Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Physiology and Pharmacology

Supervisor

Stathopulos, Peter B.

Abstract

Stromal interaction molecule 1 (STIM1) is an endo/sarcoplasmic reticulum (ER/SR) calcium (Ca2+) sensing protein that initiates cytoplasmic Ca2+ influx via store-operated calcium entry (SOCE). STIM1, in conjunction with Orai, a plasma membrane (PM) protein, function as mediators of SOCE through the formation of calcium-release activated calcium (CRAC) channels. S-Glutathionylation of STIM1 at Cys56 has been shown to evoke constitutive Ca2+ entry in DT40 cells, however no studies have carefully investigated the biophysical and structural effects of this covalent modification to the luminal domain, which are critical for understanding the molecular mechanism underlying the regulation of STIM1 and SOCE by this modification. By establishing a protocol for site-specific STIM1 S-glutathionylation using reduced glutathione (GSH) and diamide, a thiol-specific oxidant, my data have revealed that modification of STIM1, at either Cys49 or Cys56, induces thermodynamic destabilization in the Ca2+ deplete state and structural changes as measured by solvent-exposed hydrophobicity and solution NMR. Furthermore, modification at Cys56 significantly reduces Ca2+ binding affinity, as measured via intrinsic fluorescence and far-UV CD spectroscopy. This weakening in Ca2+ binding is correlated with structural perturbations localized to common regions of the core EF-SAM domain, which include the α1 helix of the canonical EF-hand, the α3 and α4 helices of the non-canonical EF-hand and α6 and α8 helices of the SAM domain. Excitingly, I also designed a novel glutathione mimic at the residue level that strongly recapitulates the S-glutathionylation structural and biophysical effects within the STIM1 luminal domain and constitutively activates SOCE when introduced into full-length STIM1. Collectively, my research has revealed novel molecular insights into S-glutathionylation-mediated STIM1 structural and functional changes, laying the groundwork for the development of SOCE modulators to be used as research tools and/or therapeutic strategies for myriad Ca2+ signaled malignancies.

Summary for Lay Audience

Store-operated calcium entry (SOCE) is a universal calcium (Ca2+) entry pathway in all eukaryotes, regulating numerous physiological processes including cell growth, proliferation, and the immune response. Upon endoplasmic reticulum (ER) Ca2+ depletion, stromal interaction molecule 1 (STIM1) initiates SOCE by binding to and activating Ca2+ channels on the plasma membrane (PM). While STIM1 Ca2+ sensing is the switch that turns SOCE ON and OFF, post-translational modifications of STIM1 can modulate this Ca2+ sensing mechanism. S-Glutathionylation of STIM1 Cys56 has been shown to evoke constitutive SOCE activation independent of ER Ca2+ store levels. Therefore, the objective of my study was to evaluate the susceptibility of STIM1 Cys to S-glutathionylation and the downstream structural mechanisms by which this modification can affect function. Using reduced glutathione (GSH) and diamide, a thiol-specific oxidant, my data elucidated the structural, biophysical and functional effects by which S-glutathionylation destabilizes the luminal STIM1 region to potentiate its function as a mediator of SOCE within mammalian cells. My work has revealed novel mechanistic insights into how STIM1 Ca2+ sensing function is mediated by S-glutathionylation, laying the groundwork for the development of SOCE modulators to be used as research tools and/or therapeutic strategies for various Ca2+ signaled diseases.

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