Characterization of Pupylation in Mycobacterium tuberculosis

描述（由申请人提供）：结核病每年在全球造成约200万人死亡。针对结核分枝杆菌（Mtb）感染的关键防御是由巨噬细胞产生一氧化氮（NO）。虽然NO控制Mtb的生长，但它很少从宿主中杀灭细菌，这表明Mtb具有抵抗NO毒性的机制。Mtb蛋白酶体是一种这样的机制，它是抵抗NO以及导致小鼠死亡所必需的。因此，我们对靶向蛋白酶体及其相关因子的药物开发感兴趣。蛋白酶体是一种多亚基的桶形复合物，可降解蛋白质。我们发现蛋白质Mpa和PafA是蛋白质降解所必需的：Mpa被认为是蛋白质伴侣蛋白进入蛋白酶体核心，PafA似乎是将原核泛素样蛋白（Pup）附着到靶向破坏的底物上所必需的。Pup代表了在任何原核生物中鉴定的第一个已知的翻译后小蛋白修饰物。关于Pup如何与其靶底物缀合知之甚少，因此我们建议使用遗传，生物化学和分子生物学技术鉴定和表征“pupylation”所需的所有蛋白质。此外，我们最近发现，pupylation是可逆的，因此，我们正在表征的过程中的“去pupylation”途径。结核分枝杆菌Pu蛋白酶体系统的阐明将有望为发现和鉴定所有细菌中的其他翻译后修饰系统奠定基础。此外，这些酶可能代表抗结核药物开发的新靶点。 公共卫生相关性：结核病治疗需要6-9个月，这一问题导致服用抗生素的依从性降低，并增加了产生耐药性的机会。广泛耐药（XDR）的M。结核病最近成为大众报章的头条新闻，已成为公众关注的问题。因此，现在需要新的结核病治疗方法，而该途径可能为药物开发提供新的靶点。

DESCRIPTION (provided by applicant): Tuberculosis kills about 2 million people globally every year. A key defense against Mycobacterium tuberculosis (Mtb) infections is the production of nitric oxide (NO) by macrophages. Although NO controls Mtb growth, it rarely sterilizes the bacterium from the host, suggesting Mtb has mechanisms to resist NO toxicity. The Mtb proteasome is one such mechanism that is required for resistance to NO as well as causing death in mice. Thus, we are interested in targeting the proteasome and its associated factors for drug development. The proteasome is a multi-subunit, barrel shaped complex that degrades proteins. We found that the proteins Mpa and PafA are required for protein degradation: Mpa is thought to chaperone proteins into the proteasome core and PafA appears to be required for the attachment of a prokaryotic ubiquitin-like protein (Pup) onto substrates targeted for destruction. Pup represents the first known post-translational small protein modifier identified in any prokaryote. Little is known about how Pup is conjugated to its target substrates thus we propose to identify and characterize all proteins required for "pupylation" using genetic, biochemical and molecular biological techniques. In addition, we have recently discovered pupylation is reversible, thus we are in the process of characterizing the "depupylation" pathway. The elucidation of the Pup-proteasome sytem of Mtb will hopefully lay the foundation for the discovery and characterization of other posttranslational modification systems in all bacteria. Furthermore, these enzymes may represent new targets for the development of anti- tuberculosis drugs. PUBLIC HEALTH RELEVANCE: Tuberculosis therapy takes 6-9 months, a problem that leads to decreased compliance for taking antibiotics and increased chances of developing drug-resistance. The rise of extensively drug resistant (XDR) strains of M. tuberculosis has become a great public concern as it has recently made headlines in the popular press. Thus the new treatments for tuberculosis are needed now, and the pupylation pathway may provide new targets for drug development.