TRIM44 interacts with and stabilizes terf, a TRIM ubiquitin E3 ligase

Terf/TRIM17 is a member of the TRIM family of proteins, which is characterized by the RING finger, B-box, and coiled-coil domains. In the present study, we found that terf interacts with TRIM44. Terf underwent ubiquitination in vitro in the presence of the E2 enzyme UbcH6; this suggests that terf exhibits E3 ubiquitin ligase activity. It was also found that terf was conjugated with polyubiquitin chains and stabilized by the proteasome inhibitor in mammalian cells; this suggested that terf rendered itself susceptible to proteasomal degradation through polyubiquitination. We also found that TRIM44 inhibited ubiquitination of terf, and thus stabilized the protein. The N-terminal region of TRIM44 contains a zinc-finger domain found in ubiquitin hydrolases (ZF UBP) and ubiquitin specific proteases (USPs). Thus, we proposed that TRIM44 may function as a new class of the “USP-like-TRIM” which regulates the activity of associated TRIM proteins.     

Emerging complexity and new roles for the RIG-I-like receptors in innate antiviral immunity

Innate immunity is critical for the control of virus infection and operates to restrict viral susceptibility and direct antiviral immunity for protection from acute or chronic viral-associated diseases including cancer. RIG-I like receptors (RLRs) are cytosolic RNA helicases that function as pathogen recognition receptors to detect RNA pathogen associated molecular patterns (PAMPs) of virus infection. The RLRs include RIG-I, MDA5, and LGP2. They function to recognize and bind to PAMP motifs within viral RNA in a process that directs the RLR to trigger downstream signaling cascades that induce innate immunity that controls viral replication and spread. Products of RLR signaling also serve to modulate the adaptive immune response to infection. Recent studies have additionally connected RLRs to signaling cascades that impart inflammatory and apoptotic responses to virus infection. Viral evasion of RLR signaling supports viral outgrowth and pathogenesis, including the onset of viral-associated cancer.     

Ectopic Expression of TRIM25 Restores RIG-I Expression and IFN Production Reduced by Multiple Enteroviruses 3Cpro

Virologica Sinica - 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...     

Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4‐2 using proteome arrays

Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2–4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4‐1) and Nedd4L (Nedd4‐2), but their global substrate recognition or differences in substrate specificity are unknown. We performed in vitro ubiquitylation and binding assays of human Nedd4‐1 and Nedd4‐2, and rat‐Nedd4‐1, using protein microarrays spotted with ～8200 human proteins. Top hits (substrates) for the ubiquitylation and binding assays mostly contain PY motifs. Although several substrates were recognized by both Nedd4‐1 and Nedd4‐2, others were specific to only one, with several Tyr kinases preferred by Nedd4‐1 and some ion channels by Nedd4‐2; this was subsequently validated in vivo. Accordingly, Nedd4‐1 knockdown or knockout in cells led to sustained signalling via some of its substrate Tyr kinases (e.g. FGFR), suggesting Nedd4‐1 suppresses their signalling. These results demonstrate the feasibility of identifying substrates and deciphering substrate specificity of mammalian E3 ligases.     

Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBC^VHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBC^VHL complex, and is probably one of the new substrates of the CBC^VHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor-binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.     

TRIM7 inhibits enterovirus replication and promotes emergence of a viral variant with increased pathogenicity

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Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBC^VHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBC^VHL complex, and is probably one of the new substrates of the CBC^VHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor-binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.     

Insights into links between autophagy and the ubiquitin system from the structure of LC3B bound to the LIR motif from the E3 ligase NEDD4

Members of the LC3/GABARAP family of ubiquitin‐like proteins function during autophagy by serving as membrane linked protein‐binding platforms. Their C‐termini are physically attached to membranes through covalent linkage to primary amines on lipids such as phosphatidylethanolamine, while their ubiquitin‐like fold domains bind “LIR” (LC3‐Interacting Region) sequences found within an extraordinarily diverse array of proteins including regulators of autophagy, adaptors that recruit ubiquitinated cargoes to be degraded, and even proteins controlling processes at membranes that are not associated with autophagy. Recently, LC3/GABARAP proteins were found to bind the ubiquitin E3 ligase NEDD4 to influence ubiquitination associated with autophagy in human cell lines. Here, we use purified recombinant proteins to define LC3B interactions with a specific LIR sequence from NEDD4, present a crystal structure showing atomic details of the interaction, and show that LC3B‐binding can steer intrinsic NEDD4 E3 ligase activity. The data provide detailed molecular insights underlying recruitment of an E3 ubiquitin ligase to phagophores during autophagy.     

Immune regulation by protein ubiquitination: roles of the E3 ligases VHL and Itch

Protein ubiquitination is an important means of posttranslational modification which plays an essential role in the regulation of various aspects of leukocyte development and function. The specificity of ubiquitin tagging to a protein substrate is determined by E3 ubiquitin ligases via defined E3-substrate interactions. In this review, we will focus on two E3 ligases, VHL and Itch, to discuss the latest progress in understanding their roles in the differentiation and function of CD4+ T helper cell subsets, the stability of regulatory T cells, effector function of CD8+ T cells, as well as the development and maturation of innate lymphoid cells. The biological implications of these E3 ubiquitin ligases will be highlighted in the context of normal and dysregulated immune responses including the control of homeostasis, inflammation, auto-immune responses and anti-tumor immunity. Further elucidation of the ubiquitin system in immune cells will help in the design of new therapeutic interventions for human immunological diseases and cancer.     

ER stress differentially regulates the stabilities of ERAD ubiquitin ligases and their substrates

Endoplasmic reticulum (ER) stress-induced accumulation of misfolded proteins in the ER stimulates the ER-associated degradation (ERAD) process. ERAD in turn eliminates those misfolded proteins. Upregulation of ubiquitination enzymes is an essential mechanism by which ER stress enhances ERAD. However, ectopic overexpression of ubiquitination enzymes often fails to increase, and sometimes, inhibits ERAD. To further understand how ER stress regulates ERAD, we studied the effects of ER stress on ubiquitin ligase (E3) gp78-mediated ERAD and on the stabilities of gp78 and another ERAD E3 Hrd1. The results showed that ER stress-inducing agent tunicamycin significantly enhanced ERAD in cells that either express endogenous or overexpress gp78. Importantly, ER stress could increase ERAD even when new protein synthesis was inhibited by cycloheximide. Surprisingly, tunicamycin treatment stabilized gp78, an established ERAD E3 and an ERAD substrate as well, for up to 8h. By contrast, ER stress had little effects on the stability of another E3 Hrd1 except that it reduced the total ubiquitination level of Hrd1. Our data suggest that ER stress differentially regulates the stabilities of ERAD E3s and their substrates, which may represent a novel mechanism by which ER stress increases ERAD.     

Mutational analysis of Mdm2 C-terminal tail suggests an evolutionarily conserved role of its length in Mdm2 activity toward p53 and indicates structural differences between Mdm2 homodimers and Mdm2/MdmX heterodimers

Mdm2 can mediate p53 ubiquitylation and degradation either in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer. The ubiquitin ligase activity of these complexes resides mainly in their respective RING finger domains and also requires adjacent C-terminal tails. So far, structural studies have failed to show significant differences between Mdm2 RING homodimers and Mdm2/MdmX RING heterodimers. Here, we report that not only the primary amino acid sequence, but also the length of the C-terminal tail of Mdm2 is highly conserved through evolution and plays an important role in Mdm2 activity toward p53. Mdm2 mutants with extended C termini do not ubiquitylate p53 despite being capable of forming Mdm2 homodimers through both RING-acidic domain and RING-RING interactions. All extended mutants also retained the ability to interact with MdmX, and this interaction led to reactivation of their E3 ubiquitin ligase activity. In contrast, only a subset of extended Mdm2 mutants was activated by the interaction with Mdm2 RING domain, suggesting that Mdm2 homodimers and Mdm2/MdmX heterodimers may not be structurally and functionally fully equivalent.     

Human and viral membrane–associated E3 ubiquitin ligases MARCH1 and MIR2 recognize different features of CD86 to downregulate surface expression

Immune-stimulatory ligands, such as major histocompatibility complex molecules and the T-cell costimulatory ligand CD86, are central to productive immunity. Endogenous mammalian membrane-associated RING-CHs (MARCH) act on these and other targets to regulate antigen presentation and activation of adaptive immunity, whereas virus-encoded homologs target the same molecules to evade immune responses. Substrate specificity is encoded in or near the membrane-embedded domains of MARCHs and the proteins they regulate, but the exact sequences that distinguish substrates from nonsubstrates are poorly understood. Here, we examined the requirements for recognition of the costimulatory ligand CD86 by two different MARCH-family proteins, human MARCH1 and Kaposi''s sarcoma herpesvirus modulator of immune recognition 2 (MIR2), using deep mutational scanning. We identified a highly specific recognition surface in the hydrophobic core of the CD86 transmembrane (TM) domain (TMD) that is required for recognition by MARCH1 and prominently features a proline at position 254. In contrast, MIR2 requires no specific sequences in the CD86 TMD but relies primarily on an aspartic acid at position 244 in the CD86 extracellular juxtamembrane region. Surprisingly, MIR2 recognized CD86 with a TMD composed entirely of valine, whereas many different single amino acid substitutions in the context of the native TM sequence conferred MIR2 resistance. These results show that the human and viral proteins evolved completely different recognition modes for the same substrate. That some TM sequences are incompatible with MIR2 activity, even when no specific recognition motif is required, suggests a more complicated mechanism of immune modulation via CD86 than was previously appreciated.     

Structural modeling of protein ensembles between E3 RING ligases and SARS-CoV-2: The role of zinc binding domains

BackgroundThe ubiquitin system is a modification process with many different cellular functions including immune signaling and antiviral functions. E3 ubiquitin ligases are enzymes that recruit an E2 ubiquitin-conjugating enzyme bound to ubiquitin in order to catalyze the transfer of ubiquitin from the E2 to a protein substrate. The RING E3s, the most abundant type of ubiquitin ligases, are characterized by a zinc (II)-binding domain called RING (Really Interesting New Gene). Viral replication requires modifying and hijacking key cellular pathways within host cells such as cellular ubiquitination. There are well-established examples where a viral proteins bind to RING E3s, redirecting them to degrade otherwise long-lived host proteins or inhibiting E3’s ubiquitination activity. Recently, three binary interactions between SARS-CoV-2 proteins and innate human immune signaling Ε3 RING ligases: NSP15-RNF41, ORF3a-TRIM59 and NSP9-MIB1 have been experimentally established.MethodsIn this work, we have investigated the mode of the previous experimentally supported NSP15-RNF41, ORF3a,-TRIM59 and NSP9-MIB1 binary interactions by in silico methodologies intending to provide structural insights of E3-virus interplay that can help identify potential inhibitors that could block SARS-CoV-2 infection of immune cells.ConclusionIn silico methodologies have shown that the above human E3 ligases interact with viral partners through their Zn(II) binding domains. This RING mediated formation of stable SARS-CoV-2-E3 complexes indicates a critical structural role of RING domains in immune system disruption by SARS-CoV-2-infection.Data AvailabilityThe data used to support the findings of this research are included within the article and are labeled with references.     

Developmental regulation of MURF ubiquitin ligases and autophagy proteins nbr1, p62/SQSTM1 and LC3 during cardiac myofibril assembly and turnover

The striated muscle-specific tripartite motif (TRIM) proteins TRIM63/MURF1, TRIM55/MURF2 and TRIM54/MURF3 can function as ubiquitin E3 ligases in ubiquitin-mediated muscle protein turnover. Despite their well-characterised roles in muscle atrophy, the dynamics of MURF expression in the development and early postnatal adaptation of striated muscle is largely unknown. Here, we show that MURF2 is expressed at the very onset of mouse cardiac differentiation at embryonic day 8.5, and represents a sensitive marker for differentiating myocardium. During cardiac development, expression shifts from the 50 kDa to the 60 kDa A-isoform, which dominates postnatally. In contrast, MURF1 shows strong postnatal upregulation and MURF3 is not significantly expressed before birth. MURF2 expression parallels that of the autophagy-associated proteins LC3, p62/SQSTM1 and nbr1. SiRNA knockdown of MURF2 in neonatal rat cardiomyocytes disrupts posttranslational microtubule modification and myofibril assembly, and is only partly compensated by upregulation of MURF3 but not MURF1. Knockdown of both MURF2 and MURF3 severely disrupts the formation of ordered Z- and M-bands, likely by perturbed tubulin dynamics. These results suggest that ubiquitin-mediated protein turnover and MURF2 in particular play an unrecognised role in the earliest steps of heart muscle differentiation, and that partial complementation of MURF2 deficiency is afforded by MURF3.     

Functional characterization of SAG/RBX2/ROC2/RNF7, an antioxidant protein and an E3 ubiquitin ligase

SAG (Sensitive to Apoptosis Gene), also known as RBX2 (RING box protein 2), ROC2 (Regulator of Cullins 2), or RNF7 (RING Finger Protein 7), was originally cloned in our laboratory as a redox inducible antioxidant protein and later characterized as the second member of the RBX/ROC RING component of the SCF (SKP1-CUL-F-box Proteins) E3 ubiquitin ligase. When acting alone, SAG scavenges oxygen radicals by forming inter- and intra- molecular disulfide bonds, whereas by forming a complex with other components of the SCF E3 ligase, SAG promotes ubiquitination and degradation of a number of protein substrates, including c-JUN, DEPTOR, HIF-1α, IκBα, NF1, NOXA, p27, and procaspase-3, thus regulating various signaling pathways and biological processes. Specifically, SAG protects cells from apoptosis, confers radioresistance, and plays an essential and non-redundant role in mouse embryogenesis and vasculogenesis. Furthermore, stress-inducible SAG is overexpressed in a number of human cancers and SAG overexpression correlates with poor patient prognosis. Finally, SAG transgenic expression in epidermis causes an early stage inhibition, but later stage promotion, of skin tumorigenesis triggered by DMBA/TPA. Given its major role in promoting targeted degradation of tumor suppressive proteins, leading to apoptosis suppression and accelerated tumorigenesis, SAG E3 ligase appears to be an attractive anticancer target.