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

项目摘要 线状肌病(NM)的特征是肌纤维中有电子致密棒，肌肉无力，缺乏 肌肉再生(Sanoudou等人，2006年；Wallgren-Pettersson等人，2011年)。已经有12个基因被 与NM密切相关(JungBluth等人，2018年)。尽管我们对NM有深入的了解，但 肌节细丝导致肌节无力，目前尚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相关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 NM连接的泛素连接酶复合体CUL3-KLHL41调节未折叠的感受器的活性 内质网的蛋白质反应(UPR)(Kim等人，2018年)。特别是，CUL3-KLHL41络合物强烈 调节C2C12肌管中UPR的PERK信号通路。UPR在肌肉中起着关键作用 生长/再生并与先天性肌病有关(Bohnert等人，2018年；Ebert等人，2012年； Miyake等人，2017；Zhang等人，2002)。然而，到目前为止，在NM中还没有研究过UPR失调。 因此，我们处于独特的地位，可以揭示CUL3、UPR和NM之间的新联系。的目标是 本申请旨在定义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接头分子和其他可能的 NM连接的分子通过UPR调节肌肉生长。这项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。为了客观地检验这一假设，我们将追求以下具体内容 目的：1)建立CUL3调节肌管UPR的机制；2)鉴定额外的CUL3 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- 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 方法和评估津贴水平的变化。对于第二个目标，我们将确定津贴 稳定的klhl41基因敲除斑马鱼品系体内调节失调。此外，我们还将采用RNAi方法 筛选51个肌肉特异性CUL3接头分子(Deshmukh等人，2015)用于PERK调节 C2C12肌管。前三名候选人将因斑马鱼的额外调节失调而接受评估。我们的建议 我们认为，研究是创新的，因为异常的普遍定期审议是一种潜在的 神经母细胞瘤的病理性萎缩是一种新的、未被发现的疾病。这一知识意义重大，因为虽然 肌节细丝很难恢复，UPR可接受药物干预。 因此，我们的研究将为NM的新的药理干预奠定基础。

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.