Cellular mechanisms leading to desminopathy: Segregation, aggregation and proteostasis imbalance of desmin mutants in muscle cells and tissue

人类2q35(DES)结蛋白基因突变可导致常染色体显性、隐性和散发性蛋白聚集性肌病和心肌病。结蛋白是一种中间丝蛋白，是横纹肌细胞肌瘤外细胞骨架的重要组成部分。我们早期的工作表明，不同的结蛋白突变在结蛋白细丝的复杂组装过程中具有不同的功能后果。然而，关于人类结蛋白的发病机制，目前尚不清楚分子发病机制是否与以下三种情况之一或组合有关：突变结蛋白的功能毒性获得、野生型结蛋白执行的功能丧失和/或结蛋白相关和无关的重要细胞成分被隔离到聚集体中。为了研究疾病机制，我们产生了R349P结蛋白敲入小鼠，它含有人类最常见的结蛋白突变R350P的同源基因。通过将这些小鼠与P53基因敲除小鼠杂交，我们进一步产生了第一个永生化杂合子和纯合子R349P结蛋白敲入肌细胞系，这些细胞系可以有效地分化为肌管。利用这些小鼠和细胞模型，我们现在的目标是澄清导致观察到的野生型和突变型结蛋白分离的细胞机制，结蛋白聚集过程本身以及与横纹肌细胞和组织中相关的蛋白平衡失衡。通过生物化学和细胞生物学相结合的方法，我们将解决以下关键问题：1.哪些细胞因子介导了功能性野生型结蛋白四聚体的形成？2.突变结蛋白是否利用了相同的一组因子，还是它们被不同的因子识别？3.哪些特定因子介导了野生型和突变型结蛋白的分选？4.蛋白分解活性如何与野生型和突变型结蛋白的特定加工相耦合？除了阐明分子病理学的最初关键步骤外，我们的研究可能为新的靶向治疗策略提供基础。最后但并非最不重要的一点是，我们的研究将为描述新的结蛋白突变体在结缔组织病中的影响奠定基础并成为一个范例。

Mutations of the human desmin gene of chromosome 2q35 (DES) cause autosomal dominant, recessive and sporadic protein aggregate myopathies and cardiomyopathies. Desmin is an intermediate filament protein, which is an essential component of the extra-sarcomeric cytoskeleton in striated muscle cells. Our earlier work demonstrated that distinct desmin mutations have different functional consequences in the complex assembly process of the desmin filaments. However, with regard to human desminopathies it is still unclear if the molecular pathogenesis is related to one or a combination of the following three scenarios: a toxic gain of function of the mutated desmin, the loss of functions executed by wildtype desmin and/or the sequestration of desmin-associated and unrelated important cellular components into the aggregates. To study the disease mechanisms, we have generated R349P desmin knock-in mice, which harbor the ortholog of the most frequent human desmin mutation R350P. By crossbreeding these mice with p53 knock-out mice, we further generated the first immortalized heterozygous and homozygous R349P desmin knock-in muscle cell lines, which can be efficiently differentiated into myotubes. Exploiting these mouse and cell models, we now aim to clarify the cellular mechanisms that cause the observed segregation of wildtype from mutant desmin, the desmin aggregation process itself as well as the associated proteostasis imbalance in striated muscle cells and tissues. By a combination of biochemical and cell biological approaches, we will address the following key questions: 1. Which cellular factors mediate the formation of functional wildtype desmin tetramers? 2. Do mutated desmins utilize the same set of factors or are they recognized by different ones? 3. Which specific factors mediate the sorting of wildtype and mutant desmin? 4. How is the proteolytic activity coupled to the specific processing of wildtype and mutant desmin? Beyond the elucidation of the initial key steps of the molecular pathology our study may provide the basis for novel targeted treatment strategies. Last but not least, our study will set the stage and serve as a paradigm for the characterization of new desmin mutants with respect to their impact in desminopathies.