CHARACTERIZATION OF THE TRAFFICKING OF THE STORE-OPERATED CA2+ ENTRY (SOCE) CHANNEL ORAI1 USING QUANTITATIVE PROTEOMICS

Abstract

Calcium (Ca2+) is a remarkable signaling ion which plays an essential role in various biological processes including birth, development, cell proliferation and cell death. Store-operated Ca2+ entry (SOCE), a pervasive Ca2+ influx pathway in cells, mediates Ca2+ influx into cells to replete Ca2+ stores and specifically activate downstream effectors, which results in a range of cellular responses including gene expression, immune cells activation, cytokine secretion and muscle development. The foremost molecular constituents of SOCE are STIM1 and ORAI1. STIM1 acts as a sensor at the ER membrane that senses stored Ca2+ levels within the ER lumen, and couples with ORAI1 at the plasma membrane (PM). ORAI1 then acts as highly selective Ca2+ channel and recycles itself at the PM, with ~40% of the total ORAI1 (T-ORAI1) pool existing at the PM in a steady state. Despite the critical role of ORAI1, the molecular machinery controlling ORAI1 trafficking remains unclear. In this study, we attempt to identify proteins that regulate Ca2+ signaling over modulation of ORAI1 trafficking using a directed quantitative proteomics approach. We first immuno-precipitate different ORAI1 pooled from cell lysate of CHO cells that express YFP-HA-ORAI1, then label the resulting peptides using dimethyl labeling for quantitative analysis using Mass Spectrometry. By comparing the protein complexes that comprise surface ORAI1(S-ORAI1) and T-ORAI1, we identified two candidates, AJUBA and DPYSL5, that may play an imperative role in ORAI1 regulation. We focused on AJUBA protein and performed knockdown experiments using siRNA, followed by YFP-HA-ORAI1 transfection and immunostaining, to study its effect on ORAI1 expression. Results show that AJUBA plays a major role in ORAI1 trafficking indicating potential signaling function downstream of ORAI1.

Date of Award	2019
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences