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Liquid-liquid phase separation has emerged as a crucial mechanism driving the formation of membraneless biomolecular condensates, which play important roles in numerous cellular processes. These condensates, found both in the nucleus and cytoplasm, are formed through multivalent, low-affinity interactions between various molecules. P62-containing condensates serve, among other functions, as proteolytic hubs for the ubiquitin-proteasome system. In this study, we investigated the dynamic interplay between nuclear p62 condensates and promyelocytic nuclear bodies (PML-NBs). We show that p62 condensates stabilize PML-NBs under both basal conditions and following exposure to arsenic trioxide which stimulates their degradation. We further show that this effect on the stability of PML-NBs is due to sequestration of their ubiquitin E3 ligase RNF4 in the p62 condensates with subsequent rapid degradation of the ligase. The sequestration of the ligase is made possible by association between the proline-rich domain of the PML protein and the PB1 domain of p62, which results in the formation of a PML-NB shell around the p62 condensates. Importantly, these hybrid structures do not undergo fusion and mixing of their contents which leaves unsolved the mechanism of sequestration of RNF4 in the condensates. These findings suggest an additional possible mechanism of PML-NB as a tumor suppressor which is mediated via interactions between different biomolecular condensates., Competing Interests: Competing interests statement:The authors declare no competing interest.

PROTAC® (proteolysis-targeting chimera) molecules induce proximity between an E3 ligase and protein-of-interest (POI) to target the POI for ubiquitin-mediated degradation. Cooperative E3-PROTAC-POI complexes have potential to achieve neo-substrate selectivity beyond that established by POI binding to the ligand alone. Here, we extend the collection of ubiquitin ligases employable for cooperative ternary complex formation to include the C-degron E3 KLHDC2. Ligands were identified that engage the C-degron binding site in KLHDC2, subjected to structure-based improvement, and linked to JQ1 for BET-family neo-substrate recruitment. Consideration of the exit vector emanating from the ligand engaged in KLHDC2's U-shaped degron-binding pocket enabled generation of SJ46421, which drives formation of a remarkably cooperative, paralog-selective ternary complex with BRD3 BD2 . Meanwhile, screening pro-drug variants enabled surmounting cell permeability limitations imposed by acidic moieties resembling the KLHDC2-binding C-degron. Selectivity for BRD3 compared to other BET-family members is further manifested in ubiquitylation in vitro, and prodrug version SJ46420-mediated degradation in cells. Selectivity is also achieved for the ubiquitin ligase, overcoming E3 auto-inhibition to engage KLHDC2, but not the related KLHDC1, KLHDC3, or KLHDC10 E3s. In sum, our study establishes neo-substrate-specific targeted protein degradation via KLHDC2, and provides a framework for developing selective PROTAC protein degraders employing C-degron E3 ligases., (© 2024. The Author(s).)

Transcription cofactor vestigial-like 3 (VGLL3), as a master regulator of female-biased autoimmunity, also functions in tumor development, while the underlying mechanisms remain largely elusive. Here, we report that VGLL3 plays an important role in DNA damage response (DDR). VGLL3 can be recruited to damage sites in a PARylation-dependent manner. VGLL3 depletion impairs the accumulation of RNF8 and RAD51 at sites of DNA damage, leading to reduced homologous recombination efficiency and increased cellular sensitivity to chemotherapeutic drugs. Mechanistically, VGLL3 can prevent CtIP from KLHL15-mediated ubiquitination and degradation through competitive binding with KLHL15 and, meanwhile, stabilize MDC1 by limiting TRIP12-MDC1 but promoting USP7-MDC1 associations for optimal RNF8 signaling initiation. Consistently, VGLL3 depletion delays tumor development and sensitizes the xenografts to etoposide treatment. Overall, our results reveal an unexpected role of VGLL3 in DDR, which is distinct from its transcriptional cofactor function and not conserved among VGLL family members.

Tsg101, a component of the endosomal sorting complex required for transport (ESCRT), is responsible for recognition of events requiring the machinery, as signaled by cargo tagging with ubiquitin (Ub), and for recruitment of downstream acting subunits to the site. Although much is known about the latter function, little is known about its role in the earlier event. The N-terminal domain of Tsg101 is a structural homologue of Ub conjugases (E2 enzymes) and the protein associates with Ub ligases (E3 enzymes) that regulate several cellular processes including virus budding. A pocket in the domain recognizes a motif, PT/SAP, that permits its recruitment. PT/SAP disruption makes budding dependent on Nedd4L E3 ligases. Using HIV-1 encoding a PT/SAP mutation that makes budding Nedd4L-dependent, we identified as critical for rescue the residues in the catalytic (HECT) domain of the E3 enzyme that lie in proximity to sites in Tsg101 that bind Ub non-covalently. Mutation of these residues impaired rescue by Nedd4L but the same mutations had no apparent effect in the context of a Nedd4 isomer, Nedd4-2s, whose N-terminal (C2) domain is naturally truncated, precluding C2-HECT auto-inhibition. Surprisingly, like small molecules that disrupt Tsg101 Ub-binding, small molecules that interfered with Nedd4 substrate recognition arrested budding at an early stage, supporting the conclusion that Tsg101-Ub-Nedd4 interaction promotes enzyme activation and regulates Nedd4 signaling for viral egress. Tsg101 regulation of E3 ligases may underlie its broad ability to function as an effector in various cellular activities, including viral particle assembly and budding.

Alkaliptosis is a type of pH-dependent cell death and plays an emerging role in tumor suppression. However, the key modulation mechanism of alkaliptosis remains largely unknown. In particular, the nucleus, as the centre of genetic and metabolic regulation, is crucial for the regulation of cellular life. It is not known whether nuclear proteins are involved in the regulation of alkaliptosis. Here, we isolated nuclear proteins to perform a proteomics that identified itchy E3 ubiquitin protein ligase (ITCH) as a natural inhibitor of alkaliptosis in human pancreatic ductal adenocarcinoma (PDAC) cells. The downregulation of ITCH protein is associated with the induction of alkaliptosis in three human PDAC cell lines (SW1990, MiaPaCa2, and PANC1). Functionally, increasing ITCH expression reduces JTC801-induced growth inhibition and cell death. In contrast, knocking down ITCH using specific shRNA increases JTC801-induced cell growth inhibition in the short or long term, resulting in increased cell death. Mechanistically, JTC801-induced ITCH inhibition blocks large tumor suppressor kinase 1 (LATS1) ubiquitination, which in turn suppresses Yes1 associated transcriptional regulator (YAP1)-dependent the transcriptional activation of solute carrier family 16 member 1 (SLC16A1), a proton-linked monocarboxylate transporter that inhibits JTC801-induced alkaliptosis. Additionally, decreased expression of ITCH is associated with longer survival times in patients with PDAC. Collectively, our results establish an ITCH-dependent pathway that regulates alkaliptotic sensitivity in PDAC cells and deepen the understanding of alkaliptosis in targeted therapy., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest or financial interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Yes-associated protein (YAP) is a central player in cancer development, with functions extending beyond its recognized role in cell growth regulation. Recent work has identified a link between YAP/transcriptional coactivator with PDZ-binding motif (TAZ) and the DNA damage response. Here, we investigated the mechanistic underpinnings of the cross-talk between DNA damage repair and YAP activity. Ku70, a key component of the nonhomologous end joining pathway to repair DNA damage, engaged in a dynamic competition with TEAD4 for binding to YAP, limiting the transcriptional activity of YAP. Depletion of Ku70 enhanced interaction between YAP and TEAD4 and boosted YAP transcriptional capacity. Consequently, Ku70 loss enhanced tumorigenesis in colon cancer and hepatocellular carcinoma (HCC) in vivo. YAP impeded DNA damage repair and elevated genome instability by inducing PARP1 degradation through the SMURF2-mediated ubiquitin-proteasome pathway. Analysis of samples from patients with HCC substantiated the link between Ku70 expression, YAP activity, PARP1 levels, and genome instability. In conclusion, this research provides insight into the mechanistic interactions between YAP and key regulators of DNA damage repair, highlighting the role of a Ku70-YAP-PARP1 axis in preserving genome stability. Significance: Increased yes-associated protein transcriptional activity stimulated by loss of Ku70 induces PARP1 degradation by upregulating SMURF2 to inhibit DNA damage, driving genome instability and tumorigenesis., (©2024 American Association for Cancer Research.)

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide. Significantly activated uridine nucleotide and fatty acid metabolism in HCC cells promote malignant proliferation and immune evasion. Herein, it is demonstrated that the tripartite motif 65 (TRIM65) E3 ubiquitin-protein ligase, O-GlcNAcylated via O-GlcNAcylation transferase, is highly expressed in HCC and facilitated metabolic remodeling to promote the accumulation of products related to uracil metabolism and palmitic acid, driving the progression of HCC. Mechanistically, it is showed that TRIM65 mediates ubiquitylation at the K44 residue of neurofibromatosis type 2 (NF2), the key protein upstream of classical Hippo signaling. Accelerated NF2 degradation inhibits yes-associated protein 1 phosphorylation, inducing aberrant activation of related metabolic enzyme transcription, and orchestrating metabolic and immune advantages. In conclusion, these results reveal a critical role for the TRIM family molecule TRIM65 in supporting HCC cell survival and highlight the therapeutic potential of targeting its E3 ligase activity to alter the regulation of proteasomal degradation., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Anaplastic thyroid carcinoma (ATC) is a highly aggressive human malignancy without effective treatment. Yes-associated protein (YAP) is a critical effector of the Hippo pathway, which is essential in thyroid carcinogenesis. However, the underlying mechanisms of aberrant YAP expression in ATC are not completely understood. Ubiquitylation-related enzyme siRNA screening identified the ubiquitin protein ligase E3 component n-recognin 1 (UBR1) as a stabilizer of YAP in ATC cells. UBR1 deficiency reduced YAP protein levels and its target gene expression. UBR1 directly interacted with YAP and promoted its monoubiquitylation, competitively suppressing its polyubiquitylation and resulting in extended protein half-life. UBR1 depletion reduced ATC cell proliferation and migration in vitro. Xenograft tumor studies also suggested that UBR1 knockdown suppressed ATC cell growth in vivo. Furthermore, exogenous YAP expression partially reversed the inhibitive effects of UBR1 depletion on ATC cell proliferation and migration. Our studies demonstrated that UBR1 directly interacts with YAP and stabilized it in a monoubiquitylation-dependent manner, consequently promoting ATC tumorigenesis, suggesting that UBR1 might be a potentially therapeutic target for ATC treatment., (© 2024. The Author(s).)

Background: There are numerous human genes associated with viral infections, and their identification in specific populations can provide suitable therapeutic targets for modulating the host immune system response and better understanding the viral pathogenic mechanisms. Many antiviral signaling pathways, including Type I interferon and NF-κB, are regulated by TRIM proteins. Therefore, the identification of TRIM proteins involved in COVID-19 infection can play a significant role in understanding the innate immune response to this virus., Methods: In this study, the expression of TRIM25 gene was evaluated in a blood sample of 330 patients admitted to the hospital (142 patients with severe disease and 188 patients with mild disease) as well as in 160 healthy individuals. The relationship between its expression and the severity of COVID-19 disease was assessed and compared among the study groups by quantitative Real-time PCR technique. The statistical analysis of the results demonstrated a significant reduction in the expression of TRIM25 in the group of patients with severe infection compared to those with mild infection. Furthermore, the impact of increased expression of TRIM25 gene in HEK-293 T cell culture was investigated on the replication of attenuated SARS-CoV-2 virus., Results: The results of Real-time PCR, Western blot for the viral nucleocapsid gene of virus, and CCID 50 test indicated a decrease in virus replication in these cells. The findings of this research indicated that the reduced expression of the TRIM25 gene was associated with increased disease severity of COVID-19 in individuals. Additionally, the results suggested the overexpression of TRIM25 gene can impress the replication of attenuated SARS-CoV-2 and the induction of beta-interferon., Conclusion: TRIM25 plays a critical role in controlling viral replication through its direct interaction with the virus and its involvement in inducing interferon during the early stages of infection. This makes TRIM25 a promising target for potential therapeutic interventions., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Shade avoidance syndrome is an important adaptive strategy. Under shade, major transcriptional rearrangements underlie the reallocation of resources to elongate vegetative structures and redefine the plant architecture to compete for photosynthesis. BBX28 is a B-box transcription factor involved in seedling de-etiolation and flowering in Arabidopsis (Arabidopsis thaliana), but its function in shade-avoidance response is completely unknown. Here, we studied the function of BBX28 using two mutant and two transgenic lines of Arabidopsis exposed to white light and simulated shade conditions. We found that BBX28 promotes hypocotyl growth under shade through the phytochrome system by perceiving the reduction of red photons but not the reduction of photosynthetically active radiation or blue photons. We demonstrated that hypocotyl growth under shade is sustained by the protein accumulation of BBX28 in the nuclei in a CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1)-dependent manner at the end of the photoperiod. BBX28 up-regulates the expression of transcription factor- and auxin-related genes, thereby promoting hypocotyl growth under prolonged shade. Overall, our results suggest the role of BBX28 in COP1 signaling to sustain the shade-avoidance response and extend the well-known participation of other members of BBX transcription factors for fine-tuning plant growth under shade., Competing Interests: Conflict of interest statement None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

TRIM32 is often aberrantly expressed in many types of cancers. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked with several human malignancies, including Kaposi's sarcoma and primary effusion lymphomas (PELs). Increasing evidence has demonstrated the crucial role of KSHV lytic replication in viral tumorigenesis. However, the role of TRIM32 in herpesvirus lytic replication remains unclear. Here, we reveal that the expression of TRIM32 is upregulated by KSHV in latency, and reactivation of KSHV lytic replication leads to the inhibition of TRIM32 in PEL cells. Strikingly, RTA, the master regulator of lytic replication, interacts with TRIM32 and dramatically promotes TRIM32 for degradation via the proteasome systems. Inhibition of TRIM32 induces cell apoptosis and in turn inhibits the proliferation and colony formation of KSHV-infected PEL cells and facilitates the reactivation of KSHV lytic replication and virion production. Thus, our data imply that the degradation of TRIM32 is vital for the lytic activation of KSHV and is a potential therapeutic target for KSHV-associated cancers., Importance: TRIM32 is associated with many cancers and viral infections; however, the role of TRIM32 in viral oncogenesis remains largely unknown. In this study, we found that the expression of TRIM32 is elevated by Kaposi's sarcoma-associated herpesvirus (KSHV) in latency, and RTA (the master regulator of lytic replication) induces TRIM32 for proteasome degradation upon viral lytic reactivation. This finding provides a potential therapeutic target for KSHV-associated cancers., Competing Interests: The authors declare no conflict of interest.

RNF214 is an understudied ubiquitin ligase with little knowledge of its biological functions or protein substrates. Here we show that the TEAD transcription factors in the Hippo pathway are substrates of RNF214. RNF214 induces non-proteolytic ubiquitylation at a conserved lysine residue of TEADs, enhances interactions between TEADs and YAP, and promotes transactivation of the downstream genes of the Hippo signaling. Moreover, YAP and TAZ could bind polyubiquitin chains, implying the underlying mechanisms by which RNF214 regulates the Hippo pathway. Furthermore, RNF214 is overexpressed in hepatocellular carcinoma (HCC) and inversely correlates with differentiation status and patient survival. Consistently, RNF214 promotes tumor cell proliferation, migration, and invasion, and HCC tumorigenesis in mice. Collectively, our data reveal RNF214 as a critical component in the Hippo pathway by forming a signaling axis of RNF214-TEAD-YAP and suggest that RNF214 is an oncogene of HCC and could be a potential drug target of HCC therapy., (© 2024. The Author(s).)

The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is attribute to the aggressive local invasion, distant metastasis and drug resistance of PDAC patients, which was strongly accelerated by epithelial-mesenchymal transition (EMT). In current study, we systematically investigate the role of ZNF263/RNF126 axis in the initiation of EMT in PDAC in vitro and vivo. ZNF263 is firstly identified as a novel transactivation factor of RNF126. Both ZNF263 and RNF126 were overexpressed in PDAC tissues, which were associated with multiple advanced clinical stages and poor prognosis of PDAC patients. ZNF263 overexpression promoted cell proliferation, drug resistance and EMT in vitro via activating RNF126 following by the upregulation of Cyclin D1, N-cad, and MMP9, and the downregulation of E-cad, p21, and p27. ZNF263 silencing contributed to the opposite phenotype. Mechanistically, ZNF263 transactivated RNF126 via binding to its promoter. Further investigations revealed that ZNF263 interacted with ZNF31 to coregulate the transcription of RNF126, which in turn promoted ubiquitination-mediated degradation of PTEN. The downregulation of PTEN activated AKT/Cyclin D1 and AKT/GSK-3β/β-catenin signaling, thereby promoting the malignant phenotype of PDAC. Finally, the coordination of ZNF263 and RNF126 promotes subcutaneous tumor size and distant liver metastasis in vivo. ZNF263, as an oncogene, promotes proliferation, drug resistance and EMT of PDAC through transactivating RNF126., (© 2024 Wiley Periodicals LLC.)

The expression of costimulatory molecules such as MHC-II, CD86 and CD83 on dendritic cells (DCs) are strongly regulated during cellular activation. Ubiquitination of some of these markers by the E3 ubiquitin ligase MARCH-I affects the maturation state of DCs and subsequently modulates immune responses. The effects of MARCH-I gene overexpression on the functional activity of human DCs is not well understood. Here, we investigate how MARCH-I, regulates maturation of DCs. We now provide evidence that MARCH-I transduced DCs secrete high levels of IL10 despite low secretion of IL 6 and IL 12 in response to LPS stimulation. They are weak stimulators of T lymphocyte cells but skewed T cell polarization toward T regulatory subset. These results exhibit that reduced expression of surface costimulatory molecules suppresses DC activation. It can be concluded that overexpression of MARCH-I gene in DCs leads to the production of tolerogenic DC., Competing Interests: Declaration of competing interest The authors declared no conflict of interest regarding the publication of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Ubiquitination plays an important role in proliferating and invasive characteristic of glioblastoma (GBM), similar to many other cancers. Tripartite motif 25 (TRIM25) is a member of the TRIM family of proteins, which are involved in tumorigenesis through substrate ubiquitination., Methods: Difference in TRIM25 expression levels between nonneoplastic brain tissue samples and primary glioma samples was demonstrated using publicly available glioblastoma database, immunohistochemistry, and western blotting. TRIM25 knockdown GBM cell lines (LN229 and U251) and patient derived GBM stem-like cells (GSCs) GBM#021 were used to investigate the function of TRIM25 in vivo and in vitro. Co-immunoprecipitation (Co-IP) and mass spectrometry analysis were performed to identify NONO as a protein that interacts with TRIM25. The molecular mechanisms underlying the promotion of GBM development by TRIM25 through NONO were investigated by RNA-seq and validated by qRT-PCR and western blotting., Results: We observed upregulation of TRIM25 in GBM, correlating with enhanced glioblastoma cell growth and invasion, both in vitro and in vivo. Subsequently, we screened a panel of proteins interacting with TRIM25; mass spectrometry and co-immunoprecipitation revealed that NONO was a potential substrate of TRIM25. TRIM25 knockdown reduced the K63-linked ubiquitination of NONO, thereby suppressing the splicing function of NONO. Dysfunctional NONO resulted in the retention of the second intron in the pre-mRNA of PRMT1, inhibiting the activation of the PRMT1/c-MYC pathway., Conclusions: Our study demonstrates that TRIM25 promotes glioblastoma cell growth and invasion by regulating the PRMT1/c-MYC pathway through mediation of the splicing factor NONO. Targeting the E3 ligase activity of TRIM25 or the complex interactions between TRIM25 and NONO may prove beneficial in the treatment of GBM., (© 2024. The Author(s).)

The function of immune cells is delicately regulated under a variety of molecular networks. Transcriptional intermediary factor 1 (TIF1) family proteins, consisting of TRIM24, TRIM28 and TRIM33, share a highly conserved RING domain that is essential for the regulation of protein ubiquitination functioning as E3 ubiquitin ligases. TIF1 family proteins are diversely expressed in different types of immune cells, and participate in the regulation of various of cellular functions including chromosome modification, DNA repair, tumor progression, and immunity. In this review, we summarized current studies on TIF1 family proteins' functions in the modulation of immune cell development, anti-infection immunity, cancer immunology, inflammation, and autoimmune diseases., (© 2024. The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd.)

Targeted protein degradation (TPD), induced by enforcing target proximity to an E3 ubiquitin ligase using small molecules has become an important drug discovery approach for targeting previously undruggable disease-causing proteins. However, out of over 600 E3 ligases encoded by the human genome, just over 10 E3 ligases are currently utilized for TPD. Here, using the affinity-directed protein missile (AdPROM) system, in which an anti-GFP nanobody was linked to an E3 ligase, we screened over 30 E3 ligases for their ability to degrade 4 target proteins, K-RAS, STK33, β-catenin, and FoxP3, which were endogenously GFP-tagged. Several new E3 ligases, including CUL2 diGly receptor KLHDC2, emerged as effective degraders, suggesting that these E3 ligases can be taken forward for the development of small-molecule degraders, such as proteolysis targeting chimeras (PROTACs). As a proof of concept, we demonstrate that a KLHDC2-recruiting peptide-based PROTAC connected to chloroalkane is capable of degrading HALO-GFP protein in cells., Competing Interests: Declaration of interests M.A.Q., A.K., P.S.S., K.-H.C., and A.I.L. are employees and shareholders of GSK. S.R. is currently an employee and shareholder of AstraZeneca., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription., Competing Interests: Declaration of interests B.L.E. has received research funding from Celgene, Deerfield, Novartis, and Calico and consulting fees from GRAIL. He is a member of the Scientific Advisory Board and shareholder for Neomorph Inc., TenSixteen Bio, Skyhawk Therapeutics, and Exo Therapeutics. A.S.S. received consulting fees from Adaptive Technologies. N.H.T. receives funding from the Novartis Research Foundation and is a member of the Scientific Advisory Board of Monte Rosa Therapeutics. K.A.D. is a consultant to Kronos Bio and Neomorph Inc. E.S.F. is a founder, Scientific Advisory Board (SAB) member, and equity holder of Civetta Therapeutics, Lighthorse Therapeutics, Proximity Therapeutics, and Neomorph, Inc. (also board of directors). B.L.E. and M.S. have received research funding from Calico Life Sciences LLC. E.S.F. is an equity holder and SAB member for Avilar Therapeutics and Photys Therapeutics and a consultant to Odyssey, Novartis, Sanofi, EcoR1 Capital, Ajax Therapeutics, and Deerfield. The Fischer lab receives or has received research funding from Deerfield, Novartis, Ajax, Interline, and Astellas., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

MORC family CW-type zinc finger 4 (MORC4) possessing nuclear matrix binding domains has been observed to be involved in multiple cancer development. By digging three gene expression omnibus (GEO) gene microarrays (GSE110223, GSE110224 and GSE24514), we found that MORC4 was overexpressed in colorectal cancer (CRC) samples (log 2 Fold change >1, p < 0.05). We aimed to investigate the role of MORC4 in CRC malignant behaviors, with an emphasis on polycomb group ring finger 1 (PCGF1)/cyclin-dependent kinase inhibitor 1A (CDKN1A) axis. Firstly, we confirmed MORC4 as an upregulated gene in 60 pairs of frozen CRC and adjacent normal samples. MORC4 overexpression increased proliferation and metastasis, and decreased apoptosis in SW480 and HT29 cells, which was diminished by the knockdown of PCGF1, a transcriptional repressor of CDKN1A (a potent cyclin-dependent kinase inhibitor). MORC4 was further identified as a novel molecule that interacted with PCGF1 via coimmunoprecipitation. MORC4 itself did not substantially suppress CDKN1A transcriptional activity, but it augmented PCGF1's effect on CDKN1A. Additionally, MORC4 acted as the substrate of HECT, C2, and WW domain-containing E3 ubiquitin protein ligase 2 (HECW2) and was degraded through ubiquitin-proteasome system. Collectively, our work suggested that MORC4 accelerated CRC progression via governing PCGF1/CDKN1A signaling., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Evidence has revealed that transcription factors play essential roles in regulation of multiple cellular processes, including cell proliferation, metastasis, EMT, cancer stem cells and chemoresistance. Dysregulated expression levels of transcription factors contribute to tumorigenesis and malignant progression. The expression of transcription factors is tightly governed by several signaling pathways, noncoding RNAs and E3 ubiquitin ligases. Cancer stem cells (CSCs) have been validated in regulation of tumor metastasis, reoccurrence and chemoresistance in human cancer. Transcription factors have been verified to participate in regulation of CSC formation, including Oct4, SOX2, KLF4, c-Myc, Nanog, GATA, SALL4, Bmi-1, OLIG2, POU3F2 and FOX proteins. In this review article, we will describe the critical role of CSC-related transcription factors. We will further discuss which E3 ligases regulate the degradation of these CSC-related transcription factors and their underlying mechanisms. We also mentioned the functions and mechanisms of EMT-associated transcription factors such as ZEB1, ZEB2, Snail, Slug, Twist1 and Twist2. Furthermore, we highlight the therapeutic potential via targeting E3 ubiquitin ligases for modulation of these transcription factors., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cell competition, the elimination of cells surrounded by more fit neighbors, is proposed to suppress tumorigenesis. Mahjong (Mahj), a ubiquitin E3 ligase substrate receptor, has been thought to mediate competition of cells mutated for lethal giant larvae (lgl), a neoplastic tumor suppressor that defines apical-basal polarity of epithelial cells. Here, we show that Drosophila cells mutated for mahjong, but not for lgl [l(2)gl], are competed because they express the bZip-domain transcription factor Xrp1, already known to eliminate cells heterozygous for ribosomal protein gene mutations (Rp/+ cells). Xrp1 expression in mahj mutant cells results in activation of JNK signaling, autophagosome accumulation, eIF2α phosphorylation and lower translation, just as in Rp/+ cells. Cells mutated for damage DNA binding-protein 1 (ddb1; pic) or cullin 4 (cul4), which encode E3 ligase partners of Mahj, also display Xrp1-dependent phenotypes, as does knockdown of proteasome subunits. Our data suggest a new model of mahj-mediated cell competition that is independent of apical-basal polarity and couples Xrp1 to protein turnover., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules., (© 2022. The Author(s).)

Prostate cancer (PCa) malignant progression is accompanied with the reprogramming of glucose metabolism. However, the genes involved in the regulation of glucose metabolism in PCa are not fully understood. Here, we propose a new method, DMRG, which constructs a weighted differential network (W-K-DN) to define the important metabolism-related genes. Based on biological knowledge and prostate cancer transcriptome data, a tripartite motif-containing 25 (TRIM25) was defined using DMRG; TRIM25 was involved in the regulation of glucose metabolism, which was verified by overexpressing or knocking down TRIM25 in PCa cell lines. Differential expression analysis of TCA cycle enzymes revealed that TRIM25 regulated isocitrate dehydrogenase 1 (IDH1) and fumarate hydratase (FH) expression. Moreover, a protein-RNA interaction network of TRIM25 revealed that TRIM25 interacted with RNA-binding proteins, including DExH-box helicase 9 and DEAD-box helicase 5, to play a role in regulating the RNA processing of metabolic enzymes, including IDH1 and FH. Furthermore, TRIM25 expression level was found to be positively correlated with Gleason scores in PCa patient tissues. In conclusion, this study provides a new method to define genes influencing tumor progression, and sheds light on the role of the defined TRIM25 in regulating glucose metabolism and promoting PCa malignancy.

Regulation of innate immune responses is essential for maintenance of immune homeostasis and development of an appropriate immunity against microbial infection. We show here that miR-3614-5p , product of the TRIM25 host gene, is induced by type I interferon (IFN-I) in several human non-immune and immune cell types, in particular in primary myeloid cells. Studies in HeLa cells showed that miR-3614-5p represses both p110 and p150 ADAR1 and reduces constitutive and IFN-induced A-to-I RNA editing. In line with this, activation of innate sensors and expression of IFN-β and the pro-inflammatory IL-6 are promoted. MiR-3614-5p directly targets ADAR1 transcripts by binding to one specific site in the 3'UTR. Moreover, we could show that endogenous miR-3614-5p is associated with Ago2 and targets ADAR1 in IFN-stimulated cells. Overall, we propose that, by reducing ADAR1, IFN-I-induced miR-3614-5p contributes to lowering the activation threshold of innate sensors. Our findings provide new insights into the role of miR-3614-5p , placing it as a potential fine tuner of dsRNA metabolism, cell homeostasis and innate immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships., (Copyright © 2022 Vuillier, Li, Black, Cruciani, Rubino, Michel and Pellegrini.)

The E3 ubiquitin ligase RNF12 plays essential roles during development, and the gene encoding it, RLIM , is mutated in the X-linked human developmental disorder Tonne-Kalscheuer syndrome (TOKAS). Substrates of RNF12 include transcriptional regulators such as the pluripotency-associated transcriptional repressor REX1. Using global quantitative proteomics in male mouse embryonic stem cells, we identified the deubiquitylase USP26 as a putative downstream target of RNF12 activity. RNF12 relieved REX1-mediated repression of Usp26 , leading to an increase in USP26 abundance and the formation of RNF12-USP26 complexes. Interaction with USP26 prevented RNF12 autoubiquitylation and proteasomal degradation, thereby establishing a transcriptional feed-forward loop that amplified RNF12-dependent derepression of REX1 targets. We showed that the RNF12-USP26 axis operated specifically in mouse testes and was required for the expression of gametogenesis genes and for germ cell differentiation in vitro. Furthermore, this RNF12-USP26 axis was disrupted by RLIM and USP26 variants found in TOKAS and infertility patients, respectively. This work reveals synergy within the ubiquitylation cycle that controls a key developmental process in gametogenesis and that is disrupted in human genetic disorders.

Homologous recombination repair (HR) is an error-free DNA damage repair pathway to maintain genome stability and a basis of gene targeting using genome-editing tools. However, the mechanisms of HR in plants are still poorly understood. Through genetic screens for DNA damage response mutants (DDRM) in Arabidopsis, we find that a plant-specific ubiquitin E3 ligase DDRM1 is required for HR. DDRM1 contains an N-terminal BRCT (BRCA1 C-terminal) domain and a C-terminal RING (really interesting new gene) domain and is highly conserved in plants including mosses. The ddrm1 mutant is defective in HR and thus is hypersensitive to DNA-damaging reagents. Biochemical studies reveal that DDRM1 interacts with and ubiquitinates the transcription factor SOG1, a plant-specific master regulator of DNA damage responses. Interestingly, DDRM1-mediated ubiquitination promotes the stability of SOG1. Consistently, genetic data support that SOG1 functions downstream of DDRM1. Our study reveals that DDRM1-SOG1 is a plant-specific module for HR and highlights the importance of ubiquitination in HR.

Hepatocellular carcinoma (HCC) is one of the largest causes of cancer-related deaths worldwide owing to the limitation of effective treatment options. The ubiquitin-proteasome system has been rapidly recognized as a frequent target of deregulation leading to cancers. Enhanced DNA damage response (DDR) promotes HCC growth and prevents chemosensitivity, and ubiquitin E3 ligases are key modulators in DDR. Therefore, a better understanding of how E3 ligases regulate cell growth and DNA damage may provide novel insights in understanding the oncogenic mechanism and improving the efficacy of DNA damage therapeutic agents. Here, we performed a high-content RNAi screening targeting 52 DDR-related E3 ligases in HCC and found that ring finger protein 4 (RNF4) was essential for HCC growth. RNF4 was highly expressed in HCC tissues, and the expression levels of RNF4 were associated with poor outcomes. RNF4 silencing significantly suppressed the cell growth, and subsequently induced G2/M arrest and apoptosis of HCC cells in vitro; RNF4 silencing also demonstrated the tumor-suppressive efficacy on HCC in vivo. Moreover, RNF4 silencing increased DNA damage, and rendered HCC cells more sensitive to DNA damage drugs and radiation. We found RNF4 functionally interacts with p62, and mechanistic analyses indicated that RNF4 silencing triggered the nuclear enrichment of p62. Moreover, the p62 nuclear targeting was required for increased DNA damage and growth suppression mediated by RNF4 silencing. Thus, our findings suggest RNF4 is essential for HCC proliferation via preventing nuclear translocation of p62. RNF4 silencing promotes DNA damage and may serve as a novel strategy to suppress cell growth and increase the sensitivity of DNA damage therapeutic agents in HCC., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Ubiquitin-conjugating enzyme E2 B (UBE2B) can form a heterodimer with ubiquitin E3 ligase RAD18. In this study, we aimed to explore new substrates of the UBE2B/RAD18 complex and their regulatory effects in ovarian cancer. Protein physical interactions were predicted using GeneMANIA. Serial sections of commercial ovarian cancer tissue arrays were used to check the protein expression of UBE2B, RAD18, and ZMYM2. Immunofluorescence staining and co-immunoprecipitation assays were performed to check their location and interactions. Cycloheximide chase assay was applied to explore the influence of UBE2B and RAD18 on ZMYM2 degradation. Xenograft tumor models were constructed to assess the influence of the UBE2B-ZMYM2 axis on in vivo tumor growth. A strong positive correlation between UBE2B and ZMYM2 and a moderate positive correlation between RAD18 and ZMYM2 were observed in 23 ovarian cancer cases. In CAOV4 and OVCAR3 cells, myc-ZMYM2 interacted with UBE2B and RAD18. UBE2B and ZMYM2 could be detected in the samples immunoprecipitated by anti-RAD18. UBE2B overexpression or knockdown did not alter ZMYM2 mRNA expression. UBE2B overexpression increased ZMYM2 monoubiquitination but reduced its polyubiquitination. RAD18 knockdown impaired UBE2B- induced ZMYM2 monoubiquitination. UBE2B overexpression significantly enhanced the stability of ZMYM2 protein, the effect of which was weakened by RAD18 knockdown. UBE2B overexpression significantly enhanced the growth of xenograft tumors derived from CAOV4 cells. ZMYM2 knockdown remarkedly suppressed tumor growth and impaired the growth-promoting effect of UBE2B overexpression. In conclusion, this study revealed a novel regulatory effect of the UBE2B/RAD18 complex on ZMYM2 monoubiquitination and stability in ovarian cancer.

REV1 is the central member of the family of TLS polymerases, which participate in various DNA damage repair and tolerance pathways and play a significant role in maintaining genomic stability. However, the role of REV1 in tumors is rarely reported. In this study, we found that the expression of REV1 was significantly upregulated in lung cancer tissues compared with matched adjacent tissues and was associated with poor prognosis. Functional experiments demonstrated that REV1 silencing decreased the growth and proliferation capacity of lung cancer cells. Mechanistically, REV1 upregulated the expression of SERTAD2 in a Rad18-dependent manner, thereby promoting lung carcinogenesis. A novel REV1 inhibitor, JH-RE-06, suppressed lung tumorigenesis in vivo and in vitro and was shown to be safe and well tolerated. Our study confirmed that REV1 is a potential diagnostic marker and therapeutic target for lung cancer and that JH-RE-06 may be a safe and efficient therapeutic agent for NSCLC., (© 2022. The Author(s).)

The use of chemical warfare agents is prohibited but they have been used in recent Middle Eastern conflicts. Their accidental exposure (e.g. arsenical lewisite) is also known and causes extensive painful cutaneous injury. However, their molecular pathogenesis is not understood. Here, we demonstrate that a nexus of stress granules (SGs), integrated stress, and RNA binding proteins (RBPs) Roquin and Reganse-1 play a key role. Lewisite and its prototype phenylarsine oxide (PAO) induce SG assembly in skin keratinocytes soon after exposure, which associate with various RBPs and translation-related proteins. SG disassembly was detected several hours after exposure. The dynamics of SG assembly-disassembly associates with the chemical insult and cell damage. Enhanced Roquin and Regnase-1 expression occurs when Roquin was recruited to SGs and Regnase-1 to the ribosome while in the disassembling SGs their expression is decreased with consequent induction of inflammatory mediators. SG-targeted protein translational control is regulated by the phosphorylation-dependent activation of eukaryotic initiation factors 2α (eIF2α). Treatment with integrated stress response inhibitor (ISRIB), which blocks eIF2α phosphorylation, impacted SG assembly dynamics. Topical application of ISRIB attenuated the inflammation and tissue disruption in PAO-challenged mice. Thus, the dynamic regulation of these pathways provides underpinning to cutaneous injury and identify translational therapeutic approach for these and similar debilitating chemicals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Srivastava, Mishra, Muzaffar, Gorbatyuk, Agarwal, Mukhtar and Athar.)

Proteolysis-targeting chimaeras (PROTACs) as well as molecular glues such as immunomodulatory drugs (IMiDs) and indisulam are drugs that induce interactions between substrate proteins and an E3 ubiquitin ligases for targeted protein degradation. Here, we develop a workflow based on proximity-dependent biotinylation by AirID to identify drug-induced neo-substrates of the E3 ligase cereblon (CRBN). Using AirID-CRBN, we detect IMiD-dependent biotinylation of CRBN neo-substrates in vitro and identify biotinylated peptides of well-known neo-substrates by mass spectrometry with high specificity and selectivity. Additional analyses reveal ZMYM2 and ZMYM2-FGFR1 fusion protein-responsible for the 8p11 syndrome involved in acute myeloid leukaemia-as CRBN neo-substrates. Furthermore, AirID-DCAF15 and AirID-CRBN biotinylate neo-substrates targeted by indisulam and PROTACs, respectively, suggesting that this approach has the potential to serve as a general strategy for characterizing drug-inducible protein-protein interactions in cells., (© 2022. The Author(s).)

Background: This study detected the methylation levels of nuclear factor-5 (NFAT5), PVT1, ribosomal protein S6 kinase A-1 (RPS6KA1), and MIB1 in patients with steroid-resistant asthma (SRA) and explored their associations with SRA., Methods: In our pilot study, we found abnormal methylation of NFAT5, PVT1, RPS6KA1, and MIB1 in SRA patients according to genome-wide methylation screening. This study expanded the sample size to further validate the results of the pilot study. Twenty patients with SRA, 20 patients with bronchial asthma, and 20 healthy volunteers constituted the SRA group, asthma control group, and healthy control group, respectively. The clinical data of all the participants were collected. Peripheral blood was taken for DNA extraction. The methylation loci and levels of NFAT5, PVT1, RPS6KA1, and MIB1 were detected using the Sequenom MassARRAY Nanodispenser RS1000. Data were processed and analyzed with SPSS 22.0 software., Results: There were 24 CpG loci detected in the NFAT5 segment 7 in the PVT1 segment, 4 in the RPS6KA1 segment, and 3 in the MIB1 segment. Among these genes, RPS6KA exhibited hypomethylation in the SRA group, which showed significant differences at the CpG&#95;1, CpG&#95;2, and CpG&#95;3 loci compared with the other groups (p < 0.05). No significant differences in the methylation levels of NFAT5, PVT1, and MIB1 were observed among the groups (p > 0.05)., Conclusions: RPS6KA1 is hypomethylated in SRA patients, which may play a role in the development of SRA via the MAPK signaling pathway. However, the influence of the methylation of NFAT5, PVT1, and MIB1 on SRA development remains to be explored., (© 2022 S. Karger AG, Basel.)

Proper dendritic morphology is fundamental to nerve signal transmission; thus, revealing the mechanism by which dendrite arborization is regulated is of great significance. Our previous studies have found that the epigenetic molecule chromodomain Y-like (CDYL) negatively regulates dendritic branching. Current research mostly focuses on the processes downstream of CDYL, whereas the upstream regulatory process has not been investigated to date. In this study, we identified an upstream regulator of CDYL, the E3 ubiquitin ligase tripartite motif-containing protein 32 (TRIM32), which promotes dendrite arborization by mediating the ubiquitylation and degradation of CDYL. By using mass spectrometry and biochemistry strategies, we proved that TRIM32 interacted with CDYL and mediated CDYL ubiquitylation modification in vivo and in vitro. Overexpressing TRIM32 decreased the protein level of CDYL, leading to an increase in the dendritic complexity of primary cultured rat neurons. In contrast, knocking down TRIM32 increased the protein level of CDYL and decreased the dendritic complexity. The truncated form of TRIM32 without E3 ligase activity (ΔRING) lost its ability to regulate dendritic complexity. Most importantly, knockdown of CDYL abolished the reduced complexity of dendrites caused by TRIM32 knockdown, indicating that the TRIM32-mediated regulation of dendritic development depends on its regulation of downstream CDYL. Hence, our findings reveal that TRIM32 could promote dendrite arborization by mediating CDYL degradation. This work initially defines a novel biological role of TRIM32 in regulating mechanisms upstream of CDYL and further presents a potential therapeutic target for the treatment of CDYL-related neurodevelopmental disorders., (© 2021 Federation of American Societies for Experimental Biology.)

The CCR4-NOT complex acts as a central player in the control of mRNA turnover and mediates accelerated mRNA degradation upon HDAC inhibition. Here, we explored acetylation-induced changes in the composition of the CCR4-NOT complex by purification of the endogenously tagged scaffold subunit NOT1 and identified RNF219 as an acetylation-regulated cofactor. We demonstrate that RNF219 is an active RING-type E3 ligase which stably associates with CCR4-NOT via NOT9 through a short linear motif (SLiM) embedded within the C-terminal low-complexity region of RNF219. By using a reconstituted six-subunit human CCR4-NOT complex, we demonstrate that RNF219 inhibits deadenylation through the direct interaction of the α-helical SLiM with the NOT9 module. Transcriptome-wide mRNA half-life measurements reveal that RNF219 attenuates global mRNA turnover in cells, with differential requirement of its RING domain. Our results establish RNF219 as an inhibitor of CCR4-NOT-mediated deadenylation, whose loss upon HDAC inhibition contributes to accelerated mRNA turnover., (© 2021. The Author(s).)

Nitrate (NO3) assimilation and signaling regulate plant growth through the relevant function of the transcription factor NIN-like Protein7 (NLP7). NO3 is also the main source for plants to produce nitric oxide (NO), which regulates growth and stress responses. NO-mediated regulation requires efficient sensing via the PROTEOLYSIS6 (PRT6)-mediated proteasome-triggered degradation of group VII of ethylene response transcription factors through the Cys/Arg N-degron pathway. The convergence of NO3 signaling and N-degron proteolysis on NO-mediated regulation remains largely unknown. Here, we investigated the functional interaction between NLP7 and PRT6 using Arabidopsis (Arabidopsis thaliana) double prt6 nlp7 mutant plants as well as complementation lines overexpressing NLP7 in different mutant genetic backgrounds. prt6 nlp7 mutant plants displayed several potentiated prt6 characteristic phenotypes, including slower vegetative growth, increased NO content, and diminished tolerance to abiotic stresses such as high-sucrose concentration, abscisic acid, and hypoxia-reoxygenation. Although NLP7 has an N-terminus that could be targeted by the N-degron proteolytic pathway, it was not a PRT6 substrate. The potential PRT6- and NO-regulated nucleocytoplasmic translocation of NLP7, which is likely modulated by posttranslational modifications, is proposed to act as a regulatory loop to control NO homeostasis and action., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Enteroviruses (EVs) 3C proteins suppress type I interferon (IFN) responses mediated by retinoid acid-inducible gene I (RIG-I), while an E3 ubiquitin ligase, tripartite motif protein 25 (TRIM25)-mediated RIG-I ubiquitination is essential for RIG-I antiviral activity. Therefore, whether the effect of EVs 3C on RIG-I is associated with TRIM25 expression is worth to be further investigated. Here, we demonstrate that 3C proteins of EV71 and coxsackievirus B3 (CVB3) reduced not only RIG-I expression but also TRIM25 expression through protease cleavage activity, while overexpression of TRIM25 restored RIG-I expression and IFN-β production reduced by 3C proteins. Further investigation confirmed that the two amino acids and functional domains in TRIM25 required for RIG-I ubiquitination and TRIM25 structural conformation were essential for the recovery of RIG-I expression. Moreover, we also observed that TRIM25 could rescue RIG-I expression reduced by 3C proteins of CVA6 and EV-D68 but not CVA16. Our findings provide an insightful interpretation of 3C-mediated host innate immune suppression and support TRIM25 as an attractive target against multiple EVs infection., (© 2021. Wuhan Institute of Virology, CAS.)

Interferon-gamma (IFN-γ) has a complex role in modulating the tumor microenvironment (TME) during renal cell carcinoma (RCC) development. To define the role of IFN-γ response genes in RCC progression, we characterized the differential gene expression, prognostic implications, and DNA variation profiles of selected IFN-γ response signatures, which exhibited a significant hazard ratio for the overall survival (OS) and progression-free survival (PFS) of papillary, chromophobia, and clear cell RCC (ccRCC) patients ( n  = 944). Prognostic nomograms were constructed to predict the outcomes for ccRCC patients, highlighting the prognostic implications of RANBP2-type and C3HC4-type zinc finger containing 1 (RBCK1). Interestingly, large-scale pan-cancer samples ( n  = 12,521) and three single-cell RNA datasets revealed that RBCK1 showed markedly differential expression between cancer and normal tissues and significantly correlated with tumor-infiltrating immune cells, tumor purity, and immune checkpoint molecules, such as PD-L1, CTLA-4, LAG-3, and TIGIT in pan-cancer samples. Notably, the TIDE score was significantly higher in the RBCK1 high group compared with the RBCK1 low group in both ccRCC and RCC cohorts. Besides, immunohistochemistry staining showed significantly elevated RBCK1 expression in tumors ( n  = 50) compared with kidney samples ( n  = 40) from a real-world cohort, Fudan University Shanghai Cancer Center (FUSCC, Shanghai). After RBCK1 expression was confirmed in ccRCC, we found a significantly decreased number of infiltrating CD4 + T cells, CD4 + FOXP3 + Treg cells, M1 macrophages, and CD56 bight/dim NK cells in the immune-cold RBCK1 high group. In addition to the distinct heterogeneous immune microenvironment, the increased expression of RBCK1 predicted a prominently worse prognosis than the RBCK1 low group for 232 ccRCC patients in the FUSCC proteomic cohort. Furthermore, after transfected with siRNA in human ccRCC cells, extraordinarily decreased cell proliferation, migration capacities, and prominently elevated apoptosis tumor cell proportion were found in the siRNA groups compared with the negative control group. In conclusion, this study identified IFN-γ response clusters, which might be used to improve the prognostic accuracy of immune contexture in the ccRCC microenvironment. Immune-cold RBCK1 high patients have pro-tumorigenic immune infiltration and significantly worse outcomes than RBCK1 low patients based on results from multi-omics to real-world data. Our discovery of novel independent prognostic indicators for RCC highlights the association between tumor alterations and immune phenotype., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer J-YZ declared a shared affiliation with several of the authors to the handling editor at the time of review. The reviewer HW declared a shared affiliation with several of the authors to the handling editor at the time of review., (Copyright © 2021 Xu, Tao, Zhu, Liu, Anwaier, Tian, Su, Shi, Huang, Wei, Li, Qu, Zhang and Ye.)

The linear ubiquitin chain assembly complex tethering motif (LUBAC-LTM) domain is composed of two different accessory LUBAC components (HOIL-1L and SHARPIN) but folds as a single globular domain. Targeted disruption of the intricate LTM-LTM interaction destabilizes LUBAC in lymphoma cells, thereby attenuating LUBAC stability, which highlights that targeting the interaction between the two LTM motifs is a promising strategy for the development of new agents against cancers that depend on LUBAC activity for their survival. To further screen for small-molecule inhibitors that can selectively disrupt the LTM-LTM interaction, it is necessary to obtain high-purity samples of the LTM domain. Ideally, such a sample would not contain any components other than the LTM itself, so that false positives (molecules binding to other parts of LUBAC) could be eliminated from the screening process. Here we report a simple strategy that enabled successful bacterial production of the isolated LUBAC LTM domain in high yield and at high purity. The strategy combines (1) structural analysis highlighting the possibility of tandem expression in the SHARPIN L ™ to HOIL-1L L ™ direction; (2) bacterial expression downstream of EGFP to efficiently monitor expression and solubility; (3) gentle low-temperature folding using autoinduction. Formation of stably folded LTM was verified by size-exclusion chromatography and heteronuclear NMR spectroscopy. From 200-ml cultures sufficient quantities (~7 mg) of high-purity protein for structural studies could be obtained. The presented strategy will be beneficial for LUBAC LTM-based drug-screening efforts and likely serve as a useful primer for similar cases, i.e., whenever a smaller folded fragment is to be isolated from a larger protein complex for site-specific downstream applications., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Nuclear factor kappa B (NF-κB) is a critical transcription factor involved in regulating cell activation, inflammation, and survival. The linear ubiquitin chain assembly complex (LUBAC) which consists of HOIL1, HOIP, and SHARPIN, catalyzes the linear ubiquitination of target proteins-a post-translational modification that is essential for NF-κB activation. Human germline pathogenic variants that dysregulate linear ubiquitination and NF-κB signaling are associated with immunodeficiency and/or autoinflammation including dermatitis, recurrent fevers, systemic inflammation and enteropathy. We previously identified MALT1 paracaspase as a novel negative regulator of LUBAC by proteolytic cleavage of HOIL1. To directly investigate the impact of HOIL1 cleavage activity on the inflammatory response, we employed a stable transduction system to express and directly compare non-cleavable HOIL1 with wild-type HOIL1 in primary HOIL1-deficient patient skin fibroblasts. We discovered that non-cleavable HOIL1 resulted in enhanced NF-κB signaling in response to innate stimuli. Transcriptomics revealed enrichment of inflammation and proinflammatory cytokine-related pathways after stimulation. Multiplexed cytokine assays confirmed a 'hyperinflammatory' phenotype in these cells. This work highlights the physiological importance of MALT1-dependent cleavage and modulation of HOIL1 on NF-κB signaling and inflammation, provides a mechanism for the autoinflammation observed in MALT1-deficient patients, and will inform the development of therapeutics that target MALT1 paracaspase and LUBAC function in treating autoinflammatory skin diseases., Competing Interests: TK was employed by Ducares/Triskelion BV. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fung, Lu, Sharma, Sharma, Saferali, Jia, Abraham, Klein, Gold, Noterangelo, Overall and Turvey.)

RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster end binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather nonspecific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments., (© 2021 Vaishali et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

IRON MAN (IMA) peptides, a family of small peptides, control iron (Fe) transport in plants, but their roles in Fe signaling remain unclear. BRUTUS (BTS) is a potential Fe sensor that negatively regulates Fe homeostasis by promoting the ubiquitin-mediated degradation of bHLH105 and bHLH115, two positive regulators of the Fe deficiency response. Here, we show that IMA peptides interact with BTS. The C-terminal parts of IMA peptides contain a conserved BTS interaction domain (BID) that is responsible for their interaction with the C terminus of BTS. Arabidopsis thaliana plants constitutively expressing IMA genes phenocopy the bts-2 mutant. Moreover, IMA peptides are ubiquitinated and degraded by BTS. bHLH105 and bHLH115 also share a BID, which accounts for their interaction with BTS. IMA peptides compete with bHLH105/bHLH115 for interaction with BTS, thereby inhibiting the degradation of these transcription factors by BTS. Genetic analyses suggest that bHLH105/bHLH115 and IMA3 have additive roles and function downstream of BTS. Moreover, the transcription of both BTS and IMA3 is activated directly by bHLH105 and bHLH115 under Fe-deficient conditions. Our findings provide a conceptual framework for understanding the regulation of Fe homeostasis: IMA peptides protect bHLH105/bHLH115 from degradation by sequestering BTS, thereby activating the Fe deficiency response., Competing Interests: The authors declare no competing interest.

In this work, we show that Not4 and Not5 from the Ccr4-Not complex modulate translation elongation dynamics and change ribosome A-site dwelling occupancy in a codon-dependent fashion. These codon-specific changes in not5Δ cells are very robust and independent of codon position within the mRNA, the overall mRNA codon composition, or changes of mRNA expression levels. They inversely correlate with codon-specific changes in cells depleted for eIF5A and positively correlate with those in cells depleted for ribosome-recycling factor Rli1. Not5 resides in punctate loci, co-purifies with ribosomes and Rli1, but not with eIF5A, and limits mRNA solubility. Overexpression of wild-type or non-complementing Rli1 and loss of Rps7A ubiquitination enable Not4 E3 ligase-dependent translation of polyarginine stretches. We propose that Not4 and Not5 modulate translation elongation dynamics to produce a soluble proteome by Rps7A ubiquitination, dynamic condensates that limit mRNA solubility and exclude eIF5A, and a moonlighting function of Rli1., Competing Interests: Declaration of interests There are no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Squalene is a triterpenoid serving as an ingredient of various products in the food, cosmetic, pharmaceutical industries. The oleaginous yeast Yarrowia lipolytica offers enormous potential as a microbial chassis for the production of terpenoids, such as carotenoid, limonene, linalool, and farnesene, as the yeast provides ample storage space for hydrophobic products. Here, we present a metabolic design that allows the enhanced accumulation of squalene in Y. lipolytica. First, we improved squalene accumulation in Y. lipolytica by overexpressing the genes ( ERG and HMG ) coding for the mevalonate pathway enzymes. Second, we increased the production of lipid where squalene is accumulated by overexpressing DGA1 (encoding diacylglycerol acyltransferase) and deleting PEX10 (for peroxisomal membrane E3 ubiquitin ligase). Third, we deleted URE2 (coding for a transcriptional regulator in charge of nitrogen catabolite repression [NCR]) to induce lipid accumulation regardless of the carbon-to-nitrogen ratio in culture media. The resulting engineered Y. lipolytica exhibited a 115-fold higher squalene content (22.0 mg/g dry cell weight) than the parental strain. These results suggest that the biological function of Ure2p in Y. lipolytica is similar to that in Saccharomyces cerevisiae, and its deletion can be utilized to enhance the production of hydrophobic target products in oleaginous yeast strains. IMPORTANCE This study demonstrated a novel strategy for increasing squalene production in Y. lipolytica. URE2, a bifunctional protein that is involved in both nitrogen catabolite repression and oxidative stress response, was identified and demonstrated correlation to squalene production. The data suggest that double deletion of PEX10 and URE2 can serve as a positive synergistic effect to help yeast cells in boosting squalene production. This discovery can be combined with other strategies to engineer cell factories to efficiently produce terpenoid in the future.

Sarcotubular myopathy (STM) is a rare autosomal recessive myopathy caused by TRIM32 gene mutations. It is predominantly characterized by the weakness of the proximal limb and mild to moderate elevation of creatine kinase (CK) levels. In this study, we describe a 50-year-old Chinese man who exhibited a proximal-to-distal weakness in the muscles of the lower limbs and who had difficulty standing up from a squat position. The symptoms gradually became more severe. He denied a history of cognitive or cardiological problems. The patient's parents and children were healthy. Histopathological examination revealed dystrophic changes and irregular slit-shaped vacuoles containing amorphous materials. Whole-exome sequencing consisting of protein-encoding regions of 19,396 genes was performed, the results of which identified one novel homozygous 2kb deletion chr9.hg19: g.119460021&#95;119461983del (exon2) in the TRIM32 gene. This was confirmed at the homozygous state with quantitative real-time PCR. Here, we present a Chinese case of STM with one novel mutation in TRIM32 and provide a brief summary of all known pathogenic mutations in TRIM32.

MicroRNA (miR)-361-5p has been studied to suppress gliomas development. Based on that, an insight into the regulatory mechanism of miR-361-5p in gliomas was supplemented from ubiquitin protein ligase E3 component N-recognin 5 (UBR5)-mediated ubiquitination of ataxia-telangiectasia mutated interactor (ATMIN). miR-361-5p, ATMIN, and UBR5 levels were clinically analyzed in gliomas tissues, which were further validated in gliomas cell lines. Loss/gain-of-function method was applied to determine the roles of miR-361-5p and UBR5 in gliomas, as to cell viability, migration, invasion, colony formation ability, and apoptosis in vitro and tumorigenesis in vivo. The relationship between miR-361-5p and UBR5 was verified and the interaction between UBR5 and ATMIN was explored. It was detected that reduced miR-361-5p and ATMIN and enhanced UBR5 levels showed in gliomas. Elevating miR-361-5p was repressive in gliomas progression. UBR5 was directly targeted by miR-361-5p. UBR5 can ubiquitinate ATMIN. miR-361-5p suppressed gliomas by regulating UBR5-mediated ubiquitination of ATMIN. Downregulating UBR5 impeded gliomas tumor growth in vivo. Upregulating miR-361-5p targets UBR5 to promote ATMIN protein expression, thus to recline the malignant phenotype of gliomas cells., (© 2021. The Author(s).)

Upon viral RNA recognition, the RIG-I signalosome continuously generates IFNs and cytokines, leading to neutrophil recruitment and inflammation. Thus, attenuation of excessive immune and inflammatory responses is crucial to restore immune homeostasis and prevent unwarranted damage, yet few resolving mediators have been identified. In the present study, we demonstrated that RTN3 is strongly upregulated during RNA viral infection and acts as an inflammation-resolving regulator. Increased RTN3 aggregates on the endoplasmic reticulum and interacts with both TRIM25 and RIG-I, subsequently impairing K63-linked polyubiquitination and resulting in both IRF3 and NF-κB inhibition. Rtn3 overexpression in mice causes an obvious inflammation resolving phenomenon when challenged with VSV, Rtn3-overexpressing mice display significantly decreased neutrophil numbers and inflammatory cell infiltration, which is accompanied by reduced tissue edema in the liver and thinner alveolar interstitium. Taken together, our findings identify RTN3 as a conserved negative regulator of immune and inflammatory responses and provide insights into the negative feedback that maintains immune and inflammatory homeostasis., Competing Interests: ZY, JW, BH, XZ, XL, EK No competing interests declared, (© 2021, Yang et al.)

A weak production of INF-β along with an exacerbated release of pro-inflammatory cytokines have been reported during infection by the novel SARS-CoV-2 virus. SARS-CoV-2 encodes several proteins able to counteract the host immune system, which is believed to be one of the most important features contributing to the viral pathogenesis and development of a severe clinical picture. Previous reports have demonstrated that SARS-CoV-2 N protein, along with some non-structural and accessory proteins, efficiently suppresses INF-β production by interacting with RIG-I, an important pattern recognition receptor (PRR) involved in the recognition of pathogen-derived molecules. In the present study, we better characterized the mechanism by which the SARS-CoV-2 N counteracts INF-β secretion and affects RIG-I signaling pathways. In detail, when the N protein was ectopically expressed, we noted a marked decrease in TRIM25-mediated RIG-I activation. The capability of the N protein to bind to, and probably mask, TRIM25 could be the consequence of its antagonistic activity. Furthermore, this interaction occurred at the SPRY domain of TRIM25, harboring the RNA-binding activity necessary for TRIM25 self-activation. Here, we describe new findings regarding the interplay between SARS-CoV-2 and the IFN system, filling some gaps for a better understanding of the molecular mechanisms affecting the innate immune response in COVID-19.

Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in H19/Igf2 locus and Oct4 promoter in mouse embryonic fibroblasts. However, nothing is known about how Kaiso influences DNA methylation at the genome level. Here we show that deficiency of Kaiso led to whole-genome hypermethylation, using Kaiso deficient human renal cancer cell line obtained via CRISPR/CAS9 genome editing. However, Kaiso serves to protect genic regions, enhancers, and regions with a low level of histone modifications from demethylation. We detected hypomethylation of binding sites for Oct4 and Nanog in Kaiso deficient cells. Kaiso immunoprecipitated with de novo DNA methyltransferases DNMT3a/3b, but not with maintenance methyltransferase DNMT1. Thus, Kaiso may attract methyltransferases to surrounding regions and modulate genome methylation in renal cancer cells apart from being methyl DNA binding protein.

The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase for linear/Met1-linked ubiquitin chain formation. One of the LUBAC components, heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L), was recently shown to catalyse oxyester bond formation between ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. Here, we present the first 3D reconstruction of human LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that this event depends on HOIL-1L catalytic activity. By cross-linking mass spectrometry showing proximity between the catalytic RING-in-between-RING (RBR) domains, a coordinated ubiquitin relay mechanism between the HOIL-1-interacting protein (HOIP) and HOIL-1L ligases is suggested. In mouse embryonic fibroblasts, these heterotypic chains were induced by TNF, which is reduced in cells expressing an HOIL-1L catalytic inactive mutant. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains by the concerted action of HOIP and HOIL-1L., Competing Interests: AR, IG, CG, AV, AS, KS, LD, ZO, KM, TC, DH, FI No competing interests declared, MK a consultant in Refeyn Ltd. SS an employee of JT Inc. PK academic founder, consultant, and shareholder in Refeyn Ltd., (© 2021, Rodriguez Carvajal et al.)