Tripeptidylpeptidase II Structure and Assembly

描述（由申请人提供）：细胞蛋白质降解是一个受调节的过程，涉及控制广泛的生物过程。泛素-蛋白酶体级联是真核细胞胞质蛋白水解的主要系统。三肽基肽酶II （Tripeptidyl peptidase II, TPP II）是一种6 MDa枯草菌素样丝氨酸肽酶，可裂解蛋白酶体产物产生MHC I类抗原肽和短肽，这些肽可被其他外肽酶用作底物。TPP II介导的内肽水解活性产生人类免疫缺陷病毒（HIV）表位。在蛋白酶体活性不理想的情况下，TPP II似乎也是一种对细胞存活至关重要的肽酶，例如在抗凋亡肿瘤细胞中观察到的情况。TPP II全息配合物是由两条扭曲的链组成的纺锤形结构，每条链含有10个二聚体；活性位点是通过二聚体的顺序结合形成的。当两个二聚体沿着生长链接触时，每个二聚体中的一个单体被激活。本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。为了支持实现这一目标，我们将通过x射线晶体学方法确定600 kDa TPP II四聚体的结构，该结构已被发现在其四个亚基中有两个保持完全的酶活性。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，反过来，将产生对组装依赖激活过程的见解。这项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电镜方法获得分辨率优于1.5 nm的6 - MDa复合物的密度图。我们将利用这一更高分辨率的基于em的图谱和TPP II四聚体的原子结构来构建6 MDa全息配合物的高分辨率模型。从四聚体的原子模型和6mda全复合物的高分辨率模型中获得的结构信息将提供分子水平上对TPP II底物进入和结合、肽水解过程以及调节复合物组装到体内一致观察到的长度的机制的见解。反过来，这一知识可能支持未来治疗某些癌症和艾滋病毒感染的治疗策略的设计。

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.