Biochemical dissection of amyloid precursor protein transport from the Golgi

 描述(由申请人提供)：这是为加州大学伯克利分校分子和细胞生物学系博士后副教授Daniel Sirkis博士提供的F32奖项的建议。Sirkis博士是一名细胞生物学家，主要研究与神经退行性疾病有关的蛋白质在细胞内的运输。F32将为Sirkis博士提供实现以下目标所需的支持：(I)获得囊泡运输事件的生物化学重建方面的经验；(Ii)学习最先进的基因组编辑技术，如CRISPR/Cas9介导的突变；(Iii)利用这些技术澄清淀粉样前体蛋白(APP)的细胞内分选与其蛋白分解产物--淀粉样蛋白(A？)的产生之间的关系，这两者都与阿尔茨海默病(AD)有关；以及(Iv)在科学交流和指导方面获得更多经验，这将是顺利过渡到独立研究事业所必需的。西尔基斯博士选择在兰迪·谢克曼博士的赞助下进行博士后研究。谢克曼博士是一名细胞生物学家，在囊泡运输和生化重建方面拥有深厚的专业知识。拟议的研究项目重点是跨高尔基网络(TGN)中APP的细胞内分选。由于TGN被认为是Aü世代的一个主要部位，这项工作有可能揭示AD的致病过程。在目标1中，西尔基斯博士将确定被发现影响阿贝生产的小GTP结合蛋白是否参与了TGN的APP交易。在目标2中，Sirkis博士将确定跨膜蛋白Sorla是否通过促进TGN或内体的APP运输而影响Aü的产生。在目标3中，Sirkis博士将确定新的AD风险因子磷脂酶D3是否通过内质网或TGN影响Aü的产生。该项目的主要目标是(I)表征新的高尔基体定位的GTP结合蛋白如何调控APP在TGN的运输；以及(Ii)阐明两个晚发性AD的遗传风险因素，这两个因素都是膜蛋白，如何影响APP的运输。如果成功，拟议的研究有望增加我们对APP分类和Aü生成的理解，并进一步阐明支撑AD的生物学过程。重要的是，Sirkis博士的F32训练将为他的独立研究生涯做好准备，该研究重点是涉及神经退化的蛋白质在细胞内的运输。

 DESCRIPTION (provided by applicant): This is a proposal for an F32 award for Dr. Daniel Sirkis, a postdoctoral associate in the Department of Molecular and Cell Biology at the University of California, Berkeley. Dr. Sirkis is a cell biologist focusing on the intracellular trafficking of proteins implicated in neurodegenerative disease. This F32 will provide Dr. Sirkis with the support required to accomplish the following goals: (i) to gain experience in the biochemical reconstitution of vesicle trafficking events; (ii) to learn state-of-the-art genome-editing techniques, such as CRISPR/Cas9-mediated mutagenesis; (iii) to use these techniques to clarify the relationship between the intracellular sorting of the amyloid precursor protein (APP and the generation of its proteolytic cleavage product, amyloid-ß (Aß), both of which have been implicated in Alzheimer's disease (AD); and (iv) to gain additional experience in scientific communication and mentoring that will be required for a smooth transition to an independent research career. Dr. Sirkis has chosen to carry out his postdoctoral studies under the sponsorship of Dr. Randy Schekman, a cell biologist with deep expertise in vesicle trafficking and biochemical reconstitution. The proposed research project focuses on the intracellular sorting of APP at the trans-Golgi network (TGN). Because the TGN has been implicated as a major site of Aß generation, this work has the potential to shed light on pathogenic processes involved in AD. In Aim 1, Dr. Sirkis will determine whether small GTP- binding proteins found to influence Aß production are involved in APP trafficking at the TGN. In Aim 2, Dr. Sirkis will determine whether the transmembrane protein SorLA, a known risk factor for late-onset AD, influences Aß production by facilitating APP transport from either the TGN or endosomes. In Aim 3, Dr. Sirkis will determine whether novel AD risk factor phospholipase D3 influences Aß production via the endoplasmic reticulum or the TGN. The overarching goals of this project are (i) to characterize how novel, Golgi-localized, GTP-binding proteins regulate the transport of APP at the TGN; and (ii) clarify how two genetic risk factors for late-onset AD, both of which are membrane proteins, affect the trafficking of APP. If successful, the proposed studies are expected to increase our understanding of APP sorting and Aß generation, and by extension, elucidate biological processes underpinning AD. Importantly, Dr. Sirkis' F32 training will prepare him for an independent research career that focuses on the intracellular trafficking of proteins involved in neurodegeneration.