Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Physiology and Pharmacology

Supervisor

Stathopulos, Peter B.

2nd Supervisor

Feng, Qingping

Co-Supervisor

Abstract

Stromal-interaction molecule 2 (STIM2) is an endoplasmic reticulum (ER) membrane-inserted Ca2+-sensing protein which, together with the plasma membrane Ca2+ channel Orai1, regulates basal Ca2+ homeostasis and store-operated Ca2+ entry (SOCE). Recent evidence suggests that S-nitrosylation, which is the covalent attachment of a nitric oxide (NO) moiety to a cysteine thiol, can attenuate the function of the paralog STIM1 protein. Compared to STIM1, STIM2 also functions as a basal Ca2+ homeostatic feedback regulator. Therefore, the objective of my study was to evaluate the susceptibility of STIM2 to S-nitrosylation and the effects that this modification has on basal Ca2+ homeostasis, which is uniquely controlled by STIM2, as well as SOCE, which is regulated by both STIM1 and STIM2. Recombinant wildtype and Cys-mutated human STIM2 Ca2+ sensing luminal domain proteins were treated with either excess NO donor, S-nitrosoglutathione (GSNO), to promote S-nitrosylation or dithiolthreitol (DTT), to maintain reduced sulfhydryls as controls, to determine the effect of S-nitrosylation on the biophysical properties of the domain. Fura-2-AM ratiometric fluorimetry was used to probe basal Ca2+ and SOCE in mammalian HEK293T cells transiently co-overexpressing enhanced green fluorescent protein fused to Orai1 (i.e. eGFP-Orai1) and mCherry tagged full length STIM2 (i.e. mChSTIM2) variants in the presence or absence of excess NO. My data revealed that excess NO thermally and thermodynamically stabilized the full luminal domain of STIM2 in a Cys-dependent manner. Structurally, I found that excess NO alters the structure of the highly conserved core Ca2+ sensing region of STIM2 despite the S-nitrosylation sites being far in sequence space. Moreover, my data suggests that all three modifiable Cys sulfhydryls within the variable N-terminal region of STIM2 can contribute to enhanced luminal domain stabilization in the presence of excess NO. Consistent with the observed stabilization in vitro, I found that the basal Ca2+ and SOCE regulating function of STIM2 was suppressed in the full-length molecular context when live cells were exposed to exogenous NO. Taken together, this study reveals novel molecular insights into S-nitrosylation-mediated regulation of STIM2 structure and function via NO exposure, and is a first step towards understanding the complex role that NO may play in the regulation of cellular Ca2+.

Summary for Lay Audience

Stromal-interaction molecule 2 (STIM2) is an endoplasmic reticulum (ER) membrane-inserted Ca2+-sensing protein, which, together with the plasma membrane Ca2+ channel Orai1, regulates basal Ca2+ homeostasis and store-operated Ca2+ entry (SOCE). Recent evidence suggests that S-nitrosylation, or the covalent attachment of nitric oxide (NO) to a cysteine thiol, can attenuate the function of Ca2+ sensing proteins by enhancing stability. Therefore, the objective of my study was to evaluate the susceptibility of STIM2 to S-nitrosylation and the effects this modification has on basal Ca2+ homeostasis and SOCE. To determine the effect S-nitrosylation has on STIM2 stability, recombinant wildtype and Cys-mutated STIM2 Ca2+-sensing luminal domain proteins were treated with the excess NO donor, S-nitrosoglutathione (GSNO), to promote S-nitrosylation, or dithiolthreitol (DTT), to maintain reduced sulfhydryls as controls. Fura-2-AM ratiometric fluorimetry was used to probe basal Ca2+ and SOCE in mammalian HEK293T cells transiently co-overexpressing enhanced green fluorescent protein fused to Orai1 (i.e. eGFP-Orai1) and mCherry tagged full-length STIM2 (i.e. mChSTIM2) variants in the presence or absence of excess NO. My data reveals S-nitrosylation stabilizes the STIM2 luminal region in a Cys-specific manner concomitant with marked structural changes. These structural changes are highly localized on one side of the Ca2+sensing region of STIM2. Furthermore, overnight incubation with GSNO to mammalian cells co-overexpressing wildtype mChSTIM2 and eGFP-Orai1 significantly attenuated both basal Ca2+ and SOCE. My study reveals novel molecular insights into S-nitrosylation-mediated regulation of STIM2 via NO exposure, and is a first step towards developing therapeutic agents to treat pathologies with aberrant Ca2+ homeostasis.

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