Role of Calpains in Plasma Membrane Repair

钙蛋白酶在质膜修复中的作用

基本信息

批准号：
7530637
负责人：
RONALD L MELLGREN
金额：
$ 20.61万
依托单位：
UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-15 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7530637
关键词：
Actins Annexins Antibodies Biological Assay Calcium Calpain Cell membrane Cell physiology Cells Class Cleaved cell Cystatins Cytoskeleton DYSF gene Derivation procedure Desmin Duchenne muscular dystrophy Endopeptidases Enzymes Event Exposure to Extracellular Matrix Failure Family member Fibroblasts Fibronectins Fusion Protein Expression Future General Population Generations Genes Goals Head Immunofluorescence Microscopy Individual Injury Intervention Investigation Laser injury Lasers Lead Lipid Bilayers Mechanics Mediating Mediator of activation protein Membrane Methodology Modality Molecular Muscle Muscle Fibers Muscular Dystrophies Myoblasts Myocardium Myopathy Natural regeneration Needles Normal Cell Organism Peptide Hydrolases Physiologic pulse Plasma Plasma Proteins Process Protective Agents Proteins Proteolysis Protocols documentation Public Health Pulse taking RNA Interference Range Recombinants Regulation Relative (related person)Resistance Role Sarcolemma Serum Proteins Site Skeletal system Small Interfering RNA Talin Technology Testing Thinking Wound Healing alpha-Fetoproteins base calpain inhibitor cell injury cell type design inhibitor/antagonist knock-down m-calpain mutant novel protective effect repaired therapy development wound

项目摘要

DESCRIPTION (provided by applicant): Cells in healthy multicellular organisms are constantly subjected to mechanical processes that damage the cell membrane. This is especially the case for skeletal and cardiac muscle, and in many of the known muscular dystrophies sarcolemmal damage is exacerbated. The broad goal of the proposed studies is to define the molecular events that underlie repair of membrane damage, focusing on the recently discovered ability of one or more calpain proteases to facilitate membrane resealing and preserve viability in wounded cells. This discovery presents a new paradigm for intervention in membrane damage, and if the proposed studies are successful could lead to novel therapies to counteract muscle damage in muscular dystrophies. The first specific aim will focus on establishing the calpain enzyme(s) required to reseal damaged plasma membrane. By use of siRNA knockdown of calpains, we will establish which of these enzymes are required for repairing myoblasts and myotubes damaged by various established protocols, including mechanical cell injury, and exposure to laser pulses. We will focus on the ubiquitously distributed <- and m-calpains. Recently, we demonstrated that the blood plasma protein fetuin A stabilizes m-calpain and facilitates plasmalemma repair. The first part of specific aim 2 will entail investigation of fetuin A interactions with m-calpain that result in its increased ability to repair wounded plasma membrane. These studies have the potential to result in derivation of the first pharmacologic agent specifically designed to aid sarcolemma repair. In the second part of specific aim 2, we will investigate the role of talin in calpain-mediated sarcolemma repair. We have shown that talin, an important mediator of cytoskeletal and plasmalemma interactions, is cleaved by calpain immediately following scrape damage to fibroblast membranes. Talin also accumulated at injury sites of damaged muscle myotubes. By expression of non-cleavable mutant talin, talin head group, and talin knockdown with siRNA, we will explore two potential roles of talin proteolysis in membrane repair: 1) rapid dismantling of the actin cytoskeleton at the site of damage to allow regeneration of a new cortical cytoskeleton during repair; 2) generation of talin head group to directly facilitate membrane reorganization during repair. Lastly, by confocal immunofluorescence microscopy and GST-fusion protein expression, we will explore the localization of calpains, activated calpains, dysferlin, talin, and annexins during sarcolemma repair, to gain information about how these putative mediators of membrane resealing may interact. PUBLIC HEALTH RELEVANCE. These studies are relevant to general public health because membrane damage occurs in many cell types in the body, and little is known about the mechanisms involved. Moreover, there is scientific evidence that some muscular dystrophies are associated with failure to repair ongoing muscle damage.

描述（由申请人提供）：健康多细胞生物体中的细胞不断地受到损害细胞膜的机械过程。对于骨骼肌和心肌来说尤其如此，并且在许多已知的肌营养不良症中，肌膜损伤加剧。拟议研究的总体目标是确定膜损伤修复背后的分子事件，重点关注最近发现的一种或多种钙蛋白酶促进膜重新密封并保持受损细胞活力的能力。这一发现提出了干预膜损伤的新范例，如果拟议的研究成功，可能会带来新的疗法来抵消肌营养不良症中的肌肉损伤。第一个具体目标将集中于建立重新密封受损质膜所需的钙蛋白酶。通过使用 siRNA 敲低钙蛋白酶，我们将确定修复因各种既定方案（包括机械细胞损伤和暴露于激光脉冲）而受损的成肌细胞和肌管需要哪些酶。我们将重点关注普遍分布的<-和m-钙蛋白酶。最近，我们证明血浆蛋白胎球蛋白 A 可以稳定 m-钙蛋白酶并促进质膜修复。具体目标 2 的第一部分将需要研究胎球蛋白 A 与 m-钙蛋白酶的相互作用，从而增强其修复受损质膜的能力。这些研究有可能衍生出第一种专门用于帮助肌膜修复的药物。在具体目标2的第二部分中，我们将研究talin在钙蛋白酶介导的肌膜修复中的作用。我们已经证明，talin 是细胞骨架和质膜相互作用的重要介质，在成纤维细胞膜刮擦损伤后立即被钙蛋白酶裂解。 Talin 还在受损肌肉肌管的损伤部位积聚。通过表达不可切割的突变体talin、talin头基和用siRNA敲低talin，我们将探索talin蛋白水解在膜修复中的两个潜在作用：1）快速拆除损伤部位的肌动蛋白细胞骨架，以允许肌动蛋白细胞骨架再生。修复期间新的皮质细胞骨架； 2) 生成talin头基团，直接促进修复过程中的膜重组。最后，通过共聚焦免疫荧光显微镜和 GST 融合蛋白表达，我们将探索肌膜修复过程中钙蛋白酶、活化钙蛋白酶、dysferlin、talin 和膜联蛋白的定位，以获得有关这些假定的膜重新密封介质如何相互作用的信息。公共卫生相关性。这些研究与一般公共卫生相关，因为体内许多细胞类型都会发生膜损伤，而人们对其所涉及的机制知之甚少。此外，有科学证据表明，某些肌营养不良症与无法修复持续的肌肉损伤有关。