Characterization of Pupylation in Mycobacterium tuberculosis

描述（由申请人提供）：全球每年约有200万人死于结核病。抗结核分枝杆菌（Mtb）感染的关键防御是巨噬细胞产生一氧化氮（NO）。虽然NO能控制结核分枝杆菌的生长，但它很少能使来自宿主的细菌绝育，这表明结核分枝杆菌具有抵抗NO毒性的机制。Mtb蛋白酶体就是这样一种机制，它是抵抗NO以及导致小鼠死亡所必需的。因此，我们对针对蛋白酶体及其相关因子进行药物开发很感兴趣。蛋白酶体是一种多亚基的桶状复合体，可以降解蛋白质。我们发现蛋白Mpa和PafA是蛋白质降解所必需的：Mpa被认为是蛋白质进入蛋白酶体核心的伴侣，而PafA似乎是原核泛素样蛋白（Pup）附着在靶向破坏底物上所必需的。Pup是已知的第一个在原核生物中发现的翻译后小蛋白修饰物。关于Pup如何与目标底物结合知之甚少，因此我们建议使用遗传，生化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pupyation是可逆的，因此我们正在描述“depupyation”途径。Mtb的pup -蛋白酶体系统的阐明有望为所有细菌中其他翻译后修饰系统的发现和表征奠定基础。此外，这些酶可能为抗结核药物的开发提供新的靶点。

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.