MOLE MECH OF THE LEGIONELLA PNEUMOPHILA EFFECTOR PROTEIN VIPF IN THE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 嗜肺军团菌是一种兼性革兰氏阴性细菌，可感染人类并导致一种被称为军团菌病的严重肺炎。细菌被巨噬细胞吞噬，并通过IV型分泌系统将效应蛋白转移到宿主细胞质中。这些效应蛋白通过几种不同的机制促进细菌在液泡状隔间中的生存和复制。VipF(液泡蛋白分选抑制蛋白F)是通过病原体效应筛选来鉴定能够改变宿主细胞蛋白运输途径的军团菌效应蛋白。对VipF一级序列的分析揭示了两个GCN5相关的乙酰基转移酶(GNAT)结构域。CHESS进行的同源模拟和小角X射线散射(SAXS)表明，VipF是一种双叶单体蛋白。GNAT是一个非常多样化的蛋白质超家族，负责催化乙酰基从乙酰辅酶A转移到受体多肽或小分子的伯胺上。在不同的生物过程中有许多关于蛋白质乙酰化的报道，但关于哪些酶负责每个修饰事件以及它在调节膜运输中的作用还知之甚少。本研究计划结合遗传学和细胞生物学，结合生物化学和结构生物学，研究VipF的酶活性及其改变宿主转运因子的机制。主要目的是确定VipF的分子结构，阐明其酶机制和乙酰化靶点，并了解酶活性如何改变酵母中的液泡蛋白分选途径。这项工作对于理解细菌效应蛋白VipF是如何帮助军团菌形成一个允许的空泡并经历正常的吞噬酶体成熟具有重要意义。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Legionella pneumophila is a facultative gram negative bacterium that can infect humans and cause a severe pneumonia known as Legionnaires¿¿" disease. The bacteria are phagocytozed by macrophages and translocate effector proteins through a Type IV secretion system into the host cytoplasm. These effector proteins facilitate the survival and replication of the bacteria in a vacuole like compartment through several different mechanisms. VipF (Vacuolar protein sorting Inhibitor Protein F) was identified by a pathogen effector screen to identify Legionella effector proteins that are able to alter host cell protein trafficking pathways. Analysis of the VipF primary sequence reveals two GCN5-related acetyltransferase (GNAT) domains. Homology modeling along with small-angle X-ray scattering (SAXS), performed at CHESS, suggest that VipF is a bilobal monomeric protein. GNATs are a very diverse superfamily of proteins that are responsible for catalyzing the transfer of an acetyl group from acetyl-CoA to a primary amine of an acceptor peptide or small molecule. There are many reports of protein acetylation in diverse biological processes but little is known about what enzymes are responsible for each modification event and what its role is in regulating membrane trafficking. This research proposal aims to study the enzymatic activity of VipF and the mechanism by which it may alter host trafficking factors through a combination of genetics and cell biology coupled with biochemistry and structural biology. The major goals are to determine the molecular structure of VipF, elucidate its enzymatic mechanism and acetylated target, and understand how the enzymatic activity alters the vacuole protein sorting pathway in yeast. This work is of fundamental importance to understand how the bacterial effector protein VipF is able aid Legionella to form a permissive vacuole that undergo normal phagolysosome maturation.