The heterodimeric ubiquitin conjugating enzyme (E2) UBC13-UEV mediates polyubiquitylation through lysine 63 of ubiquitin (K63), rather than lysine 48 (K48). This modification does not target proteins for proteasome-dependent degradation. Searching for potential regulators of this variant polyubiquitylation we have identified four proteins, namely RNF8, KIA00675, KF1, and ZNRF2, that interact with UBC13 through their RING finger domains. These domains can recruit, in addition to UBC13, other E2s that mediate canonical (K48) polyubiquitylation. None of these RING finger proteins were known previously to recruit UBC13. For one of these proteins, RNF8, we show its activity as a ubiquitin ligase that elongates chains through either K48 or K63 of ubiquitin, and its nuclear co-localization with UBC13. Thus, our screening reveals new potential regulators of non-canonical polyubiquitylation.
BackgroundSeveral pathways that control cell survival under stress, namely RNF8-dependent DNA damage recognition and repair, PCNA-dependent DNA damage tolerance and activation of NF-κB by extrinsic signals, are regulated by the tagging of key proteins with lysine 63-based polyubiquitylated chains, catalyzed by the conserved ubiquitin conjugating heterodimeric enzyme Ubc13-Uev.Methodology/Principal FindingsBy applying a selection based on in vivo protein-protein interaction assays of compounds from a combinatorial chemical library followed by virtual screening, we have developed small molecules that efficiently antagonize the Ubc13-Uev1 protein-protein interaction, inhibiting the enzymatic activity of the heterodimer. In mammalian cells, they inhibit lysine 63-type polyubiquitylation of PCNA, inhibit activation of NF-κB by TNF-α and sensitize tumor cells to chemotherapeutic agents. One of these compounds significantly inhibited invasiveness, clonogenicity and tumor growth of prostate cancer cells.Conclusions/SignificanceThis is the first development of pharmacological inhibitors of non-canonical polyubiquitylation that show that these compounds produce selective biological effects with potential therapeutic applications.
The zinc-coordinated protein motifs known as RING-finger domains, present on a class of ubiquitin ligases (E3's), recruit ubiquitin-conjugating enzymes (E2s), tethering them to substrate proteins for covalent modification with ubiquitin. Each RING-finger domain can recruit different E2s, and these interactions are frequently selective, in that certain RING-finger domains associate preferentially with certain E2s. This selectivity acquires particular biological relevance when the recruited E2s exert specialized functions. We have explored the determinants that specify the presence or absence of experimentally detectable interaction between two RING-finger domains, those on RNF11 and RNF103, and two E2s, UBC13, a specialized E2 that catalyzes ubiquitin chain elongation through Lys63 of ubiquitin, and UbcH7, which mediates polyubiquitylation through Lys48. Through the iterative use of computational predictive tools and experimental validations, we have found that these interactions and their selectivity are partly governed by the combinations of electrostatic interactions linking specific residues of the contact interfaces. Our analysis also predicts that the main determinants of selectivity of these interactions reside on the RING-finger domains, rather than on the E2s. The application of some of these rules of interaction selectivity has permitted us to experimentally manipulate the selectivity of interaction of the RING-finger domain-E2 pairs under study.
Detection of cell cycle regulators whose aberrant expression or activation influences cell cycle progression and cell proliferation is critical for the development of efficient diagnostic and therapeutic strategies. In particular Cdk1‐cyclin B1 and Cdc25 phosphatases are central players and partners that coordinate entry into mitosis and whose function is critical for cell proliferation. We have developed non‐genetically encoded fluorescent sensors which specifically recognize Cyclin B1 and Cdc25 phosphatases through molecular interfaces distinct from ATP‐binding pockets or active sites. In vitro characterization of these sensors reveals significant changes in their fluorescence upon specific detection of their target. The combination of these sensors with non‐covalent peptide‐based delivery systems enables efficient delivery into cultured mammalian cells. Characterization of the specificity and efficiency of these biosensors in a cellular environment provides promising perspectives for in vivo applications. Source of Research Support: CNRS & ANR Grant to MCM
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