Alternative assembly mechanisms of desmin disease mutants: filaments in competition with super-aggregation structures

中间丝（if）是建立动物细胞功能结构的三种丝系统之一。在肌肉中，聚乳酸蛋白构建了一个承受压力的if系统，该系统包裹着单个肌纤维，并连接到它们的中心结构锚定元素，如质膜上的Z带和costameres。人类去精蛋白基因的突变通常转化为单个氨基酸的改变，并引起严重的肌病，包括心肌病。在细胞水平上，这种疾病通常表现为大量的聚乳酸和肌细胞的肌原纤维器官的损伤。在体外组装方法中使用一组生化和生物物理技术，我们之前已经表明，根据组装和网络形成的阶段，desmin突变可以分为四组，突变会中断有序的组装过程。前三个阶段描述了(i)基本棒状四聚体蛋白复合物横向结合成单位长度细丝（ULFs）， （ii） ULFs连续纵向退火成细丝，以及（iii）随着IF的延伸而发生的径向重组（“压实”）；在第四阶段，细丝组织成功能网络。尽管第一阶段在不到一秒的时间内完成，正如停止流动实验所确定的那样，第二阶段的延伸持续了几分钟，然后被中频连续延伸的第三阶段所取代，在较短的时间内发生压实。在这里，我们建议研究四个选择的desmin突变体，它们在四个阶段中的一个阶段从正常的组装途径中衰变，以便深入了解导致本质上“有害”结构形成的分子过程。在一个专门的体外组装工作时间表中，我们将对重组蛋白进行最先进的生物物理研究，包括电子显微镜、停流实验、微流体、x射线散射和荧光相关光谱。作为补充，我们将在稳定转染的细胞系和R350P敲入小鼠建立的细胞系中研究携带各自突变蛋白的细胞，以研究新发现的if反应性小分子和药物如何随着时间的推移影响desmin聚集。此外，我们将通过免疫荧光显微镜跟踪主要细胞骨架因子的细胞重组。此外，我们将通过扫描小角x射线散射来研究聚集体形成对细胞结构组织的影响。通过这种结合的方法，我们希望深入了解哺乳动物肌肉中重要的纤维系统，包括规则组装途径的步骤顺序以及从规则组装途径偏离到聚集体形成的过程。

Intermediate filaments (IFs) represent one of three filament systems that establish the functional architecture of animal cells. In muscle, the protein desmin builds a stress-bearing IF-system that encases individual myofibers and that connects to their central structural anchorage elements such as Z bands and costameres at the plasma membrane. Mutations in the human desmin gene usually translate into single amino acid changes, and they cause severe myopathies including cardiomyopathies. At the cellular level, the disease generally manifests with massive desmin aggregates and damage of the myofibrillar apparatus of myocytes. Using a panel of biochemical and biophysical techniques in an in vitro assembly approach, we have previously shown that desmin mutations can be classified into four groups with respect to the phase of assembly and network formation, where the mutation interrupts the ordered assembly process. The first three phases describe (i) the lateral association of the basic rod-like tetrameric protein complexes into unit-length filaments (ULFs), (ii) successive longitudinal annealing of ULFs to filaments, and (iii) a radial reorganization (“compaction”) occurring as elongation of IF proceeds; (iv) in the fourth phase, filaments organize into functional networks. Whereas the first phase is complete within less than one second, as determined by stopped-flow experiments, the second phase of elongation carries on for minutes and is taken over by the third phase of continuous elongation of IF with compaction occurring in a less defined time frame. Here, we propose to study four selected desmin mutants, which decay from the normal assembly pathway in one of the four phases each, in order to get insight into the molecular process leading to the formation of essentially “noxious” structures. In a dedicated in vitro assembly work schedule, we will conduct state-of-the-art biophysical investigations of recombinant proteins, including electron microscopy, stopped-flow experiments, microfluidics, x-ray scattering and fluorescence correlation spectroscopy. In a complementing approach, we will investigate cells carrying the respective mutant proteins, both in stably transfected cell lines and in cell lines established from a R350P knock-in mouse with respect to the question how newly found IF-reactive small molecules and drugs will impact desmin aggregates over time. In addition, we will follow the cellular reorganization of major cytoskeletal factors by immunofluorescence microscopy. Moreover, we will investigate the consequences of aggregate formation for the structural organization of cells by scanning small-angle X-ray scattering. With this combined approach, we expect to get deep insight into an important filament system of mammalian muscle concerning the sequence of steps in both the regular assembly pathway and the deviation from it into aggregate formation.