelis-Menten kinetic model with cooperativity. c The value n corresponds to the number of times the experiment was performed. Conclusions Long hydrophobic peptide sequences are generally challenging to synthesize and manipulate. In the case of the analogs described here, these syntheses were particularly challenging due to the presence of C-terminal cysteines, which are known to be prone to epimerization during the repeated piperidine treatments used for Fmoc deprotection during SPPS (Han et al. 1997). This issue was indeed observed in the analysis of the crude peptides prepared here. Peptide 2a , which was synthesized using standard Wang resin showed 36% epimerization and while it was possible to obtain an enantiomerically pure peptide in the end, this represented the loss of over a third of the material synthesized. In contrast, the side chain anchoring methodology described here led to minimal epimerization prior to HPLC purification and offers simple access to peptides with a C-terminal cysteine acid using a simple saponification reaction. This strategy also gave the highest crude purity, even when compared to the amide containing peptide 3a , which was synthesized with double coupling and acetic anhydride capping, again showing the advantage of the side chain anchoring methodology. Kinetic analysis of the Ste24-catalyzed proteolytic cleavage of 33mer peptides based on the C-terminus of a -factor precursor demonstrated that there was minimal difference between peptides bearing a C-terminal methyl ester, acid or amide. From a biological perspective, this suggests that the upstream cleavage ability of Ste24 is independent of carboxylmethylation. Declarations Supplementary Information The online version contains supplementary material available at … Acknowledgements This work was supported by the National Science Foundation (NSF/CHE-1905204). Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request Author Contribution T.B. and S. B. performed the experiments and contributed to writing the manuscript. C. H. and M. D. directed the research, obtained funding and contributed to writing the manuscript and edited subsequent versions. References Ahearn IM, Court HR, Siddiqui F, et al (2021) NRAS is unique among RAS proteins in requiring ICMT for trafficking to the plasma membrane. Life Sci Alliance 4:e202000972. https://doi.org/10.26508/LSA.202000972 Albericio F, El-Faham A (2018) Choosing the Right Coupling Reagent for Peptides: A Twenty-Five-Year Journey. Org Process Res Dev 22:760–772. https://doi.org/10.1021/acs.oprd.8b00159 Anderson JL, Frase H, Michaelis S, Hrycyna CA (2005) Purification, Functional Reconstitution, and Characterization of the Saccharomyces cerevisiae Isoprenylcysteine Carboxylmethyltransferase Ste14p. 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Supplementary Files BaderSI091524.pdf Cite Share Download PDF Status: Published Journal Publication published 06 Jan, 2025 Read the published version in International Journal of Peptide Research and Therapeutics → Version 1 posted Editorial decision: Revision requested 01 Oct, 2024 Reviews received at journal 01 Oct, 2024 Reviews received at journal 30 Sep, 2024 Reviewers agreed at journal 24 Sep, 2024 Reviewers agreed at journal 24 Sep, 2024 Reviews received at journal 24 Sep, 2024 Reviews received at journal 24 Sep, 2024 Reviewers agreed at journal 24 Sep, 2024 Reviewers agreed at journal 23 Sep, 2024 Reviewers agreed at journal 23 Sep, 2024 Reviewers agreed at journal 22 Sep, 2024 Reviewers agreed at journal 22 Sep, 2024 Reviewers invited by journal 22 Sep, 2024 Editor assigned by journal 22 Sep, 2024 Submission checks completed at journal 17 Sep, 2024 First submitted to journal 15 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5094096","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":361132562,"identity":"5960c514-8329-434a-b2b9-23a9a37e930b","order_by":0,"name":"Taysir K. Bader","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Taysir","middleName":"K.","lastName":"Bader","suffix":""},{"id":361132563,"identity":"64fc8eae-e4fa-4617-9dff-f6086d113fe5","order_by":1,"name":"Shanica M. Brown","email":"","orcid":"","institution":"Purdue University","correspondingAuthor":false,"prefix":"","firstName":"Shanica","middleName":"M.","lastName":"Brown","suffix":""},{"id":361132564,"identity":"81f4aa06-22d9-4ff3-aa9f-f536244003e5","order_by":2,"name":"Christine A. Hrycyna","email":"","orcid":"","institution":"University of California, San Diego","correspondingAuthor":false,"prefix":"","firstName":"Christine","middleName":"A.","lastName":"Hrycyna","suffix":""},{"id":361132565,"identity":"351ba2d3-a49b-4d4d-9490-178e11548b21","order_by":3,"name":"Mark D. Distefano","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYDCCwyDCQIKHH8JlJkoLYwNDhY2MZAPRWg6AtJxJszE4QKwWvuPszx98bDvMY3wjO+0BQ4V1YgMhLZKHeQwbZwK1mN3I3W7AcCadsBaDwzyMzbwQLdskGNsOE6OF/WHzX5DDZoC0/CNKC4NhM9D7PAYSIC0NRGgB+WVmT4UNj8SZt9skEo6lGxPUwnf++IMPPwwk7PnbgbZ8qLGWJagFFSSQpnwUjIJRMApGAS4AACAQQTf5lbgNAAAAAElFTkSuQmCC","orcid":"","institution":"University of Minnesota","correspondingAuthor":true,"prefix":"","firstName":"Mark","middleName":"D.","lastName":"Distefano","suffix":""}],"badges":[],"createdAt":"2024-09-15 20:14:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5094096/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5094096/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10989-024-10677-9","type":"published","date":"2025-01-06T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70177233,"identity":"a4deec6b-77a4-456c-a8d8-cbabc2fe6a4f","added_by":"auto","created_at":"2024-11-29 07:45:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75588,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the 3-step posttranslational processing pathway for CaaX proteins using farnesylation as an example. The farnesyl group is shown in blue, the C-terminal -aaX tripeptide that is removed is shown in green and the methyl group donated from SAM that is transferred to the C-terminal carboxylate is shown in purple. Cellular membranes are shown in red.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5094096/v1/be689cadbde4b00c700e614c.png"},{"id":70177517,"identity":"4d3eb282-f304-47cd-b1b8-620a8503d6af","added_by":"auto","created_at":"2024-11-29 07:53:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80972,"visible":true,"origin":"","legend":"\u003cp\u003eSimilarities in protein processing to yield lamin A and yeast \u003cstrong\u003ea\u003c/strong\u003e-factor. Prelamin A is the only \u003cem\u003ebona fide\u003c/em\u003esubstrate for human ZMPSTE24 and yeast \u003cstrong\u003ea\u003c/strong\u003e-factor is known to be a substrate for the yeast homolog Ste24. Both prelamin A and \u003cstrong\u003ea\u003c/strong\u003e-factor precursor undergo similar processing: first, the three classic prenylation steps are carried out, followed by an additional upstream cleavage by either ZMPSTE24 (for prelamin A) or Ste24 (for \u003cstrong\u003ea\u003c/strong\u003e-factor). Both enzymes can act on either substrate. \u003cstrong\u003ea\u003c/strong\u003e-Factor undergoes a second cleavage step by Axl1 before being exported for mating. The function of the C-terminal prenylated fragment derived from prelamin A is unknown. The R group represents the remainder of the farnesyl chain.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5094096/v1/859f593f387473d5f71740f4.png"},{"id":70178532,"identity":"1255d729-dcbf-4ce2-b6bc-09d2ee2e4a2c","added_by":"auto","created_at":"2024-11-29 08:01:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24217,"visible":true,"origin":"","legend":"\u003cp\u003eSynthetic 33mer \u003cstrong\u003ea\u003c/strong\u003e-factor analog peptides and their corresponding C-terminal trypsin digest fragments. Three peptides were designed based on the structure of an a-factor 33mer precursor, but with varying C-termini. The corresponding C-terminal tryptic digestion fragments are also shown as they were used to evaluate the extent of epimerization of Cys. These fragments have a much better chromatographic resolution between the two epimers, allowing accurate quantitation of epimerization. Asterisks indicates epimerization prone site.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5094096/v1/d5b48ff06b600cb660f47a87.png"},{"id":70177235,"identity":"1f3cc8c5-bc25-4730-b844-e2ad9aa19a13","added_by":"auto","created_at":"2024-11-29 07:45:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":30549,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis of the 33mer \u003cstrong\u003ea\u003c/strong\u003e-factor analog peptide with a C-terminal methyl ester, \u003cstrong\u003e1a\u003c/strong\u003e. The asterisk represents an epimerization prone site.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5094096/v1/e6825f116236c9a0e1516fae.png"},{"id":70178531,"identity":"8af21b69-19b0-4787-aeac-aa6feb84ff49","added_by":"auto","created_at":"2024-11-29 08:01:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":50576,"visible":true,"origin":"","legend":"\u003cp\u003eSubstrate-based Ste24 activity assay. The upstream cleavage of \u003cstrong\u003ea\u003c/strong\u003e-factor analog peptides \u003cstrong\u003e1a\u003c/strong\u003e, \u003cstrong\u003e2a\u003c/strong\u003e, and \u003cstrong\u003e3a\u003c/strong\u003e with Ste24 liberates two peptide fragments. The N-terminal fragment (\u003cstrong\u003e13\u003c/strong\u003e) is identical for all peptides, but the C-terminal fragment is unique for each analogue (\u003cstrong\u003e14a\u003c/strong\u003e, \u003cstrong\u003e15a\u003c/strong\u003e and \u003cstrong\u003e16a\u003c/strong\u003e). Asterisk represents an epimerization prone site. In this assay, 2-aminobenzoic acid (Abz) is the fluorophore (eex = 320 nm, eem = 420 nm) that is quenched when in proximity to the 2,4-dinitrophenyl (Dnp) group, which is the case in the parent peptide. Cleavage by Ste24 uncouples the fluorophore from the quencher, resulting in an increase in Abz fluorescence that can be used to measure the activity of Ste24.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5094096/v1/0ee762688aca345e604a48a0.png"},{"id":70177239,"identity":"1816e1b6-2211-4946-a0d5-8104a6e0339a","added_by":"auto","created_at":"2024-11-29 07:45:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":185325,"visible":true,"origin":"","legend":"\u003cp\u003eTryptic digests of \u003cu\u003epurified\u003c/u\u003e 33mer peptides to evaluate enantiomeric purity. Each peptide was digested with trypsin and then analyzed by LC-MS. The resulting C-terminal 8-mer fragment resulting from cleavage at the K-G site allowed resolution of the epimeric peptides containing L-Cys or D-Cys (top chromatograms). To confirm the peak assignments for the minor epimers, the corresponding authentic standards were synthesized and co-injected with the trypsin-digested peptides (bottom chromatograms). (A) LC-MS chromatograms of peptide \u003cstrong\u003e1a\u003c/strong\u003e after trypsin digestion to produce \u003cstrong\u003e4a\u003c/strong\u003e before (top) and after (bottom) addition of authentic standard \u003cstrong\u003e4b\u003c/strong\u003e. (B) LC-MS chromatograms of peptide \u003cstrong\u003e2a\u003c/strong\u003e after trypsin digestion to produce \u003cstrong\u003e5a\u003c/strong\u003e before (top) and after (bottom) addition of authentic standard \u003cstrong\u003e5b\u003c/strong\u003e. (C) LC-MS chromatograms of peptide \u003cstrong\u003e3a\u003c/strong\u003e after trypsin digestion to produce \u003cstrong\u003e6a\u003c/strong\u003e before (top) and after (bottom) addition of authentic standard \u003cstrong\u003e6b\u003c/strong\u003e. UV absorbance was monitored at 220 nm. Gradient: 1-5 min, hold at 1% B. 5-55 min, ramp to 100% B. Note that the limit of detection was 1% in this assay.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5094096/v1/923a28c59caf38579aaf0393.png"},{"id":70177519,"identity":"6a46294e-fffd-4f4e-bc15-2245565ff0b7","added_by":"auto","created_at":"2024-11-29 07:53:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":185718,"visible":true,"origin":"","legend":"\u003cp\u003eTryptic digests of \u003cu\u003ecrude\u003c/u\u003e 33mer peptides to evaluate enantiomeric purity. Each peptide was isolated after synthesis using a Sep-Pak disposable cartridge instead of the two-stage HPLC purification. This preserved side products and impurities present in the samples after synthesis. \u0026nbsp;Each peptide was digested with trypsin and then analyzed by LC-MS. The resulting C-terminal 8-mer fragment resulting from cleavage at the K-G site allowed resolution of the epimeric peptides containing L-Cys or D-Cys (top chromatograms). To confirm the peak assignments for the minor epimers, the corresponding authentic standards were synthesized and co-injected with the trypsin-digested peptides (bottom chromatograms). (A) LC-MS chromatograms of peptide \u003cstrong\u003e1a\u003c/strong\u003e after trypsin digestion to yield \u003cstrong\u003e4a\u003c/strong\u003e before (top) and after (bottom) addition of authentic standard \u003cstrong\u003e4b\u003c/strong\u003e. (B) LC-MS chromatograms of peptide \u003cstrong\u003e2a\u003c/strong\u003e after trypsin digestion to yield \u003cstrong\u003e5a\u003c/strong\u003e before (top) and after (bottom) addition of authentic standard \u003cstrong\u003e5b\u003c/strong\u003e. (C) LC-MS chromatograms of peptide \u003cstrong\u003e3a\u003c/strong\u003e after trypsin digestion to yield \u003cstrong\u003e6a\u003c/strong\u003e before (top) and after (bottom) addition of authentic standard \u003cstrong\u003e6b\u003c/strong\u003e. UV absorbance was monitored at 220 nm. Gradient: 1-5 min, hold at 1% B. 5-55 min, ramp to 100% B.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5094096/v1/a9f5bfd17c2853873399f6c5.png"},{"id":70177237,"identity":"05600f33-95a3-402c-9ded-69148b8ce597","added_by":"auto","created_at":"2024-11-29 07:45:11","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":36255,"visible":true,"origin":"","legend":"\u003cp\u003eKinetic analysis of Ste24-catalyzed cleavage of peptides \u003cstrong\u003e1a\u003c/strong\u003e, \u003cstrong\u003e2a\u003c/strong\u003e and \u003cstrong\u003e3a\u003c/strong\u003e. (A) Data fit to Michaelis-Menten model; (B) Data fit to Michaelis-Menten model with cooperativity. Each point shown here represents the average value obtained across all data sets for a given concentration. A total of 4 data sets were obtained with \u003cstrong\u003e1a\u003c/strong\u003e, 7 data sets with \u003cstrong\u003e2a\u003c/strong\u003e and 6 data sets with \u003cstrong\u003e3a\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5094096/v1/8a992e89040d65a4fcc75574.png"},{"id":73693899,"identity":"80da6657-de7d-4b9c-bdf5-a7377b76ca7c","added_by":"auto","created_at":"2025-01-13 16:09:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1759262,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5094096/v1/44f49a56-cec0-4fa4-b961-f9ae92f7a826.pdf"},{"id":70177241,"identity":"f658fa7f-5677-49dc-8d68-bb3053b0399d","added_by":"auto","created_at":"2024-11-29 07:45:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":6110618,"visible":true,"origin":"","legend":"","description":"","filename":"BaderSI091524.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5094096/v1/8fe2cf3baf17f818c716418f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Upstream proteolysis by Ste24 does not require a C-terminal methyl ester as revealed using 33-residue a-factor precursor peptide substrates synthesized via epimerization-free methods","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProtein prenylation is a post-translational modification that links certain cytosolic proteins with a hydrophobic isoprenoid lipid. The primary purpose of this modification is to facilitate membrane localization and association, although there are examples of prenylated proteins that remain cytosolic (Casey 1992; Palsuledesai and Distefano 2015). Prenylated proteins are involved in numerous signal transduction pathways (Gelb et al. 2006; Berndt et al. 2011; Palsuledesai and Distefano 2015; Wang and Casey 2016), and often are initially synthesized with a C-terminal CaaX sequence, where C is cysteine, a is generally an aliphatic residue and X can be one of a number of amino acids. One of the most common forms of prenylation, farnesylation, occurs as the first of three enzymatic steps in the CaaX protein post-translational processing pathway (Fig. 1). The first step is the transfer of an isoprenoid moiety to the cysteine of the C-terminal CaaX sequence(Wang and Casey 2016). The isoprenoid can be either a farnesyl (3 isoprene repeats, 15 carbons) or a longer geranylgeranyl (4 isoprene repeats, 20 carbons) chain, which are added by either protein farnesyltransferase (FTase) or types 1, 2, or 3 geranylgeranyltransferase (GGTase I, II or III) respectively (Casey 1992; Palsuledesai and Distefano 2015). The second step in the pathway is the endoproteolytic removal of the aaX sequence by zinc metalloprotease sterile 24 (ZMPSTE24) or Ras converting CaaX endopeptidase 1 (Rce1) (Ma et al. 1992; Ashby et al. 1992; Trueblood et al. 2000). The final step is carboxyl methylesterification of the newly exposed C-terminal prenylcysteine by protein-\u003cem\u003eS\u003c/em\u003e-isoprenylcysteine O-methyltransferase (ICMT)(Hrycyna and Clarke 1990; Stephenson and Clarke 1990; Marr et al. 1990; Hrycyna et al. 1991; Pillingers et al. 1994; Dai et al. 1998). The resulting proteins are then shuttled to various membranes or further processed to carry out their biological functions\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZMPSTE24 is a unique membrane-bound zinc metalloprotease localized to the endoplasmic reticulum and the inner nuclear membrane (Schmidt et al. 1998; Barrowman et al. 2008). In addition to its function of endoproteolytic cleavage of aaX tripeptides from the C-termini of prenylated proteins that is similar to that of Rce1 (Schmidt et al. 1998; Porter et al. 2007), ZMPSTE24 can also catalyze a second site-specific cleavage upstream from the prenylated cysteine (Tam et al. 1998); interestingly, there is no obvious sequence similarity between the two different cleavage sites. The mechanism behind this bifunctional behavior is poorly understood, yet quite important in human health. The only \u003cem\u003ebona fide\u003c/em\u003e human substrate of ZMPSTE24 is the prenylated protein prelamin A (Corrigan et al. 2005; Barrowman et al. 2012; Casasola et al. 2016; Babatz et al. 2021). ZMPSTE24-catalyzed cleavage at two different sites of that protein leads to the release of a 15-residue prenylated peptide from the C-terminus along with the mature nuclear scaffold protein lamin A (Fig. 2). While lamin A is essential for properly forming the nuclear lamina and providing mechanical stability, the function of the 15-residue prenylated peptide product is unknown (Casasola et al. 2016). Mutations in ZMPSTE24 that prevent the second upstream cleavage result in accelerated aging progeroid diseases (Young et al. 2005; Fong et al. 2009; Yang et al. 2010; Gordon et al. 2018). Thus, a better understanding of ZMPSTE24 function and its substrate recognition would be helpful for both the development of treatments for progeroid diseases and for providing more detailed molecular insights into the process of human aging.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIntriguingly, in recent years, potentially new functions have been ascribed to ZMPSTE24 including that of a \u0026ldquo;translocon unclogger\u0026rdquo;, where misfolded proteins from the translocon are cleared during signal recognition-particle-independent protein translocation (Ast et al. 2016; Kayatekin et al. 2018). It was observed that ZMPSTE24 could clear misfolded human islet amyloid polypeptide, which is common in patients with type 2 diabetes (Kayatekin et al. 2018). This has led to the suggestion that ZMPSTE24 plays a critical role in the ER-associated degradation pathway (Avci and Lemberg 2015), and that its downstream substrates may be druggable targets (Goblirsch and Wiener 2020). Perhaps more surprisingly, some evidence suggests that ZMPSTE24 serves as an \u0026ldquo;intrinsic broad-spectrum antiviral protein\u0026rdquo; that is recruited to prevent the fusion of viral membranes and endosomal membranes (Guo et al. 2021), thus protecting against viral infections such as influenza and even SARS-CoV-2 (Fu et al. 2017; Shilagardi et al. 2022). These results cast doubt on the idea that its primary cellular function is to be CaaX protease (Goblirsch and Wiener 2020). Clearly, a better understanding of how ZMPSTE24 recognizes its substrates is critical for understanding its cellular function.\u003c/p\u003e\n\u003cp\u003eZMPSTE24 has several intriguing structural features. It consists of seven transmembrane \u0026alpha;-helices that together form a novel \u0026ldquo;\u0026alpha;-barrel,\u0026rdquo; which includes a large (\u0026gt; 12,000 \u0026Aring;\u003csup\u003e3\u003c/sup\u003e) chamber where substrate binding and catalysis take place. Proteolytic activity requires an HExxH zinc metalloprotease consensus sequence, where the two histidines and the glutamate residue bind a catalytic zinc atom (Jongeneel et al. 1989; Clark et al. 2017). For CaaX processing, it has been proposed that the endoproteolytic cleavage of the -aaX motif and carboxylmethylation by ICMT may occur before the upstream cleavage step, which would first involve substrate entry into the ZMPSTE24 cavity, -aaX cleavage, product release, ICMT-catalyzed methylation and reentry into the enzyme reaction chamber for the upstream cleavage event (Michaelis et al. 1992; Fujimura-Kamada et al. 1997; Boyartchuk et al. 1997; Tam et al. 1998; Schmidt et al. 2000; Barrowman et al. 2008; Barrowman and Michaelis 2013). However, to date, there has been no conclusive evidence to confirm the validity of that model. If carboxylmethylation is an absolute prerequisite for the upstream cleavage step, then inhibition of ICMT would lead to laminopathy symptoms similar to progeroid diseases, since unmethylated lamin A would retain the C-terminal 15 residue prenylated peptide. ICMT inhibition is currently being considered as potential therapeutic target for some Ras-based cancers (Bergo et al. 2004; Bergman et al. 2011; Ahearn et al. 2021), and thus it is essential to clarify this question. There is a possible precedent for this effect with HIV aspartyl protease inhibitor drugs, which were found to cause lipodystrophy through off-target inhibition of ZMPSTE24, thus leading to the accumulation of farnesylated prelamin A (Coffinier et al. 2007; Clarke 2007; Hudon et al. 2008; Clark et al. 2017).\u003c/p\u003e\n\u003cp\u003eSte24 is the yeast homolog of ZMPSTE24 (Michaelis and Barrowman 2012; Barrowman and Michaelis 2013; Goblirsch and Wiener 2020) and the two enzymes share significant sequence and structural similarity (Pryor et al. 2013; Clark et al. 2017; Goblirsch et al. 2020). Importantly, those similarities extend to their function as well; both enzymes can process their homolog\u0026rsquo;s substrates, and ZMPSTE24 has been found to rescue the function of yeast mutants lacking Ste24 activity (Tam et al. 1998). However, Ste24 is more amenable to purification and functional assays, making it more attractive for biochemical experiments (Boivin et al. 1993; Hrycyna and Clarke 1993). To address the question as to whether a Ste24 substrate must first undergo processing at the C-terminus before upstream cleavage can occur, a series of 33-residue \u003cstrong\u003ea\u003c/strong\u003e-factor analog precursor peptides containing a fluorescent donor-quencher pair were synthesized. These peptides incorporated either a C-terminal methyl ester to mimic the native substrate, the free acid to mimic an unmethylated peptide or an amide to mimic a differently modified C-terminus. These peptides were used as substrates for a quantitative \u003cem\u003ein vitro\u003c/em\u003e fluorescence-based proteolytic cleavage assay. The synthesis of these peptides presented several synthetic challenges due to the presence of a C-terminal methyl ester, an epimerization-prone C-terminal cysteine and an acid-sensitive farnesyl group. The synthesis of those peptides and an analysis of their cleavage at the upstream site catalyzed by Ste24 are reported here.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHPLC grade H\u003csub\u003e2\u003c/sub\u003eO and CH\u003csub\u003e3\u003c/sub\u003eCN, and sequencing grade dimethyl formamide (DMF), were purchased from Fisher Scientific. Protected amino acids, resins, O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 6-Chloro-1-hydroxybenzotriazole (Cl-HOBt) were purchased from Chem-Impex International. C-18 reverse phase Sep-Paks\u003csup\u003e\u0026Ograve;\u003c/sup\u003e (820 mg, 55-105 \u0026micro;m particle size) were purchased from Waters corporation. Sequencing-grade Trypsin was purchased from Promega and reconstituted according to the manufacturer\u0026rsquo;s specifications. Bulk \u003cem\u003eE. coli\u003c/em\u003e polar lipids were purchased from Avanti Polar Lipids.\u0026nbsp;n-Dodecyl-B-D-maltopyranoside (DDM) was purchased from Anatrace. TALON\u003csup\u003e\u0026Ograve;\u003c/sup\u003e metal affinity resin was purchased from Clontech. Amicon\u0026reg; Ultra Centrifugal Filters (30,000 MWCO) were purchased from Millipore.\u0026nbsp;All other reagents were purchased from Sigma-Aldrich and were used without further purification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.2.1.1 | Synthesis of Fmoc-\u003csub\u003eL\u003c/sub\u003e-Cys-OMe \u003cstrong\u003e7a\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFmoc-\u003csub\u003e\u0026nbsp;L\u003c/sub\u003e-cysteine hydrate (\u003cstrong\u003e7a\u003c/strong\u003e, 2 g, 5.54 mmol) was dissolved in 15 mL CH\u003csub\u003e3\u003c/sub\u003eOH, and 6 drops of concentrated HCl were added to catalyze the reaction. The solution was stirred for 24 h, affording a white slurry that was dissolved with acetone. The solvent was then removed by rotary evaporation at 25 \u0026deg;C, and the material was dried under vacuum for 5 h yielding Fmoc-\u003csub\u003e\u0026nbsp;L\u003c/sub\u003e-Cys-OMe (\u003cstrong\u003e8a\u003c/strong\u003e, 1.96 g, 99% yield) as a white solid. The \u003csup\u003e1\u003c/sup\u003eH NMR in CDCl\u003csub\u003e3,\u003c/sub\u003e was consistent with previously reported data (Diaz-Rodriguez et al. 2015).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis of Fmoc-D-Cys-OMe \u003cstrong\u003e7b\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFmoc-\u003csub\u003e\u0026nbsp;D\u003c/sub\u003e-cysteine hydrate (\u003cstrong\u003e7b\u003c/strong\u003e, 1 g, 2.77 mmol) was dissolved in 15 mL CH\u003csub\u003e3\u003c/sub\u003eOH, and 6 drops of concentrated HCl were added to catalyze the reaction. The solution was stirred for 24 h, affording a white slurry that was redissolved in acetone. The solvent was then removed by rotary evaporation at 25 \u0026deg;C, and the material was dried under vacuum for 5 h yielding Fmoc-D-Cys-OMe (\u003cstrong\u003e8b\u003c/strong\u003e, 0.97 g, 99% yield) as a white solid. The \u003csup\u003e1\u003c/sup\u003eH NMR in CDCl\u003csub\u003e3,\u003c/sub\u003e was consistent with previously reported data (Diaz-Rodriguez et al. 2015).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResin loading for the production of peptides with \u003csub\u003eL\u003c/sub\u003e-Cys-OMe C-terminus \u003cstrong\u003e9a\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo load \u003cstrong\u003e8a\u003c/strong\u003e onto the solid support, 0.82 g (1.44 mmol) of trityl-chloride resin (100-200 mesh, 1.75 mmol/g loading) was placed in a polypropylene filter syringe and washed with CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003efor 1 min 3x. Compound \u003cstrong\u003e8a\u003c/strong\u003e (1.96 g, 5.48 mmol) was then dissolved in 9 mL CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e (600 mM) along with 995 \u0026micro;L (5.74 mmol, 600 mM) DIPEA and added to the resin which was placed on a rotator for 24 h. Unreacted positions were then capped by adding 1 mL CH\u003csub\u003e3\u003c/sub\u003eOH to the solution and allowing it to rotate for 15 min. The resin was subsequently washed with CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003efor 2 min 3x and dried under a vacuum. To quantify resin loading, three samples of 10 mg were weighed and then placed in a filter syringe. Each sample was subjected to Fmoc deprotection with 1 mL 20% piperidine in DMF for 30 min. These solutions were then transferred to 25 mL volumetric flasks, and EtOH was used to wash the resins in 5 mL batches and then transferred to the same flasks until the fill lines were reached. A standard curve was then constructed consisting of Fmoc-OSu in EtOH at 1 mM, 0.75 mM, 0.5 mM, 0.25 mM, 0.125 mM, and 0.0625 mM concentrations. The absorbance of the standard curve and samples were all read in triplicate at 301 nm. A resin loading of 0.12 mmol/g was obtained.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResin loading for the production of peptides with D-Cys-OMe C-terminal \u003cstrong\u003e9b\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo load \u003cstrong\u003e8b\u003c/strong\u003e onto the solid support, 0.17 g (0.323 mmol) of trityl-chloride resin (100-200 mesh, 1.90 mmol/g loading) was placed in a polypropylene filter syringe and washed with CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003efor 1 min 3x. Compound \u003cstrong\u003e8b\u003c/strong\u003e (0.22g g, 0.616 mmol)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewas then dissolved in 2 mL CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e (final concentration 300 mM) along with 220 \u0026micro;L (1.26 mmol, final concentration 300 mM) DIPEA and added to resin which was placed on a rotator for 24 h. Unreacted positions were then capped by adding 0.5 mL CH\u003csub\u003e3\u003c/sub\u003eOH to the solution and allowing it to rotate for 15 min. The resin was subsequently washed with CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003efor 2 min 3x and dried under a vacuum. The resin was quantified in the same manner as \u003cstrong\u003e9a\u003c/strong\u003e. A resin loading of 1.49 mmol/g was obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeptide synthesis, prenylation and characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGeneral Procedure for Peptide Synthesis on Gyros PS3 automated synthesizer\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eUnprenylated precursors to peptides \u003cstrong\u003e1a\u003c/strong\u003e and \u003cstrong\u003e2a\u003c/strong\u003e were synthesized using a Gyros PS3 automated peptide synthesizer employing Fmoc/HCTU-based chemistry. Resins (0.15 mmol) for the appropriate peptide (Fmoc-Cys-OMe Trt resin for peptide \u003cstrong\u003e1a\u003c/strong\u003e, or Fmoc-Cys(Trt)-OH Wang resin (100-200 mesh) for peptide \u003cstrong\u003e2a\u003c/strong\u003e were placed in a reaction vessel, and swelled in DMF for 10 min 3x. The Fmoc group on the first amino acid was then removed using 20% piperidine in DMF for 5 min 2x. Four equiv of the subsequent amino acid were activated with an equimolar amount of HCTU in 2 mL DMF with 800 mM DIPEA and 300 mM Cl-HOBt for 3 min. This solution was then transferred to the resin, and 2 mL of DMF was used to wash the amino acid vial before being transferred to the reaction vessel, resulting in an amino acid/HCTU/Cl-HOBt concentration of 150 mM and a DIPEA concentration of 400 mM. The coupling was carried out for either 20 or 60 min with N\u003csub\u003e2\u003c/sub\u003e-mediated mixing for 1 s every 10 s. D, G, N, P, Y, K(Dnp), and Abz were coupled for 60 min. All other amino acids were coupled for 20 min. After all amino acids were coupled, a final Fmoc deprotection step was carried out and the resin was washed with CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e for 5 min (3x) and then dried in vacuo.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGeneral Procedure for Peptide Synthesis on Gyros Chorus automated synthesizer\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eUnprenylated precursor to peptide \u003cstrong\u003e3a\u003c/strong\u003e was prepared using a Gyros Chorus automated peptide synthesizer. Low-loading rink amide MBHA resin (0.2 mmol, 100-200 mesh) was placed in a polypropylene syringe with a stopcock and swelled in 5 mL DMF for 10 min 3x. The Fmoc group was removed from the resin by incubating with 5 mL 20% piperidine in DMF for 5 min 2x, then washed with 5 mL DMF for 2 min 3x. Fmoc-Cys(Trt)-OH (468 mg, 0.8 mmol, 4 equv) and 331 mg (0.8 mmol, 4 equiv) of HCTU were dissolved in 5 mL of 400 mM M DIPEA (150 mM final conc) and added to the resin, placed on a rotator and allowed to react for 1 h, after which the Ninhydrin test showed complete consumption of the amine (Kaiser et al. 1970; Vilaseca and Bardaji 1995). The resin was washed with DMF as above and placed in the instrument\u0026rsquo;s reaction vessel. The resin was then swelled in 10 mL DMF for 10 min 3x and the Fmoc group was removed using 10 mL 20% piperidine in DMF for 5 min, 2x. Subsequent amino acids (5 equiv) were activated with an equimolar amount of HCTU in 400 mM DIPEA at 150 mM and added to the resin. The coupling was carried out for 20 min, after which the resin was washed with 10 mL for 30 secs 3x, then the coupling was repeated, and the resin was washed again. After the coupling, any unreacted positions were capped using 50 % Ac\u003csub\u003e2\u003c/sub\u003eO in 400 mM DIPEA for 15 min before washing again with DMF. The Fmoc group was then removed using 10 mL of 20% piperidine for 5 min 2x before washing with DMF. K(Dnp) and Abz were also coupled manually using the same procedure as the first amino acid, and each required 1 h for reaction completion. After all amino acids were coupled, a final Fmoc deprotection step was carried out and the resin was washed with CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e for 1 min 6x and then dried in vacuo.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGeneral Procedure for Peptide Cleavage\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePeptide cleavage and global side chain deprotection were carried out by first placing an aliquot of 0.075 \u0026ndash; 0.1 mmol of the peptide on-resin in a polypropylene filter syringe with a polypropylene Luer cap. Reagent K (10 mL, 82% TFA, 2.5% ethanedithiol, 5% thioanisole, 5% phenol, and 5% H\u003csub\u003e2\u003c/sub\u003eO ) was added to the syringe and rotated for 2 h. Next, the solution was drained into a 50 mL polypropylene centrifuge tube, and TFA (10 mL) was used to wash the resin in 2 mL batches. A gentle N\u003csub\u003e2\u0026nbsp;\u003c/sub\u003estream was used to evaporate excess TFA over an additional h until approximately 2 mL of solution remained. The peptide was then precipitated by adding Et\u003csub\u003e2\u003c/sub\u003eO to the 50 mL mark and cooling in a dry ice/\u003cem\u003ei\u003c/em\u003e-PrOH bath. The peptide was then pelleted by centrifugation at 3,000 x g for 5 min. This procedure was repeated twice, with resuspension of the solid peptide in fresh Et\u003csub\u003e2\u003c/sub\u003eO through vortexing for 2 min. After the third Et\u003csub\u003e2\u003c/sub\u003eO precipitation, the tube was placed uncovered in a fume hood for 1 h to dry. Next, 3 mL of HOAc and 2 mL of H\u003csub\u003e2\u003c/sub\u003eO were added to the peptide, and incubated at rt for 10 min to allow the solid to fully dissolve. The solution was then diluted to 10 mL with H\u003csub\u003e2\u003c/sub\u003eO, flash-frozen in liquid N\u003csub\u003e2\u003c/sub\u003e, and then lyophilized. This solubilization and lyophilization process was crucial for two reasons: First, it facilitated the complete deprotection of the tryptophan side chain Boc protecting groups, which we have observed to be sluggish and results in the observation of a +44 Da side product believed to be a carbamic acid intermediate. Second, this procedure improved the solubility of the peptides in DMF for the subsequent prenylation step.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGeneral Procedure for Peptide Prenylation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll the solvents used in this procedure were sparged with N\u003csub\u003e2\u003c/sub\u003e for 3 h to deoxygenate them and prevent disulfide formation. DMF was added to the lyophilized peptide to dissolve it and then Ellman\u0026rsquo;s assay was used to quantify the amount of free thiol in the solution.(Riener et al. 2002) If the concentration was significantly higher than 1 mM, then it was adjusted to that concentration with more DMF. LC-MS analysis was used to confirm the presence of the peptide before proceeding with the reaction. Once confirmed, farnesyl bromide (5 equiv) was diluted 10-fold v/v in DMF and then added dropwise to the peptide solution. The centrifuge tube was then vortexed for 30 sec to fully dissolve the farnesyl bromide. Zn(OAc)\u003csub\u003e2\u003c/sub\u003e\u0026bull;H\u003csub\u003e2\u003c/sub\u003eO (5 equiv) were dissolved 2 M NaOAc, pH. 5.0. The buffer volume was determined based on the volume of DMF used so that the final solvent composition was 9:1 DMF/2M NaOAc buffer. Once the Zn(OAc)\u003csub\u003e2\u003c/sub\u003e\u0026bull;H\u003csub\u003e2\u003c/sub\u003eO was fully dissolved, it was added to the peptide solution, and the tube was vortexed for 30 sec before being placed on a rotator overnight. The next day, LC-MS was used to confirm the completion of the reaction (\u0026gt;90% conversion). Once complete, 5% HOAc was added to the solution to both quench the reaction and help maintain peptide solubility. The solution was then filtered through a 0.2 \u0026micro;m GHP syringe filter and purified by HPLC. It is essential to do this step promptly, or the peptide will precipitate out of the solution.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGeneral Method for LC-MS analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLC-MS analysis was performed using an Agilent 1200 series system (Windows 10, ChemStation Software, G1322A Degasser, G1312A binary pump, G1329A autosampler, G1315B diode array detector, 6130 quadrupole) equipped with a C18 column (Agilent ZORBAX 300-SB-C18, 5 \u0026mu;M, 4.6 X 250 mm). Separations were performed at a flow rate of 1 mL/min. One H\u003csub\u003e2\u003c/sub\u003eO/CH\u003csub\u003e3\u003c/sub\u003eCN solvent system containing 0.1% TFA was used, consisting of solvent A (H\u003csub\u003e2\u003c/sub\u003eO with 0.1% TFA) and solvent B (CH\u003csub\u003e3\u003c/sub\u003eCN with 0.1% TFA). Samples were filtered through a 0.2 \u0026micro;m GHP filter before injecting into the instrument. The gradient used was sample dependent and is indicated in the figure legends. Note that in samples containing DMF, a 10 min hold at 1% B at the beginning of the method before starting the gradient significantly enhanced the resolution.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGeneral Method for Two-Stage HPLC purification\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHPLC purification was performed using an Agilent 1100 series system (Windows 7, ChemStation Software, G1312A binary pump, G1329A autosampler, G1315B diode array detector, Teledyne Foxy R1 fraction collector). Samples were filtered through a 0.2 \u0026micro;m GHP syringe filter before injecting into the instrument. Purification was performed first on a preparative scale (10 - 20 mg peptide per injection, Agilent Pursuit C18, 5 \u0026mu;m, 250 \u0026times; 21.2 mm) with a 5 mL/min flow rate and using the same Solvent A/Solvent B system described above. The gradient was as follows: 1-10 min hold at 30% B, 10-70 min ramp to 100% B. Peptides \u003cstrong\u003e1\u003c/strong\u003e-\u003cstrong\u003e3\u003c/strong\u003e were then further purified on a semi-preparative scale (2 - 10 mg peptide per injection, Agilent ZORBAX 300SB-C18, 5 \u0026mu;m, 9.4 \u0026times; 250 mm) with 4 mL/min flow rate using the same solvent A/Solvent B system described above. The gradient for peptide \u003cstrong\u003e1a\u0026nbsp;\u003c/strong\u003ewas as follows: 1-5 min hold at 20% B, 10 min ramp to 45% B, 20 min ramp to 55% B, 1 min ramp to 100% B. The gradient for peptides \u003cstrong\u003e2a\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003e3a\u0026nbsp;\u003c/strong\u003ewas as follows: 1-5 min hold at 20% B, 10 min ramp to 40% B, 20 min ramp to 50% B, 1 min ramp to 100% B.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGeneral Method for Sep-Pak Purification of 33mer Peptides\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePrenylated peptides were purified using a simple Sep-Pak solid phase extraction procedure. The cartridges were first conditioned using 10 mL of 100% solvent B, followed by equilibration with 10 mL of 100% solvent A. Prenylation reaction mixtures (5 mL) containing peptides \u003cstrong\u003e1\u003c/strong\u003e-3 were then diluted 5-fold with solvent A, and loaded onto the cartridges. The cartridges were then washed using 10 mL of 100% solvent A and 10 mL of 30% solvent B, before eluting the peptide using 10 mL of 80% solvent B. The organic solvent was then removed using a gentle stream of N\u003csub\u003e2\u003c/sub\u003e before lyophilizing the peptides and redissolving them in DMSO. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis of Peptide \u003cstrong\u003e1a\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePeptide \u003cstrong\u003e1a\u003c/strong\u003e was synthesized using a PS3 automated peptide synthesizer and cleaved from the resin, prenylated, and purified by HPLC as described above. Pooled HPLC fractions were lyophilized and then redissolved in DMSO. The concentration was measured by diluting the peptide in 6 M Gdm\u0026bull;HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 360 nm (ɛ\u003csub\u003e360\u003c/sub\u003e = 17,500 M\u003csup\u003e-1\u003c/sup\u003ecm\u003csup\u003e-1\u003c/sup\u003e) (Hsu et al. 2019). ESI-MS: for C\u003csub\u003e190\u003c/sub\u003eH\u003csub\u003e289\u003c/sub\u003eN\u003csub\u003e44\u003c/sub\u003eO\u003csub\u003e56\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e3+\u003c/sup\u003e [M+3H]\u003csup\u003e3+\u003c/sup\u003e; calcd 1383.0204, found 1383.0203.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis of Peptide \u003cstrong\u003e2a\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePeptide \u003cstrong\u003e2a\u003c/strong\u003e was synthesized using a PS3 automated peptide synthesizer and cleaved from the resin, prenylated, and purified by HPLC as described above. Pooled HPLC fractions were lyophilized and then redissolved in DMSO. The concentration was measured by diluting the peptide in 6 M Gdm\u0026bull;HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 360 nm (ɛ\u003csub\u003e360\u003c/sub\u003e = 17,500 M\u003csup\u003e-1\u003c/sup\u003ecm\u003csup\u003e-1\u003c/sup\u003e) (Hsu et al. 2019). ESI-MS: for C\u003csub\u003e189\u003c/sub\u003eH\u003csub\u003e287\u003c/sub\u003eN\u003csub\u003e44\u003c/sub\u003eO\u003csub\u003e56\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e3+\u003c/sup\u003e [M+3H]\u003csup\u003e3+\u003c/sup\u003e; calcd 1378.3485, found 1378.3499.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis of Peptide \u003cstrong\u003e3a\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePeptide \u003cstrong\u003e3a\u003c/strong\u003e was synthesized using a Chorus automated peptide synthesizer and cleaved from the resin, prenylated, and purified by HPLC as described above. Pooled HPLC fractions were lyophilized and then redissolved in DMSO. Concentration was measured by diluting the peptide in 6 M Gdm\u0026bull; HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 360 nm (ɛ\u003csub\u003e360\u003c/sub\u003e = 17,500 M\u003csup\u003e-1\u003c/sup\u003ecm\u003csup\u003e-1\u003c/sup\u003e) (Hsu et al. 2019).ESI-MS: for C\u003csub\u003e189\u003c/sub\u003eH\u003csub\u003e289\u003c/sub\u003eN\u003csub\u003e45\u003c/sub\u003eO\u003csub\u003e55\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e3+\u003c/sup\u003e [M+3H]\u003csup\u003e3+\u003c/sup\u003e; calcd 1378.0205, found 1378.0190.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis of Peptide \u003cstrong\u003e4b\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePeptide \u003cstrong\u003e4b\u003c/strong\u003e was synthesized using a PS3 automated peptide synthesizer and cleaved from the resin, prenylated, and purified by HPLC as described above. Pooled HPLC fractions were lyophilized and then redissolved in DMSO. Concentration was measured by diluting the peptide in 6 M Gdm\u0026bull; HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 280 nm (ɛ\u003csub\u003e280\u003c/sub\u003e = 5,810 M\u003csup\u003e-1\u003c/sup\u003ecm\u003csup\u003e-1\u003c/sup\u003e).(Gill and von Hippel 1989) ESI-MS: for C\u003csub\u003e58\u003c/sub\u003eH\u003csub\u003e82\u003c/sub\u003eN\u003csub\u003e9\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003eS\u003csup\u003e+\u003c/sup\u003e [M+H]\u003csup\u003e+\u003c/sup\u003e; calcd 1112.5850, found 1112.5831.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis of Peptide \u003cstrong\u003e5b\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePeptide \u003cstrong\u003e5b\u003c/strong\u003e was synthesized by hydrolysis of the methyl ester of peptide \u003cstrong\u003e4b\u003c/strong\u003e as described below. After hydrolysis and reaction quenching, the peptide was isolated using the same Sep-Pak procedure described above. After lyophilizing the peptide and redissolving in DMSO, the concentration was measured by diluting the peptide in 6 M Gdm\u0026bull; HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 280 nm (ɛ\u003csub\u003e280\u003c/sub\u003e = 5,810 M\u003csup\u003e-1\u003c/sup\u003ecm\u003csup\u003e-1\u003c/sup\u003e).(Gill and von Hippel 1989) ESI-MS: for C\u003csub\u003e57\u003c/sub\u003eH\u003csub\u003e80\u003c/sub\u003eN\u003csub\u003e9\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003eS\u003csup\u003e+\u003c/sup\u003e [M+H]\u003csup\u003e+\u003c/sup\u003e; calcd 1098.2693, found 1098.2714.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis of Peptide \u003cstrong\u003e6b\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePeptide \u003cstrong\u003e6b\u003c/strong\u003e was synthesized using a Chorus automated peptide synthesizer and cleaved from the resin, prenylated, and purified by HPLC as described above. Pooled HPLC fractions were lyophilized and then redissolved in DMSO. Concentration was measured by diluting the peptide in 6 M Gdm\u0026bull; HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 280 nm (ɛ\u003csub\u003e280\u003c/sub\u003e = 5,810 M\u003csup\u003e-1\u003c/sup\u003ecm\u003csup\u003e-1\u003c/sup\u003e).(Gill and von Hippel 1989) ESI-MS: for C\u003csub\u003e57\u003c/sub\u003eH\u003csub\u003e81\u003c/sub\u003eN\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003eS\u003csup\u003e+\u003c/sup\u003e [M+H]\u003csup\u003e+\u003c/sup\u003e; calcd 1097.5853, found 1097.5868.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis of Peptide \u003cstrong\u003e11\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePeptide \u003cstrong\u003e11\u003c/strong\u003e was synthesized using a PS3 automated peptide synthesizer and cleaved from the resin and purified by HPLC as described above. Pooled HPLC fractions were lyophilized and then redissolved in DMSO. Concentration was measured by diluting the peptide in 6 M Gdm\u0026bull; HCl, 0.02 M phosphate buffer, pH 6.5, and measuring the absorbance at 310 nm (ɛ\u003csub\u003e310\u003c/sub\u003e = 2,400 M\u003csup\u003e-1\u003c/sup\u003ecm\u003csup\u003e-1\u003c/sup\u003e).(Ito et al. 2001) ESI-MS: for C\u003csub\u003e36\u003c/sub\u003eH\u003csub\u003e55\u003c/sub\u003eN\u003csub\u003e9\u003c/sub\u003eO\u003csub\u003e13\u003c/sub\u003eS\u003csup\u003e+\u003c/sup\u003e [M+H]\u003csup\u003e+\u003c/sup\u003e; calcd 853.3640, found 853.3642.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMS-MS analysis of 33mer peptides\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn order to confirm correct peptide sequence MS-MS analysis was carried out using ThermoFisher Orbitrap Fusion Lumos Tribrid Mass Spectrometer. To prevent loss of the farnesyl group in the MS\u003csup\u003e2\u003c/sup\u003e fragmentation step, data-dependent Electron Transfer Dissociation (ETD) activation was used along with EThcD collision energy type. Chromatographic separation was performed using a nano-flow 300 \u0026Aring; pore size C3 column with a 1 \u0026micro;L/min flow rate. The gradient used was as follows: 1-5 min, hold at 30% B. 5-15 min, ramp to 90% B. 1 min, ramp to 100%. Parent ions (+3, +4, and +5 charge states) were fragmented via ETD to obtain daughter ions that were primarily z and c ions. Data is summarized in Tables S1-3.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMethyl ester peptide hydrolysis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePeptides \u003cstrong\u003e1a\u003c/strong\u003e and \u003cstrong\u003e4a\u003c/strong\u003e were each hydrolyzed to their corresponding C-terminal carboxylic acids through saponification reactions. The peptide (100 \u0026micro;M) in 0.5 M NaOH with 50% v/v CH\u003csub\u003e3\u003c/sub\u003eCN for 1 h at rt. The reaction was then quenched by adding 20% HOAc, which also neutralized the base and prevented any subsequent epimerization.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTrypsin digestion\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThree aliquots of 140 \u0026micro;L of 50 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e solutions and 40 \u0026micro;L of CH\u003csub\u003e3\u003c/sub\u003eCN were prepared in 1.5 low-adhesion microcentrifuge tubes. Peptides \u003cstrong\u003e1-3\u003c/strong\u003e were added from DMSO stocks to a final concentration of 0.1 mM. Trypsin (15 \u0026micro;L of a\u0026nbsp;20 \u0026micro;g/mL stock)\u0026nbsp;was added to each tube to yield a final concentration of 1.5 \u0026micro;g/mL. The tubes were incubated at 37 ˚C overnight with rotation before subjecting to LC-MS analysis with and without the addition of the appropriate authentic standard peptides \u003cstrong\u003e4\u003c/strong\u003e-\u003cstrong\u003e6\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzymatic reactions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePlasmids and yeast strains\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe pCH1283 plasmid used for this study contains a His\u003csub\u003e10\u003c/sub\u003e-HA\u003csub\u003e3\u003c/sub\u003e tag on the N-terminus of the Ste24 gene (\u003cem\u003e2\u0026mu; URA3 P\u003csub\u003ePGK\u003c/sub\u003e-His\u003csub\u003e10\u003c/sub\u003e-HA\u003csub\u003e3\u003c/sub\u003e-Ste24\u003c/em\u003e). This plasmid was transformed into the yeast strain SM3614 that has a double deletion for endogenous Ste24 and Rce1 (\u003cem\u003eMATa trp1 leu2 ura3 his4 can1 ste24\u0026Delta;::LEU2 rce1\u0026Delta;::TRP1\u003c/em\u003e)(Tam et al. 1998).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCrude membrane preparation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo prepare crude membrane samples, a small culture of synthetic complete supplement mixture without uracil (SC-URA) was inoculated with pCH1283 WT strain and incubated overnight at 30 ⁰C. This starter culture was then used to inoculate a larger culture at a ratio of 15 mL to 1 L and grown to log phase (OD\u003csub\u003e600\u003c/sub\u003e 0.3-0.5) after which time the cultures were harvested at 4000 \u0026acute; g and the pellets stored at -80 ⁰C until needed. Cell pellets were lysed using yeast sorbitol buffer (0.3 M sorbitol, 0.1 M NaCl, 12 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1% aprotinin, 3 mM AEBSF, 1 mM DTT, 10 mM Tris-HCl, pH 7.5). Lysis buffer was added at a ratio of 1 mL to 800 OD\u0026rsquo;s culture pellets, vortexed and left on ice for 15 min. The resulting suspension was frozen and thawed twice in liquid nitrogen and then further lysed by passing through a French press twice at 18,000 psi. The solution was then centrifuged twice at 500 \u0026acute; g for 10 min to remove cell debris and then once more at 100,000 \u0026acute; g for 1 h at 4⁰C. The supernatant was removed, and the pellet resuspended in 10 mM Tris-HCl pH 7.5 and then stored at -80 ⁰C. The protein concentration of the crude membrane samples was measured using a Coomassie blue protein assay employing BSA as a standard (Sedmak and Grossberg 1977).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eProtein Purification\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCrude membrane samples were purified by first solubilizing in buffer A (0.3 M sorbitol, 0.1 M NaCl, 6 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM Tris, pH 7.5, 10% glycerol, 1% aprotinin, 2 mM AEBSF), 1% n-Dodecyl-b-D-maltopyranoside (DDM), and 20 mM imidazole. This solution was rocked at 4 ⁰C for 1 h and then centrifuged at 100,000 \u0026acute; g for 45 min. The supernatant was then added to Talon\u0026reg; metal affinity resin beads (Clontech, Inc.) and rocked at 4 ⁰C for 1 h (25 mg of protein to 1 mL of resin). The resulting resin mixture was then washed twice with buffer B (buffer A plus 40 mM imidazole and 1% DDM), once with buffer C (buffer A plus 40 mM imidazole, 1% DDM and 0.5 M KCl), and once with buffer D (buffer A plus 40 mM imidazole, 0.1% DDM and 0.5 M KCl). The protein was finally eluted using buffer E (buffer A plus 250 mM imidazole, 0.1% DDM) into a Amicon\u0026reg; Ultra Centrifugal Filter 30,000 MWCO (Millipore). The sample was then concentrated to desired volume by centrifuging at 4,000 \u0026acute; g for 20-30 min at 4 ⁰C and then stored at -80 ⁰C (Anderson et al. 2005). Protein concentration was calculated using an amido black protein assay using BSA as a standard (Schaffner and Weissmann 1973).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSDS-PAGE and immunoblot analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo assess protein purity, 1 mg of protein (diluted in 2X SDS loading buffer (0.5 M Tris-HCl, pH 6.8, 30% sucrose (w/v), 10% sodium dodecylsulfate (w/v), 3.5 M 2-mercaptoethanol and 0.1% bromophenol blue (w/v))\u0026nbsp;was loaded onto a 4\u0026ndash;15% Mini-PROTEAN\u0026reg; TGX\u0026trade; precast protein gel and run at 165 V for 45 min. The gel was then stained at rt overnight in Coomassie Blue (0.3 M Coomassie Brilliant Blue, 10% HOAc, 40% CH\u003csub\u003e3\u003c/sub\u003eOH) and then de-stained with 10% HOAc/30% CH\u003csub\u003e3\u003c/sub\u003eOH. For immunoblot analysis, 0.05 \u0026mu;g pure protein (also diluted in 2X SDS loading buffer) was loaded onto 4\u0026ndash;15% Mini-PROTEAN\u0026reg; TGX\u0026trade; a precast protein gel and run under similar conditions as described above. The resulting gel was then used to transfer the protein onto a nitrocellulose membrane (Cytiva Amersham\u0026trade; Protran\u0026trade; NC Nitrocellulose) at 100 V for 90 min. Membranes were blocked overnight with 20% milk in PBST (1x PBS buffer, 0.1% Tween-20) at 4 ⁰C, followed by treatment for 2 h with the primary antibody (mouse, anti-HA at 1:15,000) in 5% milk in PBST. After washing with PBST, the membrane was incubated for 1 h with the secondary antibody (goat-anti-mouse-HRP, 1:4,000) in 4% milk in PBST. The resulting protein bands were visualized with SuperSignal\u0026trade; West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and a GeneGnome XRQ (SynGene) instrument.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSte24 activity assay for LC-MS analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA stock of bulk \u003cem\u003eE. coli\u003c/em\u003e lipids in CHCl\u003csub\u003e3\u003c/sub\u003e was placed in a glass scintillation vial, and the chloroform was removed under vacuum in a rotary evaporator at 30 ˚C. Afterward, 150 mM Tris buffer, pH 7.5, was added to give a final lipid concentration of 10 mg/mL in order to hydrate the lipids. The water bath of the rotary evaporator was then heated to 70 ˚C with rotation but no vacuum for 30 min and then fully dissipated by sonication for 30 mins. This solution was stored at -20 ˚C until before usage, when it was further diluted to 0.625 mg/mL in Tris buffer (10 mM Tris\u0026bull;HCl, pH 7.5). A stock solution of Ste24 enzyme in DDM was diluted to 0.15 \u0026micro;g/\u0026micro;L in 10 mM Tris Buffer, pH 7.5. An aliquot (40 \u0026micro;L) of this solution was added to 80 \u0026micro;L of the aforementioned 0.625 mg/mL lipid suspension solution. Afterward, 520 \u0026micro;L of\u0026nbsp;150 mM Tris\u0026bull;HCl (pH 7.5) was added to break the detergent vesicles and translocate the enzyme into the lipid vesicles. This solution was incubated on ice for 10 min before aliquoting 160 \u0026micro;L into three low-adhesion microcentrifuge tubes and incubating at 30 ˚C for 5 min. Meanwhile, three peptide solutions containing peptides \u003cstrong\u003e1a\u003c/strong\u003e-\u003cstrong\u003e3a\u0026nbsp;\u003c/strong\u003eat 0.15 mM in 150 mM Tris\u0026bull;HCl (pH 7.5) were prepared. A 40 \u0026micro;L\u0026nbsp;aliquot\u0026nbsp;of this solution was added to the enzyme solution, and the mixture was incubated at\u0026nbsp;30 ˚C for 10 min followed by the addition of HOAc (50 \u0026micro;L) quench the reaction, and 100 \u0026micro;L of CH\u003csub\u003e3\u003c/sub\u003eCN was added to fully solubilize the reaction mixture. Each solution was subjected to LC-MS analysis without filtration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eKinetic Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFirst, a standard curve was created by plotting relative fluorescence units (RFU) versus concentration of the unquenched peptide product (peptide\u003cstrong\u003e\u0026nbsp;13\u003c/strong\u003e). To do this, 0.75 \u0026mu;g of purified Ste24 was rapidly reconstituted into 6.25 \u0026mu;g of \u003cem\u003eE.coli\u003c/em\u003e polar lipid (Avanti Polar Lipids) in 150 mM Tris-HCl, pH 7.5 and incubated on ice for 10 min. Varying concentrations of peptide \u003cstrong\u003e1a\u003c/strong\u003e (0 to 50 \u0026mu;M) were incubated with the pure WT Ste24 and fluorescence readings were obtained at 30 ⁰C. Fluorescence readings were then plotted versus concentration to create the standard curve. A calibration curve was also created by incubating equimolar amounts of peptides \u003cstrong\u003e13\u003c/strong\u003e and \u003cstrong\u003e14\u003c/strong\u003e (0 to 50 \u0026mu;M) with pure WT Ste24 protein using the same conditions as for the standard curve. This calibration curve allowed for the correction of the inner filter effect by calculating a correction factor (C). This was calculated from the ratio of RFU between the two calibration curves at each concentration of each peptide in the assay. The calculated values were then used to multiply the raw RFU units to produce corrected fluorescence values for subsequent analyses. Protein samples were prepared using standard assay conditions as described above and incubated with increasing concentrations of peptides (0 to 50 \u0026mu;M). Fluorescence values were then collected at 30 sec intervals for 1 h using excitation and emission wavelengths of 320 nm and 420 nm respectively. From the fluorescence progress curves, the initial rates were calculated using the first linear region (typically the first 10 min) and converted to specific activities using the extinction coefficient. The specific activities were corrected by multiplying by the correction factor, C, and then plotted against substrate concentration. The kinetic parameters were established by fitting specific activity and substrate concentrations to the Michaelis-Menten equation model {V = V\u003csub\u003emax\u003c/sub\u003e[S]/(K\u003csub\u003eM\u0026nbsp;\u003c/sub\u003e+ [S])} using Kaleidagraph (v5.0.3). For the cooperative model the equation V = {V = V\u003csub\u003emax\u003c/sub\u003e[S]\u003csup\u003en\u003c/sup\u003e/(K\u003csub\u003eM\u003c/sub\u003e\u003csup\u003en\u0026nbsp;\u003c/sup\u003e+ [S]\u003csup\u003en\u003c/sup\u003e)} was used. In these equations, [S] is the substrate (peptide) concentration and n is the Hill coefficient.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSubstrate design for testing Ste24 dependence on\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eC-terminal structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo probe the role of the C-terminus in ordering processing by Ste24, three peptide analogs based on the 33 residue C-terminus from the precursor of the mating pheromone \u003cstrong\u003ea\u003c/strong\u003e-factor were designed and synthesized (Fig. 3). The first, \u003cstrong\u003e1a\u003c/strong\u003e, contained a C-terminal methyl ester, modeling a substrate for Ste24 containing a fully processed C-terminus. The second peptide\u0026nbsp;analog\u0026nbsp;(\u003cstrong\u003e2a\u003c/strong\u003e) had a free C-terminal carboxylate, representing the unmethylated precursor. The final peptide, \u003cstrong\u003e3a\u003c/strong\u003e, contained an unnatural C-terminal amide; that substrate would mimic the neutral character of \u003cstrong\u003e1a\u003c/strong\u003e while eliminating the synthetically more problematic methyl ester. If carboxylmethylation is required for Ste24 cleavage at the upstream site, the methyl ester-containing peptide (\u003cstrong\u003e1a\u003c/strong\u003e) would be a significantly better Ste24 substrate than \u003cstrong\u003e2a\u003c/strong\u003e. Given its neutral charge, \u003cstrong\u003e3a\u003c/strong\u003e would also be expected to react at a rate comparable to that of \u003cstrong\u003e1a\u003c/strong\u003e. In contrast, if carboxylmethylation is not a prerequisite, then \u003cstrong\u003e2a\u003c/strong\u003e could have similar Ste24 reactivity. Peptide \u003cstrong\u003e1a\u003c/strong\u003e was synthesized using the side-chain anchoring strategy outlined in Fig. 4 while peptides \u003cstrong\u003e2a\u003c/strong\u003e and \u003cstrong\u003e3a\u003c/strong\u003e were prepared using more routine methods. To measure the activity of these substrates using an assay that avoids the use of radioactivity, each peptide was designed to contain an Abz/Dnp donor-quencher FRET pair flanking the N-terminal cleavage site with the Abz group positioned on the N-terminus and a Lys(Dnp) group located 4 residues downstream of the cleavage site; that donor-quencher pair has been used extensively in related peptides designed to probe the first cleavage that occurs C-terminal to the prenylcysteine residue (Hollander et al. 2000; Kyro et al. 2011; Hildebrandt et al. 2016, 2024; Arachea and Wiener 2017a). After Ste24 N-terminal cleavage, two peptide fragments (Fig. 5) would be liberated, including one with an unquenched Abz fluorophore, thus causing an increase in fluorescence that could be used to measure enzymatic activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of a-Factor 33mer precursor 1a with a C-termin\u003c/strong\u003e\u003cstrong\u003eal methyl ester\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo synthesize the methyl ester-containing peptide analogue \u003cstrong\u003e1a\u003c/strong\u003e, a side-chain-anchoring methodology first developed by Barany et al (Barany et al. 2003) and later optimized by Distefano and coworkers was utilized (Fig. 4) (Diaz-Rodriguez et al. 2012, 2015, 2018; Diaz-Rodriguez and Distefano 2017; Bader et al. 2019; Morstein et al. 2022). In brief, the carboxylic acid of\u0026nbsp;Fmoc-L-cysteine (\u003cstrong\u003e7a)\u003c/strong\u003e was esterified in methanolic HCl to yield methyl ester \u003cstrong\u003e8a\u003c/strong\u003e with a free side chain thiol. The thiol of\u0026nbsp;\u003cstrong\u003e8a\u003c/strong\u003e was alkylated with trityl chloride resin in the presence of DIPEA resulting in the formation of a side chain-anchored cysteine methyl ester (\u003cstrong\u003e9a\u003c/strong\u003e) suitable for SPPS. Next, the full peptide chain was elongated via automated peptide synthesis using standard HCTU/Fmoc chemistry. Single couplings (20 min) were used for all positions except D, G, N, P, Y, K(Dnp), and Abz, which were coupled for 60 mins. To suppress epimerization, those coupling reactions were supplemented with Cl-HOBt (Han et al. 1997; Montalbetti and Falque 2005; Albericio and El-Faham 2018). It is important to note that repeated exposure to HCTU can cause the development of life-threatening anaphylaxis, and so it should be handled with either a respirator or in a well-ventilated fume hood (McKnelly et al. 2020). Additionally, Cl-HOBt can be explosive under certain conditions and as a result should also be handled with care. (Wehrstedt et al. 2005; Malow et al. 2007) Once the complete peptide chain was assembled, the peptide was cleaved and globally deprotected using Reagent K, yielding the crude C-terminal methyl ester-containing peptide \u003cstrong\u003e10a\u003c/strong\u003e with a free thiol (Fig. S1). This peptide was then prenylated with farnesyl bromide under mildly acidic conditions in the presence of Zn(OAc)\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e(Fig. S2) (Xue et al. 1992; Wollack et al. 2009; Bader et al. 2019). A critical consideration for this step was to deoxygenate the solvents properly through N\u003csub\u003e2\u003c/sub\u003e sparging to prevent disulfide formation. Additionally, earlier reports used a solvent mixture containing 0.1%\u0026nbsp;TFA (Diaz-Rodriguez et al. 2012, 2015; Vervacke et al. 2014; Bader et al. 2019); this was found to decrease the reaction yield, likely due to over-acidification of the reaction and an associated decrease in the nucleophilicity of the Zn-thiolate species thought to be involved in the reaction. The original description of this reaction used 0.025% TFA, not 0.1%.(Xue et al. 1992) Improved results were observed when the reaction was buffered to pH 5.0 with sodium acetate (Wollack et al. 2009; Morstein et al. 2022). Prenylated peptide\u0026nbsp;\u003cstrong\u003e1a\u003c/strong\u003e was then purified by HPLC using a two-stage process first involving preparative scale HPLC using a broad range gradient, which resulted in purity of approximately 70% based on 220 nm integration in analytical LC-MS. This peptide was further purified to \u0026gt;95% on a semi-preparative scale using a targeted gradient ranging from 45-55% solvent B over 20 min (Table 1,\u0026nbsp;Fig. S3) to yield pure\u0026nbsp;\u003cstrong\u003e1a\u003c/strong\u003e (Fig. S4). The structure of the desired peptide was confirmed by LC-MS/MS using\u0026nbsp;ETD to minimize the loss of the farnesyl group that is common with these types of peptides. Complete coverage of all z and c type ions was observed (Table S1). The position of the farnesyl group was confirmed from analysis of the C-terminal c and z ions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Purification of 33mer peptides based on \u003cstrong\u003ea\u003c/strong\u003e-factor.\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"690\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.56522%;\"\u003e\n \u003cp\u003ePeptide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7246%;\"\u003e\n \u003cp\u003eCrude purity\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.8406%;\"\u003e\n \u003cp\u003eEpimerization in crude product\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.2609%;\"\u003e\n \u003cp\u003ePurity after initial purification\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.4783%;\"\u003e\n \u003cp\u003ePurity after final purification\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1304%;\"\u003e\n \u003cp\u003eEpimerization after final purification\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.56522%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7246%;\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.8406%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.2609%;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.4783%;\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1304%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.56522%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7246%;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.8406%;\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.2609%;\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.4783%;\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1304%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 9.56522%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.7246%;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.8406%;\"\u003e\n \u003cp\u003e\u0026lt;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.2609%;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.4783%;\"\u003e\n \u003cp\u003e99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1304%;\"\u003e\n \u003cp\u003e\u0026lt;1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of a-factor precursor analogues 2a and 3a\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeptide \u003cstrong\u003e2a\u003c/strong\u003e was synthesized using identical conditions to peptide \u003cstrong\u003e1a\u003c/strong\u003e but starting with Fmoc-Cys on Wang resin. Cleavage from resin (Fig. S1) to yield crude \u003cstrong\u003e11a\u003c/strong\u003e, prenylation (Fig. S5) and preparative HPLC purification (Fig. S6) provided pure peptide \u003cstrong\u003e2a\u003c/strong\u003e (Fig. S7). Peptide \u003cstrong\u003e3a\u003c/strong\u003e was synthesized using unloaded Rink MBHA amide resin using the same HCTU/Fmoc chemistry but without Cl-HOBt, as it was found to lower crude peptide purity due to decreased coupling efficiency (Vrettos et al. 2017; Albericio and El-Faham 2018). Double couplings (20 min) were used instead of single couplings for all positions except C, K(Dnp), and Abz, which were all coupled manually for 60 min to allow reaction monitoring using the Ninhydrin test (Kaiser et al. 1970; Vilaseca and Bardaji 1995). Also, an acetic anhydride capping step was added between each coupling cycle to prevent truncated side products that would further complicate the HPLC purification. Cleavage from resin (Fig. S1) to yield crude \u003cstrong\u003e12a\u003c/strong\u003e, prenylation (Fig. S8) and preparative HPLC purification (Fig. S9) gave pure peptide \u003cstrong\u003e3a\u003c/strong\u003e (Fig. S10). The semi-preparative HPLC purification of \u003cstrong\u003e2a\u003c/strong\u003e and \u003cstrong\u003e3a\u003c/strong\u003e was similar to that of \u003cstrong\u003e1a\u003c/strong\u003e but the targeted gradient spanned 40-50% B due to the increased polarity of analogues \u003cstrong\u003e2a\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003e3a\u0026nbsp;\u003c/strong\u003e(Fig. S5, Fig. S6). The best crude purity obtained for the unprenylated peptides was for \u003cstrong\u003e10a\u003c/strong\u003e synthesized through side chain anchoring methodology, followed by peptide \u003cstrong\u003e12a\u003c/strong\u003e, and then peptide \u003cstrong\u003e11a\u003c/strong\u003e (having the lowest crude purity (Table 1, Fig. S1\u003cstrong\u003e).\u0026nbsp;\u003c/strong\u003eThe structures of \u003cstrong\u003e2a\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003e3a\u0026nbsp;\u003c/strong\u003ewere\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003econfirmed by LC-MS/MS using\u0026nbsp;ETD (Tables S2 and S3). For \u003cstrong\u003e2a\u003c/strong\u003e, good coverage including all z ions except z1, z3, z5, z6, z13, z15, z19, and z26 was observed; those missing ions were compensated for by the presence of complementary y ions in all cases except y19. Good coverage of c ions was also observed with this peptide. When missing, those positions were confirmed by overlapping a and b ions for all positions except c12. For \u003cstrong\u003e3a\u003c/strong\u003e, excellent coverage including all z ions except z11 was obtained with y11 being observed instead. Good coverage of c ions was also observed with this peptide with missing ions being confirmed by overlapping a or b ions for all positions (b5, b6, b7, b10, b11, a13, b20, b22, b27, b33) except c12. For both \u003cstrong\u003e2a\u003c/strong\u003e and \u003cstrong\u003e3a\u003c/strong\u003e, the position of the farnesyl group was confirmed from analysis of the C-terminal c and z ions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEpimerization analysis of\u0026nbsp;a-Factor 33mer analogues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore testing the peptides for activity with the Ste24 enzyme, it was essential to determine their enantiomeric purity. C-terminal cysteines are prone to epimerization (Han et al. 1997; Kondasinghe et al. 2017), and it was unknown whether \u003cstrong\u003ea\u003c/strong\u003e-Factor containing a D-Cys would have the same reactivity as a-Factor containing L-Cys in Ste24-catalyzed proteolysis. Due to the length of these peptides, it was unlikely that there would be a detectable retention time difference between the two epimeric 33mer peptides in LC-MS analysis. Thus, each of the three peptides was subjected to trypsin digestion, resulting in much shorter 8 residue fragments containing the prenylated cysteines that should be easily resolvable via LC-MS analysis. To confirm this, authentic D-cysteine-containing standards were synthesized. Peptide \u003cstrong\u003e4b\u003c/strong\u003e (Fig. 3) was synthesized using the same procedure required to obtain peptide \u003cstrong\u003e1a\u003c/strong\u003e but starting from Fmoc-D-cysteine hydrate. Peptide \u003cstrong\u003e5b\u003c/strong\u003e (Fig. 3) was produced by hydrolyzing the methyl ester of peptide \u003cstrong\u003e4b\u003c/strong\u003e through a simple saponification reaction with NaOH. Peptide \u003cstrong\u003e6b\u003c/strong\u003e (Fig. 3) was synthesized using the same procedure as peptide \u003cstrong\u003e3a\u003c/strong\u003e but also using Fmoc-D-cysteine hydrate. Analysis of tryptic digests of peptides \u003cstrong\u003e1a, 2a,\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003e3a\u003c/strong\u003e each showed a major dominant peak in the LC-MS chromatogram for \u003cstrong\u003e4a\u003c/strong\u003e, \u003cstrong\u003e5a\u003c/strong\u003e and \u003cstrong\u003e6a\u003c/strong\u003e, respectively), along with a minor isobaric peak that integrated to 2% (\u003cstrong\u003e4b\u003c/strong\u003e from \u003cstrong\u003e1a\u003c/strong\u003e), 4% (\u003cstrong\u003e5b\u003c/strong\u003e from \u003cstrong\u003e2a\u003c/strong\u003e) and \u0026lt;1% (\u003cstrong\u003e6b\u003c/strong\u003e from \u003cstrong\u003e3a\u003c/strong\u003e). Upon co-injection with the authentic D-Cys-containing standards (\u003cstrong\u003e4b\u003c/strong\u003e, \u003cstrong\u003e5b\u003c/strong\u003e and \u003cstrong\u003e6b\u003c/strong\u003e), the minor isobaric peak grew significantly in size, suggesting that all three peptides were \u0026gt;95% enantiomerically pure (Fig. 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine whether the high enantiomeric purities of the purified peptides noted above accurately reflected the fidelity of the synthesis or were a consequence of the subsequent HPLC purification, the crude peptides were also subjected to the same trypsin digestion analysis following the prenylation reaction without the HPLC purification. Subjecting these crude peptides to tryptic digestion and subsequent LC-MS analysis showed that for the methyl ester-containing peptide \u003cstrong\u003e1a\u003c/strong\u003e, there was \u0026lt;5% epimerization in the crude peptide (Fig. 7, Table 1). A similar result was observed in the analysis of crude amide-containing peptide \u003cstrong\u003e3a\u003c/strong\u003e. In contrast, LC-MS analysis of the tryptic digest of crude carboxylic acid-containing peptide \u003cstrong\u003e2a\u003c/strong\u003e displayed two peaks, one corresponding to \u003cstrong\u003e5a\u003c/strong\u003e integrating to 64%, and a peak corresponding to the epimer \u003cstrong\u003e5b\u003c/strong\u003e integrating to 36%. Thus, substantial Cys epimerization occurred in the peptide prepared using conventional anchoring through the C-terminal carboxylate. This highlights the advantage of the side-chain anchoring methodology over traditional Wang resin for such peptides. It should be noted that side chain anchoring can also yield C-terminal acids via saponification of the methyl ester (or acidolytic cleavage of C-terminal t-butyl esters) as was done to obtain peptide \u003cstrong\u003e5b\u003c/strong\u003e. In fact, peptide \u003cstrong\u003e2a\u003c/strong\u003e was also prepared in this manner from peptide \u003cstrong\u003e1a\u003c/strong\u003e after only 1 h of incubation with NaOH at room temperature, followed by neutralization with glacial acetic acid (Fig. S11). For especially sensitive cases, bases have been identified that minimize epimerization in this hydrolytic reaction (Nicolaou et al. 2005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of peptides as substrates for Ste24\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOnce the structures and enantiomeric purities of the purified peptides were confirmed, they were subjected to Ste24-catalyzed cleavage using two \u003cem\u003ein vitro\u003c/em\u003e assays. First, to confirm that the peptides were cleaved by Ste24 at the correct site, each peptide was incubated with Ste24 for 10 min at 30 ˚C and then subjected to LC-MS analysis. The predicted cleavage products were observed for each peptide (Fig. 5, Fig. S12 (\u003cstrong\u003e1a\u003c/strong\u003e), S13 (\u003cstrong\u003e2a\u003c/strong\u003e) and S14 (\u003cstrong\u003e3a\u003c/strong\u003e)),\u0026nbsp;demonstrating that Ste24 cleaved the \u003cstrong\u003ea\u003c/strong\u003e-factor analog peptides between the Thr and Ala residues upstream of the farnesyl-modified C-terminus. Although trace levels of non-specific cleavage was observed at high concentrations of peptide, the vast majority of cleavage occurred at the target site. Next, the kinetics of the enzymatic cleavage reactions were studied using an \u003cem\u003ein vitro\u003c/em\u003e quantitative fluorescence enzymatic assay with purified and Ste24 reconstituted into membranes. In these assays, the peptide concentration was varied and the initial reaction rate was determined using the increase in Abz fluorescence as the Dnp quencher was proteolytically removed (Fig. 5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine specific activities for each peptide with Ste24, a standard curve was first generated to facilitate the conversion of raw fluorescence data from relative fluorescence units (RFU) to concentration. A calibration curve was also used to adjust for the inner filter effect resulting from the internal quenching properties of the Abz/Dnp FRET pair which occurs when using a high concentration of the fluorogenic peptide substrate (Hildebrandt et al. 2016; Arachea and Wiener 2017b). To obtain kinetic parameters for the three peptides (\u003cstrong\u003e1a\u003c/strong\u003e, \u003cstrong\u003e2a\u003c/strong\u003e and \u003cstrong\u003e3a\u003c/strong\u003e), purified Ste24 was incubated with each peptide at varying concentrations. After conversion of the fluorescence data from RFU to concentration and adjustments due to inner filter effects, the rates were plotted versus peptide concentration (Fig. 8A). The data was then fit to the Michaelis-Menten equation using a non-linear least-squares algorithm to obtain the kinetic parameters, K\u003csub\u003eM\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e (Table 2). The V\u003csub\u003emax\u003c/sub\u003e and K\u003csub\u003eM\u003c/sub\u003e values obtained for the methyl ester substrate, peptide \u003cstrong\u003e1a\u003c/strong\u003e, were 3.3 \u0026acute; 10\u003csup\u003e3\u0026nbsp;\u003c/sup\u003epmol/mg/min and 7.3 \u0026mu;M, respectively. The K\u003csub\u003eM\u003c/sub\u003e value measured here was comparable to earlier reported values of 11 \u0026micro;M (Pryor et al. 2013), measured using a different assay, and 9.1 \u0026micro;M (Hsu 2018) obtained using the fluorescence assay described here. The V\u003csub\u003emax\u003c/sub\u003e value is lower, which may be due to variability in the purity and activity of the enzyme preparations. Moreover, the relative insolubility of the peptides resulted in significant scatter in the data at concentrations above 20 \u0026micro;M making determinations of V\u003csub\u003emax\u003c/sub\u003e challenging. Overall, these data confirm that Ste24 cleaved peptide \u003cstrong\u003e1a\u003c/strong\u003e with similar efficiency as previously determined. For peptide \u003cstrong\u003e2a\u003c/strong\u003e, the C-terminal acid, the K\u003csub\u003eM\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e values obtained were 15 \u0026mu;M and 4.6 \u0026acute; 10\u003csup\u003e3\u0026nbsp;\u003c/sup\u003epmol/mg/min. Lastly, peptide \u003cstrong\u003e3a\u003c/strong\u003e, which contains a neutral C-terminal amide, yielded kinetic values of 11 \u0026mu;M and 6.1 \u0026acute; 10\u003csup\u003e3\u0026nbsp;\u003c/sup\u003epmol/mg/min for K\u003csub\u003eM\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e respectively. It is interesting to note that there appears to be some deviation from Michaelis-Menten behavior with all three peptides and a better fit was obtained by adding a cooperativity term in the equation (Fig. 8B). This type of behavior has been previously observed with other membrane-bound enzymes acting on insoluble substrates (Lister et al. 1988; Burke et al. 1995). While this analysis changed the kinetic constants, it did not reveal any major differences in the relative catalytic efficiencies detected with these different peptide substrates (Table 2). Collectively, the K\u003csub\u003eM\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e values for these three different peptides vary by a factor of 2 or less indicating that the identity of the C-terminal group does not significantly impact the rate of cleavage at the upstream site. From a biological perspective, this is a key finding that suggests that ability of Ste24 to cleave the upstream site in susbtrates that undergo CAAX processing is independent of carboxylmethylation. An important consequence of this is that inhibitors of the methyltransferase ICMT, which may be useful as therapeutic agents for cancer, should not inhibit the processing of prelamin A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. Kinetic parameters for Ste24 cleavage of peptides \u003cstrong\u003e1a\u003c/strong\u003e, \u003cstrong\u003e2a\u003c/strong\u003e and \u003cstrong\u003e3a\u003c/strong\u003e.\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePeptide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eK\u003csub\u003eM\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eK\u003csub\u003eM\u003c/sub\u003e rel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eV\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003e(nmol/min/mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eV\u003csub\u003emax\u003c/sub\u003e rel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eV\u003csub\u003emax\u003c/sub\u003e/K\u003csub\u003eM\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eV\u003csub\u003emax\u003c/sub\u003e/K\u003csub\u003eM\u003c/sub\u003e rel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHill\u003c/p\u003e\n \u003cp\u003ecoeff.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003en\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(replicates)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEster (\u003cstrong\u003e1a\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eM.M.\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003eCoop.\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e7.3 \u0026plusmn; 4.5\u003c/p\u003e\n \u003cp\u003e5.6 \u0026plusmn; 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3.3 \u0026plusmn; 0.70\u003c/p\u003e\n \u003cp\u003e2.6 \u0026plusmn; 0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2.2 \u0026plusmn; 0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAcid (\u003cstrong\u003e2a\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eM.M.\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003eCoop.\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e15 \u0026plusmn; 5.8\u003c/p\u003e\n \u003cp\u003e9.2 \u0026plusmn; 0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2.1\u003c/p\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4.6 \u0026plusmn; 0.74\u003c/p\u003e\n \u003cp\u003e3.2 \u0026plusmn; 0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.31\u003c/p\u003e\n \u003cp\u003e0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4.9 \u0026plusmn; 1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAmide(\u003cstrong\u003e3a\u003c/strong\u003e)\u003c/p\u003e\n \u003cp\u003eM.M.\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003eCoop.\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11 \u0026plusmn; 4.8\u003c/p\u003e\n \u003cp\u003e8.2 \u0026plusmn; 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6.1 \u0026plusmn; 1.0\u003c/p\u003e\n \u003cp\u003e4.7 \u0026plusmn; 0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003cp\u003e0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4.6 \u0026plusmn; 2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eValues calculated with standard Michaelis-Menten kinetic model.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003eValues calculated with Michaelis-Menten kinetic model with cooperativity.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u003c/sup\u003eThe value n corresponds to the number of times the experiment was performed.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eLong hydrophobic peptide sequences are generally challenging to synthesize and manipulate. In the case of the analogs described here, these syntheses were particularly challenging due to the presence of C-terminal cysteines, which are known to be prone to epimerization during the repeated piperidine treatments used for Fmoc deprotection during SPPS (Han et al. 1997). This issue was indeed observed in the analysis of the crude peptides prepared here. Peptide \u003cstrong\u003e2a\u003c/strong\u003e, which was synthesized using standard Wang resin showed 36% epimerization and while it was possible to obtain an enantiomerically pure peptide in the end, this represented the loss of over a third of the material synthesized. In contrast, the side chain anchoring methodology described here led to minimal epimerization prior to HPLC purification and offers simple access to peptides with a C-terminal cysteine acid using a simple saponification reaction. This strategy also gave the highest crude purity, even when compared to the amide containing peptide \u003cstrong\u003e3a\u003c/strong\u003e, which was synthesized with double coupling and acetic anhydride capping, again showing the advantage of the side chain anchoring methodology. Kinetic analysis of the Ste24-catalyzed proteolytic cleavage of 33mer peptides based on the C-terminus of \u003cstrong\u003ea\u003c/strong\u003e-factor precursor demonstrated that there was minimal difference between peptides bearing a C-terminal methyl ester, acid or amide.\u0026nbsp;From a biological perspective, this suggests that the upstream cleavage ability of Ste24 is independent\u0026nbsp;of\u0026nbsp;carboxylmethylation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e The online version contains supplementary material available at \u0026hellip;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e This work was supported by the National Science Foundation (NSF/CHE-1905204).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.B. and S. 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J Chem Educ 72:A99. https://doi.org/10.1021/ed072pA99\u003c/li\u003e\n \u003cli\u003eVrettos EI, Sayyad N, Mavrogiannaki EM, et al (2017) Unveiling and tackling guanidinium peptide coupling reagent side reactions towards the development of peptide-drug conjugates. RSC Adv 7:50519\u0026ndash;50526. https://doi.org/10.1039/c7ra06655d\u003c/li\u003e\n \u003cli\u003eWang M, Casey PJ (2016) Protein prenylation: unique fats make their mark on biology. Nat Rev Mol Cell Biol 17:110\u0026ndash;122. https://doi.org/10.1038/nrm.2015.11\u003c/li\u003e\n \u003cli\u003eWehrstedt KD, Wandrey PA, Heitkamp D (2005) Explosive properties of 1-hydroxybenzotriazoles. J Hazard Mater 126:1\u0026ndash;7. https://doi.org/10.1016/j.jhazmat.2005.05.044\u003c/li\u003e\n \u003cli\u003eWollack JW, Zeliadt NA, Mullen DG, et al (2009) Multifunctional prenylated peptides for live cell analysis. J Am Chem Soc 131:7293\u0026ndash;7303. https://doi.org/10.1021/ja805174z\u003c/li\u003e\n \u003cli\u003eXue C-B, Becker JM, Naider F (1992) Efficient regioselective isoprenylation of peptides in acidic aqueous solution using zinc acetate as catalyst. Tetrahedron Lett 33:1435\u0026ndash;1438. https://doi.org/10.1016/S0040-4039(00)91640-X\u003c/li\u003e\n \u003cli\u003eYang SH, Chang SY, Andres DA, et al (2010) Assessing the effi cacy of protein farnesyltransferase inhibitors in mouse models of progeria. J Lipid Res 51:400\u0026ndash;405. https://doi.org/10.1194/jlr.M002808\u003c/li\u003e\n \u003cli\u003eYoung SG, Fong LG, Michaelis S (2005) Thematic Review Series: Lipid Posttranslational Modifications. Prelamin A, Zmpste24, misshapen cell nuclei, and progeria\u0026mdash;new evidence suggesting that protein farnesylation could be important for disease pathogenesis. J Lipid Res 46:2531\u0026ndash;2558. https://doi.org/10.1194/jlr.R500011-JLR200\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-peptide-research-and-therapeutics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ijpr","sideBox":"Learn more about [International Journal of Peptide Research and Therapeutics](http://link.springer.com/journal/10989)","snPcode":"10989","submissionUrl":"https://submission.nature.com/new-submission/10989/3","title":"International Journal of Peptide Research and Therapeutics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"a-Factor, farnesylation, peptide epimerization, progeria, solid phase peptide synthesis, Ste24","lastPublishedDoi":"10.21203/rs.3.rs-5094096/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5094096/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProtein prenylation is a post-translational modification that links a specific cytosolic protein with a hydrophobic isoprenoid lipid resulting in membrane localization of the target protein. Following prenylation, some proteins undergo proteolytic removal of a C-terminal tripeptide. This step results in a protein terminating in a prenylcysteine residue that is subsequently methylated by the membrane-associated methyltransferase ICMT. In some cases, this proteolytic reaction is catalyzed by ZMPSTE24 in humans or Ste24 in yeast. The molecular recognition demonstrated by this family of proteases is intriguing as they also cleave at an unrelated additional site upstream from the C-terminus that has no obvious structural similarity. From a medical perspective, these events are particularly important for the posttranslational processing of protein lamin A, as mutations in ZMPSTE24 that impair its activity lead to accelerated premature aging progeroid diseases. A central question in the field regards the structure of the C-terminus of processed lamin A and whether methylation is essential for subsequent upstream cleavage. Herein, a series of 33-residue peptides based on the structure of the precursor for the peptide pheromone \u003cstrong\u003ea\u003c/strong\u003e-factor from yeast, a substrate for both Ste24 and ZMPSTE24. These peptides were synthesized with a fluorescent donor-quencher pair and incorporated either a C-terminal methyl ester to mimic the native substrate, a free acid to mimic an unmethylated peptide or an amide to mimic differently modified C-termini. Their preparation presented several synthetic challenges due to the presence of a C-terminal methyl ester, an epimerization-prone C-terminal cysteine and an acid-sensitive farnesyl group. The synthesis of those peptides and an analysis of their cleavage at the upstream site catalyzed by Ste24 are reported here. \u003cem\u003eIn vitro\u003c/em\u003e fluorescence-based proteolytic cleavage assays showed that all of these peptides were processed at similar rates, suggesting that C-terminal methylation is not a prerequisite for subsequent upstream proteolysis.\u003c/p\u003e","manuscriptTitle":"Upstream proteolysis by Ste24 does not require a C-terminal methyl ester as revealed using 33-residue a-factor precursor peptide substrates synthesized via epimerization-free methods","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-29 07:45:06","doi":"10.21203/rs.3.rs-5094096/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-01T13:04:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-01T10:02:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-30T07:49:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43040754917490440409656992499041061766","date":"2024-09-25T03:26:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16343298927407676199540721623768836584","date":"2024-09-25T01:30:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-24T12:18:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-24T10:52:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259695989057770790467567708739431989077","date":"2024-09-24T07:08:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230920491493336218817259390705429885869","date":"2024-09-23T20:23:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172840817718896238404564455008987338620","date":"2024-09-23T09:04:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252958935558225043890173305235723881808","date":"2024-09-23T03:50:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218411718866990294764792961024999728785","date":"2024-09-22T23:32:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-22T19:23:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-22T19:20:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-17T09:38:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Peptide Research and Therapeutics","date":"2024-09-15T20:11:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-peptide-research-and-therapeutics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ijpr","sideBox":"Learn more about [International Journal of Peptide Research and Therapeutics](http://link.springer.com/journal/10989)","snPcode":"10989","submissionUrl":"https://submission.nature.com/new-submission/10989/3","title":"International Journal of Peptide Research and Therapeutics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6ba30027-d31b-4cdf-9c87-17e03e1f7827","owner":[],"postedDate":"November 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-13T16:01:51+00:00","versionOfRecord":{"articleIdentity":"rs-5094096","link":"https://doi.org/10.1007/s10989-024-10677-9","journal":{"identity":"international-journal-of-peptide-research-and-therapeutics","isVorOnly":false,"title":"International Journal of Peptide Research and Therapeutics"},"publishedOn":"2025-01-06 15:57:28","publishedOnDateReadable":"January 6th, 2025"},"versionCreatedAt":"2024-11-29 07:45:06","video":"","vorDoi":"10.1007/s10989-024-10677-9","vorDoiUrl":"https://doi.org/10.1007/s10989-024-10677-9","workflowStages":[]},"version":"v1","identity":"rs-5094096","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5094096","identity":"rs-5094096","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}