Identification of novel and disease-related VCP/p97 binding partners and effects of disease-causing VCP/p97 mutations on protein interactions and protein quality control systems in Dictyostelium and mouse

人类VCP/p97基因突变导致常染色体显性“包涵体肌病伴早发性骨Paget病和额颞叶痴呆”（IBMPFD）和“肌萎缩性侧索硬化症”（ALS）变体。在第一个资助期，我们生成并鉴定了一系列VCP/p97突变盘基钢菌菌株，鉴定了VCP/p97单倍不足小鼠模型，鉴定并鉴定了新的和与疾病相关的VCP/p97结合伙伴。在第二个资助期的工作计划中，我们将扩展我们对VCP/p97的研究，特别强调IBMPFD的分子基础，采用广泛的实验程序，从生物化学和分子生物学实验，使用哺乳动物和盘基ostelium细胞的细胞生物学分析到小鼠模型。我们将进一步寻找新的VCP/p97结合伙伴，探索VCP/p97蛋白复合物内部的分子相互作用，并分析自噬组分的表达变化。我们将阐明R155C突变体VCP/p97在盘状骨菌菌株中对蛋白酶体活性、自噬通量和蛋白质聚集体组成的功能影响。我们将在新生成的R155C VCP/p97敲入小鼠模型中进行相应的实验和骨骼肌病理的详细表征。

Mutations of the human VCP/p97 gene cause autosomal dominant "Inclusion Body Myopathy with early-onset Paget disease of bone and Frontotemporal Dementia" (IBMPFD) and an "amyotrophic lateral sclerosis" (ALS) variant. In the first funding period, we generated and characterized a series of VCP/p97 mutant Dictyostelium strains, characterized a VCP/p97 haploinsufficient mouse model and identified and characterized novel and disease-relevant VCP/p97 binding partners. In the proposed work program for the second funding period, we will extend our studies on VCP/p97 with particular emphasis on the molecular basis of IBMPFD, employing a broad spectrum of experimental procedures ranging from biochemical and molecular biology experiments, cell biological assays using mammalian and Dictyostelium cells to mouse models. We will identify further novel VCP/p97 binding partners, explore the molecular interactions within VCP/p97 protein complexes and analyze expression changes in autophagy components. We will elucidate functional consequences of R155C mutant VCP/p97 in Dictyostelium strains with respect to proteasomal activity, autophagy flux, and the composition of protein aggregates. Corresponding experiments as well as a detailed characterization of the skeletal muscle pathology will be carried out in our newly generated R155C VCP/p97 knock-in mouse model.