Synthetic Reconstruction of Human Chaperone Networks in Yeast Models of Neurodegeneration

项目摘要 在老化的神经元中，关键的错误折叠蛋白聚集成聚集体是许多 神经退行性疾病。例如，TDP43的病理形式会错误定位于细胞质 在额颞叶痴呆(FTD)和其他阿尔茨海默病相关疾病中聚集 痴呆(ADRD)。被称为分子伴侣的高度复杂的酶网络对抗这些过程。 在ADRD中，尚不清楚伴侣网络如何对抗ADRD蛋白的病理形式，如 TDP43、FUS和TAU。研究伴侣网络的一个主要挑战是组合的复杂性。这个 规范的Hsp70网络由54个Hsp40、12个Hsp70和16个Hsp110基因变体组成，创造了一个景观 12,155种可能的蛋白质表达组合。Hsp40-Hsp70-Hsp110蛋白的独特组合 被认为是对复杂的人类蛋白质组中不同错误折叠的蛋白质赋予特异性。这 假说被广泛接受，但由于技术限制，它从未被直接测试过。这是NIH K99/R00 提案概述了一项计划，通过构建第一个详尽的伴侣地图来直接测试这一假说 针对ADRD相关蛋白TDP43、FUS和TAU的网络。为了实现这一目标，目标1将利用 爱德华·巴比耶里博士开发的一种新的基因技术，用于表达和研究所有12,155种可能的 人Hsp40-Hsp70-Hsp110网络在ADRD酵母模型中的组合。已确认的监护人 在目标2中，将使用人体细胞和体外试验进一步研究酵母中具有抗TDP43活性的AS。 Barbieri博士将利用人类细胞模型研究TDP43活性伴侣对细胞质TDP43的影响 在HEK-293T中聚集和评估伴侣是否恢复了天然的TDP43在mRNA剪接中的功能 细胞和神经元。利用体外生物化学，他将测量伴侣蛋白的活性，以预防和逆转 TDP43聚集。在AIM 3的R00阶段，Barbieri博士将应用伴侣网络筛查进行研究 TAU聚集和他将扩大在酵母中研究的伴侣网络，包括Hsp40和 小型HSP。最后，Barbieri博士将结合他在K99阶段学到的技能来开发一种筛查 直接在ADRD人细胞模型中联合过表达所有194个人伴侣蛋白的研究 原生环境中的蛋白质平衡网络。总之，这项提案中概述的实验将确定关键 伴侣作为ADRD的治疗靶点。Barbieri博士将在较短的时间内进行K99阶段的指导 宾夕法尼亚大学的实验室，这是一个世界级的生化实验室，拥有研究伴侣的专业知识 还有阿德勒。这是Barbieri博士获得生物化学新技能的理想培训环境。此外， Barbieri博士组建了一个咨询委员会，以提供ADRD方面的专业知识和人类 细胞分析。这些新技能将补充他目前在分子遗传学方面的专业知识，以及他概述的培训 该计划将为Barbieri博士迈向独立职位的目标提供必要的准备。

Project Summary In aging neurons, the accumulation of key misfolded proteins into aggregates is a hallmark of many neurodegenerative diseases. For example, pathological forms of TDP43 become mislocalized to the cytoplasm and accumulate in aggregates in frontotemporal dementia (FTD) and other Alzheimer Disease-related Dementias (ADRD). Highly intricate networks of enzymes called molecular chaperones combat these processes. In ADRD, it is unknown how the chaperone networks fail against pathological forms of ADRD proteins such as TDP43, FUS, and TAU. A major challenge to studying chaperone networks is the combinatorial complexity. The canonical Hsp70 network consists of 54 Hsp40, 12 Hsp70, and 16 Hsp110 gene variants, creating a landscape of 12,155 possible protein expression combinations. Unique combinations of the Hsp40-Hsp70-Hsp110 proteins are hypothesized to confer specificity for different misfolded proteins in the complex human proteome. This hypothesis is widely accepted but it has never been directly tested due to technical limitations. This NIH K99/R00 proposal outlines a plan to directly test this hypothesis by building the first exhaustive map of a chaperone network against the ADRD-associated proteins TDP43, FUS, and TAU. To achieve this goal, aim 1 will leverage a new genetic technique developed by Dr. Edward Barbieri to express and study all 12,155 possible combinations of the human Hsp40-Hsp70-Hsp110 network in yeast models of ADRD. The chaperones identified as having activity against TDP43 in yeast will be further studied in aim 2 using human cells and in vitro assays. With human cell models, Dr. Barbieri will study the effect of the TDP43-active chaperones on cytoplasmic TDP43 aggregation and assess if the chaperones restore native TDP43 function in mRNA splicing in both HEK-293T cells and neurons. Using in vitro biochemistry, he will measure chaperone activity for prevention and reversal of TDP43 aggregation. During the R00 phase in aim 3 Dr. Barbieri will apply the chaperone network screen to study the TAU aggregation and he will expand the chaperone networks studied in yeast by including Hsp40 pairs and small HSPs. Lastly, Dr. Barbieri will combine the skills he learns during the K99 phase to develop a screen for combinatorially overexpressing all 194 human chaperones directly in human cell models of ADRD to study proteostasis networks in the native context. Together, the experiments outlined in this proposal will identify key chaperones as therapeutic targets for ADRD. Dr. Barbieri will perform the K99 phase mentored in the Shorter lab at the University of Pennsylvania, a world class biochemistry lab with expertise in the study of chaperones and ADRD. This is an ideal training setting for Dr. Barbieri to acquire new skills in biochemistry. Furthermore, Dr. Barbieri assembled an advisory committee to provide expertise in ADRD and formal training in the human cell assays. The new skills will complement his current expertise in molecular genetics, and his outlined training plan will provide the necessary preparation for Dr. Barbieri to progress to his goal of an independent position.