S-Acylation Is A Key Regulator Of Orai1/Stim1-Mediated Store-Operated Calcium Entry In T Cells

Author

Savannah J. West DiazFollow

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