An ER-Associated Pathway Defines Endosomal Architecture for Controlled Cargo Transport.

Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle "cloud" and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell's periphery. By drawing the endosomal system's architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.

The CRL3gigaxonin ubiquitin ligase-USP15 pathway governs the destruction of neurofilament proteins.

Giant axonal neuropathy (GAN) is caused by mutations in the GAN gene encoding for gigaxonin (GIG), which functions as an adaptor of the CUL3-RBX1-GIG (CRL3GIG) E3 ubiquitin ligase complex. The pathological hallmark of GAN is characterized by the accumulation of densely packed neurofilaments (NFs) in the axons. However, there are fundamental knowledge gaps in our understanding of the molecular mechanisms by which the ubiquitin-proteasome system controls the homeostasis of NF proteins. Recently, the deubiquitylating enzyme USP15 was reported to play a crucial role in regulating ubiquitylation and proteasomal degradation of CRL4CRBN substrate proteins. Here, we report that the CRL3GIG-USP15 pathway governs the destruction of NF proteins NEFL and INA. We identified a specific degron called NEFLL12 degron for CRL3GIG. Notably, mutations in the C-terminal Kelch domain of GIG, represented by L309R, R545C, and C570Y, disrupted the binding of GIG to NEFL and INA, leading to the accumulation of these NF proteins. This accounts for the loss-of-function mutations in GAN patients. In addition to regulating NFs, CRL3GIG also controls actin filaments by directly targeting actin-filament-binding regulatory proteins TPM1, TPM2, TAGLN, and CNN2 for proteasomal degradation. Thus, our findings broadly impact the field by providing fundamental mechanistic insights into regulating extremely long-lived NF proteins NEFL and INA by the CRL3GIG-USP15 pathway and offering previously unexplored therapeutic opportunities to treat GAN patients and other neurodegenerative diseases by explicitly targeting downstream substrates of CRL3GIG.

USP15, activated by TFAP4 transcriptionally, stabilizes SHC1 via deubiquitination and deteriorates renal cell carcinoma.

Ubiquitin-specific peptidase 15 (USP15), a critical deubiquitinating enzyme, has been demonstrated to improve substrate stabilization by hydrolyzing the bond between the substrate and ubiquitin, and is implicated in multiple carcinogenic processes. Prompted by the information cited from The Cancer Genome Atlas (TCGA) database and the Cancer Proteogenomic Data Analysis Site (cProSite), USP15 is selectively overexpressed in clear cell renal cell carcinoma (ccRCC) samples. We aimed to investigate the function of USP15 on ccRCC malignant features, which was emphasized in its deubiquitination of SHC adaptor protein 1 (SHC1). The overexpression of USP15 promoted the capacity of proliferation, migration, and invasion in ccRCC CAKI1 and 769-P cells, and these malignant biological properties were diminished by USP15 deletion in 786-O cells. USP15 accelerated tumor growth and lung metastasis in vivo. In addition, deubiquitinase USP15 was further identified as a new protector for SHC1 from degradation by the ubiquitination pathway, the post-translational modification. In sequence, transcription factor activating enhancer binding protein 4 (TFAP4) was shown to be partly responsible for USP15 expression at the level of transcription, as manifested by the chromatin immunoprecipitation and pull-down assay. Based on the in vitro and in vivo data, we postulate that USP15 regulated by TFAP4 transcriptionally deteriorates ccRCC malignant biological properties via stabilizing SHC1 by deubiquitination.

PRDM1 promotes the ferroptosis and immune escape of thyroid cancer by regulating USP15-mediated SELENBP1 deubiquitination.

BACKGROUND: The deubiquitinating enzyme Ubiquitin-specific peptidase 15 (USP15) is upregulated in various cancers and promotes tumor progression by increasing the expression of several oncogenes. This project is designed to explore the role and mechanism of USP15 in thyroid cancer (TC) progression. METHODS: Selenium-binding protein 1 (SELENBP1), USP15, CCL2/5, CXCL10/11, IL-4, and TGF-ß1 mRNA levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR). SELENBP1, USP15, GPX4, IL-10, Arg-1, Granzyme B, TNF-α, and PR domain zinc finger protein 1 (PRDM1) protein levels were examined by western blot assay. Fe+ level, malondialdehyde (MDA), and lipid-ROS levels were determined using special kits. The proportion of CD11b+CD206+ positive cells was detected using a flow cytometry assay. The role of SELENBP1 on TC cell growth was examined using a xenograft tumor model in vivo. After GeneMANIA prediction, the interaction between USP15 and SELENBP1 was verified using Co-immunoprecipitation (CoIP) assay. The binding between PRDM1 and USP15 promoter was predicted by JASPAR and validated using Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. RESULTS: SELENBP1 was increased in TC subjects and cell lines, and its knockdown repressed TC cell proliferation, migration, invasion, immune escape, and induced ferroptosis in vitro, as well as blocked tumor growth in vivo. In mechanism, USP15 interacted with SELENBP1 and maintained its stabilization by removing ubiquitin. Meanwhile, the upregulation of USP15 was induced by the transcription factor PRDM1. CONCLUSION: USP15 transcriptionally mediated by PRDM1 might boost TC cell malignant behaviors through deubiquitinating SELENBP1, providing a promising therapeutic target for TC treatment.

ISGylation-independent protection of cell growth by USP18 following interferon stimulation.

Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signalling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation, a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non-catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity.

USP15 antagonizes CRL4CRBN-mediated ubiquitylation of glutamine synthetase and neosubstrates.

Targeted protein degradation by the ubiquitin-proteasome system represents a new strategy to destroy pathogenic proteins in human diseases, including cancer and neurodegenerative diseases. The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide have revolutionized the treatment of patients with multiple myeloma (MM) and other hematologic malignancies, but almost all patients eventually develop resistance to IMiDs. CRBN, a substrate receptor of CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase, is a direct target for thalidomide teratogenicity and antitumor activity of IMiDs (now known as Cereblon E3 ligase modulators: CELMoDs). Despite recent advances in developing potent CELMoDs and CRBN-based proteolysis-targeting chimeras (PROTACs), many questions apart from clinical efficacy remain unanswered. CRBN is required for the action of IMiDs, but its protein expression levels do not correlate with intrinsic resistance to IMiDs in MM cells, suggesting other factors involved in regulating resistance to IMiDs. Our recent work revealed that the CRL4CRBN-p97 pathway is required for degradation of natural substrate glutamine synthetase (GS) and neosubstrates. Here, I show that USP15 is a key regulator of the CRL4CRBN-p97 pathway to control stability of GS and neosubstrates IKZF1, IKZF3, CK1-α, RNF166, GSPT1, and BRD4, all of which are crucial drug targets in different types of cancer. USP15 antagonizes ubiquitylation of CRL4CRBN target proteins, thereby preventing their degradation. Notably, USP15 is highly expressed in IMiD-resistant cells, and depletion of USP15 sensitizes these cells to lenalidomide. Inhibition of USP15 represents a valuable therapeutic opportunity to potentiate CELMoD and CRBN-based PROTAC therapies for the treatment of cancer.

PLOD2 promotes colorectal cancer progression by stabilizing USP15 to activate the AKT/mTOR signaling pathway.

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) has been reported as an oncogenic gene, affecting various malignant tumors, including endometrial carcinoma, osteosarcoma, and gastric cancer. These effects are mostly due to the enhanced deposition of collagen precursors. However, more studies need to be conducted on how its lysyl hydroxylase function affects cancers like colorectal carcinoma (CRC). Our present results showed that PLOD2 expression was elevated in CRC, and its higher expression was associated with poorer survival. Overexpression of PLOD2 also facilitated CRC proliferation, invasion, and metastasis in vitro and in vivo. In addition, PLOD2 interacted with USP15 by stabilizing it in the cytoplasm and then activated the phosphorylation of AKT/mTOR, thereby promoting CRC progression. Meanwhile, minoxidil was demonstrated to downregulate the expression of PLOD2 and suppress USP15, and the phosphorylation of AKT/mTOR. Our study reveals that PLOD2 plays an oncogenic role in colorectal carcinoma, upregulating USP15 and subsequently activating the AKT/mTOR pathway.

Alleviative effect of microRNA-497 on diabetic neuropathic pain in rats in relation to decreased USP15.

The current study tries to discuss the functional role of microRNA-497 (miR-497) in diabetic neuropathic pain (DNP) and the related downstream mechanism. Bioinformatics analysis was implemented for the identification of differentially expressed miRNAs and genes. DNP was simulated in rats through intraperitoneal injection of streptozotocin. The expression patterns of miR-497, USP15, NRF2, and G6PD were then determined. The binding of miR-497 and USP15 was confirmed. Using gain- and loss-of-function assays, we analyzed the critical role of miR-497-mediated USP15 in DNP through the NRF2/G6PD axis. Downregulated miR-497 and elevated USP15 were observed in the dorsal root ganglion neurons isolated from spinal cord tissues of STZ-induced DNP rats. miR-497 could alleviate DNP, which was associated with suppression of USP15, a confirmed target of miR-497. USP15 enhanced the degradation and ubiquitination of NRF2 and induced G6PD expression, leading to the progression of DNP. We highlighted the crucial role of miR-497-mediated USP15 in DNP through the NRF2/G6PD axis. 1. miR-497 is downregulated in DRG neurons from spinal cord tissues of STZ-induced DNP rats. 2. miR-497 inhibits the expression of USP15, thereby alleviating STZ-induced DNP in rats. 3. USP15 promotes ubiquitination and degradation of NRF2, reducing the expression of G6PD. 4. miR-497 alleviates STZ-induced DNP in rats by regulating the USP15/NRF2/G6PD axis.

Inhibition of USP15 ameliorates high-glucose-induced oxidative stress and inflammatory injury in podocytes through regulation of the Keap1/Nrf2 signaling.

Ubiquitin-specific peptidase 15 (USP15) is implicated in the pathogenesis of numerous diseases. However, whether USP15 plays a role in diabetic nephropathy remains undetermined. This project was designed to determine the potential role of USP15 in mediating high glucose (HG)-induced podocyte injury, a key event in the pathogenesis of diabetic nephropathy. We found that USP15 levels were elevated in podocytes after HG stimulation. Inhibition of USP15 led to decreases in HG-evoked apoptosis, oxidative stress, and inflammation in podocytes. Further investigation showed that inhibition of USP15 enhanced the activation of NF-E2-related factor 2 (Nrf2) and expression of Nrf2 target genes in HG-simulated podocytes. Moreover, depletion of Kelch-like ECH-associated protein 1 (Keap1) diminished the regulatory effect of USP15 inhibition on Nrf2 activation. In addition, Nrf2 suppression reversed USP15-inhibition-induced protective effects in HG-injured podocytes. Taken together, these data indicate that USP15 inhibition protects podocytes from HG-induced injury by enhancing Nrf2 activation via Keap1.

KIF15 upregulation promotes leiomyosarcoma cell growth via promoting USP15-mediated DEK deubiquitylation.

Kinesin Family Member 15 (KIF15) is a plus end-directed microtubule motor, which exerts complex regulations in cancer biology. This study aimed to explore the functional role of KIF15 in leiomyosarcoma (LMS). Bioinformatic analysis was carried out using data from The Cancer Genome Atlas (TCGA)-Sarcoma (SARC). LMS cell lines SK-UT-1 and SK-LMS-1 were used as in vitro cell models. Results showed that LMS patients with high KIF15 expression had significantly worse survival than the low KIF15 expression counterparts. KIF15 knockdown slowed, while KIF15 overexpression increased the proliferation of SK-UT-1 and SK-LMS-1 cells. Co-IP assay confirmed mutual interaction between endogenous KIF15 and DEK (encoded by DEK proto-oncogene). KIF15 knockdown facilitated DEK degradation, while KIF15 overexpression slowed DEK degradation. In ubiquitination assay, a significant increase in DEK polyubiquitylation was observed when KIF15 expression was suppressed. USP15 physically interacted with both DEK and KIF15 in the cells. USP15 knockdown decreased DEK protein stability and canceled KIF15-mediated DEK stabilization. USP15 overexpression enhanced DEK stability, the effect of which was impaired by KIF15 knockdown. USP15 overexpression reduced DEK polyubiquitination. USP15 knockdown increased DEK polyubiquitination and canceled the effect of KIF15 overexpression on reducing DEK polyubiquitination. DEK overexpression enhanced the proliferation of SK-UT-1 and SK-LMS-1 cells. DEK knockdown decreased cell proliferation and canceled the effect of KIF15 overexpression on cell proliferation. In conclusion, this study revealed a novel mechanism that KIF15 enhances LMS cell proliferation via preventing DEK protein from degradation by increasing USP15 mediated deubiquitylation.

The Multifaceted Roles of USP15 in Signal Transduction.

Ubiquitination and deubiquitination are protein post-translational modification processes that have been recognized as crucial mediators of many complex cellular networks, including maintaining ubiquitin homeostasis, controlling protein stability, and regulating several signaling pathways. Therefore, some of the enzymes involved in ubiquitination and deubiquitination, particularly E3 ligases and deubiquitinases, have attracted attention for drug discovery. Here, we review recent findings on USP15, one of the deubiquitinases, which regulates diverse signaling pathways by deubiquitinating vital target proteins. Even though several basic previous studies have uncovered the versatile roles of USP15 in different signaling networks, those have not yet been systematically and specifically reviewed, which can provide important information about possible disease markers and clinical applications. This review will provide a comprehensive overview of our current understanding of the regulatory mechanisms of USP15 on different signaling pathways for which dynamic reverse ubiquitination is a key regulator.

Inhibition of USP15 Prevent Glutamate-Induced Oxidative Damage by Activating Nrf2/HO-1 Signaling Pathway in HT22 Cells.

Oxidative stress has been identified as the significant mediator in epilepsy, which is a chronic disorder in central nervous system. About 30% of epilepsy patients are refractory to antiepileptic drug treatment. However, the underlying mechanism of oxidative damage in epilepsy needs further investigation. In our study, we first find that ubiquitin-specific peptidase 15 (USP15) expression was upregulated in a pentylenetetrazole (PTZ) kindled rat model of epilepsy. Silencing USP15 protected against glutamate-mediated neuronal cell death, and inhibited the high expression levels of cleaved caspase-3. Knockout of USP15 significantly reduced intracellular reactive oxygen species (ROS) levels and enhanced superoxide dismutase (SOD) activity in HT22 cells under the exposure to glutamate treatment. Furthermore, USP15 inhibition induced nuclear factor erythroid-derived 2-related factor2 (Nrf2) nuclear translocation and promoted protein expression level of heme oxygenase (HO-1). Taken together, our findings first reveal a role of USP15 in the pathogenesis of epilepsy, and silencing USP15 in vitro protects against glutamate-mediated cytotoxicity in HT22 cells. Pharmacological inhibition of USP15 may alleviate epileptic seizures via fighting against oxidative damage, providing a novel antiepileptic target.

The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel.

Ubiquitin-specific protease 15 (USP15) regulates important cellular processes, including transforming growth factor ß (TGF-ß) signaling, mitophagy, mRNA processing, and innate immune responses; however, structural information on USP15's catalytic domain is currently unavailable. Here, we determined crystal structures of the USP15 catalytic core domain, revealing a canonical USP fold, including a finger, palm, and thumb region. Unlike for the structure of paralog USP4, the catalytic triad is in an inactive configuration with the catalytic cysteine ∼10 Å apart from the catalytic histidine. This conformation is atypical, and a similar misaligned catalytic triad has so far been observed only for USP7, although USP15 and USP7 are differently regulated. Moreover, we found that the active-site loops are flexible, resulting in a largely open ubiquitin tail-binding channel. Comparison of the USP15 and USP4 structures points to a possible activation mechanism. Sequence differences between these two USPs mainly map to the S1' region likely to confer specificity, whereas the S1 ubiquitin-binding pocket is highly conserved. Isothermal titration calorimetry monoubiquitin- and linear diubiquitin-binding experiments showed significant differences in their thermodynamic profiles, with USP15 displaying a lower affinity for monoubiquitin than USP4. Moreover, we report that USP15 is weakly inhibited by the antineoplastic agent mitoxantrone in vitro A USP15-mitoxantrone complex structure disclosed that the anthracenedione interacts with the S1' binding site. Our results reveal first insights into USP15's catalytic domain structure, conformational changes, differences between paralogs, and small-molecule interactions and establish a framework for cellular probe and inhibitor development.

USP15 inhibits HPV16 E6 degradation and catalytically inactive USP15 has reduced inhibitory activity.

High-risk human papillomaviruses (HPVs) possess transforming activity leading to development of the cancer, including oropharyngeal, anal, penile, vulvar, vaginal, and cervical cancer. The stability of E6 is essential for its complete function as an oncoprotein. Using the yeast two-hybrid system, we identified ubiquitin-specific protease 15 (USP15) as an HPV16 E6-interacting protein. USP15 cleaves polyubiquitin chains of HPV16 E6 and/or ubiquitin precursors. Our results indicate that USP15 could increase the level of HPV16 E6 by inhibiting E6 degradation. USP15 inhibited the degradation of HPV16 E6 in dose-dependent manner. In contrast, catalytically inactive mutants of USP15 had a reduced inhibitory effect on E6 degradation. In particular, USP15 mutants of all three cysteine boxes and the NHL mutant of the KRF box had a drastically reduced inhibitory effect on HPV16 E6 degradation. In addition, HPV16 E6 mRNA was not induced by USP15; therefore, HPV16 E6 appears to be post-translationally regulated. These results suggest that USP15 has the ability to stabilize E6 as a deubiquitinating enzyme, and as an oncoprotein affects biological functions in infected human cells.

USP15 attenuates IGF-I signaling by antagonizing Nedd4-induced IRS-2 ubiquitination.

Insulin receptor substrates (IRSs) are phosphorylated by IGF-I receptor tyrosine kinase in a ligand-dependent manner. In turn, they bind to and activate effector proteins such as PI3K, leading to various cell responses including cell proliferation. We had reported that ubiquitin ligase Nedd4 induces mono-ubiquitination of IRS-2, thereby enhancing IRS-2 tyrosine phosphorylation, leading to increased IGF signaling and mitogenic activity. Here we show that ubiquitin-specific protease 15 (USP15) antagonizes the effect of Nedd4 on IRS-2. We identified USP15 as a protein that preferentially bound to IRS-2 when IRS-2 was conjugated with ubiquitin. In HEK293 cells, Nedd4 overexpression induced IRS-2 ubiquitination, which was decreased by USP15 co-expression while increased by USP15 knockdown. Nedd4 overexpression enhanced IGF-I-dependent IRS-2 tyrosine phosphorylation, and USP15 co-expression suppressed it. Conversely, USP15 knockdown increased IRS-2 tyrosine phosphorylation and downstream signaling in prostate cancer PC-3 cells. We concluded that USP15 attenuates IGF-I signaling by antagonizing Nedd4-induced IRS-2 ubiquitination.

The U4/U6 recycling factor SART3 has histone chaperone activity and associates with USP15 to regulate H2B deubiquitination.

Post-translational modifications of histone proteins produce dynamic signals that regulate the structure and function of chromatin. Mono-ubiquitination of H2B in the histone tail (at Lys-123 in yeast or Lys-120 in humans) is a conserved modification that has been implicated in the regulation of transcription, replication, and DNA repair processes. In a search for direct effectors of ubH2B, we identified a deubiquitinating enzyme, Usp15, through affinity purification with a nonhydrolyzable ubH2B mimic. In the nucleus, Usp15 indirectly associates with the ubH2B E3 ligase, RNF20/RNF40, and directly associates with a component of the splicing machinery, SART3 (also known as TIP110 or p110). These physical interactions place Usp15 in the vicinity of actively transcribed DNA. Importantly we found that SART3 has previously unrecognized histone chaperone activities. SART3, but not the well-characterized histone chaperone Nap1, enhances Usp15 binding to ubH2B and facilitates deubiquitination of ubH2B in free histones but not in nucleosomes. These results suggest that SART3 recruits ubH2B, which may be evicted from DNA during transcription, for deubiquitination by Usp15. In light of the function played by SART3 in U4/U6 di-snRNP formation, our discovery points to a direct link between eviction-coupled erasure of the ubiquitin mark from ubH2B and co-transcriptional pre-mRNA splicing.

USP15 promotes the progression of papillary thyroid cancer by regulating HMGB1 stability through its deubiquitination.

Purpose: Papillary thyroid cancer (PTC) stands as one of the most prevalent types of thyroid cancers, characterized by a propensity for in-situ recurrence and distant metastasis. The high mobility group protein (HMGB1), a conserved nuclear protein, plays a pivotal role in carcinogenesis by stimulating tumor cell growth and migration. Nevertheless, the underlying mechanism driving aberrant HMGB1 expression in PTC necessitates further elucidation. Materials and methods: Our study unraveled the impact of low and overexpression of USP15 on the proliferation, invasion, and metastasis of PTC cells. Through a comprehensive array of molecular techniques, we uncovered the intricate relationship between HMGB1 and USP15 in the progression of PTC. Results: In this study, we identified USP15, a deubiquitinase in the ubiquitin-specific proteases family, as a true deubiquitylase of HMGB1 in PTC. USP15 was shown to interact with HMGB1 in a deubiquitination activity-dependent manner, deubiquitinating and stabilizing HMGB1. USP15 depletion significantly decreased PTC cell proliferation, migration, and invasion. In addition, the effects induced by USP15 depletion could be rescued by further HMGB1 overexpression. But when HMGB1 is knocked down, even overexpression of USP15 could not promote the progression of PTC cells. Conclusion: In essence, our discoveries shed light on the previously uncharted catalytic role of USP15 as a deubiquitinating enzyme targeting HMGB1, offering a promising avenue for potential therapeutic interventions in the management of PTC.

USP15 inhibits hypoxia-induced IL-6 signaling by deubiquitinating and stabilizing MeCP2.

Methyl-CpG binding protein 2 (MeCP2) is an important X-linked DNA methylation reader and a key heterochromatin organizer. The expression level of MeCP2 is crucial, as indicated by the observation that loss-of-function mutations of MECP2 cause Rett syndrome, whereas an extra copy spanning the MECP2 locus results in MECP2 duplication syndrome, both being progressive neurodevelopmental disorders. Our previous study demonstrated that MeCP2 protein expression is rapidly induced by renal ischemia-reperfusion injury (IRI) and protects the kidney from IRI through transcriptionally repressing the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 signaling pathway. However, the mechanisms underlying the upregulation of MeCP2 have remained elusive. Here, by using two hypoxia cell models, hypoxia and reoxygenation and cobalt chloride stimulation, we confirmed that the removal of lysine 48-linked ubiquitination from MeCP2 prevented its proteasome-dependent degradation under hypoxic conditions. Through unbiased screening based on a deubiquitinating enzymes library, we identified ubiquitin-specific protease 15 (USP15) as a stabilizer of MeCP2. Further studies revealed that USP15 could attenuate hypoxia-induced MeCP2 degradation by cleaving lysine 48-linked ubiquitin chains from MeCP2, primarily targeting its C-terminal domain. Consistently, USP15 inhibited hypoxia-induced signal transducer and activator of transcription 3 activation, resulting in reduced transcription of IL-6 downstream genes. In summary, our study reveals an important role for USP15 in the maintenance of MeCP2 stability and the regulation of IL-6 signaling.

miR-26a exerts broad-spectrum antiviral effects via the enhancement of RIG-I-mediated type I interferon response by targeting USP15.

IMPORTANCE: miR-26a serves as a potent positive regulator of type I interferon (IFN) responses. By inhibiting USP15 expression, miR-26a promotes RIG-I K63-ubiquitination to enhance type I IFN responses, resulting in an active antiviral state against viruses. Being an intricate regulatory network, the activation of type I IFN responses could in turn suppress miR-26a expression to avoid the disordered activation that might result in the so-called "type I interferonopathy." The knowledge gained would be essential for the development of novel antiviral strategies against viral infection.

K48-linked deubiquitination of VGLL4 by USP15 enhances the efficacy of tumor immunotherapy in triple-negative breast cancer.

Immunotherapy based on PD-1/PD-L1 antagonists has been demonstrated to be efficacious in inducing tumor remission in patients with triple-negative breast cancer (TNBC). However, tumor immune evasion caused by the PD-1/PD-L1 pathway inhibits the immunotherapeutic effect of PD-1/PD-L1 inhibitors against TNBC. Therefore, identifying potential targets for blocking the PD-1/PD-L1 pathway is a compelling strategy for TNBC treatment. Here, we discovered that VGLL4 could inhibit PD-L1 transcription by suppressing STAT3 activation, thereby enhancing the efficacy of anti-PD-1 antibody immunotherapy in TNBC. Low expression of USP15, a deubiquitinating enzyme of VGLL4, was associated with reduced CD8+ T cell infiltration and poor prognosis in TNBC patients. USP15 was found to inhibit PD-L1 transcription, leading to increased CD8+ T cell infiltration and thus enhancing the efficacy of TNBC immunotherapy. Furthermore, SART3 regulated VGLL4 stability and PD-L1 transcription by influencing the nuclear translocation of USP15. In conclusion, our study provides new insights into the biological regulation of PD-L1, identifies a previously unrecognized regulator of this critical immune checkpoint, and highlights potential therapeutic targets for overcoming immune evasion in TNBC.