Structural studies of the M. tuberculosis proteasome and proteasomal ATPase

描述(申请人提供)：全球每年有150-200万人死于结核病。一种有效的疫苗或化疗尚未开发出来。最近，通过大规模的转座子突变筛选，结核分枝杆菌(Mtb)蛋白酶体和蛋白酶体ATPase(MPA)被发现是结核分枝杆菌抵抗一氧化氮(NO)来源的杀戮所必需的。NO是由宿主免疫系统产生的，用于控制结核分枝杆菌感染。蛋白酶体和甲孕酮似乎通过降解暴露于NO后的蛋白质来保护Mtb免受NO的伤害。因此，MPA和Mtb蛋白酶体有望成为抗结核化疗药物开发的靶点。MTB蛋白酶体对合成的小肽具有独特的广谱底物特异性，其蛋白分解活性可被MLN-273抑制，MLN-273是针对人蛋白酶体的抗骨髓瘤药物Bortezomib的类似物。Mtb蛋白酶体独特的底物结合特性可用于开发Mtb特异性抑制剂。甲孕酮与真核蛋白酶体中的ATPase同源，可能为Mtb蛋白酶体的降解提供蛋白质底物。突变的MPA蛋白只缺少最后两个氨基酸，保持了ATPase活性，但不能保护Mtb免受亚硝酸盐的伤害。我们建议使用冷冻电子显微镜(Cryo-EM)和X射线结晶学相结合的方法来研究Mtb蛋白酶体和Mpa的结构。我们的全面结构研究(1)将揭示Mtb蛋白酶体广泛底物专一性的结构基础；(2)将揭示二肽基硼酸盐MLN-273的抑制机制；(3)将阐明与ATP水解循环相关的MPA的构象变化；(4)将为长期存在的问题提供答案，即对称错配的六倍ATPase寡聚体如何激活七倍蛋白酶体；(5)将有助于更好地了解真细菌蛋白酶体的生物学；以及(6)将为基于结构的抗结核化疗药物的开发奠定基础。

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.