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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

Combined hepatocellular cholangiocarcinoma (cHCC-CC) is a rare primary liver malignancy that comprises features of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). Due to the rarity of this tumor, the treatment of choice has not yet been defined. For resectable disease, liver resection is the mainstay treatment. However, most patients relapse or display advanced disease and were not surgical candidates. Although the majority of patients are either primarily or secondarily treated in palliative intent, no guideline recommendations or prospective trial reports exist to allow reliable evaluation of debated treatment options. We review different locoregional or medical treatment options for advanced combined hepatocellular cholangiocarcinoma (cHCC-CC) in the neoadjuvant, adjuvant, or palliative setting and discuss the possibility of predictive biomarker-guided therapeutic options.

Introduction: S100A1 is a type of S100 protein with two EF-hand motif. It has a unique characteristic of interacting with multiple proteins of contractile machinery & mitochondria. S100A1 protein able to govern proper systolic & diastolic performance of cardiomyocyte interacting, respectively, with SERCA2A & RYR2. S100A1 level decreases dramatically as a consequence of cellular cues such as oxidative-stress or activation of cellular proteasomes. Selective reconstitution of reduced S100A1 level by AAV9-gene transfer rescued failing heart. Because of the unique ability of S100A1 to interact with myriads of proteins with multiple function, we aimed to dissect the structure-function relationship of the domains of S100A1. Methods & results: The C-terminus domain of S100A1 (S100A1ct) equally recapitulated the inotropic & other benefits of the S100A1-FL in experimental model of heart failure & identified as the bioactive lead domain of S100A1. To elucidate the function of the S100A1 without the C-terminus, here after, referred as S100A1ΔCT, we adenoviraly expressed S100A1ΔCT, & as a control, either expressing GFP or S100A1-FL. We, inadvertently, discovered that S100A1ΔCT could not be detected at protein level. PCR analysis & sequencing proved the presence S100A1ΔCT mRNA transcript. Bioinformatics revealed the presence of SUMO interaction motif (SIM) with in the C-terminus & multiple ubiquitination residues on S100A1ΔCT, suggesting the SIM domain of the S100A1 is crucial for stability. Inhibition of cellular proteasome lead to the detection of the S100A1ΔCT protein in cells over expressing S100A1ΔCT. We overexpressed S100a1ΔCT, deletion mutants having part of the SIM motif (S100a1-SIM - ), the entire motif (S100a1-SIM + ) & the S100a1-FL. Whereas the overexpression of S100A1-SIM + & S100A1-FL were detected, S100A1ΔCT & S100A1-SIM - could not be detected at protein level. Conclusion: The C-terminus of S100A1 is essential for stability of S100A1, besides, its potent inotropic function. Taken together, we deciphered the previously unrecognized posttranslational modification governed S100A1 regulation, thus providing new insight into the development of novel therapeutic strategies for heart failure via posttranslational manipulation of S100A1.