Modulating amyloid-β 42 aggregation and neurotoxicity by Kunitz domains and their derived peptides

Modulating amyloid-β 42 aggregation and neurotoxicity by Kunitz domains and their derived peptides | 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 Modulating amyloid-β 42 aggregation and neurotoxicity by Kunitz domains and their derived peptides Maya Rabinovich, Shiran Lacham-Hartman, Niv Papo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8603888/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Alzheimer’s disease is associated with the aggregation of amyloid‑β42 (Aβ42) into species of varying sizes, with intermediate oligomers being the most neurotoxic. We recently reported that amyloid precursor protein inhibitor (APPI), a Kunitz-type protein, and a cyclic peptide derived from its β-domain reduced Aβ42-mediated neurotoxicity, the former by reducing Aβ42 aggregation and formation of toxic Aβ42 oligomers, and the latter by promoting Aβ42 aggregation to form fibrils rather than the neurotoxic Aβ42 oligomers. To address the question of whether these two inhibition mechanisms are controlled by the structure or the amino acid sequence of the protein/peptide, we exploited three Kunitz-type proteins, bikunin, bovine pancreatic trypsin inhibitor (BPTI) and tissue factor pathway inhibitor (TFPI) – chosen for their similar β-strand-rich structures but different sequences to one another and to APPI – and also short peptides that mimic their β-domains, in either cyclic or linear conformation. In-vitro studies showed that the formation of Aβ42 aggregates was reduced by the three Kunitz-type proteins and by their derived cyclic peptides, but not by the linear counterparts of the cyclic peptides. In SH-SY5Y neuroblastoma cells, the Kunitz-type proteins and the cyclic (but not the linear) peptides reduced the intracellular and extracellular accumulation of Aβ42 aggregates, respectively. Both the Kunitz-type proteins and the cyclic peptides inhibited Aβ42-induced mitochondrial membrane depolarization and reduced Aβ42-mediated apoptosis and cell death. Overall, this study thus reveals the potential of the β-hairpin structure, whether as a segment within the Kunitz-type proteins or isolated as a cyclic peptide, to interact with Aβ42, thereby reducing Aβ42 aggregation and hence its neurotoxicity. Alzheimer's disease Aβ42 aggregation BPTI TFPI bikunin β-hairpin Kunitz-type proteins neurotoxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION Alzheimer’s disease (AD) – currently the most common neurodegenerative disease – is a significant public health concern, affecting millions of people worldwide and increasing with age from 40% of those older than 85 years (1). Although the etiology of AD remains a subject of intense study, it is widely accepted that AD is associated with the extracellular and intracellular accumulation of aggregations of β -amyloid (A β ) peptide fragments of amyloid precursor protein (APP). These fragments are generated by sequential cleavage by β -secretase at the ectodomain of APP and then by γ-secretase at its transmembrane domain (2). Among the resultant cleavage fragments, the 42-amino-acid-containing A β fragment (designated A β 42) is a particularly neurotoxic isoform, by virtue of its propensity to misfold and to aggregate into amyloid plaques in the brain, and it is thus a key player in the pathology of AD (3,4). The nucleation of disordered monomeric A β 42 into neurotoxic oligomeric aggregates and ordered β -sheet-structured fibrils (5) results from the interaction between the two hydrophobic segments within the A β 42 sequence, namely, the central hydrophobic core (residues 16–22) and the C-terminal region (residues 30–42), which fold into a β -hairpin structure (6–8). Numerous studies have sought to block the aggregation of A β 42 (as a means to prevent/retard the onset of AD) by using a variety of inhibitors, with focus in recent years on two main types of proteinaceous molecule—sequence-based peptides, i.e., those with hydrophobic sequences that are the same or very similar to the native sequence of A β 42 (9–11), and structure-based peptides, i.e., those with a β -strand structure (12–14). In an example of the first approach, treatment of A β 42 with the short hydrophobic peptide LPYFD-amide (a derivative of residues 17–21 of the central hydrophobic core of A β 42) reduced A β 42 aggregation and its associated toxicity in neuronal cells and in mice (15,16). By applying the second approach, a similar inhibitory effect was obtained by a different peptide (LYFGA), also mimicking the hydrophobic core of A β 42 (i.e., residues 17–21) but having a β -strand structure that was generated by chemically cross-linking Tyr at position 2 and Gly at position 4 (17). In yet another approach that appeared to offer promise for the generation of peptides inhibiting A β 42 aggregation, ring-like peptide structures were designed and tested (18). A representative example is provided by a 23-residue bicyclic peptide in which three cysteine residues enabled double cyclization to produce a molecule with two central regions, LGIKI and TSVYHA, that bind the C-terminal region of A β 42 at residues 31–36 and 38–42, respectively (19). Nonetheless, although this bicyclic peptide remodeled A β 42 aggregation, producing shorter, thicker, and non-fibrillar structures, which reduced A β 42-associated toxicity in a Caenorhabditis elegans model, its interaction with the monomeric form of A β 42 was weak and transient and showed no inhibitory effect on pre-formed fibrils (19). In parallel, studies on cyclic peptides that were specifically designed to mimic the β -hairpin motif in A β 42 showed that they were indeed able to reduce A β 42 aggregation and toxicity (20–23). For example, Yamin et al. synthesized a 13-mer cyclic peptide with hairpin structure that interacts with A β 42(24). Although they showed that the cyclic peptide modulated A β 42 aggregation by inhibiting the formation of oligomers and fibrils while promoting the generation of non-fibrillar aggregates (24), the lack of cellular toxicity assays leaves the conclusions of the study somewhat limited. In summary, although cyclic peptides appear to have potential as blockers of A β 42 aggregation, work to date on the effect on A β 42 aggregation of the amino acid sequence or the structure of these peptides has not produced definitive conclusions that may ultimately be leveraged in the design of AD therapeutics. A starting point for addressing the above issues is an examination of the structure and activity of human APP. This protein has three major isoforms that are produced as a result of alternative splicing, namely, APP695, APP751, and APP770. The latter two isoforms each contain a 58-amino acid extracellular subunit known as a Kunitz protease inhibitor or as an amyloid precursor protein inhibitor (APPI) (25–27). The two APPI-containing isoforms, APP751 and APP770, are expressed in multiple tissues (e.g., glia cells, skin, and lungs), while the APPI-deficient isoform, APP695, is expressed primarily in neurons (28,29). Of note, previous studies have reported a significant increase in mRNA and protein levels of the APPI-containing isoforms in AD brains, but the involvement of APPI in AD progression has yet to be elucidated (30,31). Thus, to date, APPI has been studied predominantly for its function as a serine protease inhibitor that targets proteases, such as mesotrypsin, whose catalytic activity plays roles in various malignancies and in pancreatitis (32–34). Nonetheless, two recent papers from our laboratory have investigated the role of APPI in the formation A β 42 aggregates (35,36). The first showed that a cyclic β -hairpin peptide derived from the β -domain of APPI (but not its linear counterpart) reduced the toxicity of A β 42 and enhanced the formation of mature Aβ42 fibrils from intermediate, toxic oligomeric A β 42 species (35) . The second study demonstrated the ability of both extra- and intracellular full-length APPI to reduce A β 42 aggregate formation and hence to decrease the cellular toxicity mediated by both extra- and intracellular A β 42 (36). These two studies thus revealed an apparent contradiction in that the full-length APPI protein reduced A β 42 aggregation and the formation of toxic A β 42 oligomers, but the cyclic APPI peptide enhanced A β 42 aggregation, forming fibrils rather than neurotoxic A β 42 oligomers and thereby ameliorating A β 42-mediated neurotoxicity. To further investigate this issue and in line with previous reports highlighting the potential of cyclic β -hairpin mimetics in AD therapeutics, we sought to address the open question of whether it is a structural element or the specific sequence of the cyclic APPI fragment that interacts with A β 42 to enhance its aggregation and hence to attenuate A β 42 neurotoxicity. To this end, we investigated the putative inhibitory activities of three other Kunitz-type proteins, namely, tissue factor pathway inhibitor (TFPI), bovine pancreatic trypsin inhibitor (BPTI) and bikunin(37), and their derived β -domain peptides in cyclic or linear conformation. The rationale for choosing these three Kunitz-type proteins was that they have similar lengths (58 amino acids) and structures ( β -hairpin domain with two antiparallel β -sheets and one helical region) to APPI [investigated in our previous studies (1,36)], but different sequences to one another and to APPI, both in the full-length protein and within the β -hairpin region (37–40). Herein, we demonstrate that, like the β -domain of APPI(36), the β -domains of the three Kunitz-type proteins interact with A β 42, thereby modulating its aggregation mechanism. Using thioflavin T (ThT) fluorescence assays, circular dichroism (CD) spectroscopy, and transmission electron microscopy (TEM) imaging, we show that both the Kunitz-type proteins, at a molar ratio of 64:1 (A β 42:Kunitz), and the Kunitz-derived cyclic peptides with a β -hairpin structure, at a molar ratio of 1:2 (A β 42:cyclic peptide), reduce the formation of A β 42 aggregates. In contrast, the Kunitz-derived linear peptides do not result in a significant reduction in A β 42 aggregation. Similarly, treatment of SH-SY5Y neuroblastoma cells with the Kunitz-type proteins and their derived cyclic peptides, but not their linear counterparts, reduce the intracellular and extracellular accumulation of A β 42 aggregates, respectively, as assessed by confocal microscopy. Notably, the Kunitz-type proteins and the cyclic peptides prevent A β 42-mediated mitochondrial membrane depolarization and reduce A β 42-mediated apoptosis and cell death. Taken together, the findings of this study show that the combination of the amino acid sequence of the Kunitz-type proteins and the β -hairpin structure present in both Kunitz-type proteins and cyclic peptides impacts A β 42 aggregation and that the β -hairpin structure is responsible for reducing neurotoxicity—irrespective of the amino acid sequence. RESULTS The Inhibitory Effect of the Kunitz-Type Proteins on A β 42 Aggregation in Vitro is Similar to that of their Derived Cyclic Peptides. To examine the effect of the Kunitz-type proteins and their derived peptides on A β 42 aggregation, it was first necessary to produce and purify BPTI, TFPI, and bikunin. The three proteins were expressed and purified as described previously (42,43) (see Methods), and for TFPI and bikunin representative results for the different stages of the procedure are given in the Supporting Information ( Figures S1 and S2, respectively). Yields of 15 mg, 7.4 mg, and 10 mg per 1 L of yeast culture were obtained for BPTI, TFPI, and bikunin, respectively. The ability of the Kunitz-type proteins to inhibit aggregation of A β 42 was then investigated by incubating A β 42 for 16 h in the absence or presence of the proteins and monitoring the aggregation levels of untreated or treated A β 42 ( Figure 1A ). As expected, the highest aggregation signal was observed for A β 42 alone, consistent with its intrinsic tendency to self-assemble first into oligomers and aggregates and then into fibrils (5), while a decrease in aggregation was observed when A β 42 was incubated with the Kunitz-type proteins. With the aims to study the effect of the β -domain of Kunitz-type proteins on A β 42 aggregation and to determine whether it is the structure or the sequence of the domains that influences the aggregation, we designed three linear peptides and their three cyclic counterparts that were derived from the β -hairpin domains of the three native Kunitz-type proteins, BPTI, TFPI and bikunin ( Figure S3A ), as follows: Three 18-mer linear peptides were derived from the full β -hairpin sequences of BPTI, bikunin and TFPI ( Figure S3B ); and three 20-mer cyclic peptides were designed such that they had the same sequences as the linear peptides, but with two cysteine residues added at the N- and C-termini to form a disulfide bridge, creating a cyclic structure ( Figure S3C ) and thereby mimicking the structure of the β -domain in the Kunitz-type proteins (41). In addition, to prevent potential undesired interactions, the internal cysteine residue in the sequences of the cyclic peptides was replaced with serine, and to better mimic the β -domains in the native Kunitz-type protein sequences, the N-termini of all six peptides were acetylated and the C-termini were amidated. To confirm both the formation of the disulfide bridge within the cyclic peptides and the correct molar weights of the linear and cyclic peptides, all six peptides were analyzed by mass spectrometry ( Figure S4 ). The spectra showed good agreement between theoretical and experimental values; for example, the calculated molar weight of the cyclic BPTI peptide, taking into consideration the formation of the disulfide bridge and a consequent reduction of 2 Da, was 2399.81 Da, which matched the experimentally obtained molar weight of 2399.78 Da ( Figure S4B ). Next, the ability of the cyclic and linear peptides to inhibit the aggregation of A β 42 was investigated by incubating A β 42 for 16 h in the absence or presence of the peptides. Monitoring of the aggregation levels of untreated and treated A β 42 revealed a decrease in aggregation upon incubation of A β 42 with the cyclic peptides but not the linear peptides ( Figure 1B,C ). Of note, while the cyclic BPTI peptide was a potent inhibitor of A β 42 aggregation, the linear BPTI peptide was the only linear peptide that enhanced A β 42 aggregation. Moreover, neither the three Kunitz-type proteins per se nor their derived peptides gave any significant aggregation signals (cyclic TFPI gave a low signal), indicating that these proteins/peptides do not undergo self-aggregation ( Figure 1 ). The Ability of the Kunitz-Type Proteins to Reduce the β -Sheet and Fibrillar Contents of A β 42 is Retained by the Cyclic Peptides. We then aimed to explore – at the molecular and macroscopic levels – the inhibition of A β 42 aggregation by the Kunitz-type proteins and their derived peptides. To this end, we first recorded the CD spectra of samples of A β 42 (50 µ M) post 18 h of incubation in the absence and presence of the Kunitz-type proteins (781.25 nM) at a molar ratio of 64:1 (A β 42:protein) or the peptides (100 µ M) at a molar ratio of 1:2 (A β 42:peptides). The CD spectrum of A β 42 revealed a negative peak at 216 nm and a positive peak at 195 nm—both characteristic of a β -sheet secondary structure (42), which is commonly regarded as the signature of A β 42 aggregates (43,44). Reductions in β -sheet structures and increases in random secondary structures were observed upon incubation of A β 42 with the cyclic peptides and to a lesser extent with the linear peptides and the Kunitz-type proteins ( Figure 2 ). The CD spectra of control cyclic and linear peptides and Kunitz-type proteins revealed predominantly random β -sheet structures at insignificant levels ( Figure S5 ). To complement the molecular level structural results, samples taken from the CD experiment (18 h post incubation) were analyzed by TEM. Consistent with the high ThT signal and β -sheet content of A β 42 and with the findings of previous studies (45,46), the TEM images showed that untreated A β 42 formed elongated and branched fibrils ( Figure 3A ). The TEM images of A β 42 treated with Kunitz-type proteins revealed a decrease in both the abundance and the branching of the fibrils, with a tendency toward shorter and unbranched fibrils ( Figure 3B ). TEM images of A β 42 samples incubated with the cyclic peptides revealed a dramatic reduction in the quantity of aggregates, with the fibrils being shorter and less entangled ( Figure 3C ). In contrast, in the presence of the linear peptides, only a slight decrease in the quantity of A β 42 aggregates was observed, with the species appearing more densely packed and tangled ( Figure 3D ). The above findings were confirmed by dot blot analysis with an anti-amyloid fibril antibody, which showed a strong reduction in the quantity of A β 42 fibrils upon 18 h of incubation with the proteins and peptides, in the order: cyclic peptides > Kunitz-type proteins > linear peptides ( Figure 4 ). The Kunitz-Type Proteins Reduce Intracellular A β 42 Aggregate Formation in SH‑SY5Y Cells. To determine whether bikunin, BPTI, and TFPI can reduce the formation of A β 42 aggregates in cells, SH-SY5Y cells were co-transfected with either the Aβ42-GFP genetic construct and an empty plasmid or with Aβ42-GFP together with each Kunitz-type protein gene ( bikunin , BPTI , or TFPI ). Confocal microscopy imaging performed 48 h post-transfection revealed a high accumulation of green fluorescence protein (GFP) in inclusion bodies in the absence of a Kunitz-type protein, indicating the presence of A β 42 aggregates (47). In contrast, when Kunitz-type proteins were co-expressed with A β 42-GFP, the observed GFP signal was spread throughout the cells ( Figure 5 A ). In addition, western blot analysis confirmed that intracellular A β 42-GFP expression levels did not change upon co-expression with the Kunitz-type proteins ( Figure 5B ). Extracellular Cyclic Peptides Inhibit the Internalization and Accumulation of A β 42 in SH‑SY5Y Cells. Studies have shown that extracellular A β 42 interacts with the cell membrane and is internalized into the cell via multiple uptake mechanisms (48,49). To follow the effect of extracellular cyclic and linear peptides on the internalization and accumulation of A β 42 in cells, SH-SY5Y cells were treated with HiLyte™ Fluor 488 -labeled A β 42 (designated A β 42-488) in the presence or absence of cyclic or linear peptides and visualized using confocal microscopy. The cells treated with A β 42-488 alone exhibited strong internal fluorescence signals, indicating internalization of the extracellular A β 42 ( Figure 6A ). Upon exposure of the cells to a mixture of A β 42-488 and each of the cyclic peptides at molar ratio of 1:2 (A β 42:peptides), internalization and accumulation of A β 42 in the cells was reduced ( Figure 6A ). In contrast, confocal microscopy images of the cells following treatment with mixtures of A β 42-488 and linear peptides revealed the presence of inclusion bodies within the cells, which indicated that the accumulation of A β 42 had not been affected by the presence of the linear peptides ( Figure 6B ). Based on the inhibitory activity observed for the Kunitz-type proteins and the cyclic peptides but not the linear peptides, our subsequent cellular assays were performed only with the Kunitz-type proteins and the cyclic peptides. The Kunitz-Type Proteins and Cyclic Peptides Enhance the Viability of SH-SY5Y Cells Exposed to Extracellular A β 42. To further investigate the ability of Kunitz-type proteins and cyclic peptides to reduce extracellular A β 42-mediated cytotoxicity in SH-SY5Y cells, we performed cell-based assays to assess cell viability and apoptosis. The XTT viability assay revealed an average of ~30% reduction in viability vs. control cells for SH-SY5Y cells exposed for 48 h to pre-formed A β 42 aggregates ( Figure 7A ). However, SH-SY5Y cells treated with mixtures of A β 42 and Kunitz-type proteins (pre-incubated together for 18 h at 37 ºC before exposure to the cells) exhibited improved viability, reaching complete recovery (normalized to control cells) for bikunin and BPTI treatments and ~90% for the TFPI treatment ( Figure 7A ). Similarly, treating SH-SY5Y cells with a mixture of A β 42 and cyclic peptides (pre-incubated for 18 h at 37 ºC) improved cell viability compared to control cells, reaching 86% for cyclic bikunin and complete recovery for both cyclic BPTI and cyclic TFPI ( Figure 7B ). Notably, exposure of the cells to the Kunitz-type proteins and the cyclic peptides alone did not significantly affect cell viability ( Figure 7C,D ). We also sought to investigate the effect of our proteins/cyclic peptides on A β 42-induced apoptosis, since previous studies have reported that most of the accumulated intraneuronal A β may be attributed to the uptake of extracellular A β fragments, which induce a neuronal apoptosis cascade (50,51) by promoting uncontrolled elevation of cytosolic Ca 2+ levels (52) and impairing mitochondrial redox activity (53). Thus, to evaluate the ability of the Kunitz-type proteins and the cyclic peptides to reduce A β 42-induced apoptosis in SH-SY5Y cells, treated and untreated cells were stained with Annexin-V APC and SYTOX Green (54,55), and the apoptotic stages were analyzed by flow cytometry. Figure 8A shows that 98% of the control cells were healthy (negative for both Annexin-V APC and SYTOX Green) and 42.2% of cells exposed to extracellular A β 42 aggregates for 24 h were in early-stage apoptosis (positive for Annexin-V APC and negative for SYTOX Green), whereas cells treated with mixtures of A β 42 and Kunitz-type proteins (pre-incubated together) revealed a reduction in cell toxicity, with only 17.8%, 31.1%, and 29.5% of cells in the early apoptotic stage following treatment with A β 42 together with bikunin, BPTI, or TFPI, respectively ( Figure 8B , left panels). Treatment with Kunitz-type proteins alone, as control groups, showed that approximately 85% of the cells were healthy living cells, with less than 1% in a late apoptosis stage and less than 15% of cells in an early apoptosis stage ( Figure 8B , right panels). A more marked reduction in A β 42-induced apoptosis was obtained for the cyclic peptides, with only 8.86%, 9.47%, and 7.59% of cells in early-stage apoptosis for cyclic bikunin, cyclic BPTI, or cyclic TFPI, respectively ( Figure 8C , left panels). These results indicate an approximately 30% decrease in early apoptotic cells compared to those treated with extracellular A β 42 aggregates ( Figure 8A , right panel). Notably, approximately 85% of the control SH-SY5Y cells treated with cyclic peptides alone were healthy, and < 9% were in an early apoptotic stage ( Figure 8C , right panels). The Kunitz-Type Proteins and Cyclic Peptides Suppress the Mitochondrial Membrane Depolarization Potential Mediated by A β 42 in SH-SY5Y Cells . Previous studies have shown that A β 42 impairs mitochondrial function by disrupting the membrane potential and the electron transport chain, thereby generating reactive oxygen species and damaging mitochondrial DNA and proteins and ultimately leading to neuronal apoptosis (56–58). Thus, following the viability and apoptosis assays showing that both the Kunitz-type proteins and the cyclic peptides suppressed A β 42-mediated apoptosis and cell death in SH-SY5Y cells, we sought to examine the effect of those proteins and peptides on the mitochondrial membrane potential of intact SH-SY5Y cells. Mitochondrial damage was assessed by measuring the changes in mitochondrial polarization resulting from exposure to A β 42 aggregates, pre-formed in the presence or absence of Kunitz-type proteins and cyclic peptides. As shown in Figure 9, cells that were exposed to pre-formed A β 42 aggregates for 48 h exhibited an average of ~40% reduction in mitochondrial membrane potential compared to untreated cells. The addition of the Kunitz-type proteins improved mitochondrial function and reduced the changes in mitochondrial membrane potential, which reached complete recovery in SH-SY5Y cells treated with mixtures of A β 42 and bikunin or TFPI, and 97% in cells treated with A β 42 and BPTI ( Figure 9A ). Similarly, the reduction in mitochondrial membrane potential was almost abolished in cells treated with A β 42 and cyclic peptides, with improvements of 95%, 98%, and 96% in cells treated with A β 42 and cyclic bikunin, cyclic BPTI, and cyclic TFPI, respectively, compared to untreated cells ( Figure 9B ). We observed that both Kunitz-type proteins and cyclic peptides did not induce significant changes in mitochondrial membrane potential ( Figure 9C,D ). DISCUSSION Studies have shown Kunitz-type proteins to be implicated in the etiology of AD, although open questions remain regarding their roles in the development of AD and the generation of A β aggregates. For example, it was shown that TFPI, which is abundant in the brains of AD patients (59), is elevated in the frontal cortex and blood plasma of AD patients and, notably, that TFPI is localized in some A β plaques in AD brains (60,61). Less was known about another Kunitz-type protein, APPI, until our recent studies of the protein and of a peptide sequence taken from its β -hairpin domain demonstrated that both are potent inhibitors of A β 42-induced neuronal cell toxicity, but act via different mechanisms: the full-length protein reduces A β 42 aggregation, whereas its β -hairpin domain enhances aggregation (35,36). To investigate these differences in modes of inhibition and to elucidate whether the inhibition mechanism depends on the structure or on the sequence of the peptides – or perhaps on both – we extended our work on APPI (35,36) to other Kunitz-type proteins, namely, TFPI, BPTI and bikunin, and the short cyclic peptides that mimic their β -hairpin domains and their unstructured linear peptide counterparts. Integrating the findings of the current study with those of our previous study on APPI reveals that a β -hairpin motif of the interacting cyclic peptide is a prerequisite for conferring inhibition (cyclic TFPI, BPTI and bikunin peptides) or enhancement (the cyclic APPI peptide) of A β 42 aggregation, whereas the lack of a β -hairpin loop in the interacting linear peptide may either not affect (linear APPI, TFPI and bikunin) or enhance (linear BPTI) A β 42 aggregation. Moreover, for any particular β -hairpin sequence in a peptide, the specific amino acid composition may finetune the level of inhibition of A β 42 aggregation. An analysis of the three studies together also shows that all four different Kunitz-type protein family members, which have a similar fold (i.e., an α/β fold with two antiparallel β -sheets and one helical region) but different sequences (with only ~25% similarity) (32,37,62), inhibit the aggregation of A β 42. An examination of the mechanism of A β 42 inhibition indicates that the β -sheet domain of the α/β fold is the segment that interacts with A β 42 to modulate its aggregation pathway, probably through hydrophobic interactions with A β 42, as observed for other peptide-based β -sheet breakers designed to inhibit or reverse the misfolding of A β 42 into β -sheet-rich structures (63–65), which is a key pathological event in neurodegenerative diseases, such as AD (7,66). By binding to the central hydrophobic core of A β 42 aggregates, probably via their β -hairpin peptide domain, the Kunitz proteins disrupt the stability of the aggregated A β 42, preventing further fibril formation and thereby reducing neurotoxicity. It is possible that – like APPI – bikunin, BPTI, and TFPI proteins inhibit the A β 42 'on-pathway,' in which unordered monomeric A β 42 self-assembles to form toxic oligomeric intermediates that promote further elongation of A β 42 into mature amyloid fibrils (67,68). The potential of β -hairpin-like cyclic peptides to inhibit 'on-pathway' A β 42 aggregation has been examined in previous studies using synthetic peptides (not related to the A β 42 sequence) (23,24) or peptides comprising the self-assembly region of A β 42 (69). However, in view of the lack of a comprehensive study, the role played by the cyclic conformation in these inhibitory activities of the peptides remains unclear. Consistent with our premise that the β -sheet domain interacts with A β 42, Costa et al. demonstrated that A β 42 aggregation and cytotoxicity can be mitigated by the β -hairpin domain of transthyretin (TTR), either as a segment within TTR (70,71) or as an isolated cyclic peptide mimetic derived from the A β -TTR binding interface (72). Similar to our current study, the above authors also showed that the linear versions of the peptides exhibited only minor inhibitory activity, whereas significant inhibition of A β 42-induced toxicity was achieved only with the cyclic peptides and at concentrations comparable to those observed for the Kunitz-derived cyclic peptides (20-40 µ M). In the current study, an examination of the inhibition of A β 42 aggregation by the linear peptides revealed that the effects of these peptides differed from those of the cyclic peptides: In particular, linear BPTI markedly increased aggregation, while both linear TFPI and linear bikunin exerted only a minor effect on A β 42 aggregation. The reduction in the β -sheet content of A β 42 aggregates treated with the linear peptides, as shown by the CD spectra, may be a result of the formation of large insoluble fibrils (73,74). Indeed, TEM images of A β 42 treated with linear peptides showed the formation of large aggregates with a morphology different from that of the aggregates of untreated A β 42, with the former species appearing to be more densely packed and tangled than in untreated A β 42. A comparison between cyclic peptides and their linear versions has also been undertaken in other studies (17,75). For example, Jha et al., who tested a 10-mer cyclic peptide and its linear counterpart, showed that the cyclic peptide reduced both aggregation and β -sheet content of A β 42 in vitro and mitigated A β 42-induced toxicity in SH-SY5Y cells, whereas the linear peptide had no effect on A β 42 aggregation in vitro and or in cells (76). Reinforcing the findings that the three Kunitz-type proteins (i.e., bikunin, BPTI, and TFPI) and their derived cyclic peptides – but not their linear counterparts – inhibited A β 42 aggregation in vitro, our results in cells indicate that treatment with the Kunitz-type proteins and their derived cyclic peptides also led to a reduction in the β -sheet content of A β 42; namely, a reduction in the formation of A β 42 aggregates (as shown in the ThT assay and TEM images) was visualized using confocal microscopy of SH-SY5Y cells, where the internalization of extracellular A β 42 aggregates was reduced upon interaction with the cyclic peptides but not with the linear peptides. In contrast, as shown in one of our previous studies, the interaction of the cyclic APPI peptide with A β 42 enhanced A β 42 aggregation, thereby leading to the formation of more fibrils and less toxic A β 42 oligomers (35). The explanation for the above differences may lie in the ability of the cyclic peptides (whether cyclic APPI or the cyclic bikunin, BPTI, and TFPI peptides) to interfere with the interaction of A β 42 with the cell membrane due to structural changes induced in A β 42 upon interaction with the peptides (77). These structural changes, in turn, led to reduced A β 42-induced cytotoxicity in SH‑SY5Y cells, as reflected in a reduction of both apoptotic processes and mitochondrial dysfunction. Taken together, these findings led us to posit that, like the Kunitz-type proteins, all four cyclic peptides inhibited the A β 42 'on-pathway' mechanism, thereby reducing the quantity of toxic oligomers that subsequently nucleate to form A β fibrils. The study's collective results confirm that both the specific amino acid sequence and the β -hairpin structure found in Kunitz-type proteins and cyclic peptides are critical for altering A β 42 aggregation but the only the β -hairpin structure is essential for mitigating neurotoxicity. This study also reveals the potential of members of the Kunitz-type protein family and their derived cyclic peptides to reduce the neurotoxicity of A β 42 aggregates either by decreasing formation of toxic oligomers or by enhancing the formation of A β 42 fibrils, both resulting in disruption of the 'on-pathway' mechanism of A β 42 oligomerization (78). We emphasize that it is the alterations to the aggregation pathway of A β 42 – whether enhancing or impeding aggregation – controlled by the β -hairpin domain (either as a sub-domain within a Kunitz-type protein or as an isolated cyclic peptide) and its amino acid sequence, that lead to a reduction in the quantity of the toxic 'on-pathway' oligomers, which subsequently elongate and nucleate to form A β fibrils. METHODS BPTI, Bikunin and TFPI Expression and Purification. BPTI, bikunin and TFPI genes were separately cloned into pPICK9K plasmids as described previously (79,80). The plasmids were transformed into Pichia pastoris strain GS115 and then inoculated into and allowed to stand overnight in 5 ml of BMGY medium (1% yeast extract, 2% peptone, 0.23% potassium phosphate monobasic, 1.18% potassium phosphate dibasic, 1.34% yeast nitrogen base, 4×10 -5 % biotin and 1% glycerol). Thereafter, the cells were transferred to 50 ml of BMGY, followed by scaling up to 500 ml of BMGY. For protein expression, the cells were resuspended in 500 ml of BMMY (same as BMGY, but with 0.5% methanol instead of glycerol) and grown for three days, with 2% methanol being added every 24 h to maintain induction. Following four days of induction, the cultures were centrifuged at 4,700×g for 20 min, and the supernatants containing the secreted proteins were adjusted to 10 mM imidazole and 0.5 M NaCl at pH 8.0, followed by incubation for 1 h at 4 °C and then centrifugation at 4,700×g for 20 min. Next, the supernatants were filtered through 0.22- μ m Stericup bottle-top filters (Millipore, MA, USA). The filtered supernatants were loaded onto 5-ml HisTrap columns (GE Healthcare, Piscataway, NJ, USA) at a flow rate of 0.9 ml/min for 16 h, washed with a washing buffer (20 mM sodium phosphate, 0.5 M NaCl, and 10 mM imidazole; pH 8.0), and eluted with an elution buffer (as for washing buffer but with 0.5 M imidazole) using ÄKTA™ start (GE Healthcare). As the final step for BPTI, bikunin and TFPI purification, SEC was performed on Superdex 75 16/600 columns (GE Healthcare) equilibrated with PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 ; pH 7.4), at a flow rate of 1 ml/min on ÄKTA start. The purity of the proteins was confirmed by 15% SDS-PAGE gel under non-reducing conditions. The concentrations of the proteins were determined with a BCA Protein Assay Kit (Thermo Fisher, MA, USA) according to manufacturer's instructions. The molar weights of the pure proteins, validated using a MALDI-TOF (Bruker, Billerica, MA, USA) mass spectrometer (The Ilse Katz Institute for Nanoscale Science and Technology, BGU), were 9.68, 9.31, and 9.71 kDa for BPTI, bikunin, and TFPI, respectively. Synthetic Peptides. Synthetic A β (1–42) (A β 42) was purchased from AnaSpec (Fremont, CA, USA) and from GL Biochem (Shanghai, China). Fluorescently labeled A β 42-Hylight 488 (A β 42-488) was purchased from AnaSpec (Fremont, CA, USA). The unlabeled A β 42 from AnaSpec was dissolved in hexafluoroisopropanol (HFIP; Sigma-Aldrich, Israel) to a final concentration of 221 µ M. The required quantity of synthetic A β 42 peptide from AnaSpec was taken, followed by evaporation of the HFIP for 10 h. The synthetic A β 42 peptide from GL Biochem was dissolved in 10 mM NaOH, and the concentration was determined by NanoDrop spectrophotometer (Thermo Fisher, MA, USA) using an extinction coefficient of 1490 M −1 cm −1 . A β 42-488 was dissolved in 10 mM NaOH to final concentration of 205 µ M. The synthetic peptides, cyclic TFPI (CMKRFFFNIFTRQSEEFIYC), linear TFPI (MKRFFFNIFTRQSEEFIY), cyclic BPTI (CIIRYFYNAKAGLSQTFVYC), linear BPTI (IIRYFYNAKAGLSQTFVY), cyclic bikunin (CIQLWAFDAVKGKSVLFPYC), and linear bikunin (IQLWAFDAVKGKSVLFPY), were synthesized and divided into 1-mg aliquots by GL Biochem. For all experiments, except the CD measurements, the linear and cyclic peptides were dissolved in 1 ml of DMSO. The concentrations of the stock solutions were determined according to the Mw of the synthetic peptides (Figure S4). Pre-Incubation of the Peptides. For all experiments (except ThT assays and CD measurements), A β 42 was dissolved in phosphate buffer (20 mM sodium phosphate, pH 7.4, 150 mM NaCl) in the presence or absence of the peptides or Kunitz-type proteins and incubated in a Thermo Shaker incubator for 18 h (300 rpm, orbital shaking) at 37 °C. Cell Culture. SH-SY5Y neuroblastoma cells were grown at 37 °C and 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM; Sartorius, Göttingen, Germany) supplemented with 10% tetracycline-free fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 2 mM l‑glutamine (Gibco), and penicillin (100 units/ ml)/streptomycin (0.1 mg/ml) (Gibco). Detection of A β 42 Aggregation by using the ThT Assay. The aggregation of A β 42 was determined using the fluorescent dye ThT, as follows. A β 42, 4 µ M, and ThT (Sigma-Aldrich, Israel), 10 µ M, were incubated in the absence or presence of 62.5 nM Kunitz-type proteins (BPTI, TFPI or bikunin) or 8 µ M linear or cyclic peptides in 350 µ l of phosphate buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4). Aliquots of 100 μ l of reaction mixture were added to each well of a black 96-well plate (Greiner Bio-One, Germany), and the fluorescence intensity was determined at 5-min intervals, for 16 h at 37 °C, with constant orbital shaking at 205 rpm. The fluorescence signals were detected using BioTek Synergy H1 microplate reader (Winooski, Vermont, USA) with excitation and emission wavelengths of 440 and 485 nm, respectively. Each experiment was performed in triplicate. CD Spectra. The linear and cyclic peptides were dissolved in phosphate buffer and mixed with 50% acetonitrile to a concentration of 2000 µ M. Samples of 50 µ M A β 42 in the absence or presence of Kunitz-type proteins (781.25 nM) or linear or cyclic peptides (100 µ M) were added to 200 µ l of diluted phosphate buffer (5 mM sodium phosphate, 37.5 mM NaCl, pH 7.4) in order to maintain an appropriate high-tension (HT) voltage of the instrument. The samples were incubated in a Thermo Shaker Incubator for 18 h (500 rpm, orbital shaking) at 37 °C. The pre-incubated peptide samples were scanned using a Jasco J-715 spectropolarimeter (Jasco, Japan). The spectrum of each sample was recorded three times at 25 °C in a range between 195–260 nm using a quartz cuvette with a path length of 1 mm, a scanning speed of 50 nm/min, and a data interval of 0.5 nm. The spectral scans were averaged to smooth the data curves. For baseline correction, protein-free phosphate buffer was used. TEM Imaging of A β 42 Aggregates. Samples of A β 42 with Kunitz-type proteins, cyclic peptides, or linear peptides taken from the above CD assay post 18 h of incubation were investigated using TEM. For this purpose, 2.5 µ l of each sample was deposited on a carbon-coated copper 300-mesh grid. After 1 min, excess liquid was carefully removed with filter paper Then, 2% uranyl acetate in doubly distilled water was added to each sample, and after 1 min any excess of uranyl acetate solution was carefully removed using a filter paper. . Images were acquired using a Tecnai G2 12 BioTWIN (FEI, ThermoFisher Scientific, Paisley, UK) TEM with an acceleration voltage of 120 kV (The Ilse Katz Institute for Nanoscale Science and Technology, BGU). Different magnifications were used for visualization, depending on the size of the aggregates, and multiple fields were analyzed in each grid. Dot Blot Analysis. Samples from the CD measurements of A β 42 with Kunitz-type proteins, cyclic peptides, or linear peptides were analyzed. Each sample (5 µ l) was loaded onto two nitrocellulose membranes (Bio-Rad, Hercules, California, USA), which were blocked with 5% skim milk (Sigma-Aldrich, Israel) in Tris-buffered saline with 0.1% Tween (TBST; Biolab, Israel), and then further washed with TBST three times for 5 min each. The first membrane was incubated overnight with rabbit amyloid fibril polyclonal antibody (Thermo Fisher, Israel) diluted 1:1000 in PBS. The second membrane was incubated overnight with mouse anti-A β 42 antibody (Abcam, Cambridge, UK) diluted 1:1000 in PBS to confirm that the same amount of A β 42 had been loaded in each sample. After washing, the membranes were incubated for 1 h with HRP-linked anti-mouse secondary antibody (Cell Signaling, Danvers, MA, USA) and anti-rabbit secondary antibody (Cell Signaling), respectively. The chemiluminescence signals were detected using a Fusion FX imaging system (Vilber, Germany). The experiments were performed in triplicate. Mammalian Expression Plasmids. For expression in SH-SY5Y cells, A β 42-GFP, BPTI, bikunin, and TFPI were cloned into a pPHAGE2 vector under the control of the human cytomegalovirus promoter. Aβ42-GFP was cloned as described previously (81). BPTI , bikunin , and TFPI genes were cloned into the pHAGE2 plasmid using NotI and BamHI restriction enzymes (New England Biolabs, MA, USA) as described previously (36). The pPHAGE2 plasmid was used as a mammalian vector for expressing BPTI, bikunin, and TFPI linked to blue fluorescent protein via an internal ribosome entry site in SH-SH5Y cells under the control of the human cytomegalovirus promoter. Confocal Microscopy Imaging. In all experiments, SH-SY5Y cells were seeded at a density of 10 4 cells per well in 1-cm micro-slides (ibidi, Gräfelfing, Germany). For intracellular expression of Kunitz-type proteins (i.e., BPTI, bikunin, and TFPI) and A β 42-GFP, the cells were transfected one day after seeding with either 1 µ g of a mixture of A β 42-GFP plasmid and empty plasmids or a mixture of A β 42-GFP and Kunitz-type gene containing pPHAGE plasmids by using the jetOPTIMUS transfection reagent (Polyplus, Illkrich, France), according to the manufacturer’s manual. After 48 h of incubation, the cells were washed with PBS and fixed by incubation with 4% paraformaldehyde (PFA) in PBS for 30 min. Cell imaging was performed using Olympus FV1000 confocal microscope with a long-working distance ×60/1.35 numerical aperture oiled-immersion objective [The National Institute for Biotechnology in the Negev (NIBN), BGU]. For extracellular treatments with A β 42 and cyclic or linear peptides, the cells were treated with pre-incubated samples of 1 µ M A β 42-488 in the absence or presence of 2 µ M cyclic or linear peptides. After the incubation, the cells were washed with PBS and fixed by incubation with 4% PFA in PBS for 30 min. Cells imaging was performed using a Zeiss LSM 880 confocal microscope with a ×40 objective (The Ilse Katz Institute for Nanoscale Science and Technology, BGU). Detection of A β 42-GFP Expression in SH-SY5Y Cells. SH-SY5Y cells (3 ´10 5 cells per well) were seeded in a six-well tissue culture plate (Greiner, Sigma-Aldrich, Israel). For transient expression of A β 42-GFP in the presence or absence of the Kunitz-type proteins, the cells were transiently transfected on the following day using the jetOPTIMUS transfection reagent (Polyplus) according to the manufacturer’s manual. Each well was transfected with 1 µ g of pPHAGE-A β 42-GFP and 1 µ g of pHAGE2-empty, pHAGE2-BPTI, pHAGE2-bikunin, or pHAGE2-TFPI. After 48 h, the medium was removed, and the cells were incubated for 20 min with RIPA buffer (EMD Millipore Corp., Billerica, MA, USA) and protease inhibitor cocktail (APE×BIO, Boston, MA, USA) for cell lysis. The cell cultures were centrifuged, and protein concentrations were determined in the supernatants by the BCA assay (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Each lysate (50 µ g) was loaded onto a 15% SDS-PAGE and then transferred to a nitrocellulose membrane for western blot analysis. The membrane was blocked with 5% skim milk and incubated overnight with mouse anti-A β 42 antibody diluted 1:1000 in PBS (Abcam). After washing, the membrane was incubated for 1 h with HRP-linked anti-mouse secondary antibody (Cell Signaling, Danvers, MA, USA), and chemiluminescence signals were detected. XTT Cell Viability Assay. SH-SY5Y cells (10 4 cells per well) were seeded in 96-well tissue culture plate (Greiner, Sigma-Aldrich, Israel). After 24 h, the cells were treated with pre-incubated samples (see details in ‘pre-incubation of the peptides’ subsection) of 10 µ M A β 42 in the presence or absence of the Kunitz-type proteins (156.25 nM) or the cyclic peptides (8 µ M). The viability of the cells was assessed 48 h post-treatment by using an XTT-based kit (Cell Signaling) according to the manufacturer’s protocol. The absorbance was read at a wavelength of 450 nm using a BioTek Synergy H1 microplate reader. Three repetitions were performed for each treatment. The values were normalized to the control group (untreated cells). Mitochondrial Membrane Depolarization Potential Measurements. To measure the changes in the mitochondrial membrane potential in SH-SY5Y cells, 10 4 cells/well were seeded in a black-clear bottomed 96-well tissue culture plate (Greiner, Sigma-Aldrich, Israel). The cells were treated for 48 h with pre-incubated samples (see details in ‘pre-incubation of the peptides’ subsection) of 10 µ M A β 42 in the absence or presence of the Kunitz-type proteins (156.25 nM) or the cyclic peptides (8 µ M). The changes in the mitochondrial membrane potential were assessed using TMRE-Mitochondrial Membrane Potential Assay Kit (Abcam), according to the manufacturer’s protocol. The fluorescence intensity, which indicated mitochondrial potential, was detected using TECAN infinite M200 microplate reader (Männedorf, Switzerland) with excitation and emission wavelengths of 549 and 575 nm, respectively. Each experiment was performed in triplicate. The values were normalized to the control group (untreated cells). Flow Cytometry Analysis of Apoptotic Cells. SH-SY5Y cells (3×10 4 cells per well) were seeded in a 24-well plate and incubated for 18 h with 15 µ M pre-formed A β 42 aggregates in the absence or presence of 250 nM Kunitz-type proteins (BPTI, TFPI and bikunin) or 30 µ M cyclic peptides (see details in the ‘pre-incubation of the peptides’ subsection). Untreated cells served as a negative control. After 24 h of incubation, the cells were harvested, washed, and stained with Annexin V-APC and SYTOX Green using an Apoptosis Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The cells were analyzed using a BD FACS Canto II (BD Biosciences, Erembodegem, Belgium). The following cell populations were analyzed: intact cells (Annexin V-APC negative and SYTOX Green negative), early apoptotic cells (Annexin V-APC positive and SYTOX Green negative), late apoptotic cells (annexin V-APC and SYTOX Green positive), and necrotic cells (annexin V-APC negative and SYTOX Green positive). Statistical Analysis. All data were analyzed statistically with GraphPad Prism, version 8.00, for Windows (La Jolla, CA, USA). Data are shown as means ± SD. Statistical significance between the control group and different treatments was determined by an unpaired Student’s t-test, with statistical significance * p < 0.05; ** p < 0.01; *** p < 0.001. Abbreviations A β amyloidβ A β 42 amyloid β 1–42 AD Alzheimer's disease APP amyloid precursor protein APPI amyloid precursor protein inhibitor BPTI bovine pancreatic trypsin inhibitor CD circular dichroism DMEM Dulbecco's modified Eagle's medium GFP green fluorescent protein HFIP 1,1,1,3,3,3-hexafluoro-2-propanol PBS phosphate-buffered saline TEM transmission electron microscope TFPI tissue factor pathway inhibitor ThT thioflavin T TMRE tetramethylrhodamine,ethyl ester TTR transthyretin XTT 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide. Declarations Funding This work was supported by the Rosetrees Trust (OoR2022/100004), Israel Science Foundation (grant number 1437/24), and the Worldwide Cancer Research (grant number 20-0238) to NP. Competing Interests The authors declare that they have no conflict of interest with respect to publication of this paper. Author Contributions M.R. and N.P. designed the research; M.R. and S.L.H. performed the research; M.R. and N.P. analyzed the data; M.R. and N.P. wrote the paper. All authors edited the manuscript and approved the final version. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics Declaration This study does not require ethics approval. References Beydoun MA, Beydoun HA, Gamaldo AA, Teel A, Zonderman AB, Wang Y (2014) Epidemiologic studies of modifiable factors associated with cognition and dementia: systematic review and meta-analysis. 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Biochem J 475(19):3087–3103 Supplementary Files image1.png GRAPHICAL ABSTRACT Cyclic peptides derived from Kunitz protein β-domains demonstrate neuroprotective properties by suppressing the formation of Aβ42 fibrils, inhibiting Aβ42-induced membrane depolarization, and attenuating Aβ42-mediated apoptosis and cytotoxicity in SH-SY5Y cells. The figure was prepared with BioRender.com. RabinovichetalKunitzAb42SuppInfoCMLS14.1.26.docx Cite Share Download PDF Status: Posted Version 1 posted 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-8603888","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":577155422,"identity":"82b5990f-a0db-4ddd-b1b8-2508eae0ac21","order_by":0,"name":"Maya Rabinovich","email":"","orcid":"","institution":"Ben-Gurion University of the Negev","correspondingAuthor":false,"prefix":"","firstName":"Maya","middleName":"","lastName":"Rabinovich","suffix":""},{"id":577155423,"identity":"39c125d0-d964-43d5-b283-d22b24175f8a","order_by":1,"name":"Shiran Lacham-Hartman","email":"","orcid":"","institution":"Ben-Gurion University of the 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12:42:50","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":221818,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8603888/v1/9a93593c94e2a2ae224c8071.html"},{"id":100791821,"identity":"942088f1-3ca3-4262-9e9f-13aea13cb136","added_by":"auto","created_at":"2026-01-21 12:42:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":175209,"visible":true,"origin":"","legend":"\u003cp\u003eAbility of the Kunitz-type proteins and their derived cyclic peptides to inhibit A\u003cem\u003eβ\u003c/em\u003e42 aggregation. A\u003cem\u003eβ\u003c/em\u003e42 aggregation was monitored for 16 h by using the ThT assay, as follows. Mixtures of 4 \u003cem\u003eµ\u003c/em\u003eM A\u003cem\u003eβ\u003c/em\u003e42 with (A) 62.5 nM Kunitz proteins at a molar ratio of 64:1 (A\u003cem\u003eβ\u003c/em\u003e42:protein), (B) 8 \u003cem\u003eµ\u003c/em\u003eM cyclic peptides at a molar ratio of 1:2 (A\u003cem\u003eβ\u003c/em\u003e42:peptide), or (C) 8 \u003cem\u003eµ\u003c/em\u003eM linear peptides at a molar ratio of 1:2 (A\u003cem\u003eβ\u003c/em\u003e42:peptide) dissolved in phosphate buffer (20 mM sodium phosphate, pH 7.4, 150 mM NaCl) were incubated with 10 \u003cem\u003eµ\u003c/em\u003eM ThT for 37 °C with constant shaking. The fluorescence was read using BioTek Synergy H1 microplate reader (Winooski, Vermont, USA) using excitation and emission wavelengths of 440 and 485 nm, respectively.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8603888/v1/a29c18ea7fbe05a96feeaee4.png"},{"id":100791848,"identity":"769e5912-2c1b-4e43-8df6-0f9cb12cb314","added_by":"auto","created_at":"2026-01-21 12:42:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110529,"visible":true,"origin":"","legend":"\u003cp\u003eKunitz-type proteins and their derived peptides reduce \u003cem\u003eβ\u003c/em\u003e-sheet content in A\u003cem\u003eβ\u003c/em\u003e42. (A) Kunitz-type proteins, 781.25 nM,(B) cyclic peptides, 100 \u003cem\u003eµ\u003c/em\u003eM, or (C) linear peptides, 100 \u003cem\u003eµ\u003c/em\u003eM, at a molar ratio of 1:2 (A\u003cem\u003eβ\u003c/em\u003e42:peptide) were incubated with A\u003cem\u003eβ\u003c/em\u003e42, 50 \u003cem\u003eµ\u003c/em\u003eM, for 18 h. CD spectra were acquired over the range 195–260 nm.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8603888/v1/07094799db5a28aec316a3c7.png"},{"id":100791825,"identity":"0a495f03-598c-444d-8eb9-cd146d00bf3c","added_by":"auto","created_at":"2026-01-21 12:42:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2092249,"visible":true,"origin":"","legend":"\u003cp\u003eThe Kunitz-type proteins and their derived cyclic peptides reduce the formation of A\u003cem\u003eβ\u003c/em\u003e42 aggregates.\u003cstrong\u003e \u003c/strong\u003eTEM images of samples of A\u003cem\u003eβ\u003c/em\u003e42 (50 \u003cem\u003eµ\u003c/em\u003eM)incubated for 18 h in the absence of proteins or peptides (A) or in the presence of (B) the Kunitz-type proteins (781.25 nM), (C) the cyclic peptides (100 \u003cem\u003eµ\u003c/em\u003eM), or (D) the linear peptides (100 \u003cem\u003eµ\u003c/em\u003eM). White arrows indicate A\u003cem\u003eβ\u003c/em\u003e42 fibrils. Insets show magnified views of the boxedregions. Scale bar, 500 nm.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8603888/v1/bceb645959f438e8efdf621f.png"},{"id":100791837,"identity":"f2540076-cdd6-45ce-8558-04d04013cb4b","added_by":"auto","created_at":"2026-01-21 12:42:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":168686,"visible":true,"origin":"","legend":"\u003cp\u003eThe Kunitz-type proteins and their derived peptides reduce the quantity of A\u003cem\u003eβ\u003c/em\u003e42 fibrils. Dot blot analysis of samples of A\u003cem\u003eβ\u003c/em\u003e42, 50 \u003cem\u003eµ\u003c/em\u003eM, incubated for 18 h in the absence or in the presence of (A) the Kunitz-type proteins, 781.25 nM, (B) the cyclic peptides, 100 \u003cem\u003eµ\u003c/em\u003eM, or (C) the linear peptides, 100 \u003cem\u003eµ\u003c/em\u003eM. The quantity of A\u003cem\u003eβ\u003c/em\u003e42 fibrils was assessed using anti-amyloid fibril antibody. The total quantity of A\u003cem\u003eβ\u003c/em\u003e42 in each sample was detected by blotting with anti-A\u003cem\u003eβ\u003c/em\u003e42 antibody.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8603888/v1/88ef0778b38967832aac5580.png"},{"id":100797070,"identity":"fba386bd-01b6-4bb5-af9b-f3ccdf9a6106","added_by":"auto","created_at":"2026-01-21 13:47:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":822464,"visible":true,"origin":"","legend":"\u003cp\u003eThe Kunitz-type proteins reduce intracellular A\u003cem\u003eβ\u003c/em\u003e42 aggregate formation in SH-SY5Y cells.\u003cstrong\u003e \u003c/strong\u003e(A) Confocal microscopy images of SH-SY5Y cells expressing A\u003cem\u003eβ\u003c/em\u003e42-GFP or coexpressing A\u003cem\u003eβ\u003c/em\u003e42-GFP and the Kunitz-type proteins. Images were acquired with an Olympus FV1000 confocal microscope with a ×60 objective. White arrows indicate A\u003cem\u003eβ\u003c/em\u003e42 aggregates. Scale bar, 50 \u003cem\u003eµ\u003c/em\u003em. (B) Lysates of cells transiently expressing A\u003cem\u003eβ\u003c/em\u003e42-GFP or both A\u003cem\u003eβ\u003c/em\u003e42-GFP and a Kunitz-type protein, followed by western blot analysis using an anti-A\u003cem\u003eβ\u003c/em\u003e42 antibody.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8603888/v1/2b0d9178f5d989f1a8b925f6.png"},{"id":100797513,"identity":"412837f6-bf18-45f1-90d9-e6a74f2bc6ea","added_by":"auto","created_at":"2026-01-21 13:49:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1800030,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of extracellular A\u003cem\u003eβ\u003c/em\u003e42 internalization and accumulation by the cyclic peptides. SH‑SY5Y cells were treated with samples of 1 \u003cem\u003eµ\u003c/em\u003eM A\u003cem\u003eβ\u003c/em\u003e42 labeled with HyLyte Fluor 488 that had been pre-incubated for 18 h in the absence or presence of 2 \u003cem\u003eµ\u003c/em\u003eM (A) cyclic peptides and (B) linear peptides. Confocal microscopy images of the cells were acquired with a Zeiss LSM 880 confocal microscope. Arrows inicate inclusion bodies. Scale bar, 50 \u003cem\u003eµ\u003c/em\u003em.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8603888/v1/d51475b8fb9f9a54958fc65e.png"},{"id":100791838,"identity":"ebc6c62d-a96f-4eb2-a710-a42bfba380f4","added_by":"auto","created_at":"2026-01-21 12:42:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":93628,"visible":true,"origin":"","legend":"\u003cp\u003eThe Kunitz-type proteins and their derived cyclic peptides reduce A\u003cem\u003eβ\u003c/em\u003e42 toxicity in SH‑SY5Y cells.\u003cstrong\u003e \u003c/strong\u003eXTT cell viability assay of SH-SY5Y cells after 48 h of treatment with: (A,B) 10 \u003cem\u003eµ\u003c/em\u003eM A\u003cem\u003eβ\u003c/em\u003e42 that had been pre-incubated for 18 h at 37 ºC in the absence or presence of 156.25 nM Kunitz-type proteins or 20 \u003cem\u003eµ\u003c/em\u003eM cyclic peptides, respectively, or (C,D) 156.25 nM Kunitz-type proteins or 20 \u003cem\u003eµ\u003c/em\u003eM cyclic peptides, respectively. Statistical analysis (n = 3) was performed by an unpaired Student's t-test. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003e p \u003c/em\u003e\u0026lt; 0.01; *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8603888/v1/5f781fb888b39fab97a7e7b9.png"},{"id":100796965,"identity":"07a26ff8-9e06-40a8-8e83-46e8a43b8a8f","added_by":"auto","created_at":"2026-01-21 13:46:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":778304,"visible":true,"origin":"","legend":"\u003cp\u003eApoptotic processes induced by extracellular A\u003cem\u003eβ\u003c/em\u003e42 aggregates were reduced by the Kunitz-type proteins and cyclic peptides.\u003cstrong\u003e \u003c/strong\u003eFlow cytometry analysis of Annexin-V- and SYTOX-Green-stained SH-SY5Y cells after 24 h of incubation with A\u003cem\u003eβ\u003c/em\u003e42 (15 \u003cem\u003eµ\u003c/em\u003eM) that had been pre-incubated for 18 h at 37 ºC in the absence (A) or presence of (B) Kunitz-type proteins (250 nM) or (C) cyclic peptides (30 \u003cem\u003eµ\u003c/em\u003eM).\u003cstrong\u003e \u003c/strong\u003eUntreated cells served as a negative control.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-8603888/v1/069c501e648de7b56290c533.png"},{"id":100791828,"identity":"512dd278-55fd-489e-b08f-04a2e06499c5","added_by":"auto","created_at":"2026-01-21 12:42:50","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":71735,"visible":true,"origin":"","legend":"\u003cp\u003eSuppression of the A\u003cem\u003eβ\u003c/em\u003e42-mediated mitochondrial membrane depolarization potential (ΔΨm) by the Kunitz-type proteins and the cyclic peptides in SH-SY5Y cells treated with: (A, B) 10 \u003cem\u003eμ\u003c/em\u003eM A\u003cem\u003eβ\u003c/em\u003e42 that had been pre-incubated for 18 h at 37 ºC in the absence or presence of 156.25 nM Kunitz-type proteins or 20 \u003cem\u003eµ\u003c/em\u003eM cyclic peptides, respectively; or (C,D) 156.25 nM of each Kunitz-type protein \u0026nbsp;or \u0026nbsp;20 \u003cem\u003eµ\u003c/em\u003eM of each cyclic peptide, respectively. Changes in ΔΨm were assessed after 48 h by using tetramethylrhodamine ethyl ester (TMRE), which is an indicator of mitochondrial function in living cells. Statistical analysis (n = 3) was performed by an unpaired Student's t-test. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ** p \u0026lt; 0.01; *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-8603888/v1/295ece67a0eac7878598582c.png"},{"id":105566251,"identity":"3b9b5600-aa29-4402-b76f-b9f6a13e0c09","added_by":"auto","created_at":"2026-03-27 12:55:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6872745,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8603888/v1/6beb9766-2437-4ace-9b00-ba93ae86d4dd.pdf"},{"id":100791822,"identity":"2b3ff817-9d5e-481e-ab0a-95c0c5851ff5","added_by":"auto","created_at":"2026-01-21 12:42:49","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":969914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGRAPHICAL ABSTRACT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCyclic peptides derived from Kunitz protein β-domains demonstrate neuroprotective properties by suppressing the formation of Aβ42 fibrils, inhibiting Aβ42-induced membrane depolarization, and attenuating Aβ42-mediated apoptosis and cytotoxicity in SH-SY5Y cells. The figure was prepared with BioRender.com.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8603888/v1/b324ce408e0e129d604fdf5d.png"},{"id":100796922,"identity":"1cc749b1-130a-4f9f-9dd0-82edfdb9522b","added_by":"auto","created_at":"2026-01-21 13:46:45","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2271846,"visible":true,"origin":"","legend":"","description":"","filename":"RabinovichetalKunitzAb42SuppInfoCMLS14.1.26.docx","url":"https://assets-eu.researchsquare.com/files/rs-8603888/v1/69a9fce394d48ca29d4fef00.docx"}],"financialInterests":"","formattedTitle":"Modulating amyloid-β 42 aggregation and neurotoxicity by Kunitz domains and their derived peptides","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) \u0026ndash; currently the most common neurodegenerative disease \u0026ndash; is a significant public health concern, affecting millions of people worldwide and increasing with age from \u0026lt; 1% of people younger than 60 years to \u0026gt; 40% of those older than 85 years (1). Although the etiology of AD remains a subject of intense study, it is widely accepted that AD is associated with the extracellular and intracellular accumulation of aggregations of \u003cem\u003e\u0026beta;\u003c/em\u003e-amyloid (A\u003cem\u003e\u0026beta;\u003c/em\u003e) peptide fragments of amyloid precursor protein (APP). These fragments are generated by sequential cleavage by \u003cem\u003e\u0026beta;\u003c/em\u003e-secretase at the ectodomain of APP and then by \u0026gamma;-secretase at its transmembrane domain (2). Among the resultant cleavage fragments, the\u0026nbsp;42-amino-acid-containing A\u003cem\u003e\u0026beta;\u003c/em\u003e fragment (designated A\u003cem\u003e\u0026beta;\u003c/em\u003e42) is a particularly neurotoxic isoform, by virtue of its propensity to misfold and to aggregate into amyloid plaques in the brain, and it is thus a key player in the pathology of AD (3,4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe nucleation of disordered monomeric A\u003cem\u003e\u0026beta;\u003c/em\u003e42 into neurotoxic oligomeric aggregates and ordered \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet-structured fibrils (5) results from the interaction between the two hydrophobic segments within the A\u003cem\u003e\u0026beta;\u003c/em\u003e42 sequence, namely, the central hydrophobic core (residues 16\u0026ndash;22) and the C-terminal region (residues 30\u0026ndash;42), which fold into a \u003cem\u003e\u0026beta;\u003c/em\u003e-hairpin structure (6\u0026ndash;8). Numerous studies have sought to block the aggregation of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 (as a means to prevent/retard the onset of AD) by using a variety of inhibitors, with focus in recent years on two main types of proteinaceous molecule\u0026mdash;sequence-based peptides, i.e., those\u0026nbsp;with hydrophobic sequences that are the same or very similar to the native sequence of\u0026nbsp;A\u003cem\u003e\u0026beta;\u003c/em\u003e42 (9\u0026ndash;11), and structure-based peptides, i.e., those with a\u003cem\u003e\u0026nbsp;\u0026beta;\u003c/em\u003e-strand structure (12\u0026ndash;14). In an example of the first approach, treatment of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 with the short hydrophobic peptide LPYFD-amide (a derivative of residues 17\u0026ndash;21 of the central hydrophobic core of A\u003cem\u003e\u0026beta;\u003c/em\u003e42) reduced A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation and its associated toxicity in neuronal cells and in mice (15,16). By applying the second approach, a similar inhibitory effect was obtained by a different peptide (LYFGA), also mimicking the hydrophobic core of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 (i.e., residues 17\u0026ndash;21) but having a \u003cem\u003e\u0026beta;\u003c/em\u003e-strand structure that was generated by chemically cross-linking Tyr at position 2 and Gly at position 4 (17).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn yet another approach that appeared to offer promise for the generation of peptides inhibiting A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation, ring-like peptide structures were designed and tested (18). A representative example is provided by a 23-residue bicyclic peptide in which three cysteine residues enabled double cyclization to produce a molecule with two central regions, LGIKI and TSVYHA, that bind the C-terminal region of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 at residues 31\u0026ndash;36 and 38\u0026ndash;42, respectively (19). Nonetheless, although this bicyclic peptide remodeled A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation, producing shorter, thicker, and non-fibrillar structures, which reduced A\u003cem\u003e\u0026beta;\u003c/em\u003e42-associated toxicity in a \u003cem\u003eCaenorhabditis\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eelegans\u0026nbsp;\u003c/em\u003emodel, its interaction with the monomeric form of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 was weak and transient and showed no inhibitory effect on pre-formed fibrils (19). In parallel, studies on cyclic peptides that were specifically designed to mimic the \u003cem\u003e\u0026beta;\u003c/em\u003e-hairpin motif in A\u003cem\u003e\u0026beta;\u003c/em\u003e42 showed that they were indeed able to reduce A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation and toxicity (20\u0026ndash;23). For example, Yamin et al. synthesized a 13-mer cyclic peptide with hairpin structure that interacts with A\u003cem\u003e\u0026beta;\u003c/em\u003e42(24). Although they showed that the cyclic peptide modulated A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation by inhibiting the formation of oligomers and fibrils while promoting the generation of non-fibrillar aggregates (24), the lack of cellular toxicity assays leaves the conclusions of the study somewhat limited.\u0026nbsp;In summary, although cyclic peptides appear to have potential as blockers of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation, work to date on the effect on A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation of the amino acid sequence or the structure of these peptides has not produced definitive conclusions that may ultimately be leveraged in the design of AD therapeutics.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA starting point for addressing the above issues is an examination of the structure and activity of human APP. This protein has three major isoforms that are produced as a result of alternative splicing, namely, APP695, APP751, and APP770. The latter two isoforms each contain a 58-amino acid extracellular subunit known as a Kunitz protease inhibitor or as an amyloid precursor protein inhibitor (APPI) (25\u0026ndash;27).\u0026nbsp;The two APPI-containing isoforms, APP751 and APP770, are expressed in multiple tissues (e.g., glia cells, skin, and lungs), while the APPI-deficient isoform, APP695, is expressed primarily in neurons (28,29). Of note, previous studies have reported a significant increase in mRNA and protein levels of the APPI-containing isoforms in AD brains, but the involvement of APPI in AD progression has yet to be elucidated\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(30,31).\u003c/p\u003e\n\u003cp\u003eThus, to date, APPI has been studied predominantly for its function as a serine protease inhibitor that targets proteases, such as mesotrypsin, whose catalytic activity plays roles in various malignancies and in pancreatitis (32\u0026ndash;34). Nonetheless, two recent papers from our laboratory have investigated the role of APPI in the formation A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregates (35,36). The first showed\u0026nbsp;that\u0026nbsp;a cyclic \u003cem\u003e\u0026beta;\u003c/em\u003e-hairpin peptide derived from the \u003cem\u003e\u0026beta;\u003c/em\u003e-domain of APPI\u0026nbsp;(but not its linear counterpart) reduced the toxicity of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 and enhanced the formation of mature A\u0026beta;42 fibrils from intermediate, toxic oligomeric A\u003cem\u003e\u0026beta;\u003c/em\u003e42 species (35)\u0026nbsp;.\u0026nbsp;The second study demonstrated the ability of both extra- and intracellular full-length APPI to reduce A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregate formation and hence to decrease the cellular toxicity mediated by both extra- and intracellular A\u003cem\u003e\u0026beta;\u003c/em\u003e42 (36). These two studies thus revealed an apparent contradiction in that the full-length APPI protein reduced A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation and the formation of toxic A\u003cem\u003e\u0026beta;\u003c/em\u003e42 oligomers, but the cyclic APPI peptide enhanced A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation, forming fibrils rather than neurotoxic A\u003cem\u003e\u0026beta;\u003c/em\u003e42 oligomers and thereby ameliorating A\u003cem\u003e\u0026beta;\u003c/em\u003e42-mediated neurotoxicity.\u003c/p\u003e\n\u003cp\u003eTo further investigate this issue and in line with previous reports highlighting the potential of cyclic \u003cem\u003e\u0026beta;\u003c/em\u003e-hairpin mimetics in AD therapeutics, we sought to address the open question of\u0026nbsp;whether it is a structural element or the specific sequence of the cyclic APPI fragment that interacts with A\u003cem\u003e\u0026beta;\u003c/em\u003e42 to enhance its aggregation and hence to attenuate A\u003cem\u003e\u0026beta;\u003c/em\u003e42 neurotoxicity. To this end, we\u0026nbsp;investigated the putative inhibitory activities of three other Kunitz-type proteins, namely, tissue factor pathway inhibitor (TFPI), bovine pancreatic trypsin inhibitor (BPTI) and bikunin(37), and their derived \u003cem\u003e\u0026beta;\u003c/em\u003e-domain\u0026nbsp;peptides in cyclic or linear conformation. The rationale for choosing these three Kunitz-type proteins was that they have similar lengths (58 amino acids) and structures (\u003cem\u003e\u0026beta;\u003c/em\u003e-hairpin domain with\u0026nbsp;two antiparallel \u003cem\u003e\u0026beta;\u003c/em\u003e-sheets and one helical region) to APPI [investigated in our previous studies (1,36)], but different sequences to one another and to APPI, both in the full-length protein and\u0026nbsp;within the \u003cem\u003e\u0026beta;\u003c/em\u003e-hairpin region (37\u0026ndash;40).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHerein, we demonstrate that, like the \u003cem\u003e\u0026beta;\u003c/em\u003e-domain of APPI(36), the \u003cem\u003e\u0026beta;\u003c/em\u003e-domains of the three Kunitz-type proteins interact with A\u003cem\u003e\u0026beta;\u003c/em\u003e42, thereby\u0026nbsp;modulating its\u0026nbsp;aggregation mechanism. Using thioflavin T (ThT)\u0026nbsp;fluorescence assays,\u0026nbsp;circular dichroism (CD) spectroscopy, and transmission electron microscopy (TEM) imaging, we show that both the Kunitz-type proteins, at a molar ratio of 64:1 (A\u003cem\u003e\u0026beta;\u003c/em\u003e42:Kunitz), and the Kunitz-derived cyclic peptides with a \u003cem\u003e\u0026beta;\u003c/em\u003e-hairpin structure, at a molar ratio of 1:2 (A\u003cem\u003e\u0026beta;\u003c/em\u003e42:cyclic peptide), reduce the formation of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregates. In contrast, the Kunitz-derived linear peptides do not result in a significant reduction in A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation. Similarly, treatment of SH-SY5Y neuroblastoma cells with the Kunitz-type proteins and their derived cyclic peptides, but not their linear counterparts, reduce the intracellular and extracellular accumulation of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregates, respectively, as assessed by confocal microscopy. Notably, the Kunitz-type proteins and the cyclic peptides prevent A\u003cem\u003e\u0026beta;\u003c/em\u003e42-mediated mitochondrial membrane depolarization and reduce A\u003cem\u003e\u0026beta;\u003c/em\u003e42-mediated apoptosis and cell death.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaken together, the findings of this study show that the combination of the amino acid sequence\u0026nbsp;of the Kunitz-type proteins and the \u003cem\u003e\u0026beta;\u003c/em\u003e-hairpin structure present in both Kunitz-type proteins and cyclic peptides impacts A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation and that the \u003cem\u003e\u0026beta;\u003c/em\u003e-hairpin structure is responsible for reducing neurotoxicity\u0026mdash;irrespective of the amino acid sequence.\u0026nbsp;\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eThe Inhibitory Effect of the Kunitz-Type Proteins on A\u003cem\u003e\u0026beta;\u003c/em\u003e42 Aggregation in Vitro is Similar to that of their Derived Cyclic Peptides. \u003c/strong\u003eTo examine the effect of the Kunitz-type proteins and their derived peptides on A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation, it was first necessary to produce and purify BPTI, TFPI, and bikunin. The three proteins were expressed and purified as described previously (42,43) (see Methods), and for TFPI and bikunin representative results for the different stages of the procedure are given in the Supporting Information (\u003cstrong\u003eFigures S1\u003c/strong\u003e and\u003cstrong\u003e S2,\u003c/strong\u003e respectively). Yields of 15 mg, 7.4 mg, and 10 mg per 1 L of yeast culture were obtained for BPTI, TFPI, and bikunin, respectively.\u003c/p\u003e\n\u003cp\u003eThe ability of the Kunitz-type proteins to inhibit aggregation of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 was then investigated by incubating A\u003cem\u003e\u0026beta;\u003c/em\u003e42 for 16 h in the absence or presence of the proteins and monitoring the aggregation levels of untreated or treated A\u003cem\u003e\u0026beta;\u003c/em\u003e42 (\u003cstrong\u003eFigure 1A\u003c/strong\u003e). As expected, the highest aggregation signal was observed for A\u003cem\u003e\u0026beta;\u003c/em\u003e42 alone, consistent with its intrinsic tendency to self-assemble first into oligomers and aggregates and then into fibrils (5), while a decrease in aggregation was observed when A\u003cem\u003e\u0026beta;\u003c/em\u003e42 was incubated with the Kunitz-type proteins.\u003c/p\u003e\n\u003cp\u003eWith the aims to study the effect of the \u003cem\u003e\u0026beta;\u003c/em\u003e-domain of Kunitz-type proteins on A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation and to determine whether it is the structure or the sequence of the domains that influences the aggregation, we designed three linear peptides and their three cyclic counterparts that were derived from the \u003cem\u003e\u0026beta;\u003c/em\u003e-hairpin domains of the three native Kunitz-type proteins, BPTI, TFPI and bikunin (\u003cstrong\u003eFigure S3A\u003c/strong\u003e), as follows: Three 18-mer linear peptides were derived from the full \u003cem\u003e\u0026beta;\u003c/em\u003e-hairpin sequences of BPTI, bikunin and TFPI (\u003cstrong\u003eFigure S3B\u003c/strong\u003e); and three 20-mer cyclic peptides were designed such that they had the same sequences as the linear peptides, but with two cysteine residues added at the N- and C-termini to form a disulfide bridge, creating a cyclic structure (\u003cstrong\u003eFigure S3C\u003c/strong\u003e) and thereby mimicking the structure of the \u003cem\u003e\u0026beta;\u003c/em\u003e-domain in the Kunitz-type proteins (41). In addition, to prevent potential undesired interactions, the internal cysteine residue in the sequences of the cyclic peptides was replaced with serine, and to better mimic the \u003cem\u003e\u0026beta;\u003c/em\u003e-domains in the native Kunitz-type protein sequences, the N-termini of all six peptides were acetylated and the C-termini were amidated.\u003c/p\u003e\n\u003cp\u003eTo confirm both the formation of the disulfide bridge within the cyclic peptides and the correct molar weights of the linear and cyclic peptides, all six peptides were analyzed by mass spectrometry (\u003cstrong\u003eFigure\u0026nbsp;S4\u003c/strong\u003e). The spectra showed good agreement between theoretical and experimental values; for example, the calculated molar weight of the cyclic BPTI peptide, taking into consideration the formation of the disulfide bridge and a consequent reduction of 2 Da, was 2399.81 Da, which matched the experimentally obtained molar weight of 2399.78 Da (\u003cstrong\u003eFigure\u0026nbsp;S4B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eNext, the ability of the cyclic and linear peptides to inhibit the aggregation of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 was investigated by incubating A\u003cem\u003e\u0026beta;\u003c/em\u003e42 for 16 h in the absence or presence of the peptides. Monitoring of the aggregation levels of untreated and treated A\u003cem\u003e\u0026beta;\u003c/em\u003e42 revealed a decrease in aggregation upon incubation of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 with the cyclic peptides but not the linear peptides (\u003cstrong\u003eFigure 1B,C\u003c/strong\u003e). Of note, while the cyclic BPTI peptide was a potent inhibitor of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation, the linear BPTI peptide was the only linear peptide that enhanced A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation. Moreover, neither the three Kunitz-type proteins per se nor their derived peptides gave any significant aggregation signals (cyclic TFPI gave a low signal), indicating that these proteins/peptides do not undergo self-aggregation (\u003cstrong\u003eFigure\u0026nbsp;1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Ability of the Kunitz-Type Proteins to Reduce the \u003cem\u003e\u0026beta;\u003c/em\u003e-Sheet and Fibrillar Contents of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 is Retained by the Cyclic Peptides.\u003c/strong\u003e We then aimed to explore \u0026ndash; at the molecular and macroscopic levels \u0026ndash; the inhibition of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregation by the Kunitz-type proteins and their derived peptides. To this end, we first recorded the CD spectra of samples of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 (50 \u003cem\u003e\u0026micro;\u003c/em\u003eM) post 18 h of incubation in the absence and presence of the Kunitz-type proteins (781.25 nM) at a molar ratio of 64:1 (A\u003cem\u003e\u0026beta;\u003c/em\u003e42:protein) or the peptides (100 \u003cem\u003e\u0026micro;\u003c/em\u003eM) at a molar ratio of 1:2 (A\u003cem\u003e\u0026beta;\u003c/em\u003e42:peptides). The CD spectrum of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 revealed a negative peak at 216 nm and a positive peak at 195 nm\u0026mdash;both characteristic of a \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet secondary structure (42), which is commonly regarded as the signature of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregates (43,44). Reductions in \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet structures and increases in random secondary structures were observed upon incubation of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 with the cyclic peptides and to a lesser extent with the linear peptides and the Kunitz-type proteins (\u003cstrong\u003eFigure 2\u003c/strong\u003e). The CD spectra of control cyclic and linear peptides and Kunitz-type proteins revealed predominantly random\u003cem\u003e \u0026beta;\u003c/em\u003e-sheet structures at insignificant levels (\u003cstrong\u003eFigure S5\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo complement the molecular level structural results, samples taken from the CD experiment (18 h post incubation) were analyzed by TEM. Consistent with the high ThT signal and \u003cem\u003e\u0026beta;\u003c/em\u003e-sheet content of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 and with the findings of previous studies (45,46), the TEM images showed that untreated A\u003cem\u003e\u0026beta;\u003c/em\u003e42 formed elongated and branched fibrils (\u003cstrong\u003eFigure 3A\u003c/strong\u003e). The TEM images of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 treated with Kunitz-type proteins revealed a decrease in both the abundance and the branching of the fibrils, with a tendency toward shorter and unbranched fibrils (\u003cstrong\u003eFigure\u0026nbsp;3B\u003c/strong\u003e). TEM images of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 samples incubated with the cyclic peptides revealed a dramatic reduction in the quantity of aggregates, with the fibrils being shorter and less entangled (\u003cstrong\u003eFigure 3C\u003c/strong\u003e). In contrast, in the presence of the linear peptides, only a slight decrease in the quantity of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregates was observed, with the species appearing more densely packed and tangled (\u003cstrong\u003eFigure\u0026nbsp;3D\u003c/strong\u003e). The above findings were confirmed by dot blot analysis with an anti-amyloid fibril antibody, which showed a strong reduction in the quantity of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 fibrils upon 18 h of incubation with the proteins and peptides, in the order: cyclic peptides \u0026gt; Kunitz-type proteins \u0026gt; linear peptides (\u003cstrong\u003eFigure 4\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe \u003c/strong\u003e\u003cstrong\u003eKunitz-Type Proteins Reduce Intracellular A\u003cem\u003e\u0026beta;\u003c/em\u003e42 Aggregate Formation in SH‑SY5Y Cells.\u003c/strong\u003e To determine whether bikunin, BPTI, and TFPI can reduce the formation of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregates in cells, SH-SY5Y cells were co-transfected with either the \u003cem\u003eA\u0026beta;42-GFP\u003c/em\u003e genetic construct and an empty plasmid or with \u003cem\u003eA\u0026beta;42-GFP\u003c/em\u003e together with each Kunitz-type protein gene (\u003cem\u003ebikunin\u003c/em\u003e, \u003cem\u003eBPTI\u003c/em\u003e, or \u003cem\u003eTFPI\u003c/em\u003e). Confocal microscopy imaging performed 48 h post-transfection revealed a high accumulation of green fluorescence protein (GFP) in inclusion bodies in the absence of a Kunitz-type protein, indicating the presence of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregates (47). In contrast, when Kunitz-type proteins were co-expressed with A\u003cem\u003e\u0026beta;\u003c/em\u003e42-GFP, the observed GFP signal was spread throughout the cells (\u003cstrong\u003eFigure 5\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e). In addition, western blot analysis confirmed that intracellular A\u003cem\u003e\u0026beta;\u003c/em\u003e42-GFP expression levels did not change upon co-expression with the Kunitz-type proteins (\u003cstrong\u003eFigure\u0026nbsp;5B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtracellular Cyclic Peptides Inhibit the Internalization and Accumulation of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 in SH‑SY5Y Cells.\u003c/strong\u003e Studies have shown that extracellular A\u003cem\u003e\u0026beta;\u003c/em\u003e42 interacts with the cell membrane and is internalized into the cell via multiple uptake mechanisms (48,49). To follow the effect of extracellular cyclic and linear peptides on the internalization and accumulation of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 in cells, SH-SY5Y cells were treated with HiLyte\u0026trade; Fluor\u0026nbsp;\u003cem\u003e488\u003c/em\u003e-labeled A\u003cem\u003e\u0026beta;\u003c/em\u003e42 (designated A\u003cem\u003e\u0026beta;\u003c/em\u003e42-488) in the presence or absence of cyclic or linear peptides and visualized using confocal microscopy. The cells treated with A\u003cem\u003e\u0026beta;\u003c/em\u003e42-488 alone exhibited strong internal fluorescence signals, indicating internalization of the extracellular A\u003cem\u003e\u0026beta;\u003c/em\u003e42 (\u003cstrong\u003eFigure 6A\u003c/strong\u003e). Upon exposure of the cells to a mixture of A\u003cem\u003e\u0026beta;\u003c/em\u003e42-488 and each of the cyclic peptides at molar ratio of 1:2 (A\u003cem\u003e\u0026beta;\u003c/em\u003e42:peptides), internalization and accumulation of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 in the cells was reduced (\u003cstrong\u003eFigure 6A\u003c/strong\u003e). In contrast, confocal microscopy images of the cells following treatment with mixtures of A\u003cem\u003e\u0026beta;\u003c/em\u003e42-488 and linear peptides revealed the presence of inclusion bodies within the cells, which indicated that the accumulation of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 had not been affected by the presence of the linear peptides (\u003cstrong\u003eFigure 6B\u003c/strong\u003e). Based on the inhibitory activity observed for the Kunitz-type proteins and the cyclic peptides but not the linear peptides, our subsequent cellular assays were performed only with the Kunitz-type proteins and the cyclic peptides.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Kunitz-Type Proteins and Cyclic Peptides Enhance the Viability of SH-SY5Y Cells Exposed to Extracellular A\u003cem\u003e\u0026beta;\u003c/em\u003e42.\u003c/strong\u003e To further investigate the ability of Kunitz-type proteins and cyclic peptides to reduce extracellular A\u003cem\u003e\u0026beta;\u003c/em\u003e42-mediated cytotoxicity in SH-SY5Y cells, we performed cell-based assays to assess cell viability and apoptosis. The XTT viability assay revealed an average of ~30% reduction in viability vs. control cells for SH-SY5Y cells exposed for 48 h to pre-formed A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregates (\u003cstrong\u003eFigure 7A\u003c/strong\u003e). However, SH-SY5Y cells treated with mixtures of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 and Kunitz-type proteins (pre-incubated together for 18 h at 37 \u0026ordm;C before exposure to the cells) exhibited improved viability, reaching complete recovery (normalized to control cells) for bikunin and BPTI treatments and ~90% for the TFPI treatment (\u003cstrong\u003eFigure 7A\u003c/strong\u003e). Similarly, treating SH-SY5Y cells with a mixture of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 and cyclic peptides (pre-incubated for 18 h at 37 \u0026ordm;C) improved cell viability compared to control cells, reaching 86% for cyclic bikunin and complete recovery for both cyclic BPTI and cyclic TFPI (\u003cstrong\u003eFigure 7B\u003c/strong\u003e). Notably, exposure of the cells to the Kunitz-type proteins and the cyclic peptides alone did not significantly affect cell viability (\u003cstrong\u003eFigure 7C,D\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe also sought to investigate the effect of our proteins/cyclic peptides on A\u003cem\u003e\u0026beta;\u003c/em\u003e42-induced apoptosis, since previous studies have reported that most of the accumulated intraneuronal A\u003cem\u003e\u0026beta;\u003c/em\u003e may be attributed to the uptake of extracellular A\u003cem\u003e\u0026beta;\u003c/em\u003e fragments, which induce a neuronal apoptosis cascade (50,51) by promoting uncontrolled elevation of cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e levels (52) and impairing mitochondrial redox activity (53). Thus, to evaluate the ability of the Kunitz-type proteins and the cyclic peptides to reduce A\u003cem\u003e\u0026beta;\u003c/em\u003e42-induced apoptosis in SH-SY5Y cells, treated and untreated cells were stained with Annexin-V APC and SYTOX Green (54,55), and the apoptotic stages were analyzed by flow cytometry. \u003cstrong\u003eFigure 8A\u003c/strong\u003e shows that 98% of the control cells were healthy (negative for both Annexin-V APC and SYTOX Green) and 42.2% of cells exposed to extracellular A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregates for 24 h were in early-stage apoptosis (positive for Annexin-V APC and negative for SYTOX Green), whereas cells treated with mixtures of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 and Kunitz-type proteins (pre-incubated together) revealed a reduction in cell toxicity, with only 17.8%, 31.1%, and 29.5% of cells in the early apoptotic stage following treatment with A\u003cem\u003e\u0026beta;\u003c/em\u003e42 together with bikunin, BPTI, or TFPI, respectively (\u003cstrong\u003eFigure 8B\u003c/strong\u003e, left panels). Treatment with Kunitz-type proteins alone, as control groups, showed that approximately 85% of the cells were healthy living cells, with less than 1% in a late apoptosis stage and less than 15% of cells in an early apoptosis stage (\u003cstrong\u003eFigure 8B\u003c/strong\u003e, right panels). A more marked reduction in A\u003cem\u003e\u0026beta;\u003c/em\u003e42-induced apoptosis was obtained for the cyclic peptides, with only 8.86%, 9.47%, and 7.59% of cells in early-stage apoptosis for cyclic bikunin, cyclic BPTI, or cyclic TFPI, respectively (\u003cstrong\u003eFigure 8C\u003c/strong\u003e, left panels). These results indicate an approximately 30% decrease in early apoptotic cells compared to those treated with extracellular A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregates (\u003cstrong\u003eFigure 8A\u003c/strong\u003e, right panel). Notably, approximately 85% of the control SH-SY5Y cells treated with cyclic peptides alone were healthy, and \u0026lt; 9% were in an early apoptotic stage (\u003cstrong\u003eFigure 8C\u003c/strong\u003e, right panels).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Kunitz-Type Proteins and Cyclic Peptides Suppress the Mitochondrial Membrane Depolarization Potential Mediated by A\u003cem\u003e\u0026beta;\u003c/em\u003e42 in SH-SY5Y Cells\u003c/strong\u003e. Previous studies have shown that A\u003cem\u003e\u0026beta;\u003c/em\u003e42 impairs mitochondrial function by disrupting the membrane potential and the electron transport chain, thereby generating reactive oxygen species and damaging mitochondrial DNA and proteins and ultimately leading to neuronal apoptosis (56\u0026ndash;58). Thus, following the viability and apoptosis assays showing that both the Kunitz-type proteins and the cyclic peptides suppressed A\u003cem\u003e\u0026beta;\u003c/em\u003e42-mediated apoptosis and cell death in SH-SY5Y cells, we sought to examine the effect of those proteins and peptides on the mitochondrial membrane potential of intact SH-SY5Y cells. Mitochondrial damage was assessed by measuring the changes in mitochondrial polarization resulting from exposure to A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregates, pre-formed in the presence or absence of Kunitz-type proteins and cyclic peptides. As shown in Figure 9, cells that were exposed to pre-formed A\u003cem\u003e\u0026beta;\u003c/em\u003e42 aggregates for 48 h exhibited an average of ~40% reduction in mitochondrial membrane potential compared to untreated cells. The addition of the Kunitz-type proteins improved mitochondrial function and reduced the changes in mitochondrial membrane potential, which reached complete recovery in SH-SY5Y cells treated with mixtures of A\u003cem\u003e\u0026beta;\u003c/em\u003e42 and bikunin or TFPI, and 97% in cells treated with A\u003cem\u003e\u0026beta;\u003c/em\u003e42 and BPTI (\u003cstrong\u003eFigure\u0026nbsp;9A\u003c/strong\u003e). Similarly, the reduction in mitochondrial membrane potential was almost abolished in cells treated with A\u003cem\u003e\u0026beta;\u003c/em\u003e42 and cyclic peptides, with improvements of 95%, 98%, and 96% in cells treated with A\u003cem\u003e\u0026beta;\u003c/em\u003e42 and cyclic bikunin, cyclic BPTI, and cyclic TFPI, respectively, compared to untreated cells (\u003cstrong\u003eFigure\u0026nbsp;9B\u003c/strong\u003e). We observed that both Kunitz-type proteins and cyclic peptides did not induce significant changes in mitochondrial membrane potential (\u003cstrong\u003eFigure 9C,D\u003c/strong\u003e).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eStudies have shown Kunitz-type proteins to be implicated in the etiology of AD, although open questions remain regarding their roles in the development of AD and the generation of A\u003cem\u003eβ\u003c/em\u003e aggregates. For example, it was shown that TFPI, which is abundant in the brains of AD patients (59), is elevated in the frontal cortex and blood plasma of AD patients and, notably, that TFPI is localized in some A\u003cem\u003eβ\u003c/em\u003e plaques in AD brains (60,61). Less was known about another Kunitz-type protein, APPI, until our recent studies of the protein and of a peptide sequence taken from its \u003cem\u003eβ\u003c/em\u003e-hairpin domain demonstrated that both are potent inhibitors of A\u003cem\u003eβ\u003c/em\u003e42-induced neuronal cell toxicity, but act via different mechanisms: the full-length protein reduces A\u003cem\u003eβ\u003c/em\u003e42 aggregation, whereas its \u003cem\u003eβ\u003c/em\u003e-hairpin domain enhances aggregation (35,36). To investigate these differences in modes of inhibition and to elucidate whether the inhibition mechanism depends on the structure or on the sequence of the peptides – or perhaps on both – we extended our work on APPI (35,36) to other Kunitz-type proteins, namely, TFPI, BPTI and bikunin, and the short cyclic peptides that mimic their \u003cem\u003eβ\u003c/em\u003e-hairpin domains and their unstructured linear peptide counterparts.\u003c/p\u003e\n\u003cp\u003eIntegrating the findings of the current study with those of our previous study on APPI reveals that a \u003cem\u003eβ\u003c/em\u003e-hairpin motif of the interacting cyclic peptide is a prerequisite for conferring inhibition (cyclic TFPI, BPTI and bikunin peptides) or enhancement (the cyclic APPI peptide) of A\u003cem\u003eβ\u003c/em\u003e42 aggregation, whereas the lack of a \u003cem\u003eβ\u003c/em\u003e-hairpin loop in the interacting linear peptide may either not affect (linear APPI, TFPI and bikunin) or enhance (linear BPTI) A\u003cem\u003eβ\u003c/em\u003e42 aggregation. Moreover, for any particular \u003cem\u003eβ\u003c/em\u003e-hairpin sequence in a peptide, the specific amino acid composition may finetune the level of inhibition of A\u003cem\u003eβ\u003c/em\u003e42 aggregation. An analysis of the three studies together also shows that all four different Kunitz-type protein family members, which have a similar fold (i.e., an \u003cem\u003eα/β\u003c/em\u003e fold with two antiparallel \u003cem\u003eβ\u003c/em\u003e-sheets and one helical region) but different sequences (with only ~25% similarity) (32,37,62), inhibit the aggregation of A\u003cem\u003eβ\u003c/em\u003e42.\u003c/p\u003e\n\u003cp\u003eAn examination of the mechanism of A\u003cem\u003eβ\u003c/em\u003e42 inhibition indicates that the \u003cem\u003eβ\u003c/em\u003e-sheet domain of the \u003cem\u003eα/β\u003c/em\u003e fold is the segment that interacts with A\u003cem\u003eβ\u003c/em\u003e42 to modulate its aggregation pathway, probably through hydrophobic interactions with A\u003cem\u003eβ\u003c/em\u003e42, as observed for other peptide-based \u003cem\u003eβ\u003c/em\u003e-sheet breakers designed to inhibit or reverse the misfolding of A\u003cem\u003eβ\u003c/em\u003e42 into \u003cem\u003eβ\u003c/em\u003e-sheet-rich structures (63–65), which is a key pathological event in neurodegenerative diseases, such as AD (7,66). By binding to the central hydrophobic core of A\u003cem\u003eβ\u003c/em\u003e42 aggregates, probably via their \u003cem\u003eβ\u003c/em\u003e-hairpin peptide domain, the Kunitz proteins disrupt the stability of the aggregated A\u003cem\u003eβ\u003c/em\u003e42, preventing further fibril formation and thereby reducing neurotoxicity. It is possible that – like APPI – bikunin, BPTI, and TFPI proteins inhibit the A\u003cem\u003eβ\u003c/em\u003e42 'on-pathway,' in which unordered monomeric A\u003cem\u003eβ\u003c/em\u003e42 self-assembles to form toxic oligomeric intermediates that promote further elongation of A\u003cem\u003eβ\u003c/em\u003e42 into mature amyloid fibrils (67,68).\u003c/p\u003e\n\u003cp\u003eThe potential of \u003cem\u003eβ\u003c/em\u003e-hairpin-like cyclic peptides to inhibit 'on-pathway' A\u003cem\u003eβ\u003c/em\u003e42 aggregation has been examined in previous studies using synthetic peptides (not related to the A\u003cem\u003eβ\u003c/em\u003e42 sequence) (23,24) or peptides comprising the self-assembly region of A\u003cem\u003eβ\u003c/em\u003e42 (69). However, in view of the lack of a comprehensive study, the role played by the cyclic conformation in these inhibitory activities of the peptides remains unclear. Consistent with our premise that the \u003cem\u003eβ\u003c/em\u003e-sheet domain interacts with A\u003cem\u003eβ\u003c/em\u003e42, Costa et al. demonstrated that A\u003cem\u003eβ\u003c/em\u003e42 aggregation and cytotoxicity can be mitigated by the \u003cem\u003eβ\u003c/em\u003e-hairpin domain of transthyretin (TTR), either as a segment within TTR (70,71) or as an isolated cyclic peptide mimetic derived from the A\u003cem\u003eβ\u003c/em\u003e-TTR binding interface (72). Similar to our current study, the above authors also showed that the linear versions of the peptides exhibited only minor inhibitory activity, whereas significant inhibition of A\u003cem\u003eβ\u003c/em\u003e42-induced toxicity was achieved only with the cyclic peptides and at concentrations\u0026nbsp;comparable to those observed for the Kunitz-derived cyclic peptides (20-40 \u003cem\u003eµ\u003c/em\u003eM).\u003c/p\u003e\n\u003cp\u003eIn the current study, an examination of the inhibition of A\u003cem\u003eβ\u003c/em\u003e42 aggregation by the linear peptides revealed that the effects of these peptides differed from those of the cyclic peptides: In particular, linear BPTI markedly increased aggregation, while both linear TFPI and linear bikunin exerted only a minor effect on A\u003cem\u003eβ\u003c/em\u003e42 aggregation. The reduction in the \u003cem\u003eβ\u003c/em\u003e-sheet content of A\u003cem\u003eβ\u003c/em\u003e42 aggregates treated with the linear peptides, as shown by the CD spectra, may be a result of the formation of large insoluble fibrils (73,74). Indeed, TEM images of A\u003cem\u003eβ\u003c/em\u003e42 treated with linear peptides showed the formation of large aggregates with a morphology different from that of the aggregates of untreated A\u003cem\u003eβ\u003c/em\u003e42, with the former species appearing to be more densely packed and tangled than in untreated A\u003cem\u003eβ\u003c/em\u003e42. A comparison between cyclic peptides and their linear versions has also been undertaken in other studies (17,75). For example, Jha et al., who tested a 10-mer cyclic peptide and its linear counterpart, showed that the cyclic peptide reduced both aggregation and \u003cem\u003eβ\u003c/em\u003e-sheet content of A\u003cem\u003eβ\u003c/em\u003e42 in vitro and mitigated A\u003cem\u003eβ\u003c/em\u003e42-induced toxicity in SH-SY5Y cells, whereas the linear peptide had no effect on A\u003cem\u003eβ\u003c/em\u003e42 aggregation in vitro and or in cells (76).\u003c/p\u003e\n\u003cp\u003eReinforcing the findings that the three Kunitz-type proteins (i.e., bikunin, BPTI, and TFPI) and their derived cyclic peptides – but not their linear counterparts – inhibited A\u003cem\u003eβ\u003c/em\u003e42 aggregation in vitro, our results in cells indicate that treatment with the Kunitz-type proteins and their derived cyclic peptides also led to a reduction in the \u003cem\u003eβ\u003c/em\u003e-sheet content of A\u003cem\u003eβ\u003c/em\u003e42; namely, a reduction in the formation of A\u003cem\u003eβ\u003c/em\u003e42 aggregates (as shown in the ThT assay and TEM images) was visualized using confocal microscopy of SH-SY5Y cells, where the internalization of extracellular A\u003cem\u003eβ\u003c/em\u003e42 aggregates was reduced upon interaction with the cyclic peptides but not with the linear peptides. In contrast, as shown in one of our previous studies, the interaction of the cyclic APPI peptide with A\u003cem\u003eβ\u003c/em\u003e42 enhanced A\u003cem\u003eβ\u003c/em\u003e42 aggregation, thereby leading to the formation of more fibrils and less toxic A\u003cem\u003eβ\u003c/em\u003e42 oligomers (35). The explanation for the above differences may lie in the ability of the cyclic peptides (whether cyclic APPI or the cyclic bikunin, BPTI, and TFPI peptides)\u0026nbsp;to interfere with the interaction of A\u003cem\u003eβ\u003c/em\u003e42 with the cell membrane due to structural changes induced in A\u003cem\u003eβ\u003c/em\u003e42 upon interaction with the peptides (77). These structural changes, in turn, led to reduced A\u003cem\u003eβ\u003c/em\u003e42-induced cytotoxicity in SH‑SY5Y cells, as reflected in a reduction of both apoptotic processes and mitochondrial dysfunction. Taken together, these findings led us to posit that, like the Kunitz-type proteins, all four cyclic peptides inhibited the A\u003cem\u003eβ\u003c/em\u003e42 'on-pathway' mechanism, thereby reducing the quantity of toxic oligomers that subsequently nucleate to form A\u003cem\u003eβ\u003c/em\u003e fibrils.\u003c/p\u003e\n\u003cp\u003eThe study's collective results confirm that both the specific amino acid sequence and the \u003cem\u003eβ\u003c/em\u003e-hairpin structure found in Kunitz-type proteins and cyclic peptides are critical for altering A\u003cem\u003eβ\u003c/em\u003e42 aggregation but the only the \u003cem\u003eβ\u003c/em\u003e-hairpin structure is essential for mitigating neurotoxicity. This study also reveals the potential of members of the Kunitz-type protein family and their derived cyclic peptides to reduce the neurotoxicity of A\u003cem\u003eβ\u003c/em\u003e42 aggregates either by decreasing formation of toxic oligomers or by enhancing the formation of A\u003cem\u003eβ\u003c/em\u003e42 fibrils, both resulting in disruption of the 'on-pathway' mechanism of A\u003cem\u003eβ\u003c/em\u003e42 oligomerization (78). We emphasize that it is the alterations to the aggregation pathway of A\u003cem\u003eβ\u003c/em\u003e42 – whether enhancing or impeding aggregation – controlled by the \u003cem\u003eβ\u003c/em\u003e-hairpin domain (either as a sub-domain within a Kunitz-type protein or as an isolated cyclic peptide) and its amino acid sequence, that lead to a reduction in the quantity of the toxic 'on-pathway' oligomers, which subsequently elongate and nucleate to form A\u003cem\u003eβ\u003c/em\u003e fibrils.\u0026nbsp;\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eBPTI, Bikunin and TFPI Expression and Purification.\u003c/strong\u003e BPTI, bikunin and TFPI genes were separately cloned into pPICK9K plasmids as described previously (79,80). The plasmids were transformed into \u003cem\u003ePichia pastoris\u003c/em\u003e\u0026nbsp;strain GS115 and then inoculated into and allowed to stand overnight in 5 ml of BMGY medium (1% yeast extract, 2% peptone, 0.23% potassium phosphate monobasic, 1.18% potassium phosphate dibasic, 1.34% yeast nitrogen base, 4×10\u003csup\u003e-5\u003c/sup\u003e% biotin and 1% glycerol). Thereafter, the cells were transferred to 50 ml of BMGY, followed by scaling up to 500 ml of BMGY. For protein expression, the cells were resuspended in 500 ml of BMMY (same as BMGY, but with 0.5% methanol instead of glycerol) and grown for three days, with 2% methanol being added every 24 h to maintain induction. Following four days of induction, the cultures were centrifuged at 4,700×g for 20 min, and the supernatants containing the secreted proteins were adjusted to 10 mM imidazole and 0.5 M NaCl at pH 8.0, followed by incubation for 1 h at 4 °C and then centrifugation at 4,700×g for 20 min. Next, the supernatants were filtered through 0.22-\u003cem\u003eμ\u003c/em\u003em Stericup bottle-top filters (Millipore, MA, USA). The filtered supernatants were loaded onto 5-ml HisTrap columns (GE Healthcare, Piscataway, NJ, USA) at a flow rate of 0.9 ml/min for 16 h, washed with a washing buffer (20 mM sodium phosphate, 0.5 M NaCl, and 10 mM imidazole; pH 8.0), and eluted with an elution buffer (as for washing buffer but with 0.5 M imidazole) using ÄKTA™ start (GE Healthcare). As the final step for BPTI, bikunin and TFPI purification, SEC was performed on Superdex 75 16/600 columns (GE Healthcare) equilibrated with PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, and 2 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e; pH 7.4), at a flow rate of 1 ml/min on ÄKTA start. The purity of the proteins was confirmed by 15% SDS-PAGE gel under non-reducing conditions. The concentrations of the proteins were determined with a BCA Protein Assay Kit (Thermo Fisher, MA, USA) according to manufacturer's instructions. The molar weights of the pure proteins, validated using a MALDI-TOF (Bruker, Billerica, MA, USA) mass spectrometer (The Ilse Katz Institute for Nanoscale Science and Technology, BGU), were 9.68, 9.31, and 9.71 kDa for BPTI, bikunin, and TFPI, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthetic Peptides.\u003c/strong\u003e Synthetic A\u003cem\u003eβ\u003c/em\u003e(1–42) (A\u003cem\u003eβ\u003c/em\u003e42) was purchased from AnaSpec (Fremont, CA, USA) and from GL Biochem (Shanghai, China). Fluorescently labeled A\u003cem\u003eβ\u003c/em\u003e42-Hylight 488 (A\u003cem\u003eβ\u003c/em\u003e42-488) was purchased from AnaSpec (Fremont, CA, USA). The unlabeled A\u003cem\u003eβ\u003c/em\u003e42 from AnaSpec was dissolved in hexafluoroisopropanol (HFIP; Sigma-Aldrich, Israel) to a final concentration of 221 \u003cem\u003eµ\u003c/em\u003eM. The required quantity of synthetic A\u003cem\u003eβ\u003c/em\u003e42 peptide from AnaSpec was taken, followed by evaporation of the HFIP for 10 h. The synthetic A\u003cem\u003eβ\u003c/em\u003e42 peptide from GL Biochem was dissolved in 10 mM NaOH, and the concentration was determined by NanoDrop spectrophotometer (Thermo Fisher, MA, USA) using an extinction coefficient of 1490 M\u003csup\u003e−1\u003c/sup\u003e cm\u003csup\u003e−1\u003c/sup\u003e. A\u003cem\u003eβ\u003c/em\u003e42-488 was dissolved in 10 mM NaOH to final concentration of 205 \u003cem\u003eµ\u003c/em\u003eM. The synthetic peptides, cyclic TFPI (CMKRFFFNIFTRQSEEFIYC), linear TFPI (MKRFFFNIFTRQSEEFIY), cyclic BPTI (CIIRYFYNAKAGLSQTFVYC), linear BPTI (IIRYFYNAKAGLSQTFVY), cyclic bikunin (CIQLWAFDAVKGKSVLFPYC), and linear bikunin (IQLWAFDAVKGKSVLFPY), were synthesized and divided into 1-mg aliquots by GL Biochem. For all experiments, except the CD measurements, the linear and cyclic peptides were dissolved in 1 ml of DMSO. The concentrations of the stock solutions were determined according to the Mw of the synthetic peptides (Figure S4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePre-Incubation of the Peptides.\u003c/strong\u003e For all experiments (except ThT assays and CD measurements), A\u003cem\u003eβ\u003c/em\u003e42 was dissolved in phosphate buffer (20 mM sodium phosphate, pH 7.4, 150\u0026nbsp;mM NaCl) in the presence or absence of the peptides or Kunitz-type proteins and incubated in a Thermo Shaker incubator for 18 h (300 rpm, orbital shaking) at 37 °C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture.\u0026nbsp;\u003c/strong\u003eSH-SY5Y neuroblastoma cells were grown at 37 °C and 5% CO\u003csub\u003e2\u003c/sub\u003e in Dulbecco’s modified Eagle’s medium (DMEM; Sartorius, Göttingen, Germany) supplemented with 10% tetracycline-free fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 2 mM l‑glutamine (Gibco), and penicillin (100 units/ ml)/streptomycin (0.1 mg/ml) (Gibco).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of A\u003cem\u003eβ\u003c/em\u003e42 Aggregation by using the ThT Assay.\u003c/strong\u003e The aggregation of A\u003cem\u003eβ\u003c/em\u003e42 was determined using the fluorescent dye ThT, as follows. A\u003cem\u003eβ\u003c/em\u003e42, 4 \u003cem\u003eµ\u003c/em\u003eM, and ThT (Sigma-Aldrich, Israel), 10 \u003cem\u003eµ\u003c/em\u003eM, were incubated in the absence or presence of 62.5 nM Kunitz-type proteins (BPTI, TFPI or bikunin) or 8 \u003cem\u003eµ\u003c/em\u003eM linear or cyclic peptides in 350 \u003cem\u003eµ\u003c/em\u003el of phosphate buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4). Aliquots of 100 \u003cem\u003eμ\u003c/em\u003el of reaction mixture were added to each well of a black 96-well plate (Greiner Bio-One, Germany), and the fluorescence intensity was determined at 5-min intervals, for 16 h at 37 °C, with constant orbital shaking at 205 rpm. The fluorescence signals were detected using BioTek Synergy H1 microplate reader (Winooski, Vermont, USA) with excitation and emission wavelengths of 440 and 485 nm, respectively. Each experiment was performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD Spectra.\u003c/strong\u003e The linear and cyclic peptides were dissolved in phosphate buffer and mixed with 50% acetonitrile to a concentration of 2000 \u003cem\u003eµ\u003c/em\u003eM. Samples of 50 \u003cem\u003eµ\u003c/em\u003eM A\u003cem\u003eβ\u003c/em\u003e42 in the absence or presence of Kunitz-type proteins (781.25 nM) or linear or cyclic peptides (100 \u003cem\u003eµ\u003c/em\u003eM) were added to 200 \u003cem\u003eµ\u003c/em\u003el of diluted phosphate buffer (5 mM sodium phosphate, 37.5 mM NaCl, pH 7.4) in order to maintain\u0026nbsp;an appropriate high-tension (HT) voltage of the instrument. The samples were incubated in a Thermo Shaker Incubator for 18 h (500 rpm, orbital shaking) at 37\u0026nbsp;°C. The pre-incubated peptide samples were scanned using a Jasco J-715 spectropolarimeter (Jasco, Japan). The spectrum of each sample was recorded three times at 25 °C in a range between 195–260 nm using a quartz cuvette with a path length of 1 mm, a scanning speed of 50 nm/min, and a data interval of 0.5 nm. The spectral scans were averaged to smooth the data curves. For baseline correction, protein-free phosphate buffer was used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTEM Imaging of A\u003cem\u003eβ\u003c/em\u003e42 Aggregates.\u003c/strong\u003e Samples of A\u003cem\u003eβ\u003c/em\u003e42 with Kunitz-type proteins, cyclic peptides, or linear peptides taken from the above CD assay post 18 h of incubation were investigated using TEM. For this purpose, 2.5 \u003cem\u003eµ\u003c/em\u003el of each sample was deposited on a carbon-coated copper 300-mesh grid. After 1 min, excess liquid was carefully removed with filter paper Then, 2% uranyl acetate in doubly distilled water was added to each sample,\u0026nbsp;and after 1 min any excess of uranyl acetate solution was carefully removed using a filter paper. . Images were acquired using a Tecnai G2 12 BioTWIN (FEI, ThermoFisher Scientific, Paisley, UK) TEM with an acceleration voltage of 120\u0026nbsp;kV (The Ilse Katz Institute for Nanoscale Science and Technology, BGU). Different magnifications were used for visualization, depending on the size of the aggregates, and multiple fields were analyzed in each grid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDot Blot Analysis.\u003c/strong\u003e Samples from the CD measurements of A\u003cem\u003eβ\u003c/em\u003e42 with Kunitz-type proteins, cyclic peptides, or linear peptides were analyzed. Each sample (5 \u003cem\u003eµ\u003c/em\u003el) was loaded onto two nitrocellulose membranes (Bio-Rad, Hercules, California, USA), which were blocked with 5% skim milk (Sigma-Aldrich, Israel) in Tris-buffered saline with 0.1% Tween (TBST; Biolab, Israel), and then further washed with TBST three times for 5 min each. The first membrane was incubated overnight with rabbit amyloid fibril polyclonal antibody (Thermo Fisher, Israel) diluted 1:1000 in PBS. The second membrane was incubated overnight with mouse anti-A\u003cem\u003eβ\u003c/em\u003e42 antibody (Abcam, Cambridge, UK) diluted 1:1000 in PBS to confirm that the same amount of A\u003cem\u003eβ\u003c/em\u003e42 had been loaded in each sample. After washing, the membranes were incubated for 1 h with HRP-linked anti-mouse secondary antibody (Cell Signaling, Danvers, MA, USA) and anti-rabbit secondary antibody (Cell Signaling), respectively. The chemiluminescence signals were detected using a Fusion FX imaging system (Vilber, Germany). The experiments were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMammalian Expression Plasmids.\u0026nbsp;\u003c/strong\u003eFor expression in SH-SY5Y cells, A\u003cem\u003eβ\u003c/em\u003e42-GFP, BPTI, bikunin, and TFPI were cloned into a pPHAGE2 vector under the control of the human cytomegalovirus promoter. \u003cem\u003eAβ42-GFP\u003c/em\u003e was cloned as described previously (81). \u003cem\u003eBPTI\u003c/em\u003e, \u003cem\u003ebikunin\u003c/em\u003e, and \u003cem\u003eTFPI\u003c/em\u003e genes were cloned into the pHAGE2 plasmid using NotI and BamHI restriction enzymes (New England Biolabs, MA, USA) as described previously (36). The pPHAGE2 plasmid was used as a mammalian vector for expressing BPTI, bikunin, and TFPI linked to blue fluorescent protein via an internal ribosome entry site in SH-SH5Y cells under the control of the human cytomegalovirus promoter.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfocal Microscopy Imaging.\u003c/strong\u003e In all experiments, SH-SY5Y cells were seeded at a density of 10\u003csup\u003e4\u003c/sup\u003e cells per well in 1-cm micro-slides (ibidi, Gräfelfing, Germany). For intracellular expression of Kunitz-type proteins (i.e., BPTI, bikunin, and TFPI) and A\u003cem\u003eβ\u003c/em\u003e42-GFP, the cells were transfected one day after seeding with either 1 \u003cem\u003eµ\u003c/em\u003eg of a mixture of A\u003cem\u003eβ\u003c/em\u003e42-GFP\u0026nbsp; plasmid\u0026nbsp;and empty plasmids or a mixture of A\u003cem\u003eβ\u003c/em\u003e42-GFP and Kunitz-type gene containing \u0026nbsp;pPHAGE plasmids by using the jetOPTIMUS transfection reagent (Polyplus, Illkrich, France), according to the manufacturer’s manual. After 48 h of incubation, the cells were washed with PBS and fixed by incubation with 4% paraformaldehyde (PFA) in PBS for 30 min. Cell imaging was performed using Olympus FV1000 confocal microscope with a long-working distance ×60/1.35 numerical aperture oiled-immersion objective [The National Institute for Biotechnology in the Negev (NIBN), BGU].\u003c/p\u003e\n\u003cp\u003eFor extracellular treatments with A\u003cem\u003eβ\u003c/em\u003e42 and cyclic or linear peptides, the cells were treated with pre-incubated samples of 1 \u003cem\u003eµ\u003c/em\u003eM A\u003cem\u003eβ\u003c/em\u003e42-488 in the absence or presence of 2 \u003cem\u003eµ\u003c/em\u003eM cyclic or linear peptides. After the incubation, the cells were washed with PBS and fixed by incubation with 4% PFA in PBS for 30 min. Cells imaging was performed using a Zeiss LSM 880 confocal microscope with a ×40 objective (The Ilse Katz Institute for Nanoscale Science and Technology, BGU).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of A\u003cem\u003eβ\u003c/em\u003e42-GFP Expression in SH-SY5Y Cells.\u003c/strong\u003e SH-SY5Y cells (3\u0026nbsp;´10\u003csup\u003e5\u003c/sup\u003e cells per well) were seeded in a six-well tissue culture plate (Greiner, Sigma-Aldrich, Israel). For transient expression of A\u003cem\u003eβ\u003c/em\u003e42-GFP in the presence or absence of the Kunitz-type proteins, the cells were transiently transfected on the following day using the jetOPTIMUS transfection reagent (Polyplus) according to the manufacturer’s manual. Each well was transfected with 1 \u003cem\u003eµ\u003c/em\u003eg of pPHAGE-A\u003cem\u003eβ\u003c/em\u003e42-GFP and 1 \u003cem\u003eµ\u003c/em\u003eg of pHAGE2-empty, pHAGE2-BPTI, pHAGE2-bikunin, or pHAGE2-TFPI. After 48 h, the medium was removed, and the cells were incubated for 20 min with RIPA buffer (EMD Millipore Corp., Billerica, MA, USA) and protease inhibitor cocktail (APE×BIO, Boston, MA, USA) for cell lysis. The cell cultures were centrifuged, and protein concentrations were determined in the supernatants by the BCA assay (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Each lysate (50 \u003cem\u003eµ\u003c/em\u003eg) was loaded onto a 15% SDS-PAGE and then transferred to a nitrocellulose membrane for western blot analysis. The membrane was blocked with 5% skim milk and incubated overnight with mouse anti-A\u003cem\u003eβ\u003c/em\u003e42 antibody diluted 1:1000 in PBS (Abcam). After washing, the membrane was incubated for 1 h with HRP-linked anti-mouse secondary antibody (Cell Signaling, Danvers, MA, USA), and chemiluminescence signals were detected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXTT Cell Viability Assay.\u0026nbsp;\u003c/strong\u003eSH-SY5Y cells (10\u003csup\u003e4\u003c/sup\u003e cells per well) were seeded in 96-well tissue culture plate (Greiner, Sigma-Aldrich, Israel). After 24 h, the cells were treated with pre-incubated samples (see details in ‘pre-incubation of the peptides’ subsection) of 10 \u003cem\u003eµ\u003c/em\u003eM A\u003cem\u003eβ\u003c/em\u003e42 in the presence or absence of the Kunitz-type proteins (156.25 nM) or the cyclic peptides (8 \u003cem\u003eµ\u003c/em\u003eM). The viability of the cells was assessed 48 h post-treatment by using an XTT-based kit (Cell Signaling) according to the manufacturer’s protocol. The absorbance was read at a wavelength of 450 nm using a BioTek Synergy H1 microplate reader. Three repetitions were performed for each treatment. The values were normalized to the control group (untreated cells).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial Membrane Depolarization Potential Measurements.\u003c/strong\u003e To measure the changes in the mitochondrial membrane potential in SH-SY5Y cells, 10\u003csup\u003e4\u003c/sup\u003e cells/well were seeded in a black-clear bottomed 96-well tissue culture plate (Greiner, Sigma-Aldrich, Israel). The cells were treated for 48 h with pre-incubated samples (see details in ‘pre-incubation of the peptides’ subsection) of 10 \u003cem\u003eµ\u003c/em\u003eM A\u003cem\u003eβ\u003c/em\u003e42 in the absence or presence of the Kunitz-type proteins (156.25 nM) or the cyclic peptides (8 \u003cem\u003eµ\u003c/em\u003eM). The changes in the mitochondrial membrane potential were assessed using TMRE-Mitochondrial Membrane Potential Assay Kit (Abcam), according to the manufacturer’s protocol. The fluorescence intensity, which indicated mitochondrial potential, was detected using TECAN infinite M200 microplate reader (Männedorf, Switzerland) with excitation and emission wavelengths of 549 and 575 nm, respectively. Each experiment was performed in triplicate. The values were normalized to the control group (untreated cells).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow Cytometry Analysis of Apoptotic Cells.\u003c/strong\u003e SH-SY5Y cells (3×10\u003csup\u003e4\u003c/sup\u003e cells per well) were seeded in a 24-well plate and incubated for 18 h with 15 \u003cem\u003eµ\u003c/em\u003eM pre-formed A\u003cem\u003eβ\u003c/em\u003e42 aggregates in the absence or presence of 250 nM Kunitz-type proteins (BPTI, TFPI and bikunin) or 30 \u003cem\u003eµ\u003c/em\u003eM cyclic peptides (see details in the ‘pre-incubation of the peptides’ subsection). Untreated cells served as a negative control. After 24 h of incubation, the cells were harvested, washed, and stained with Annexin V-APC and SYTOX Green using an Apoptosis Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The cells were analyzed using a BD FACS Canto II (BD Biosciences, Erembodegem, Belgium). The following cell populations were analyzed: intact cells (Annexin V-APC negative and SYTOX Green negative), early apoptotic cells (Annexin V-APC positive and SYTOX Green negative), late apoptotic cells (annexin V-APC and SYTOX Green positive), and necrotic cells (annexin V-APC negative and SYTOX Green positive).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis.\u003c/strong\u003e All data were analyzed statistically with GraphPad Prism, version 8.00, for Windows (La Jolla, CA, USA). Data are shown as means ± SD. Statistical significance between the control group and different treatments was determined by an unpaired Student’s t-test, with statistical significance *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eA\u003cem\u003eβ\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eamyloidβ\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eA\u003cem\u003eβ\u003c/em\u003e42\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eamyloid \u003cem\u003eβ\u003c/em\u003e 1\u0026ndash;42\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlzheimer's disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAPP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eamyloid precursor protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAPPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eamyloid precursor protein inhibitor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBPTI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebovine pancreatic trypsin inhibitor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecircular dichroism\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco's modified Eagle's medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGFP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egreen fluorescent protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHFIP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1,1,1,3,3,3-hexafluoro-2-propanol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etransmission electron microscope\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etissue factor pathway inhibitor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eThT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethioflavin T\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTMRE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etetramethylrhodamine,ethyl ester\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTTR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etransthyretin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eXTT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Rosetrees Trust (OoR2022/100004), Israel Science Foundation (grant number 1437/24), and the Worldwide Cancer Research (grant number 20-0238) to NP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest with respect to publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.R. and N.P. designed the research; M.R. and S.L.H. performed the research; M.R. and N.P. analyzed the data; M.R. and N.P. wrote the paper. All authors edited the manuscript and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not require ethics approval.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBeydoun MA, Beydoun HA, Gamaldo AA, Teel A, Zonderman AB, Wang Y (2014) Epidemiologic studies of modifiable factors associated with cognition and dementia: systematic review and meta-analysis. BMC Public Health 14(1):643\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHampel H, Hardy J, Blennow K, Chen C, Perry G, Kim SH et al (2021) The Amyloid-β Pathway in Alzheimer\u0026rsquo;s Disease. 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Biochem J 475(19):3087\u0026ndash;3103\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer's disease, Aβ42 aggregation, BPTI, TFPI, bikunin, β-hairpin, Kunitz-type proteins, neurotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-8603888/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8603888/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Alzheimer’s disease is associated with the aggregation of amyloid‑β42 (Aβ42) into species of varying sizes, with intermediate oligomers being the most neurotoxic. We recently reported that amyloid precursor protein inhibitor (APPI), a Kunitz-type protein, and a cyclic peptide derived from its β-domain reduced Aβ42-mediated neurotoxicity, the former by reducing Aβ42 aggregation and formation of toxic Aβ42 oligomers, and the latter by promoting Aβ42 aggregation to form fibrils rather than the neurotoxic Aβ42 oligomers. To address the question of whether these two inhibition mechanisms are controlled by the structure or the amino acid sequence of the protein/peptide, we exploited three Kunitz-type proteins, bikunin, bovine pancreatic trypsin inhibitor (BPTI) and tissue factor pathway inhibitor (TFPI) – chosen for their similar β-strand-rich structures but different sequences to one another and to APPI – and also short peptides that mimic their β-domains, in either cyclic or linear conformation. In-vitro studies showed that the formation of Aβ42 aggregates was reduced by the three Kunitz-type proteins and by their derived cyclic peptides, but not by the linear counterparts of the cyclic peptides. In SH-SY5Y neuroblastoma cells, the Kunitz-type proteins and the cyclic (but not the linear) peptides reduced the intracellular and extracellular accumulation of Aβ42 aggregates, respectively. Both the Kunitz-type proteins and the cyclic peptides inhibited Aβ42-induced mitochondrial membrane depolarization and reduced Aβ42-mediated apoptosis and cell death. Overall, this study thus reveals the potential of the β-hairpin structure, whether as a segment within the Kunitz-type proteins or isolated as a cyclic peptide, to interact with Aβ42, thereby reducing Aβ42 aggregation and hence its neurotoxicity.","manuscriptTitle":"Modulating amyloid-β 42 aggregation and neurotoxicity by Kunitz domains and their derived peptides","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 12:42:44","doi":"10.21203/rs.3.rs-8603888/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d107389d-8122-4cbe-9daa-b200c8d568b8","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-26T07:08:02+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 12:42:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8603888","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8603888","identity":"rs-8603888","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}