P5377A SUMO1-SIM motif within the C-terminal alpha-helical domain of S100A1 prevents its proteasomal degradation in cardiomyocytes
Authors: Jebessa Z H , Zhao J , Schneider A , Busch M , Katus H A , Most P 
Published: October 2019

S100A1, a EF-hand calcium (Ca2+) sensor protein, predominantly expressed in cardiomyocytes, improves contractile performance and energy metabolism targeting activity of downstream key factors such as SERCA2a and F1ATPase. Vice versa, decreased cardiac S100A1 expression in heart failure thereby accelerates the progression to contractile and energetic failure. Although previous studies showed that transcriptional inhibition may contribute to the loss of S100A1 expression, little is known with respect to its post-translational stability in cardiomyocytes. As such, advanced understanding of the molecular mechanisms that may regulate S100A1 degradation may be of interest to target its stability in the failing heart.

In search of protein stabilizing motifs, we identified a Small Ubiquitin-related Modifier1 (SUMO1) interaction motif (SIM) in the S100A1 C-terminal domain extending from AA 69–87 as well as multiple ubiquitylatable Lysin-residues within the N-terminal domain by an in silico structural analysis. Given the role of the C-terminal domain of S100A1 in accommodating target binding and activity modulation, we hypothesized that these residues may convey a dual function and also contribute to S100A1 protein stability. To answer this question, we generated different Myc-tagged and non-tagged C-terminal deletion mutants (Myc-S100a1ΔCTs/S100a1ΔCTs) within the SIM motif encompassing AA 1–74 and AA 1–83 besides the full length AA 1–94 (S100A1 -FL). Constructs packaged into bicistronic recombinant Adenovirus Serotype 5 also contained GFP to control for sufficient transduction. Ad5-mediated gene delivery of S100a1-Fl to cell culture resulted in robust S100A1-FL protein expression. In contrast, delivery of the truncated S100a1 mutants did not yield detectable S100A1ΔCT protein variants, whereas the presence of mRNA from all S100a1ΔCT variants by PCR excluded transcriptional inhibition or blockade as a potential cause for the failure to detect S100A1ΔCT protein variants. When a proteasomal inhibitor (MG-132) was then used, S100A1ΔCT variants were detected at protein level in tested models indicating that proteasomal degradation may target SIM deleted S100A1 ΔCT variants. This notion was corroborated by unchanged expression levels of FL-S100A1 as well as deletion C-terminal mutants containing the entire SIM motif. Our novel results indicate that the C-terminal domain of S100A1 may contain a SIM motif which shields S100A1 protein against proteasomal degradation. SUMOylation analysis as well as site-directed mutagenesis of the motif is subsequently required to determine conditions where S100A1 SUMOylation may be decreased (i.e. heart failure) and which specific AA residues may convey this action. Overall, the S100A1 C-terminus may play a role in target recognition as well as in the regulation of S100A1's protein stability. In vivo studies will be required to provide the ultimate proof for this novel hypothesis.

Acknowledgement/Funding

DZHK