Biochemical dissection of amyloid precursor protein transport from the Golgi

 描述（由申请人提供）：这是一份为加州大学伯克利分校分子和细胞生物学系博士后研究员丹尼尔·瑟基斯博士提供的F32奖的提案。Sirkis博士是一位细胞生物学家，专注于神经退行性疾病中蛋白质的细胞内运输。该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化重建经验;（ii）学习最先进的基因组编辑技术，如CRISPR/Cas9介导的诱变;（iii）获得最先进的基因组编辑技术，如CRISPR/Cas9介导的诱变。（iii）使用这些技术来阐明淀粉样前体蛋白的细胞内分选与蛋白质的细胞内分选之间的关系。（APP及其蛋白水解裂解产物淀粉样蛋白（AAPs）的产生，这两者都与阿尔茨海默病（AD）有关;以及（iv）获得科学交流和指导方面的额外经验，这是顺利过渡到独立研究生涯所需的。Sirkis博士选择在Randy Schekman博士的赞助下进行博士后研究，Randy Schekman博士是一位细胞生物学家，在囊泡运输和生化重建方面具有深厚的专业知识。拟议的研究项目重点关注跨高尔基体网络（TGN）中APP的细胞内排序。由于TGN已被认为是产生AD的主要位点，因此这项工作有可能阐明AD中涉及的致病过程。在目标1中，Sirkis博士将确定是否发现影响AAPs产生的小GTP结合蛋白参与TGN的APP运输。在目标2中，Sirkis博士将确定跨膜蛋白SorLA（迟发性AD的已知风险因素）是否通过促进APP从TGN或内体转运来影响Ablation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ApoE的产生。本项目的主要目标是：（i）研究高尔基体定位的GTP结合蛋白如何调控APP在TGN的转运;以及（ii）阐明晚发性AD的两个遗传风险因素（均为膜蛋白）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分选和AAPs生成的理解，并通过扩展，阐明支持AD的生物过程。Sirkis博士的F32培训将为他的独立研究生涯做好准备，该研究的重点是参与神经变性的蛋白质的细胞内运输。

 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.