Tripeptidylpeptidase II Structure and Assembly

描述（由申请人提供）：细胞蛋白质降解是一种受调控的过程，参与控制广泛的生物学过程。泛素-蛋白酶体级联反应是真核细胞胞浆蛋白质降解的主要系统。三肽基肽酶II（TPP II）是一种6 MDa的枯草杆菌蛋白酶样丝氨酸肽酶，其切割蛋白酶体产物以产生可被其他外肽酶用作底物的MHC I类抗原肽和短肽。TPP II介导的内溶肽活性产生人免疫缺陷病毒（HIV）表位Nef。TPP II似乎也是一种在蛋白酶体活性次优的情况下对细胞存活至关重要的肽酶，例如在抗肿瘤细胞中观察到的。TPP II全复合物是由两条扭曲的链组成的纺锤形结构，每条链包含10个二聚体;活性位点通过二聚体的顺序缔合形成。当两个二聚体沿着生长的链接触时，来自每个二聚体的一个单体被活化。这项研究计划的长期目标是了解TPP II介导的肽解、复合物组装和组装依赖性激活的分子机制。在实现这一目标的支持，我们将确定，通过X-射线晶体学方法，600 kDa的TPP II四聚体的结构，已被发现保留其四个亚基中的两个完全酶活性。四聚体的高分辨率结构将提供两个活性亚基和两个非活性亚基的结构细节，这反过来将深入了解组装依赖性激活的过程。本工作还将包括与抑制剂结合的四聚体的结构测定。我们还建议使用单粒子冷冻EM方法获得6 MDa复合物的密度图，分辨率优于1.5 nm。我们将使用这种更高分辨率的基于EM的地图和TPP II四聚体的原子结构来构建6 MDa全息复合物的高分辨率模型。从四聚体的原子模型中获得的结构信息以及6 MDa全复合物的高分辨率模型将提供对TPP II底物进入和结合、肽水解过程以及调节复合物组装至体内一致观察到的长度所涉及的机制的分子水平洞察。反过来，这些知识可以支持设计未来治疗某些癌症和艾滋病毒感染的治疗策略。

DESCRIPTION (provided by applicant): Cellular protein degradation is a regulated process involved in the control of a broad array of biological processes. The ubiquitin-proteasome cascade is the major system for proteolysis of cytosolic proteins in eukaryotic cells. Tripeptidyl peptidase II (TPP II), a 6 MDa subtilisin-like serine peptidase, cleaves proteasome products to produce MHC class I antigenic peptides and short peptides that can be used as substrates by other exo-peptidases. TPP II mediated endo-peptidolytic activity generates the human immunodeficiency virus (HIV) epitope, Nef. TPP II also appears to be a peptidase critical for cell survival in cases where proteasome activity is sub-optimal, such as has been observed in apoptosis-resistant tumor cells. The TPP II holo-complex is a spindle-shaped structure composed of two twisted strands, each containing 10 dimers; active sites are formed through the sequential association of dimers. As two dimers come into contact along a growing strand, one monomer from each dimer becomes activated. The long-range objective of this research proposal is to understand the molecular mechanism of TPP II mediated peptidolysis, complex assembly, and assembly-dependent activation. In support of achieving this objective we will determine, by x-ray crystallographic methods, the structure of the 600 kDa TPP II tetramer, which has been found to retain full enzymatic activity in two of its four subunits. A high-resolution structure of the tetramer will provide structural details of the two active and two inactive subunits which, in turn, will yield insight into the process of assembly-dependent activation. This work will also include the structure determination of the tetramer with inhibitors bound. We also propose to obtain a density map of the 6 MDa complex to a resolution better than 1.5 nm using single particle cryo-EM methods. We will use this higher resolution EM-based map and the atomic structure of the TPP II tetramer to construct a high-resolution model of the 6 MDa holo-complex. The structural information gained from the atomic model of the tetramers together with a high resolution model of the 6 MDa holo-complex will provide molecular level insight into TPP II substrate access and binding, the peptidolytic process, and the mechanisms involved in regulating assembly of the complex to the length consistently observed in vivo. This knowledge, in turn, may support the design of future therapeutic strategies for the treatment of certain cancers and HIV infection.