Date Approved

11-6-2023

Embargo Period

11-6-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Molecular Cell Biology and Neuroscience

College

Rowan-Virtua School of Translational Biomedical Engineering & Sciences

Advisor

Darren Boehning, Ph.D.

Committee Member 1

Askar Akimzhanov, Ph.D.

Committee Member 2

Val Carabetta, Ph.D.

Committee Member 3

Mouyid Islam, Ph.D.

Committee Member 4

Amanda Fakira, Ph.D.

Committee Member 5

Jim Holaska, Ph.D.

Keywords

dhhc enzymes;orai1;palmitoylation;S-acylation;stim1;store-operated calcium entry

Subject(s)

Calcium

Disciplines

Chemistry | Life Sciences | Medicine and Health Sciences

Abstract

Calcium plays a pivotal role in many physiological functions in cells. Cytosolic calcium levels are finely tuned by calcium ion channels, pumps, and intracellular organelles. Store-operated calcium entry (SOCE) is when depletion of endoplasmic reticulum (ER) calcium stores activates a calcium sensor known as stromal interaction molecule 1 (STIM1). Activation of STIM1 leads to a conformational change from a compact state to an extended state. This extended state of STIM1 allows it to bind to a calcium channel in the plasma membrane (PM) known as Orai1. The binding of Orai1 and STIM1 leads to opening of Orai1 channels and calcium entry into the cell. This is known as the calcium-release activated calcium (CRAC) current. Multiple subunits of Orai1 and STIM1 coalesce together at the ER:PM junctions to form oligomers known as “puncta”. Many cellular stimuli lead to store-depletion, most of which are mediated by inositol 1,4,5 trisphosphate (IP3)-mediated calcium release thorough the IP3 receptor calcium channels in the ER. Despite being discovered over two decades ago, the precise mechanism of how these two proteins in distinct subdomains in the cells colocalize to promote calcium entry remains elusive. A predominant model for how STIM1 activates Orai1 in the current field is known as a “diffusion-trap model”, which postulates that the extended conformation of STIM1 upon store-depletion traps Orai1 in the plasma membrane by passive diffusion. However, this model has several shortcomings. Upon store-depletion, Orai1 and STIM1 show a directed movement toward membrane subdomains enriched in cholesterol and sphingolipids (also known as lipid rafts). The non-random assembly of Orai1/STIM1 puncta in the membrane suggests that additional mechanisms other than random diffusion mediate puncta formation. S-acylation is the reversible addition of a lipid moiety to cytosolic cysteine residues mediated by a set of enzymes known as palmitoyl acyltransferases (PATs). These enzymes are also known as DHHC enzymes owing to the aspartate-histidine-histidine-cysteine residues in their catalytic site. S-acylation regulates many protein functions such as stability, activity, trafficking, and recruitment to membrane subdomains. In our previous research endeavors, we found that some signaling proteins involved in T cell receptor pathway undergo S-acylation upon T cell activation. Activation of T cells is also known to induce ER store-depletion through IP3R activity. Depilated mice carry a deletion of phenylalanine residue at 233 (ΔF233) in the DHHC21 enzyme. In these mice, many components of the T cell receptor complex cannot undergo S-acylation. Based on these observations, we hypothesized that Orai1 and STIM1 proteins undergo DHHC21-mediated S-acylation to promote SOCE. Here, we show that both Orai1 and STIM1 undergo S-acylation upon ER store-depletion. Using cysteine mutant versions of Orai1 and STIM1, we also show that these proteins that cannot undergo S-acylation and have deficits in puncta formation and SOCE. Using the cells obtained from depilated mice, we show DHHC21 mediates the S-acylation of STIM1. Our data show that S-acylation of Orai1 and STIM1 regulates CRAC channel formation and SOCE.

Download

Share

COinS