Characterization of the Exercise-induced Orai1 Proteome in Skeletal Muscle

运动诱导的骨骼肌 Orai1 蛋白质组的表征

• 批准号: 10604393
• 负责人: Robert T Dirksen
• 金额: $ 20.33万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-06 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10604393
• 关键词: Acute; Adult; Aging; Antibodies; Binding Proteins; Biology; Biotinylation; Buffers; C-terminal; Calsequestrin; Coupling; Data; Development; Epitopes; Exercise; Fatigue; Fiber; Functional disorder; Genes; Human; Incidence; Knock-in Mouse; Knockout Mice; Left; Mediating; Membrane; Molecular; Motor; Mus; Muscle; Muscle Contraction; Muscle Development; Muscle Fatigue; Muscle Fibers; Muscle Weakness; Muscle function; Muscular Dystrophies; Mutation; Myopathy; N-terminal; Outcome; Pathway interactions; Permeability; Play; Production; Protein Isoforms; Proteins; Proteome; Proteomics; Research; Resistance; Rest; Rewards; Role; Running; Sarcomeres; Sarcoplasmic Reticulum; Skeletal Muscle; Small Interfering RNA; Specific qualifier value; Spleen; Subcellular structure; Surface; Testing; Tubular Aggregate Myopathies; Tubular formation; Vesicle; Wild Type Mouse; endurance exercise; extracellular; frontier; gain of function mutation; glycosylation; high reward; high risk; induced pluripotent stem cell; insight; knock-down; mouse model; novel; recruit; sarcopenia; sedentary; sensor; skeletal; tool

Store-operated Ca2+ entry (SOCE) in skeletal muscle is mediated by coupling between Stim1 Ca2+ sensors in the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the transverse tubule (TT). SOCE activity is also modulated by calsequestrin-1 (Casq1), the primary SR Ca2+ buffer in muscle. SOCE enhances muscle development, limits fatigue, and promotes fatigue-resistant type I fiber specification. On the other hand, SOCE dysfunction contributes to muscle weakness in aging, exacerbates muscular dystrophy, and mutations in Stim1, Orai1, and Casq1 all result in a myopathy characterized by the presence of tubular aggregates. Thus, Orai1-dependent SOCE activity plays a critical role in both normal muscle function and disease. We recently found that acute exercise drives the formation of new SR-TT junctions (Ca2+ entry units, CEUs). CEUs promote Orai1-dependent constitutive and store-operated Ca2+ entry that enhances SR store refilling, Ca2+ release, and force production during repetitive stimulation. Interestingly, CEUs are also constitutively present in sedentary mice that lack Casq1 and wild type mice after 1 month of voluntary wheel running (VWR). For this project, we developed knock-in mice with a V5-3xHA epitope tag on the extreme Orai1 C-terminus (Orai1V5HA/+ mice). Using these mice, we identified two distinct Orai1 isoforms (short and long) in skeletal muscle (both being heavily glycosylated), but only one isoform in spleen. Our preliminary data demonstrate the feasibility of using Orai1V5HA/+ mice to identify the Orai1 interacting proteins in muscle at rest and after acute exercise. Parallel studies will be conducted using N-terminal and C-terminal Orai1-TurboBioID tandem constructs following introduction in muscle of inducible, muscle-specific Orai1 knockout mice. The proposed “high risk/high reward” studies will employ cutting-edge, non-biased proteomic approaches (HA-IP, proximity biotinylation) to identify changes in Orai1 interacting proteins (“Orai1 interactome”) in skeletal muscle under conditions that promote CEU formation and SOCE activity (acute exercise, VWR, and Casq1 deficiency). We will use these discoveries, research tools, and cutting-edge approaches to determine the molecular mechanisms that coordinate Orai1-dependent CEU formation, stabilization and disassembly. We hypothesize that: 1) acute exercise drives Orai1 protein interactions that trigger a macromolecular motor to drive dynamic and reversible CEU formation and 2) challenges to SR Ca2+ store content (prolonged VWR, Casq1 deficiency) promote Orai1 protein interactions that stabilize CEUs. Aim 1 will interrogate the molecular mechanism that coordinates dynamic, exercise-dependent CEU formation by characterizing the Orai1 interactome in muscle at rest and after acute exercise. Aim 2 will characterize the Orai1 interactome in muscle under conditions that favor the stable formation of CEUs (long-term VWR and Casq1 deficiency). These studies will delineate the molecular and cellular mechanisms that coordinate CEU formation/stabilization and SOCE function in skeletal muscle.

骨骼肌中存储操纵的 Ca2+ 进入 (SOCE) 由 Stim1 Ca2+ 传感器之间的耦合介导 横管 (SOCE) 中的肌浆网 (SR) 和 Ca2+ 通透性 Orai1 通道。 活性还受到肌肉中主要 SR Ca2+ 缓冲剂 calsequestrin-1 (Casq1) 的调节。 另一方面，促进肌肉发育、限制疲劳并促进抗疲劳的 I 型纤维规格。 SOCE 功能障碍会导致衰老时的肌肉无力，加剧肌营养不良和突变 Stim1、Orai1 和 Casq1 均会导致以存在管状聚集体为特征的肌病。 Orai1 依赖性 SOCE 活性在正常肌肉功能和疾病中都起着至关重要的作用。 我们最近发现，剧烈运动会促进新的 SR-TT 连接（Ca2+ 输入单位，CEU）的形成。 CEU 促进 Orai1 依赖的组成型和钙池操作的 Ca2+ 进入，从而增强 SR 池的再填充， 重复刺激过程中 Ca2+ 的释放和力的产生。 缺乏 Casq1 的久坐小鼠和野生型小鼠在自愿轮跑 (VWR) 1 个月后存在。 在这个项目中，我们开发了在 Orai1 C 末端带有 V5-3xHA 表位标签的敲入小鼠 （Orai1V5HA/+ 小鼠），我们在骨骼中发现了两种不同的 Orai1 亚型（短和长）。 肌肉（两者都被高度糖基化），但在脾脏中只有一种同种型，我们的初步数据证明了这一点。 使用 Orai1V5HA/+ 小鼠鉴定静息时和急性发作后肌肉中的 Orai1 相互作用蛋白的可行性 将使用 N 端和 C 端 Orai1-TurboBioID 串联进行平行研究。 在引入可诱导的肌肉特异性 Orai1 敲除小鼠的肌肉后构建。 “高风险/高回报”研究将采用尖端、无偏见的蛋白质组学方法（HA-IP、邻近 生物素化）来识别骨骼肌中 Orai1 相互作用蛋白（“Orai1 相互作用组”）的变化 促进 CEU 形成和 SOCE 活动的条件（急性运动、VWR 和 Casq1 缺乏）。 我们将利用这些发现、研究工具和尖端方法来确定分子 协调 Orai1 依赖的 CEU 形成、稳定和分解的机制。 1）急性运动驱动Orai1蛋白相互作用，触发大分子马达驱动动态 和可逆的 CEU 形成以及 2) 对 SR Ca2+ 储存内容的挑战（VWR 延长、Casq1 缺乏） 促进稳定 CEU 的 Orai1 蛋白相互作用，目的 1 将探究其分子机制。 通过表征肌肉中的 Orai1 相互作用组来协调动态、运动依赖性 CEU 形成 目标 2 将在有利于休息和剧烈运动的条件下表征肌肉中的 Orai1 相互作用组。 CEU 的稳定形成（长期 VWR 和 Casq1 缺乏）这些研究将描述分子机制。 以及协调骨骼肌中 CEU 形成/稳定和 SOCE 功能的细胞机制。

项目成果

Voluntary wheel running mitigates disease in an Orai1 gain-of-function mouse model of tubular aggregate myopathy.

自愿跑轮可减轻 Orai1 功能获得性小鼠管状聚集性肌病模型的疾病。

• 发表时间: 2023-09-29
• 作者: O'Connor, Thomas N;Zhao, Nan;Orciuoli, Haley M;Brasile, Alice;Pietrangelo, Laura;He, Miao;Groom, Linda;Leigh, Jennifer;Mahamed, Zahra;Liang, Chen;Malik, Sundeep;Protasi, Feliciano;Dirksen, Robert T
• 通讯作者: Dirksen, Robert T

Constitutive assembly of Ca2+ entry units in soleus muscle from calsequestrin knockout mice.

来自 calsequestrin 敲除小鼠的比目鱼肌中 Ca2 进入单元的组成性组装。

• 发表时间: 2022-12-05
• 作者: Michelucci, Antonio;Pietrangelo, Laura;Rastelli, Giorgia;Protasi, Feliciano;Dirksen, Robert T;Boncompagni, Simona
• 通讯作者: Boncompagni, Simona