Biology subjects

Stathopulos, P. B.

Publications and source records attributed to Stathopulos, P. B..

3 recordsLinked to original sources

bioRxiv2023

Defining the SPCA2C interactome identifies unique links to store-operated calcium entry

Calcium (Ca2+) is critical for normal cell function and several protein networks are required for Ca2+ signaling. In the pancreas, regulated changes in cytosolic Ca2+ allow for the exocytosis of zymogen granules and altered Ca2+ signaling underlies pancreatic pathologies. Previously, our laboratory showed a pancreas-specific isoform of secretory pathway Ca2+-ATPase 2 (SPCA2C) affects multiple pathways involved in Ca2+ homeostasis. The goal of this study was to define the SPCA2C interactome that contributes to these processes. Using proximity-dependent biotin identification, BioID, we expressed SPCA2C-BirA*HA in HEK293 cells with constitutive Orai1 expression. In silico modeling of SPCA2C showed a highly dynamic cytosolic C-terminus, revealing a putative site for interactions and was selected for BirA* fusion. 150 candidate SPCA2C interactors were identified. Gene Ontology and KEGG Pathway analyses supported localization of SPCA2C to the endoplasmic reticulum as well as function in Ca2+ signaling and suggested roles in vesicular transport. SPCA2C interactions with stromal interaction molecule 1 (STIM1), extended synaptotagmin-1 (ESYT1), and aspartate beta-hydroxylase (ASPH) were validated. Coiled-coil domain containing protein 47 (CCDC47) ranked as a high confidence SPCA2C-interactor which was confirmed through immunoprecipitation and co-localization with SPCA2C. CCDC47 interactions with SPCA2C, STIM1 and Orai1 were maintained following deletion of the CCDC47 luminal and cytosolic domains while deletion of only the coiled-coil domain altered CCDC47 localization and decreased interactions with STIM1 and Orai1. Overall, this study defines several novel protein interactions for SPCA2C and suggests it may be involved in affecting CCDC47, STIM1 and Orai1 function.

cell biology↗

bioRxiv2020

p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis.

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation and STIM1 (stromal interaction molecule-1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile -motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) interactions and enhanced SERCA pump activity leading to increased Store Operated Calcium Entry (SOCE). In the pancreatic AR42J cells expressing the p.E152K variant, Ca2+-signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation. Summary statementp.E152K-STIM1 variant found in pancreatitis patients leads to intracellular changes in calcium homeostasis through SERCA interaction, enabling intracellular trypsin activation and pancreatic acinar cell death.

cell biology↗

bioRxiv2018

S-Nitrosylation of STIM1 by neuronal nitric oxide synthase inhibits store-operated Ca2+ entry

Store-operated Ca2+ entry (SOCE) mediated by stromal interacting molecule-1 (STIM1) and Orai1 represents a major route of Ca2+ entry in mammalian cells and is initiated by STIM1 oligomerization in the endoplasmic or sarcoplasmic reticulum (ER/SR). However, the effects of nitric oxide (NO) on STIM1 function are unknown. Neuronal NO synthase (nNOS) is located in the SR of cardiomyocytes. Here, we show that STIM1 is susceptible to S-nitrosylation. nNOS deficiency or inhibition enhanced Ca2+ release-activated Ca2+ channel current (ICRAC) and SOCE in cardiomyocytes. Consistently, NO donor S-nitrosoglutathione (GSNO) inhibited STIM1 puncta formation and ICRAC in HEK293 cells, but this effect was absent in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Furthermore, NO donors caused Cys49 and Cys56-specific structural changes associated with reduced protein backbone mobility, increased thermal stability and suppressed Ca2+-depletion-dependent oligomerization of the luminal Ca2+-sensing region of STIM1. Collectively, our data show that S-nitrosylation of STIM1 suppresses oligomerization via enhanced luminal domain stability and rigidity, and inhibits SOCE in cardiomyocytes.

physiology↗