Structural studies of the M. tuberculosis proteasome and proteasomal ATPase

描述（由申请人提供）：全球每年有150万至200万人死于结核病。有效的疫苗或化疗尚未开发。最近，通过大规模转座诱变筛选，发现结核分枝杆菌（Mtb）蛋白酶体和蛋白酶体atp酶（Mpa）是结核分枝杆菌抵抗一氧化氮（NO）杀伤所必需的。NO由宿主免疫系统产生以控制结核杆菌感染。蛋白酶体和Mpa似乎通过降解暴露于NO后的蛋白质来保护Mtb免受NO的侵害。因此，Mpa和Mtb蛋白酶体可能是开发抗结核化疗药物的有希望的靶点。Mtb蛋白酶体对小合成肽具有独特的广泛底物特异性，其蛋白水解活性被MLN-273抑制，MLN-273是一种针对人蛋白酶体的抗骨髓瘤药物硼替佐米的类似物。利用结核分枝杆菌蛋白酶体独特的底物结合特性，可以开发结核分枝杆菌特异性抑制剂。Mpa与与真核蛋白酶体一起发挥作用的atp酶是同源的，并且可能展开被Mtb蛋白酶体降解的蛋白质底物。突变的Mpa蛋白只缺少其最后两个氨基酸，保留atp酶活性，但不能保护结核分枝杆菌免受亚硝酸盐的侵害。我们建议使用低温电子显微镜（cryo-EM）和x射线晶体学相结合的方法来研究Mtb蛋白酶体和Mpa的结构。我们全面的结构研究(1)将揭示Mtb蛋白酶体广泛底物特异性的结构基础；(2)揭示硼酸二肽基MLN-273的抑制机制；(3)阐明与ATP水解循环相关的Mpa构象变化；(4)将回答一个长期存在的问题，即对称错配的六倍atp酶寡聚物如何激活七倍蛋白酶体，(5)将有助于更好地理解真菌性蛋白酶体生物学，(6)将为基于结构的抗结核化疗开发奠定基础。

DESCRIPTION (provided by applicant): Tuberculosis kills 1.5-2 million people globally every year. An effective vaccine or chemotherapy has yet to be developed. Recently, through a large-scale transposon mutagenesis screening, the Mycobacterium tuberculosis (Mtb) proteasome and the proteasomal ATPase (Mpa) were found to be required for Mtb resistance to killing by a source of nitric oxide (NO). NO is produced by the host immune system to control Mtb infections. Proteasome and Mpa appear to protect Mtb against NO by degrading proteins after exposure to NO. Thus, Mpa and the Mtb proteasome may be promising targets for the development of anti-Tb chemotherapeutics. Mtb proteasome has unique broad substrate specificity towards small synthetic peptides, and its proteolytic activity is inhibited by MLN-273, an analog of the anti-myeloma drug bortezomib that targets human proteasome. The unique substrate binding property of Mtb proteasome can be exploited to develop Mtb specific inhibitors. Mpa is homologous to ATPases that function with the eukaryotic proteasome, and likely unfolds the protein substrate for processive degradation by Mtb proteasome. A mutant Mpa protein missing only its last two amino acids retains ATPase activity, yet fails to protect Mtb against nitrite. We propose to use a combination of cryo-electron microscopy (cryo-EM) and X-ray crystallography to investigate the structures of Mtb proteasome and Mpa. Our comprehensive structural studies (1) will reveal the structural basis of the Mtb proteasome's broad substrate specificity; (2) will uncover the inhibition mechanism of the dipeptidyl boronate MLN-273; (3) will elucidate the conformational changes of Mpa associated with the ATP hydrolysis cycle; (4) will provide answer to the long standing question as to how a symmetry mismatched six-fold ATPase oligomer activates the seven-fold proteasome, (5) will contribute to a better understanding of the eubacterial proteasome biology, and (6) will set the stage for the structure-based anti-TB chemotherapeutic development.