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TRIM25 interacts with and polyubiquitinates G3BP (A) Western blot of TRIM25 KO 293T cells transfected with myc-G3BP1/2 and FLAG-TRIM25-WT, -R54P, or -PTAA. Lysates were subjected to FLAG IP. Data are representative of three independent experiments. (B) Western blot of TRIM25 KO and TRIM25-WT, -R54P, or -PTAA inducible cells transfected with myc-G3BP1/2 and HA-Ub-WT in the presence of 1 μg/mL dox. (C) Western blot of TRIM25-WT inducible cells transfected with myc-G3BP1/2 and HA-Ub-WT, -K48, or -K63 in the presence of 1 μg/mL dox. (B-C) Lysates were subjected to myc IP. Data are representative of three independent experiments.
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The tripartite motif (TRIM) family of E3 ubiquitin ligases is well known for its roles in antiviral restriction and innate immunity regulation, in addition to many other cellular pathways. In particular, TRIM25-mediated ubiquitination affects both carcinogenesis and antiviral response. While individual substrates have been identified for TRIM25, it...
Citations
... 11,12 The discovery of new ligands for E3 ligases, particularly those with non-degradative ubiquitination activity, presents a promising strategy to activate alternative cellular outcomes for disease-related proteins. 13 We sought to assess whether the E3 ligase TRIM25, reported to catalyse the formation of both Lys48-and Lys63-linked ubiquitin chains, 14 could be liganded and repurposed for targeted protein ubiquitination. TRIM25 is a member of the TRIM family of RING-type E3 ligases and comprises a canonical Nterminal tripartite motif (TRIM) and a variable C-terminal PRYSPRY substrate binding domain (Fig. 1A). ...
The tripartite motif (TRIM) family of RING-type E3 ligases catalyses the formation of many different types of ubiquitin chains, and as such, plays important roles in diverse cellular functions, ranging from immune regulation to cancer signalling pathways. Few ligands have been discovered for TRIM E3 ligases, and these E3s are under-represented in the rapidly expanding field of induced proximity. Here we present the identification of a novel covalent ligand for the PRYSPRY substrate binding domain of TRIM25. We employ covalent fragment screening coupled with high-throughput chemistry direct-to-biology optimisation to efficiently elaborate covalent fragment hits. We demonstrate that our optimised ligand enhances the in vitro auto-ubiquitination activity of TRIM25 and engages TRIM25 in live cells. We also present the X-ray crystal structure of TRIM25 PRYSPRY in complex with this covalent ligand. Finally, we incorporate our optimised ligand into heterobifunctional proximity-inducing compounds and demonstrate the in vitro targeted ubiquitination of a neosubstrate by TRIM25.
... The TRIM family, known for its diverse roles in immune regulation and antiviral defense, has been extensively studied in relation to MAVS and the RIG-I pathway. For example, TRIM25, a TRIM family member, exhibited antiviral activity (40)(41)(42), has been shown to ubiquitinate RIG-I (43), thereby promoting its activation and enhanc ing downstream antiviral signaling. Additionally, TRIM38 has been shown to nega tively regulate RIG-I activation, thereby modulating inflammatory responses during (44). ...
Activation of the innate immune response is essential for host cells to restrict the dissemination of invading viruses and other pathogens. Proteins belonging to the tripartite motif (TRIM) family are key effectors in antiviral innate immunity. Among these, TRIM22, a RING-type E3 ubiquitin ligase, has been recognized as a significant regulator in the pathogenesis of various diseases. In the present study, we identified TRIM22 as a critical modulator of mitochondrial antiviral signaling protein (MAVS) activation. Loss of TRIM22 function led to reduced production of type I interferons (IFNs) in response to viral infection such as influenza A virus (IAV) or vesicular stomatitis virus (VSV), thereby facilitating viral replication. Mechanistically, TRIM22 was found to enhance retinoic acid-inducible gene I (RIG-I)-mediated signaling through the catalysis of Lys63-linked polyubiquitination of MAVS, which, in turn, activated the TANK-binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) pathway, driving IFN-β production. Additionally, TRIM22 was shown to inhibit the assembly of the MAVS-NLRX1 inhibitory complex, further amplifying innate immune responses. Our findings also demonstrated that RNA virus infection upregulated TRIM22 expression via the nuclear translocation of ELF3, a transcription factor that activates TRIM22 gene expression. This regulatory loop underscores the role of TRIM22 in modulating the type I IFN pathway, providing critical insights into the host’s antiviral defense mechanisms. Our research highlights the potential of targeting the ELF3-TRIM22-MAVS axis as a therapeutic strategy for enhancing antiviral immunity and preventing RNA virus infections.
IMPORTANCE
Interferon (IFN)-mediated antiviral responses are crucial for the host’s defense against foreign pathogens and are regulated by various signaling pathways. The tripartite motif (TRIM) family, recognized for its multifaceted roles in immune regulation and antiviral defense, plays a significant part in this process. In our study, we explored the important role of TRIM22, a protein that helped regulate the host’s immune response to viral infections. We found that TRIM22 enhances the Lys63-linked polyubiquitination of mitochondrial antiviral signaling protein (MAVS), which was essential for producing type I interferons. Interestingly, we discovered that the expression of TRIM22 increases after an RNA virus infection, due to a transcription factor ELF3, which moved into the nucleus of cells to activate TRIM22 transcription. This created a feedback loop that strengthens the role of TRIM22 in modulating the type I IFN pathway. By uncovering these mechanisms, we aimed to enhance our understanding of how the immune system works and provide insights that could lead to innovative antiviral therapies.
... We hypothesized that TRIM25 might provide a necessary scaffolding function to promote the stability of M3.1 complexes, or alternatively, that TRIM25 ubiquitin ligase activity might be necessary for NF-κB induction by M3.1. To test the role of TRIM25 catalytic activity, we generated two TRIM25 mutants with disrupted ubiquitin ligase activity: C50S/C53S, which blocks zinc finger coordination, and R54P, which prevents the interaction with E2 conjugating enzymes (25,40). First, we tested if the mutations affected the interaction between TRIM25 and M3.1. ...
Effector-triggered immunity (ETI) is a form of pathogen sensing that involves detection of pathogen-encoded virulence factors or 'effectors'. To discover novel ETI pathways in mammals, we developed a screening approach in which individual virulence factors are expressed in human monocytes and transcriptional responses are assessed by RNA-seq. Using this approach, we identify a poxvirus effector, myxoma virus M3.1, which elicits an anti-viral NF-κB response. We find that NF-κB is unleashed by an ETI pathway that senses M3.1 attack of two anti-viral complexes: ZAP and TBK1. NF-κB activation occurs because the proteins inhibited by M3.1 (N4BP1, ZC3H12A, and TBK1) are negative regulators of NF-κB. Our results illustrate how negative regulators can function as pathogen sensors and establish a systematic approach for the discovery of ETI pathways.
... A lysine-to-arginine (KtoR) fUBE2D3-HA-Ub probe was generated to minimize autoubiquitination and degradation of the probe. TRIM25, an E3 ligase known to pair with UBE2D3 that exhibits autoubiquitination activity 35,36,39,57 , was also blotted, and a clearly visible labeled band was observed. UBE2D3 was blotted, and levels of overexpressed WT fUBE2D3-HA-Ub probe were relatively low compared to levels of endogenous UBE2D3 (Fig. 1c). ...
The E2 ubiquitin (Ub)-conjugating enzyme primarily determines Ub conjugation as Ub-isopeptide (lysine), Ub-oxyester (serine/threonine) or Ub-thioester (cysteine). However, E2-specific Ub conjugation profiles within cells remain elusive. Here we developed the fusion E2–Ub-R74G profiling (FUSEP) strategy to access E2-specific Ub conjugation profiles in cells with amino acid resolution. The probe-specific leucine-glycine-glycine-glycine-modified Ub remnant enables systematic studies of non-lysine Ub conjugation and provides site-specific information. Multiple E2 enzymes were found to be involved in non-lysine ubiquitination. Profiling with UBE2D3–Ub-R74G probes identified a post-translational modification, tyrosine ubiquitination, in human Cullin-1, a scaffold protein for Cullin-RING E3 Ub ligases. This modification is distinct from lysine ubiquitination. A single-pass membrane-bound E3 ligase, RNF149, was identified to pair with UBE2D3 to regulate pyroptosis by ubiquitinating apoptosis-associated speck-like protein ASC. The availability of this toolbox paves the way for uncovering E2-specific Ub conjugation profiles and identifying previously unknown E3 Ub ligases for potential therapeutic applications.
... To date, only a few E3 ubiquitin ligases (E3) and deubiquitinases (DUB) have been identified as host factor or restriction factor for alphaviruses. TRIM25, a well-studied E3 that participates in host antiviral innate immunity [22], also acts as a co-factor of zinc-finger antiviral protein (ZAP) to promote ZAP-mediated inhibition of alphaviruses [23][24][25]. In mosquito cells, an E3 ubiquitin ligase scaffolding protein, AcCullin3 was identified as pro-viral factor of CHIKV replication [26]. ...
Alphaviruses are mosquito borne RNA viruses that are a reemerging public health threat. Alphaviruses have a broad host range, and can cause diverse disease outcomes like arthritis, and encephalitis. The host ubiquitin proteasome system (UPS) plays critical roles in regulating cellular processes to control the infections with various viruses, including alphaviruses. Previous studies suggest alphaviruses hijack UPS for virus infection, but the molecular mechanisms remain poorly characterized. In addition, whether certain E3 ubiquitin ligases or deubiquitinases act as alphavirus restriction factors remains poorly understood. Here, we employed a cDNA expression screen to identify E3 ubiquitin ligase TRIM32 as a novel intrinsic restriction factor against alphavirus infection, including VEEV-TC83, SINV, and ONNV. Ectopic expression of TRIM32 reduces alphavirus infection, whereas depletion of TRIM32 with CRISPR-Cas9 increases infection. We demonstrate that TRIM32 inhibits alphaviruses through a mechanism that is independent of the TRIM32-STING-IFN axis. Combining reverse genetics and biochemical assays, we found that TRIM32 interferes with genome translation after membrane fusion, prior to replication of the incoming viral genome. Furthermore, our data indicate that the monoubiquitination of TRIM32 is important for its antiviral activity. Notably, we also show two TRIM32 pathogenic mutants R394H and D487N, related to Limb-girdle muscular dystrophy (LGMD), have a loss of antiviral activity against VEEV-TC83. Collectively, these results reveal that TRIM32 acts as a novel intrinsic restriction factor suppressing alphavirus infection and provides insights into the interaction between alphaviruses and the host UPS.
... Full-length TRIM25 (gift from Dr. Jae U. Jung at Cleveland Clinic Lerner Research Institute) [65] was cloned into pcDNA3.1-myc as previously described [66]. The ZAP positive selection mutants, PAR binding deficient Q668R mutant, and N658G/K/S/D mutants were generated by the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA) or synthesized as a gene block (Twist Bioscience, South San Francisco, CA) with ClaI and NotI restriction sites and ligated into the ePB vector. ...
... 1μg/mL puromycin was added 48 hours post-transfection to select for ZAP KO HEK293T cells that have incorporated the ePB transposon. Our ZAPS WT and ZAPL WT cell lines were made by selecting single cell clones that follow two criteria: 1) robustly express ZAP following 24 hours of 1μg/mL doxycycline treatment, and 2) recapitulate differential alphaviral sensitivities (S2 Fig) similar to previously generated bulk cell lines with inducible ZAP expression [7,66]. The mutant ZAP cell lines in this study were bulk cells that survived after puromycin selection. ...
... SINV (Toto1101) [67], SINV expressing luciferase (Toto1101/Luc and Toto1101/Luc:ts6) [39], SINV expressing enhanced green fluorescent protein (EGFP) (TE/5'2J/GFP) [68], RRV expressing EGFP (gift from Dr. Mark Heise, University of North Carolina) [69], ONNV expressing EGFP (gift from Dr. Steve Higgs, Kansas State University) [70], CHIKV vaccine strain 181/clone 25 expressing EGFP (gift from Scott Weaver, The University of Texas Medical Branch at Galveston) [70], VEEV vaccine strain TC-83 expressing EGFP (gift from Dr. Ilya Frolov, University of Alabama at Birmingham), and HIV-1 Bru ΔEnv pseudotyped with the glycoprotein from vesicular stomatitis virus have been previously described [8,66,72]. All alphaviral stocks were generated and titered in BHK-21 cells [39]. ...
The host interferon pathway upregulates intrinsic restriction factors in response to viral infection. Many of them block a diverse range of viruses, suggesting that their antiviral functions might have been shaped by multiple viral families during evolution. Host-virus conflicts have led to the rapid adaptation of host and viral proteins at their interaction hotspots. Hence, we can use evolutionary genetic analyses to elucidate antiviral mechanisms and domain functions of restriction factors. Zinc finger antiviral protein (ZAP) is a restriction factor against RNA viruses such as alphaviruses, in addition to other RNA, retro-, and DNA viruses, yet its precise antiviral mechanism is not fully characterized. Previously, an analysis of 13 primate ZAP orthologs identified three positively selected residues in the poly(ADP-ribose) polymerase-like domain. However, selective pressure from ancient alphaviruses and others likely drove ZAP adaptation in a wider representation of mammals. We performed positive selection analyses in 261 mammalian ZAP using more robust methods with complementary strengths and identified seven positively selected sites in all domains of the protein. We generated ZAP inducible cell lines in which the positively selected residues of ZAP are mutated and tested their effects on alphavirus replication and known ZAP activities. Interestingly, the mutant in the second WWE domain of ZAP (N658A) is dramatically better than wild-type ZAP at blocking replication of Sindbis virus and other ZAP-sensitive alphaviruses due to enhanced viral translation inhibition. The N658A mutant is adjacent to the previously reported poly(ADP-ribose) (PAR) binding pocket, but surprisingly has reduced binding to PAR. In summary, the second WWE domain is critical for engineering a more potent ZAP and fluctuations in PAR binding modulate ZAP antiviral activity. Our study has the potential to unravel the role of ADP-ribosylation in the host innate immune defense and viral evolutionary strategies that antagonize this post-translational modification.
... TRIM25 harbors a "404-PTFG-407" motif between its coiled-coil and PRY/SPRY domains, which displays some degree of similarity with the previously identified G3BP-binding motif, ΦxFG 35-38 (where Φ is a hydrophobic residue and X is any residue). Indeed, both alanine substitution (PTFG AAAA ) and deletion of the PTFG motif (ΔPTFG) abolished the interaction between TRIM25 and G3BP1 in immunoprecipitation (IP) experiments, like previous reports 36 (Fig. 3b, c). Intriguingly, single or double replacement of the four amino acids in the PTFG motif to alanine abolished the interaction with G3BP1, highlighting the importance of the integrity of the PTFG sequence (Fig. S3c). ...
Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25’s ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.
... OASL, a member of the IFN-I-induced antiviral protein OAS family, can inhibit RNA virus replications in chickens (Rong et al., 2018;Ghosh et al., 2019). Other antiviral factors like TRIM25, Mx1, ADAR, GBP, PKR, and IFIT5 have also been reported (Kanwal and Mahmood, 2014;Dai et al., 2020;Sarute and Ross, 2020;Diaz-Beneitez et al., 2022;Labeau et al., 2022;Yang et al., 2022a). However, PKR shows a slight downregulation upon 4/91 vaccine infection at 7 dpi. ...
The gamma coronavirus infectious bronchitis virus (IBV) is known to cause an acute and highly contagious infectious disease in poultry. Here, this study aimed to investigate the impact of virulent or avirulent IBV infection on the avian host by conducting proteomics with data-independent acquisition mass spectrometry (DIA-MS) in the kidneys of IBV-infected chickens. The results revealed 267, 489, and 510 differentially expressed proteins (DEPs) in the chicken kidneys at 3, 5, and 7 days postinfection (dpi), respectively, when infected with the GD17/04 strain, which is a highly nephrogenic strain and belongs to the 4/91 genotype. In contrast, the attenuated 4/91 vaccine resulted in the identification of 144, 175, and 258 DEPs at 3, 5, and 7 dpi, respectively. Functional enrichment analyses indicated distinct expression profiles between the 2 IBV strains. Upon GD17/04 infection, metabolic pathways respond initially in the early stage (3 dpi) and immune-related signaling pathways respond in the middle and late stages (5 and 7 dpi). The 4/91 vaccine elicited a completely opposite response compared to the GD17/04 infection. Among all DEPs, 62 immune-related DEPs were focused on and found to be mainly enriched in the type I interferon (IFN-I) signaling pathway and involved in humoral and cellular immunity. Notably, key molecules in the IFN-I signaling pathway including MDA5, LGP2, and TBK1 may serve as regulatory targets of IBV. Overall, this study highlights similarities and discrepancies in the patterns of protein expression at different stages of infection with virulent and avirulent IBV strains, with the IFN-I signaling pathway emerging as a critical response to IBV infection.
... Dysregulated protein abundance and quality control is central to nearly all human diseases, spanning cardiovascular disease 1,2 , cancer 3-5 , neurodegeneration [6][7][8] , and viral infections 9,10 . The latter is particularly noteworthy given both the recent COVID-19 pandemic and the preponderance of mechanisms by which host proteostasis processes are co-opted by pro-viral factors [11][12][13][14][15] . ...
Protein homeostasis is tightly regulated, with damaged or misfolded proteins quickly eliminated by the proteasome and autophagosome pathways. By co-opting these processes, targeted protein degradation technologies enable pharmacological manipulation of protein abundance. Recently, cysteine-reactive molecules have been added to the degrader toolbox, which offer the benefit of unlocking the therapeutic potential of ‘undruggable’ protein targets. The proteome-wide impact of these molecules remains to be fully understood and given the general reactivity of many classes of cysteine-reactive electrophiles, on- and off-target effects are likely. Using chemical proteomics, we identified a cysteine-reactive small molecule degrader of the SARS-CoV-2 non-structural protein 14 (nsp14), which effects degradation through direct modification of cysteines in both nsp14 and in host chaperones together with activation of global cell stress response pathways. We find that cysteine-reactive electrophiles increase global protein ubiquitylation, trigger proteasome activation, and result in widespread aggregation and depletion of host proteins, including components of the nuclear pore complex. Formation of stress granules was also found to be a remarkably ubiquitous cellular response to nearly all cysteine-reactive compounds and degraders. Collectively, our study sheds light on complexities of covalent target protein degradation and highlights untapped opportunities in manipulating and characterizing proteostasis processes via deciphering the cysteine-centric regulation of stress response pathways.
... UBE2L6 encodes UBCH8, which is also implicated in ubiquitination reactions for protein degradation via the UPS [103]. Histone deacetylation inhibitors (e.g., LBH589) increase UBCH8 levels, favoring the degradation of a mutated form of fms related receptor tyrosine kinase 3 (FLT3) associated with acute myeloid leukemia [104]. These data suggest that alterations in other posttranslational modifications may also affect protein ISGylation in the cancer context. ...
Interferon (IFN)-stimulated gene 15 (ISG15) is a member of the ubiquitin-like (UBL) protein family that can modify specific proteins via a catalytic process called ISGylation. This posttranslational modification can modulate the stability of the ISGylated proteins and protein-protein interactions. Some proteins modified by ISG15 have been identified in malignant neoplasms, suggesting the functional relevance of ISGylation in cancer. This review discusses the ISGylated proteins reported in malignant neoplasms that suggest the potential of ISG15 as a biomarker and therapeutic target in cancer.
