SLC7A11 is a cystine transporter and ferroptosis inhibitor. How the stability of SLC7A11 is coordinately regulated in response to environmental cystine by which E3 ligase and deubiquitylase (DUB) remains elusive. Here, we report that neddylation inhibitor MLN4924 increases cystine uptake by causing SLC7A11 accumulation, via inactivating Cullin-RING ligase-3 (CRL-3). We identified KCTD10 as the substrate-recognizing subunit of CRL-3 for SLC7A11 ubiquitylation, and USP18 as SLC7A11 deubiquitylase. Upon cystine deprivation, the protein levels of KCTD10 or USP18 are decreased or increased, respectively, contributing to SLC7A11 accumulation. By destabilizing or stabilizing SLC7A11, KCTD10, or USP18 inversely regulates the cystine uptake and ferroptosis. Biologically, MLN4924 combination with SLC7A11 inhibitor Imidazole Ketone Erastin (IKE) enhanced suppression of tumor growth. In human breast tumor tissues, SLC7A11 levels were negatively or positively correlated with KCTD10 or USP18, respectively. Collectively, our study defines how SLC7A11 and ferroptosis is coordinately regulated by the CRL3KCTD10/E3-USP18/DUB axis, and provides a sound rationale of drug combination to enhance anticancer efficacy.

UNASSIGNED: Long non-coding RNAs are important regulators in cancer biology and function either as tumor suppressors or as oncogenes. Their dysregulation has been closely associated with tumorigenesis. LINC00265 is upregulated in lung adenocarcinoma and is a prognostic biomarker of this cancer. However, the mechanism underlying its function in cancer progression remains poorly understood.
UNASSIGNED: Here, the regulatory role of LINC00265 in lung adenocarcinoma was examined using lung cancer cell lines, clinical samples, and xenografts.
UNASSIGNED: We found that high levels of LINC00265 expression were associated with shorter overall survival rate of patients, whereas knockdown of LINC00265 inhibited proliferation of cancer cell lines and tumor growth in xenografts. Western blot and flow cytometry analyses indicated that silencing of LINC00265 induced autophagy and apoptosis. Moreover, we showed that LINC00265 interacted with and stabilized the transcriptional co-repressor Switch-independent 3a (SIN3A), which is a scaffold protein functioning either as a tumor repressor or as an oncogene in a context-dependent manner. Silencing of SIN3A also reduced proliferation of lung cancer cells, which was correlated with the induction of autophagy. These observations raise the possibility that LINC00265 functions to promote the oncogenic activity of SIN3A in lung adenocarcinoma.
UNASSIGNED: Our findings thus identify SIN3A as a LINC00265-associated protein and should help to understand the mechanism underlying LINC00265-mediated oncogenesis.

Liver fibrosis is a persistent damage repair response triggered by various injury factors, which leads to an abnormal accumulation of extracellular matrix within liver tissue samples. The current clinical treatment of liver fibrosis is currently ineffective; therefore, elucidating the mechanism of liver fibrogenesis is of significant importance. Herein, the function and related mechanisms of lncRNA Snhg12 within hepatic fibrosis were investigated. Snhg12 expression was shown to be increased in mouse hepatic fibrotic tissue samples, and Snhg12 knockdown suppressed hepatic pathological injury and down-regulated the expression levels of fibrosis-associated proteins. Mechanistically, Snhg12 played a role in the early activation of mouse hepatic stellate cells (mHSCs) based on bioinformatics analysis, and Snhg12 was positively correlated with Igfbp3 expression. Further experimental results demonstrated that Snhg12 knockdown impeded mHSCs proliferation and activation and also downregulated the protein expression of Igfbp3. Snhg12 could interact with IGFBP3 and boost its protein stability, and overexpression of Igfbp3 partially reversed the inhibition of mHSCsproliferation and activation by the knockdown of Snhg12. In conclusion, LncRNA Snhg12 mediates liver fibrosis by targeting IGFBP3 and promoting its protein stability, thereby promoting mHSC proliferation and activation. Snhg12 has been identified as an underlying target for treating liver fibrosis.

The ORF9b protein, derived from the nucleocapsid\'s open-reading frame in both SARS-CoV and SARS-CoV-2, serves as an accessory protein crucial for viral immune evasion by inhibiting the innate immune response. Despite its significance, the precise regulatory mechanisms underlying its function remain elusive. In the present study, we unveil that the ORF9b protein of SARS-CoV-2, including emerging mutant strains like Delta and Omicron, can undergo ubiquitination at the K67 site and subsequent degradation via the proteasome pathway, despite certain mutations present among these strains. Moreover, our investigation further uncovers the pivotal role of the translocase of the outer mitochondrial membrane 70 (TOM70) as a substrate receptor, bridging ORF9b with heat shock protein 90 alpha (HSP90α) and Cullin 5 (CUL5) to form a complex. Within this complex, CUL5 triggers the ubiquitination and degradation of ORF9b, acting as a host antiviral factor, while HSP90α functions to stabilize it. Notably, treatment with HSP90 inhibitors such as GA or 17-AAG accelerates the degradation of ORF9b, leading to a pronounced inhibition of SARS-CoV-2 replication. Single-cell sequencing data revealed an up-regulation of HSP90α in lung epithelial cells from COVID-19 patients, suggesting a potential mechanism by which SARS-CoV-2 may exploit HSP90α to evade the host immunity. Our study identifies the CUL5-TOM70-HSP90α complex as a critical regulator of ORF9b protein stability, shedding light on the intricate host-virus immune response dynamics and offering promising avenues for drug development against SARS-CoV-2 in clinical settings.

Pathological cardiac hypertrophy is one of the major risk factors of heart failure and other cardiovascular diseases. However, the mechanisms underlying pathological cardiac hypertrophy remain largely unknown. Here, we identified the first evidence that TNFAIP3 interacting protein 3 (TNIP3) was a negative regulator of pathological cardiac hypertrophy. We observed a significant upregulation of TNIP3 in mouse hearts subjected to transverse aortic constriction (TAC) surgery and in primary neonatal rat cardiomyocytes stimulated by phenylephrine (PE). In Tnip3-deficient mice, cardiac hypertrophy was aggravated after TAC surgery. Conversely, cardiac-specific Tnip3 transgenic (TG) mice showed a notable reversal of the same phenotype. Accordingly, TNIP3 alleviated PE-induced cardiomyocyte enlargement in vitro. Mechanistically, RNA-sequencing and interactome analysis were combined to identify the signal transducer and activator of transcription 1 (STAT1) as a potential target to clarify the molecular mechanism of TNIP3 in pathological cardiac hypertrophy. Via immunoprecipitation and Glutathione S-transferase assay, we found that TNIP3 could interact with STAT1 directly and suppress its degradation by suppressing K48-type ubiquitination in response to hypertrophic stimulation. Remarkably, preservation effect of TNIP3 on cardiac hypertrophy was blocked by STAT1 inhibitor Fludaradbine or STAT1 knockdown. Our study found that TNIP3 serves as a novel suppressor of pathological cardiac hypertrophy by promoting STAT1 stability, which suggests that TNIP3 could be a promising therapeutic target of pathological cardiac hypertrophy and heart failure.

Alternative splicing plays a crucial role in increasing the diversity of mRNAs expressed in the genome. Serine/arginine-rich splicing factor 3 (SRSF3) is responsible for regulating the alternative splicing of its own mRNA and ensuring that its expression is balanced to maintain homeostasis. Moreover, the exon skipping of SRSF3 leads to the production of a truncated protein instead of a frameshift mutation that generates a premature termination codon (PTC). However, the precise regulatory mechanism involved in the splicing of SRSF3 remains unclear. In this study, we first established a platform for coexpressing full-length SRSF3 (SRSF3-FL) and SRSF3-PTC and further identified a specific antibody against the SRSF3-FL and truncated SRSF3 (SRSF3-TR) proteins. Next, we found that exogenously overexpressing SRSF3-FL or SRSF3-PTC failed to reverse the effects of digoxin, caffeine, or both in combination on this molecule and its targets. Endoplasmic reticulum-related pathways, transcription factors, and chemicals such as palmitic acid and phosphate were found to be involved in the regulation of SRSF3 expression. The downregulation of SRSF3-FL by palmitic acid and phosphate was mediated via different regulatory mechanisms in HeLa cells. In summary, we provide new insights into the altered expression of the SRSF3-FL and SRSF3-TR proteins for the identification of the functions of SRSF3 in cells.

The TBX1 gene plays a critical role in the development of 22q11.2 deletion syndrome (22q11.2DS), a complex genetic disorder associated with various phenotypic manifestations. In this study, we performed in-silico analysis to identify potentially deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) within the TBX1 gene and evaluate their functional and structural impact on 22q11.2DS. A comprehensive analysis pipeline involving multiple computational tools was employed to predict the pathogenicity of nsSNPs. This study assessed protein stability and explored potential alterations in protein-protein interactions. The results revealed the rs751339103(C>A), rs780800634(G>A), rs1936727304(T>C), rs1223320618(G>A), rs1248532217(T>C), rs1294927055 (C>T), rs1331240435 (A>G, rs1601289406 (A>C), rs1936726164 (G>A), and rs911796187(G>A) with a high-risk potential for affecting protein function and stability. These nsSNPs were further analyzed for their impact on post-translational modifications and structural characteristics, indicating their potential disruption of molecular pathways associated with TBX1 and its interacting partners. These findings provide a foundation for further experimental studies and elucidation of potential therapeutic targets and personalized treatment approaches for individuals affected by 22q11.2DS.

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BACKGROUND: There is growing evidence indicating that deubiquitinating enzymes may contribute to tumor progression and can serve as promising therapeutic targets.
METHODS: The overexpression of deubiquitinase OTUD6B in lung adenocarcinoma (LUAD) and its adjacent tissues was analyzed by immunohistochemistry and TCGA/GO database. Survival analysis further supported OTUD6B as a potential target for LUAD treatment. We assessed the effect of OTUD6B on LUAD cell growth using cell viability assays and conducted TUNEL staining, migration, and invasion experiments to investigate the impact of OTUD6B on the apoptosis and metastasis of LUAD cells. Additionally, we established a transplanted tumor model in nude mice to validate our findings in vivo. Finally, using IP mass spectrometry and co-IP experiments, we screened and confirmed the influence of RIPK1 as a substrate of OTUD6B in LUAD.
RESULTS: OTUD6B is highly overexpressed in human LUAD and predicts poor prognosis in LUAD patients. OTUD6B knockdown inhibited the proliferation of LUAD cells and enhanced apoptosis and inhibited metastasis in LUAD cells suppressed. A549 xenografts revealed that OTUD6B deletion can slow down tumour growth. Additionally, OTUD6B can bind to RIPK1, reduce its ubiquitination level and increase its protein stability.
CONCLUSIONS: Our results suggest that OTUD6B is a promising clinical target for LUAD treatment and that targeting OTUD6B may constitute an effective anti-LUAD strategy.

p53 regulates multiple signaling pathways and maintains cell homeostasis under conditions of DNA damage and oxidative stress. Although USP7 has been shown to promote p53 stability via deubiquitination, the USP7-p53 activation mechanism has remained unclear. Here, we propose that DNA damage induces reactive oxygen species (ROS) production and activates ATM-CHK2, and CHK2 then phosphorylates USP7 at S168 and T231. USP7 phosphorylation is essential for its deubiquitination activity toward p53. USP7 also deubiquitinates CHK2 at K119 and K131, increasing CHK2 stability and creating a positive feedback loop between CHK2 and USP7. Compared to peri-tumor tissues, thyroid cancer and colon cancer tissues show higher CHK2 and phosphorylated USP7 (S168, T231) levels, and these levels are positively correlated. Collectively, our results uncover a phosphorylation-deubiquitination positive feedback loop involving the CHK2-USP7 axis that supports the stabilization of p53 and the maintenance of cell homeostasis.