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

Abstract

Stromal interaction molecule−1 and −2 (STIM1/2) are endoplasmic reticulum (ER) membrane-inserted calcium (Ca 2+) sensing proteins that, together with Orai1-composed Ca 2+ channels on the plasma membrane (PM), regulate intracellular Ca 2+ levels. Recent evidence suggests that S-nitrosylation of the luminal STIM1 Cys residues inhibits store operated Ca 2+ entry (SOCE). However, the effects of thiol modifications on STIM2 during nitrosative stress and their role in regulating basal Ca 2+ levels remain unknown. Here, we demonstrate that the nitric oxide (NO) donor nitrosoglutathione (GSNO) thermodynamically stabilizes the STIM2 Ca 2+ 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 ca 2+ 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 Ca 2+ and Soce regulation. Stromal-interaction molecules (STIM)s are endoplasmic reticulum (ER) membrane-inserted calcium (Ca 2+) sensors that respond to fluctuations in luminal stored Ca 2+ levels 1,2. In Homo sapiens, two STIM homologs exist: stromal interaction molecule−1 (STIM1) and −2 (STIM2) 3. Upon ER luminal Ca 2+ store depletion, human STIM1 undergoes structural changes that promote oligomerization and translocation to ER-plasma membrane (PM) junctions 4-6. At these junctions, STIM1 interacts with Orai1 proteins, which are the pore-forming subunits of Ca 2+ release activated Ca 2+ (CRAC) channels 7-9. The direct interaction of STIM1 with Orai1 facilitates gating of the CRAC channels to induce the influx of extracellular Ca 2+ into the cytosol 10-12 , otherwise known as store operated Ca 2+ entry (SOCE) 13,14. The human STIM2 paralog similarly regulates SOCE; however, STIM2 is less efficient than STIM1 in this cellular process 15-18. Instead, STIM2 is more intimately involved in the regulation of basal Ca 2+ levels 15. Interestingly, in lower order eukaryotes such as Drosophila melanogaster and Caenorhabditis elegans, only one STIM gene product has been identified 3. The highly conserved EF-hand and sterile α-motif (EF-SAM) domains of STIMs are the core luminal protein machinery that sense ER Ca 2+ changes 17,19,20 , while the cytosolic STIM-Orai1-activating-region (SOAR) coiled-coils are the highly conserved cytosolic domains that couple to and open Orai1 channels 11,12. Compared

STIM and Orai Mediated Regulation of Calcium Signaling in Age-Related Diseases

“…Like STIM1, STIM2 has cysteine residues in its luminal domain; two of them Cys53, and Cys60 are conserved with STIM1, and one Cys15 is unique to STIM2. All three residues can be S-nitrosylated leading to a synergistic stabilization of the EF-SAM region, reduced basal cytosolic Ca 2+ and lower STIM2-mediated SOCE ( Novello et al, 2020 ). In contrast to STIM1, STIM2 constitutively clusters at the ER-PM junctions in both mobile and immobile clusters with changes in both IP 3 R function and ER Ca 2+ levels being the driving factors contributing to the increases or decreases of immobile clusters of STIM2 in ER-PM junctions ( Ahmad et al, 2022 ).…”

“…They also showed that upon the induction of oxidative stress the oxidation of Cys313 on STIM2 is modified promoting a reduction in SOCE through the prevention of STIM2 oligomerization. It has also been shown that in response to the NO donor, nitrosoglutathione, STIM2 was S-nitrosylated at cysteines 15, 53, and 60 in HEK cells which was required for the stabilization of STIM2 ( Novello et al, 2020 ). Like STIM isoforms, Orai isoforms are also subject to redox regulation.…”

Análisis transcriptómico del remodelado de la homeostasis del calcio intracelular en cáncer de colon

“…Su extremo N-terminal se sitúa en el lumen del RE, donde se localiza el sitio de unión a Ca 2+ , llamado motivo-𝛼 Esteril EF (SAM). En cuanto a su extremo C-terminal, da a la cara citosólica y presenta tres segmentos hélices arrolladas o coiled-coil (CC1-3) seguidos de una región flexible (figura 2.15) (Hooper et al (2013), Fahrner et al (2017, Novello et al (2020), Grabmayr, Romanin, and Fahrner (2021), Tiffner et al (2021), Berlansky et al (2021)). A continuación, se desarrollan en más detalle las diferentes regiones y dominios de estas proteínas, comenzando desde el extremo N-terminal (figura 2.15).…”

“…A continuación, se desarrollan en más detalle las diferentes regiones y dominios de estas proteínas, comenzando desde el extremo N-terminal (figura 2.15). Además, se explicarán las diferencias entre las dos isoformas más convencionales, STIM1 y STIM2.2, y las implicaciones funcionales que estas tienen que, probablemente, prevengan la sobrecarga de Ca 2+ mediada por STIM2.2 (Hooper et al (2013), Fahrner et al (2017, Novello et al (2020), Grabmayr, Romanin, and Fahrner (2021), Tiffner et al (2021), Berlansky et al (2021)).…”