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：
9979481
负责人：
Jinoh Kim
金额：
$ 16.38万
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 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9979481
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.

项目摘要 Nemaline肌病（NM）的标志是肌纤维中的电子致密杆，肌肉无力和缺乏肌肉再生（Sanoudou等，2006； Wallgren-Pettersson等，2011）。十二个基因已经与NM密切相关（Jungbluth等，2018）。尽管我们对NM有深入的了解，肉瘤细丝会导致肌肉无力，目前尚不清楚这些结构缺陷如何触发肌肉萎缩和肌肉再生。因此，迫切需要确定 NM连接分子会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）在人类发展中的作用。我们最近发现了一本小说 CUL3-KLHL41的活性是NM连接的泛素连接酶复合物，它调节了展开的传感器 ER的蛋白质反应（UPR）（Kim等，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在肌肉中起关键作用生长/再生并与先天性肌病有关（Bohnert等，2018； Ebert等，2012； Miyake等人，2017年； Zhang等，2002）。但是，到现在为止，NM尚未在NM中检查UPR失调。因此，我们处于独特的位置，可以揭示Cul3，UPR和NM之间的新联系。目的该应用是为了定义CUL3-KLHL41和其他Cul3适配器分子如何（即KLHL40，KBTBD13，等等）调节肌肉中的UPR。我们的中心假设是Cul3适配器分子以及其他可能 NM连接的分子通过UPR调节肌肉生长。拟议研究基础的基本原理是，一旦我们实现了目标，我们将能够为发病机理，诊断和 NM的新治疗方法。为了客观地检验该假设，我们将追求以下特定的特定目的：1）建立CUL3调节Myotub中UPR的机制； 2）确定其他CUL3 突变引起斑马鱼的肌病的衔接基因。在第一个目标下，我们将确定肌肉 - 调节C2C12肌管中UPR的CUL3-KLHL41的特定底物。我们将采用验证的RNAi 方法论并评估PERK水平的变化。为了第二个目标，我们将确定振作稳定的KLHL41敲除斑马鱼线的体内失调。此外，我们将采用RNAi方法屏幕51肌肉特异性推定的Cul3适配器分子（Deshmukh等，2015）用于PERK调节 C2C12肌管。将评估斑马鱼中的PERK失调的前三名候选人。我们提出的在我们看来，研究具有创新性，因为异常UPR是一种基本机制的观念 NM中的病理萎缩是新的且未探索的。这些知识很重要，因为尽管肌膜的细丝很难恢复，UPR适合药理干预措施。因此，我们的研究将为NM的新药理干预奠定基础。