Synergistic stabilization by nitrosoglutathione-induced thiol modifications in the stromal interaction molecule-2 luminal domain suppresses basal and store operated calcium entry

Matthew J Novello,Jinhui Zhu,Peter B Stathopulos,Mengqi Zhang,Qingping Feng

doi:10.1038/s41598-020-66961-3

Matthew J Novello, Jinhui Zhu + Show 3 more

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https://doi.org/10.1038/s41598-020-66961-3

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Journal: Scientific Reports	Publication Date: Jun 23, 2020
Citations: 4	License type: open-access

Affiliation: Western University, University of Ottawa

Abstract

Stromal interaction molecule−1 and −2 (STIM1/2) are endoplasmic reticulum (ER) membrane-inserted calcium (Ca2+) sensing proteins that, together with Orai1-composed Ca2+ channels on the plasma membrane (PM), regulate intracellular Ca2+ levels. Recent evidence suggests that S-nitrosylation of the luminal STIM1 Cys residues inhibits store operated Ca2+ entry (SOCE). However, the effects of thiol modifications on STIM2 during nitrosative stress and their role in regulating basal Ca2+ levels remain unknown. Here, we demonstrate that the nitric oxide (NO) donor nitrosoglutathione (GSNO) thermodynamically stabilizes the STIM2 Ca2+ sensing region in a Cys-specific manner. We uncovered a remarkable synergism in this stabilization involving the three luminal Cys of STIM2, which is unique to this paralog. S-Nitrosylation causes structural perturbations that converge on the face of the EF-hand and sterile α motif (EF-SAM) domain, implicated in unfolding-coupled activation. In HEK293T cells, enhanced free basal cytosolic Ca2+ and SOCE mediated by STIM2 overexpression could be attenuated by GSNO or mutation of the modifiable Cys located in the luminal domain. Collectively, we identify the Cys residues within the N-terminal region of STIM2 as modifiable targets during nitrosative stress that can profoundly and cooperatively affect basal Ca2+ and SOCE regulation.

Highlights

Stromal interaction molecule−1 and −2 (STIM1/2) are endoplasmic reticulum (ER) membrane-inserted calcium (Ca2+) sensing proteins that, together with Orai1-composed Ca2+ channels on the plasma membrane (PM), regulate intracellular Ca2+ levels
Recent evidence indicates that S-nitrosylation of the variable N-terminal region of human stromal interaction molecule−1 (STIM1) enhances the stability of the full luminal domain and inhibits SOCE27,28
The ability of this approach to efficiently promote GSNO-mediated modification of the STIM1 luminal Cys residues was previously confirmed by solution nuclear magnetic resonance (NMR) spectroscopy[28]

Summary

Introduction

Stromal interaction molecule−1 and −2 (STIM1/2) are endoplasmic reticulum (ER) membrane-inserted calcium (Ca2+) sensing proteins that, together with Orai1-composed Ca2+ channels on the plasma membrane (PM), regulate intracellular Ca2+ levels. We recently demonstrated that two Cys residues within the variable N-terminal region of STIM1 (Cys[49] and Cys56) can undergo S-nitrosylation, stabilize the luminal domain of STIM1 and inhibit SOCE27,28 While these two Cys residues are conserved between STIM vertebrate homologs (Cys[53] and Cys[60] in STIM2), STIM2 contains an additional Cys, non-existent in STIM1, within its variable N-terminal region (Cys15)[29], which may serve as an added regulatory input site after thiol modifications (Fig. 1a). We identify the Cys residues within the N-terminal region of STIM2 as modifiable targets during nitrosative stress that can profoundly affect the Ca2+ sensing function of the molecule in the regulation of basal cytosolic Ca2+ and SOCE

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