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  • Sumoylation is involved in regulating protein protein intera

    2021-10-21

    Sumoylation is involved in regulating protein-protein interactions, subcellular protein localization, protein stability, and transcriptional factor activity (reviewed in [29]). The small ubiquitin-like modifier (SUMO) protein is covalently attached to a lysine residue in a Ψ-K-X-D/E consensus sequence (where Ψ is a large hydrophobic residue and X represents any amino acid) on the substrate protein, via an enzymatic cascade similar to ubiquitination in which a battery of E1-activating enzymes (SAE1/SAE2), an E2-conjugating enzyme (Ubc9), and SUMO E3 ligases participate. In comparison, SUMO-mediated protein interactions are based on a non-covalent interaction employing a specialized SUMO-interacting motif (SIM) within the protein sequence of the binding partner [30]. To date, the best-characterized class of SIMs consists of a hydrophobic core ((V/I)X(V/I)(V/I)) flanked by a negatively charged cluster of Protease Inhibitor Library [[31], [32], [33]]. Recent studies favored the concept that the SIM functions as a receptor for the SUMO moiety of SUMO-modified substrate during the downstream effects of sumoylation. The presence of the SIM in a large number of transcription factors and coregulatory partners implies its importance in the control of eukaryotic gene expressions [34]. However, how sumoylation and SIMs act together in modulating gene transcription is not well defined. In addition, SUMO-SIM interactions are also involved in the assembly of large macromolecular structures, including PML nuclear bodies [35], ribosomes [36], nucleoli [37], and kinetochores [38]. Our previous studies showed that the SIM of the multifunctional protein Daxx is essential for binding to SUMO-modified PML and for its targeting to PML nuclear bodies [39]. This same SIM also mediates SUMO-dependent repression of androgen receptor (AR) and glucocorticoid receptor (GR) transcription [39,40].
    Materials and methods
    Results
    Discussion The MR is a member of the nuclear receptor superfamily and acts as a ligand-dependent transcription factor that physiologically regulates salt and water homeostasis in epithelial cells. Previous studies demonstrated that coregulatory molecules (coactivators and corepressors) play pivotal roles in mediating the functions of nuclear receptors. In addition, sumoylation has emerged as an important regulatory mechanism in controlling transcriptional activities of nuclear receptors [46]. Indeed, sumoylation represses the transcription activation activity of the MR [23,27]. FAF1, an apoptosis regulatory molecule, was previously reported to interact with the MR and potentiate transcriptional activity of the MR in neural cells [16]. However, the molecular mechanism underlying FAF1-regulated transcription remains uncertain. Herein, we defined two SIMs within FAF1 and showed that they were crucial for interactions with and transrepression of the sumoylated MR. We provided further evidence suggesting that the mechanisms of FAF1/SIM-mediated MR transrepression involves disrupting the MR N/C-interaction and promoting MR ubiquitination and degradation. Our work provides mechanistic insights into the role of FAF1 in SUMO-dependent transcriptional control and ubiquitin-mediated proteolysis. In this report, we isolated SUMO-1 and Ubc9 as FAF1-interacting proteins (Fig. 1). Our study clearly demonstrated that FAF1 interacts with SUMO-1 in a non-covalent manner. This is quite surprising because Ubc9 is the sole SUMO E2 enzyme known to covalently conjugate SUMO to target substrates. In general, SUMO conjugation (sumoylation) is mediated by the recognition of Ubc9 at the consensus sequence ΨKx(D/E) (where Ψ is a hydrophobic residue) of target proteins, allowing the Ubc9-SUMO thioester complex to catalyze formation of an isopeptide bond between SUMO and the target lysine residue. Additionally, sumoylation can also be mediated through SIMs, which can interact with SUMO of the Ubc9-SUMO thioester complex, enabling it to conjugate SUMO to a target lysine. Interestingly, the catalysis-independent functions of Ubc9 were reported. For example, Ubc9 can act as a transcriptional coactivator [28] or corepressor [47], a regulator of protein shuttling [48] or protein turnover [49]. Previous studies indicated that Ubc9 serves as a coactivator for the MR by forming a complex with the N-terminal MR and SRC-1, independent of its E2 SUMO-conjugating activity and the sumoylation status of the MR [28]. Here, we demonstrated that FAF1 mediates SUMO-dependent MR transcriptional repression. It is likely that FAF1 binds to Ubc9 and sequesters it from the MR/co-activator complex, and thereby represses MR-mediated transcription. Spatiotemporal regulation of these interactions remains to be clarified. Notably, a recent report showed that Drosophila FAF1 (Caspar) is sumoylated at K551 and is associated with inflammatory responses [50]. However, we showed that FAF1 was not sumoylated in both in vitro and in vivo sumoylation assays. It is possible that the extents of SUMO modifications of different FAF1 family members are diverse and may vary in different conditions or cell types. Notably, the authors showed that numerous sumoylated proteins are co-purified along with Caspar during affinity purification, consistent with our findings that FAF1 can precipitate multiple sumoylated proteins in cells (Fig. 2G). In light of our results that FAF1 possesses SIMs, it is envisioned that FAF1 plays fundamental roles in mediating biological functions of sumoylated proteins.