The Regulation of Orai1 and STIM1 by Dynamic S-Acylation

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Abstract Store operated calcium entry is an essential process for proper T cell activation and development. The two proteins that facilitate this process are Orai1 and STIM1. Orai1 is localized to the plasma membrane (PM). STIM1 is localized to the endoplasmic reticulum (ER) membrane, and has a calcium sensing EF hand within the ER lumen. Upon T cell receptor activation, ER calcium stores are depleted, activating STIM1, which then undergoes an elongating conformational change, and binds to and forms complexes with Orai1 at ER-PM junctions, resulting in calcium entry into the cell. The formation of these complexes has been shown to be critical for proper T cell signaling, however, the underlying molecular mechanisms that regulate the localization of Orai1 and STIM1 to ER-PM junctions are still unknown. We have found that both Orai1 and STIM1 are S- acylated. S-acylation is the post-translational lipidation of cysteine residues by the DHHC family of protein acyltransferases, and has been found to regulate protein localization and trafficking. Therefore, this project aims to define the role of S-acylation of Orai1 and STIM1 on proper localization and calcium channel function, and determine the DHHC enzymes responsible for S-acylation. The essential role of S-acylation of Orai1/STIM1 will be evaluated using biochemical and advanced live cell imaging approaches. These complementary techniques will define the importance of S-acylation of Orai1 and STIM1 on localization to ER- PM junctions, store operated calcium entry, and downstream T cell signaling. This will be studied in two specific aims. In Aim 1, we will determine the requirement of Orai1 and STIM1 S-acylation on complex formation and store operated calcium entry. Utilizing high resolution microscopy techniques and functional assays, we will test the hypothesis that rapid and transient T cell receptor-induced S-acylation of Orai1 and STIM1 is essential for Orai1/STIM1 complex formation and downstream store operated calcium entry. In Aim 2, we will establish what enzymes mediate Orai1 and STIM1 S-acylation and evaluate their effects on downstream T cell receptor signaling and effector function. Upon identification of the enzymes responsible for Orai1/STIM1 S-acylation, we will use biochemical approaches to determine the effect of the enzymes on T cell proliferation and differentiation. As both gain-of-function and loss-of-function mutations in Orai1 and STIM1 cause diseases such as Störmorken syndrome and severe combined immunodeficiency, this research will also describe potential novel therapeutic targets.