Targeting protein s-acylation during Tubular Aggregate Myopathy

Dr. Amado Carreras Sureda, University of Geneva

Abstract (lay summary see below)

Activation of skeletal muscle contraction consists of different steps where depolarization of the plasma membrane is coupled to the release of calcium (Ca2+) ion on the sarcoplasmic reticulum (SR) leading to muscular contraction. Replenishing of the intracellular SR Ca2+ stores is critical to prevent muscular fatigue, a process accomplished by the Store Operated Ca2+ Entry (SOCE) machinery. SOCE is triggered by depletion of intracellular Ca2+ stores and sensed by stomal interacting molecules 1 and 2 (STIM), in the lumen of the SR, prompting them to oligomerize and relocate to sites of contact with the plasma membrane (PM). In the PM STIM gate the Orai1 channels flooding the cytosol with Ca2+ which is then pumped into the SR, de-oligomerising STIM, filling the SR and preventing Ca2+ overloading. Mutations in STIM and Orai proteins are causative of severe human diseases, including a rare debilitating myopathy named Tubular Aggregate Myopathy (TAM), for which no cure is currently available. TAM is caused by a variety of autosomal dominant mutations in either STIM1 or Orai1 that result in a channel gain of function (GoF), thereafter reducing SOCE activity has therapeutic value to TAM patients. We have recently identified a novel pathway to modulate SOCE by promoting s-acylation of Orai1 on Cysteine 143. This work demonstrated with cellular and genetic approaches, that Orai1 needs to be s-acylated in order to promote SOCE. However, drugs that prevent de-acylation, severely impair SOCE, suggesting that: i) s-acylation machinery plays a pivotal role for SOCE not explained solely by Orai1 s-acylation and ii) targeting s-acylation, aiming to reduce SOCE, has therapeutic value for TAM patients. Thereafter we propose to study s-acylation of the SOCE machinery during muscular physiological processes with the idea to target it in the context of TAM disease. We plan to understand what is the s-acylation status of muscular cells during differentiation and ion channel stimulation. On a second phase we will explore how ORAI1 and STIM proteins are s-acylated in order to have a full mechanistic model of SOCE. Acylation and de-acylation experiments together with Cysteine mutations will be used to stablish hierarchies on how s-acylation is molecularly targeting SOCE. Finally, we plan to explore how different drugs that target s-acylation impact SOCE cell lines bearing STIM and Orai1 TAM mutations. This will be further expanded in human and mouse primary cultures and in vivo assays using a STIM1 Knock in mouse model bearing the TAM mutation R304W. Overall, by using genetic and chemical targeting in cell lines, human primary myoblasts, and a TAM genetic mouse model we propose to study and target s-acylation in muscular cells, aiming to explore therapeutic solutions for TAM patients.

Lay summary

La contraction des muscles squelettiques induite par la libération de calcium du réticulum sarcoplasmique active un influx calcique capacitif médié par les protéines STIM1 et ORAI1. Des mutations de STIM1 et ORAI1 sont associées à une maladie rare, la myopathie à agrégats tubulaire (TAM), causée par des élévations anormales de calcium dans les muscles. Nous avons récemment identifié qu'une modification lipidique (S-acylation) augmente l’activité des canaux ORAI1. Nous souhaitons établir le rôle de ce nouveau mode de régulation dans la contraction musculaire et valider son ciblage thérapeutique pour corriger les déséquilibres calciques causant la TAM. Nous proposons d'utiliser des lignées cellulaires, des cellules primaires et un modèle murin de TAM pour étudier et cibler la S-acylation des protéines STIM et ORAI1 dans le but de développer de nouveaux outils thérapeutiques bénéfiques pour les patients souffrant de TAM.