Modelling proteostasis in dilated cardiomyopathy - the role of BAG3 and the HSP70 system

扩张型心肌病的蛋白质稳态建模 - BAG3 和 HSP70 系统的作用

• 批准号: 402866991
• 负责人: Professorin Dr. Janine Kirstein
• 金额: --
• 依托单位: Leibniz-Institut für Alternsforschung - Fritz-Lipmann-Institut e.V. (FLI)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/402866991?language=en
• 关键词: Modelling; proteostasis; dilated; cardiomyopathy; role

Studying genetically determined, primary cardiomyopathies is an auspicious approach for establishing basic disease mechanisms and novel therapeutic options in myocardial disease. One such potential molecular target is Bcl-2-associated athanogene 3 (BAG3) that is mutated in primary dilated cardiomyopathy (DCM). BAG3 serves as nucleotide exchange factor (NEF) for heat shock protein 70 family members (HSP70s), which regulate protein folding and degradation pathways.In this proposal we aim to explore the molecular mechanism how mutation of the BAG3 BAG domain affect the biochemical function of constitutive HSPA8, perturb protein homeostasis (proteostasis) in cardiomyocytes, and finally lead to DCM. We hypothesize that DCM missense mutations within the BAG3 BAG domain specifically disturb the NEF function of BAG3. Thus, we will explore biochemically the activity of the BAG3/HSPA8 complex using in vitro chaperone assays. Specifically, we will model a novel missense mutation from a large DCM family in isogenic induced pluripotent stem cells (iPSC). Using these iPSCs we will differentiate cardiomyocytes that we further analyze for protein aggregate formation, proteostasis, autophagy, and proteasome activity. The molecular network of co-chaperones associated with HSPA8 is not well understood in cardiomyocytes. We will use proteome data from iPSC derived cardiomyocytes and in vitro chaperone assays to characterize the composition as well as activity of the cardiac specific HSPA8 complex. As we observe a considerable phenotypic variability in patients of this DCM family, we will utilize iPSCs from patients and control individuals in proteome and RNAseq expression studies to search for cardio protective factors. Lastly, we aim to characterize the BAG3 associated heart phenotype on pathophysiological and molecular level in a transgenic zebrafish model. The analysis of specific BAG3 missense mutations will help to explore the molecular mechanism how BAG3 affects proteostasis in post-mitotic cardiomyocytes and consequently induces DCM. Our long-term goal is to translate our mechanistic findings and technical toolbox into therapeutic approaches targeting DCM.

研究基因决定的原发性心肌病是建立心肌病基本发病机制和新的治疗选择的有利途径。一个这样的潜在分子靶点是Bcl-2相关的产气基因3（BAG 3），其在原发性扩张型心肌病（DCM）中突变。BAG 3作为热休克蛋白70家族成员（HSP 70 s）的核苷酸交换因子（NEF），调节蛋白质的折叠和降解途径，本研究旨在探讨BAG 3 BAG结构域突变如何影响组成型HSPA 8的生化功能，扰乱心肌细胞内蛋白质稳态（proteostasis），最终导致DCM的分子机制。我们假设BAG 3 BAG结构域内的DCM错义突变特异性地干扰BAG 3的NEF功能。因此，我们将探索生物化学的活性的BAG 3/HSPA 8复合物使用体外伴侣测定。具体来说，我们将在同基因诱导多能干细胞（iPSC）中从一个大型DCM家族中模拟一种新的错义突变。使用这些iPSC，我们将分化心肌细胞，我们进一步分析蛋白质聚集体形成，蛋白质稳态，自噬和蛋白酶体活性。在心肌细胞中，与HSPA 8相关的辅分子伴侣的分子网络还没有很好地理解。我们将使用来自iPSC衍生的心肌细胞的蛋白质组数据和体外伴侣测定来表征心脏特异性HSPA 8复合物的组成和活性。由于我们在该DCM家族的患者中观察到相当大的表型变异性，我们将在蛋白质组和RNAseq表达研究中利用来自患者和对照个体的iPSC来寻找心脏保护因子。最后，我们的目标是在转基因斑马鱼模型中在病理生理和分子水平上表征BAG 3相关的心脏表型。对BAG 3错义突变的分析将有助于探讨BAG 3影响有丝分裂后心肌细胞蛋白质稳态从而诱导DCM的分子机制。我们的长期目标是将我们的机制发现和技术工具箱转化为针对DCM的治疗方法。