Klinikum Grosshadern, Department of Neurology, Ludwig-Maximilians-University of Munich, Munich, Germany.
J Neuropathol Exp Neurol. 2010 Apr;69(4):415-24. doi: 10.1097/NEN.0b013e3181d71305.
Mutations in the intermediate filament protein desmin cause a distinct class of myofibrillar myopathies that are characterized by deposition of aggregated desmin. To assess the effect of different disease-associated mutations at the molecular level, we applied confocal single-particle fluorescence spectroscopy. We studied the de novo aggregation properties of desmin in vitro and the aggregation state of desmin in homogenates of transfected cells rendering purification unnecessary. We detected divergent assembly patterns for 3 different desmin missense mutations. R350P-desmin showed a strong inhibition of assembly formation that was associated with a reduced level of tetramers and an increase in dimers in native cell extracts. E413K-desmin formed hyperstable tetramers. For R454W-desmin, there were subtle effects on assembly at the dimer and tetramer levels by single-particle spectroscopy that are not detectable by classical fluorescence microscopy. We also found that R350P-desmin efficiently interacts with the wild-type protein resulting in a dominant-negative effect on desmin assembly. Taken together, these results provide a molecular basis for a detailed functional classification of mutations in the desmin gene. The findings may also have implications for diagnostic and therapeutic strategies for primary desminopathies based on the different molecular events that disrupt physiological filament formation.
中间丝蛋白结蛋白的突变导致一类独特的肌纤维原性肌病，其特征是聚集的结蛋白沉积。为了在分子水平上评估不同疾病相关突变的影响，我们应用共焦单颗粒荧光光谱法。我们研究了结蛋白在体外的从头聚集特性以及转染细胞匀浆中的聚集状态，从而无需进行纯化。我们检测到 3 种不同结蛋白错义突变的不同组装模式。R350P-结蛋白表现出强烈的组装形成抑制作用，这与四聚体水平降低和天然细胞提取物中二聚体增加有关。E413K-结蛋白形成超稳定的四聚体。对于 R454W-结蛋白，单颗粒光谱法在二聚体和四聚体水平上对组装的细微影响在经典荧光显微镜下无法检测到。我们还发现 R350P-结蛋白有效地与野生型蛋白相互作用，从而对结蛋白组装产生显性负效应。总之，这些结果为结蛋白基因突变的详细功能分类提供了分子基础。这些发现也可能对基于破坏生理纤维形成的不同分子事件的原发性结蛋白病的诊断和治疗策略具有重要意义。
