K63-linked Polyubiquitination Research Articles

Ubiquitin-induced RNF168 condensation promotes DNA double-strand break repair

Rapid accumulation of repair factors at DNA double-strand breaks (DSBs) is essential for DSB repair. Several factors involved in DSB repair have been found undergoing liquid-liquid phase separation (LLPS) at DSB sites to facilitate DNA repair. RNF168, a RING-type E3 ubiquitin ligase, catalyzes H2A.X ubiquitination for recruiting DNA repair factors. Yet, whether RNF168 undergoes LLPS at DSB sites remains unclear. Here, we identified K63-linked polyubiquitin-triggered RNF168 condensation which further promoted RNF168-mediated DSB repair. RNF168 formed liquid-like condensates upon irradiation in the nucleus while purified RNF168 protein also condensed in vitro. An intrinsically disordered region containing amino acids 460-550 was identified as the essential domain for RNF168 condensation. Interestingly, LLPS of RNF168 was significantly enhanced by K63-linked polyubiquitin chains, and LLPS largely enhanced the RNF168-mediated H2A.X ubiquitination, suggesting a positive feedback loop to facilitate RNF168 rapid accumulation and its catalytic activity. Functionally, LLPS deficiency of RNF168 resulted in delayed recruitment of 53BP1 and BRCA1 and subsequent impairment in DSB repair. Taken together, our finding demonstrates the pivotal effect of LLPS in RNF168-mediated DSB repair.

Triptolide induced spermatogenesis dysfunction via ferroptosis activation by promoting K63-linked GPX4 polyubiquitination in spermatocytes

Triptolide (TP) is a major bioactive compound derived from Tripterygium wilfordii Hook. F. (TwHF) known for its medicinal properties, but it also exhibits potential toxic effects. It has been demonstrated to induce severe male reproductive toxicity, yet the precise mechanism behind this remains unclear, which limits its broad clinical application. This study aimed to investigate the mechanisms underlying testicular damage and spermatogenesis dysfunction induced by TP in mice, using both mouse models and the spermatocyte-derived cell line GC-2spd. In the present study, it was found that TP displayed significant testicular microstructure damaged and spermatogenesis defects including lower concentration and abnormal morphology by promoting ROS formation, MDA production and restraining GSH level, glutathione peroxidase 4 (GPX4) expression in vivo. Furthermore, Ferrostatin-1 (FER-1), a ferroptosis inhibitor, was found to significantly reduce the accumulation of lipid peroxidation, alleviate testicular microstructural damage, and enhance spermatogenic function in mice. Besides, notably decreased cell viability, collapsed mitochondrial membrane potential, and elevated DNA damage were observed in vitro. The above-mentioned phenomenon could be reversed by pre-treatment of FER-1, indicating that ferroptosis participated in the TP-mediated spermatogenesis dysfunction. Mechanistically, TP could enhance GPX4 ubiquitin degradation via triggering K63-linked polyubiquitination of GPX4, thereby stimulating ferroptosis in spermatocytes. Functionally, GPX4 deletion intensified ferroptosis and exacerbated DNA damage in GC-2 cells, while GPX4 overexpression mitigated ferroptosis induced by TP. Overall, these findings for the first time indicated a vital role of ferroptosis in TP induced-testicular injury and spermatogenic dysfunction through promoting GPX4 K63-linked polyubiquitination, which hopefully offers a potential therapeutic avenue for TP-related male reproductive damage. In addition, this study also provides a theoretical foundation for the improved clinical application of TP or TwHF in the future.

Deubiquitination of aryl hydrocarbon receptor (AhR) by USP21 negatively regulates Th17 cells differentiation.

Aryl hydrocarbon receptor (AhR) is a key transcription factor that modulates the differentiation of T helper 17 (Th17) cells. How AhR is regulated at the post-translational level in Th17 cells remains largely unclear. Here we identify USP21 as a newly defined deubiquitinase of AhR. We demonstrate that USP21 interacts with and stabilizes AhR by removing the K48-linked polyubiquitin chains from AhR. Interestingly, USP21 inhibits the transcriptional activity of AhR in a deubiquitinating-dependent manner. USP21 deubiquitinates AhR at the K432 residue, and the maintenance of ubiquitination on this site is required for the intact transcriptional activity of AhR. Moreover, the deficiency of USP21 promotes the differentiation of Th17 cells both in vitro and in vivo. Consistently, adoptive transfer of USP21 deficient naïve CD4+ T cells elicits more severe colitis in Rag1-/- recipients. Therefore, our study reveals a novel mechanism in which USP21 deubiquitinates AhR and negatively regulates the differentiation of Th17 cells.

UBE2C-induced crosstalk between mono- and polyubiquitination of SNAT2 promotes lymphatic metastasis in bladder cancer.

Ubiquitination plays an essential role in protein stability, subcellular localization, and interactions. Crosstalk between different types of ubiquitination results in distinct biological outcomes for proteins. However, the role of ubiquitination-related crosstalk in lymph node (LN) metastasis and the key regulatory factors controlling this process have not been determined. Using high-throughput sequencing, we found that ubiquitin-conjugating enzyme E2 C (UBE2C) was overexpressed in bladder cancer (BCa) and was strongly associated with an unfavorable prognosis. Overexpression of UBE2C increased BCa lymphangiogenesis and promoted LN metastasis both in vitro and in vivo. Mechanistically, UBE2C mediated sodium-coupled neutral amino acid transporter 2 (SNAT2) monoubiquitination at lysine 59 to inhibit K63-linked polyubiquitination at lysine 33 of SNAT2. Crosstalk between monoubiquitination and K63-linked polyubiquitination increased SNAT2 membrane protein levels by suppressing epsin 1-mediated (EPN1-mediated) endocytosis. SNAT2 facilitated glutamine uptake and metabolism to promote VEGFC secretion, ultimately leading to lymphangiogenesis and LN metastasis in patients with BCa. Importantly, inhibition of UBE2C significantly attenuated BCa lymphangiogenesis in a patient-derived xenograft model. Our results reveal the mechanism by which UBE2C mediates crosstalk between the monoubiquitination and K63-linked polyubiquitination of SNAT2 to promote BCa metastasis and identify UBE2C as a promising target for treating LN-metastatic BCa.

TRIM65 deficiency alleviates renal fibrosis through NUDT21-mediated alternative polyadenylation.

Chronic kidney disease (CKD) is a major global health concern and the third leading cause of premature death. Renal fibrosis is the primary process driving the progression of CKD, but the mechanisms behind it are not fully understood, making treatment options limited. Here, we find that the E3 ligase TRIM65 is a positive regulator of renal fibrosis. Deletion of TRIM65 results in a reduction of pathological lesions and renal fibrosis in mouse models of kidney fibrosis induced by unilateral ureteral obstruction (UUO)- and folic acid. Through screening with a yeast-hybrid system, we identify a new interactor of TRIM65, the mammalian cleavage factor I subunit CFIm25 (NUDT21), which plays a crucial role in fibrosis through alternative polyadenylation (APA). TRIM65 interacts with NUDT21 to induce K48-linked polyubiquitination of lysine 56 and proteasomal degradation, leading to the inhibition of TGF-β1-mediated SMAD and ERK1/2 signaling pathways. The degradation of NUDT21 subsequently altered the length and sequence content of the 3'UTR (3'UTR-APA) of several pro-fibrotic genes including Col1a1, Fn-1, Tgfbr1, Wnt5a, and Fzd2. Furthermore, reducing NUDT21 expression via hydrodynamic renal pelvis injection of adeno-associated virus 9 (AAV9) exacerbated UUO-induced renal fibrosis in the normal mouse kidneys and blocked the protective effect of TRIM65 deletion. These findings suggest that TRIM65 promotes renal fibrosis by regulating NUDT21-mediated APA and highlight TRIM65 as a potential target for reducing renal fibrosis in CKD patients.

E3 ubiquitin ligase TRIM31 alleviates dopaminergic neurodegeneration by promoting proteasomal degradation of VDAC1 in Parkinson's Disease model.

Mitochondrial dysfunction plays a pivotal role in the pathogenesis of Parkinson's disease (PD). As a mitochondrial governor, voltage-dependent anion channel 1 (VDAC1) is critical for cell survival and death signals and implicated in neurodegenerative diseases. However, the mechanisms of VDAC1 regulation are poorly understood and the role of tripartite motif-containing protein 31 (TRIM31), an E3 ubiquitin ligase which is enriched in mitochondria, in PD remains unclear. In this study, we found that TRIM31-/- mice developed age associated motor defects and dopaminergic (DA) neurodegeneration spontaneously. In addition, TRIM31 was markedly reduced both in nigrostriatal region of PD mice induced by MPTP and in SH-SY5Y cells stimulated by MPP+. TRIM31 deficiency significantly aggravated DA neurotoxicity induced by MPTP. Mechanistically, TRIM31 interacted with VDAC1 and catalyzed the K48-linked polyubiquitination to degrade it through its E3 ubiquitin ligase activity. In conclusion, we demonstrated for the first time that TRIM31 served as an important regulator in DA neuronal homeostasis by facilitating VDAC1 degradation through the ubiquitin-proteasome pathway. Our study identified TRIM31 as a novel potential therapeutic target and pharmaceutical intervention to the interaction between TRIM31 and VDAC1 may provide a promising strategy for PD.

Disrupting CCDC137-mediated LZTS2 and β-TrCP interaction in the nucleus inhibits hepatocellular carcinoma development via β-catenin and AKT.

Hepatocellular carcinoma (HCC) is a highly heterogeneous solid tumor, with its biological characteristics intricately linked to the activation of oncogenes. This research specifically explored CCDC137, a molecule within the CCDC family exhibiting the closest association with HCC. Our investigation aimed to unravel the role, underlying mechanisms, and potential therapeutic implications of CCDC137 in the context of HCC. We observed a close correlation between elevated CCDC137 expression and poor prognosis in HCC patients, along with a promotive effect on HCC progression in vitro and in vivo. Mechanistically, we identified LZTS2, a negative regulator of β-catenin, as the binding protein of CCDC137. CCDC137 facilitated K48-linked poly-ubiquitination of LZTS2 at lysine 467 via recruiting E3 ubiquitin ligase β-TrCP in the nucleus, triggering AKT phosphorylation and activation of β-catenin pathway. Moreover, the 1-75 domain of CCDC137 was responsible for the formation of the CCDC137-LZTS2-β-TrCP complex. Subsequently, designed peptides targeting the 1-75 domain of CCDC137 to disrupt CCDC137-LZTS2 interaction demonstrated efficacy in inhibiting HCC progression. This promising outcome was further supported by HCC organoids and patient-derived xenograft (PDX) models, underscoring the potential clinical utility of the peptides. This study elucidated the mechanism of the CCDC137-LZTS2-β-TrCP protein complex in HCC and offered clinically significant therapeutic strategies targeting this complex.

MiR-17∼92 is involved in NF-κB activation via targeting the ubiquitin-editing proteins to mediate RIP1 complex polyubiquitinations in ABC-DLBCL

Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoma characterized by constitutive NF-κB activation, but whether miR-17∼92 contributes to this activation remains unclear. Herein, we sought to evaluate the role of miR-17∼92 in the process of NF-κB activation in ABC-DLBCL. We found that the expression of miR-17∼92 primary transcript was positively correlated with NF-κB activity, miR-17∼92 activated the NF-κB signaling in ABC-DLBCL, and its over-expression promoted ABC-DLBCL cell growth, accelerated cell G1 to S phase transition and enhanced cell resistance to NF-κB inhibitor. Importantly, miR-17∼92 promoted NF-κB activation through directly targeting multiple ubiquitin-editing regulators to lead to increase the K63-linked polyubiquitination and decrease the K48-linked polyubiquitination of RIP1 complex in ABC-DLBCL. We further found that miR-17∼92 selectively activated IκB-α and NF-κB p65 but not NF-κB p52/p100, and high miR-17∼92 expression was also associated with poorer outcome in ABC-DLBCL patients. Overall, our results showed that miR-17∼92 selectively activated the canonical NF-κB signaling via targeting ubiquitin-editing regulators to lead to constitutively NF-κB activation and poorer outcome in ABC-DLBCL. These findings uncovered an innovative function of miR-17∼92 and previously unappreciated regulatory mechanism of NF-κB activation in ABC-DLBCL. Targeting miR-17∼92 may thus provide a novel bio-therapeutic strategy for ABC-DLBCL patients.

Characterization and noncovalent inhibition of the K63-deubiquitinase activity of SARS-cov-2 PLpro

SARS-CoV-2 papain-like protease (PLpro) could facilitate viral replication and host immune evasion by respectively hydrolyzing viral polyprotein and host ubiquitin conjugates, thereby rendering itself as an important antiviral target. Yet few noncovalent PLpro inhibitors of SARS-CoV-2 have been reported with improved directed towards pathogenic deubiquitinating activities inhibition. Herein, we report that coronavirus PLpro proteases have distinctive substrate bias and are conserved to deubiquitylate K63-linked polyubiquitination, thereby attenuating host type I interferon response. We identify a noncovalent compound specifically optimized towards halting the K63-deubiquitinase activity of SARS-CoV-2 PLpro, but not other coronavirus (CoV) counterparts or host deubiquitinase. Contrasting with GRL-0617, a SARS-CoV-1 PLpro inhibitor, SIMM-036 is 50-fold and 7-fold (half maximal inhibitory concentration (IC50)) more potent to inhibit viral replication during SARS-CoV-2 infection and restore the host interferon-β (IFN-β) response in human angiotensin-converting enzyme 2 (hACE2)-HeLa cells, respectively. Structure-activity relationship (SAR) analysis further reveals the importance of BL2 groove of PLpro, which could determine the selectivity of K63-deubiquitinase activity of the enzyme.

The m6A methyltransferase METTL3 drives neuroinflammation and neurotoxicity through stabilizing BATF mRNA in microglia.

Persistent neuroinflammation and progressive neuronal loss are defining features of acute brain injury including traumatic brain injury (TBI) and cerebral stroke. Microglia, the most abundant type of brain-resident immune cells, continuously surveil the environment and play a central role in shaping the inflammatory state of the central nervous system (CNS). In the study, we discovered that the protein expression of METTL3 (a m6A methyltransferase) was upregulated in inflammatory microglia independent of increased Mettl3 gene transcription following TBI in both human and mouse subjects. Subsequently, we identified TRIP12, a HECT-domain E3 ubiquitin ligase, as a negative regulator of METTL3 protein expression by facilitating METTL3 K48-linked polyubiquitination. Importantly, selective ablation of Mettl3 inhibited microglial pathogenic activities, diminished neutrophil infiltration, rescued neuronal loss and facilitated functional recovery post-TBI. Using MeRIP-seq and CUT&Tag sequencing, we identified that METTL3 promoted the expression of Basic Leucine Zipper Transcriptional Factor ATF-Like (BATF), which in turn directly bound to a cohort of characteristic inflammatory cytokines and chemokine genes. Enhanced activities of BATF in microglia elicited TNF-dependent neurotoxicity and can also promote neutrophil recruitment through releasing CXCL2. Pharmacological inhibition of METTL3 using a BBB-penetrating drug-loaded nano-system showed satisfactory therapeutic effects in both TBI and stroke mouse models. Collectively, our findings identified METTL3-m6A-BATF axis as a potential therapeutic target for terminating detrimental neuroinflammation and progressive neuronal loss following acute brain injury. METTL3 protein is significantly up-regulated in inflammatory microglia due to the decreased proteasomal degradation mediated by TRIP12 and ERK-USP5 pathways. METTL3 stabilized BATF mRNA stability and promoted BATF expression through the m6A-IGF2BP2-dependent mechanism. Elevated expression of BATF elicits a pro-inflammatory gene program in microglia, and aggravates neuroinflammatory response including local immune responses and peripheral immune cell infiltration. Genetic deletion or pharmaceutically targeting METTL3-BATF axis suppressed microglial pro-inflammatory activities and promoted neurological recovery following TBI and stroke.

Multiple E3 ligases act as antiviral factors against SARS-CoV-2 via inducing the ubiquitination and degradation of ORF9b.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) open reading frame 9b (ORF9b) antagonizes the antiviral type I and III interferon (IFN) responses and is ubiquitinated and degraded via the ubiquitin-proteasome pathway. However, E3 ubiquitin ligases that mediate the polyubiquitination and degradation of ORF9b remain unknown. In this study, we identified 14 E3 ligases that specifically bind to SARS-CoV-2 ORF9b. Specifically, three E3 ligases, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, induced K48-linked polyubiquitination and degradation of ORF9b, thereby attenuating ORF9b-mediated inhibition of the IFN response and SARS-CoV-2 replication. Moreover, each E3 ligase performed this function independent of the other two E3 ligases. Therefore, the three E3 ligases identified in this study as anti-SARS-CoV-2 host factors provide novel molecular insight into the virus-host interaction.IMPORTANCEUbiquitination is an important post-translational modification that regulates multiple biological processes, including viral replication. Identification of E3 ubiquitin ligases that target viral proteins for degradation can provide novel targets for antagonizing viral infections. Here, we identified multiple E3 ligases, including HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, that ubiquitinated and induced the degradation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 9b (ORF9b), an interferon (IFN) antagonist, thereby enhancing IFN production and attenuating SARS-CoV-2 replication. Our study provides new possibilities for drug development targeting the interaction between E3 ligases and ORF9b.

Glucose deprivation triggers DCAF1-mediated inactivation of Rheb-mTORC1 and promotes cancer cell survival

Low glucose is a common microenvironment for rapidly growing solid tumors, which has developed multiple approaches to survive under glucose deprivation. However, the specific regulatory mechanism remains largely elusive. In this study, we demonstrate that glucose deprivation, while not amino acid or serum starvation, transactivates the expression of DCAF1. This enhances the K48-linked polyubiquitination and proteasome-dependent degradation of Rheb, inhibits mTORC1 activity, induces autophagy, and facilitates cancer cell survival under glucose deprivation conditions. This study identified DCAF1 as a new cellular glucose sensor and uncovered new insights into mechanism of DCAF1-mediated inactivation of Rheb-mTORC1 pathway for promoting cancer cell survival in response to glucose deprivation.

RNF216 Inhibits the Replication of H5N1 Avian Influenza Virus and Regulates the RIG-I Signaling Pathway in Ducks.

The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-β expression during virus infection, the expression level of IFN-β in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks.

SIAH2 suppresses c-JUN pathway by promoting the polyubiquitination and degradation of HBx in hepatocellular carcinoma.

As an important protein encoded by hepatitis B virus (HBV), HBV X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). It has been shown that seven in absentia homologue 1 (SIAH1) could regulates the degradation of HBx through the ubiquitin-proteasome pathway. However, as a member of SIAH family, the regulatory effects of SIAH2 on HBx remain unclear. In this study, we first confirmed that SIAH2 could reduce the protein levels of HBx depending on its E3 ligase activity. Moreover, SIAH2 interacted with HBx and induced its K48-linked polyubiquitination and proteasomal degradation. Furthermore, we provided evidence that SIAH2 inhibits HBx-associated HCC cells proliferation by regulating HBx. In conclusion, our study identified a novel role for SIAH2 in promoting HBx degradation and SIAH2 exerts an inhibitory effect in the proliferation of HBx-associated HCC through inducing the degradation of HBx. Our study provides a new idea for the targeted degradation of HBx and may have great huge significance into providing novel evidence for the targeted therapy of HBV-infected HCC.

Dynamic modulation of nodulation factor receptor levels by phosphorylation-mediated functional switch of a RING-type E3 ligase during legume nodulation

The precise control of receptor levels is crucial for initiating cellular signaling transduction in response to specific ligands; however, such mechanisms regulating nodulation factor (NF) receptor (NFR)-mediated perception of NFs to establish symbiosis remain unclear. In this study, we unveil the pivotal role of the NFR-interacting RING-type E3 ligase 1 (NIRE1) in regulating NFR1/NFR5 homeostasis to optimize rhizobial infection and nodule development in Lotus japonicus. We demonstrated that NIRE1 has a dual function in this regulatory process. It associates with both NFR1 and NFR5, facilitating their degradation through K48-linked polyubiquitination before rhizobial inoculation. However, following rhizobial inoculation, NFR1 phosphorylates NIRE1 at a conserved residue, Tyr-109, inducing a functional switch in NIRE1, which enables NIRE1 to mediate K63-linked polyubiquitination, thereby stabilizing NFR1/NFR5 in infected root cells. The introduction of phospho-dead NIRE1Y109F leads to delayed nodule development, underscoring the significance of phosphorylation at Tyr-109 in orchestrating symbiotic processes. Conversely, expression of the phospho-mimic NIRE1Y109E results in the formation of spontaneous nodules in L. japonicus, further emphasizing the critical role of the phosphorylation-dependent functional switch in NIRE1. In summary, these findings uncover a fine-tuned symbiotic mechanism that a single E3 ligase could undergo a phosphorylation-dependent functional switch to dynamically and precisely regulate NF receptor protein levels.

A computational approach for structural and functional analyses of disease-associated mutations in the human CYLD gene

Tumor suppressor cylindromatosis protein (CYLD) regulates NF-κB and JNK signaling pathways by cleaving K63-linked poly-ubiquitin chain from its substrate molecules and thus preventing the progression of tumorigenesis and metastasis of the cancer cells. Mutations in CYLD can cause aberrant structure and abnormal functionality leading to tumor formation. In this study, we utilized several computational tools such as PANTHER, PROVEAN, PredictSNP, PolyPhen-2, PhD-SNP, PON-P2, and SIFT to find out deleterious nsSNPs. We also highlighted the damaging impact of those deleterious nsSNPs on the structure and function of the CYLD utilizing ConSurf, I-Mutant, SDM, Phyre2, HOPE, Swiss-PdbViewer, and Mutation 3D. We shortlisted 18 high-risk nsSNPs from a total of 446 nsSNPs recorded in the NCBI database. Based on the conservation profile, stability status, and structural impact analysis, we finalized 13 nsSNPs. Molecular docking analysis and molecular dynamic simulation concluded the study with the findings of two significant nsSNPs (R830K, H827R) which have a remarkable impact on binding affinity, RMSD, RMSF, radius of gyration, and hydrogen bond formation during CYLD-ubiquitin interaction. The principal component analysis compared native and two mutants R830K and H827R of CYLD that signify structural and energy profile fluctuations during molecular dynamic (MD) simulation. Finally, the protein–protein interaction network showed CYLD interacts with 20 proteins involved in several biological pathways that mutations can impair. Considering all these in silico analyses, our study recommended conducting large-scale association studies of nsSNPs of CYLD with cancer as well as designing precise medications against diseases associated with these polymorphisms.

OTUB1 contributes to the stability and function of Influenza A virus NS2.

The influenza A virus (IAV) consists of 8 single-stranded, negative-sense viral RNA (vRNA) segments. After infection, vRNA is transcribed, replicated, and wrapped by viral nucleoprotein (NP) to form viral ribonucleoprotein (vRNP). The transcription, replication, and nuclear export of the viral genome are regulated by the IAV protein, NS2, which is translated from spliced mRNA transcribed from viral NS vRNA. This splicing is inefficient, explaining why NS2 is present in low abundance after IAV infection. The levels of NS2 and its subsequent accumulation are thought to influence viral RNA replication and vRNP nuclear export. Here we show that NS2 is ubiquitinated at the K64 and K88 residues by K48-linked and K63-linked polyubiquitin (polyUb) chains, leading to the degradation of NS2 by the proteasome. Additionally, we show that a host deubiquitinase, OTUB1, can remove polyUb chains conjugated to NS2, thereby stabilizing NS2. Accordingly, knock down of OTUB1 by siRNA reduces the nuclear export of vRNP, and reduces the overall production of IAV. These results collectively demonstrate that the levels of NS2 in IAV-infected cells are regulated by a ubiquitination-deubiquitination system involving OTUB1 that is necessary for optimal IAV replication.

Molecular screening of phytocompounds targeting the interface between influenza A NS1 and TRIM25 to enhance host immune responses

BackgroundInfluenza A virus causes severe respiratory illnesses, especially in developing nations where most child deaths under 5 occur due to lower respiratory tract infections. The RIG-I protein acts as a sensor for viral dsRNA, triggering interferon production through K63-linked poly-ubiquitin chains synthesized by TRIM25. However, the influenza A virus's NS1 protein hinders this process by binding to TRIM25, disrupting its association with RIG-I and preventing downstream interferon signalling, contributing to the virus's evasion of the immune response. MethodsIn our study we used structural-based drug designing, molecular simulation, and binding free energy approaches to identify the potent phytocompounds from various natural product databases (>100,000 compounds) able to inhibit the binding of NS1 with the TRIM25. ResultsThe molecular screening identified EA-8411902 and EA-19951545 from East African Natural Products Database, NA-390261 and NA-71 from North African Natural Products Database, SA-65230 and SA- 4477104 from South African Natural Compounds Database, NEA- 361 and NEA- 4524784 from North-East African Natural Products Database, TCM-4444713 and TCM-6056 from Traditional Chinese Medicines Database as top hits. The molecular docking and binding free energies results revealed that these compounds have high affinity with the specific active site residues (Leu95, Ser99, and Tyr89) involved in the interaction with TRIM25. Additionally, analysis of structural dynamics, binding free energy, and dissociation constants demonstrates a notably stronger binding affinity of these compounds with the NS1 protein. Moreover, all selected compounds exhibit exceptional ADMET properties, including high water solubility, gastrointestinal absorption, and an absence of hepatotoxicity, while adhering to Lipinski’s rule. ConclusionOur molecular simulation findings highlight that the identified compounds demonstrate high affinity for specific active site residues involved in the NS1-TRIM25 interaction, exhibit exceptional ADMET properties, and adhere to drug-likeness criteria, thus presenting promising candidates for further development as antiviral agents against influenza A virus infections.

African swine fever virus MGF505-6R attenuates type I interferon production by targeting STING for degradation.

African swine fever (ASF) is an acute hemorrhagic and devastating infectious disease affecting domestic pigs and wild boars. It is caused by the African swine fever virus (ASFV), which is characterized by genetic diversity and sophisticated immune evasion strategies. To facilitate infection, ASFV encodes multiple proteins to antagonize host innate immune responses, thereby contributing to viral virulence and pathogenicity. The molecular mechanisms employed by ASFV-encoded proteins to modulate host antiviral responses have not been comprehensively elucidated. In this study, it was observed that the ASFV MGF505-6R protein, a member of the multigene family 505 (MGF505), effectively suppressed the activation of the interferon-beta (IFN-β) promoter, leading to reduced mRNA levels of antiviral genes. Additional evidence has revealed that MGF505-6R antagonizes the cGAS-STING signaling pathway by interacting with the stimulator of interferon genes (STING) for degradation in the autophagy-lysosomal pathway. The domain mapping revealed that the N-terminal region (1-260aa) of MGF505-6R is the primary domain responsible for interacting with STING, while the CTT domain of STING is crucial for its interaction with MGF505-6R. Furthermore, MGF505-6R also inhibits the activation of STING by reducing the K63-linked polyubiquitination of STING, leading to the disruption of STING oligomerization and TANK binding kinase 1 (TBK1) recruitment, thereby impairing the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3). Collectively, our study elucidates a novel strategy developed by ASFV MGF505-6R to counteract host innate immune responses. This discovery may offer valuable insights for further exploration of ASFV immune evasion mechanisms and antiviral strategies.

Roquin-2 promotes oxidative stress-induced cell death by ubiquitination-dependent degradation of TAK1

Reactive oxygen species (ROS) are highly reactive and their accumulation causes oxidative damage to cells. Cells maintain survival upon mild oxidative stress with anti-oxidative systems, such as the kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system. On the other hand, upon severe oxidative stress, cells undergo regulated cell death, including apoptosis, for eliminating damaged cells. To execute efficient cell death, cells need to turn off the anti-oxidant systems, while triggering cell death. However, it remains unknown how cells orchestrate these two conflicting systems under excessive oxidative stress.Herein, we show that when cells are exposed to excessive oxidative damage, an E3 ubiquitin ligase Roquin-2 (also known as RC3H2) plays a key role in switching cell fate from survival to death by terminating activation of transforming growth factor-β-activated kinase 1 (TAK1), a positive regulator for Nrf2 activation. Roquin-2 interacted with TAK1 via four cysteine residues in TAK1 (C96, C302, C486, and C500) that are susceptible to oxidative stress and participate in oligomer formation via disulfide bonds, promoting K48-linked polyubiquitination and degradation of TAK1. Nrf2 was inactivated upon lethal oxidative stress in wild-type mouse embryonic fibroblast (MEF) cells, whereas it sustained activation and conferred resistance to Roquin-2 deficient cells, which was reversed by pharmacological or genetic inhibition of TAK1. These data demonstrate that in response to excessive ROS exposure, Roquin-2 promotes ubiquitination and degradation of TAK1 to suppress Nrf2 activation, and thereby contributes to an efficient cell death, providing insight into the pathogenesis of oxidative stress-related diseases, including cancer.