Ubiquitin-specific Protease Research Articles

NRF1 Transcriptionally Regulates USP1-Mediated Deubiquitination of AURKA to Facilitate Osteosarcoma Progression.

The poor prognosis of osteosarcoma (OS) lead to the low survival rate of OS patients. Numerous researches have shown that nuclear factor erythroid 2-like 1 (NRF1) can regulate the development and progression of cancer, but it has not yet been documented in OS. Therefore, the impact of NRF1 on OS progression and specific mechanisms will be investigated in this study. This may provide new ideas for the treatment of OS. The expressions of NRF1, ubiquitin-specific protease 1 (USP1), and aurora kinase A (AURKA) were determined by RT-qPCR and Western blot. CCK-8, colony formation, EdU staining, and Transwell assay were used to detect cell viability, proliferation, and invasion. Apoptosis and cell cycle were detected by flow cytometry. The deubiquitination of AURKA by USP1 was investigated by Co-IP and Western blot. The binding of NRF1 and USP1 promoter was explored by ChIP and dual-luciferase assay. A subcutaneous tumorigenic model in nude mice was used to further explore the effects of NRF1/USP1/AURKA axis on OS in vivo. NRF1, USP1, and AURKA were highly expressed in OS. The proliferation and invasion of OS cells were suppressed and apoptosis enhanced by silencing NRF1 expression. Moreover, knockdown of NRF1 inhibited tumor growth in vivo. USP1 increased AURKA stability by mediating AURKA deubiquitination. NRF1 bound to the promoter of USP1, so as to transcriptionally activate USP1 expression. USP1 overexpression reversed the effects of NRF1 knockdown on USP1 and AURKA expression and on OS cells trends and in vivo tumor growth. NRF1 transcriptionally modulated USP1, which facilitates deubiquitination of AURKA and thereby fostering OS progression.

Hao-Fountain syndrome protein USP7 controls neuronal differentiation via BCOR-ncPRC1.1.

Pathogenic variants in the ubiquitin-specific protease 7 (USP7) gene cause a neurodevelopmental disorder called Hao-Fountain syndrome. However, it remains unclear which of USP7's pleiotropic functions are relevant for neurodevelopment. Here, we present a combination of quantitative proteomics, transcriptomics, and epigenomics to define the USP7 regulatory circuitry during neuronal differentiation. USP7 activity is required for the transcriptional programs that direct both the differentiation of embryonic stem cells into neural stem cells and the neuronal differentiation of SH-SY5Y neuroblastoma cells. USP7 controls the dosage of the Polycomb monubiquitylated histone H2A lysine 119 (H2AK119ub1) ubiquitin ligase complexes ncPRC1.1 and ncPRC1.6. Loss-of-function experiments revealed that BCOR-ncPRC1.1, but not ncPRC1.6, is a key effector of USP7 during neuronal differentiation. Indeed, BCOR-ncPRC1.1 mediates a major portion of USP7-dependent gene regulation during this process. Besides providing a detailed map of the USP7 regulome during neurodifferentiation, our results suggest that USP7- and ncPRC1.1-associated neurodevelopmental disorders involve dysregulation of a shared epigenetic network.

Drosophila ubiquitin-specific peptidase 14 stabilizes the PERIOD protein by regulating a ubiquitin ligase SLIMB

The circadian clock orchestrates behavior and physiology through the oscillation of key clock proteins like PERIOD (PER). Here, we investigate the role of ubiquitin-specific peptidase 14 (USP14) in modulating PER stability and circadian rhythms in Drosophila. We find that overexpression of USP14 in clock cells reduces PER protein levels without altering its mRNA levels whereas USP14 knockdown increases PER protein levels, suggesting that USP14 regulates PER post-translationally. Interestingly, despite these alterations in PER levels, neither USP14 overexpression nor knockdown significantly impacts circadian behavioral rhythms, likely because of slight effects on PER levels in small ventral lateral neurons (sLNvs). Further analysis shows that USP14 physically interacts with Supernumerary Limbs (SLIMB), a protein involved in PER degradation. Moreover, reducing slimb expression mitigates the effects of USP14 on PER protein stability. Mass spectrometry identifies two ubiquitination sites on PER (Lys1117 and Lys1118) critical for its degradation. Expression of PER1117A, 1118A mutant in per01 background impairs circadian rhythm strength. In conclusion, this study demonstrates that Drosophila USP14 indirectly modulates PER protein stability by affecting SLIMB and highlights the critical role of specific ubiquitination sites on PER in maintaining circadian rhythms.

X-linked ubiquitin-specific peptidase 11 (USP11) increases susceptibility to Cushing’s disease in women

The incidence of pituitary adrenocorticotropic hormone (ACTH)-secreting PitNETs, commonly known as ACTH PitNETs, is significantly higher in females; however, the underlying causes for this gender disparity remain unclear. In this study, we analyzed the expression of deubiquitinating enzymes in functioning ACTH PitNETs from both male and female subjects using RNA sequencing and identified USP11 as a potential susceptibility factor contributing to the higher prevalence of these PitNETs in females. Further investigation revealed that USP11 expression is markedly elevated in female functioning ACTH PitNETs, with levels significantly higher than those observed in male PitNETs and normal pituitary tissue. Experimental data indicate that USP11 promotes the transcription of proopiomelanocortin (POMC) and the secretion of ACTH. In contrast, knockdown of USP11 leads to a substantial reduction in both POMC transcription and ACTH secretion, as demonstrated in both in vitro and in vivo models. Mechanistically, we found that USP11 facilitates the deubiquitination of the key transcription factor TPIT in functioning ACTH PitNETs, enhancing its protein stability and thereby promoting POMC transcription and ACTH secretion. Additionally, virtual screening identified Lomitapide and Nicergoline as potential inhibitors of USP11, reducing POMC expression and ACTH secretion. Thus, USP11 emerges as a potential therapeutic target, and drugs aimed at inhibiting its function could benefit women with Cushing’s disease.

Ubiquitin-specific peptidase 14 promotes neuron injury by stabilizing acyl-CoA synthetase long-chain family member 4 through deubiquitination

Objective: Ubiquitin-specific peptidase 14 (USP14) may be a target for stroke treatment. Our study aims to explore the molecular mechanism of USP14 in the stroke process. Material and Methods: A stroke cell model was constructed using oxygen–glucose deprivation/reoxygenation (OGD/R)-induced SK-N-SH cells, and cell growth was assessed using cell counting kit 8 assay, EdU assay, and flow cytometry. Proinflammatory cytokine levels were tested through an enzyme-linked immunosorbent assay. The levels of USP14 and acyl-CoA synthetase long-chain family member 4 (ACSL4) were determined through Western blot and quantitative real-time polymerase chain reaction, whereas the interaction of USP14 and ACS14 was evaluated by co-immunoprecipitation assay. Results: OGD/R-induced SK-N-SH cell injury by enhancing ferroptosis and the knockdown of USP14 inhibited OGD/R-induced cell inflammation, apoptosis, and ferroptosis. Moreover, USP14 enhanced ACSL4 protein expression through deubiquitination. ACSL4 silencing mitigated neuron injury, and ACSL4 upregulation abolished USP14 knockdown-mediated inhibition of neuron injury. Conclusion: USP14 can enhance neuron injury through stabilizing ACSL4 protein expression.

Discovery and Optimization of a Series of Novel Morpholine-Containing USP1 Inhibitors.

Ubiquitin-specific protease 1 (USP1), a well-known member of the deubiquitinating enzymes, serves as a key regulator in DNA damage repair (DDR) processes. Herein, we utilized ring-opening and cyclization strategies based on KSQ-4279 to design a novel series of USP1 inhibitors featuring a morpholine scaffold. Notably, compound 38-P2 exhibited a more potent enzymatic and cellular inhibition activity compared to KSQ-4279. Mechanistically, 38-P2 was characterized as a selective, reversible, and noncompetitive USP1 inhibitor. 38-P2 efficiently activated the DDR pathway, induced cell cycle arrest and cell apoptosis, and inhibited cell survival. Importantly, it enhanced the sensitivity of olaparib-resistant cells to olaparib and showed a synergetic effect with andrographolide in BRCA-proficient cancer cells. Furthermore, 38-P2 had favorable pharmacokinetic profiles and good safety properties in vitro and in vivo. In the MDA-MB-436 xenograft model, 38-P2 displayed significant, dose-dependent antitumor efficacy. Overall, these findings indicate that 38-P2 is a promising lead compound for further drug development.

Tumor-Intrinsic SIRPA Drives Pyroptosis Evasion in Head and Neck Cancer.

Pyroptosis, a gasdermin-mediated immunogenic cell death, has been shown to elicit adaptive antitumor immune responses, thereby augmenting the response to cancer immunotherapy when pyroptosis is therapeutically activated. However, despite increased gasdermin E (GSDME) expression, significant pyroptosis remains elusive in certain tumor types, and the underlying regulatory mechanisms are poorly understood. In this study, we observed high signal regulatory protein α1 (SIRPA) expression in head and neck squamous cell carcinoma (HNSCC) cells, a target in cancer immunotherapy. Intriguingly, SIRPA inhibition markedly augmented pyroptosis activity in tumor tissues and modulated tumor growth in a HNSCC mouse model. Subsequent investigations revealed that SIRPA knockout upregulated GSDME expression and potentiated cisplatin-induced pyroptosis in cancer cells. Integrative transcriptomics and metabolomics analysis suggested that the SIRPA knockout profoundly altered protein ubiquitination and augmented argininosuccinic acid levels in cancer cells. Specifically, we demonstrated that ubiquitin-specific peptidase 18 (USP18), a deubiquitinating enzyme, targets GSDME for deubiquitination and that USP18 knockdown suppressed cisplatin-induced pyroptosis. Notably, we found that succinylation of GSDME, which is mediated by succinyl-CoA, promotes GSDME cleavage without affecting caspase-3 activation. Further experiments indicated that SIRPA expression in tumor cells can decrease the antitumor efficacy of chemotherapy and immunotherapy in HNSCC mouse models. In summary, our findings reveal a novel mechanism of pyroptosis evasion in HNSCC, whereby tumor-intrinsic SIRPA enhances GSDME ubiquitylation and inhibits its succinylation. These insights suggest that inhibiting SIRPA expression may improve the efficacy of immunotherapy for HNSCC by inducing pyroptosis.

Variable transduction of thyroid hormone signaling in structures of the mouse brain

L-thyroxine (L-T4) monotherapy is the standard treatment for hypothyroidism, administered daily to normalize TSH levels. Once absorbed, T4 is converted to T3 to alleviate most symptoms. However, this treatment abnormally elevates plasma T4 levels in over 50% of patients. Using L-T4-treated Thyroid Hormone (TH) Action Indicator mice, which express a T3-regulated luciferase (Luc) reporter, we examined whether these T4 elevations disrupt TH signaling. Hypothyroid mice exhibited reduced Luc expression across brain regions, and L-T4 treatment failed to restore T3 signaling uniformly. There was also variability in the activity of type 2 deiodinase (D2), the enzyme that generates most brain T3. Intracerebroventricular T4 administration achieved higher elevation of Luc expression in the mediobasal hypothalamus compared to the cortex, and studies on cultured cortical astrocytes and hypothalamic tanycytes revealed cell-type-specific responses to T4. In tanycytes, exposure to T4 sustained D2 activity, leading to progressive T3 signaling, whereas in astrocytes, T4 exposure triggered a drop in D2 activity, limiting T3 production through a ubiquitin-dependent, self-limiting mechanism. The sustained D2 activity in tanycytes was linked to rapid deubiquitination by USP33, as confirmed using a ubiquitin-specific protease (USP) pan-inhibitor and USP33 knockout mice. In conclusion, the brain's response to L-T4 treatment is heterogeneous, influenced by cell-specific regulation of D2-mediated T3 production. While cortical astrocytes exhibit limited T3 signaling due to D2 ubiquitination, tanycytes coexpressing USP33 amplify T3 signaling by rescuing ubiquitinated D2 from proteasomal degradation. These findings provide mechanistic insights into the limitations of L-T4 therapy and highlight the need for tailored approaches to managing hypothyroidism.

Cardiomyocyte Foxp1‐Specific Deletion Promotes Post‐injury Heart Regeneration via Targeting Usp20‐HIF1ɑ‐Hand1 Signaling Pathway

AbstractThe adult mammalian heart has limited regenerative capacity to replace lost tissue after a major injury. Forkhead box P1 (Foxp1) regulates embryonic cardiomyocyte proliferation and heart development. However, whether Foxp1 participates in postnatal‐injury cardiomyocyte proliferation and heart regeneration remains unclear. This study demonstrates that Foxp1 is downregulated at border zone cardiomyocytes of both neonatal apical resection and adult myocardial infarction. Analysis of the Single‐cell transcriptome database reveals reduced Foxp1 expression in the cardiomyocyte population with high regenerative capacity. Cardiomyocyte‐Foxp1 loss‐of‐function significantly promotes, whereas cardiomyocytes‐Foxp1 gain‐of‐function suppresses cardiomyocyte proliferation. Mechanistically, Foxp1 directly regulates ubiquitin specific peptidase 20 (USP20), a de‐ubiquitinase that prevents hypoxia inducible factor 1ɑ (HIF1α) degradation. Thus, Foxp1 regulates HIF1α and downstream heart and neural crest derivatives expressed 1 (Hand1) to control the cardiomyocyte proliferation via metabolic transition from fatty acid oxidation to glycolysis. Finally, cardiac type troponin T2 (cTnT)‐promoter‐driven adeno‐associated virus 9 (AAV9) for Hand1 induction in cardiomyocytes significantly promoted cardiac regeneration and functional recovery. These findings may provide novel molecular strategies to promote heart regeneration and therapeutic interventions for heart failure.

USP7 deficiency promotes diabetic wound healing by repressing GATA3-mediated pro-inflammatory macrophage polarization.

The polarization of inflammatory macrophages is an important factor contributing to delay wound healing during in diabetic foot ulcers (DFU). In this study, the role of ubiquitin-specific protease 7 (USP7) in regulating macrophage polarization during DFU progression was investigated. Gene and protein expression levels were assessed using qRT-PCR and western blot. In vitro and in vivo diabetes mellitus (DM) models were established by HG treatment and STZ injection, respectively. HUVEC viability, migration, and angiogenesis were detected by CCK8 assay, wound healing assay, and tube formation assay, respectively. Flow cytometry was employed to analyze the levels of macrophage polarization markers. Co-IP assay was performed to analyze the interaction between USP7 and GATA3. Our results demonstrated that USP7 was overexpressed in ulcer margin tissues of DFU patients, wound tissues of DFU mice, and HG-treated macrophages. Functionally, USP7 deficiency inhibited macrophage M1 polarization and promoted wound healing in DFU mice. In vitro, USP7 knockdown promoted HUVEC proliferation, migration, and angiogenesis by inducing M2 macrophage polarization and inhibiting M1 macrophage polarization under HG condition. Mechanistically, USP7 could stabilized GATA3 protein in macrophages by deubiquitinating GATA3. Moreover, the effects of USP7 knockdown on HUVEC function and macrophage polarization under HG condition were partially reversed by GATA3 overexpression. USP7 silencing enhanced wound healing in DFU by inhibiting M1 macrophage polarization and promoting M2 macrophage polarization through mediating GATA3 deubiquitylation.

USP14 is crucial for proteostasis regulation and α-synuclein degradation in human SH-SY5Y dopaminergic cells.

Ubiquitin specific protease-14 (USP14) is critical for controlling proteostasis disturbed in human disorders, including Parkinson's disease (PD). Here we investigated USP14 in the regulation of α-synuclein (α-syn) degradation via the proteasome and autophagy. α-Syn and pS129 α-syn were elevated in USP14 gene-deleted SH-SY5Y dopaminergic cells with decreased proteasome activity. However, autophagy and coordinated lysosomal expression and regulation pathways were elevated in USP14 lacking cells with higher levels of the transcription factor TFEB. There was an increase in reactive oxidative species (ROS) and elongated mitochondria in USP14 deficient cells and counteracting oxidative stress decreased α-syn levels. Phosphoproteomics revealed that USP14 is phosphorylated at residue S143 that reduces its binding to the proteasome. Re-expression of wild-type and phospho-mimetic S143D-USP14 mutant lowered ROS and α-syn levels in USP14 lacking cells. USP14 is a promising factor to consider in PD to target α-syn through its regulation of proteasomes and oxidative stress in dopaminergic neurons.

USP13: A therapeutic target for combating tumorigenesis and antitumor therapy resistance.

Ubiquitin-specific peptidase 13 (USP13) has emerged as a key regulator of proteins critical to the hallmarks of cancer, playing an essential role in cellular regulation. This deubiquitinating enzyme, often overexpressed in malignancies, wields its molecular scissors precisely, snipping ubiquitin tags to rescue oncoproteins from degradation. Our review highlights the dual role of USP13 in cancer biology: while it predominantly fuels tumor growth and metastasis, USP13 occasionally functions as a tumor suppressor. USP13 is as a formidable factor in cancer therapy, fortifying tumors against an arsenal of treatments. It bolsters DNA repair mechanisms, ignites prosurvival autophagy, and even reprograms cell lineages to evade targeted therapies. However, USP13 is also a promising target in the treatment of cancer. We highlight burgeoning strategies to neutralize USP13, from small molecule inhibitors to innovative protein degraders, which may disarm cancer resistance mechanisms. We also offer suggestions for future USP13 research, emphasizing the need for structural insights and more potent inhibitors. This review highlights the critical role of USP13 in cancer and underscores its potential as a therapeutic target for advancing cancer treatment.

Ubiquitin-specific peptidase 11 selectively interacts with and deubiquitination-dependently stabilizes diacylglycerol kinase δ to maintain cellular glucose uptake

Diacylglycerol kinase δ (DGKδ) phosphorylates diacylglycerol and converts it into phosphatidic acid. DGKδ contributes to glucose uptake as one of its cellular functions. However, detail mechanisms underlying the regulation of DGKδ protein stability remain unelucidated. Herein, we identified ubiquitin-specific peptidase 11 (USP11) in the DGKδ protein complex by DGKδ-interactome analysis. By mapping analysis, we clarified that a wider region of USP11, including the catalytic domain 1 region, and both the C1 domains and catalytic subdomain-a of DGKδ mainly contributed to their association. Cellular dysfunction of USP11 by mitoxiantrone (a USP11-specific inhibitor) or siRNA knockdown markedly decreased DGKδ protein levels. Additionally, we found that DGKδ ubiquitination was increased by USP11 dysfunction, and cumulative ubiquitination was reduced by rescue manipulation. Functionally, USP11 dysfunction reduced cellular glucose uptake. Altogether, our findings provide the first evidence that USP11 deubiquitination-dependently stabilizes DGKδ to maintain cellular glucose uptake.

Patchouli alcohol from Pogostemon cablin Benth inhibits H1N1 infection by repressing inflammasome and proptosis by targeting ubiquitin specific peptidase USP18.

Influenza virus infection can cause lung inflammation and viral pneumonia in patients. Patchouli alcohol (PA), a tricyclic sesquiterpene derived from Pogostemonis Herba, has been shown to alleviate inflammation in various diseases. However, the molecular mechanism by which patchouli exerts its anti-inflammatory effects, particularly its role in mitigating influenza virus induced inflammation and pneumonia during H1N1 viral infection, remains largely unclear. Herein, we found that PA considerably reduced body weight loss, lung pathological index and attenuated lung histological damage in H1N1-infected mice. Mechanistically, PA reduced the production and secretion of inflammatory cytokines via inhibition of the NF-κB-signaling pathway and blocking inflammasome-mediated proptosis. Additionally, proteomic analysis identified several potential targets of PA, with ubiquitin-specific peptidase 18 (USP18) emerging as a key candidate. Further investigation revealed that PA binds to USP18, enhancing its stability and increasing its transcriptional and translational expression. Overall, our results emphasize the anti-inflammatory effects of PA during influenza virus infection. PA may alleviate lung inflammation and damage by targeting USP18, offering a potential therapeutic strategy for treating influenza-induced lung complications.

Deubiquitinase USP9X controls Wnt signaling for CNS vascular formation and barrier maintenance.

Deubiquitinating enzymes play crucial roles in various cellular activities, yet their involvement in central nervous system (CNS) vascularization and barrier function remains elusive. Canonical Wnt signaling is essential for proper CNS vascularization and barrier maintenance. Using a loss-of-function screening for Wnt-signaling activity, we identified ubiquitin-specific peptidase 9 X-linked (USP9X) as a key regulator in brain endothelial cells (BECs). Endothelium-specific Usp9x knockout mice exhibit reduced Wnt-signaling activity, compromising CNS vascularization and barrier function during development. Activation of Wnt signaling rescues these defects. Mechanistically, we identified β-catenin as a direct substrate of USP9X, with USP9X catalyzing K48 polyubiquitin chains to stabilize β-catenin. In pathological mouse models of impaired CNS vascular barrier function, including intracerebral hemorrhage and an oxygen-induced retinopathy, loss of Usp9x intensifies barrier disruption, accentuating defects. This finding implicates USP9X as a critical regulator of CNS vascularization and barrier function through Wnt signaling, offering insights into CNS disease implications.

Redox-Induced Stabilization of AMBRA1 by USP7 Promotes Intestinal Oxidative Stress and Colitis Through Antagonizing DUB3-Mediated NRF2 Deubiquitination.

Inflammatory bowel disease (IBD) is associated with oxidative stress and redox signaling disruption. It is recently reported that proautophagic autophagy/beclin-1 regulator 1 (AMBRA1) is a positive modulator of the NF-κB pathway that promotes intestinal inflammation. However, its effect on intestinal redox state and whether AMBRA1 is regulated by oxidative stress remain unknown. In this study, it is found that AMBRA1 functions as a pro-oxidative factor that increases oxidative stress in intestinal epithelial cells (IECs) in vitro and in vivo. Mechanistically, the N-terminal F1 domain is required for AMBRA1 to competitively interact with the N-terminal domain of NRF2, thereby antagonizing the interaction between deubiquitinating protein 3 (DUB3) and NRF2, suppressing DUB3-mediated NRF2 deubiquitination, and leading to NRF2 degradation. In response to H2O2 stimulation, the interaction between AMBRA1 and ubiquitin-specific protease 7 (USP7) is enhanced, facilitating USP7 to deubiquitinate AMBRA1 at K83 and K86 and stabilize AMBRA1. Notably, the USP7 inhibitor, P5091, inhibits oxidative stress and colitis in vivo. Elevated AMBRA1 expression in inflamed colon tissues from ulcerative colitis patients is negatively correlated with decreased NRF2 protein levels. Overall, this study identifies AMBRA1 as a pro-oxidative factor in IECs and provides a redox-modulating therapeutic strategy for targeting USP7/AMBRA1 in IBD.

Usp7 contributes to the tail regeneration of planarians via Islet/Wnt1 axis.

Regeneration plays a key role in energy recycling and homeostasis maintenance. Planarians, as ideal model animals for studying regeneration, stem cell proliferation, and apoptosis, have the strong regenerative abilities. Considerable evidence suggests that ubiquitin plays an important role in maintaining homeostasis and regulating regeneration, but the function of Ubiquitin specific proteases 7 (Usp7) on regeneration in planarians remains elusive. We identified an evolutionarily conserved gene, Usp7, and utilized RNA interference (RNAi), Quantitative real-time PCR (qRT-PCR), Whole-mount immunofluorescence, Tunnel, Whole-mount in situ hybridization (WISH), and western blotting to detect the function of Usp7 during the planarian regeneration. In this study, we found that the regenerative trunk fragments in the Usp7 RNAi worms could not regenerate missing tails; meanwhile, the level of cell proliferation was decreased, while cell apoptosis was increased. Furthermore, the expression of Islet was inhibited in the Usp7 RNAi worms during planarian regeneration. The hybridization signal of wnt1/P-1 exhibited the dot-like pattern at the posterior of the regenerating planarians after Usp7 RNAi at regenerative 1day (R 1 d). However, the concentrated expression pattern wnt1/P-1 dramatically declined at regenerative 3days (R 3 d) and disappeared at regenerative 7days (R 7 d). In addition, activating the Wnt pathway partially rescued regenerative defects induced by inhibition of Usp7. Collectively, Usp7 is necessary for tissue regeneration and tail blastema formation partially by regulating the cell proliferation and apoptosis during planarian regeneration. It could also promote the posterior polarity reconstruction of the regenerative planarians via the Islet/Wnt1 axis.

Corneal stromal cells from patients with keratoconus exhibit alterations in the ESCRT-dependent machinery responsible for multivesicular body formation.

Previous studies have reported that exosomes produced by corneal stromal cells from keratoconus patients exhibit a molecular content distinct from those produced by cells from healthy donors. This study investigates differences in the expression of ESCRT components, regarded as the most critical mechanism in exosome biogenesis. The study included analysis of transcription levels of system-encoding genes using qRT-PCR reactions, as well as semiquantitative protein determination through immunocytochemistry. Of the 34 molecules analyzed, mRNA downregulation was observed in 8 in pathological cells. In keratoconus, genes encoding STAM2 from the ESCRT-0 complex and VPS37A, VPS37C, VPS37D and UBAP1 from the ESCRT-I complex were found to be underexpressed, although VPS37D could not be confirmed at the protein level. Additionally, two other expression alterations affected the ESCRT-III complex, involving the core protein CHMP4C and the regulatory protein CHMP1B. Finally, deregulation of the ubiquitin-specific peptidase UBPY was observed. Most changes identified in this study affected specific isoforms, which could suggest functional diversification and differences in cargo recognition in the context of pathology. Altogether, these findings suggest that the previously reported alteration in the molecular content of exosomes produced by stromal cells in keratoconus may be, at least partially, due to disruptions in the exosome synthesis machinery.

Caspase 3-specific cleavage of ubiquitin-specific peptidase 48 enhances drug-induced apoptosis in AML

ABSTRACT Dysfunction or dysregulation of deubiquitination is closely related to the initiation and development of multiple cancers. Targeted regulation of deubiquitination has been recognized as an important strategy in tumor therapy. However, the mechanism by which drugs regulate deubiquitinase is not clear. Here, we identified ubiquitin-specific peptidase 48 (USP48), a member of the ubiquitin-specific protease family highly expressed in various tumors, as a specific substrate for the activated caspase-3. During drug induced apoptosis of AML cells, activated caspase-3 cleaves USP48 through recognizing the conservative motif DEQD located at 611–614 sites of human USP48. Subsequent analysis showed that the cleavage USP48 N-terminal fragment which contains catalytic active domain is easily degraded by ubiquitination. Meanwhile knockdown experiment showed that inhibiting the expression of USP48 could also promotes apoptosis and enhance the efficacy of chemotherapy drugs. Altogether, these results suggest that targeting USP48 may represent a novel therapeutic strategy in AML.

Ubiquitin-specific protease 7 exacerbates acute pancreatitis progression by enhancing ATF4-mediated autophagy.

Acute pancreatitis (AP) is a serious inflammatory disease with high incidence rate and mortality. It was confirmed that overactivation of autophagy in acinar cells can increase the risk of AP. Nevertheless, the regulatory mechanism of autophagy in AP is unclear. The role of ubiquitin-specific peptidase 7 (USP7) in controlling autophagy during AP development was examined in this study. AR42J cells were subjected to caerulein to establish a cell model of AP. ELISA utilized to assess IL-6, IL-1β, and TNF-α secretion levels. Cell viability and death were detected using CCK8 assay and flow cytometry, respectively. The interaction between USP7 and ATF4 was analyzed by Co-IP assay. USP7 and ATF4 were abnormally overexpressed in AP patients and cellular models. Loss of function of USP7 increased cell viability, but alleviated cell death and secretions of inflammatory cytokines including IL-6, IL-1β, and TNF-α in AP cellular models. Importantly, autophagy level was activated in AP cells, and could be repressed after USP7 knockdown, and rapamycin treatment greatly diminished the beneficial functions mediated by USP7 downregulation in AP cells. Mechanically, ATF4, an activator of stress autophagy in AP, was proved to be a deubiquitination modification target downstream of USP7, and its protein stability was weakened after USP7 reduction. ATF4 upregulation abolished the protective effect of USP7 silencing on caerulein-induced autophagy, inflammation, and cell injury in AR42J cells. USP7 knockdown reduced inflammation and cell injury during AP progression by inhibiting ATF4-mediated autophagy activation.