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

结蛋白疾病突变体的替代组装机制：与超聚集结构竞争的细丝

• 批准号: 429958739
• 负责人: Professor Dr. Harald Herrmann
• 金额: --
• 依托单位: Institut für Röntgenphysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/429958739?language=en
• 关键词: Alternative; assembly; mechanisms; desmin; disease

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.

中间纤维（IF）代表了建立动物细胞功能结构的三种纤维系统之一。在肌肉中，蛋白结蛋白构建了一个承载应力的IF系统，该系统包裹单个肌纤维，并连接到它们的中央结构锚定元件，如质膜上的Z带和中肋。人类结蛋白基因的突变通常转化为单个氨基酸的变化，它们会导致严重的肌病，包括心肌病。在细胞水平上，该疾病通常表现为大量结蛋白聚集和肌细胞肌原纤维装置的损伤。在体外组装方法中使用一组生化和生物物理技术，我们先前已经表明，结蛋白突变可以分为四组，相对于组装和网络形成的阶段，突变中断有序的组装过程。前三个阶段描述（i）基本棒状四聚体蛋白质复合物横向缔合成单位长度细丝（ULF），（ii）ULF连续纵向退火成细丝，和（iii）随着IF的伸长进行而发生的径向重组（“压实”）;（iv）在第四阶段，细丝组织成功能网络。而第一阶段是在不到一秒内完成，如通过停流实验所确定的，伸长的第二阶段持续数分钟，并由IF的连续伸长的第三阶段接管，其中压实在较不确定的时间范围内发生。在这里，我们建议研究四个选定的结蛋白突变体，从正常的组装途径中的四个阶段之一，每个衰减，以深入了解分子过程，导致形成基本上“有害”的结构。在专门的体外组装工作计划中，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。在一个补充的方法，我们将调查携带各自的突变蛋白的细胞，无论是在稳定转染的细胞系和细胞系建立从R350 P基因敲入小鼠的问题，如何新发现的IF反应性小分子和药物将影响结蛋白聚集体随着时间的推移。此外，我们将通过免疫荧光显微镜观察主要细胞骨架因子的细胞重组。此外，我们将调查聚集体形成的后果，通过扫描小角度X射线散射的细胞的结构组织。通过这种结合的方法，我们期望深入了解哺乳动物肌肉的一个重要的细丝系统，该系统涉及常规组装途径中的步骤顺序和从其到聚集体形成的偏离。