Ubiquitin-specific Protease Research Articles

Esculin alleviates lipopolysaccharide (LPS)-induced pneumonia by regulating the USP7/MAPK14 axis.

Pneumonia is a serious and life-threatening lung inflammation with high morbidity and mortality. Accumulating evidence has suggested that esculin, a derivative of coumarin, possesses potent anti-inflammatory effects. This study is designed to explore the pharma role and underlying mechanism of esculin against lipopolysaccharides (LPS)-induced pneumonia. TC-1 cells were stimulated by LPS to mimic the inflammatory injury model in vitro. Cell viability, proliferation, and apoptosis were determined using MTT assay, 5-ethynyl-2'-deoxyuridine assay, and flow cytometry. Interleukin-1β and tumor necrosis factor α levels were analyzed using an enzyme-linked immunosorbent assay. Reactive oxygen species and superoxide dismutase were examined using special assay kits. Macrophage polarization was detected using flow cytometry. Mitogen-activated protein kinase 14 (MAPK14) level was detected by real-time quantitative polymerase chain reaction. MAPK14 and ubiquitin-specific protease 7 (USP7) protein levels were determined using western blot assay. After Ubibrowser database prediction, the interaction between USP7 and MAPK14 was verified using a Co-immunoprecipitation assay. The biological role of esculin was verified in LPS-challenged ALI mice in vivo. Here, we found that esculin significantly relieved LPS-induced TC-1 cell proliferation inhibition, and apoptosis, inflammatory response, oxidative stress, and M1-type macrophage polarization promotion. MAPK14 and USP7 expressions were enhanced in LPS-treated TC-1 cells, which was partly abolished by esculin treatment. Overexpressing MAPK14 attenuated the repression of esculin on LPS-triggered TC-1 cell injury. At the molecular level, USP7 interacted with MAPK14 and maintained its stability by removing ubiquitin. Moreover, esculin repressed the progression of pneumonia in vivo by regulating MAPK14. Taken together, esculin exposure could mitigate LPS-induced TC-1 cell injury partly by targeting the USP7/MAPK14 axis, providing a better understanding of the role of esculin in the anti-inflammatory therapeutics for pneumonia.

USP13 regulates ferroptosis in chicken follicle granulosa cells by deubiquitinating ATG7

The development and maturation of follicles are intricately linked to egg production and reproductive performance of chickens. Granulosa cells death directly affects the development and maturation of follicles, thereby impacting the reproductive performance of hens. Ferroptosis is a new type of cell death, it is unknown how it affects the growth and development of chicken follicles. In this study, RNA-seq analysis revealed significant differences in the expression of ferroptosis-related genes between normal follicles and atretic follicles, suggesting a potential role for ferroptosis in follicle growth and development. In addition, we found that ubiquitin-specific protease 13 (USP13) was significantly upregulated in atrophic follicles. Overexpression of USP13 results in depletion of glutathione (GSH), peroxidation of lipids, accumulation of iron, and activation of ferroptosis in chicken granulosa cells. In contrast, USP13 knockdown significantly inhibited ferroptosis events. Mechanistically, USP13 prevents the degradation of autophagy related 7 (ATG7) by deubiquitinating it, thereby enhancing the stability of ATG7 protein and ultimately promoting ferroptosis. In conclusion, this study elucidates the crucial role of the USP13-ATG7 axis in regulating ferroptosis in chicken follicle granulosa cells, thereby presenting a novel avenue for molecular breeding research in chickens.

Multi-regulatory potency of USP1 on inflammasome components promotes pyroptosis in thyroid follicular cells and contributes to the progression of Hashimoto's thyroiditis

BackgroundInflammatory diseases are often initiated by the activation of inflammasomes triggered by pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs), which mediate pyroptosis. Although pyroptosis resulting from aberrant inflammasome triggering in thyroid follicular cells (TFCs) has been observed in Hashimoto's thyroiditis (HT) patients, the underlying mechanisms remain largely unknown. Given the extensive involvement of protein ubiquitination and deubiquitination in inflammatory diseases, we aimed to investigate how deubiquitinating enzymes regulate thyroid follicular cell pyroptosis and HT pathogenesis.MethodsOur study specifically investigated the role of Ubiquitin-specific peptidase 1 (USP1), a deubiquitinase (DUB), in regulating the inflammasome components NLRP3 and AIM2, which are crucial in pyroptosis. We conducted a series of experiments to elucidate the function of USP1 in promoting pyroptosis associated with inflammasomes and the progression of HT. These experiments involved techniques such as USP1 knockdown or inhibition, measurement of key pyroptosis indicators including caspase-1, caspase-1 p20, and GSDMD-N, and examination of the effects of USP1 abrogation on HT using a mouse model. Furthermore, we explored the impact of USP1 on NLRP3 transcription and its potential interaction with p65 nuclear transportation.ResultsOur findings provide compelling evidence indicating that USP1 plays a pivotal role in promoting inflammasome-mediated pyroptosis and HT progression by stabilizing NLRP3 and AIM2 through deubiquitination. Furthermore, we discovered that USP1 modulates the transcription of NLRP3 by facilitating p65 nuclear transportation. Knockdown or inhibition of USP1 resulted in weakened cell pyroptosis, as evidenced by reduced levels of caspase-1 p20 and GSDMD-N, which could be restored upon AIM2 overexpression. Remarkably, USP1 abrogation significantly ameliorated HT in the mice model, likely to that treating mice with pyroptosis inhibitors VX-765 and disulfiram.ConclusionsOur study highlights a regulatory mechanism of USP1 on inflammasome activation and pyroptosis in TFCs during HT pathogenesis. These findings expand our understanding of HT and suggest that inhibiting USP1 may be a potential treatment strategy for managing HT.

USP24 promotes hepatocellular carcinoma tumorigenesis through deubiquitinating and stabilizing TRAF2

Ubiquitin-specific peptidase 24 (USP24), a member of the deubiquitinase family, plays an important role in tumor regulation. However, the role of USP24 in Hepatocellular carcinoma（HCC）is unknown. The aim of our study was to explore the role of USP24 in HCC to seek new therapeutic targets for HCC. In this study, we found that USP24 was aberrantly upregulated in HCC tissues and predicted poor prognosis. USP24 markedly promoted HCC proliferation and progression in vitro and in vivo. Mechanistically, USP24 binds to tumor necrosis factor receptor-associated factor 2(TRAF2) and inhibits its degradation, thereby promoting the accumulation of TRAF2. Upregulation of TRAF2 activated protein kinase B/nuclear factor kappa-B (AKT/ NF-κB) signaling pathway and promoted HCC cell survival. In addition, USP24 positively correlated with programmed cell death ligand 1(PD-L1) expression in HCC, highlighting the clinical significance of USP24 activation in tumor immune evasion. Deletion of USP24 enhanced the tumor-killing ability of CD8+ T cells. Deletion of USP24 combined with anti-PD-1 antibody significantly enhanced the efficacy of HCC immunotherapy. Taken together, USP24 can be employed as a promising target to restrain tumor growth and increase the efficacy of HCC immunotherapy.

USP25 Promotes the Antimycobacterial Response of Macrophages Through Stabilizing B-Raf and C-Raf.

Ubiquitin-specific peptidase 25 (USP25) is one of the best-characterized deubiquitinating enzymes and plays a vital regulatory role in various biological processes, especially in cancer development and immune regulation. However, the exact role of USP25 and its underlying mechanisms in macrophage activation and immunogenicity during Mycobacterium tuberculosis infection remain unclear. In this study, we found that M tuberculosis infection induced USP25 expression in human and mouse macrophages. In particular, USP25 expression is elevated in multiple cell types, especially monocytes, in patients with tuberculosis. Additionally, USP25 deficiency in macrophages and mice resulted in compromised immunity against M tuberculosis infection, accompanied by reduced expressions of various proinflammatory cytokines and chemokines. Mechanistically, USP25 in macrophages promoted the activation of the ERK signaling pathway through deubiquitination and stabilization of B-Raf and C-Raf. These findings collectively suggest the critical roles of USP25 in M tuberculosis infection and its potential as a therapeutic target.

USP9X promotes LPS-induced fibroblast cell apoptosis, inflammation and oxidative stress by regulation of TBL1XR1 deubiquitination

Abstract Background Ubiquitination and deubiquitination are involved in the progression of human diseases, including acute pneumonia. In this study, we aimed to explore the functions of ubiquitin-specific peptidase 9X-linked (USP9X) in lipopolysaccharide (LPS)-treated WI-38 cells. Methods WI-38 cells were treated with LPS to induce the cellular damage and inflammation. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay and 5-ethynyl-2’-deoxyuridine (EdU) assay were performed to examine the proliferation of LPS-treated WI-38 cells. Flow cytometry analysis was conducted to detect LPS-treated WI-38 cell apoptosis. ELISA kits were utilized to determine the concentrations of inflammatory factors (IL-1β and TNF-α). Superoxide dismutase (SOD) activity and reactive oxygen species (ROS) level were examined with related kits. Ubibrowser (http://ubibrowser.bio-it.cn/ubibrowser/), ubiquitination assay and Co-Immunoprecipitation (Co-IP) assay demonstrated the interaction between USP9X and transducin β-like 1X related protein 1 (TBL1XR1). qRT-PCR assay and western blot assay were manipulated to determine the expression of USP9X and TBL1XR1. TBL1XR1 and USP9X knockdown experiments were conducted to explore their functions on LPS-induced WI-38 cell injury and inflammation. Results TBL1XR1 expression was upregulated in LPS-treated WI-38 cells. TBL1XR1 knockdown promoted cell proliferation and repressed apoptosis, inflammation and oxidative stress in LPS-treated WI-38 cells. Moreover, USP9X deubiquitinated TBL1XR1 to regulate TBL1XR1 expression. USP9X knockdown restored the effects of LPS on WI-38 cell proliferation, apoptosis, inflammation and oxidative stress, but these effects of USP9X knockdown were further abolished by TBL1XR1 overexpression. Additionally, USP9X promoted the NF-κB signaling pathway by the deubiquitination of TBL1XR1. Conclusion USP9X promoted the apoptosis, inflammation and oxidative stress of LPS-stimulated WI-38 cells through the deubiquitination of TBL1XR1.

Ubiquitin-Specific Protease 22 Plays a Key Role in Increasing Extracellular Vesicle Secretion and Regulating Cell Motility of Lung Adenocarcinoma.

Tumor-derived extracellular vesicles (EVs) are potential biomarkers for tumors, but their reliable molecular targets have not been identified. The previous study confirms that ubiquitin-specific protease 22 (USP22) promotes lung adenocarcinoma (LUAD) metastasis in vivo and in vitro. Moreover, USP22 regulates endocytosis of tumor cells and localizes to late endosomes. However, the role of USP22 in the secretion of tumor cell-derived EVs remains unknown. In this study, it demonstrates that USP22 increases the secretion of tumor cell-derived EVs and accelerates their migration and invasion, invadopodia formation, and angiogenesis via EV transfer. USP22 enhances EV secretion by upregulating myosin IB (MYO1B). This study further discovers that USP22 activated the SRC signaling pathway by upregulating the molecule KDEL endoplasmic reticulum protein retention receptor 1 (KDELR1), thereby contributing to LUAD cell progression. The study provides novel insights into the role of USP22 in EV secretion and cell motility regulation in LUAD.

The role of RNA viruses in human cancers

Many RNA viruses have been reported to be oncogenic (or carcinogenic) in a variety of animal and human cancers. The increase in the incidence and prevalence of cancer-causing viruses in human populations can be known as a key precursor to the development of various cancers. The retrovirus family and Hepatitis C virus (HCV) are also reported to cause cancer. Viral oncoproteins such as Tax of HTLV 1 interacts with cellular ubiquitination complex such as cyclindromatosis tumor suppressor, ubiquitin-specific proteases 7, 11, 15 and 20, A-20 and signal-transducing adaptor molecule binding protein-like-1 in order to improve the cellular signaling pathways. The viral oncoproteins binding to DUB, leading to proliferation of virus-infected cells and cell transformation. Proto-oncogenes (c-onc genes) are the cellular form of v-onc genes. The activation of c-onc genes leads to cell growth. C-onc genes are transformed into an oncogenic form by viral infection. C-onc genes play some roles such as protein kinases, growth factors, growth factor receptors, and DNA binding proteins. The study of transforming retroviruses and their oncogenes and the multiple mechanisms deployed by other RNA viruses to use the growth-suppressive and proapoptotic function of tumor suppressor genes has been added to our current understanding of cancer biology. Oncogenic RNA viruses are important experimental models to study molecular investigation such as cellular networks, including the discovery of oncogenes and tumor suppressors. Understanding of different strategies of RNA viruses as well as the function of their proteins helps to make more extensive plans regarding the adoption of follow-up, prevention and treatment strategies in cancer patients caused by viral origin.

Ubiquitin-specific protease UBP14 stabilizes HY5 by deubiquitination to promote photomorphogenesis in Arabidopsis thaliana

Transcription factor ELONGATED HYPOCOTYL5 (HY5) is the central hub for seedling photomorphogenesis. E3 ubiquitin (Ub) ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) inhibits HY5 protein accumulation through ubiquitination. However, the process of HY5 deubiquitination, which antagonizes E3 ligase-mediated ubiquitination to maintain HY5 homeostasis has never been studied. Here, we identified that Arabidopsis thaliana deubiquitinating enzyme, Ub-SPECIFIC PROTEASE 14 (UBP14) physically interacts with HY5 and enhances its protein stability by deubiquitination. The da3-1 mutant lacking UBP14 function exhibited a long hypocotyl phenotype, and UBP14 deficiency led to the failure of rapid accumulation of HY5 during dark to light. In addition, UBP14 preferred to stabilize nonphosphorylated form of HY5 which is more readily bound to downstream target genes. HY5 promoted the expression and protein accumulation of UBP14 for positive feedback to facilitate photomorphogenesis. Our findings thus established a mechanism by which UBP14 stabilizes HY5 protein by deubiquitination to promote photomorphogenesis in A. thaliana.

Active protein ubiquitination regulates xylem vessel functionality.

Xylem vessels function in the long-distance conduction of water in land plants. The NAC transcription factor VASCULAR-RELATED NAC-DOMAIN7 (VND7) is a master regulator of xylem vessel cell differentiation in Arabidopsis (Arabidopsis thaliana). We previously isolated suppressor of ectopic xylem vessel cell differentiation induced by VND7 (seiv) mutants. Here, we report that the responsible genes for seiv3, seiv4, seiv6, and seiv9 are protein ubiquitination-related genes encoding PLANT U-BOX46 (PUB46), an uncharacterized F-BOX protein (FBX), PUB36, and UBIQUITIN-SPECIFIC PROTEASE1 (UBP1), respectively. We also found decreased expression of genes downstream of VND7 and abnormal xylem transport activity in the seiv mutants. Upon VND7 induction, ubiquitination levels from 492 and 180 protein groups were upregulated and downregulated, respectively. VND7 induction resulted in the ubiquitination of proteins for cell wall biosynthesis and protein transport, whereas such active protein ubiquitination did not occur in the seiv mutants. We detected the ubiquitination of three lysine residues in VND7: K94, K105, and K260. Substituting K94 with arginine significantly decreased the transactivation activity of VND7, suggesting that the ubiquitination of K94 is crucial for regulating VND7 activity. Our findings highlight the crucial roles of target protein ubiquitination in regulating xylem vessel activity.

Abstract Or110: Inflammation-induced endothelial cell activation and angiogenic sprouting are downmodulated by ubiquitin-specific peptidase 20

The deubiquitinase ubiquitin-specific peptidase 20 (USP20) reverses lysine-63 ubiquitination of the signaling adaptor TRAF6 and suppresses nuclear-factor-κB (NFκB) activation induced by proatherogenic stimuli in vascular smooth muscle cells (SMCs). Dephosphorylation of USP20 at Ser 334 augments USP20/TRAF6 binding and USP20-mediated TRAF6 deubiquitination, consequently decreasing NFκB signaling. SMC USP20 activity also attenuates atherosclerosis in Ldlr -/- mice. NFκB is a key mediator of vascular endothelial cell (EC) activation and inflammation-driven angiogenesis, which promotes growth and rupture of atherosclerotic plaques. We tested the hypothesis that USP20 attenuates EC NFκB signaling and decreases angiogenesis, with both in vitro and ex vivo models to study the role of USP20 in EC activation and ensuing angiogenesis. We analyzed experimental data by one-way ANOVA followed by Holm-Šídák's multiple comparisons test. Cytokine-induced NFκB activity was elevated in primary ECs isolated from Usp20 -/- mice as compared with ECs from congenic wild type (WT) mice (N=3, p<0.001). Assessed by scratch-wound healing and spheroid assays, respectively, migration and angiogenesis of mouse coronary endothelial cells (MCECs) transduced with recombinant adenoviruses was (a) increased by inactive USP20 or phospho-mimetic USP20(S334D) (migration, N=4, P<0.05; angiogenesis, N=6, P<0.01), and (b) decreased by transduction with WT USP20 or phospho-resistant USP20(S334A) (migration, N=4, P< 0.05; angiogenesis, N=6, P<0.01). Additionally, chemical inhibition of NFκB activation with TPCA-1 (which inhibits the upstream kinase IKK-2) attenuated MCEC migration (N=4, P<0.0001) and angiogenesis (N=6, P<0.005), as compared with untreated cells. Angiogenesis assessed by aortic ring sprouting was increased in Usp20 -/- compared with WT specimens (males, N=3, P<0.0001; females, N=2, P<0.0001), and it was also suppressed by TPCA-1 (males, N=2, WT; N=3 Usp20 -/- , P<0.0001; females, N=2 WT; N=3, Usp20 -/- P<0.0005). Angiogenesis was enhanced in aortic rings from USP20(S334D) CRISPR/Cas9 gene-edited mice (males, N=3, P<0.01; females, N=3 WT & N=4, USP20(S334D) P<0.0001), but reduced in aortic rings from USP20(S334A) mice (males, N=3, P<0.05; females, N=3 WT & N=5 USP20(S334A) P<0.0001). We conclude that preserving EC USP20 activity by preventing phosphorylation of USP20 on Ser 334 diminishes endothelial activation and angiogenic sprouting, which can decrease atherosclerosis.

USP19 Stabilizes TAK1 to Regulate High Glucose/Free Fatty Acid-induced Dysfunction in HK-2 Cells.

Obesity-induced kidney injury contributes to the development of diabetic nephropathy (DN). Here, we identified the functions of ubiquitin-specific peptidase 19 (USP19) in HK-2 cells exposed to a combination of high glucose (HG) and free fatty acid (FFA) and determined its association with TGF-beta-activated kinase 1 (TAK1). HK-2 cells were exposed to a combination of HG and FFA. USP19 mRNA expression was detected by quantitative RT-PCR (qRT-PCR), and protein analysis was performed by immunoblotting (IB). Cell growth was assessed by Cell Counting Kit-8 (CCK-8) viability and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assays. Cell cycle distribution and apoptosis were detected by flow cytometry. The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation (Co-IP) assays and IB. In HG+FFA-challenged HK-2 cells, USP19 was highly expressed. USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells. Moreover, USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1 (PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species (ROS) generation in HK-2 cells. Mechanistically, USP19 stabilized the TAK1 protein through deubiquitination. Importantly, increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells. The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1, providing a potential therapeutic strategy for combating DN.

The Deubiquitinase USP22-Stabilized COL17A1 Promotes Lung Adenocarcinoma Progression.

Lung adenocarcinoma (LUAD) is a highly aggressive and rapidly fatal malignancy worldwide. Collagen XVII (COL17A1) has been implicated in various protumorigenic processes. However, the functions and mechanisms of COL17A1 in LUAD progression still remain elusive. COL17A1 and ubiquitin-specific protease 22 (USP22) mRNA analysis was performed by quantitative PCR, and their protein levels were detected by immunoblotting and immunohistochemistry. The functional influence was evaluated by determining cell viability, proliferation, apoptosis, invasion, migration, and ferroptosis invitro, as well as xenograft growth invivo. Co-immunoprecipitation (Co-IP) and IP experiments were used to examine the USP22/COL17A1 interaction and COL17A1 deubiquitination. Cycloheximide treatment was used to analyze COL17A1 protein stability. COL17A1 and USP22 were upregulated in human LUAD tissues and cell lines. Functionally, COL17A1 knockdown acted for the suppression of LUAD cell growth, invasion, and migration as well as promotion of cell apoptosis and ferroptosis invitro. COL17A1 knockdown could diminish the tumorigenicity of LUAD cells invivo. Mechanistically, USP22 stabilized and upregulated COL17A1 by enhancing the deubiquitination of COL17A1. Additionally, reexpression of COL17A1 could reverse USP22 silencing-induced phenotype changes of LUAD cells invitro. Our findings demonstrate that USP22-stabilized COL17A1 possesses oncogenic activity in LUAD. We propose that USP22 and COL17A1 would be potential targets for the establishment of therapeutic approaches against LUAD.

Identification of USP2 as a novel target to induce degradation of KRAS in myeloma cells

Inducing the degradation of KRAS represents a novel strategy to combat cancers with KRAS mutation. In this study, we identify ubiquitin-specific protease 2 (USP2) as a novel deubiquitinating enzyme of KRAS in multiple myeloma (MM). Specifically, we demonstrate that gambogic acid (GA) forms a covalent bond with the Cysteine 284 residue of USP2 through an allosteric pocket, inhibiting its deubiquitinating activity. Inactivation or knockdown of USP2 leads to the degradation of KRAS, resulting in the suppression of MM cell proliferation in vitro and in vivo. Conversely, overexpressing USP2 stabilizes KRAS and partially abrogates GA-induced apoptosis in MM cells. Furthermore, elevated USP2 levels may be associated with poorer prognoses in MM patients. These findings highlight the potential of the USP2/KRAS axis as a therapeutic target in MM, suggesting that strategically inducing KRAS degradation via USP2 inhibition could be a promising approach for treating cancers with KRAS mutations.

Ubiquitin-specific protease 22 controls melanoma metastasis and vulnerability to ferroptosis through targeting SIRT1/PTEN/PI3K signaling.

Metastasis is a major contributing factor that affects the prognosis of melanoma patients. Nevertheless, the underlying molecular mechanisms involved in melanoma metastasis are not yet entirely understood. Here, we identified ubiquitin-specific protease 22 (USP22) as a pro-oncogenic protein in melanoma through screening the survival profiles of 52 ubiquitin-specific proteases (USPs). USP22 demonstrates a strong association with poor clinical outcomes and is significantly overexpressed in melanoma. Ablation of USP22 expression remarkably attenuates melanoma migration, invasion, and epithelial-mesenchymal transition in vitro and suppresses melanoma metastasis in vivo. Mechanistically, USP22 controls melanoma metastasis through the SIRT1/PTEN/PI3K pathway. In addition, we conducted an United States Food and Drug Administration-approved drug library screening and identified topotecan as a clinically applicable USP22-targeting molecule by promoting proteasomal degradation of USP22. Finally, we found that both pharmacological and genetic silence of USP22 sensitize RSL3-induced ferroptosis through suppressing the PI3K/Akt/mTOR pathway and its downstream SCD, and ferroptosis inhibitor could partly rescued the decreased lung metastasis by topotecan in vivo. Overall, our findings reveal a prometastatic role of USP22 and identify topotecan as a potent USP22-targeting drug to limit melanoma metastasis.

AB002. DNA methylation-regulated genes contribute to temozolomide (TMZ) resistance by scaffolding paraspeckle proteins.

Temozolomide (TMZ) resistance in glioblastoma (GBM) remains a challenge in clinical treatment and the mechanism is largely unknown. Emerging evidence shows that epigenetic modifications including DNA methylation and non-coding RNA were involved in diverse biological processes, including therapeutic resistance. However, the underlying mechanisms by which DNA methylation-mediated non-coding RNA regulates TMZ resistance remain poorly characterized. RNA microarray and DNA methylation chips of TMZ-resistant and parental GBM cells were performed for the gain of unreported long non-coding RNA HSD52. Quantitative reverse transcription polymerase chain reaction (PCR) and fluorescence in situ hybridization assays were used to detect HSD52 levels in GBM cells and tissues. The investigation into HSD52's impact on TMZ resistance was conducted utilizing both in vitro assays and intracranial xenograft mouse models. The mechanism of HSD52 expression and its relationships with paraspeckle proteins, non-POU domain-containing octamer-binding protein (NONO) and splicing factor proline/glutamine rich (SFPQ), as well as alpha-thalassemia mental retardation X-linked (ATRX) mRNA were determined by pyrosequencing assay, chromatin immunoprecipitation, chromatin isolation by RNA purification, RNA immunoprecipitation, RNA pulldown, immunofluorescence, and western blot assays. HSD52 was highly expressed in high-grade glioma and TMZ-resistant GBM cells. Phosphorylated p38 mitogen-activated protein kinase (p38 MAPK)/ubiquitin specific peptidase 7 (USP7) axis mediates H3 ubiquitination, impairs the interaction between H3K23ub and DNA methyltransferase 1 (DNMT1) and the recruitment of DNMT1 at the HSD52 promoter to attenuate DNA methylation, which makes the transcription factor 12 (TCF12) more accessible to the promoter region to regulate HSD52 expression. Further analysis showed that HSD52 can serve as a scaffold to promote the interaction between NONO and SFPQ, and then increase the paraspeckle assembly and activate the paraspeckle/ataxia telangiectasia mutated (ATM) kinase pathway in GBM cells. In addition, HSD52 forms an RNA-RNA duplex with ATRX mRNA, and facilitates the association of heteromer of SFPQ and NONO with RNA duplex, thus leading to the increase of ATRX mRNA stability and level. In clinical patients, HSD52 is required for TMZ resistance and GBM recurrence. Our results reveal that HSD52 in GBM could serve as a therapeutic target to overcome TMZ resistance, enhancing the clinical benefits of TMZ chemotherapy.

UBE2S facilitates glioblastoma progression through activation of the NF-κB pathway via attenuating K11-linked ubiquitination of AKIP1

BackgroundRapid proliferation is a hallmark of glioblastoma multiforme (GBM) and a major contributor to its recurrence. Aberrant ubiquitination has been implicated in various diseases, including cancer. In our preliminary studies, we identified Ubiquitin-conjugating enzyme E2S (UBE2S) as a potential glioma biomarker, exhibiting close associations with glioma grade and protein phosphatase 1, regulatory subunit 105 (Ki67) expression levels. However, the underlying molecular mechanisms remained elusive. NF-κB is an important signaling pathway that promotes GBM proliferation. Direct intervention targeting NF-κB has not yielded the expected results, prompting the exploration of new molecules for regulating NF-κB as a new direction. MethodsThis study employed methods including yeast two-hybrid and immunoprecipitation to uncover the interaction between UBE2S and A kinase interacting protein 1 (AKIP1). Laser confocal microscopy was used to observe the localization of UBE2S and AKIP1. Dual luciferase reporter genes were utilized to observe the activation of NF-κB. ResultsOur findings demonstrate that UBE2S deficiency significantly impedes GBM progression, both in vitro and in vivo. Mechanistically, UBE2S plays a crucial role in recruiting Ubiquitin Specific Peptidase 15 (USP15), facilitating the removal of K11-linked ubiquitination on AKIP1. This action enhances AKIP1 stability within the GBM context. The resulting increase in AKIP1 levels further augments nuclear factor kappa-B (NF-κB) transcriptional activity, leading to the upregulation of downstream genes regulated by the NF-κB pathway, thereby promoting GBM progression. ConclusionsIn summary, our findings reveal the role of the UBE2S/AKIP1-NF-κB axis in regulating GBM progression and provide novel evidence supporting UBE2S as a potential drug target for GBM.

Exosomal circ-0100519 promotes breast cancer progression via inducing M2 macrophage polarisation by USP7/NRF2 axis.

Breast cancer (BC) is one of the most prevalent malignant tumours that threatens women health worldwide. It has been reported that circular RNAs (circRNAs) play an important role in regulating tumour progression and tumour microenvironment (TME) remodelling. Differentially expression characteristics and immune correlations of circRNAs in BC were verified using high-throughput sequencing and bioinformatic analysis. Exosomes were characterised by nanoparticle transmission electron microscopy and tracking analysis. The biological function of circ-0100519 in BC development was demonstrated both in vitro and in vivo. Western blotting, RNA pull-down, RNA immunoprecipitation, flow cytometry, and luciferase reporter were conducted to investigate the underlying mechanism. Circ-0100519 was significant abundant in BC tumour tissues and related to poor prognosis. It can be encapsulated into secreted exosomes, thereby promoting BC cell invasion and metastasis via inducing M2-like macrophages polarisation.Mechanistically, circ-0100519 acted as a scaffold to enhance the interaction between the deubiquitinating enzyme ubiquitin-specific protease 7 (USP7) and nuclear factor-like 2 (NRF2) in macrophages, inducing the USP7-mediated deubiquitination of NRF2. Additionally,HIF-1α could function as an upstream effector to enhance circ-0100519 transcription. Our study revealed that exosomal circ-0100519 is a potential biomarker for BC diagnosis and prognosis, and the HIF-1α inhibitor PX-478 may provide a therapeutic target for BC.

De novo variants in ATXN7L3 lead to developmental delay, hypotonia and distinctive facial features.

Deubiquitination is crucial for the proper functioning of numerous biological pathways, such as DNA repair, cell cycle progression, transcription, signal transduction and autophagy. Accordingly, pathogenic variants in deubiquitinating enzymes (DUBs) have been implicated in neurodevelopmental disorders and congenital abnormalities. ATXN7L3 is a component of the DUB module of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex and two other related DUB modules, and it serves as an obligate adaptor protein of three ubiquitin-specific proteases (USP22, USP27X or USP51). Through exome sequencing and by using GeneMatcher, we identified nine individuals with heterozygous variants in ATXN7L3. The core phenotype included global motor and language developmental delay, hypotonia and distinctive facial characteristics, including hypertelorism, epicanthal folds, blepharoptosis, a small nose and mouth, and low-set, posteriorly rotated ears. To assess pathogenicity, we investigated the effects of a recurrent nonsense variant [c.340C>T; p.(Arg114Ter)] in fibroblasts of an affected individual. ATXN7L3 protein levels were reduced, and deubiquitylation was impaired, as indicated by an increase in histone H2Bub1 levels. This is consistent with the previous observation of increased H2Bub1 levels in Atxn7l3-null mouse embryos, which have developmental delay and embryonic lethality. In conclusion, we present clinical information and biochemical characterization supporting ATXN7L3 variants in the pathogenesis of a rare syndromic neurodevelopmental disorder.

Autoinhibition of ubiquitin-specific protease 8: Insights into domain interactions and mechanisms of regulation

Ubiquitin-specific proteases (USPs) are a family of multi-domain deubiquitinases (DUBs) with variable architectures, some containing regulatory auxiliary domains. Among the USP family, all occurrences of intramolecular regulation presently known are autoactivating. USP8 remains the sole exception as its putative WW-like domain, conserved only in vertebrate orthologs, is autoinhibitory. Here, we present a comprehensive structure-function analysis describing the autoinhibition of USP8 and provide evidence of the physical interaction between the WW-like and catalytic domains. The solution structure of full-length USP8 reveals an extended, monomeric conformation. Coupled with DUB assays, the WW-like domain is confirmed to be the minimal autoinhibitory unit. Strikingly, autoinhibition is only observed with the WW-like domain in cis and depends on the length of the linker tethering it to the catalytic domain. Modelling of the WW:CD complex structure and mutagenesis of interface residues suggests a novel binding site in the S1 pocket. To investigate the interplay between phosphorylation and USP8 autoinhibition, we identify AMP-activated protein kinase as a highly selective modifier of S718 in the 14-3-3 binding motif. We show that 14-3-3γ binding to phosphorylated USP8 potentiates autoinhibition in a WW-like domain-dependent manner by stabilizing an autoinhibited conformation. These findings provide mechanistic details on the autoregulation of USP8 and shed light on its evolutionary significance.