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

人类染色体2q35 （DES）特异蛋白基因突变可引起常染色体显性、隐性和散发性蛋白聚集性肌病和心肌病。Desmin是一种中间纤维蛋白，是横纹肌细胞肌外细胞骨架的重要组成部分。我们早期的工作表明，不同的特异蛋白突变在特异蛋白细丝的复杂组装过程中具有不同的功能后果。然而，对于人类desmin病，尚不清楚其分子发病机制是否与以下三种情况中的一种或组合有关：突变的desmin毒性获得功能，野生型desmin执行功能的丧失和/或desmin相关和不相关的重要细胞成分被隔离到聚集体中。为了研究这种疾病的发病机制，我们培育了R349P基因敲入小鼠，这些小鼠与最常见的人类基因突变R350P具有同源性。通过将这些小鼠与p53敲除小鼠杂交，我们进一步获得了第一个永活的杂合子和纯合子R349P敲除蛋白肌肉细胞系，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，我们现在的目标是阐明导致野生型和突变型desmin分离的细胞机制，desmin聚集过程本身以及横纹肌细胞和组织中相关的蛋白酶平衡失衡。通过生物化学和细胞生物学方法的结合，我们将解决以下关键问题：1。哪些细胞因子介导功能性野生型四聚体的形成？2. 变异的基因是利用同一组因子，还是被不同的因子识别？3. 哪些特定因素介导了野生型和突变型desmin的分选？4. 蛋白水解活性是如何与野生型和突变型desmin的特定加工相结合的？除了阐明分子病理学的初始关键步骤外，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是，我们的研究将为新的幻视蛋白突变体在幻视病中的影响的表征奠定基础，并作为一个范例。

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.