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

项目摘要 线状体肌病（NM）的标志是肌纤维中的电子致密棒、肌无力和缺乏肌张力。 肌肉再生（Sanoudou等人，2006年; Wallen-Pettersson等人，2011年）。12个基因 与NM密切相关（Jungbluth等人，2018年）。尽管我们对NM有着先进的理解， 肌节细丝导致肌节无力，但仍不清楚这些结构缺陷如何触发 肌肉萎缩和肌肉再生缺陷。因此，迫切需要确定 NM连接的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。我们最近发现了一本小说 CUL 3-KLHL 41是一种NM连接的泛素连接酶复合物，它调节未折叠的 ER的蛋白质反应（UPR）（Kim等，2018年）。特别地，CUL 3-KLHL 41复合物强烈地 调节C2 C12肌管中UPR的PERK信号通路。普遍定期审议在肌肉方面发挥着关键作用， 生长/再生并与先天性肌病有关（博纳特等人，2018; Ebert等人，二〇一二年; Miyake等人，2017年; Zhang等人，2002年）。然而，UPR失调尚未在NM检查到现在。 因此，我们处于一个独特的位置，以揭示CUL 3，UPR和NM之间的新联系。的目标 本申请是为了定义CUL 3-KLHL 41和其它CUL 3衔接分子（即，KLHL40、KBTBD13、 等等）。调节肌肉中的UPR。我们的中心假设是，CUL 3衔接分子和可能的其他 NM连接的分子通过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、诊断和治疗提供一个新的概念。 NM的新治疗方法。为了客观地检验这个假设，我们将进行以下具体的研究。 目的：1）建立CUL 3调控肌管UPR的机制; 2）鉴定CUL 3的其他功能 突变导致斑马鱼肌病的适配基因。在第一个目标下，我们将识别肌肉- C2 C12肌管中调节UPR的CUL 3-KLHL 41的特异性底物。我们将使用经过验证的RNAi技术 方法和评估的PERK水平的变化。对于第二个目标，我们将确定PERK 在稳定的klhl 41敲除斑马鱼系中的体内失调。此外，我们将采用RNAi方法， 为了筛选51种肌肉特异性推定的CUL 3衔接子分子（Deshmukh等，2015年）， C2 C12肌管。前三名候选人将在斑马鱼中评估PERK失调。我们提出的 我们认为，研究是创新的，因为普遍定期审议异常是一种潜在的机制， NM中的病理性萎缩是新的和未探索的。这一知识是重要的，因为尽管 由于肌节的细丝难以恢复，因此UPR适合于药物干预。 因此，本研究将为NM的新的药物干预奠定基础。

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.