Cbl-b E3 ligase-mediated neddylation and activation of PARP-1 induces vascular calcification

Cbl-b E3 ligase-mediated neddylation and activation of PARP-1 induces vascular calcification | 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 Article Cbl-b E3 ligase-mediated neddylation and activation of PARP-1 induces vascular calcification Hyun Kook, Duk-Hwa Kwon, Sera Shin, Yoon Seok Nam, Nakwon Choe, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3939434/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Oct, 2024 Read the published version in Experimental & Molecular Medicine → Version 1 posted 10 You are reading this latest preprint version Abstract Aims: Vascular calcification (VC) refers to the accumulation of mineral deposits on the walls of arteries and veins, and it is closely associated with increased mortality in cardiovascular disease, particularly among high-risk patients with diabetes and chronic kidney diseases (CKD). Neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) is an ubiquitin-like protein that plays a pivotal role in various cellular functions, primarily through its conjugation to target proteins and subsequent relay of biological signals. However, the role of NEDDylation in VC has not been investigated. Methods and Results: In our study, we observed that MLN4924, an inhibitor of the NEDD8-activating E1 enzyme, effectively impedes progress of VC. By LC-MS/MS analysis, we identified that poly(ADP-ribose) polymerase 1 (PARP-1) is subjected to NEDD8 conjugation, leading to an increase in PARP-1 activity during VC. Subsequently, we uncovered that the PARP-1 NEDDylation is mediated by the E3 ligase Cbl proto-oncogene B (Cbl-b) and is reversed by the NEDD8-specific protease 1 (NEDP-1) during VC. Furthermore, Cbl-b C373 peptide effectively mitigates the inactive form of E3 ligase activity of Cbl-b, ultimately preventing VC. Conclusions: These findings provide compelling evidence that the NEDD8-dependent activation of PARP-1 represents a novel mechanism underlying vascular calcification and suggests a promising new therapeutic target for VC. Biological sciences/Molecular biology/Post-translational modifications/Neddylation Health sciences/Diseases/Cardiovascular diseases/Vascular diseases/Calcification NEDD8 PARP-1 Cbl-b NEDP-1 Vascular calcification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Vascular calcification pertains to the abnormal accumulation of calcium within the walls of blood vessels, leading to a reduction in their elasticity and functionality, as well as an elevated risk of cardiovascular events like heart attack and stroke 1 . This process is intricate and influenced by multiple factors, often originated from various underlying conditions, including aging, diabetes, chronic kidney disease, and atherosclerosis 2 . Vascular calcification manifests in two distinct forms: intimal and medial calcification. Intimal calcification occurs within the inner layer of blood vessels and is closely associated with atherosclerosis 3 , whereas medial calcification affects the middle layer of vessels and is linked to aging and conditions like chronic kidney disease 4 . Currently, treatment options for vascular calcification are limited, primarily concentrating on the management of the causes that contribute to its development. NEDD8, which stands for Neural precursor cell Expressed Developmentally Downregulated 8, is a small protein similar to ubiquitin. It regulates the activity and stability of specific proteins through a process known as neddylation 5 , 6 . Similar to ubiquitination, NEDD8 binds to its target proteins by forming an isopeptide chain between its C-terminal glycine residue (Gly76) and a lysine residue on targeted proteins. This attachment occurs through a series of enzymatic reactions involving NEDD8-activating enzyme E1, NEDD8-conjugating enzyme E2, and NEDD8-ligase enzymes E3 6 . Importantly, this process is reversible, and NEDD8 can be detached from its targets by a family of enzymes known as NEDD8-specific proteases (NEDP1) 7 , 8 . NEDD8 plays a pivotal role in a variety of cellular processes, including the cell cycle regulation, DNA damage response, and protein degradation in diverse tissues 6 , 9 , 10 . Targeting the NEDD8 pathway has emerged as a potential therapeutic strategy for cancer treatment 11 , 12 . Several small molecule inhibitors of the NEDD8-activating enzyme and NEDD8-conjugating enzyme have been developed, such as MLN4924/Pevonedistat 11, 13 , 14 and UBE2M-DCN1 inhibitors 15 . These inhibitors are currently undergoing evaluation in clinical trials, marking a significant development in the field of cancer therapy 16 – 19 . Poly(ADP-ribose) polymerase-1 (PARP-1) is a key member of the PARP family, which comprises 18 members and contributes to nearly 90% of cellular PARP activity 19 , 20 . PARP-1 plays a pivotal role in various cellular processes, including DNA damage repair, transcription, and cell death signaling 20 , 21 . Numerous studies have provided insights into the multifaceted roles of PARP-1, extending its influence to a wide array of diseases. These encompass neurodegenerative disorders 22 , chronic inflammation 23 , cardiovascular diseases 24 and cancer 25 . Several studies have also demonstrated that PARP-1 instigates an osteogenic transition of vascular smooth muscle cells (VSMCs) by upregulating pivotal transcription factors such as Runx2 and NF-κB 26 , 27 . Inhibition of PARP-1 has exhibited protective effects against mineralization and calcification. Notably, the activity of PARP-1 is influenced by oxidative stress and DNA damage, which, in turn, foster vascular calcification 28 , 29 . Our research has unveiled a novel association between NEDD8 and PARP-1 under VC conditions. Despite the recognized involvement of PARP-1 activity in osteogenic calcification, the precise regulatory mechanisms governing its activity during the progression of VC remain to be elucidated. In the present study, we have delved into the significance of NEDD8 in the context of vascular calcification. Our investigation has unequivocally showcased that NEDD8 plays a pivotal role in the promotion of vascular calcification, and conversely, the inhibition of NEDD8 serves as a protective measure against this process. Within the conditions conducive to vascular calcification, our observations have revealed an activation of PARP-1 due to the interaction facilitated by NEDD8 poly-chains. This interaction is mediated through the E3 ligase activity of Cbl-b. Our findings compellingly suggest that Cbl-b orchestrates the process of PARP-1 neddylation, consequently modulating its activity in the course of vascular calcification. MATERIAL and METHODS All experimental procedures were approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee and followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978). Induction of vascular calcification The RVSMCs and A10 cells are cultured in growth medium and changed with calcification medium containing 2 mM or 4 mM inorganic phosphate (pH 7.4) for up to 3 days or 6 days. The medium was changed every 2 days. Calcium deposition in VSMCs was determined after washing with 1x PBS. Wild-type C57BL/6 male mice were used for induction of vascular calcification by administration of vitamin D 3 as described previously 30 , 31 . Vitamin D 3 (Cholecalciferol) in 70 µl of absolute ethanol was mixed with 500 µl Cremophor (Alkamuls EL-620, Sigma St. Louis, MO, USA) for 15 minutes at RT and then combined with 6.2 mL sterilized water containing 250 mg of dextrose for 15 minutes at RT. The mice were injected with a dose of vitamin D 3 (5x10 5 IUkg − 1 day) subcutaneously for 3 days and maintained for 6 days to induce vascular calcification. All animals were killed by inhalated CO 2 at chamber, and the aortas were isolated. All experiments were performed using male mice at 8–10 weeks of age. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Chonnam National University (CNU-IACUC-HA-2020-17 and CNU-IACUC-H-2021-39). Measurement of calcium deposition The A10 Cells and tissues were decalcified with 0.6N HCl at 4°C for 24 hours. The calcium content of the HCl supernatants was determined colorimetrically using the QuantiChrom calcium assay kit (QuantiChromTM Calcium Assay Kit, BioAssay Systems, Hayward, USA) according to the manufacturer’s instructions. Briefly, 5 µL of the samples was transferred to a 96-well plate and 200 µl working reagent was added (1:1, solution A and B). Mixed samples were briefly incubated for 3 min and absorbance was measured at 570 nm by using an ELx808 absorbance reader (BTELX808, BioTek Instruments, Winooski, VT, USA). And next, the cells were washed 3 times with 1xPBS and lysed with 0.1N NaOH/0/1% SDS to extract proteins. The calcium content was then normalized to the total protein amount, whereas that of the tissues was normalized to tissue dry weight. Alizarin Red S staining and quantification The A10 cells were washed with 1xPBS and fixed with 10% formalin for 30 min at RT. After being washed three time with distilled water, the cells were stained with 40 mM alizarin red S solution (pH 4.2, Sigma-Aldrich) for 30 min at room temperature and washed with distilled water to remove nonspecific staining. For quantification of alizarin red S staining, the stained cells were destained with 10% cetylpyridinium chloride (CPC) in 10 mM sodium phosphate buffer (pH 7.4) for 30 min at room temperature. The absorbance was then measured at 450 nm, and the values were normalized using a 10% CPC standard solution. To determine arterial calcification, the aorta was collected and fixed with 10% formalin for overnight at 4℃. The arterial tissue sample was placed in 2% KOH for overnight at RT and stained with 0.3% Alizarin red S in 1.6% KOH for 2 days at RT. After staining, the tissue was placed in preservation solution containing glycerin, 70% ethanol, and benzyl alcohol (2:2:1 ratio) for 3 hours at room temperature, followed by a 10 min incubation at 37℃. Proximity ligation assay (PLA) Proximity ligation assay (PLA) was performed using the Duolink® In Situ Red Starter Kit Mouse/Rabbit (Sigma-Aldrich) according to the manufactures’ introductions. Before PLA starting, the cells were fixed with 4% formaldehyde for 60 minutes and washed 2 times with 1xPBS. The Cells were permeabilized with 0.2% Triton X-100 in PBS for 10 minutes and then blocked with Duolink blocking solution in a heated humidity chamber for 60 minutes at 37℃. The cells were incubated overnight with anti-PARP-1 and anti-NEDD8 at 4℃, followed by treatment with Duolink PLA plus/minus probes for 60 minutes in a heated humidity chamber at 37℃. After washing 2 times with 1x wash buffer A, PLA signals were generated following reaction with ligation for 30 minutes and subsequent amplification for 90 minutes at 37℃. Slides were mounted using ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher) and photographed by confocal fluorescent microscopy (DE/LSM700, Cal Zeiss Microscopy) Administration of MLN4924, si-Cbl-b or Cblb C373 peptide to mice MLN4924 (10mg kg − 1 per day), Cbl-b C373 (10 mg kg − 1 per day), vehicle or scramble was intraperitoneally injected into mice. For siRNA injections, 50ug of siRNA was diluted in 10% glucose, mixed with in vivo-jetPEI (polyplus-transfection SA, Illkirch, France) also diluted in 10% glucose, and then incubated for 15 minutes at RT, followed by a brief vortex. si-Cbl-b (50µg per day) or scramble was injected into the mice’s tail vein after equilibration at RT. All of these reagents were administered at the same dose every 2 days for 6 days after vitamin D 3 injection until the animals were sacrificed. PARP-1 activity assay The cells and tissues were lysed with 0.5% NP-40 solution to extract protein and incubated with anti-PARP-1overnight at 4°C. Then PARP-1 activity was measured using PARP univseral colorimetric assay kit, following by manufacture protocol. Briefly, histone coated plate was activated with 1xPARP buffer for 30 min at RT. After remove 1xPARP buffer, the well plate was added samples and PARP-HSA enzyme and incubated for 10 min at room temperature. Prepared 1X PARP Cocktail buffer containing PARP cocktail, activated DNA was distributed into the wells and incubated at RT for 1 hour and added Strep-HRP working solution after washing 2 times with 1X PBS + 0.1% Triton X-100 (200 µL/well) followed by 2 washes with 1X PBS. The wells were incubated with pre-warmed TACS-Sapphire in the dark for 15 min at RT and added .2 M HCl to stop reaction and read the absorbance at 450nm by using an ELx808 absorbance reader (BioTek Instruments). Liquid chromatography-mass spectrometry analysis NEDD8-conjugated proteins were determined by peptide-precipitation and LC-MS/MS analysis. RVSMC cells were treated with high phosphate and MLN4924 (1µM) for 6 days. The extracted proteins were immunoprecipitated with anti-NEDD8 overnight at 4°C and next NEDD8-conjugated proteins were separated on gradient SDS-PAGE gels (8%-15%) and visualized by coomassie brilliant blue staining method. Specific bands in the Pi-treated lane which Pi lane were presented, but it was diminished by MLN4924 treatement were cut and LC-MS/MS was performed. Nano LC-MS/MS analysis was performed at the Korea Basic Science Institute (Biomedical Omics Research Center, Ochang, Korea). The gels were destained with a 50% acetonitrile containing 10 mmol NH 4 HCO 3 and were then rinsed a few times with distilled water to stop the destining reaction. The gels were inculated with 10 mmole dithiothreitol and 100 mmol ammonium bicarbonate at 56℃ to reduce the proteins, followed by 100 nmol iodoacetamide to alkylate the cysteines. The gels were then washed with three volumes of distilled water by vortexing and completely dried in a speed vacuum concentrator for 20 minutes. The dried gels were rehydrated with 12.5mg/mL trypsin in 50 mmol NH 4 HCO 3 solution and digestion was by 37°C incubation for overnight. Digested protein samples were speed-vaccum dried and the dissolved in 20µl of water containing 0.1% formic acid and LC-MS/MS were analyzed by using Hybrid FT-ETD Mass sepectrometer system (Thermo Fisher Scientific). Histology and Immunohistochemistry Tissue samples were fixed with 4% paraformaldehyde and embedded in paraffin. Cross-sections (8µm) were prepared and visualized by Alizarin red S staining and immunohistochemistry to evaluate vascular calcification and to analyze protein expression, respectively. Proximity ligation assay was performed after fixation, retrieval and permeabilization to detect binding between NEDD8 and PARP-1. The following primary antibodies were used for the immunostaining including PLA assay: NEDD8 (1:100, Cell signaling), PARP-1 (1:100, Santa cruz). The microscopic images were captured by an Axio Scan.Z1 scanner (Cal Zeiss Microscopy, GmbH, Jena, Germany) and a laser scanning microscope (DE/LSM700, Cal Zeiss Microscopy. Statistical analysis SPSS software (version 27.0, IBM Corp, Chicago, IL) was used for all statistical analyses. Each experiment was performed in triplicate, where appropriate. For two independent groups, two-tailed unpaired Student’s t-test or nonparametric Mann-Whitney U test was applied after checking for a normal distribution. In the case of more than two groups, one-way analysis of variance (ANOVA) or two-way ANOVA with post hoc tests was used depending on the number of main effects. Data are presented as the mean ± standard error and determined at the p-value than 0.05 as statistical significance. RESULTS MLN4924 inhibits Pi-induced calcium deposition in VSMCs. To investigate the potential involvement of NEDD8 in vascular calcification (VC), we initially assessed the effect of MLN4924 on Pi-induced calcium deposition in VSMCs. Treatment with MLN4924 resulted in a significant dose-dependent reduction in Pi-induced calcium deposition (Fig. 1 a). Alizarin red S staining revealed that mineralization, characteristic of Pi-induced VC, was effectively inhibited by MLN4924 treatment (Fig. 1 b, c). Pi treatment led to decrease in the expression of SM22a and SMA, genes associated with the smooth muscle differentiation, whereas upregulating the expression of osteogenic genes RUNX2 and ALP. However, these changes were counteracted by MLN4924 treatment (Fig. 1 d). In addition, we assessed cell viability using the MTT assay and observed that MLN4924 did not affect cell viability in both Pi-treated VSMCs (Supplementary Fig. 1a). We further assessed whether Pi can induce the NEDDylation by western blot analysis. Formation of poly-NEDD8 was increased in high phosphate-treated VSMCs (Fig. 1 e). Overexpression of NEDD8 potentiated Pi-induced calcium deposition in VSMCs (Fig. 1 f and Supplementary Fig. 1b), while transfection of NEDD8-ΔGG, a mutant incapable of conjugation, inhibited Pi-induced calcium deposition (Fig. 1 g and Supplementary Fig. 1c). These findings collectively suggest that the NEDD8 pathway plays a significant role in VC processes, while MLN4924 effectively prevents Pi-induced VC. NEDD8 is conjugated PARP-1 in VC To undercover which proteins undergo NEDD8-mediated modifications, we conducted immunoprecipitation-based proteomic analysis using an anti-NEDD8 antibody, followed by the affinity purification and liquid chromatography-mass spectrometry. In Pi-treated RVSMCs, NEDD8-conjugated proteins were isolated and the NEDDylation was confirmed through Coomassie blue staining. The gel bands that increased in the Pi lane and decreased in the lane with Pi and MLN4924 were cut out and subjected to LC-MS/MS to identify the NEDDylated proteins (Fig. 2 a). We have identified NEDD8-conjugated candidate proteins, and used the PANTHER tool (PANTHERdb.org) to examine gene ontology (GO) analysis, which provided a comprehensive overview of the biological processes and molecular functions regulated by NEDDylation under the VC condition. Supplementary Fig. 2a illustrates the major cellular components, biological process, and molecular functions. According to the enriched GO annotation, the cellular components can be divided into two categories: cellular anatomical entity and protein-containing complex. Additionally, the identified NEDD8-conjugated candidate proteins were categorized into several major biological processes including cellular process, biological regulation and response to stimulus as well as diverse molecular function encompassing binding, catalytic activity, and transcription regulator activity. Further assessment of these identified proteins through bioinformatics analysis led to the selection of several proteins including HSP90, PARP-1, EEF2, NBR1 and DDB1 as NEDD8-conjugated candidate (Supplementary Fig. 2b). Among these NEDDylated candidate proteins, we decided to focuse on Poly (ADP-ribose) polymerase-1 (PARP-1) as a candidate protein in the context of vascular calcification. Although the roles of PARP-1 in vascular calcification were known, the significance of its posttranslational neddylation had not been extensively investigated. Therefore, we aimed to elucidate the role of neddylation of PARP-1 in the context of vascular calcification. To validate the presence of NEDD8-conjugated PARP-1, we confirmed the interaction between PARP-1 and endogenous NEDD8. This interaction resulted in smear bands on the Western blots of Pi-treated VSMCs (Fig. 2 b). Overexpression of NEDD8 led to the appearance of a higher molecular weight band for PARP-1, indicative of NEDDylation. This smear pattern was abolished when using NEDD8ΔGG, a conjugation-defective mutant due to a Gly-75/76 deletion (Fig. 2 c). These findings established that poly-neddylation of PARP-1 with NEDD8 is intricately associated with vascular calcification. Visualization of NEDD8-PARP-1 interaction Proximity ligation assay (PLA) is useful for the visualization of interacting proteins 32 . The PLA assay revealed that NEDD8 binds to PARP-1 in the nucleus. However, this binding was disrupted when MLN4924 was treated (Fig. 2 d). Furthermore, MLN4924 treatment attenuated Pi-induced PARP-1 neddylation and Runx2 expression (Fig. 2 e). NEDDylation of PARP-1 Affects PARP-1 Activity and VC We found that PARP-1 NEDDylation was increased in vascular calcification. Given that PARP-1 is associated with vascular calcification and its enzymatic activation involves poly (ADP)-ribosylation (PARylation) 28 and then PARP-1 catalyze the addition of poly (ADP-ribose) (PAR) to substrate proteins via cleavage of NAD + 33 , it is curious whether PARP-1 NEDDylation can affect the enzymatic activation of PARP-1 in association with vascular calcification. Thus, we explored whether PARP-1 NEDDylation affects its enzymatic activity in VC. PAR polymer expression was detected by its ability to bind to boronate shown in Fig. 2 f. Pi treatment induced PAR polymer expression, evidenced by the smearing of boronate-conjugated precipitates (second lane). This smearing was diminished by MLN4924 treatment, which confirms its generation through NEDDylation. Cobb et al., reported that following DNA damage-induced VC, Runx2 undergoes PARylation dependent on PARP-1 activity 34 . As shown in Fig. 2 f (bottom band), Runx2 was pulled down as a PARylated protein, which was blocked by MLN4924 treatment. Direct measurement of PARP-1 activity using a colorimetric assay (PARP universal colorimetric assay methods) revealed that Pi-induced PARP-1 activity was blunted by MLN4924 treatment (Fig. 2 g). These findings underscore the dependency of PARP-1 activation on PARP-1 neddylation during the progression of VC. MLN4924 mitigates VD 3 -induced VC In pursuit of evaluating the therapeutic prospects of NEDDylation reversal in vascular calcification in vivo, we introduced a mouse model of vitamin D 3 -induced calcification and administered intraperitoneal injections of MLN4924 (10mg/kg) every other day for six days. The experimental timeline is outlined in Fig. 3 a. For calcification assessment, we employed alizarin red S staining of the entire aorta. The results presented in Fig. 3 b demonstrate that MLN4924 treatment effectively counteracted vitamin D 3 -induced VC when compared with the control group. Calcium levels were quantified in both arteries and serum of mice. Remarkably, administration of MLN4924 resulted in a substantial reduction in calcium deposition within the arteries of vitamin D 3 -treated mice, as shown in Fig. 3 c. However, no significant alteration was observed in serum calcium levels in vitamin D 3 -treated mice upon MLN4924 treatment (Fig. 3 d), Importantly, NEDD8-conjugated PARP-1 induced by vitamin D 3 , along with its enzymatic activity indicated by poly(ADP-ribose) expression were effectively suppressed by MLN4924 treatment (Fig. 3 e, f). Additionally, MLN4924 administration attenuated PARP-1 activity in mice subjected to vitamin D 3 injection (Fig. 3 g). As visualized by Alizarin Red S staining, vitamin D 3 -induced calcium deposition, appeared as reddish regions in the aortic media wall, was conspicuously absent by MLN4924 administration (Fig. 3 h). Moreover, the interaction of PARP-1 with NEDD8 induced by vitamin D 3 was blunted by MLN4924 (Fig. 3 i). Collectively, these findings firmly establish the efficacy of MLN4924 in ameliorating vascular calcification through the inhibition of PARP-1 NEDDylation. Furthermore, these findings underscore MLN4924 as a promising candidate for potential therapeutic interventions against VC. Cbl-b mediates NEDD8 binding to PARP-1 in VC Given that many posttranslational modifications involve the final conjugation of small molecular moieties, facilitated by E1, E2, and E3 ligases 35 , with target specificity often guided by E3 ligases, we directed our attention toward deciphering the specific E3 ligase responsible for PARP-1 neddylation. Mammalian cells house several hundred E3 ligases, with CBL-3, RBX1, and Fbxo11 serving as representative E3 ligases that oversee NEDDylation 12 . Consequently, we next embarked on identification of the specific E3 ligase involved in PARP-1 neddylation in the context of VC. For the identification of dysregulated E3 ligase, we employed our previous microarray analysis (GSE74755) results of rat VSMCs treated with Pi 30 . (Supplementary Fig. 3a). Baculoviral IAP Repeat Containing 3 (Birc3), Casitas B–lineage lymphoma protein b (Cbl-b), Ring-Box 1 (Rbx1), Mouse double minute 2 homolog (MDM2), Mouse double minute 4 homolog (MDM4), Ring Finger protein 7 (Rnf7), and Ring Finger Protein 111 (Rnf111) were dysregulated. Among candidates, we previously reported that MDM2 mediates ubiquitination of HDAC1 during the VC process 30 , Thus we have excluded MDM2 as a E3 ligase for the neddylation process of PARP-1. Next, we quantified mRNA level changes of the dysregulated E3 ligase genes using quantitative real-time PCR in Pi-induced VSMCs. Notably, Birc3, Cbl-b, Rbx1, and Rnf111 were significantly upregulated (Supplementary Fig. 3b). Subsequently, using siRNA to knockdown these E3 ligases, we assessed the effect on PARP1- neddylation. Remarkably, only the deletion of Cbl-b effectively attenuated PARP-1 neddylation, whereas the loss of other E3 ligases like Birc3, Rbx1, and Rnf111, had negligible effects on PARP-1 neddylation (Fig. 4 a and Supplementary Fig. 4a). PLA further showed the dissociation of NEDD8 from PARP-1 upon Cbl-b knockdown (Fig. 4 b). Immunoprecipitation analysis confirmed the interaction between Cbl-b and PARP-1 (Fig. 4 c and Supplementary Fig. 4b). Knocking-down of Cbl-b had no effect on the viability of A10 cells in Pi. (Supplementary Fig. 4c). Given the observation of PARP-1 neddylation influencing its activity in vascular calcification, we proceeded to explore whether Cbl-b played a role in regulating PARP-1 activity. Indeed, Cbl-b knockdown significantly dampened Pi-induced PARP-1 activity in VSMCs (Fig. 4 d). Role of Cbl-b in VC and its mechanistic Insights Because the role of Cbl-b in VC had not been previously reported, we proceeded to evaluate its effects by overexpression of HA-Cbl-b in VSMCs. Intriguingly, overexpression of Cbl-b significantly potentiated Pi-induced calcium deposition in a dose-dependent manner (Supplementary Fig. 4d). Furthermore, protein expression of Cbl-b was increased in a time-dependent manner bothe in VSMCs treated with Pi (Supplementary Fig. 4e) and vitamin D 3 -induced VC mouse models (Supplementary Fig. 4f). Notably, knockdown of Cbl-b significantly reduced the intensity of alizarin red S staining in Pi-induced VC (Supplementary Fig. 4g, h). Consistently, the augmentation of calcium deposition induced by Pi was blocked by si-Cbl-b transfection (Fig. 4 e). Next, we evaluated the effects of Cbl-b knockdown by siRNA on VC in mouse models. We intravenously injected 50µg of si-Cbl-b, combined with a transfection reagent, twice during the VC induction periods following three consecutive days of vitamin D 3 injection into mice (Fig. 4 f). The knockdown of Cbl-b was successfully achieved in various tissues, including the aorta, brain, kidney, liver and lung (Supplementary Fig. 5a). This led to a reduction in VD 3 -induced calcium accumulation in the aorta, but not in serum levels (Fig. 4 g and Supplementary Fig. 5b). Additionally, the loss of Cbl-b in VC resulted in blunted PARP-1 neddylation and Runx2 expression (Fig. 4 h). As anticipated, the mineralization of vascular smooth muscle from the aorta in the Vitamin D 3 group was inhibited following the injection with si-Cbl-b (Fig. 4 i). Collectively, these results underscore the pivotal role of the E3 ligase Cbl-b in mediating PARP-1 neddylation and its regulatory role in VC. Cbl-b E3 ligase activity governs PARP-1 Neddylation in VC Given that Cbl-b E3 ligase belongs to the RING finger family, which engages E2 enzymes to facilitate substrate ubiquitination 36 , we examined whether E3 ligase activity of Cbl-b is essential for PARP-1 neddylation. It was previously reported that C373 and W400 of Cbl-b are critical for its E3 ligase activity 37 . We generated these functionally inert forms of Cbl-b that lacks E3 ligase activity and next subjected them to immunoprecipitation with anti-PARP-1 antibody. As anticipated, high Pi exposure potentiated PARP-1 neddylation (Fig. 5 a) and consequently, PARP-1 activity (Fig. 5 b). These effects were pronounced upon transfection with Cbl-b WT, but not Cbl-b C373A or Cbl-b W400A. Interestingly, contrary to Cbl-b WT, both Cbl-b C373A and Cbl-b W400A failed to increase in mineralization and calcium deposition (Fig. 5 c, d). Besides, Cbl-b WT and Cbl-b E3 ligase activity dead mutants (C373A and W400A) did not alter cell viability in Pi with A10 cells (Supplementary Fig. 6a) Next, we interrogated whether blocking of E3 ligase activity via a peptide spanning crucial residue C373 and W400 could impede PARP-1 neddylation and, consequently, vascular calcification. Sequence analysis of different species revealed that the C373 and W400 site of Cbl-b are highly conserved (Supplementary Fig. 6b). To visualize the localization of these peptides, fluorescein isothiocyanate-conjugated nuclear localization signal (NLS) sequence 38 were added. The synthetic peptide C373, but not W400, effectively entered the nucleus in VSMCs (Supplementary Fig. 6c). Cbl-b C373 peptide did not affect cell viability in Pi with A10 cells (Supplementary Fig. 6d). In the subsequent experiments, we utilized C373 peptide. NEDD8 conjugated with PARP-1 is cleaved by treatment of Cbl-b C373 peptide in VSMCs under Pi conditions (Fig. 5 e). Moreover, both PARP-1 neddylation and its activity were also blunted by C373 in Pi-induced VC (Fig. 5 f, h). Further corroborating these findings, the Cbl-b C373 peptide significantly attenuated the propensity for calcium deposition and alizarin red S staining in Pi-treated VSMCs (Fig. 5 i, k). Taken together, these observations underscore the pivotal role of Cbl-b E3 ligase activity, particularly at residue C373, in promoting PARP-1 neddylation within the context of vascular calcification. Additionally, the C373-spanning peptide emerges as a potential candidate for thwarting vascular calcification, offering novel therapeutic avenues for its prevention or treatment. Alleviation of Vitamin D3-induced VC through Cbl-b C373 peptide To further validate the therapeutic potential of the Cbl-b C373-blocking peptide, we examined its effects in VD3-induced VC models. Mice were intraperitoneally administered with Cbl-b C373 peptide (1mg/kg/day) every two day, following a single injection of vitamin D 3 in mice. After 9-day of experimental period, mice were sacrificed and assessed for VC effects (Fig. 6 a). The uptake of the C373 peptide into the mouse aorta was observed through FITC fluorescence, as shown in Supplementary Fig. 7. Treatment with Cbl-b C373 peptide effectively attenuated the formation of calcified nodules in aorta of vitamin D 3 -induced VC, compared to the scramble group (Fig. 6 b). Calcium content analysis demonstrated that increased calcium deposition induced by vitamin D3 was significantly counteracted by Cbl-b C373 peptide treatment in aorta, but not in serum (Fig. 6 c, d). Remarkably, the enhancement of PARP-1 neddylation and PARP-1 enzyme activities such as PAR polymer, instigated by vitamin D 3 , were effectively mitigated by Cbl-b C373-blocking peptide within the aorta, compared to the scramble control in mice (Fig. 6 e, f). Evidently, the distinct calcium deposition characteristic of vitamin D 3 presence was entirely abrogated following the administration of the Cbl-b C373 peptide (Fig. 6 g, left panel). Additionally, the typical interaction between PARP-1 and NEDD8 elicited by vitamin D 3 was efficiently hindered by the Cbl-b C373 peptide, as illustrated by Fig. 6 g (right panel). Taken together, these findings underscore the efficacy of the Cbl-b C373-blocking peptide in vitamin D 3 -induced vascular calcification through the disruption of PARP-1 neddylation. PARP-1 neddylation is counteracted by NEDP-1 in VC The dynamic nature of NEDDylation is well-established, encompassing a reversible process facilitated by NEDP-1, an NEDD8-specific protease 1 39 . In light of this, we embarked on investigating whether NEDP-1-mediated de-NEDDylation could effectively reverse PARP-1 neddylation in VC. Thus, we designed a study centered on the NEDP-1. As anticipated, the introduction of ectopically expressed Cbl-b augmented the conjugation of NEDD8 with PARP-1. Nevertheless, this enhancement was nullified by the overexpression of NEDP-1 (Fig. 7 a). For deeper insights, we scutinized the effect of NEDP-1 on PARP-1 neddylation and Poly (ADP-ribose)ylation in Pi-induced VSMCs. Remarkably, the overexpression of NEDP-1 resulted in the suppression of Pi-induced PARP-1 neddylation and poly(ADP)-ribosylation (Fig. 7 b, c). Furthermore, the increase in the activity of PARP-1 evident in the Pi-induced VC model was effectively down-regulated by the overexpression of NEDP-1 (Fig. 7 d). Until now, the role of NEDP-1 in vascular calcification has not been reported. Therefore, we sought to investigate this. The expression of NEDP-1 expression gradually decreased in vascular calcification both in vitro and in vivo models (Supplementary Fig. 8a, b). NEDP-1 overexpression did not affect cell viability in VSMCs (Supplementary Fig. 8c). Notably, the transient overexpression of NEDP-1 in VSMCs via transfection significantly exerted a dose-dependent migratory effect on Pi-induced calcium deposition (Fig. 7 e and Supplementary Fig. 8d) and mineralization in VSMCs (Fig. 7 f, g). Conversely, the depletion of NEDP-1 significantly amplified the accumulation of calcium stimulated by Pi in a dose dependent manner (Supplementary Fig. 8e, f). Given the function of NEDP-1 in dissociating NEDD8 from target proteins, we postulated that NEDP-1 might exert an antagonistic effect against the functional interplay of neddylated PARP-1 and E3 ligase activity of Cbl-b in the milieu of VC. Armed with this supposition, we proceeded to investigate whether functionality of either PARP-1 or Cbl-b could be effectively counteracted by NEDP-1. Evidently, the overexpression of either PARP-1 or Cbl-b led to an elevation of calcium content and mineralization in the presence of Pi in VSMCs. Notably, this exacerbation was significantly dampened by the simultaneous overexpression of NEDP-1 in a dose dependent manner (Fig. 7 h-k). Collectively, these findings underscore the role of NEDP-1 as a pivotal conteractive element against PARP-1 neddylation in VC. DISCUSSION Here, we describe how the NEDD8 modifier is increased in response to vascular calcification stimuli and conjugated with PARP-1, promoting vascular calcification. PARP-1 neddylation is mediated by the E3 ligase Cbl-b and is deneddylated by NEDP-1. Consequently, inhibiting PARP-1 neddylation could be potential therapeutic stgrategies for alleviating vascular calcification (Fig. 8 ). Post-translational modifications (PTMs) play crucial roles in various biological processes, including vascular calcification. Runx2, a major transcription factor, can undergo diverse PTM by such as phosphorylation, acetylation and ubiquitination and also PARylation in genotoxic stress during vascular calcification We also have previously reported that E3 ligase MDM2 mediated HDAC1 ubiquitination induces vascular calcification 30 . Both MSX1 and MSX2 act as upstream transcription regulator of MDM2 31 . Numerous studies have reported that PARP-1 undergoes diverse posttranslational modifications such sumoylation, ubiquitination and acetylation 40 – 42 . In this study, we define neddylation as a novel modification of PARP-1 and a critical regulatory mechanism for vascular calcification. To identife genuin neddylation substrates, some criteria is reqiured for the charaterization of NEDD8 substrates 6 . Herein, we provides a serieses of data to prove that PARP-1 is a substrate for NEDD8: (i) NEDD8 is covalently attached to PARP-1; (ii) endogenou detection of PARP-1 neddylation; (iii) PARP-1 neddylation depends on an activatin enzyme (E1) and MLN4924 inhibits PARP-1 neddylation; (iv) we identified Cbl-b as a specific ligase for PARP-1 neddylation; (v) we identified NEDP-1 as deneddylase for PARP-1 in vivo; (vi) formation by NEDD8 of a poly-neddylation chain on PARP-1; (vii) PARP-1 neddylation regulates PARylation as downstream. Collectively, based on these findings, we conclude that during vascular calcification, PARP-1 ia a genuine subjected to neddylation. To date, several researchers have studied the correlation between PARP-1 and vascular calcification. Wang et al., reported that PARP-1 could promote the osteogenic transition of VSMCs via the JAK2/STAT3/miR-204/Runx2 pathway 29 . Other studies demonstrated that oxidative DNA damage is a key driver of vascular calcification and that PARP-1 is activated at the site of such calcification 43 . Although, PARP-1 is activated by various stimuli and conditions associated with vascular calcification. However, how to regulate PARP-1 is poorly understood in vascular calcification. Herein we provide evidence that PARP-1 is conjugated with NEDD8 which in turn activates PARP-1 activity. This activation subsequently induce PARylation and promotes vascular calcification. Nonetheless, it still remains unclear which lysine residues on PARP-1 NEDD8 binds to. This necessitates further investigation into this aspect of PARP-1 neddylation activation. Cbl-b is a member of the Cbl family proteins, which consists of three homologues known as c-Cbl, Cbl-b, Cbl-3. Cbl-b is predominantly expressed in T-cells and marcrophages plaques. Studies have reported that Cbl-b regulates both innate and adaptive immune cell responses through immune T cell activation 44 , 45 . In atherosclerosis, genetic deficency of Cbl-b aggravated atherosclerosis in ApoE-/- mice by recruiting CD8 + T-cells to the plaque 46 , 47 . In this study, we found that Cbl-b is upregulated and acts as a specific key mediator of PARP-1 neddylation in vascular calcifcation. This process depends on the E3 ligase catalytic activity of Cbl-b. Catalytically inhibitors such as overexpressing the plasmid construct of inactive mutant Cbl-b C373A and blocking peptide targeting residue 373 (C373), inhibit PARP-1 neddylation mediated by Cbl-b. Furthermore, inhibition of E3 ligase catalytic activity of Cbl-b hampers calcim accumulation and minalization during vascular calcification progession. Therefore, we propose that Cbl-b C373 blocking peptides may be therapeutic agents for vascular calcification. In addition to this, other upregulated E3 ligases such as Birc3, Rbx1, and Rnf111 do not affect PARP-1 neddylation but may possibly serve as regulators in vascular calcficiation In conclusion, this study suggests that Cbl-b acts as a critical mediator of NEDD8-conjugated PARP-1 neddylation under vascular calcification. PARP-1 neddylation plays a modulatory role in PARP-1 neddylation-mediated PARylation by regulating catalytic activity of PARP-1. Therefore, treatment with inhibitors of PARP-1 neddylation such as MLN4924 and Cbl-b C373 blocking peptides, can block PARylation, thereby ameliorating vascular calcification. Our findings provide insights into the prevention and treatment of a variety of cardiovascular disease related to vascular calcification. Declarations Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2022R1A4A2000767, 2022R1I1A1A01053681, RS-2023-00208097, and RS-2023-002374089). Author contributions Duk-Hwa Kwon: Conceptualization; Data curation; Formal analysis; Validation; Investigation; Mothology; Funding acquisition; Writing-original draft. Sera Shin: Data curation; Validation; Visualization. Yoon Seok Nam: Conceptualization; Formal analysis. Nakwon Choe: Data curation. Methodology. Younwoon Lim: Data curation. Anna Jeong: Data curation. Methodology. Yun-Gyeong Lee: Data curation. Young-Kook Kim: Formal analysis; Supervision. Hyun Kook: Conceptualization; Validation, Funding acquisition, Writing-original draft. 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Additional Declarations (Not answered) Supplementary Files KwonetalSupplementalData.docx Cite Share Download PDF Status: Published Journal Publication published 01 Oct, 2024 Read the published version in Experimental & Molecular Medicine → Version 1 posted Editorial decision: revise 26 Mar, 2024 Review # 2 received at journal 26 Mar, 2024 Review # 1 received at journal 25 Mar, 2024 Reviewer # 2 agreed at journal 09 Mar, 2024 Reviewer # 1 agreed at journal 07 Mar, 2024 Reviewers invited by journal 07 Mar, 2024 Submission checks completed at journal 20 Feb, 2024 First submitted to journal 20 Feb, 2024 Unknown event 12 Feb, 2024 Editor assigned by journal 08 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-3939434","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":273985594,"identity":"051808a9-eb5d-4de5-9ff0-f9431df2a8e0","order_by":0,"name":"Hyun Kook","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYBACCQYGZmYQg5+BByHK2ECMFskGkrUYHCBWi2R782HjwrbDecbHzx58XLjDLpqB/fADxpl7cGuR5jmWnDyz7XCx2Zm8ZOOZZ5JzG3jSDBg3PMOtRU4ix/gwb9vhxG03eMykeduYcxsYchgYHxwgQsvmGWAt9bkN/G/wa5EGakkGadkgAdZyOLdBAmjLBjxaJHuOJRvznEtPnHEmx9iYt+14bpvEM4ODM/BokTjefFiap8w6sb/9jOFj3rbq3H7+5IcPe/BoAQNGNiQOiE1IAxD8IaxkFIyCUTAKRjAAAOvOTr0idGwEAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-0740-1806","institution":"Chonnam National University Medical School","correspondingAuthor":true,"prefix":"","firstName":"Hyun","middleName":"","lastName":"Kook","suffix":""},{"id":273985595,"identity":"6d34e54d-a5ba-44a9-8e15-2b79ce569a80","order_by":1,"name":"Duk-Hwa Kwon","email":"","orcid":"","institution":"Chonnam National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Duk-Hwa","middleName":"","lastName":"Kwon","suffix":""},{"id":273985596,"identity":"ec17db2f-b97b-4371-90d0-a4fe84d6e196","order_by":2,"name":"Sera Shin","email":"","orcid":"","institution":"Chonnam National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Sera","middleName":"","lastName":"Shin","suffix":""},{"id":273985597,"identity":"7f67d206-6df1-4e13-9cdf-a00bea616d86","order_by":3,"name":"Yoon Seok Nam","email":"","orcid":"","institution":"Chonnam National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Yoon","middleName":"Seok","lastName":"Nam","suffix":""},{"id":273985598,"identity":"49cf98e4-5b7e-48ce-8364-c9802f5b93a8","order_by":4,"name":"Nakwon Choe","email":"","orcid":"","institution":"Chonnam National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Nakwon","middleName":"","lastName":"Choe","suffix":""},{"id":273985599,"identity":"338f6e5e-6383-4a4e-903b-0f6517d777c5","order_by":5,"name":"Yongwoon Lim","email":"","orcid":"","institution":"Chonnam National University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yongwoon","middleName":"","lastName":"Lim","suffix":""},{"id":273985600,"identity":"beeac9a4-3e81-4b8b-abec-8e12ae7e995f","order_by":6,"name":"Anna Jeong","email":"","orcid":"","institution":"Chonnam National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Jeong","suffix":""},{"id":273985601,"identity":"3bb2d99b-f6f8-43ed-b593-2bc30b29b015","order_by":7,"name":"Yun-Gyeong Lee","email":"","orcid":"","institution":"Chonnam National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Yun-Gyeong","middleName":"","lastName":"Lee","suffix":""},{"id":273985602,"identity":"6359aea2-42dc-4df8-96de-890feff429fe","order_by":8,"name":"Young-Kook Kim","email":"","orcid":"https://orcid.org/0000-0001-6434-2235","institution":"Chonnam National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Young-Kook","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2024-02-08 09:22:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3939434/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3939434/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s12276-024-01322-y","type":"published","date":"2024-10-01T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52452829,"identity":"85d2e36e-9360-47c6-af22-f94aa3a04ed4","added_by":"auto","created_at":"2024-03-11 19:17:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1236683,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMLN4924 inhibits Pi-induced vascular calcification.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Treatment with MLN4924 (0.001uM, 0.01uM, 0.1uM, 1uM) inhibited Pi (4mM)-induced calcium deposition in A10 cells. n=4~12 per group, independent experiments. (b) A10 cells were treated with Pi and MLN4924 under the aforementioned conditions. Representative Alizarin Red S stained VSMCs showed that mineralization was blocked by MLN4924 in Pi-treated A10 cells. Scale bar, 10mm. (c) Quantification of alizarin red S staining was measured. n=6 per group, independent experiments. (d) mRNA levels of smooth muscle marker genes (Sma and Acta) and osteogenic-related marker genes (Runx2 and Alp) were measured in A10 cells treated with Pi and MLN4924. n=6 per group, independent experiments. (e) NEDD8 immunoblotting was performed after treatment with Pi and MLN4924 in A10 cells. The red arrow indicates free NEDD8. (f-g) The contents of calcium accumulation were measured. Overexpression of NEDD8 potentiated Pi-induced calcium deposition in A10 cells. n=8 per group, independent experiments. (f). Inhibition of NEDD8 conjugation with NEDD8 ΔGG blunted Pi-induced calcium deposition. n=8 per group, independent experiments. (g). Data are shown as mean ± SEM. Statistical significance was tested using ANOVA with Turkey HSD and Dunnett T3.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3939434/v1/a3b80511940a4313d516c8cb.jpg"},{"id":52452835,"identity":"7aa6c963-fad2-4802-b4bb-cba097ea87d0","added_by":"auto","created_at":"2024-03-11 19:17:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1397864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNEDD8 is conjugated PARP-1 in VC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) A gel stained with Coomassie blue for mass spectrometry analysis. Cell extracts from A10 cells treated with Pi (4mM) and MLN4924 (0.1μM and 1μM), were immunopurified with anti-NEDD8 and loaded onto SDS-PAGE. Bands indicated by arrows were then digested with trypsin and analyzed by mass spectrometry. Scale bar, 5mm. (b) Cell lysates from Pi-treated A10 cells were immunoprecipitated with anti-NEDD8 and immobilized with anti-PARP-1. PARP-1 neddylation increased in Pi-induced VC. (c) Whole cell lysate from 293T cells transfected with the indicated constructs were subjected to Ni-NTA Pull-down. PARP-1 neddylation was blunted in NEDD8 ΔGG, compared with NEDD8 WT. (d) Proximity Ligase Assay (PLA) was performed. NEDD8-conjugated PARP-1 is dissociated by MLN4924 (1μM). Scale bar=20μM. (e) Immunoprecipitation with anti-PARP-1. Pi-induced PARP-1 neddylation is attenuated by MLN4924 (1μM) in A10 cells. (f-g) PARP-1 activity was measured by detecting poly(ADP)-ribosylation (PAR) polymer (f) and conducting a PARP-1 enzyme activity assay (g). Pi-induced PARP-1 activity was blunted by MLN4924 treatment in A10 cells. n=6 per group, independent experiments. A.U., arbitrary units. Scale bar, 5mm. Data are represented as mean ± SEM. Statistical significance was tested using ANOVA with Turkey HSD.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3939434/v1/2cf8d035a18abd89c5a76505.jpg"},{"id":52452826,"identity":"837ff1e2-2650-4b56-98ae-9392829b41ce","added_by":"auto","created_at":"2024-03-11 19:17:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1818172,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMLN4924 ameliorates VD\u003c/strong\u003e\u003csub\u003e3\u003c/sub\u003e\u003cstrong\u003e-induced VC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Treatment timeline for MLN4924 (10mg/kg/day) with vitamin D\u003csub\u003e3\u003c/sub\u003e (5x10\u003csup\u003e5\u003c/sup\u003e IU/kg/day) in mice (b) Whole aorta with alizarin red S staining. MLN4924 mitigated VD3-induced calcification. Scale bar, 10mm. (c-d) Measurement of Ca\u003csup\u003e2+\u003c/sup\u003e levels in aorta (c) and serum (d) after MLN4924 treatment in vitamin D\u003csub\u003e3\u003c/sub\u003e-injected mice. n=9~11 per group, independent experiments. Treatment of MLN4924 blunted Ca\u003csup\u003e2+\u003c/sup\u003e accumulation in aorta, but not in serum, from VD3-induced VC. (e) PARP-1 neddylation is increased by injection of VD\u003csub\u003e3\u003c/sub\u003e, but it was blocked by MLN4924. (f) Boronic acid bead-pull down assay were performed. PARP-1 activity, along with PAR polymer is blunted by MLN4924. (g) PARP-1 enzyme activity was measured. (h) Alizarin red S staining in cross section of the aorta. Scale bar, 200mM. (i) Proximity Ligation Assay (PLA) with anti-NEDD8 and anti-PARP-1 was performed on cross-sections. Scale bar, 20mM, \u0026nbsp;Data are represented as mean ± SEM. Statistical significance was tested using ANOVA with Bonferroni.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3939434/v1/611b80178ab09c0f4f4557dc.jpg"},{"id":52452827,"identity":"08f98068-f64c-44cc-ac6a-6892fa56e7dc","added_by":"auto","created_at":"2024-03-11 19:17:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1364318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eE3 ligase Cbl-b mediates PARP-1 neddylation in Pi-induced VC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Immunoblot analysis of immunoprecipitation with anti-NEDD8 from Pi-treated A10 cells following the depletion of Birc3, Cbl-b, Rbx1 and Rnf111. (b) PLA assay was performed with anti-NEDD8 and anti-PARP-1 in transfection with siCbl-b and Pi in A10 cells. scale bar=20µM.\u003c/p\u003e\n\u003cp\u003e(c) PARP-1 activity was analyzed in Pi-treated A10 cells with Cbl-b knockdown. n=5 per group, independent experiments (d) Endogeous PARP-1 is interacted with Cbl-b in A10 cells treated with Pi. (e) Calcium content wa measured. Knockdown of Cbl-b inhibited Pi-induced calcium accumulation in a dose dependent manner. n=4~7 per group, independent experiments (f) Experimental procedure timeline. Mice were treated with si-Cbl-b or scramble via tail vein injection twice during VC induction period following vitamin D\u003csub\u003e3\u003c/sub\u003e injection (5x10\u003csup\u003e5\u003c/sup\u003eIU/kg/day). (g) Calcium assay. Vitamin D3-increased calcium accumulation is inhibited by Cbl-b knockdown in the aorta (h) Immunoprecipitation with anti-NEDD8 was performed. Vitamin D3-induced PARP-1 neddylation is blunted by knockdown of Cbl-b. (i) Vascular calcification in the aorta was determined by alizarin red S staining. Representative images are shown. Scale bar, 200mM. \u0026nbsp;Statistical significance was tested using ANOVA with Turkey HSD and Bonferroni.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3939434/v1/b6b498a2989b82fc7259b729.jpg"},{"id":52452839,"identity":"096e3dca-2160-4716-9671-a49e827d0a3a","added_by":"auto","created_at":"2024-03-11 19:17:56","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1583499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCbl-b E3 ligase activity at K373 promotes PARP-1 neddylation in Pi-induced VC. \u003c/strong\u003e(a) PARP-1 neddylation in Pi-treated A10 cells is dependent on Cbl-b E3 ligase activity. (b) PARP-1 activity was determined using anti-PARP-1. Mutants with inactive Cbl-b E3 ligase activity decreased PARP-1 activity in Pi-treated A10 cells. n=4 per group, independent experiments. (c) Calcium contents were measured. Pi increased calcium accumulation, but it was blocked by inactive Cbl-b E3 ligase mutants (C373A and W400A). n=11 per group, independent experiments. (d) Alizarin red S staining was performed. Scale bar, 10mm. (e) PLA assay. NEDD8 was associated with PARP-1 under Pi conditions, but it was dissociated by the Cbl-b C373 peptide. (f) Immunoblot analysis was performed using immunoprecipitation with anti-PARP-1. (g) Boronic acid-pull down was performed to detect PAR polymer. (h) PARP-1 activity was analyzed. Pi-induced PARP-1 activity was attenuated by Cbl-b C373. n=4 per group, independent experiments. (i) Calcium assay. Cbl-b C373 inhibited Pi-induced calcium deposition in A10 cells. n=6 per group, independent experiments. (j) Calcification was determined by Alizarin Red S staining. Scale bar, 10mm. (k) Quantification of Alizarin Red S staining was performed. n=6 per group, independent experiments. Data are represented as mean ± SEM. Statistical significance was tested using ANOVA with Tukey HSD and Dunnett T3.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3939434/v1/95fae707e8465c27d946ed24.jpg"},{"id":52452840,"identity":"0b550d64-a36f-48dd-ab4f-dd3cb33c7e1d","added_by":"auto","created_at":"2024-03-11 19:17:56","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1472823,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdministration of Cbl-b C373 peptide mitigates vitamin D\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-induced VC. \u003c/strong\u003e(a) Experimental procedure for administering C373 peptide (1mg/kg/day) via intraperitoneal injection to mice with vitamin D\u003csub\u003e3\u003c/sub\u003e-induced VC. (b) Alizarin Red S staining of the entire aorta in mice. Scale bar, 10mm. (c-d) Aortic Ca\u003csup\u003e2+\u003c/sup\u003e (c) and serum Ca\u003csup\u003e2+\u003c/sup\u003e (d) levels were measured. Administration of C373 peptide reduced vitamin D\u003csub\u003e3\u003c/sub\u003e-induced calcium accumulation. n=6 per group, independent experiments. (e) Immunoprecipitation with anti-NEDD8 was used to detect PARP-1 neddylation. PARP-1 neddylation was diminished by administration of C373 peptide in the aorta of VD\u003csub\u003e3\u003c/sub\u003e-induced VC mice. (f) PARP-1 activity was detected by PAR polymer with a boronate pull-down assay. C373 peptide blunted vitamin D\u003csub\u003e3\u003c/sub\u003e-induced PARP-1 activity. (g) Histology: alizarin red S staining (left panel scale bar, 200mM) and proximity ligation assay (PLA) with anti-NEDD8 and anti-PARP-1 (right panel, scale bar, 20mM) were performed on cross-sections. Vitamin D\u003csub\u003e3\u003c/sub\u003e increased vascular calcification, but it was inhibited by C373 peptide. The administration of the Cbl-b C373 peptide led to the dissociation of NEDD8 conjugated with PARP-1. Data are represented as mean ± SEM. Statistical significance was tested using ANOVA with Tukey HSD and Dunnett T3.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3939434/v1/05b486653896dbf3aeb7dfb0.jpg"},{"id":52452836,"identity":"a4311d40-0d8b-47c6-8543-ee854f80e59d","added_by":"auto","created_at":"2024-03-11 19:17:56","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1561357,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePARP-1 deneddylation by NEDP-1 attenuates VC. \u003c/strong\u003e(a) Extracted cell lysates from 293T cells, with transfected indicated plasmid DNAs were subjected to immunoprecipitation with anti-Flag. Overexpression of NEDP-1 resulted in the deneddylation of PARP-1. (b) Transfection of NEDP-1 into A10 cells followed by treatment with Pi blocked Pi-induced PARP-1 neddylation. (c) Overexpression of NEDP-1 and Pi treatment in A10 cells led to decreased expression of PAR polymer. (d) PARP-1 activity was measured under the same conditions. n=5 per group. (e) Calcium contents were measured. n=8 per group, independent experiments. (f) Cells were stained with alizarin red S. Representative whole well image is shown. Scale bar, 10mm. (g) The extent of alizarin red S staining was quantified. n=6 per group. (h) Pi-treated A10 cells were co-transfected with Flag-PARP-1 and NEDP-1-V5. Calcium content was measured. PARP-1 potentiated Pi-induced calcium deposition, which was blocked by overexpression of NEDP-1. n=12 per group, independent experiments. (i) Alizarin res S staining was performed under the same conditions well. Scale bar, 10mm. (j) A10 cells were co-transfected with HA-Cbl-b and NEDP-1-V5. Cbl-b also potentiated Pi-induced calcium deposition, which was also blocked by NEDP-1 overexpression. n=8 per group, independent experiments. (k) Alizarin red S staining under the same condition. Scale bar, 10mm. Data are shown as mean ±SEM. Statistical significance was tested using ANOVA with Tukey HSD and Dunnett T3.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3939434/v1/1c193bc60faf9867e1526c84.jpg"},{"id":52452815,"identity":"0e8c2702-aaee-4803-bba8-bd4406fe245b","added_by":"auto","created_at":"2024-03-11 19:17:51","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2377992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThis diagram illustrates how PARP-1 activity, facilitated by NEDD8-conjugated PARP-1, induces vascular calcification. \u003c/strong\u003eA diverse range of stimuli, such as inorganic phosphate (Pi) or vitamin D3, drives NEDD8 conjugation with PARP-1, which is mediated by Cbl-b, and this process is reversed by NEDP-1. Notably, the neddylation of PARP-1 leads to an increase in PARP-1 activity, which contributes to the progression of vascular calcification (VC). However, the NEDD8-activating E1 enzyme inhibitor, MLN4924, effectively impedes the progression of VC. Additionally, a C373 peptide derived from Cbl-b shows promise in preventing VC by mitigating the inactive form of Cbl-b's E3 ligase activity. Therefore, we propose that targeting the NEDD8-dependent activation of PARP-1 could be a potentially effective therapeutic approach for VC.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3939434/v1/a9b65ea2837110e7bf07cedb.jpg"},{"id":65671416,"identity":"861c4636-358e-43dd-b9a6-3768c6921541","added_by":"auto","created_at":"2024-10-01 07:11:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13655261,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939434/v1/3760b8c8-8a4f-4ef5-869b-abae563cda60.pdf"},{"id":52452849,"identity":"1d3f4839-e848-4e97-bb65-5f705c9deba7","added_by":"auto","created_at":"2024-03-11 19:17:58","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":990717,"visible":true,"origin":"","legend":"","description":"","filename":"KwonetalSupplementalData.docx","url":"https://assets-eu.researchsquare.com/files/rs-3939434/v1/c232fc9223f2beee6ab2e904.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Cbl-b E3 ligase-mediated neddylation and activation of PARP-1 induces vascular calcification","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eVascular calcification pertains to the abnormal accumulation of calcium within the walls of blood vessels, leading to a reduction in their elasticity and functionality, as well as an elevated risk of cardiovascular events like heart attack and stroke\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. This process is intricate and influenced by multiple factors, often originated from various underlying conditions, including aging, diabetes, chronic kidney disease, and atherosclerosis\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Vascular calcification manifests in two distinct forms: intimal and medial calcification. Intimal calcification occurs within the inner layer of blood vessels and is closely associated with atherosclerosis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, whereas medial calcification affects the middle layer of vessels and is linked to aging and conditions like chronic kidney disease\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Currently, treatment options for vascular calcification are limited, primarily concentrating on the management of the causes that contribute to its development.\u003c/p\u003e \u003cp\u003eNEDD8, which stands for Neural precursor cell Expressed Developmentally Downregulated 8, is a small protein similar to ubiquitin. It regulates the activity and stability of specific proteins through a process known as neddylation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Similar to ubiquitination, NEDD8 binds to its target proteins by forming an isopeptide chain between its C-terminal glycine residue (Gly76) and a lysine residue on targeted proteins. This attachment occurs through a series of enzymatic reactions involving NEDD8-activating enzyme E1, NEDD8-conjugating enzyme E2, and NEDD8-ligase enzymes E3\u003csup\u003e6\u003c/sup\u003e. Importantly, this process is reversible, and NEDD8 can be detached from its targets by a family of enzymes known as NEDD8-specific proteases (NEDP1)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. NEDD8 plays a pivotal role in a variety of cellular processes, including the cell cycle regulation, DNA damage response, and protein degradation in diverse tissues\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Targeting the NEDD8 pathway has emerged as a potential therapeutic strategy for cancer treatment\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Several small molecule inhibitors of the NEDD8-activating enzyme and NEDD8-conjugating enzyme have been developed, such as MLN4924/Pevonedistat\u003csup\u003e11, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and UBE2M-DCN1 inhibitors\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These inhibitors are currently undergoing evaluation in clinical trials, marking a significant development in the field of cancer therapy\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePoly(ADP-ribose) polymerase-1 (PARP-1) is a key member of the PARP family, which comprises 18 members and contributes to nearly 90% of cellular PARP activity \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. PARP-1 plays a pivotal role in various cellular processes, including DNA damage repair, transcription, and cell death signaling\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Numerous studies have provided insights into the multifaceted roles of PARP-1, extending its influence to a wide array of diseases. These encompass neurodegenerative disorders\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, chronic inflammation\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, cardiovascular diseases \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and cancer\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Several studies have also demonstrated that PARP-1 instigates an osteogenic transition of vascular smooth muscle cells (VSMCs) by upregulating pivotal transcription factors such as Runx2 and NF-κB\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Inhibition of PARP-1 has exhibited protective effects against mineralization and calcification. Notably, the activity of PARP-1 is influenced by oxidative stress and DNA damage, which, in turn, foster vascular calcification\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Our research has unveiled a novel association between NEDD8 and PARP-1 under VC conditions. Despite the recognized involvement of PARP-1 activity in osteogenic calcification, the precise regulatory mechanisms governing its activity during the progression of VC remain to be elucidated.\u003c/p\u003e \u003cp\u003eIn the present study, we have delved into the significance of NEDD8 in the context of vascular calcification. Our investigation has unequivocally showcased that NEDD8 plays a pivotal role in the promotion of vascular calcification, and conversely, the inhibition of NEDD8 serves as a protective measure against this process. Within the conditions conducive to vascular calcification, our observations have revealed an activation of PARP-1 due to the interaction facilitated by NEDD8 poly-chains. This interaction is mediated through the E3 ligase activity of Cbl-b. Our findings compellingly suggest that Cbl-b orchestrates the process of PARP-1 neddylation, consequently modulating its activity in the course of vascular calcification.\u003c/p\u003e"},{"header":"MATERIAL and METHODS","content":"\u003cp\u003eAll experimental procedures were approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee and followed the National Institutes of Health \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e (NIH Publication No. 8023, revised 1978).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInduction of vascular calcification\u003c/h2\u003e \u003cp\u003eThe RVSMCs and A10 cells are cultured in growth medium and changed with calcification medium containing 2 mM or 4 mM inorganic phosphate (pH 7.4) for up to 3 days or 6 days. The medium was changed every 2 days. Calcium deposition in VSMCs was determined after washing with 1x PBS.\u003c/p\u003e \u003cp\u003eWild-type C57BL/6 male mice were used for induction of vascular calcification by administration of vitamin D\u003csub\u003e3\u003c/sub\u003e as described previously\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Vitamin D\u003csub\u003e3\u003c/sub\u003e (Cholecalciferol) in 70 \u0026micro;l of absolute ethanol was mixed with 500 \u0026micro;l Cremophor (Alkamuls EL-620, Sigma St. Louis, MO, USA) for 15 minutes at RT and then combined with 6.2 mL sterilized water containing 250 mg of dextrose for 15 minutes at RT. The mice were injected with a dose of vitamin D\u003csub\u003e3\u003c/sub\u003e (5x10\u003csup\u003e5\u003c/sup\u003e IUkg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eday) subcutaneously for 3 days and maintained for 6 days to induce vascular calcification. All animals were killed by inhalated CO\u003csub\u003e2\u003c/sub\u003e at chamber, and the aortas were isolated. All experiments were performed using male mice at 8\u0026ndash;10 weeks of age. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Chonnam National University (CNU-IACUC-HA-2020-17 and CNU-IACUC-H-2021-39).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of calcium deposition\u003c/h3\u003e\n\u003cp\u003eThe A10 Cells and tissues were decalcified with 0.6N HCl at 4\u0026deg;C for 24 hours. The calcium content of the HCl supernatants was determined colorimetrically using the QuantiChrom calcium assay kit (QuantiChromTM Calcium Assay Kit, BioAssay Systems, Hayward, USA) according to the manufacturer\u0026rsquo;s instructions. Briefly, 5 \u0026micro;L of the samples was transferred to a 96-well plate and 200 \u0026micro;l working reagent was added (1:1, solution A and B). Mixed samples were briefly incubated for 3 min and absorbance was measured at 570 nm by using an ELx808 absorbance reader (BTELX808, BioTek Instruments, Winooski, VT, USA). And next, the cells were washed 3 times with 1xPBS and lysed with 0.1N NaOH/0/1% SDS to extract proteins. The calcium content was then normalized to the total protein amount, whereas that of the tissues was normalized to tissue dry weight.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAlizarin Red S staining and quantification\u003c/h2\u003e \u003cp\u003eThe A10 cells were washed with 1xPBS and fixed with 10% formalin for 30 min at RT. After being washed three time with distilled water, the cells were stained with 40 mM alizarin red S solution (pH 4.2, Sigma-Aldrich) for 30 min at room temperature and washed with distilled water to remove nonspecific staining. For quantification of alizarin red S staining, the stained cells were destained with 10% cetylpyridinium chloride (CPC) in 10 mM sodium phosphate buffer (pH 7.4) for 30 min at room temperature. The absorbance was then measured at 450 nm, and the values were normalized using a 10% CPC standard solution.\u003c/p\u003e \u003cp\u003eTo determine arterial calcification, the aorta was collected and fixed with 10% formalin for overnight at 4℃. The arterial tissue sample was placed in 2% KOH for overnight at RT and stained with 0.3% Alizarin red S in 1.6% KOH for 2 days at RT. After staining, the tissue was placed in preservation solution containing glycerin, 70% ethanol, and benzyl alcohol (2:2:1 ratio) for 3 hours at room temperature, followed by a 10 min incubation at 37℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eProximity ligation assay (PLA)\u003c/h2\u003e \u003cp\u003eProximity ligation assay (PLA) was performed using the Duolink\u0026reg; In Situ Red Starter Kit Mouse/Rabbit (Sigma-Aldrich) according to the manufactures\u0026rsquo; introductions. Before PLA starting, the cells were fixed with 4% formaldehyde for 60 minutes and washed 2 times with 1xPBS. The Cells were permeabilized with 0.2% Triton X-100 in PBS for 10 minutes and then blocked with Duolink blocking solution in a heated humidity chamber for 60 minutes at 37℃. The cells were incubated overnight with anti-PARP-1 and anti-NEDD8 at 4℃, followed by treatment with Duolink PLA plus/minus probes for 60 minutes in a heated humidity chamber at 37℃. After washing 2 times with 1x wash buffer A, PLA signals were generated following reaction with ligation for 30 minutes and subsequent amplification for 90 minutes at 37℃. Slides were mounted using ProLong\u0026trade; Diamond Antifade Mountant with DAPI (Thermo Fisher) and photographed by confocal fluorescent microscopy (DE/LSM700, Cal Zeiss Microscopy)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAdministration of MLN4924, si-Cbl-b or Cblb C373 peptide to mice\u003c/h2\u003e \u003cp\u003eMLN4924 (10mg kg\u0026thinsp;\u0026minus;\u0026thinsp;1 per day), Cbl-b C373 (10 mg kg\u0026thinsp;\u0026minus;\u0026thinsp;1 per day), vehicle or scramble was intraperitoneally injected into mice. For siRNA injections, 50ug of siRNA was diluted in 10% glucose, mixed with in vivo-jetPEI (polyplus-transfection SA, Illkirch, France) also diluted in 10% glucose, and then incubated for 15 minutes at RT, followed by a brief vortex. si-Cbl-b (50\u0026micro;g per day) or scramble was injected into the mice\u0026rsquo;s tail vein after equilibration at RT. All of these reagents were administered at the same dose every 2 days for 6 days after vitamin D\u003csub\u003e3\u003c/sub\u003e injection until the animals were sacrificed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePARP-1 activity assay\u003c/h2\u003e \u003cp\u003eThe cells and tissues were lysed with 0.5% NP-40 solution to extract protein and incubated with anti-PARP-1overnight at 4\u0026deg;C. Then PARP-1 activity was measured using PARP univseral colorimetric assay kit, following by manufacture protocol. Briefly, histone coated plate was activated with 1xPARP buffer for 30 min at RT. After remove 1xPARP buffer, the well plate was added samples and PARP-HSA enzyme and incubated for 10 min at room temperature. Prepared 1X PARP Cocktail buffer containing PARP cocktail, activated DNA was distributed into the wells and incubated at RT for 1 hour and added Strep-HRP working solution after washing 2 times with 1X PBS\u0026thinsp;+\u0026thinsp;0.1% Triton X-100 (200 \u0026micro;L/well) followed by 2 washes with 1X PBS. The wells were incubated with pre-warmed TACS-Sapphire in the dark for 15 min at RT and added .2 M HCl to stop reaction and read the absorbance at 450nm by using an ELx808 absorbance reader (BioTek Instruments).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eLiquid chromatography-mass spectrometry analysis\u003c/h2\u003e \u003cp\u003eNEDD8-conjugated proteins were determined by peptide-precipitation and LC-MS/MS analysis. RVSMC cells were treated with high phosphate and MLN4924 (1\u0026micro;M) for 6 days. The extracted proteins were immunoprecipitated with anti-NEDD8 overnight at 4\u0026deg;C and next NEDD8-conjugated proteins were separated on gradient SDS-PAGE gels (8%-15%) and visualized by coomassie brilliant blue staining method. Specific bands in the Pi-treated lane which Pi lane were presented, but it was diminished by MLN4924 treatement were cut and LC-MS/MS was performed.\u003c/p\u003e \u003cp\u003eNano LC-MS/MS analysis was performed at the Korea Basic Science Institute (Biomedical Omics Research Center, Ochang, Korea). The gels were destained with a 50% acetonitrile containing 10 mmol NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e and were then rinsed a few times with distilled water to stop the destining reaction. The gels were inculated with 10 mmole dithiothreitol and 100 mmol ammonium bicarbonate at 56℃ to reduce the proteins, followed by 100 nmol iodoacetamide to alkylate the cysteines. The gels were then washed with three volumes of distilled water by vortexing and completely dried in a speed vacuum concentrator for 20 minutes. The dried gels were rehydrated with 12.5mg/mL trypsin in 50 mmol NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e solution and digestion was by 37\u0026deg;C incubation for overnight. Digested protein samples were speed-vaccum dried and the dissolved in 20\u0026micro;l of water containing 0.1% formic acid and LC-MS/MS were analyzed by using Hybrid FT-ETD Mass sepectrometer system (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eHistology and Immunohistochemistry\u003c/h2\u003e \u003cp\u003eTissue samples were fixed with 4% paraformaldehyde and embedded in paraffin. Cross-sections (8\u0026micro;m) were prepared and visualized by Alizarin red S staining and immunohistochemistry to evaluate vascular calcification and to analyze protein expression, respectively. Proximity ligation assay was performed after fixation, retrieval and permeabilization to detect binding between NEDD8 and PARP-1. The following primary antibodies were used for the immunostaining including PLA assay: NEDD8 (1:100, Cell signaling), PARP-1 (1:100, Santa cruz). The microscopic images were captured by an Axio Scan.Z1 scanner (Cal Zeiss Microscopy, GmbH, Jena, Germany) and a laser scanning microscope (DE/LSM700, Cal Zeiss Microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSPSS software (version 27.0, IBM Corp, Chicago, IL) was used for all statistical analyses. Each experiment was performed in triplicate, where appropriate. For two independent groups, two-tailed unpaired Student\u0026rsquo;s t-test or nonparametric Mann-Whitney U test was applied after checking for a normal distribution. In the case of more than two groups, one-way analysis of variance (ANOVA) or two-way ANOVA with post hoc tests was used depending on the number of main effects. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error and determined at the p-value than 0.05 as statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eMLN4924 inhibits Pi-induced calcium deposition in VSMCs.\u003c/b\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo investigate the potential involvement of NEDD8 in vascular calcification (VC), we initially assessed the effect of MLN4924 on Pi-induced calcium deposition in VSMCs. Treatment with MLN4924 resulted in a significant dose-dependent reduction in Pi-induced calcium deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Alizarin red S staining revealed that mineralization, characteristic of Pi-induced VC, was effectively inhibited by MLN4924 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). Pi treatment led to decrease in the expression of SM22a and SMA, genes associated with the smooth muscle differentiation, whereas upregulating the expression of osteogenic genes RUNX2 and ALP. However, these changes were counteracted by MLN4924 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). In addition, we assessed cell viability using the MTT assay and observed that MLN4924 did not affect cell viability in both Pi-treated VSMCs (Supplementary Fig.\u0026nbsp;1a).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further assessed whether Pi can induce the NEDDylation by western blot analysis. Formation of poly-NEDD8 was increased in high phosphate-treated VSMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Overexpression of NEDD8 potentiated Pi-induced calcium deposition in VSMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef and Supplementary Fig.\u0026nbsp;1b), while transfection of NEDD8-ΔGG, a mutant incapable of conjugation, inhibited Pi-induced calcium deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg and Supplementary Fig.\u0026nbsp;1c). These findings collectively suggest that the NEDD8 pathway plays a significant role in VC processes, while MLN4924 effectively prevents Pi-induced VC.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eNEDD8 is conjugated PARP-1 in VC\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo undercover which proteins undergo NEDD8-mediated modifications, we conducted immunoprecipitation-based proteomic analysis using an anti-NEDD8 antibody, followed by the affinity purification and liquid chromatography-mass spectrometry. In Pi-treated RVSMCs, NEDD8-conjugated proteins were isolated and the NEDDylation was confirmed through Coomassie blue staining. The gel bands that increased in the Pi lane and decreased in the lane with Pi and MLN4924 were cut out and subjected to LC-MS/MS to identify the NEDDylated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). We have identified NEDD8-conjugated candidate proteins, and used the PANTHER tool (PANTHERdb.org) to examine gene ontology (GO) analysis, which provided a comprehensive overview of the biological processes and molecular functions regulated by NEDDylation under the VC condition. Supplementary Fig.\u0026nbsp;2a illustrates the major cellular components, biological process, and molecular functions. According to the enriched GO annotation, the cellular components can be divided into two categories: cellular anatomical entity and protein-containing complex. Additionally, the identified NEDD8-conjugated candidate proteins were categorized into several major biological processes including cellular process, biological regulation and response to stimulus as well as diverse molecular function encompassing binding, catalytic activity, and transcription regulator activity. Further assessment of these identified proteins through bioinformatics analysis led to the selection of several proteins including HSP90, PARP-1, EEF2, NBR1 and DDB1 as NEDD8-conjugated candidate (Supplementary Fig.\u0026nbsp;2b). Among these NEDDylated candidate proteins, we decided to focuse on Poly (ADP-ribose) polymerase-1 (PARP-1) as a candidate protein in the context of vascular calcification. Although the roles of PARP-1 in vascular calcification were known, the significance of its posttranslational neddylation had not been extensively investigated. Therefore, we aimed to elucidate the role of neddylation of PARP-1 in the context of vascular calcification.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate the presence of NEDD8-conjugated PARP-1, we confirmed the interaction between PARP-1 and endogenous NEDD8. This interaction resulted in smear bands on the Western blots of Pi-treated VSMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Overexpression of NEDD8 led to the appearance of a higher molecular weight band for PARP-1, indicative of NEDDylation. This smear pattern was abolished when using NEDD8ΔGG, a conjugation-defective mutant due to a Gly-75/76 deletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). These findings established that poly-neddylation of PARP-1 with NEDD8 is intricately associated with vascular calcification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eVisualization of NEDD8-PARP-1 interaction\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eProximity ligation assay (PLA) is useful for the visualization of interacting proteins\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The PLA assay revealed that NEDD8 binds to PARP-1 in the nucleus. However, this binding was disrupted when MLN4924 was treated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Furthermore, MLN4924 treatment attenuated Pi-induced PARP-1 neddylation and Runx2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNEDDylation of PARP-1 Affects PARP-1 Activity and VC\u003c/h2\u003e \u003cp\u003eWe found that PARP-1 NEDDylation was increased in vascular calcification. Given that PARP-1 is associated with vascular calcification and its enzymatic activation involves poly (ADP)-ribosylation (PARylation)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and then PARP-1 catalyze the addition of poly (ADP-ribose) (PAR) to substrate proteins via cleavage of NAD\u003csup\u003e+\u0026thinsp;33\u003c/sup\u003e, it is curious whether PARP-1 NEDDylation can affect the enzymatic activation of PARP-1 in association with vascular calcification. Thus, we explored whether PARP-1 NEDDylation affects its enzymatic activity in VC. PAR polymer expression was detected by its ability to bind to boronate shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef. Pi treatment induced PAR polymer expression, evidenced by the smearing of boronate-conjugated precipitates (second lane). This smearing was diminished by MLN4924 treatment, which confirms its generation through NEDDylation. Cobb et al., reported that following DNA damage-induced VC, Runx2 undergoes PARylation dependent on PARP-1 activity\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef (bottom band), Runx2 was pulled down as a PARylated protein, which was blocked by MLN4924 treatment. Direct measurement of PARP-1 activity using a colorimetric assay (PARP universal colorimetric assay methods) revealed that Pi-induced PARP-1 activity was blunted by MLN4924 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). These findings underscore the dependency of PARP-1 activation on PARP-1 neddylation during the progression of VC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMLN4924 mitigates VD\u003csub\u003e3\u003c/sub\u003e-induced VC\u003c/h2\u003e \u003cp\u003eIn pursuit of evaluating the therapeutic prospects of NEDDylation reversal in vascular calcification in vivo, we introduced a mouse model of vitamin D\u003csub\u003e3\u003c/sub\u003e-induced calcification and administered intraperitoneal injections of MLN4924 (10mg/kg) every other day for six days. The experimental timeline is outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. For calcification assessment, we employed alizarin red S staining of the entire aorta. The results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb demonstrate that MLN4924 treatment effectively counteracted vitamin D\u003csub\u003e3\u003c/sub\u003e-induced VC when compared with the control group. Calcium levels were quantified in both arteries and serum of mice. Remarkably, administration of MLN4924 resulted in a substantial reduction in calcium deposition within the arteries of vitamin D\u003csub\u003e3\u003c/sub\u003e-treated mice, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec. However, no significant alteration was observed in serum calcium levels in vitamin D\u003csub\u003e3\u003c/sub\u003e-treated mice upon MLN4924 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), Importantly, NEDD8-conjugated PARP-1 induced by vitamin D\u003csub\u003e3\u003c/sub\u003e, along with its enzymatic activity indicated by poly(ADP-ribose) expression were effectively suppressed by MLN4924 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f). Additionally, MLN4924 administration attenuated PARP-1 activity in mice subjected to vitamin D\u003csub\u003e3\u003c/sub\u003e injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). As visualized by Alizarin Red S staining, vitamin D\u003csub\u003e3\u003c/sub\u003e-induced calcium deposition, appeared as reddish regions in the aortic media wall, was conspicuously absent by MLN4924 administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). Moreover, the interaction of PARP-1 with NEDD8 induced by vitamin D\u003csub\u003e3\u003c/sub\u003e was blunted by MLN4924 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). Collectively, these findings firmly establish the efficacy of MLN4924 in ameliorating vascular calcification through the inhibition of PARP-1 NEDDylation. Furthermore, these findings underscore MLN4924 as a promising candidate for potential therapeutic interventions against VC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCbl-b mediates NEDD8 binding to PARP-1 in VC\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGiven that many posttranslational modifications involve the final conjugation of small molecular moieties, facilitated by E1, E2, and E3 ligases\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, with target specificity often guided by E3 ligases, we directed our attention toward deciphering the specific E3 ligase responsible for PARP-1 neddylation. Mammalian cells house several hundred E3 ligases, with CBL-3, RBX1, and Fbxo11 serving as representative E3 ligases that oversee NEDDylation\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Consequently, we next embarked on identification of the specific E3 ligase involved in PARP-1 neddylation in the context of VC. For the identification of dysregulated E3 ligase, we employed our previous microarray analysis (GSE74755) results of rat VSMCs treated with Pi\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. (Supplementary Fig.\u0026nbsp;3a). Baculoviral IAP Repeat Containing 3 (Birc3), Casitas B\u0026ndash;lineage lymphoma protein b (Cbl-b), Ring-Box 1 (Rbx1), Mouse double minute 2 homolog (MDM2), Mouse double minute 4 homolog (MDM4), Ring Finger protein 7 (Rnf7), and Ring Finger Protein 111 (Rnf111) were dysregulated. Among candidates, we previously reported that MDM2 mediates ubiquitination of HDAC1 during the VC process\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, Thus we have excluded MDM2 as a E3 ligase for the neddylation process of PARP-1. Next, we quantified mRNA level changes of the dysregulated E3 ligase genes using quantitative real-time PCR in Pi-induced VSMCs. Notably, Birc3, Cbl-b, Rbx1, and Rnf111 were significantly upregulated (Supplementary Fig.\u0026nbsp;3b). Subsequently, using siRNA to knockdown these E3 ligases, we assessed the effect on PARP1- neddylation. Remarkably, only the deletion of Cbl-b effectively attenuated PARP-1 neddylation, whereas the loss of other E3 ligases like Birc3, Rbx1, and Rnf111, had negligible effects on PARP-1 neddylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;4a). PLA further showed the dissociation of NEDD8 from PARP-1 upon Cbl-b knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Immunoprecipitation analysis confirmed the interaction between Cbl-b and PARP-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and Supplementary Fig.\u0026nbsp;4b). Knocking-down of Cbl-b had no effect on the viability of A10 cells in Pi. (Supplementary Fig.\u0026nbsp;4c). Given the observation of PARP-1 neddylation influencing its activity in vascular calcification, we proceeded to explore whether Cbl-b played a role in regulating PARP-1 activity. Indeed, Cbl-b knockdown significantly dampened Pi-induced PARP-1 activity in VSMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRole of Cbl-b in VC and its mechanistic Insights\u003c/h2\u003e \u003cp\u003eBecause the role of Cbl-b in VC had not been previously reported, we proceeded to evaluate its effects by overexpression of HA-Cbl-b in VSMCs. Intriguingly, overexpression of Cbl-b significantly potentiated Pi-induced calcium deposition in a dose-dependent manner (Supplementary Fig.\u0026nbsp;4d). Furthermore, protein expression of Cbl-b was increased in a time-dependent manner bothe in VSMCs treated with Pi (Supplementary Fig.\u0026nbsp;4e) and vitamin D\u003csub\u003e3\u003c/sub\u003e-induced VC mouse models (Supplementary Fig.\u0026nbsp;4f). Notably, knockdown of Cbl-b significantly reduced the intensity of alizarin red S staining in Pi-induced VC (Supplementary Fig.\u0026nbsp;4g, h). Consistently, the augmentation of calcium deposition induced by Pi was blocked by si-Cbl-b transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Next, we evaluated the effects of Cbl-b knockdown by siRNA on VC in mouse models. We intravenously injected 50\u0026micro;g of si-Cbl-b, combined with a transfection reagent, twice during the VC induction periods following three consecutive days of vitamin D\u003csub\u003e3\u003c/sub\u003e injection into mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). The knockdown of Cbl-b was successfully achieved in various tissues, including the aorta, brain, kidney, liver and lung (Supplementary Fig.\u0026nbsp;5a). This led to a reduction in VD\u003csub\u003e3\u003c/sub\u003e-induced calcium accumulation in the aorta, but not in serum levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg and Supplementary Fig.\u0026nbsp;5b). Additionally, the loss of Cbl-b in VC resulted in blunted PARP-1 neddylation and Runx2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). As anticipated, the mineralization of vascular smooth muscle from the aorta in the Vitamin D\u003csub\u003e3\u003c/sub\u003e group was inhibited following the injection with si-Cbl-b (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). Collectively, these results underscore the pivotal role of the E3 ligase Cbl-b in mediating PARP-1 neddylation and its regulatory role in VC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCbl-b E3 ligase activity governs PARP-1 Neddylation in VC\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGiven that Cbl-b E3 ligase belongs to the RING finger family, which engages E2 enzymes to facilitate substrate ubiquitination\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, we examined whether E3 ligase activity of Cbl-b is essential for PARP-1 neddylation. It was previously reported that C373 and W400 of Cbl-b are critical for its E3 ligase activity\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. We generated these functionally inert forms of Cbl-b that lacks E3 ligase activity and next subjected them to immunoprecipitation with anti-PARP-1 antibody. As anticipated, high Pi exposure potentiated PARP-1 neddylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) and consequently, PARP-1 activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). These effects were pronounced upon transfection with Cbl-b WT, but not Cbl-b C373A or Cbl-b W400A. Interestingly, contrary to Cbl-b WT, both Cbl-b C373A and Cbl-b W400A failed to increase in mineralization and calcium deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d). Besides, Cbl-b WT and Cbl-b E3 ligase activity dead mutants (C373A and W400A) did not alter cell viability in Pi with A10 cells (Supplementary Fig.\u0026nbsp;6a)\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we interrogated whether blocking of E3 ligase activity via a peptide spanning crucial residue C373 and W400 could impede PARP-1 neddylation and, consequently, vascular calcification. Sequence analysis of different species revealed that the C373 and W400 site of Cbl-b are highly conserved (Supplementary Fig.\u0026nbsp;6b). To visualize the localization of these peptides, fluorescein isothiocyanate-conjugated nuclear localization signal (NLS) sequence \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e were added. The synthetic peptide C373, but not W400, effectively entered the nucleus in VSMCs (Supplementary Fig.\u0026nbsp;6c). Cbl-b C373 peptide did not affect cell viability in Pi with A10 cells (Supplementary Fig.\u0026nbsp;6d). In the subsequent experiments, we utilized C373 peptide. NEDD8 conjugated with PARP-1 is cleaved by treatment of Cbl-b C373 peptide in VSMCs under Pi conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Moreover, both PARP-1 neddylation and its activity were also blunted by C373 in Pi-induced VC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, h). Further corroborating these findings, the Cbl-b C373 peptide significantly attenuated the propensity for calcium deposition and alizarin red S staining in Pi-treated VSMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei, k). Taken together, these observations underscore the pivotal role of Cbl-b E3 ligase activity, particularly at residue C373, in promoting PARP-1 neddylation within the context of vascular calcification. Additionally, the C373-spanning peptide emerges as a potential candidate for thwarting vascular calcification, offering novel therapeutic avenues for its prevention or treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAlleviation of Vitamin D3-induced VC through Cbl-b C373 peptide\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo further validate the therapeutic potential of the Cbl-b C373-blocking peptide, we examined its effects in VD3-induced VC models. Mice were intraperitoneally administered with Cbl-b C373 peptide (1mg/kg/day) every two day, following a single injection of vitamin D\u003csub\u003e3\u003c/sub\u003e in mice. After 9-day of experimental period, mice were sacrificed and assessed for VC effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The uptake of the C373 peptide into the mouse aorta was observed through FITC fluorescence, as shown in Supplementary Fig.\u0026nbsp;7. Treatment with Cbl-b C373 peptide effectively attenuated the formation of calcified nodules in aorta of vitamin D\u003csub\u003e3\u003c/sub\u003e-induced VC, compared to the scramble group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Calcium content analysis demonstrated that increased calcium deposition induced by vitamin D3 was significantly counteracted by Cbl-b C373 peptide treatment in aorta, but not in serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d). Remarkably, the enhancement of PARP-1 neddylation and PARP-1 enzyme activities such as PAR polymer, instigated by vitamin D\u003csub\u003e3\u003c/sub\u003e, were effectively mitigated by Cbl-b C373-blocking peptide within the aorta, compared to the scramble control in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, f).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEvidently, the distinct calcium deposition characteristic of vitamin D\u003csub\u003e3\u003c/sub\u003e presence was entirely abrogated following the administration of the Cbl-b C373 peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, left panel). Additionally, the typical interaction between PARP-1 and NEDD8 elicited by vitamin D\u003csub\u003e3\u003c/sub\u003e was efficiently hindered by the Cbl-b C373 peptide, as illustrated by Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg (right panel). Taken together, these findings underscore the efficacy of the Cbl-b C373-blocking peptide in vitamin D\u003csub\u003e3\u003c/sub\u003e-induced vascular calcification through the disruption of PARP-1 neddylation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003ePARP-1 neddylation is counteracted by NEDP-1 in VC\u003c/h2\u003e \u003cp\u003eThe dynamic nature of NEDDylation is well-established, encompassing a reversible process facilitated by NEDP-1, an NEDD8-specific protease 1\u003csup\u003e39\u003c/sup\u003e. In light of this, we embarked on investigating whether NEDP-1-mediated de-NEDDylation could effectively reverse PARP-1 neddylation in VC. Thus, we designed a study centered on the NEDP-1. As anticipated, the introduction of ectopically expressed Cbl-b augmented the conjugation of NEDD8 with PARP-1. Nevertheless, this enhancement was nullified by the overexpression of NEDP-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). For deeper insights, we scutinized the effect of NEDP-1 on PARP-1 neddylation and Poly (ADP-ribose)ylation in Pi-induced VSMCs. Remarkably, the overexpression of NEDP-1 resulted in the suppression of Pi-induced PARP-1 neddylation and poly(ADP)-ribosylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, c). Furthermore, the increase in the activity of PARP-1 evident in the Pi-induced VC model was effectively down-regulated by the overexpression of NEDP-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUntil now, the role of NEDP-1 in vascular calcification has not been reported. Therefore, we sought to investigate this. The expression of NEDP-1 expression gradually decreased in vascular calcification both in vitro and in vivo models (Supplementary Fig.\u0026nbsp;8a, b). NEDP-1 overexpression did not affect cell viability in VSMCs (Supplementary Fig.\u0026nbsp;8c). Notably, the transient overexpression of NEDP-1 in VSMCs via transfection significantly exerted a dose-dependent migratory effect on Pi-induced calcium deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee and Supplementary Fig.\u0026nbsp;8d) and mineralization in VSMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef, g). Conversely, the depletion of NEDP-1 significantly amplified the accumulation of calcium stimulated by Pi in a dose dependent manner (Supplementary Fig.\u0026nbsp;8e, f).\u003c/p\u003e \u003cp\u003eGiven the function of NEDP-1 in dissociating NEDD8 from target proteins, we postulated that NEDP-1 might exert an antagonistic effect against the functional interplay of neddylated PARP-1 and E3 ligase activity of Cbl-b in the milieu of VC. Armed with this supposition, we proceeded to investigate whether functionality of either PARP-1 or Cbl-b could be effectively counteracted by NEDP-1. Evidently, the overexpression of either PARP-1 or Cbl-b led to an elevation of calcium content and mineralization in the presence of Pi in VSMCs. Notably, this exacerbation was significantly dampened by the simultaneous overexpression of NEDP-1 in a dose dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh-k). Collectively, these findings underscore the role of NEDP-1 as a pivotal conteractive element against PARP-1 neddylation in VC.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eHere, we describe how the NEDD8 modifier is increased in response to vascular calcification stimuli and conjugated with PARP-1, promoting vascular calcification. PARP-1 neddylation is mediated by the E3 ligase Cbl-b and is deneddylated by NEDP-1. Consequently, inhibiting PARP-1 neddylation could be potential therapeutic stgrategies for alleviating vascular calcification (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePost-translational modifications (PTMs) play crucial roles in various biological processes, including vascular calcification. Runx2, a major transcription factor, can undergo diverse PTM by such as phosphorylation, acetylation and ubiquitination and also PARylation in genotoxic stress during vascular calcification We also have previously reported that E3 ligase MDM2 mediated HDAC1 ubiquitination induces vascular calcification\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Both MSX1 and MSX2 act as upstream transcription regulator of MDM2\u003csup\u003e31\u003c/sup\u003e. Numerous studies have reported that PARP-1 undergoes diverse posttranslational modifications such sumoylation, ubiquitination and acetylation\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In this study, we define neddylation as a novel modification of PARP-1 and a critical regulatory mechanism for vascular calcification. To identife genuin neddylation substrates, some criteria is reqiured for the charaterization of NEDD8 substrates\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Herein, we provides a serieses of data to prove that PARP-1 is a substrate for NEDD8: (i) NEDD8 is covalently attached to PARP-1; (ii) endogenou detection of PARP-1 neddylation; (iii) PARP-1 neddylation depends on an activatin enzyme (E1) and MLN4924 inhibits PARP-1 neddylation; (iv) we identified Cbl-b as a specific ligase for PARP-1 neddylation; (v) we identified NEDP-1 as deneddylase for PARP-1 in vivo; (vi) formation by NEDD8 of a poly-neddylation chain on PARP-1; (vii) PARP-1 neddylation regulates PARylation as downstream. Collectively, based on these findings, we conclude that during vascular calcification, PARP-1 ia a genuine subjected to neddylation.\u003c/p\u003e \u003cp\u003eTo date, several researchers have studied the correlation between PARP-1 and vascular calcification. Wang et al., reported that PARP-1 could promote the osteogenic transition of VSMCs via the JAK2/STAT3/miR-204/Runx2 pathway\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Other studies demonstrated that oxidative DNA damage is a key driver of vascular calcification and that PARP-1 is activated at the site of such calcification \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Although, PARP-1 is activated by various stimuli and conditions associated with vascular calcification. However, how to regulate PARP-1 is poorly understood in vascular calcification. Herein we provide evidence that PARP-1 is conjugated with NEDD8 which in turn activates PARP-1 activity. This activation subsequently induce PARylation and promotes vascular calcification. Nonetheless, it still remains unclear which lysine residues on PARP-1 NEDD8 binds to. This necessitates further investigation into this aspect of PARP-1 neddylation activation.\u003c/p\u003e \u003cp\u003eCbl-b is a member of the Cbl family proteins, which consists of three homologues known as c-Cbl, Cbl-b, Cbl-3. Cbl-b is predominantly expressed in T-cells and marcrophages plaques. Studies have reported that Cbl-b regulates both innate and adaptive immune cell responses through immune T cell activation\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In atherosclerosis, genetic deficency of Cbl-b aggravated atherosclerosis in ApoE-/- mice by recruiting CD8\u003csup\u003e+\u003c/sup\u003e T-cells to the plaque\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In this study, we found that Cbl-b is upregulated and acts as a specific key mediator of PARP-1 neddylation in vascular calcifcation. This process depends on the E3 ligase catalytic activity of Cbl-b. Catalytically inhibitors such as overexpressing the plasmid construct of inactive mutant Cbl-b C373A and blocking peptide targeting residue 373 (C373), inhibit PARP-1 neddylation mediated by Cbl-b. Furthermore, inhibition of E3 ligase catalytic activity of Cbl-b hampers calcim accumulation and minalization during vascular calcification progession. Therefore, we propose that Cbl-b C373 blocking peptides may be therapeutic agents for vascular calcification. In addition to this, other upregulated E3 ligases such as Birc3, Rbx1, and Rnf111 do not affect PARP-1 neddylation but may possibly serve as regulators in vascular calcficiation\u003c/p\u003e \u003cp\u003eIn conclusion, this study suggests that Cbl-b acts as a critical mediator of NEDD8-conjugated PARP-1 neddylation under vascular calcification. PARP-1 neddylation plays a modulatory role in PARP-1 neddylation-mediated PARylation by regulating catalytic activity of PARP-1. Therefore, treatment with inhibitors of PARP-1 neddylation such as MLN4924 and Cbl-b C373 blocking peptides, can block PARylation, thereby ameliorating vascular calcification. Our findings provide insights into the prevention and treatment of a variety of cardiovascular disease related to vascular calcification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2022R1A4A2000767, 2022R1I1A1A01053681, RS-2023-00208097, and RS-2023-002374089).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuk-Hwa Kwon: Conceptualization; Data curation; Formal analysis; Validation; Investigation; Mothology; Funding acquisition; Writing-original draft. Sera Shin: Data curation; Validation; Visualization. Yoon Seok Nam: Conceptualization; Formal analysis. Nakwon Choe: Data curation. Methodology. Younwoon Lim: Data curation. Anna Jeong: Data curation. Methodology. Yun-Gyeong Lee: Data curation. Young-Kook Kim: Formal analysis; Supervision. Hyun Kook: Conceptualization; Validation, Funding acquisition, Writing-original draft.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary information is available at available Experimental \u0026amp; Molecular Medicine’s website (http://www.nature.com/emm/)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLeopold JA. Vascular calcification: Mechanisms of vascular smooth muscle cell calcification. \u003cem\u003eTrends Cardiovasc Med.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 267-274 (2015).\u003c/li\u003e\n\u003cli\u003eNicoll R, Henein M. 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The immunology of atherosclerosis. \u003cem\u003eNat Rev Nephrol.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 368-380 (2017).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"NEDD8, PARP-1, Cbl-b, NEDP-1, Vascular calcification","lastPublishedDoi":"10.21203/rs.3.rs-3939434/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3939434/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eAims:\u003c/strong\u003e Vascular calcification (VC) refers to the accumulation of mineral deposits on the walls of arteries and veins, and it is closely associated with increased mortality in cardiovascular disease, particularly among high-risk patients with diabetes and chronic kidney diseases (CKD). Neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) is an ubiquitin-like protein that plays a pivotal role in various cellular functions, primarily through its conjugation to target proteins and subsequent relay of biological signals. However, the role of NEDDylation in VC has not been investigated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods and Results:\u003c/strong\u003e In our study, we observed that MLN4924, an inhibitor of the NEDD8-activating E1 enzyme, effectively impedes progress of VC. By LC-MS/MS analysis, we identified that poly(ADP-ribose) polymerase 1 (PARP-1) is subjected to NEDD8 conjugation, leading to an increase in PARP-1 activity during VC. Subsequently, we uncovered that the PARP-1 NEDDylation is mediated by the E3 ligase Cbl proto-oncogene B (Cbl-b) and is reversed by the NEDD8-specific protease 1 (NEDP-1) during VC. Furthermore, Cbl-b C373 peptide effectively mitigates the inactive form of E3 ligase activity of Cbl-b, ultimately preventing VC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e These findings provide compelling evidence that the NEDD8-dependent activation of PARP-1 represents a novel mechanism underlying vascular calcification and suggests a promising new therapeutic target for VC.\u003c/p\u003e","manuscriptTitle":"Cbl-b E3 ligase-mediated neddylation and activation of PARP-1 induces vascular calcification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-11 19:17:18","doi":"10.21203/rs.3.rs-3939434/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-03-26T07:23:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-03-26T05:58:08+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-03-25T06:29:05+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-03-09T08:35:57+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-03-07T23:20:01+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-03-07T13:17:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-21T00:57:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2024-02-20T11:04:40+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2024-02-13T01:19:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-08T09:19:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"69098aa2-3d2c-4729-a323-b305aa1e8343","owner":[],"postedDate":"March 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28878589,"name":"Biological sciences/Molecular biology/Post-translational modifications/Neddylation"},{"id":28878590,"name":"Health sciences/Diseases/Cardiovascular diseases/Vascular diseases/Calcification"}],"tags":[],"updatedAt":"2024-10-01T07:11:18+00:00","versionOfRecord":{"articleIdentity":"rs-3939434","link":"https://doi.org/10.1038/s12276-024-01322-y","journal":{"identity":"experimental-and-molecular-medicine","isVorOnly":false,"title":"Experimental \u0026 Molecular Medicine"},"publishedOn":"2024-10-01 04:00:00","publishedOnDateReadable":"October 1st, 2024"},"versionCreatedAt":"2024-03-11 19:17:18","video":"","vorDoi":"10.1038/s12276-024-01322-y","vorDoiUrl":"https://doi.org/10.1038/s12276-024-01322-y","workflowStages":[]},"version":"v1","identity":"rs-3939434","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3939434","identity":"rs-3939434","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}