Metabolic defects promote pathogenesis in a Drosophila model of muscular dystrophy

项目总结 肌营养不良症是一组不同类型的肌病，其特征是进行性的 骨骼肌和心肌的变性。一种类型的营养不良会导致肌肉无力和 肌肉力量被称为2H型肢体肌营养不良症(LGMD2H)。LGMD2H是由突变引起的 在E3泛素连接酶蛋白TRIM32中。在细胞周期调控中发现了不同的TRIM32底物， 神经元分化、肌肉生理学和肿瘤发生。Trim32/-基因敲除小鼠的世代 是研究LGMD2H肌肉退行性变的关键进展。然而，TRIM32和TRIM32的普遍表达 在这些突变小鼠中存在的多效性表型并没有阐明TRIM32在LGMD2H发病机制中的作用。 由于这些原因，显然需要替代模型来充分理解肌肉内在的作用。 TRIM32.我们是第一个发表果蝇LGMD2H模型的研究小组。Thin(Tn)中的突变， 编码果蝇TRIM32，表现为退行性肌肉表型，运动能力缺陷。 这些特征概括了LGMD2H患者存在的组织学和活动度缺陷。TRIM32是 其特征是N-末端的环结构域和C-末端的NHL(NCL-1，HT2A，LIN-41)重复。致病 导致LGMD2H的等位基因位于NHL区域，该区域被预测为介导蛋白质相互作用。 已有报道称，NHL结构域可防止果蝇LGMD2H的肌肉退化 模特。 在这里，我们确定了果蝇NHL区域的结构，并将其与哺乳动物进行了比较 NHL域。这两种结构的叠加表明，果蝇的NHL区 TRIM32是了解非霍奇金淋巴瘤功能的可靠模型。使用蛋白质组学方法来鉴定能够 物理上与NHL结构域相互作用，我们发现TRIM32-NHL与糖酵解蛋白结合。此外，损失 果蝇的TRIM32改变了这些酶的亚细胞定位。建议的总体目标是 研究是使用我们的果蝇LGMD2H模型来确定TRIM32是如何以及为什么需要代谢的 糖酵解肌肉的维持。我们将通过完成两个具体目标来实现这一目标。首先，我们将 生物化学和遗传学表征肌肉组织中的TRIM32-糖酵解复合体。第二个目标是 评估致病基因TRIM32突变的肌肉特异性表达在体外和体内的后果 糖酵解蛋白水平、肌节定位和新陈代谢。我们的更简单的果蝇模型 卫星细胞或获得性免疫可消除与肌肉再生相关的并发症 在哺乳动物模型中推动疾病进展的免疫细胞的渗透。总体而言，完成这些 AIMS将是理解新陈代谢如何调节以维持健康肌肉组织的重要一步。

PROJECT SUMMARY Muscular dystrophies are a heterogeneous group of myopathic disorders characterized by the progressive degeneration of skeletal and cardiac muscle. One type of dystrophy that leads to muscle weakness and a loss of muscle strength is called Limb-Girdle Muscular Dystrophy type 2H (LGMD2H). LGMD2H is caused by a mutation in the E3 ubiquitin ligase protein TRIM32. Diverse TRIM32 substrates have been identified in cell cycle regulation, neuronal differentiation, muscle physiology, and tumorigenesis. The generation of a Trim32-/- knockout mouse was a key advance in studying LGMD2H muscle degeneration. However, the ubiquitous expression of TRIM32 and pleiotropic phenotypes present in these mutant mice did not clarify the role of TRIM32 in LGMD2H pathogenesis. For these reasons, it is clear that alternative models are needed to fully understand the muscle-intrinsic role of TRIM32. We were the first group to publish a Drosophila model for LGMD2H. Mutations in thin (tn), which encodes for Drosophila TRIM32, exhibit a degenerative muscle phenotype and are defective in locomotor ability. These features recapitulate the histological and mobility defects present in LGMD2H patients. TRIM32 is characterized by an N-terminal RING domain and C-terminal NHL (Ncl-1, HT2A, Lin-41) repeats. Pathogenic alleles that cause LGMD2H are located within the NHL region, which is predicted to mediate protein interactions. It has already been reported that the NHL domain prevents muscle degeneration in our Drosophila LGMD2H model. Herein we determined the structure of the Drosophila NHL region and compared this to the mammalian NHL domain. The superimposition of these two structures demonstrate that the NHL region of Drosophila TRIM32 is a faithful model to understand NHL function. Using a proteomics approach to identify proteins that physically interact with the NHL domain, we find that TRIM32-NHL binds to glycolytic proteins. Moreover, loss of Drosophila TRIM32 alters the subcellular localization of these enzymes. The overall objective of the proposed research is to use our Drosophila LGMD2H model to determine how and why TRIM32 is required for the metabolic maintenance of glycolytic muscles. We will pursue this goal by completing two specific aims. First, we will biochemically and genetically characterize the TRIM32-glycolytic complex in muscle tissue. The second aim will assess the in vitro and in vivo consequences of muscle-specific expression of pathogenic TRIM32 mutations on glycolytic protein levels, sarcomeric localization, and metabolism. Our simpler Drosophila model that is devoid of satellite cells or adaptive immunity eliminates complications associated with muscle regeneration and the infiltration of immune cells that drive disease progression in mammalian models. Collectively, completion of these aims will be a major step forward in understanding how metabolism is regulated to maintain healthy muscle tissue.