The TDP-43 is originally a nuclear protein but translocates to the cytoplasm in the pathological condition. TDP-43, as an RNA-binding protein, consists of two RNA Recognition Motifs (RRM1 and RRM2). RRMs are known to involve both protein-nucleotide and protein-protein interactions and mediate the formation of stress granules. Thus, they assist the entire TDP-43 protein with participating in neurodegenerative and cancer diseases. Consequently, they are potential therapeutic targets. Protein-observed and ligand-observed nuclear magnetic resonance (NMR) spectroscopy were used to uncover the small molecule inhibitors against the tandem RRM of TDP-43. We identified three hits weakly binding the tandem RRMs using the ligand-observed NMR fragment-based screening. The binding topology of these hits is then depicted by chemical shift perturbations (CSP) of the 15N-labeled tandem RRM and RRM2, respectively, and modeled by the CSP-guided High Ambiguity Driven biomolecular DOCKing (HADDOCK). These hits mainly bind to the RRM2 domain, which suggests the druggability of the RRM2 domain of TDP-43. These hits also facilitate further studies regarding the hit-to-lead evolution against the TDP-43 RRM domain.
MEX‐3C, a novel RNA binding E3 ubiquitin ligases, contains two N‐terminal heterogeneous nuclear ribonucleoprotein K homology (KH) domains and C‐terminal Ring finger domain. Recent evidence has suggested that human MEX‐3C has a strong bondage with carcinogenesis and the MEX‐3C‐mediated ubiquitination of RIG‐I is essential for the antiviral innate immune response. Moreover, the Ring finger domain of MEX‐3C could regulate the degradation of HLA‐A2 (an MHC‐I allotype) mRNA with a novel mechanism. However, the structural basis for the ubiquitination catalyzed by hMEX‐3C Ring finger domain remains evasive. In this study, we solved the crystal structure of dimeric Ring finger domain of hMEX‐3C and compared it with the complex structure of MDM2/MDMX–UbcH5b–Ub. Our ubiquitination assay demonstrated that the Ring finger domain of hMEX‐3C acts as a ubiquitin E3 ligase in vitro, cooperating with specific E2 to mediate ubiquitination. Then, we identified several key residues in Ring finger domain of hMEX‐3C possibly involved in the interaction with E2–Ub conjugate and analyzed the E3 ligase activities of wild type and mutants at key sites. Additionally, zinc chelation experiments indicated that the intact structural stability is essential for the self‐ubiquitination activity of the Ring finger domain of hMEX‐3C. Taken together, our studies provided new insight into the mechanism of the Ring finger domain of hMEX‐3C that may play an important role in eliciting antiviral immune responses and therapeutic interventions.
Interleukin-17 (IL-17) is a pro-inflammatory cytokine, participating in innate and adaptive immune responses, that plays an important role in host defense, autoimmune diseases, tissue regeneration, metabolic regulation, and tumor progression. Post-translational modifications (PTMs) are crucial for protein function, stability, cellular localization, cellular transduction, and cell death. However, PTMs of IL-17 receptor A (IL-17RA) have not been investigated. Here, we showed that human IL-17RA was targeted by F-box and WD repeats domain containing 11 (FBXW11) for ubiquitination, followed by proteasome-mediated degradation. We used bioinformatics tools and biochemical techniques to determine that FBXW11 ubiquitinated IL-17RA through a lysine 27-linked polyubiquitin chain, targeting IL-17RA for proteasomal degradation. Domain 665-804 of IL-17RA was critical for interaction with FBXW11 and subsequent ubiquitination. Our study demonstrates that FBXW11 regulates IL-17 signaling pathways at IL-17RA level.
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