一个吵闹的邻居:Lpg2149的纯化和功能表征

Ashley M. Holahan
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引用次数: 0

摘要

泛素化是一种标记蛋白质的过程,用于各种细胞信号通路,即蛋白质降解和其他过程。这些途径在广泛的细胞过程中是必不可少的,包括防御入侵病原体的机制。泛素化过程普遍存在于包括植物和动物在内的所有真核生物中,因此在细胞稳态中起着至关重要的作用。近年来,有更多的研究发现,原核载体蛋白可以在自身没有泛素系统的情况下劫持泛素化系统。MavC,也称为lpg2147 (Gan, Nakayasu, Hollenbeck, & Luo, 2019;Puvar et al., 2020;Valleau等人,2018)被发现是一种泛素化酶,通过绕过通常的E1, E2, E3泛素化途径使UbE2N泛素化。MavC在嗜肺军团菌感染中起重要作用,而嗜肺军团菌是军团病的元凶。通过早期的分子生物学分析,已经发现与MavC在同一位点上的邻近基因编码lpg2149,该基因被表征为抑制MavC的功能。鉴于该蛋白的新颖性,本研究项目旨在实现lpg2149的克隆、表达和纯化,以便通过x射线晶体学捕获其抑制复合体中的lpg2149。尝试用MavC对lpg2149进行结晶,得到其结构。通过本项目对lpg2149的优化生产,可以更好地了解嗜肺乳杆菌的发病机制,有助于我们对嗜肺乳杆菌感染调控的理解。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A Nosy Neighbor: Purification and Functional Characterization of Lpg2149
Ubiquitination is a process that marks proteins for various cell-signaling pathways, namely protein degradation and other processes. Th ese pathways are essential in a wide array of cellular processes, including defense mechanisms against invading pathogens. Th e ubiquitination process is universally found in all eukaryotic organisms, including plants and animals, and thus plays a vital role in cellular homeostasis. Recently, more discoveries have been made on prokaryotic eff ector proteins that hijack the ubiquitination system even when they do not possess a ubiquitin system of their own. MavC, also known as lpg2147 (Gan, Nakayasu, Hollenbeck, & Luo, 2019; Puvar et al., 2020; Valleau et al., 2018), has been found to be a ubiquitinating enzyme that ubiquitinates UbE2N by bypassing the usual E1, E2, E3 ubiquitination pathway. MavC plays an important role in the infection of Legionella pneumophila, which is the culprit of Legionnaires’ disease. Th rough earlier molecular biology analysis, it has been discovered that a neighboring gene on the same locus as MavC encodes lpg2149, which has been characterized to inhibit MavC’s function. Given the novelty of this protein, this research project aims to achieve cloning, expression, and purifi cation of lpg2149 so that in its inhibitory complex lpg2149 can be captured by X-ray crystallography. An attempt was made to crystallize and obtain the structure of lpg2149 with MavC. With the optimization of lpg2149 production demonstrated in this project, a better understanding of L. pneumophila pathogenesis can be obtained, which would help in our understanding of regulating L. pneumophila infection.
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