Author ORCID Identifier
0000-0002-2106-7680
Date of Graduation
1-2023
Document Type
Dissertation (PhD)
Program Affiliation
Biochemistry and Molecular Biology
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Askar Akimzhanov, PhD
Committee Member
Darren Boehning, PhD
Committee Member
Ilya Levental, PhD
Committee Member
Michael Lorenz, PhD
Committee Member
Kartik Venkatachalam, PhD
Committee Member
Michael Zhu, PhD
Abstract
Orai1 and STIM1 proteins are the essential components of the Ca2+ release activated Ca2+ (CRAC) channel which is required for store-operated Ca2+ entry (SOCE) in T cells and subsequent signaling events leading to T cell activation, proliferation, and differentiation. Plasma membrane (PM)-localized Orai1 is the pore-forming subunit of the CRAC channel, and STIM1 is the Ca2+ sensor localized to the endoplasmic reticulum (ER) membrane in quiescent T cells. T cell receptor (TCR) stimulation leads to depletion of ER Ca2+ stores resulting in Ca2+ no longer being bound to STIM1. This activates STIM1 by triggering a lengthening conformational change which allows it to bind Orai1 at ER-PM junctions within the immunological synapse. Binding of STIM1 to Orai1 activates the CRAC channel resulting in SOCE. Although the functional importance of these events is well-known, the molecular mechanism supporting timely and efficient recruitment of both Orai1 and STIM1 to the ER-PM junctions remains poorly understood. We have found that, among several other TCR machinery proteins, both Orai1 and STIM1 are rapidly and transiently S-acylated upon TCR stimulation. Furthermore, using electrophysiology studies and Fura-2 Ca2+ imaging, we demonstrated that both Orai1 and STIM1 require S-acylation to form active CRAC channels and mediate SOCE. Lastly, our studies using total internal reflection fluorescence microscopy showed that a lack of S-acylation of either Orai1 or STIM1 resulted in significantly reduced Orai1/STIM1 colocalization as evident from the diminished PM puncta formation. These data describe a novel form of SOCE regulation in T cells that may provide new therapeutic avenues to treat diseases caused by dysfunctional CRAC channels, such as severe combined immunodeficiency and Störmorken syndrome.
Keywords
S-acylation, calcium signaling, T cells, palmitoylation, DHHC enzymes