EF-hand Calcium Research Articles

S100A1's single cysteine is an indispensable redox switch for the protection against diastolic calcium waves in cardiomyocytes.

The EF-hand calcium (Ca2+) sensor protein S100A1 combines inotropic with antiarrhythmic potency in cardiomyocytes (CMs). Oxidative posttranslational modification (ox-PTM) of S100A1's conserved, single-cysteine residue (C85) via reactive nitrogen species (i.e., S-nitrosylation or S-glutathionylation) has been proposed to modulate conformational flexibility of intrinsically disordered sequence fragments and to increase the molecule's affinity toward Ca2+. Considering the unknown biological functional consequence, we aimed to determine the impact of the C85 moiety of S100A1 as a potential redox switch. We first uncovered that S100A1 is endogenously glutathionylated in the adult heart in vivo. To prevent glutathionylation of S100A1, we generated S100A1 variants that were unresponsive to ox-PTMs. Overexpression of wild-type (WT) and C85-deficient S100A1 protein variants in isolated CM demonstrated equal inotropic potency, as shown by equally augmented Ca2+ transient amplitudes under basal conditions and β-adrenergic receptor (βAR) stimulation. However, in contrast, ox-PTM defective S100A1 variants failed to protect against arrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca2+ waves and ryanodine receptor 2 (RyR2) hypernitrosylation during βAR stimulation. Despite diastolic performance failure, C85-deficient S100A1 protein variants exerted similar Ca2+-dependent interaction with the RyR2 than WT-S100A1. Dissecting S100A1's molecular structure-function relationship, our data indicate for the first time that the conserved C85 residue potentially acts as a redox switch that is indispensable for S100A1's antiarrhythmic but not its inotropic potency in CMs. We, therefore, propose a model where C85's ox-PTM determines S100A1's ability to beneficially control diastolic but not systolic RyR2 activity.NEW & NOTEWORTHY S100A1 is an emerging candidate for future gene-therapy treatment of human chronic heart failure. We aimed to study the significance of the conserved single-cysteine 85 (C85) residue in cardiomyocytes. We show that S100A1 is endogenously glutathionylated in the heart and demonstrate that this is dispensable to increase systolic Ca2+ transients, but indispensable for mediating S100A1's protection against sarcoplasmic reticulum (SR) Ca2+ waves, which was dependent on the ryanodine receptor 2 (RyR2) nitrosylation status.

P5377A SUMO1-SIM motif within the C-terminal alpha-helical domain of S100A1 prevents its proteasomal degradation in cardiomyocytes

Abstract 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

Emerging Roles of Neuronal Ca2+ Sensor-1 in Cardiac and Neuronal Tissues: A Mini Review.

The EF-hand calcium (Ca2+)-binding protein, neuronal Ca2+ sensor-1 (NCS-1/frequenin), is predominantly expressed in neuronal tissues and plays a crucial role in neuronal functions, including synaptic transmission and plasticity. NCS-1 has diverse functional roles, as elucidated in the past 15 years, which include the regulation of phosphatidylinositol 4-kinase IIIβ (PI-4K-β) and several ion channels such as voltage-gated K+ and Ca2+ channels, the D2 dopamine receptors, and inositol 1,4,5-trisphosphate receptors (InsP3Rs). Functional analyses demonstrated that NCS-1 enhances exocytosis and neuronal survival after injury, as well as promotes learning and memory in mice. NCS-1 is also expressed in the heart including the Purkinje fibers (PFs) of the conduction system. NCS-1 interacts with KV4 K+ channels together with dipeptidyl peptidase-like protein-6 (DPP-6), and this macromolecule then composes the transient outward current in PFs and contributes to the repolarization of PF action potential, thus being responsible for idiopathic arrhythmia. Moreover, NCS-1 expression was reported to be significantly high at the immature stage and at hypertrophy in adults. That report demonstrated that NCS-1 positively regulates cardiac contraction in immature hearts by increasing intracellular Ca2+ signals through interaction with InsP3Rs. With the related signals, NCS-1 activates nuclear Ca2+ signals, which would be a mechanism underlying hormone-induced cardiac hypertrophy. Furthermore, NCS-1 contributes to stress tolerance in cardiomyocytes by activating mitochondrial detoxification pathways, with a key role in Ca2+-dependent pathways. In this review, we will discuss recent findings supporting the functional significance of NCS-1 in the brain and heart and will address possible underlying molecular mechanisms.

Abstract 582: Determining and Modeling of the Cardiac Protein-Protein Interaction Network of the Inotropic Factor S100A1 by AP-MS/MS

Background: S100A1 is an EF-hand calcium (Ca 2+ ) sensor protein with predominant expression in the mammalian heart. Although its outstanding influence on cardiac performance is well described, only few target molecules are known so far, such as the sarcoplasmic reticulum Ca 2+ ATPase (SERCA2a). Objective and Results: The discrepancy between the known interactors and the diversity of S100A1`s functions brings up the question which key targets are undetected so far and can, in addition to the known ones, explain the importance of S100A1 for the heart function. S100A1 knock-out (SKO -/- ) mouse heart extracts and lysates of isolated SKO -/- cardiomyocytes were subjected to HPLC S100A1 affinity-purification (AP) columns coupled to tandem mass spectrometry (MS) followed by network modeling of the interactome, to identify novel and to confirm previously known targets in the heart. Human recombinant S100A1 protein AP columns were used to trap EGTA- and Ca 2+ -dependent interactions. Subsequent MS and computational analyses revealed more than 90 novel cardiac interactors for S100A1 in a strict Ca 2+ -dependent manner, while previously known targets were confirmed. We showed that helix 4 of S100A1 (S100A1ct) alone can improve main parameters for the heart performance and might thereby function as a therapeutic biologic. Investigating the molecular mechanisms of S100A1`s interactions with its target molecules might help to understand this effect and unveil relevant regions for other therapeutic effects. In order to uncover the mechanisms of the various interactions, docking experiments were performed to gain an understanding, which areas of S100A1 and S100A1ct might be relevant for the biological functions on selected targets. Thus we detected a region on SERCA2a where S100A1 as well as S100A1ct might compete with the SERCA2a inhibitor phospholamban and by that may improve the SERCA2a activity. These in silico findings will be combined with experimental investigations of the mode of interaction by Cross-link MS experiments. Conclusion: Combining target screening via AP-MS/MS with computer-supported epitope mapping provides a powerful strategy to understand the molecular action of known therapies and by that helps to develop new treatments against heart failure.

A sensitive biomarker panel to distinguish pancreaticobiliary malignancies from benign disease.

238 Background: Biliary strictures present a diagnostic challenge to differentiate benign disease from hepatopancreaticobiliary (HPB) malignancies. Cytology from Endoscopic retrograde cholangiopancreatography (ERCP) or Endoscopic ultrasound with fine needle aspiration (EUS-FNA) have both been plagued by poor sensitivity and high false negative rates. In a review of over 80 adjunct molecular biology tests that have been attempted on ERCP and EUS-FNA samples, four immunohistochemistry biomarkers with sensitivities ranging 74-80% were identified; von Hippel Lindau loss of expression (VHL), over-expression of insulin-like growth factor 2 mRNA-binding Protein 3 (IMP3), and EF-hand Calcium 2+ binding S100 subfamily members A4 (S100A4) and P (S100P). We sought to determine if these results could be validated in a tumor explant model and furthermore if combining these tests into a biomarker panel could boost overall diagnostic sensitivity to 100%. Methods: Tumor tissue and normal surrounding pancreas from 27 pancreaticoduodenectomy specimens were selected by an experienced pathologist, subjected to immunohistochemistry staining with VHL, IMP3, S100A4, S100P, and the intensity and percent of cells staining was graded. Using ROC curve analysis, threshold criteria were chosen for each biomarker to differentiate between tumor and normal pancreas. Biomarkers were then evaluated as a panel for their ability to discriminate malignant from benign specimens. Results: Individual sensitivity of VHL, IMP3, S100A4, and S100P were found to be 75.0%, 79.2%, 45.8%, and 0%. When VHL, IMP3, and S100A4 were grouped into a panel, they were able to distinguish cancer from normal tissue with a sensitivity of 100% and a specificity of 96%. S100P was dispensable in our assay and only VHL, IMP3, and S100A4 staining were required to achieve 100% sensitivity. Conclusions: A panel of three biomarkers, VHL, IMP3, and S100A4, were able to distinguish pancreatic cancer from surrounding normal tissue with 100% sensitivity in our tumor explant model. Prospective studies on patient biopsy specimens are required to further validate the clinical use of this biomarker panel.

Extra EF Hand Unit (DX) Mediated Stabilization and Calcium Independency of α-Amylase

It is the common feature of α-amylases that calcium ion is required for their structural integrity and thermal stability. All amylases have at least one Ca(2+) per molecule; therefore amino acids involved in calcium binding are specific and conserved. In this study, sequence analysis revealed the presence of EF-hand-like motif in calcium-binding loop of Bacillus megaterium WHO (BMW)-amylase that was previously isolated from BMW. The EF-hand motif and its variants (EF-hand-like motif) are the most common calcium-binding motifs found in a large number of protein families. To investigate the effect of calcium ion on the thermal stability and activity of BMW-amylase, we used site-directed mutagenesis to replace histidine 58 with Asp (D), Ile (I), Tyr (Y), Phe (F), and Arg (R) at the seventh position of EF-hand-like motif. Upon the addition of an extra DX unit to the calcium-binding loop in H58D variant, thermal stability, catalytic activity, and chelating power of the enzyme improved due to higher affinity toward calcium. H58D variant demonstrated calcium independency compared to the wild type and other created mutants. Conformational changes in the presence and absence of Ca(2+) were monitored using fluorescence technique.