Dysregulation of the unfolded protein response of the ER in nemaline myopathy

线状肌病中 ER 未折叠蛋白反应的失调

基本信息

批准号：
10249222
负责人：
Jinoh Kim
金额：
$ 18.69万
依托单位：
IOWA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10249222
关键词：
ACTA1 gene ATF6 gene Affect Atrophic BTB/POZ Domain Biology CUL3 gene Candidate Disease Gene Cell Death Cell Survival Cessation of life Collagen Complex Data Defect Diagnosis Disease Distal Muscular Dystrophies Electrons Endoplasmic Reticulum FDA approved Fibroblasts Foundations Gene Mutation Genes Glean Goals Growth Human Human Development Incidence Intervention Knock-out Knowledge Link Live Birth Methodology Microarray Analysis Mission Modeling Mus Muscle Muscle Fibers Muscle Weakness Muscular Atrophy Musculoskeletal Diseases Mutation Myopathy Natural regeneration Nemaline Myopathies Pathogenesis Pathologic Pharmaceutical Preparations Pharmacology Play Population Positioning Attribute Prevalence Prevention Procollagen Proteins Proteomics Public Health RNA Interference Regulation Research Rod Role Sarcomeres Signal Pathway Signal Transduction Structure Tertiary Protein Structure Testing Thin Filament United States National Institutes of Health Zebrafish base congenital myopathy craniofacial disease-causing mutation in vivo innovation muscle regeneration novel overexpression postnatal prenatal response satellite cell sensor skeletal disorder treatment strategy ubiquitin ligase

项目摘要

Project Summary Hallmarks of nemaline myopathy (NM) are electron dense rods in myofibers, muscle weakness, and lack of muscle regeneration (Sanoudou et al., 2006; Wallgren-Pettersson et al., 2011). Twelve genes have been closely linked to NM (Jungbluth et al., 2018). Despite our advanced understanding of NM that defects of the sarcomeric thin filament cause sarcomeric weakness, it still remains unclear how these structural flaws trigger muscle atrophy and defective muscle regeneration. There is, therefore, an urgent need to identify the mechanisms by which the NM-linked molecules influence muscle growth and survival. Our long-term goal is to understand roles of the endoplasmic reticulum (ER) in human development. We recently uncovered a novel activity of CUL3-KLHL41, a NM-linked ubiquitin ligase complex, that it regulates the sensors of the unfolded protein response (UPR) of the ER (Kim et al., 2018). In particular, the CUL3-KLHL41 complex strongly regulates the PERK signaling pathway of the UPR in C2C12 myotubes. The UPR plays a critical role in muscle growth/regeneration and has been implicated in congenital myopathies (Bohnert et al., 2018; Ebert et al., 2012; Miyake et al., 2017; Zhang et al., 2002). However, UPR dysregulation has not been examined in NM until now. Thus, we are in a unique position to reveal a new connection among CUL3, the UPR, and NM. The objective of this application is to define how CUL3-KLHL41 and other CUL3 adaptor molecules (i.e., KLHL40, KBTBD13, etc.) regulate the UPR in muscles. Our central hypothesis is that CUL3 adaptor molecules and possibly other NM-linked molecules regulate muscle growth via the UPR. The rationale that underlies the proposed research is that once we achieve the goal we will be able to provide a new concept for pathogenesis, diagnosis, and new treatment approaches for NM. To objectively test the hypothesis, we will pursue the following specific aims: 1) Establish the mechanism by which CUL3 regulates the UPR in myotubes; 2) Identify additional CUL3 adaptor genes whose mutations cause a myopathy in zebrafish. Under the first aim, we will identify muscle- specific substrates of CUL3-KLHL41 that regulates the UPR in C2C12 myotubes. We will employ proven RNAi methodology and evaluate changes in the levels of PERK. For the second aim, we will determine PERK dysregulation in vivo in stable klhl41 knockout zebrafish lines. Additionally, we will employ RNAi methodology to screen 51 muscle-specific putative CUL3 adaptor molecules (Deshmukh et al., 2015) for PERK regulation in C2C12 myotubes. Top three candidates will be evaluated for PERK dysregulation in zebrafish. Our proposed research is innovative, in our opinion, because the notion that aberrant UPR is an underlying mechanism of pathological atrophy in NM is new and unexplored. This knowledge is significant because while defects of the thin filaments of the sarcomere are difficult to restore, the UPR is amenable to pharmacological interventions. Thus, our research will lay a foundation for new pharmacological interventions of NM.

项目概要线状肌病 (NM) 的标志是肌纤维中存在电子致密杆、肌肉无力和缺乏肌肉再生（Sanoudou 等人，2006；Wallgren-Pettersson 等人，2011）。十二个基因被与 NM 密切相关（Jungbluth 等人，2018）。尽管我们对 NM 有深入的了解，但肌节细丝导致肌节无力，目前仍不清楚这些结构缺陷是如何触发的肌肉萎缩和肌肉再生缺陷。因此，迫切需要确定 NM 连接分子影响肌肉生长和存活的机制。我们的长期目标是了解内质网 (ER) 在人类发育中的作用。最近我们发现了一本小说 CUL3-KLHL41（一种 NM 连接的泛素连接酶复合物）的活性，它调节未折叠的传感器 ER 的蛋白质反应 (UPR)（Kim 等人，2018）。特别是，CUL3-KLHL41 复合物强烈调节 C2C12 肌管中 UPR 的 PERK 信号通路。 UPR在肌肉中起着至关重要的作用生长/再生，并与先天性肌病有关（Bohnert et al., 2018；Ebert et al., 2012；三宅等人，2017；张等人，2002）。然而，迄今为止，UPR 失调尚未在 NM 中得到研究。因此，我们处于一个独特的位置来揭示 CUL3、UPR 和 NM 之间的新联系。的目标该应用程序旨在定义 CUL3-KLHL41 和其他 CUL3 接头分子（即 KLHL40、KBTBD13、等）调节肌肉中的 UPR。我们的中心假设是 CUL3 接头分子和可能的其他分子 NM 连接分子通过 UPR 调节肌肉生长。拟议研究的基本原理一旦我们实现了这一目标，我们将能够为发病机制、诊断和治疗提供新的概念。 NM 的新治疗方法。为了客观地检验假设，我们将采取以下具体措施目的：1）建立CUL3调节肌管UPR的机制； 2) 识别额外的CUL3 其突变导致斑马鱼肌病的衔接基因。在第一个目标下，我们将确定肌肉- CUL3-KLHL41 的特定底物调节 C2C12 肌管中的 UPR。我们将采用经过验证的 RNAi 方法并评估 PERK 水平的变化。对于第二个目标，我们将确定PERK 稳定的 klhl41 敲除斑马鱼系体内的失调。此外，我们将采用 RNAi 方法筛选 51 个肌肉特异性推定 CUL3 接头分子（Deshmukh 等人，2015），用于 PERK 调节 C2C12 肌管。将评估前三名候选人的斑马鱼 PERK 失调情况。我们提出的我们认为，这项研究具有创新性，因为异常 UPR 是一种潜在机制的概念 NM 中的病理性萎缩是新的且未经探索的。这些知识很重要，因为虽然缺陷肌节的细丝很难恢复，UPR 适合药物干预。因此，我们的研究将为 NM 新的药理干预奠定基础。