Unanchored Polyubiquitin Research Articles

Unraveling the Immune Regulatory Functions of USP5: Implications for Disease Therapy.

Ubiquitin-specific protease 5 (USP5) belongs to the ubiquitin-specific protease (USP) family, which uniquely recognizes unanchored polyubiquitin chains to maintain the homeostasis of monoubiquitin chains. USP5 participates in a wide range of cellular processes by specifically cleaving isopeptide bonds between ubiquitin and substrate proteins or ubiquitin itself. In the process of immune regulation, USP5 affects important cellular signaling pathways, such as NF-κB, Wnt/β-catenin, and IFN, by regulating ubiquitin-dependent protein degradation. These pathways play important roles in immune regulation and inflammatory responses. In addition, USP5 regulates the activity and function of immunomodulatory signaling pathways via the deubiquitination of key proteins, thereby affecting the activity of immune cells and the regulation of immune responses. In the present review, the structure and function of USP5, its role in immune regulation, and the mechanism by which USP5 affects the development of diseases by regulating immune signaling pathways are comprehensively overviewed. In addition, we also introduce the latest research progress of targeting USP5 in the treatment of related diseases, calling for an interdisciplinary approach to explore the therapeutic potential of targeting USP5 in immune regulation.

K29-linked free polyubiquitin chains affect ribosome biogenesis and direct ribosomal proteins to the intranuclear quality control compartment

Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs) Ubp2 and Ubp14, and E3 ligases Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the intranuclear quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with ribosomopathies.

MAVS-loaded unanchored Lys63-linked polyubiquitin chains activate the RIG-I-MAVS signaling cascade.

The adaptor molecule MAVS forms prion-like aggregates to govern the RIG-I-like receptor (RLR) signaling cascade. Lys63 (K63)-linked polyubiquitination is critical for MAVS aggregation, yet the underlying mechanism and the corresponding E3 ligases and deubiquitinating enzymes (DUBs) remain elusive. Here, we found that the K63-linked polyubiquitin chains loaded on MAVS can be directly recognized by RIG-I to initiate RIG-I-mediated MAVS aggregation with theprerequisite of the CARDRIG-I-CARDMAVS interaction. Interestingly, many K63-linked polyubiquitin chains attach to MAVS via an unanchored linkage. We identified Ube2N as a major ubiquitin-conjugating enzyme for MAVS and revealed that Ube2N cooperates with the E3 ligase Riplet and TRIM31 to promote the unanchored K63-linked polyubiquitination of MAVS. In addition, we identified USP10 as a direct DUB that removes unanchored K63-linked polyubiquitin chains from MAVS. Consistently, USP10 attenuates RIG-I-mediated MAVS aggregation and the production of type I interferon. Mice with a deficiency in USP10 show more potent resistance to RNA virus infection. Our work proposes a previously unknown mechanism for the activation of the RLR signaling cascade triggered by MAVS-attached unanchored K63-linked polyubiquitin chains and establishes the DUB USP10 and the E2:E3 pair Ube2N-Riplet/TRIM31 as a specific regulatory system for the unanchored K63-linked ubiquitination and aggregation of MAVS upon viral infection.

A pan-cancer analysis of the role of USP5 in human cancers

Posttranslational modifications (PTM) such as acetylation, deubiquitination, and phosphorylation of proteins, play important roles in various kinds of cancer progression. Ubiquitin-specific proteinase 5 (USP5), a unique member of deubiquitinating enzymes (DUBs) which recognizes unanchored polyubiquitin specifically, could regulate the stability of many tumorigenesis-associated proteins to influence cancer initiation and progression. However, the diverse biological significance of USP5 in pan-cancer has not been systematically and comprehensively studied. Here, we explored the role of USP5 in pan-cancer using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) database, and we also acquired and analyzed data via various software and web platforms such as R, GEPIA2.0, HPA, TISIDB, cBioPortal, UALCAN, TIMER 2.0, CancerSEA and BioGRID. USP5 expression was high in most cancers and differed significantly in different molecular and immune subtypes of cancers. In addition, USP5 had certain diagnostic value in multiple cancers, and high expression of USP5 generally predicted poor prognosis for cancer patients. We also found that the most frequent genetic alterations type of USP5 was mutation, and the DNA methylation level of USP5 decreased in various cancers. Furthermore, USP5 expression correlated with cancer-associated fibroblasts (CAFs), endothelial cells (EC) and genetic markers of immunodulators in cancers. Moreover, the result from single cell sequencing showed that USP5 could regulate several tumor biological behaviors such as apoptosis, DNA damage and metastasis. Gene enrichment analysis indicated “spliceosome” and “RNA splicing” may be the critical mechanism for USP5 to involve in cancer. Taken together, our study elucidates the biological significance of USP5 in the diagnosis, prognosis and immune in human pan-cancer.

Modulation of virus-induced neuroinflammation by the autophagy receptor SHISA9 in mice.

Microglia and astrocytes are subgroups of brain glia cells that support and protect neurons within the central nervous system (CNS). At early stages of viral infection in the CNS, they are predominant responding cells and lead to recruitment of peripheral immune cells for viral clearance. Inhibitor of nuclear factor κB kinase subunit epsilon (IKKi) is critical for type I interferon signalling and inflammation, which modulate heterogenic immune responses during CNS infection. Balanced autophagy is vital to maintain brain integrity, yet regulation of autophagy and immune activity within brain glia cells is poorly understood. Here we identify SHISA9 as an autophagy cargo receptor that mediates the autophagy-dependent degradation of IKKi during herpes simplex virus type 1 infection. IKKi is recognized by SHISA9 through unanchored K48-linked poly-ubiquitin chains and bridged to autophagosome membrane components GABARAPL1. Single-cell RNA sequencing analysis shows that SHISA9 has temporal characteristics while modulating both antiviral and inflammatory responses in microglia and astrocytes at different stages during viral infection. We found that Shisa9-/- mice are highly susceptible to herpes simplex virus encephalitis, have pathogenic astrocytes and display more severe neuroinflammation compared with wild-type mice. Taken together, our study unravels a critical role of selective autophagy by orchestrating immune heterogeneity of different CNS resident cells through the SHISA9-IKKi axis.

The RNA helicase DHX16 recognizes specific viral RNA to trigger RIG-I-dependent innate antiviral immunity.

Type I interferons (IFN-I) are essential to establish antiviral innate immunity. Unanchored (or free) polyubiquitin (poly-Ub) has been shown to regulate IFN-I responses. However, few unanchored poly-Ub interactors are known. To identify factors regulated by unanchored poly-Ub in a physiological setting, we developed an approach to isolate unanchored poly-Ub from lung tissue. We identified the RNA helicase DHX16 as a potential pattern recognition receptor (PRR). Silencing of DHX16 in cells and in vivo diminished IFN-I responses against influenza virus. These effects extended to members of other virus families, including Zika and SARS-CoV-2. DHX16-dependent IFN-I production requires RIG-I and unanchored K48-poly-Ub synthesized by the E3-Ub ligase TRIM6. DHX16 recognizes a signal in influenza RNA segments that undergo splicing and requires its RNA helicase motif for direct, high-affinity interactions with specific viral RNAs. Our study establishes DHX16 as a PRR that partners with RIG-I for optimal activation of antiviral immunity requiring unanchored poly-Ub.

Role of ubiquitin-specific protease 5 in the inflammatory response of chronic periodontitis.

The systemic inflammatory response caused by chronic periodontitis is a risk factor for multiple diseases. Ubiquitin-specific protease 5 (USP5) is a kind of deubiquitinase which mainly responsible for dissociating unanchored polyubiquitin. However, the functions of USP5 in chronic periodontitis have not been reported. Chronic periodontitis patients were recruited, and their periodontal samples were collected. The levels of USP5, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in gingival crevicular fluid were evaluated by ELISA. The expression of USP5, TNF-α, IL-6, and IL-1β in human periodontal ligament stem cells (PDLSCs) was estimated by qRT-PCR assay. The activation of STAT3 signaling was examined by Western blot. USP5 was upregulated in the gingival crevicular fluid and gingival tissues of chronic periodontitis patients. USP5 expression was positively correlated with the expression of proinflammatory factors. USP5 knockdown and deubiquitinase inhibitor inhibited LPS-induced inflammatory responses in PDLSCs. Suppressing USP5 inhibited STAT3 signaling in PDLSCs. Suppression deubiquitinase USP5 inhibits the inflammatory response of chronic periodontitis by suppressing STAT3 signaling.

Human Norovirus NTPase Antagonizes Interferon-β Production by Interacting With IkB Kinase ε.

Human norovirus (HuNoV) is the leading cause of epidemic acute gastroenteritis worldwide. Type I interferons (IFN)-α/β are highly potent cytokines that are initially identified for their essential roles in antiviral defense. It was reported that HuNoV infection did not induce IFN-β expression but was controlled in the presence of IFN-β in human intestinal enteroids and a gnotobiotic pig model, suggesting that HuNoV has likely developed evasion countermeasures. In this study, we found that a cDNA clone of GII.4 HuNoV, the predominantly circulating genotype worldwide, inhibits the production of IFN-β and identified the viral NTPase as a key component responsible for such inhibition. HuNoV NTPase not only inhibits the activity of IFN-β promoter but also the mRNA and protein production of IFN-β. Additional studies indicate that NTPase inhibits the phosphorylation and nuclear translocation of interferon-regulatory factor-3 (IRF-3), leading to the suppression of IFN-β promoter activation. Mechanistically, NTPase interacts with IkB kinase ε (IKKε), an important factor for IRF-3 phosphorylation, and such interaction blocks the association of IKKε with unanchored K48-linked polyubiquitin chains, resulting in the inhibition of IKKε phosphorylation. Further studies demonstrated that the 1-179 aa domain of NTPase which interacts with IKKε is critical for the suppression of IFN-β production. Our findings highlight the role of HuNoV NTPase in the inhibition of IFN-β production, providing insights into a novel mechanism underlying how HuNoV evades the host innate immunity.

Modification of small ubiquitin-related modifier 2 (SUMO2) by phosphoubiquitin in HEK293T cells.

Additional complexity in the post-translational modification of proteins by ubiquitin is achieved by ubiquitin phosphorylation, for example within PINK1-parkin mediated mitophagy. We performed a preliminary proteomic analysis to identify proteins differentially modified by ubiquitin in HEK293T, compared to phosphomimetic ubiquitin (Ser65Asp), and identified small ubiquitin-related modifier 2 (SUMO2) as a candidate. By transfecting SUMO2 and its C-terminal-GG deletion mutant, along with phosphomimetic ubiquitin, we confirm that ubiquitin modifies SUMO2, rather than vice versa. Further investigations revealed that transfected SUMO2 can also be conjugated by endogenous phospho-Ser65-(poly)ubiquitin in HEK293T cells, pointing to a previously unappreciated level of complexity in SUMO2 modification, and that unanchored (substrate-free) polyubiquitin chains may also be subject to phosphorylation.

A novel combination therapy targeting ubiquitin-specific protease 5 in MYCN-driven neuroblastoma

Histone deacetylase (HDAC) inhibitors are effective in MYCN-driven cancers, because of a unique need for HDAC recruitment by the MYCN oncogenic signal. However, HDAC inhibitors are much more effective in combination with other anti-cancer agents. To identify novel compounds which act synergistically with HDAC inhibitor, such as suberanoyl hydroxamic acid (SAHA), we performed a cell-based, high-throughput drug screen of 10,560 small molecule compounds from a drug-like diversity library and identified a small molecule compound (SE486-11) which synergistically enhanced the cytotoxic effects of SAHA. Effects of drug combinations on cell viability, proliferation, apoptosis and colony forming were assessed in a panel of neuroblastoma cell lines. Treatment with SAHA and SE486-11 increased MYCN ubiquitination and degradation, and markedly inhibited tumorigenesis in neuroblastoma xenografts, and, MYCN transgenic zebrafish and mice. The combination reduced ubiquitin-specific protease 5 (USP5) levels and increased unanchored polyubiquitin chains. Overexpression of USP5 rescued neuroblastoma cells from the cytopathic effects of the combination and reduced unanchored polyubiquitin, suggesting USP5 is a therapeutic target of the combination. SAHA and SE486-11 directly bound to USP5 and the drug combination exhibited a 100-fold higher binding to USP5 than individual drugs alone in microscale thermophoresis assays. MYCN bound to the USP5 promoter and induced USP5 gene expression suggesting that USP5 and MYCN expression created a forward positive feedback loop in neuroblastoma cells. Thus, USP5 acts as an oncogenic cofactor with MYCN in neuroblastoma and the novel combination of HDAC inhibitor with SE486-11 represents a novel therapeutic approach for the treatment of MYCN-driven neuroblastoma.

Isoleucine 44 Hydrophobic Patch Controls Toxicity of Unanchored, Linear Ubiquitin Chains through NF-κB Signaling.

Ubiquitination is a post-translational modification that regulates cellular processes by altering the interactions of proteins to which ubiquitin, a small protein adduct, is conjugated. Ubiquitination yields various products, including mono- and poly-ubiquitinated substrates, as well as unanchored poly-ubiquitin chains whose accumulation is considered toxic. We previously showed that transgenic, unanchored poly-ubiquitin is not problematic in Drosophila melanogaster. In the fruit fly, free chains exist in various lengths and topologies and are degraded by the proteasome; they are also conjugated onto other proteins as one unit, eliminating them from the free ubiquitin chain pool. Here, to further explore the notion of unanchored chain toxicity, we examined when free poly-ubiquitin might become problematic. We found that unanchored chains can be highly toxic if they resemble linear poly-ubiquitin that cannot be modified into other topologies. These species upregulate NF-κB signaling, and modulation of the levels of NF-κB components reduces toxicity. In additional studies, we show that toxicity from untethered, linear chains is regulated by isoleucine 44, which anchors a key interaction site for ubiquitin. We conclude that free ubiquitin chains can be toxic, but only in uncommon circumstances, such as when the ability of cells to modify and regulate them is markedly restricted.

Regulation of RIG-I antiviral function by the RNA helicase DHX16 and unanchored polyubiquitin chains

Abstract The type-I interferon (IFN-I) pathway mediates antiviral responses and is regulated to balance pathogen clearance while mitigating host damage. Our group recently showed that unanchored K48-linked poly-ubiquitin (K48 poly-ub) chains associate with the IKKɛ kinase to induce an optimal IFN-I mediated antiviral response. To identify other factors that may be regulated by unanchored poly-ub, we developed a novel method to isolate these chains from influenza A virus (IAV)-infected mouse lungs. Combining this approach with mass spectrometry analysis we identified DHX16, an RNA helicase previously known only as a pre-mRNA splicing factor. Next-Generation sequencing identified several IFN-stimulated genes whose expression was significantly downregulated in DHX16 knockdown cells. Consistent with this, in vitro and in vivo knockdown of DHX16 during IAV infection reduced IFN-I responses leading to increased IAV replication. DHX16-dependent IFN induction requires its intact RNA helicase motif and DHX16 ATPase activity is enhanced upon addition of unanchored K48 poly-ub in the presence of viral RNA, suggesting DHX16 first recognizes viral RNA and unanchored poly-ub regulate its activity. Furthermore, DHX16-induced IFN also requires RIG-I and this occurs in a RNA-dependent manner. Finally, we identified TRIM6 as the E3-Ub ligase that catalyzes the synthesis of unanchored K48 poly-Ub that positively regulate DHX16 activity. In conclusion, we uncovered a novel mechanism by which unanchored poly-Ub and DHX16 contribute to viral RNA recognition by RIG-I to induce an optimal IFN-I mediated antiviral response

Oligomerization of RIG-I and MDA5 2CARD domains.

The innate immune system is the first line of defense against invading pathogens. The retinoic acid-inducible gene I (RIG-I) like receptors (RLRs), RIG-I and melanoma differentiation-associated protein 5 (MDA5), are critical for host recognition of viral RNAs. These receptors contain a pair of N-terminal tandem caspase activation and recruitment domains (2CARD), an SF2 helicase core domain, and a C-terminal regulatory domain. Upon RLR activation, 2CARD associates with the CARD domain of MAVS, leading to the oligomerization of MAVS, downstream signaling and interferon induction. Unanchored K63-linked polyubiquitin chains (polyUb) interacts with the 2CARD domain, and in the case of RIG-I, induce tetramer formation. However, the nature of the MDA5 2CARD signaling complex is not known. We have used sedimentation velocity analytical ultracentrifugation to compare MDA5 2CARD and RIG-I 2CARD binding to polyUb and to characterize the assembly of MDA5 2CARD oligomers in the absence of polyUb. Multi-signal sedimentation velocity analysis indicates that Ub4 binds to RIG-I 2CARD with a 3:4 stoichiometry and cooperatively induces formation of an RIG-I 2CARD tetramer. In contrast, Ub4 and Ub7 interact with MDA5 2CARD weakly and form complexes with 1:1 and 2:1 stoichiometries but do not induce 2CARD oligomerization. In the absence of polyUb, MDA5 2CARD self-associates to forms large oligomers in a concentration-dependent manner. Thus, RIG-I and MDA5 2CARD assembly processes are distinct. MDA5 2CARD concentration-dependent self-association, rather than polyUb binding, drives oligomerization and MDA5 2CARD forms oligomers larger than tetramer. We propose a mechanism where MDA5 2CARD oligomers, rather than a stable tetramer, function to nucleate MAVS polymerization.

Discovery of Small Molecule Antagonists of the USP5 Zinc Finger Ubiquitin-Binding Domain.

USP5 disassembles unanchored polyubiquitin chains to recycle free monoubiquitin, and is one of the 12 ubiquitin specific proteases featuring a zinc finger ubiquitin-binding domain (ZnF-UBD). This distinct structural module has been associated with substrate positioning or allosteric modulation of catalytic activity, but its cellular function remains unclear. We screened a chemical library focused on the ZnF-UBD of USP5, crystallized hits in complex with the protein, and generated a preliminary structure-activity relationship, which enables the development of more potent and selective compounds. This work serves as a framework for the discovery of a chemical probe to delineate the function of USP5 ZnF-UBD in proteasomal degradation and other ubiquitin signaling pathways in health and disease.

Autophagy induction in atrophic muscle cells requires ULK1 activation by TRIM32 through unanchored K63-linked polyubiquitin chains.

Optimal autophagic activity is crucial to maintain muscle integrity, with either reduced or excessive levels leading to specific myopathies. LGMD2H is a muscle dystrophy caused by mutations in the ubiquitin ligase TRIM32, whose function in muscles remains not fully understood. Here, we show that TRIM32 is required for the induction of muscle autophagy in atrophic conditions using both in vitro and in vivo mouse models. Trim32 inhibition results in a defective autophagy response to muscle atrophy, associated with increased ROS and MuRF1 levels. The proautophagic function of TRIM32 relies on its ability to bind the autophagy proteins AMBRA1 and ULK1 and stimulate ULK1 activity via unanchored K63-linked polyubiquitin. LGMD2H-causative mutations impair TRIM32's ability to bind ULK1 and induce autophagy. Collectively, our study revealed a role for TRIM32 in the regulation of muscle autophagy in response to atrophic stimuli, uncovering a previously unidentified mechanism by which ubiquitin ligases activate autophagy regulators.

Induction of innate antiviral immunity through a novel pattern recognition receptor, the RNA helicase DHX16, is regulated by unanchored K48-linked poly-ubiquitin chains

Abstract The type-I interferon (IFN-I) pathway mediates antiviral responses and is regulated to balance pathogen clearance while mitigating host damage. Unanchored K48-linked poly-ubiquitin (K48 poly-ub) chains, which associate with targets through non-covalent interactions, have been shown by our group to be a critical regulatory signal in IKKe kinase-mediated antiviral immunity. To identify other components requiring unanchored poly-ub, we developed a novel method to isolate these chains from influenza virus (IAV)-infected mouse lungs. Using this approach combined with mass spectrometry analysis we identified DHX16, an RNA helicase and pre-mRNA splicing component. The role of DHX16 in innate immunity and regulation of its activity have not been described. We found that during IAV infection, the levels of unanchored K48 poly-ub that associate with DHX16 are significantly increased and these DHX16-interacting chains are synthetized by the E3-ubiquitin ligase TRIM6, which we have previously shown to be critical in IFN-I and antiviral responses. Next-Generation sequencing identified a number of IFN-stimulated genes whose expression was significantly downregulated in DHX16 knockdown cells. Consistent with this, overexpression of DHX16 induced IRF3 phosphorylation, which can be further enhanced upon IAV infection. Furthermore, the ATPase activity of DHX16 is enhanced upon addition of unanchored K48 poly-ub only in the presence of viral RNA suggesting DHX16 first recognizes viral RNA and unanchored poly-ub regulate its activity. Together these data suggest DHX16 acts as a novel pattern recognition receptor involved in the IFN-I pathway and its activity is regulated by unanchored K48 poly-ub.

Unanchored ubiquitin chains do not lead to marked alterations in gene expression in Drosophila melanogaster.

ABSTRACTThe small protein modifier ubiquitin regulates various aspects of cellular biology through its chemical conjugation onto proteins. Ubiquitination of proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Ubiquitin chains can be attached onto other proteins or can exist as unanchored species, i.e. free from another protein. Unanchored ubiquitin chains are thought to be deleterious to the cell and rapidly disassembled into mono-ubiquitin. We recently examined the toxicity and utilization of unanchored poly-ubiquitin in Drosophila melanogaster. We found that free poly-ubiquitin species are largely innocuous to flies and that free poly-ubiquitin can be controlled by being degraded by the proteasome or by being conjugated onto another protein as a single unit. Here, to explore whether an organismal defense is mounted against unanchored chains, we conducted RNA-Seq analyses to examine the transcriptomic impact of free poly-ubiquitin in the fly. We found ∼90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was essentially devoid of ubiquitin-, proteasome-, or autophagy-related components. The seeming absence of a large and multipronged response to unanchored poly-ubiquitin supports the conclusion that these species need not be toxic in vivo and underscores the need to re-examine the role of free ubiquitin chains in the cell.

Biochemical properties and in planta effects of NopM, a rhizobial E3 ubiquitin ligase

Nodulation outer protein M (NopM) is an IpaH family type three (T3) effector secreted by the nitrogen-fixing nodule bacterium Sinorhizobium sp. strain NGR234. Previous work indicated that NopM is an E3 ubiquitin ligase required for an optimal symbiosis between NGR234 and the host legume Lablab purpureus Here, we continued to analyze the function of NopM. Recombinant NopM was biochemically characterized using an in vitro ubiquitination system with Arabidopsis thaliana proteins. In this assay, NopM forms unanchored polyubiquitin chains and possesses auto-ubiquitination activity. In a NopM variant lacking any lysine residues, auto-ubiquitination was not completely abolished, indicating noncanonical auto-ubiquitination of the protein. In addition, we could show intermolecular ubiquitin transfer from NopM to C338A (enzymatically inactive NopM form) in vitro Bimolecular fluorescence complementation analysis provided clues about NopM-NopM interactions at plasma membranes in planta NopM, but not C338A, expressed in tobacco cells induced cell death, suggesting that E3 ubiquitin ligase activity of NopM induced effector-triggered immunity responses. Likewise, expression of NopM in Lotus japonicus caused reduced nodule formation, whereas expression of C338A showed no obvious effects on symbiosis. Further experiments indicated that serine residue 26 of NopM is phosphorylated in planta and that NopM can be phosphorylated in vitro by salicylic acid-induced protein kinase (NtSIPK), a mitogen-activated protein kinase (MAPK) of tobacco. Hence, NopM is a phosphorylated T3 effector that can interact with itself, with ubiquitin, and with MAPKs.

Molecular mechanism of influenza A NS1-mediated TRIM25 recognition and inhibition

RIG-I is a viral RNA sensor that induces the production of type I interferon (IFN) in response to infection with a variety of viruses. Modification of RIG-I with K63-linked poly-ubiquitin chains, synthesised by TRIM25, is crucial for activation of the RIG-I/MAVS signalling pathway. TRIM25 activity is targeted by influenza A virus non-structural protein 1 (NS1) to suppress IFN production and prevent an efficient host immune response. Here we present structures of the human TRIM25 coiled-coil-PRYSPRY module and of complexes between the TRIM25 coiled-coil domain and NS1. These structures show that binding of NS1 interferes with the correct positioning of the PRYSPRY domain of TRIM25 required for substrate ubiquitination and provide a mechanistic explanation for how NS1 suppresses RIG-I ubiquitination and hence downstream signalling. In contrast, the formation of unanchored K63-linked poly-ubiquitin chains is unchanged by NS1 binding, indicating that RING dimerisation of TRIM25 is not affected by NS1.

Purification of Unanchored Polyubiquitin Chains from Influenza Virions.

Influenza A virus (IAV) is an enveloped virus with a segmented single-stranded negative-strand RNA genome. In general, the role of virally encapsidated host cell proteins in the viral life cycle is unclear. The virion contains abundant ubiquitin molecules some of which have been identified as unanchored polyubiquitin chains. These ubiquitin chains have been postulated to play a role in recruiting histone deacetylase 6 (HDAC6) to the cytosolic surface of late endosomes (LEs), promoting IAV uncoating via aggresome processing-a cellular machinery that disposes of protein waste. HDAC6, a class II HDAC, is unusual because it resides mostly in the cytosol instead of the nucleus. It is a unique protein consisting of two catalytic domains (CDs) and a zinc-finger ubiquitin-binding domain (ZnF-UBP) close to its C-terminus. This ZnF-UBP recognizes the unconjugated ubiquitin C-terminus (di-Gly motif) with very high affinity. Biochemical analyses showed that free di-Gly motifs are present in the form of unanchored ubiquitin inside IAV virions. These motifs are exposed following low pH-triggered viral fusion at the LEs and attract HDAC6 transiently to the cytosolic surface of vesicles. The binding of the two components promotes viral uncoating via HDAC6 interaction with cellular motor proteins dynein and myosin II and the viral M1 capsid. The cellular mechanism involved is related to aggresome processing, a pathway that promotes degradation of misfolded protein aggregates. K63-linked ubiquitin chains are thought to be the trigger for aggresome processing, though it is still not clear whether such types of chains are prevalent within the IAV capsid. Here, we present methods using purified ZnF-UBP domain of HDAC6 to immunoprecipitate viral unanchored ubiquitin chains, which can then be used for further biochemical analyses of ubiquitin chain linkage.