Targeting deubiquitinase OTUB1 protects vascular smooth muscle cells in atherosclerosis by modulating PDGFRβ

Abstract

Atherosclerosis is a chronic artery disease that causes various types of cardiovascular dysfunction. Vascular smooth muscle cells (VSMCs), the main components of atherosclerotic plaque, switch from contractile to synthetic phenotypes during atherogenesis. Ubiquitylation is crucial in regulating VSMC phenotypes in atherosclerosis, and it can be reversely regulated by deubiquitinases. However, the specific effects of deubiquitinases on atherosclerosis have not been thoroughly elucidated. In this study, RNAi screening in human aortic smooth muscle cells was performed to explore the effects of OTU family deubiquitinases, which revealed that silencing OTUB1 inhibited PDGF-BB-stimulated VSMC phenotype switch. Further in vivo studies using Apoe^−/− mice revealed that knockdown of OTUB1 in VSMCs alleviated atherosclerosis plaque burden in the advanced stage and led to a stable plaque phenotype. Moreover, VSMC proliferation and migration upon PDGF-BB stimulation could be inhibited by silencing OTUB1 in vitro. Unbiased RNA-sequencing data indicated that knocking down OTUB1 influenced VSMC differentiation, adhesion, and proliferation. Mass spectrometry of ubiquitinated protein confirmed that proteins related to cell growth and migration were differentially ubiquitylated. Mechanistically, we found that OTUB1 recognized the K707 residue ubiquitylation of PDGFRβ with its catalytic triad, thereby reducing the K48-linked ubiquitylation of PDGFRβ. Inhibiting OTUB1 in VSMCs could promote PDGFRβ degradation via the ubiquitin–proteasome pathway, so it was beneficial in preventing VSMCs’ phenotype switch. These findings revealed that knocking down OTUB1 ameliorated VSMCs’ phenotype switch and atherosclerosis progression, indicating that OTUB1 could be a valuable translational therapeutic target in the future.

Keywords

atherosclerosis / vascular smooth muscle cell / ubiquitylation / deubiquitinase / OTUB1 / PDGFRβ

1 Introduction

Atherosclerosis, illustrated by lipid accumulation and plaque formation in arteries, is the major pathological process of various cardiovascular diseases (CVDs) and cerebrovascular diseases [1–3]. Atherosclerotic plaque comprises vascular smooth muscle cells (VSMCs), which play a prominent role in atherogenesis and the evolution of early- and advanced-stage atherosclerosis [4–6]. In healthy arteries, VSMCs are necessary for vessels’ normal functions, including blood pressure regulation, flow distribution, and vessel tone [4]. In atherosclerotic arteries, VSMCs undergo a phenotype switch from contractile to dedifferentiated synthetic phenotypes, which is considered one of the main pathological processes of atherosclerosis. Phenotypically switched VSMCs secrete a variety of extracellular matrix (ECM) proteins and pro-inflammatory mediators; they are prone to proliferate and migrate, promoting atherosclerosis progression [4,7]. Therefore, therapeutically inhibiting the phenotype switch of VSMCs may provide an opportunity to mitigate atherosclerosis.

Ubiquitylation is a crucial post-translational modification (PTM) that can lead to ubiquitin–proteasome-dependent protein degradation. It was found to be involved in the regulation of VSMCs’ phenotype switch during atherosclerosis development [8–10]. For instance, Grootaert et al. discovered that ubiquitin ligase CHIP helps maintain SIRT6 protein stability and protects VSMCs from switching to a senescence-associated secretive phenotype [11]. Ye et al. discovered that E3-ubiquitin ligase β-TRCP modulates REST ubiquitylation, so it is necessary for the phenotype switch process of VSMCs [12]. Deubiquitinases can cleave polyubiquitin chains or remove ubiquitin from substrates, thereby protecting proteins from ubiquitin–proteasome-dependent degradation, which is mostly realized through K48-linked ubiquitylation [13,14]. Researchers have now identified approximately 100 human deubiquitinases, which are divided into seven superfamilies [13,15,16]. The ovarian tumor protease (OTU) superfamily deubiquitinases have emerged as critical mediators in cell proliferation, inflammatory responses, and DNA damage response [17–20]. Interestingly, a member of OTU deubiquitinase, OTUD7B (Cezanne), has been proven to regulate VSMCs’ phenotype switch by deubiquitylating KLF4 [21]. Whether other OTU deubiquitinases modulate VSMCs’ phenotype switch remains to be investigated.

As a core member of the Otubain subfamily in the OTU superfamily, OTUB1 is known to play critical roles in cell proliferation, migration, and inflammation [16,17]. OTUB1 enhances breast cancer proliferation and epirubicin resistance by targeting FOXM1 [22]; it also maintains the stability of PD-L1, which can mediate immune invasion and promote tumor growth [23]. OTUB1 was found to prevent liver inflammation by stabilizing c-IAP1 [18] and inhibiting RIG-I-dependent immune signaling [24], but it can also activate NF-κB signaling in dendritic cells [17]. Considering that VSMCs’ phenotype switch is closely related to excessive cell proliferation and response to inflammatory stimuli, we hypothesized that OTUB1 may modulate VSMC pathophysiology during atherosclerosis.

Here, to study the functions of OTUB1, we constructed an atherosclerosis mouse model to analyze plaque formation and the components of plaques. Human aortic smooth muscle cells (HASMCs) were used to investigate the phenotype changes and the molecular mechanisms after depleting OTUB1. We found that knocking down Otub1 ameliorated plaque progression and helped stabilize atherosclerotic plaque, whereas the phenotype switches of HASMCs from contractile to synthetic phenotypes were altered after silencing OTUB1. Mechanistically, OTUB1 increased the stability of PDGFRβ by removing K48-linked ubiquitylation, thereby inhibiting VSMCs’ phenotype switch. Therefore, these data revealed that OTUB1 is a promising novel potential target for atherosclerosis and CVD treatment.

2 Materials and methods

2.1 Animal experiments

All animal experiments were approved by the ethics committee at Zhongshan Hospital, Fudan University, and they were performed according to the local relevant guidelines. The experiments conformed to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Animal experiments were performed in the animal laboratory of Zhongshan Hospital, Fudan University (Shanghai, China). The mice were maintained under specific pathogen-free conditions on a 12:12 h light/dark cycle, with controlled temperature (22 °C ± 2 °C) and humidity (55% ± 5%). The mice were given free access to water and food.

C57BL/6 background male apolipoprotein E-deficient (Apoe^−/−) mice (23.3 ± 2.4 g) were purchased from GemPharmatech (China) at 6 weeks of age. The mice were randomly allocated to early- or late-stage atherosclerosis groups and fed a high-fat diet (HFD) with 40% kcal fat, 1.25% cholesterol, and 0.5% cholic acid (D12109C, Research Diets, USA) for 6 (early-stage atherosclerosis group) or 16 weeks (late-stage atherosclerosis group). To selectively knock down Otub1 in VSMCs, we randomly assigned the mice to the normal control (NC) and Otub1 knockdown groups, receiving 1×10¹² viral titer/mL shOtub1 AAV9 (Hanbio Technology, China) with Tagln-promoter or control AAV9 (empty vector with Tagln-promoter) in a total volume of 100 μL/mice by tail vein injection. The target site sequences for interfering Otub1 were as follows: Forward: GCUUCACUGAAUUCACAAUTT, Reverse: AUUGUGAAUUCAGUGAAGCTT. The mice were then fed an HFD for 6 weeks before sacrifice.

At the endpoint of the experiment, all mice were euthanized in accordance with the local relevant policy in Zhongshan Hospital, Fudan University. Euthanasia was performed using deep anesthesia by inhaling an overdose of isoflurane (5%) (RWD, China). The aortas and hearts were then isolated after perfusion with PBS.

2.2 Cell culture

Primary human aortic smooth muscle cells (HASMCs) were purchased from ScienCell (Cat. #6110, USA) and maintained in smooth muscle cell medium (SMCM, Cat. #1101, ScienCell, USA) supplemented with 2% FBS, 1% SMCGS, and 1% penicillin/streptomycin. Considering that the phenotype markers (OPN and ACTA2) and OTUB1 expression were relatively stable from the 1st to 7th passages, experiments were performed with HASMCs from the 4th to 7th passages (Fig. S1). HEK293T and MOVAS cells were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology (Cat. #ZQ0033, China). HEK293T and MOVAS cells were maintained in Dulbecco’s modified eagle medium (Cat. #11995-065, Thermo Fisher, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. All the cells were maintained at 37 °C in a humidified incubator with 5% CO₂. In separate experiments, cells were treated with 50 μg/mL cycloheximide (CHX, Cat. #M4879, AbMole, USA), 10 μM Z-Leu-Leu-Leu-al (MG132, Cat. #HY-13259, MedChemExpress, USA), or 10 ng/mL recombinant human PDGF-BB following 12 h of serum-free starvation (Cat. #AD-100-14B, PeproTech, USA) for the indicated time.

2.3 Plasmid construction, siRNA synthesis, and transfection

Human PDGFRβ and OTUB1 cDNA clones were purchased from Hanbio, China. The overexpression plasmids and their mutants were cloned into pcDNA3.1 or lentivirus vectors. All constructs were confirmed by DNA sequencing. The duplex siRNA targeting PDGFRβ and OTU family deubiquitinases were purchased from GenePharma (China), and the sequences are provided in Table S1. HASMCs were seeded at 1×10⁵ cells/well in 12-well-plates or 1×10⁶ cells in 10 cm wells, and cell transfection was conducted with Lipofectamine 3000 Reagent (Cat. #L3000015, ThermoFisher, USA) according to the provider’s protocols. The culture medium was replaced 6 h after transfection, and cells were processed to subsequent experiments after 24 h unless overwise mentioned.

2.4 Cell migration study

In vitro cell migration was conducted with Boyden chamber transwell migration assay and scratch assay. For the Boyden chamber migration assay, cultured cells were transfected with siRNA or plasmids for 24 h, resuspended, and counted. Subsequently, 5×10⁴ cells were seeded into the upper section of a 24-transwell plate. Cells were allowed to migrate to the filter’s lower side media for 24 h. The migrated cells in the bottom chamber were stained with crystal violet dye (Cat. #C0121, Beyotime, China) and analyzed by microscopy (Olympus, Japan). For the scratch assay, we seeded approximately 1×10⁵ cells/well HASMCs into six-well plates, cultured them until the cells reached 70% confluence, and transfected them with the indicated plasmids or siRNA. After 48 h, the culture medium was replaced with SMCM supplemented with 0.2% FBS. After 12 h, the wound was made by scratching the cell monolayer with a pipette tip. The scratched cells were analyzed under microscopy (Olympus, Japan) at 0 and 24 h after scratching.

2.5 Protein extraction and Western blot

Gross mouse aortas or HASMCs were lysed in ice-cold RIPA lysis buffer (Beyotime, China) supplemented with a proteasome inhibitor cocktail (Cat. #P1005, Beyotime, China) for 20 min. The protein concentration was measured with a BCA Protein Assay Kit (Cat. #P0007, Beyotime, China). Extracted proteins were separated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA), blocked in 5% bovine serum albumin (BSA) in 0.1% Tween 20/TBS for 1 h, and incubated overnight with the following primary antibodies at the indicated dilutions: anti-GAPDH (1:2000; Cat. #5174, Cell Signaling Technology, USA), anti-OTUB1 (1:1000; Cat. #175200, Abcam, USA), anti-PDGFRβ (1:1000; Cat. #3169, Cell Signaling Technology, USA), anti-c-CBL (1:1000; Cat. #25818-1-AP, ProteinTech, China), anti-K48-linked-ubiquitin (1:1000; Cat. #ab140601, Abcam, USA), anti-Ubiquitin (1:1000; Cat. #PT-5798, PTM BIO, China), anti-K63-linked-ubiquitin (1:1000; Cat. #ab179434, Abcam, USA), anti-SLC3A2 (1:2000; Cat. #15193-1-AP, ProteinTech, China), anti-SLC7A5 (1:500; #13752-1-AP, ProteinTech, China), anti-ACTA2 (1:2000; Cat. #19245, Cell Signaling Technology, USA), anti-OPN (1:1000; Cat. #22952-1-AP, ProteinTech, China), anti-TAGLN (1:1000; Cat. #10493-1-AP, ProteinTech, China), and horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, USA). Signals were detected with the Luminata^TM Forte Western HRP Substrate (#WBLUF0500, Millipore, USA).

2.6 Oil-Red O staining

Frozen aortic root tissues in OCT cryostat embedding compound (Tissue-Tek, USA) were sliced into 10 pieces of 8 μm cryosections from the origin of the aortas. For morphometric analyses, slices of the same level from each tissue were assessed. In this work, 0.5% Oil-Red O (ORO; Cat. #A600395, Sangon, China) staining was performed to measure the extent of atherosclerotic plaque. The lesion area fraction was calculated by dividing the ORO-stained lesion area by the total area of the aortic wall and expressed as a percentage. For gross observation of the ORO-positive area in the aortas, samples were processed to ORO staining after being fixed with 4% paraformaldehyde, and photos were obtained by a camera (Canon, Japan). ImageJ software was used to analyze the ORO-positive area.

2.7 H&E and Masson’s trichrome staining

Mouse aortic root tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced into 10 pieces of 3 μm paraformaldehyde-fixed sections from the origin of the aortas. For morphometric analyses, slices of the same level from each tissue were assessed. Hematoxylin/eosin (H&E) staining was performed with a modified H&E stain kit (Cat. #G1121, Solarbio, China) to perform morphological analysis. The necrotic core area was divided by the total lesion area and shown as a percentage. To detect collagen components, we conducted Masson’s trichrome staining using a modified Masson’s trichrome stain kit (Cat. #G1346, Solarbio, China) following the manufacturer’s instructions. Fibrous cap thicknesses were assessed by measuring cap thickness at four sites at plaque shoulders (2 sites at each side) and two sites at the midpoint for each lesion from Masson’s trichrome staining. The average cap thickness of atherosclerotic lesions on three aortic valves was analyzed to obtain an average cap thickness per sample. Images were analyzed with ImageJ software. For the calculation of collagen composition, the collagen area was divided by the total lesion area and expressed as a percentage.

2.8 Immunofluorescence staining

Cells were washed twice with freshly prepared PBS and then fixed with freshly prepared 4% paraformaldehyde for 20 min. For 5-ethynyl-2’-deoxyuridine (EdU) staining, cells were incubated in 50 μM EdU (Cat. #C00003, RiboBio, China) for 2 h before harvest. Mouse aortic root tissues were processed and sliced into 8 μm cryosections. The cells were then fixed. Tissue sections were permeabilized with 0.5% Triton X-100 for 10 min, blocked with 5% BSA (Cat. #A1933, Sigma, USA) in PBS for 1 h, and incubated with primary antibodies (ACTA2, Cat. #19245, Cell Signaling Technology, USA and Cat. #CL488-14395, ProteinTech, China; F4/80, Cat. #sc-377009, Santa Cruz, USA; Ki-67, Cat. #ab15580, Abcam, USA; OTUB1, Cat. #orb158068, Biorbyt, China; PDGFRβ, Cat. #ab69506, Abcam, USA; c-CBL, Cat. #ab32027, Abcam, USA) at 4 °C overnight. The cells were then washed three times with ice-cold PBS and incubated with fluorescence-labeled secondary antibodies (Alexa Fluor 488, Cat. #A-11034, ThermoFisher, USA; Alexa Fluor 546, Cat. #A-11003 and #A-11035, ThermoFisher, USA; Alexa Fluor 647, Cat. #A-21235 and Cat. #A-21244, ThermoFisher, USA) for 1 h at room temperature. To detect the location of c-CBL in HASMCs, VSMC marker ACTA2 (green) was detected using a CoraLite® Plus 488-conjugated fluorescent antibody (Cat. #CL488-14395, ProteinTech, China).

The atherosclerotic plaque vulnerability index was calculated as follows: (macrophage staining % + lipid staining %)/(SMC staining % + collagen staining %) [25].

For cell proliferation assays, EdU staining was performed following the instructions of the manufacturer (Cell-Light EdU Apollo567 In Vitro Kit, Cat. #C10310-1, RiboBio, China). For cell apoptosis analyses, TUNEL assay was conducted using an In Situ Cell Death TMR Kit (Cat. #12156792910, Roche, USA) following the manufacturer’s guide. After staining nuclei with DAPI for 10 min, samples were observed under a fluorescence microscope (Leica, Germany) or scanned under a confocal laser scanning microscope (Olympus, Japan).

2.9 RNA isolation, reverse transcription, and qRT-PCR

Total RNA was isolated and purified from cell or tissue samples using TRIzol reagent (Cat. #15596018, Invitrogen, USA) following the manufacturer’s protocol. The concentration and purity of each isolated RNA sample were confirmed with NanoDrop 3300 (Thermo Fisher Scientific, USA). The PrimeScript^TM RT reagent Kit with gDNA eraser (Cat. #RR047, TAKARA, China) was used to synthesize cDNA from 1 μg of isolated RNA. The cDNA was then processed for qRT-PCR using SYBR Premix Ex Taq^TM (Cat. #RR820, TAKARA, China) on a CFX96 qPCR system (Bio-rad, USA) following the manufacturer’s instructions. Primer sequences are available in Table S2.

2.10 Co-immunoprecipitation (co-IP)

HASMCs or 293T cells were lysed in ice-cold 1× cell lysis buffer (Cat. #abs9116, Absin, China) containing PMSF proteasome inhibitor (Cat. #36978, ThermoFisher, USA) for 30 min. The cell lysates were centrifuged and incubated on a sample mixer with indicated primary antibodies (OTUB1, Cat. #175200, Abcam, USA; PDGFRβ, Cat. #3169, Cell Signaling Technology, USA; c-CBL, Cat. #ab32027, Abcam, USA) at 4 °C overnight. Following the manufacturer’s instructions, the samples were immunoprecipitated with an IP/co-IP kit (Cat. #abs955, Absin, China). Rabbit IgG antibody (Cat. #172730, Abcam, USA) was used as control.

2.11 mRNA library construction and RNA sequencing

For the mRNA sequencing of HASMCs, the extracted RNA integrity was assessed by a Bioanalyzer 2100 (Agilent, USA) with RIN number > 7.0 and confirmed by denaturing agarose gel electrophoresis. Poly(A) RNA was purified from 1 μg of total RNA using Dynabeads Oligo (dT) 25-61005 (Thermo Fisher, USA) twice before it was fragmented into small pieces using Magnesium RNA Fragmentation Module (Cat. #e6150, NEB, USA). The RNA fragments were reverse-transcribed using SuperScript II Reverse Transcriptase (Invitrogen, Cat. #1896649, USA) and processed to synthesize U-labeled second-stranded DNAs with Escherichia coli DNA polymerase I (Cat. #m0209, NEB, USA), RNase H (Cat. #m0297, NEB, USA), and dUTP solution (Cat. #R0133, Thermo Fisher, USA). An A-base was added to the blunt ends of each strand to prepare them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads (Cat. #e6150, Beckman, USA). After the heat-labile UDG enzyme (Cat. #m0280, NEB, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified by PCR. The average insert size for the final cDNA library was 300 ± 50 bp. Finally, the 2 × 150 bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 (LC-Bio Technology Co., Ltd., China) was performed following the vendor’s recommended protocol. The RNA sequencing data of HASMCs were deposited to NCBI Gene Expression Omnibus (GEO) database with the accession number GSE232121.

2.12 Sample preparation for mass spectrometry (MS) analysis

For the proteome analysis of ubiquitylated proteins, HASMCs were transfected with siRNAs (the three replicates were separately treated) and harvested after 48 h. Cells were lysed in ice-cold urea lysis buffer containing 8 mol/L urea, 100 mmol/L Tris-HCl (pH 8.5), 150 mmol/L NaCl, and 50 μmol/L PR-619 and then supplemented with protease inhibitors. After centrifugation, the protein concentration was determined using a BCA protein assay kit (Cat. #23227, ThermoFisher, USA). The lysed protein was digested with trypsin following the protocols by Wisniewski et al. [26]. The peptide fractions were reconstituted in IP buffer (100 mmol/L NaCl, 1 mmol/L EDTA, 50 mmol/L Tris-HCl, and 0.5% NP-40, pH 8.0), centrifuged, and incubated with pre-washed K-GG ubiquitin remnant motif antibody bead conjugate (Cat. #PTM-1104, PTM Biolabs, China). The samples were gently washed with IP buffer and ddH₂O to remove unspecific binding peptides before eluting the peptides with 0.1% trifluoroacetic acid three times. The resulting peptides were completely dried and desalted with C18 ZipTips (Cat. #ZTC18S, Merck, USA) following the manufacturer’s instructions.

2.13 LC-MS/MS assay

The peptide fractions were dissolved in liquid chromatography mobile phase A and separated by a NanoElute (Bruker, Germany) UPLC system. After being separated by the UPLC system, the peptide was injected into the capillary ion source for ionization and then analyzed by timsTOF Pro (Bruker, Germany) MS. The data were acquired using the parallel cumulative serial fragmentation (PASEF) mode. After primary mass spectra were collected, the secondary mass spectra were scanned in the PASEF mode ten times. Statistical analysis was then performed using the R software environment. For Gene Ontology (GO) enrichment analysis, GO annotation was obtained from UniProt, and the significance of the enrichment of a specific term was determined using Fisher’s exact test. P values were corrected using the Benjamini and Hochberg FDR. Data with P < 0.05 and |log₂ (fold change)| > 1.5 were considered significant.

2.14 Statistical analysis

Statistical analyses were performed with Prism8 software (GraphPad, USA). An unpaired two-tailed t-test was used to compare data between two groups unless otherwise mentioned. For experiments involving more than two groups, data were analyzed with one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SEM unless otherwise mentioned. P < 0.05 was considered statistically significant.

3 RESULTS

3.1 OTUB1 facilitates the phenotype switch of VSMCs

The ovarian tumor protease (OTU) superfamily deubiquitinases are the second-largest deubiquitinase family, and they are known to play essential roles in tumorigenesis, inflammation, and infectious diseases [13,27,28], which leads us to hypothesize that OTU deubiquitinases may regulate the pathophysiology of VSMCs in atherosclerosis. To investigate this hypothesis, we transfected primary HASMCs with non-targeting control siRNAs (si-NC) or effective siRNAs targeting Otubains and OTUD subfamily deubiquitinases (Fig.1, 1B, and S2), to examine whether contractile (TAGLN and ACTA2) or synthetic (OPN and BMP2) markers are dysregulated after stimulation with platelet-derived growth factor-BB (PDGF-BB), which can induce a contractile-to-synthetic phenotype switch. As illustrated in Fig.1–1F, instead of other deubiquitinases, knocking down OTUB1 sufficiently increased TAGLN and ACTA2 and simultaneously significantly decreased OPN and BMP2 expression in HASMCs after PDGF-BB stimulation. These results demonstrated the protective role of OTUB1 depletion in VSMC phenotype transition.

To further investigate the roles of OTUB1 in atherosclerosis, we analyzed a GEO dataset GSE57691 and found that OTUB1 expression increased in atherosclerotic human aortic occlusive disease specimens compared with healthy donor specimens (Fig.2) [29]. In addition, OTUB1 expression in Apoe^−/− mouse aortas increased in the HFD-induced atherosclerosis group, and PDGF-BB stimulation could increase OTUB1 protein expression in vitro in HASMCs (Fig. S3B and S3C). Together, these findings indicated that OTUB1 potentially played an important role in atherosclerosis.

3.2 OTUB1 is required for PDGF-BB-induced proliferation, phenotype switching, and migration of HASMCs

OTUB1 has been shown to modulate proliferation and migration in tumor cells and can regulate the TGFβ, NF-κB, and mTOR pathways [30–33], but whether it influences the biological and pathological processes of VSMCs in atherosclerosis has not been elucidated. To investigate this hypothesis, we knocked down OTUB1 with siRNAs in HASMCs, treated them with PDGF-BB or solvent after 24 h, and harvested cells after 48 h of incubation. We then assessed cell proliferation, migration, and phenotypic switch in detail. EdU and Ki-67 staining revealed that OTUB1 depletion ameliorated HASMCs’ proliferation induced by PDGF-BB, which was also indicated by CCK-8 assay results and Western blot on markers related to proliferation (Fig.3–2D, S4A, and S4B). Changes in the expression of contractile (ACTA2 and TAGLN) and synthetic (OPN) VSMC markers, as well as the accelerated migration rate of HASMCs induced by PDGF-BB, were also significantly reduced after OTUB1 depletion (Fig.3–2G and S4C–S4F). To avoid the off-target effects of siRNAs, we used another pair of siRNAs (si-OTUB1 #1) and the siRNAs used in other parts of this study (si-OTUB1 #2) to repeat the experiments and reached similar results in the proliferation, phenotype switch, and migration of HASMCs (Fig. S5). We identified that knocking down OTUB1 helped maintain HASMCs in a contractile phenotype.

To provide comprehensive evidence of the mechanisms underlying the effects of OTUB1 on HASMCs, we conducted unbiased RNA sequencing on HASMCs transfected with si-OTUB1 and treated with PDGF-BB (Fig.3). The differentially expressed protein-coding genes (DEGs) were categorized using GO analyses, showing that the DEGs were related to cell differentiation, adhesion, proliferation, angiogenesis, and ECM organization (Fig.3). Moreover, Kyoto Encyclopedia of Genes and Genomes analysis revealed that focal adhesion, cell adhesion molecules, and ECM–receptor interaction, all of which are critical mediators of atherogenesis, were altered significantly after OTUB1 knockdown (Fig.3). Interestingly, further analyses using gene set enrichment analysis showed that the DEGs were negatively enriched in the DNA replication, TGF-β pathway, and cell cycle pathways (Fig. S6). These data suggested that OTUB1 was an important mediator of VSMC pathophysiology after PDGF-BB stimulation.

3.3 OTUB1 deficiency enhances global ubiquitylation and causes dysregulation of atherogenesis-related proteins’ ubiquitylation

We then surmised that the regulatory roles of OTUB1 in atherosclerosis and VSMC pathophysiology were exerted through deubiquitylation. Comparing ubiquitylated protein profiles from MS analysis data between wild-type and OTUB1-knockdown HASMCs, we identified 478 proteins whose ubiquitylation was significantly differentially regulated by OTUB1 and analyzed their ubiquitylation motif using modification motif (MoMo) (Fig.2–3C) [34]. Among the 163 proteins with significantly elevated ubiquitylation after depleting the deubiquitinase OTUB1, we found that PDGFRβ, SLC3A2, and SLC7A5 were implicated in atherogenesis, as indicated by previous studies (Fig.2) [35–38]. Moreover, genes encoding the differentially ubiquitylated proteins were categorized with GO, and biological processes including lipid transport, cell growth, cell migration, and cell motility, were significantly affected (Fig.2 and 3F).

OTUB1 displays marked specificity to remove K48- and K63-linked ubiquitin chains [39]. K48-linked ubiquitylation processes proteins to degradation via the ubiquitin–proteasome system [40,41], whereas K63-linked ubiquitylation is related to protein localization and protein–protein interactions [42]. Thus, we conducted in vitro validation in PDGF-BB-treated HASMCs using Western blot to assess whether the global ubiquitin levels changed. Our results showed that basal total- and K48-linked ubiquitylation levels increased significantly in OTUB1-depleted HASMCs, whereas K63-linked ubiquitylation levels were relatively unchanged (Fig. S7). These results indicated that OTUB1 mainly deteriorated K48-linked ubiquitylation in VSMCs.

3.4 OTUB1 restrains proteasome degradation of PDGFRβ

We then investigated whether OTUB1 can influence the stability of proteins with altered ubiquitylation levels. We first focused on the three proteins implicated in atherosclerosis whose ubiquitylation levels were elevated after knocking down OTUB1 from our MS data, namely, PDGFRβ, SLC3A2, and SLC7A5 (Fig.2). Surprisingly, qPCR quantification showed no difference in mRNA expression (Fig. 4A–4C), and OTUB1 knockdown significantly reduced the protein level of PDGFRβ but had no significant effect on SCL3A2 and SLC7A5 (Fig. 4D), suggesting that OTUB1 positively regulated PDGFRβ protein stability. By contrast, OTUB1 overexpression (OE) enhanced PDGFRβ protein expression (Fig. 4E). Although dysregulation of ubiquitylation could alter the localization of substrates occasionally [13], we did not observe a difference in PDGFRβ subcellular trafficking between control or OTUB1 knockdown HASMCs (Fig. S8).

Co-IP was performed to further confirm that OTUB1 regulates PDGFRβ’s ubiquitination. Consistent with the MS results, ubiquitin chains conjugated to endogenous PDGFRβ increased significantly after OTUB1 knockdown. Notably, K48-linked polyubiquitin changed in parallel with the total ubiquitin level, but K63-linked polyubiquitin stayed unchanged, revealing that K48-linked ubiquitylation was selectively removed from PDGFRβ (Fig. 4F–4H).

To reveal the regulatory roles of OTUB1 on the stability and degradation of PDGFRβ, we transfected HASMCs with control siRNA or si-OTUB1 at 48 h before treating them with PDGF-BB to induce PDGFRβ degradation, as well as translation inhibitor cycloheximide (CHX) for 0–8 h to inhibit de novo protein synthesis. PDGFRβ loss was significantly accelerated in cells transfected with si-OTUB1 (Fig. 4I). By contrast, PDGFRβ expression in HASMCs overexpressing OTUB1 stayed relatively steady (Fig. 4J). These results revealed that OTUB1 inhibited PDGFRβ degradation in HASMCs.

To further confirm that OTUB1 modulates the process via the proteasome degradation system, we transfected HASMCs with control siRNA or si-OTUB1 while incubating with or without 10 μM proteasome inhibitor MG132. Cells were then harvested at different time points. OTUB1 knockdown in untreated HASMCs led to an accelerated decline of PDGFRβ level, but MG132 treatment abrogated the destabilization effect, revealing that OTUB1 modulated PDGFRβ expression through the proteasome degradation system (Fig. 4K and 4L). These results supported the conclusion that OTUB1 modulated the ubiquitin–proteasome degradation process of PDGFRβ.

3.5 OTUB1 interacts with PDGFRβ and regulates its expression through K48-linked ubiquitylation

To further elucidate the mechanisms by which OTUB1 modulates PDGFRβ, we performed immunoprecipitation using antibodies against PDGFRβ in HASMC cell lyses to examine the effects of OTUB1 on its ubiquitylation.

The interaction between OTUB1 and PDGFRβ was then investigated. Reciprocal in vitro co-IP with antibodies against OTUB1 followed by PDGFRβ Western blot staining showed that PDGFRβ was efficiently precipitated by an antibody against OTUB1 but not by control IgG antibody, and vice versa (Fig.5 and 5B), confirming the binding between OTUB1 and PDGFRβ in HASMCs.

Meanwhile, considering that the only known E3 ligase of PDGFRβ from UbiNet and Ubibrowser databases is c-CBL [43–45], and c-CBL is also highly expressed in VSMCs (Fig. S9A), we further investigated whether c-CBL is indispensable for the interaction between and PDGFRβ. Surprisingly, depleting c-CBL inhibited PDGFRβ from being immunoprecipitated by OTUB1, and vice versa (Fig.5). Additionally, knocking down c-CBL inhibited si-OTUB1’s pro-ubiquitylation effects on PDGFRβ (Fig. S9B and S9C). These results indicated that E3 ligase c-CBL is necessary for OTUB1’s deubiquitination on PDGFRβ.

Moreover, we specified the lysine site where PDGFRβ was ubiquitylated and degraded. UbiNet, PLMD, and Ubibrowser websites predicted that PDGFRβ could be ubiquitylated in the lysine 707 residue (K707), which was the only verified ubiquitylation site of c-CBL on PDGFRβ (Table S3) [45,46]. Therefore, we replaced the lysine 707 residue with inactive arginine to the PDGFRβ-OE plasmid (PDGFRβ^K707R) and expressed PDGFRβ^K707R in OTUB1-depleted HASMCs, which led to less significant degradation of PDGFRβ, compared with wild-type PDGFRβ (Fig.5). In addition, overexpressing OTUB1 in PDGFRβ^K707R-transfected cells resulted in a less significant decrease (lane 3 and lane 6, compared with lane 2 and lane 5) in total ubiquitin and K48-linked ubiquitin compared with PDGFRβ^WT (Fig.5 and 5F). These results further confirmed the importance of the K707 site in PDGFRβ for its deubiquitination by OTUB1 in HASMCs.

OTUB1 deubiquitinates its substrates via two mechanisms: catalyzes ubiquitin chains through its catalytic center in the C-terminal of the OTU domain or inhibits E2 ubiquitin-conjugating enzymes through its N-terminal ubiquitin binding motif [23,47]. In the catalytic mechanism, OTUB1 has a conserved triad as its catalytic center, which is composed of aspartate 88 (D88), cysteine 91 (C91), and histidine 265 (H265). To reveal the mechanisms underlying OTUB1’s activity on PDGFRβ, we constructed five mutants of OTUB1, namely, catalytically inactive OTUB1^D88A, OTUB1^C91S, OTUB1^H265A, OTUB1^ASA (D88A/C91S/H265A), and an N-terminal-truncated mutant (ΔN; Fig.5). Ubiquitin chains conjugated to PDGFRβ increased in the ASA, D88A, and C91S groups but not in the ΔN or H265A mutants (Fig.5 and 5I), showing that OTUB1 deubiquitinated PDGFRβ via its catalytic triad.

3.6 PDGFRβ is necessary for OTUB1-mediated HASMC phenotypic switch

As an essential regulator in pathological vascular remodeling, PDGFRβ promotes VSMC proliferation, differentiation, migration, and inflammation [38,48], and its degradation occurs after being polyubiquitinated [49]. Therefore, to confirm that PDGFRβ is necessary for the regulative roles of OTUB1 in VSMCs, we performed rescue experiments in OTUB1-depleted HASMCs, which were treated with PDGF-BB for 48 h. By overexpressing PDGFRβ in HASMCs, the decrease in proliferative HASMCs induced by knocking down OTUB1 was compromised (Fig.6 and 6B). We also found that the increase in contractile gene repertoire (TAGLN and ACTA2) and the decrease in a synthetic marker (OPN) induced by knocking down OTUB1 were ameliorated in protein and mRNA levels (Fig.6–6E). Additionally, the Boyden chamber migration assay and scratch assay indicated that the decelerated migration rates induced by OTUB1 knockdown were significantly compromised by overexpressing PDGFRβ (Fig.6–6I). Therefore, we confirmed that PDGFRβ is critical for OTUB1’s regulative roles in VSMCs.

3.7 OTUB1 silencing prevents atherogenesis and stabilizes atherosclerotic plaque in vivo

Considering that OTUB1 modulates the VSMC phenotype after PDGF-BB treatment and is a potential translational therapeutic target for atherosclerosis, we investigated whether knocking down Otub1 in atherosclerotic mice could delay atherogenesis. Six-week-old male Apoe^−/− mice were fed an HFD for 12 or 22 weeks to generate early- or late-stage atherosclerosis models (Fig.7). With an effective shRNA targeting mice Otub1 (Fig. S10A), the atherosclerotic mice were injected with Tagln-shOtub1 AAV9 at 6 weeks before sacrifice to selectively knock down VSMC Otub1 and explore whether OTUB1 is a therapeutic target for atherosclerosis. In agreement with the in vitro results, Western blot analyses showed that PDGFRβ expression decreased in Tagln-shOtub1-treated mice aortas (Fig.7 and 7C), confirming that OTUB1 positively regulated PDGFRβ protein expression in vivo. Consistent with in vitro study results, in shOtub1-AAV9-infected aortas, the basal total- and K48-linked ubiquitylation levels increased significantly, whereas the K63-linked ubiquitylation levels remained steady (Figs. S10B and S10C). However, we did not observe an alteration in c-CBL expression after Otub1-silencing, indicating that c-CBL expression was not modulated by OTUB1 (Fig. S10D and S10E).

Although no statistically significant difference was observed in the aortic plaque size indicated by ORO staining of early-stage atherosclerotic mice, the Otub1 knockdown group had a smaller atherosclerotic plaque burden in the late stage in the aortic roots and gross observation of aortas compared with that in the early stage (Fig.7, 7E, and S11). Masson’s trichrome staining showed that the Otub1 knockdown group’s atherosclerotic plaque had a higher proportion of collagen compared with the treatment group (Fig.7 and 7G). Interestingly, as revealed by Masson’s trichrome staining and H&E staining, larger and thicker fibrous caps and smaller necrotic core areas were found in Tagln-shOtub1-treated mice than in the knockdown group (Fig.7 and 7J), indicating a more stable plaque phenotype. Macrophage infiltration, which is positively correlated with plaque destabilization and could be exacerbated by VSMC PDGFRβ signaling [37,50,51], decreased in Otub1 knockdown in both early- and late-stage atherosclerotic mice, and the plaque vulnerability index in the Otub1 knockdown group was elevated (Fig.7–7M). This result could at least partly explain OTUB1’s plaque destabilization effect. Considering that plaque stability is also closely related to VSMC apoptosis and OTUB1 could promote apoptosis [52], we performed TdT-mediated dUTP nick end labeling (TUNEL) staining on aortic plaque and HASMCs and found that VSMCs’ apoptosis decreased significantly in the Otub1 knockdown group in vivo and in vitro (Fig. S12), suggesting that the decrease in VSMC apoptosis may contribute to enhanced plaque stability, but the specific molecular mechanisms remain to be investigated in the future. Meanwhile, the contractile markers (ACTA2 and TAGLN) were highly expressed in the Otub1 knockdown mice, whereas OPN expression decreased after Otub1 knockdown (Fig. S13). Taken together, these results proved that knocking down Otub1 in VSMCs could therapeutically delay atherogenesis and help stabilize atherosclerotic plaque, especially in advanced atherosclerotic mice.

4 Discussion

In this study, we identified OTUB1 as a novel and important mediator of atherosclerosis progression and VSMC phenotypic switching. The in vivo importance of OTUB1 was illustrated by a low plaque burden and a stable plaque phenotype after its depletion, especially in advanced atherosclerosis. Our unbiased RNA-seq results also showed that knocking down OTUB1 in VSMCs influenced its atherogenesis-related signaling pathways. Moreover, with MS of ubiquitylated protein and IP assays, we demonstrated that OTUB1 deubiquitinated PDGFRβ. Thus, knocking down OTUB1 prevented the sensitivity of VSMCs to PDGF-BB-induced proliferation, migration, and dedifferentiation. Mechanistically, we revealed that OTUB1 removed the K48-linked ubiquitin chains on PDGFRβ and regulated its degradation via the ubiquitin–proteasome system in VSMCs.

VSMCs play a critical role in different stages of atherogenesis, from pre-atherosclerosis diffuse intimal thickening to the formation of fibroatheroma in late atherosclerosis [4]. During atherosclerosis progression, VSMCs lose their VSMC-specific contractile protein markers, including TAGLN and ACTA2, and transform to a proliferative and synthetic state. After phenotypic switching, VSMCs could migrate into the intimal, where they may also re-differentiate into a contractile phenotype [2]. However, according to recent studies, phenotypic switching of VSMCs is not a binary process because contractile VSMCs can trans-differentiate to a spectrum of other phenotypes, including mesenchymal-like, fibroblast-like, macrophage-like, osteogenic-like, and adipocyte-like cells [53,54]. In this study, we found that OTUB1 silencing hindered the loss of contractile markers in VSMCs and inhibited the induction of synthetic markers, but evidence of whether it influences other phenotypes is still lacking. Therefore, further studies using lineage tracing or single-cell RNA-sequencing are necessary to investigate whether knocking down OTUB1 leads VSMCs to transform into other phenotypes. In this study, although VSMC migration was reduced in si-OTUB1-treated VSMCs in vitro, we surprisingly found that in vivo plaque stability was enhanced after Otub1 knockdown. The plaque stabilization effect may be due to the alleviation of macrophage infiltration, which could be regulated by VSMC PDGFR signaling [51]. si-OTUB1 also ameliorated VSMC apoptosis in HASMCs and contributed to plaque stabilization. In the past decade, researchers found that OTUB1 promotes cell apoptosis in osteosarcoma and lung cancer by regulating p53 protein stability and activity [52,55], but the mechanisms through which OTUB1 modulates VSMC apoptosis in atherosclerotic plaque should be investigated in the future.

Emerging evidence suggests that deubiquitinases can target key molecules in pathways related to atherogenesis and play essential roles in the modulation of atherosclerosis progression. For instance, USP20 was reported to regulate TNFR1 signaling pathway activation by directly deubiquitinating RIPK1, which leads to an attenuation of atherogenic signaling in VSMCs [56]. Moreover, OTU family deubiquitinase A20 can inhibit TBK1 activation in VSMCs and vascular endothelial cells, thereby negatively regulating the atherogenic STAT1/IFNγ signaling pathway [57]. In this study, we used siRNAs targeting Otubains and OTUD family deubiquitinases, which have small catalytic centers, and revealed that inhibiting OTUB1 is sufficient to inhibit VSMCs’ phenotypic switch. However, the roles of deubiquitinases from other families remain to be investigated. Our findings revealed for the first time that OTUB1 played a key role in the post-transcriptional regulation of atherogenic protein PDGFRβ in VSMCs, indicating that OTUB1 could be targeted for therapeutic purposes toward atherosclerosis. We confirmed the binding between OTUB1 and PDGFRβ in HASMCs via co-IP assay, but future colocalization analyses using immunofluorescent staining and PLA assay will be needed to further confirm their interaction. Further study is necessary to understand the effect of OTUB1 on other cell types in atherosclerotic plaque, including endothelial cells and macrophages.

PDGFRβ, which plays a crucial role in early- and late-stage atherosclerosis, is highly expressed in VSMCs in atherosclerotic vessels [58]. PDGFRβ is known to regulate atherosclerosis development via mediating local inflammation and stimulating VSMC involvement [37,48]. Thus, the stability of PDGFRβ is of great importance for atherosclerosis development. In general, upon ligand binding by PDGF and autophosphorylation, PDGFRβ is polyubiquitinated and internalized, leading to its degradation [49,59]. Sarri et al. found that deubiquitinases USP4 and USP17 can modulate the trafficking of PDGFRβ, but the deubiquitinases that regulate PDGFRβ protein levels are currently unknown [60]. Interestingly, although reducing PDGFRβ expression by mediating its degradation can ameliorate atherosclerosis development, genetic deletion of PDGFRβ in VSMCs was recently found to be detrimental to plaque stability [61], suggesting that a relatively low level of PDGFRβ is necessary for the normal functions of plaque VSMCs.

Previously, Takayama et al. reported that ubiquitin E3-ligase c-CBL can accelerate the ubiquitylation and degradation of PDGFRβ [43], whereas Guo et al. found that altered c-CBL expression leads to altered PDGFRβ ubiquitylation, which is mediated by PDGF-BB [62]. In the current study, we identified for the first time that depleting c-CBL hindered the interaction between OTUB1 and PDGFRβ, and mutating c-CBL’s ubiquitylation site K707 abrogated OTUB1’s deubiquitination effects on PDGFRβ, suggesting that c-CBL was necessary for the interaction between OTUB1 and PDGFRβ.

The current study revealed that knocking down Otub1 had a stronger protective effect in advanced- than early-stage atherosclerosis. In general, VSMCs’ invasion into atherosclerotic plaque mostly occurs in the later phase of atherogenesis, which can lead to the different responses between advanced- and early-stage atherosclerosis observed here [37]. Another possible mechanism is that senescent cells accumulate with atherosclerosis development, resulting in chronic inflammation and a different local milieu in advanced atherosclerosis [63]. Consistent with our findings, previous reports showed that anti-plaque stabilizing treatments including IL-1β and peptide Ac2-26, which are protective in advanced atherosclerosis, may not be as effective in early-stage atherosclerosis because the main pathological process in early stage is plaque formation [64,65]. Although we proved that selectively knocking down OTUB1 in VSMCs protected against atherosclerosis, whether global loss of OTUB1 in vivo will be beneficial or detrimental to atherosclerosis remains unknown. As plaque development and destabilization are long-term processes, further experiments are needed to investigate the effect of prolonged OTUB1 loss on atherogenesis.

From a translational aspect, the data presented in this study established that OTUB1 was highly expressed in human atheroma VSMCs, and the expression increased in unstable plaque. We found that OTUB1 was a key regulator of VSMCs’ function in atherosclerosis through deubiquitinating PDGFRβ, suggesting that OTUB1 was a promising therapeutic target. Although our in vivo experiments indicated that targeting OTUB1 could slow down the progress of atherosclerosis, the experiments were conducted with a Tagln-promoted AAV9 vector, whose clinical safety still needs to be further confirmed. Future investigations on how to precisely and safely target OTUB1 in certain stages and cell types of atherosclerosis are warranted to translate these findings into clinical practice.

However, another limitation of this study is that the in vivo study was only performed in mice. Experiments in large animals could help enhance the credibility of the findings. Additionally, morphometric analyses were performed with slices of the same level from each aortic tissue to minimize errors caused by manual operation, but some angular difference is still unavoidable because of our technical limitations in small animal experiments. Moreover, the findings would benefit from future flow cytometry studies using atherosclerotic aortas, which can accurately reflect PDGFRβ expression and ubiquitylation level in aortic VSMCs.

Conclusively, our current study identified that OTUB1 could significantly regulate the pathophysiology of VSMCs during atherosclerosis, and PDGFRβ is a novel target of deubiquitinase OTUB1. Moreover, we provided experimental evidence of the interaction between PDGFRβ and OTUB1. The regulatory mechanism of OTUB1 on PDGFRβ is removing K48-linked polyubiquitylation via its catalytical mechanism. In the future, targeting OTUB1 in VSMCs may be a potential therapeutic target for atherosclerosis.

References

[1] Libby P. The changing landscape of atherosclerosis. Nature 2021; 592(7855): 524–533

[2] Björkegren JLM, Lusis AJ. Atherosclerosis: recent developments. Cell 2022; 185(10): 1630–1645

[3] Hetherington I, Totary-Jain H. Anti-atherosclerotic therapies: milestones, challenges, and emerging innovations. Mol Ther 2022; 30(10): 3106–3117

[4] Basatemur GL, Jørgensen HF, Clarke MCH, Bennett MR, Mallat Z. Vascular smooth muscle cells in atherosclerosis. Nat Rev Cardiol 2019; 16(12): 727–744

[5] Miano JM, Fisher EA, Majesky MW. Fate and state of vascular smooth muscle cells in atherosclerosis. Circulation 2021; 143(21): 2110–2116

[6] Golforoush P, Yellon DM, Davidson SM. Mouse models of atherosclerosis and their suitability for the study of myocardial infarction. Basic Res Cardiol 2020; 115(6): 73

[7] Swiatlowska P, Sit B, Feng Z, Marhuenda E, Xanthis I, Zingaro S, Ward M, Zhou X, Xiao Q, Shanahan C, Jones GE, Yu CH, Iskratsch T. Pressure and stiffness sensing together regulate vascular smooth muscle cell phenotype switching. Sci Adv 2022; 8(15): eabm3471

[8] Borgo C, D’Amore C, Sarno S, Salvi M, Ruzzene M. Protein kinase CK2: a potential therapeutic target for diverse human diseases. Signal Transduct Target Ther 2021; 6(1): 183

[9] Herrmann J, Soares SM, Lerman LO, Lerman A. Potential role of the ubiquitin-proteasome system in atherosclerosis: aspects of a protein quality disease. J Am Coll Cardiol 2008; 51(21): 2003–2010

[10] Demasi M, Laurindo FR. Physiological and pathological role of the ubiquitin-proteasome system in the vascular smooth muscle cell. Cardiovasc Res 2012; 95(2): 183–193

[11] Grootaert MOJ, Finigan A, Figg NL, Uryga AK, Bennett MR. SIRT6 protects smooth muscle cells from senescence and reduces atherosclerosis. Circ Res 2021; 128(4): 474–491

[12] Ye M, Guo X, Wang H, Wang Y, Qian X, Deng H, Wang W, Yang S, Ni Q, Chen J, Lv L, Zhao Y, Xue G, Li Y, Zhang L. Mutual regulation between β-TRCP mediated REST protein degradation and Kv1.3 expression controls vascular smooth muscle cell phenotype switch. Atherosclerosis 2020; 313: 102–110

[13] Lange SM, Armstrong LA, Kulathu Y. Deubiquitinases: from mechanisms to their inhibition by small molecules. Mol Cell 2022; 82(1): 15–29

[14] Mevissen TE, Hospenthal MK, Geurink PP, Elliott PR, Akutsu M, Arnaudo N, Ekkebus R, Kulathu Y, Wauer T, El Oualid F, Freund SM, Ovaa H, Komander D. OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis. Cell 2013; 154(1): 169–184

[15] Clague MJ, Urbé S, Komander D. Breaking the chains: deubiquitylating enzyme specificity begets function. Nat Rev Mol Cell Biol 2019; 20(6): 338–352

[16] Liao Y, Yang M, Wang K, Wang Y, Zhong B, Jiang N. Deubiquitinating enzyme OTUB1 in immunity and cancer: good player or bad actor?. Cancer Lett 2022; 526: 248–258

[17] Mulas F, Wang X, Song S, Nishanth G, Yi W, Brunn A, Larsen PK, Isermann B, Kalinke U, Barragan A, Naumann M, Deckert M, Schlüter D. The deubiquitinase OTUB1 augments NF-κB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13. Cell Mol Immunol 2021; 18(6): 1512–1527

[18] Koschel J, Nishanth G, Just S, Harit K, Kröger A, Deckert M, Naumann M, Schlüter D. OTUB1 prevents lethal hepatocyte necroptosis through stabilization of c-IAP1 during murine liver inflammation. Cell Death Differ 2021; 28(7): 2257–2275

[19] Bonacci T, Suzuki A, Grant GD, Stanley N, Cook JG, Brown NG, Emanuele MJ. Cezanne/OTUD7B is a cell cycle-regulated deubiquitinase that antagonizes the degradation of APC/C substrates. EMBO J 2018; 37(16): e98701

[20] Luo Q, Wu X, Zhao P, Nan Y, Chang W, Zhu X, Su D, Liu Z. OTUD1 Activates caspase-independent and caspase-dependent apoptosis by promoting AIF nuclear translocation and MCL1 degradation. Adv Sci (Weinh) 2021; 8(8): 2002874

[21] Yang GS, Zheng B, Qin Y, Zhou J, Yang Z, Zhang XH, Zhao HY, Yang HJ, Wen JK. Salvia miltiorrhiza-derived miRNAs suppress vascular remodeling through regulating OTUD7B/KLF4/NMHC IIA axis. Theranostics 2020; 10(17): 7787–7811

[22] Karunarathna U, Kongsema M, Zona S, Gong C, Cabrera E, Gomes AR, Man EP, Khongkow P, Tsang JW, Khoo US, Medema RH, Freire R, Lam EW. OTUB1 inhibits the ubiquitination and degradation of FOXM1 in breast cancer and epirubicin resistance. Oncogene 2016; 35(11): 1433–1444

[23] Zhu D, Xu R, Huang X, Tang Z, Tian Y, Zhang J, Zheng X. Deubiquitinating enzyme OTUB1 promotes cancer cell immunosuppression via preventing ER-associated degradation of immune checkpoint protein PD-L1. Cell Death Differ 2021; 28(6): 1773–1789

[24] Jahan AS, Biquand E, Muñoz-Moreno R, Le Quang A, Mok CK, Wong HH, Teo QW, Valkenburg SA, Chin AWH, Man Poon LL, Te Velthuis A, García-Sastre A, Demeret C, Sanyal S. OTUB1 is a key regulator of RIG-I-dependent immune signaling and is targeted for proteasomal degradation by influenza A NS1. Cell Rep 2020; 30(5): 1570–1584.e6

[25] Liu S, Jiang X, Cui X, Wang J, Liu S, Li H, Yang J, Zhang C, Zhang W. Smooth muscle-specific HuR knockout induces defective autophagy and atherosclerosis. Cell Death Dis 2021; 12(4): 385

[26] Wiśniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat Methods 2009; 6(5): 359–362

[27] Shin D, Bhattacharya A, Cheng YL, Alonso MC, Mehdipour AR, van der Heden van Noort GJ, Ovaa H, Hummer G, Dikic I. Bacterial OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection. Elife 2020; 9: e58277

[28] Schubert AF, Nguyen JV, Franklin TG, Geurink PP, Roberts CG, Sanderson DJ, Miller LN, Ovaa H, Hofmann K, Pruneda JN, Komander D. Identification and characterization of diverse OTU deubiquitinases in bacteria. EMBO J 2020; 39(15): e105127

[29] Biros E, Gäbel G, Moran CS, Schreurs C, Lindeman JH, Walker PJ, Nataatmadja M, West M, Holdt LM, Hinterseher I, Pilarsky C, Golledge J. Differential gene expression in human abdominal aortic aneurysm and aortic occlusive disease. Oncotarget 2015; 6(15): 12984–12996

[30] Herhaus L, Al-Salihi M, Macartney T, Weidlich S, Sapkota GP. OTUB1 enhances TGFβ signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3. Nat Commun 2013; 4(1): 2519

[31] Li Y, Yang JY, Xie X, Jie Z, Zhang L, Shi J, Lin D, Gu M, Zhou X, Li HS, Watowich SS, Jain A, Yun Jung S, Qin J, Cheng X, Sun SC. Preventing abnormal NF-κB activation and autoimmunity by Otub1-mediated p100 stabilization. Cell Res 2019; 29(6): 474–485

[32] Zhu Q, Fu Y, Li L, Liu CH, Zhang L. The functions and regulation of Otubains in protein homeostasis and diseases. Ageing Res Rev 2021; 67: 101303

[33] Ruiz-Serrano A, Monné Rodríguez JM, Günter J, Sherman SPM, Jucht AE, Fluechter P, Volkova YL, Pfundstein S, Pellegrini G, Wagner CA, Schneider C, Wenger RH, Scholz CC. OTUB1 regulates lung development, adult lung tissue homeostasis, and respiratory control. FASEB J 2021; 35(12): e22039

[34] Cheng A, Grant CE, Noble WS, Bailey TL. MoMo: discovery of statistically significant post-translational modification motifs. Bioinformatics 2019; 35(16): 2774–2782

[35] Baumer Y, McCurdy S, Alcala M, Mehta N, Lee BH, Ginsberg MH, Boisvert WA. CD98 regulates vascular smooth muscle cell proliferation in atherosclerosis. Atherosclerosis 2017; 256: 105–114

[36] Goetzl EJ, Schwartz JB, Mustapic M, Lobach IV, Daneman R, Abner EL, Jicha GA. Altered cargo proteins of human plasma endothelial cell-derived exosomes in atherosclerotic cerebrovascular disease. FASEB J 2017; 31(8): 3689–3694

[37] He C, Medley SC, Hu T, Hinsdale ME, Lupu F, Virmani R, Olson LE. PDGFRβ signalling regulates local inflammation and synergizes with hypercholesterolaemia to promote atherosclerosis. Nat Commun 2015; 6(1): 7770

[38] Papadopoulos N, Lennartsson J. The PDGF/PDGFR pathway as a drug target. Mol Aspects Med 2018; 62: 75–88

[39] Iglesias-Gato D, Chuan YC, Jiang N, Svensson C, Bao J, Paul I, Egevad L, Kessler BM, Wikström P, Niu Y, Flores-Morales A. OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo. Mol Cancer 2015; 14(1): 8

[40] Han X, Ren C, Lu C, Qiao P, Yang T, Yu Z. Deubiquitination of MYC by OTUB1 contributes to HK2 mediated glycolysis and breast tumorigenesis. Cell Death Differ 2022; 29(9): 1864–1873

[41] Goncharov T, Niessen K, de Almagro MC, Izrael-Tomasevic A, Fedorova AV, Varfolomeev E, Arnott D, Deshayes K, Kirkpatrick DS, Vucic D. OTUB1 modulates c-IAP1 stability to regulate signalling pathways. EMBO J 2013; 32(8): 1103–1114

[42] Martínez-Férriz A, Ferrando A, Fathinajafabadi A, Farràs R. Ubiquitin-mediated mechanisms of translational control. Semin Cell Dev Biol 2022; 132: 146–154

[43] Takayama Y, May P, Anderson RG, Herz J. Low density lipoprotein receptor-related protein 1 (LRP1) controls endocytosis and c-CBL-mediated ubiquitination of the platelet-derived growth factor receptor β (PDGFR β). J Biol Chem 2005; 280(18): 18504–18510

[44] Wang X, Li Y, He M, Kong X, Jiang P, Liu X, Diao L, Zhang X, Li H, Ling X, Xia S, Liu Z, Liu Y, Cui CP, Wang Y, Tang L, Zhang L, He F, Li D. UbiBrowser 2.0: a comprehensive resource for proteome-wide known and predicted ubiquitin ligase/deubiquitinase-substrate interactions in eukaryotic species. Nucleic Acids Res 2022; 50(D1): D719–D728

[45] Li Z, Chen S, Jhong JH, Pang Y, Huang KY, Li S, Lee TY. UbiNet 2.0: a verified, classified, annotated and updated database of E3 ubiquitin ligase-substrate interactions. Database (Oxford) 2021; 2021: baab010

[46] Xu H, Zhou J, Lin S, Deng W, Zhang Y, Xue Y. PLMD: an updated data resource of protein lysine modifications. J Genet Genomics 2017; 44(5): 243–250

[47] Wiener R, Zhang X, Wang T, Wolberger C. The mechanism of OTUB1-mediated inhibition of ubiquitination. Nature 2012; 483(7391): 618–622

[48] Zhang Y, Wang Y, Zhang L, Xia L, Zheng M, Zeng Z, Liu Y, Yarovinsky T, Ostriker AC, Fan X, Weng K, Su M, Huang P, Martin KA, Hwa J, Tang WH. Reduced platelet miR-223 induction in Kawasaki disease leads to severe coronary artery pathology through a miR-223/PDGFRβ vascular smooth muscle cell axis. Circ Res 2020; 127(7): 855–873

[49] Reddi AL, Ying G, Duan L, Chen G, Dimri M, Douillard P, Druker BJ, Naramura M, Band V, Band H. Binding of Cbl to a phospholipase Cgamma1-docking site on platelet-derived growth factor receptor beta provides a dual mechanism of negative regulation. J Biol Chem 2007; 282(40): 29336–29347

[50] Hamid T, Xu Y, Ismahil MA, Rokosh G, Jinno M, Zhou G, Wang Q, Prabhu SD. Cardiac mesenchymal stem cells promote fibrosis and remodeling in heart failure: role of PDGF signaling. JACC Basic Transl Sci 2022; 7(5): 465–483

[51] Doran AC, Meller N, McNamara CA. Role of smooth muscle cells in the initiation and early progression of atherosclerosis. Arterioscler Thromb Vasc Biol 2008; 28(5): 812–819

[52] Saldana M, VanderVorst K, Berg AL, Lee H, Carraway KL. Otubain 1: a non-canonical deubiquitinase with an emerging role in cancer. Endocr Relat Cancer 2019; 26(1): R1–R14

[53] Yap C, Mieremet A, de Vries CJM, Micha D, de Waard V. Six shades of vascular smooth muscle cells illuminated by KLF4 (Krüppel-like factor 4). Arterioscler Thromb Vasc Biol 2021; 41(11): 2693–2707

[54] Durham AL, Speer MY, Scatena M, Giachelli CM, Shanahan CM. Role of smooth muscle cells in vascular calcification: implications in atherosclerosis and arterial stiffness. Cardiovasc Res 2018; 114(4): 590–600

[55] Sun XX, Challagundla KB, Dai MS. Positive regulation of p53 stability and activity by the deubiquitinating enzyme Otubain 1. EMBO J 2012; 31(3): 576–592

[56] Jean-Charles PY, Wu JH, Zhang L, Kaur S, Nepliouev I, Stiber JA, Brian L, Qi R, Wertman V, Shenoy SK, Freedman NJ. USP20 (ubiquitin-specific protease 20) inhibits TNF (tumor necrosis factor)-triggered smooth muscle cell inflammation and attenuates atherosclerosis. Arterioscler Thromb Vasc Biol 2018; 38(10): 2295–2305

[57] Moll HP, Lee A, Minussi DC, da Silva CG, Csizmadia E, Bhasin M, Ferran C. A20 regulates atherogenic interferon (IFN)-γ signaling in vascular cells by modulating basal IFNβ levels. J Biol Chem 2014; 289(45): 30912–30924

[58] Karvinen H, Rutanen J, Leppänen O, Lach R, Levonen AL, Eriksson U, Ylä-Herttuala S. PDGF-C and -D and their receptors PDGFR-α and PDGFR-β in atherosclerotic human arteries. Eur J Clin Invest 2009; 39(4): 320–327

[59] Rorsman C, Tsioumpekou M, Heldin CH, Lennartsson J. The ubiquitin ligases c-Cbl and Cbl-b negatively regulate platelet-derived growth factor (PDGF) BB-induced chemotaxis by affecting PDGF receptor β (PDGFRβ) internalization and signaling. J Biol Chem 2016; 291(22): 11608–11618

[60] Sarri N, Wang K, Tsioumpekou M, Castillejo-López C, Lennartsson J, Heldin CH, Papadopoulos N. Deubiquitinating enzymes USP4 and USP17 finetune the trafficking of PDGFRβ and affect PDGF-BB-induced STAT3 signalling. Cell Mol Life Sci 2022; 79(2): 85

[61] Newman AAC, Serbulea V, Baylis RA, Shankman LS, Bradley X, Alencar GF, Owsiany K, Deaton RA, Karnewar S, Shamsuzzaman S, Salamon A, Reddy MS, Guo L, Finn A, Virmani R, Cherepanova OA, Owens GK. Multiple cell types contribute to the atherosclerotic lesion fibrous cap by PDGFRβ and bioenergetic mechanisms. Nat Metab 2021; 3(2): 166–181

[62] Guo X, Nie L, Esmailzadeh L, Zhang J, Bender JR, Sadeghi MM. Endothelial and smooth muscle-derived neuropilin-like protein regulates platelet-derived growth factor signaling in human vascular smooth muscle cells by modulating receptor ubiquitination. J Biol Chem 2009; 284(43): 29376–29382

[63] Childs BG, Baker DJ, Wijshake T, Conover CA, Campisi J, van Deursen JM. Senescent intimal foam cells are deleterious at all stages of atherosclerosis. Science 2016; 354(6311): 472–477

[64] Gomez D, Baylis RA, Durgin BG, Newman AAC, Alencar GF, Mahan S, St Hilaire C, Müller W, Waisman A, Francis SE, Pinteaux E, Randolph GJ, Gram H, Owens GK. Interleukin-1β has atheroprotective effects in advanced atherosclerotic lesions of mice. Nat Med 2018; 24(9): 1418–1429

[65] Fredman G, Kamaly N, Spolitu S, Milton J, Ghorpade D, Chiasson R, Kuriakose G, Perretti M, Farokzhad O, Tabas I. Targeted nanoparticles containing the proresolving peptide Ac2-26 protect against advanced atherosclerosis in hypercholesterolemic mice. Sci Transl Med 2015; 7(275): 275ra20

RIGHTS & PERMISSIONS

Higher Education Press