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S100A1 is a small EF-type Ca2+sensor protein that belongs to the multigenic S100 protein family. It is abundantly expressed in cardiomyocytes (CMs) and has been described as a key regulator of CM performance due to its unique ability to interact with structural contractile proteins, regulators of cardiac Ca2+cycling, and mitochondrial proteins. However, our understanding of the molecular mechanisms regulating S100A1 protein levels is limited. We used the bioinformatics tool GPS-SUMO2.0 to identify a putative SUMO interacting motif (SIM) on S100A1. Consistently, a S100A1:SUMO interaction assay showed a Ca2+-dependent interaction of S100A1 with SUMO proteins. In neonatal rat ventricular myocytes (NRVM) and COS1 cells, S100A1 protein abundance increased in the presence of overexpressed SUMO1 without affecting the S100A1 mRNA transcript. We then generated S100A1 truncation mutants, where the SIM motif was removed by truncation or in which the core residues of the SIM motif (residues 77-79) were deleted or replaced by alanine. In COS1 cells and NRVM, overexpression of these S100A1 mutants led to elevated S100A1 mutant mRNA levels but failed to produce respective protein levels. Protein expression of these mutants could be rescued from degradation by addition of the proteasome inhibitor MG-132. By using an information-driven approach to dock the three-dimensional structures of S100A1 and SUMO, we predict a novel interaction mode between the SIM in S100A1 and SUMO. This study shows an important role of SUMO:SIM-mediated protein:protein interaction in the regulation of post-translational protein stability, and provides mechanistic insights into the indispensability of the core SIM for S100A1 post-translational stability.

Background & Objectives: S100A1 protein is a 10 kDa Ca 2+ sensor & abundantly expressed in cardiomyocytes (CMs) of vertebrates. Marked reduction in S100A1 level is hallmark of diseased heart, vice versa, addition of S100A1 by gene transfer rescues diseased heart contractile dysfunction. Since our understanding of the molecular circuits that may contribute to the regulation of S100A1 protein levels are still scarce, we conducted a hybrid computational structural & experimental approach to unveil underlying molecular residues & mechanisms that may control S100A1’s protein stability. Methods & Results: We employed in silico computational prediction & molecular docking tools, respectively, to inform experimental approaches in order to characterize residues within the 94 amino acid (aa) of S100A1. A web server-based GPS-SUMO 2.0 analysis of the human S100A1 sequence unveiled a putative SUMO interacting (SIM) motif (76-VVLVA-80) within the alpha-helical C-terminus of S100A1. Restrained docking with HADDOCK predicted a molecular interaction between SUMO-1 & the SIM-lined groove of the Ca2+-bound (holo) S100A1 homodimer that presents a potential novel type of interaction mode. We then performed an in vitro S100A1-SUMO interaction assay in the presence of 1 mM Ca 2+ or 1 mM EGTA & the assay revealed calcium dependent specific S100A1-SUMO proteins interaction. Overexpression of S100A1 together with SUMO1 increases S100A1 protein abundance in CMs & COS1 cells without changing the mRNA level of S100A1. Overexpression assays in COS1 cells & CMs involving S100A1 truncation mutant lacking SIM motif (S100A1-1-74) or site directed mutagenesis deleted (DSIM) or Alanine replaced SIM of S100A1 showed that S100A1 lacking SIM motif either via truncation & deletion or Alanine substitution led to, respectively, absence of detection or massively reduced overexpression of S100A1 protein without affecting the mRNA overexpression of the mutants. The aforementioned mutants could be rescued at protein level in CMs & COS1 cells by addition of proteasome inhibitor-MG-132. Conclusion: Here we describe a yet unrecognized post-translational molecular checkpoint for S100A1’s protein stability involving a SIM-mediated interaction between S100A1 & SUMO

Gordon Holmes syndrome (GDHS) is an adult-onset neurodegenerative disorder characterized by ataxia and hypogonadotropic hypogonadism. GDHS is caused by mutations in the gene encoding the RING-between-RING (RBR)-type ubiquitin ligase RNF216, also known as TRIAD3. The molecular pathology of GDHS is not understood, although RNF216 has been reported to modify several substrates with K48-linked ubiquitin chains, thereby targeting them for proteasomal degradation. We identified RNF216 in a bioinformatical screen for putative SUMO-targeted ubiquitin ligases and confirmed that a cluster of predicted SUMO-interaction motifs (SIMs) indeed recognizes SUMO2 chains without targeting them for ubiquitination. Surprisingly, purified RNF216 turned out to be a highly active ubiquitin ligase that exclusively forms K63-linked ubiquitin chains, suggesting that the previously reported increase of K48-linked chains after RNF216 overexpression is an indirect effect. The linkage-determining region of RNF216 was mapped to a narrow window encompassing the last two Zn-fingers of the RBR triad, including a short C-terminal extension. Neither the SIMs nor a newly discovered ubiquitin-binding domain in the central portion of RNF216 contributes to chain specificity. Both missense mutations reported in GDHS patients completely abrogate the ubiquitin ligase activity. For the R660C mutation, ligase activity could be restored by using a chemical ubiquitin loading protocol that circumvents the requirement for ubiquitin-conjugating (E2) enzymes. This result suggests Arg-660 to be required for the ubiquitin transfer from the E2 to the catalytic cysteine. Our findings necessitate a re-evaluation of the previously assumed degradative role of RNF216 and rather argue for a non-degradative K63 ubiquitination, potentially acting on SUMOylated substrates.