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Insights into the Regulation of Store-operated Calcium Entry

  • Author / Creator
    Prins, Daniel S
  • Calcium (Ca2+) ions serve a crucial role in numerous intracellular signaling pathways, controlling physiological functions as diverse as cell proliferation, immune system function, and muscular contraction. As such, the cell has evolved many different mechanisms to precisely control the movement of Ca2+ ions into and out of various cellular compartments. One such pathway is called store-operated Ca2+ entry (SOCE), which couples an initial depletion of endoplasmic reticulum (ER) luminal Ca2+ stores with Ca2+ influx across the plasma membrane into the cytoplasm. SOCE is controlled by two major proteins, STIM1 and Orai1. STIM1 senses ER luminal Ca2+ levels and transduces this signal across the ER membrane by migrating to subplasmalemmal punctae, where it activates Orai1, a plasma membrane Ca2+ channel that allows Ca2+ flux across the plasma membrane. SOCE is of key importance to human health; as such, a more complete understanding of how the cell controls SOCE will yield deeper insights into how the pathway might be manipulated for therapeutic purposes. The research in this thesis focuses on novel cellular mechanisms to control SOCE, specifically focusing on protein-protein interactions of STIM1. We used a variety of biochemical and cell biological techniques to discover novel protein binding partners of STIM1 and to elucidate the functional consequences of these interactions. First, we discovered that ERp57, an ER luminal oxidoreductase, binds to the luminal domain of STIM1. ERp57 binding is dependent upon two highly conserved cysteine residues within STIM1 and this binding serves to inhibit the initiation of SOCE. We hypothesize that ERp57-STIM1 interactions may act as a brake upon SOCE to prevent its overactivation. Importantly, the triggering of SOCE is known to occur within the ER lumen, and our results were the first published description of a protein-protein interaction within the ER luminal domain of STIM1. Second, we discovered that the cytoplasmic domain of STIM1 is subject to calpain cleavage. This cleavage occurs in healthy cells to regulate the abundance of STIM1: inhibition of calpain cleavage increases STIM1 levels. Additionally, STIM1 cleavage is upregulated during programmed cell death. We hypothesize that the interplay between STIM1 and calpains is a mechanism to control the cellular abundance of STIM1 and that perturbations of this mechanism may lead to dysregulation of STIM1 protein levels. Finally, we discovered that a previously described STIM1-calnexin interaction is of key importance in immune cells. Calnexin-deficient T cells exhibit severely impaired SOCE, which may be due to impaired movement of STIM1 to subplasmalemmal punctae or increased susceptibility to calpain cleavage. Calnexin's effects on SOCE seem to be specific to immune cells, suggesting that the calnexin-STIM1 interaction could be one mechanism by which the immune system strengthens SOCE, where a strong, sustained Ca2+ influx is so important. Overall, the research presented in this thesis describes three unique protein binding partners of STIM1 and explains how each interaction serves to regulate SOCE. Any alterations in these interactions may underlie dysregulated SOCE in disease; conversely, interventions targeting these protein binding partners could serve as methods to manipulate SOCE for therapeutic outcomes.

  • Subjects / Keywords
  • Graduation date
    2015-11
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3J960N2P
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Biochemistry
  • Supervisor / co-supervisor and their department(s)
    • Michalak, Marek (Biochemistry)
  • Examining committee members and their departments
    • Lytton, Jonathan (Biochemistry & Molecular Biology, University of Calgary)
    • Fliegel, Larry (Biochemistry)
    • Eitzen, Gary (Cell Biology)
    • Touret, Nicolas (Biochemistry)