NMR Structural Studies of Ubiquitin Receptor Protein Complexes

描述（由申请人提供）：蛋白酶体的蛋白质降解必须受到严格调控，因为它控制从细胞周期进展到细胞死亡的事件。26 S蛋白酶体由20 S催化核心颗粒（CP）组成，其两端被19 S调节颗粒（RP）封端。尽管CP含有负责蛋白质底物蛋白水解的酶活性，但蛋白质降解的承诺由RP组分决定，RP组分在底物进入CP之前识别和处理底物。对于大多数蛋白酶体底物，泛素化是其降解的先决条件，因为它们与蛋白酶体的初始相互作用是通过RP泛素受体S5a和Rpn 13。底物随后被三种去泛素化酶去泛素化，并被ATP酶的异六聚环解折叠。这些活动准备底物进入一个狭窄的室，导致蛋白酶体的CP的催化中心。由于并不是所有的泛素化蛋白质对接到蛋白酶体最终降解，有很大的兴趣在了解的RP底物加工的机械细节。我们的长期目标是确定底物如何被蛋白酶体的19S调节颗粒加工。通过对Rpn 13和S5a的研究，我们将这个建议集中在这个过程的第一阶段，即底物识别和去泛素化。我们研究了Rpn13是如何对接到蛋白酶体和追求的初步数据表明，它被激活的这种定位。我们已经设计了一种新的策略来研究Rpn 13和S5a的RP的背景下，并测试其协调结合到泛素化底物的工作模型。为了实现我们的研究目标，我们使用功能测定，包括那些测试纯化的蛋白酶体物种的泛素结合能力和泛素链解偶联的Uch 37，以及各种生物物理方法，特别是NMR光谱。最终，拟议研究的完成将提供Rpn 13和S5a所承担的不同角色的基本信息，这些角色不仅仅是将泛素化蛋白质对接到蛋白酶体中。

DESCRIPTION (provided by applicant): Protein degradation by the proteasome must be tightly regulated, as it controls events ranging from cell cycle progression to cell death. The 26S proteasome is composed of a 20S catalytic core particle (CP) that is capped at either end by a 19S regulatory particle (RP). Whereas the CP contains the enzymatic activity responsible for proteolyzing protein substrates, the commitment to protein degradation is determined by RP components, which recognize and process substrates prior to their passage into the CP. For most proteasome substrates, ubiquitination is prerequisite to their degradation, as their initial interaction with the proteasome is through RP ubiquitin receptors, S5a and Rpn13. Substrates are subsequently deubiquitinated by three deubiquitinating enzymes and unfolded by a heterohexomeric ring of ATPases. These activities prepare substrates for entry through a narrow chamber leading to the catalytic center of the proteasome's CP. Since not all ubiquitinated proteins that dock into the proteasome are ultimately degraded, there is a great deal of interest in understanding the mechanistic details of substrate processing in the RP. Our long-term goal is to define how substrates are processed by the 19S regulatory particle of the proteasome. We focus this proposal on the first stage of this process, namely substrate recognition and deubiquitination, through our studies of Rpn13 and S5a. We study how Rpn13 is docked into the proteasome and pursue preliminary data that indicates it to be activated by this localization. We have devised a novel strategy to study Rpn13 and S5a in the context of the RP and test a working model of their coordinated binding to ubiquitinated substrates. To achieve our research goals, we use functional assays, including those that test the ubiquitin binding capacity of purified proteasome species and ubiquitin chain deconjugation by Uch37, as well as a variety of biophysical methods, especially NMR spectroscopy. Ultimately, the completion of the proposed research will provide fundamental information on distinct roles assumed by Rpn13 and S5a that extend beyond simply docking ubiquitinated proteins into the proteasome.