USF1 transcriptionally activates USP14 to drive atherosclerosis by promoting EndMT through NLRC5/Smad2/3 axis.

BACKGROUND: Endothelial-to-Mesenchymal Transformation (EndMT) plays key roles in endothelial dysfunction during the pathological progression of atherosclerosis; however, its detailed mechanism remains unclear. Herein, we explored the biological function and mechanisms of upstream stimulating factor 1 (USF1) in EndMT during atherosclerosis. METHODS: The in vivo and in vitro atherosclerotic models were established in high fat diet-fed ApoE-/- mice and ox-LDL-exposed human umbilical vein endothelial cells (HUVECs). The plaque formation, collagen and lipid deposition, and morphological changes in the aortic tissues were evaluated by hematoxylin and eosin (HE), Masson, Oil red O and Verhoeff-Van Gieson (EVG) staining, respectively. EndMT was determined by expression levels of EndMT-related proteins. Target molecule expression was detected by RT-qPCR and Western blotting. The release of pro-inflammatory cytokines was measured by ELISA. Migration of HUVECs was detected by transwell and scratch assays. Molecular mechanism was investigated by dual-luciferase reporter assay, ChIP, and Co-IP assays. RESULTS: USF1 was up-regulated in atherosclerosis patients. USF1 knockdown inhibited EndMT by up-regulating CD31 and VE-Cadherin, while down-regulating α-SMA and vimentin, thereby repressing inflammation, and migration in ox-LDL-exposed HUVECs. In addition, USF1 transcriptionally activated ubiquitin-specific protease 14 (USP14), which promoted de-ubiquitination and up-regulation of NLR Family CARD Domain Containing 5 (NLRC5) and subsequent Smad2/3 pathway activation. The inhibitory effect of sh-USF1 or sh-USP14 on EndMT was partly reversed by USP14 or NLRC5 overexpression. Finally, USF1 knockdown delayed atherosclerosis progression via inhibiting EndMT in mice. CONCLUSION: Our findings indicate the contribution of the USF1/USP14/NLRC5 axis to atherosclerosis development via promoting EndMT, which provide effective therapeutic targets.

USP18 enhances the resistance of BRAF-mutated melanoma cells to vemurafenib by stabilizing cGAS expression to induce cell autophagy.

This study aims to discern the possible molecular mechanism of the effect of ubiquitin-specific peptidase 18 (USP18) on the resistance to BRAF inhibitor vemurafenib in BRAF V600E mutant melanoma by regulating cyclic GMP-AMP synthase (cGAS). The cancer tissues of BRAF V600E mutant melanoma patients before and after vemurafenib treatment were collected, in which the protein expression of USP18 and cGAS was determined. A BRAF V600E mutant human melanoma cell line (A2058R) resistant to vemurafenib was constructed with its viability, apoptosis, and autophagy detected following overexpression and depletion assays of USP18 and cGAS. Xenografted tumors were transplanted into nude mice for in vivo validation. Bioinformatics analysis showed that the expression of cGAS was positively correlated with USP18 in melanoma, and USP18 was highly expressed in melanoma. The expression of cGAS and USP18 was up-regulated in cancer tissues of vemurafenib-resistant patients with BRAF V600E mutant melanoma. Knockdown of cGAS inhibited the resistance to vemurafenib in A2058R cells and the protective autophagy induced by vemurafenib in vitro. USP18 could deubiquitinate cGAS to promote its protein stability. In vivo experimentations confirmed that USP18 promoted vemurafenib-induced protective autophagy by stabilizing cGAS protein, which promoted resistance to vemurafenib in BRAF V600E mutant melanoma cells. Collectively, USP18 stabilizes cGAS protein expression through deubiquitination and induces autophagy of melanoma cells, thereby promoting the resistance to vemurafenib in BRAF V600E mutant melanoma.

A proteome-scale map of the SARS-CoV-2-human contactome.

Understanding the mechanisms of coronavirus disease 2019 (COVID-19) disease severity to efficiently design therapies for emerging virus variants remains an urgent challenge of the ongoing pandemic. Infection and immune reactions are mediated by direct contacts between viral molecules and the host proteome, and the vast majority of these virus-host contacts (the 'contactome') have not been identified. Here, we present a systematic contactome map of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with the human host encompassing more than 200 binary virus-host and intraviral protein-protein interactions. We find that host proteins genetically associated with comorbidities of severe illness and long COVID are enriched in SARS-CoV-2 targeted network communities. Evaluating contactome-derived hypotheses, we demonstrate that viral NSP14 activates nuclear factor κB (NF-κB)-dependent transcription, even in the presence of cytokine signaling. Moreover, for several tested host proteins, genetic knock-down substantially reduces viral replication. Additionally, we show for USP25 that this effect is phenocopied by the small-molecule inhibitor AZ1. Our results connect viral proteins to human genetic architecture for COVID-19 severity and offer potential therapeutic targets.

Inhibition of USP14 enhances anti-tumor effect in vemurafenib-resistant melanoma by regulation of Skp2.

BACKGROUND: The mutation of BRAF V600E often occurred in melanoma and results in tumorigenesis. BRAF mutation drives hyperactivation of the RAF-MAPK-ERK pathway. The acquired drug resistance upon prolonged use of BRAF inhibitors (such as vemurafenib) still remains the main obstacle. Previously, we have found that E3 ligase Skp2 over-expresses vemurafenib-resistant melanoma cells, and knockdown of Skp2 enhances the anti-tumor effect of vemurafenib. Interestingly, the literature has reported that the selective USP14/UCHL5 inhibitor b-AP15 displays great potential in melanoma therapy; however, the molecular mechanism still remains unknown. METHODS: In vitro, the effect of the combination regimen of vemurafenib (Vem, PLX4032) and b-AP15 on vem-sensitive and vem-resistant melanoma has been investigated by wound healing, colony formation, transwell invasion assay, flow cytometry, lysosome staining, and ROS detection. In vivo, the combination effect on vem-resistant melanoma has been evaluated with a nude mice xenograft tumor model. GST-pulldown and co-immunoprecipitation (co-IP) assays have been applied to investigate the interactions between USP14, UCHL5, and Skp2. Cycloheximide (CHX) assay and ubiquitination assays have been used to explore the effect of USP14 on Skp2 protein half-life and ubiquitination status. RESULTS: In the present study, we have revealed that repression of USP14 sensitizes vemurafenib resistance in melanoma through a previously unappreciated mechanism that USP14 but not UCHL5 stabilizes Skp2, blocking its ubiquitination. K119 on Skp2 is required for USP14-mediated deubiquitination and stabilization of Skp2. Furthermore, the mutated catalytic activity amino acid cysteine (C) 114 on USP14 abrogates stabilization of Skp2. Stabilization of Skp2 is required for USP14 to negatively regulate autophagy. The combination regimen of Skp2 inhibitor vemurafenib and USP14/UCHL5 inhibitor b-AP15 dramatically inhibits cell viability, migration, invasion, and colony formation in vemurafenib-sensitive and vemurafenib-resistant melanoma. Vemurafenib and b-AP15 hold cells in the S phase thus leading to apoptosis as well as the formation of the autophagic vacuole in vemurafenib-resistant SKMEL28 cells. The enhanced proliferation effect of USP14 and Skp2 is mainly due to a more effective reduction of cell apoptosis and autophagy. Further evaluation of various protein alterations has revealed that the increased expression of cleaved-PARP, LC3, and decreased Ki67 are more obvious in the combination of vemurafenib and b-AP15 treatment than those in single-drug treatment. Moreover, the co-treatment of vemurafenib and b-AP15 dramatically inhibits the growth of vemurafenib-resistant melanoma xenograft in vivo. Collectively, our findings have demonstrated that the combination of Skp2 inhibitor and USP14 inhibitor provides a new solution for the treatment of BRAF inhibitor resistance melanoma.

UCHL1 Impairs Periodontal Ligament Stem Cell Osteogenesis in Periodontitis.

Periodontitis comprises a series of inflammatory responses resulting in alveolar bone loss. The suppression of osteogenesis of periodontal ligament stem cells (PDLSCs) by inflammation is responsible for impaired alveolar bone regeneration, which remains an ongoing challenge for periodontitis therapy. Ubiquitin C-terminal hydrolase L1 (UCHL1) belongs to the family of deubiquitinating enzymes, which was found to play roles in inflammation previously. In this study, the upregulation of UCHL1 was identified in inflamed PDLSCs isolated from periodontitis patients and in healthy PDLSCs treated with tumor necrosis factor-α or interleukin-1ß, and the higher expression level of UCHL1 was accompanied with the impaired osteogenesis of PDLSCs. Then UCHL1 was inhibited in PDLSCs using the lentivirus or inhibitor, and the osteogenesis of PDLSCs suppressed by inflammation was rescued by UCHL1 inhibition. Mechanistically, the negative effect of UCHL1 on the osteogenesis of PDLSCs was attributable to its negative regulation of mitophagy-dependent bone morphogenetic protein 2/Smad signaling pathway in periodontitis-associated inflammation. Furthermore, a ligature-induced murine periodontitis model was established, and the specific inhibitor of UCHL1 was administrated to periodontitis mice. The histological results showed increased active osteoblasts on alveolar bone surface and enhanced alveolar bone regeneration when UCHL1 was inhibited in periodontitis mice. Besides, the therapeutic effects of UCHL1 inhibition on ameliorating periodontitis were verified, as indicated by less bone loss and reduced inflammation. Altogether, our study proved UCHL1 to be a key negative regulator of the osteogenesis of PDLSCs in periodontitis and suggested that UCHL1 inhibition holds promise for alveolar bone regeneration in periodontitis treatment.

USP9X prevents AGEs-induced upregulation of FN and TGF-ß1 through activating Nrf2-ARE pathway in rat glomerular mesangial cells.

Oxidative stress is a key pathological factor for diabetic renal fibrosis by activating TGF-ß/Smad pathway in glomerular mesangial cells (GMCs) to promote the synthesis of extracellular matrix such as fibronectin (FN). Nuclear factor-E2-related factor (Nrf2)- anti-oxidant response element (ARE) anti-oxidative pathway has crucial renoprotective effects, and inhibiting ubiquitin-mediated degradation of Nrf2 delays diabetic renal fibrosis development. Ubiquitin-specific protease 9X (USP9X) has close relationship with oxidative stress and TGF-ß/Smad pathway, but whether it regulate diabetic renal fibrosis remains unclarified. Here, we found that advanced glycation-end products (AGEs) dose- and time-dependently reduced the protein expression and deubiquitinase activity of USP9X in GMCs. USP9X overexpression attenuated AGEs-induced upregulation of FN, TGF-ß1, and Collagen Ⅳ, three fibrosis-related marker proteins, in a deubiquitinase activity-dependent manner. While USP9X depletion with siRNAs further promoted the expressions of those proteins in AGEs-treated GMCs. Under AGEs treatment conditions, USP9X overexpression markedly increased the total and nuclear levels, ARE-binding ability, and transcriptional activity of Nrf2, upregulated the protein expressions of Nrf2 downstream genes HO-1 and NQO1, and eventually reduced the excessive production of ROS. Overexpression of the deubiquitinase catalytically inactive USP9X-C1556S mutant failed to exert such effects. Silencing Nrf2 abolished the renoprotective effects of USP9X. Further study showed that upon AGEs stimulation, Nrf2 transferred into the nucleus and the interaction between USP9X and Nrf2 was weakened. AGEs also increased Nrf2 ubiquitination level, and overexpression of USP9X, instead of USP9X-C1556S, significantly reduced the ubiquitination level of Nrf2. Taken together, USP9X reduced Nrf2 ubiquitination level and promoted Nrf2-ARE pathway activation to prevent the accumulation of extracellular matrix, eventually alleviated the pathological process of diabetic renal fibrosis.

USP9X promotes the progression of hepatocellular carcinoma by regulating beta-catenin.

Hepatocellular carcinoma (HCC) is among the malignant tumors with highest mortality. The role of USP9X in the carcinogenesis of HCC has not yet been determined. In this study, USP9X was found significantly highly expressed in the intratumor tissues. Expression of intratumor USP9X was associated with tumor size and microvascular invasion while USP9X is independent risk factor of HCC disease-free survival and overall survival. In vitro studies revealed that knockdown of USP9X significantly inhibited the proliferation of HCC cells. Mechanically, USP9X promotes HCC cell proliferation by regulating the expression of beta-catenin. The results of the present study demonstrated that high expression of USP9X in intratumoral cells is associated with poor HCC prognosis, which may serve as a potential target for an adjuvant therapy.

Versatility of USP18 in physiology and pathophysiology.

Ubiquitin-specific peptidase 18 (USP18) is a multifunctional protein and its roles are still being investigated. This enzyme removes ubiquitin-like molecules from their substrates and the only known interferon-stimulated gene 15 (ISG15) specific protease. Apart from its enzymatic function, it also inhibits interferon type I and III signalling pathways. USP18 is known to regulate multiple processes, such as: cell cycle, cell signalling and response to viral and bacterial infections. Moreover, it contributes to the development of several autoimmune diseases and carcinogenesis, and recently was described as a cardiac remodelling inhibitor. This review summarizes the current knowledge on USP18 functions, highlighting its contribution to the development of heart failure, given the fact that this disease's etiology is now considered to be inflammatory in nature.

Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells.

USP14 is a cysteine protease deubiquitinase associated with the proteasome and plays important catalytic and allosteric roles in proteasomal degradation. USP14 inhibition has been considered a therapeutic strategy for accelerating degradation of aggregation-prone proteins in neurodegenerative diseases and for inhibiting proteasome function to induce apoptotic cell death in cancers. Here we studied the effects of USP14 inhibition in mammalian cells using small molecule inhibitors and an inactive USP14 mutant C114A. Neither the inhibitors nor USP14 C114A showed consistent or significant effects on the level of TDP-43, tau or α-synuclein in HEK293T cells. However, USP14 C114A led to a robust accumulation of ubiquitinated proteins, which were isolated by ubiquitin immunoprecipitation and identified by mass spectrometry. Among these proteins we confirmed that ubiquitinated ß-catenin accumulated in the cells expressing USP14 C114A with immunoblotting and immunoprecipitation experiments. The proteasome binding domain of USP14 C114A is required for its effect on ubiquitinated proteins. UCHL5 is the other cysteine protease deubiquitinase associated with the proteasome. Interestingly, the inactive mutant of UCHL5 C88A also caused an accumulation of ubiquitinated proteins in HEK293T cells but did not affect ß-catenin, demonstrating USP14 but not UCHL5 has a specific effect on ß-catenin. We used ubiquitin immunoprecipitation and mass spectrometry to identify the accumulated ubiquitinated proteins in UCHL5 C88A expressing cells which are mostly distinct from those identified in USP14 C114A expressing cells. Among the identified proteins are well established proteasome substrates and proteasome subunits. Besides ß-catenin, we also verified with immunoblotting that UCHL5 C88A inhibits its own deubiquitination and USP14 C114A inhibits deubiquitination of two proteasomal subunits PSMC1 and PSMD4. Together our data suggest that USP14 and UCHL5 can deubiquitinate distinct substrates at the proteasome and regulate the ubiquitination of the proteasome itself which is tightly linked to its function.

Innervation and functional characteristics of connective tissues, especially elastic fibers, in human fetal thoracic intervertebral articular capsule and its surroundings.

The articular capsules between the thoracic vertebrae, which have physiologically different functions from those of other levels of the vertebrae, have yet to be subjected to neuro-anatomical and fine structural analysis. In the present study, we analyzed serial frozen sections of decalcified thoracic vertebrae in human fetuses, and identified the articular capsule tissue with its unique distribution of elastic fibers. The fine structure of the elastic fibers was studied by transmission electron microscopy. In the early-stage fetus, the fibrous membrane forming the lateral intervertebral articular capsule contained abundant thin elastic fibers consisting of microfibrils. In the late-stage fetus, the lateral capsule of fibrous membrane was occupied by thick elastic fibers. A medial articular capsule, namely the ligamenta flava, contained numerous thick elastic fibers in both early and late-stage fetuses. The distributional differences in nerve fibers between early and late-stage fetuses were determined by immunostaining, using antibodies raised against protein gene product 9.5 (PGP 9.5; ubiquitin carboxyl-terminal hydrolase). Innervation by PGP 9.5 immunoreactive fibers was limited to the areas of the articular capsules near the blood vessels, which may indicate their functional relation with blood flow. No PGP 9.5 immunoreactive fibers were found in the ligamenta flava of the late-stage fetus. Innervation might be directly involved in the development of the intervertebral articular capsules in normal human fetuses.