Function and Assembly of Eukaryotic Proteasomes

描述(申请人提供)：多蛋白质机器控制许多复杂的生物过程，如DNA复制、RNA加工和蛋白质降解。26S蛋白酶体负责真核生物中的大部分蛋白质降解，是许多细胞活动所必需的。蛋白酶体活性异常影响许多人类疾病，包括癌症、神经退行性疾病和糖尿病。像许多多亚单位催化复合体一样，蛋白酶体形成环状结构，底物通过该结构，允许多种酶活性在底物上顺序作用。蛋白酶体由一个圆柱形的20S蛋白酶体核心颗粒(CP)和一个19S调节颗粒(RP)在一端或两端组成。每个RP可分为LID亚复合体和碱基亚复合体。CP有两个外杂七聚体1环，夹着一对2环，这对环容纳了中央蛋白水解室。RP碱基包括六种不同的ATPase，它们形成一个六聚体环；RP结合并展开底物，并将它们驱动到CP内部。这些大型复合体是如何在体内组装的，人们对此知之甚少。这对于CP是正确的，对于~19亚基RP更是如此。蛋白酶体组装似乎是一个跨物种保守的有序过程。由于蛋白酶体非常丰富，必须以高保真的方式进行组装，以避免过度积累非生产性和潜在有毒的旁路中间体。CP和RP组装都依赖于专门的组装伴侣。这笔赠款的长期目标是描绘真核26S蛋白酶体生物发生的途径。蛋白酶体已成为抗癌治疗和其他治疗的重要靶点。干扰其组装将为药物干预提供一种新的途径。拟议的实验结合了遗传学、生化、细胞生物学和生物物理学的方法，主要集中在模式真核生物酿酒酵母上，它的26S蛋白酶体与人类的复合体非常相似。第一次更新的主要目标将是破译CP组装因子(目标1)和四个RP组装伴侣(RAC)(目标2)的组装促进机制。这两组因素都是在这笔赠款的上一个周期中发现的。目标3旨在确定RP盖的中间体在体内形成的是什么，以及这些中间体是否作为组装中间体。盖底连接将是需要分析的关键步骤。最后，目标4考虑了蛋白酶体在细胞中组装的位置以及蛋白酶体如何在细胞内重新定位以响应生长条件的变化的问题。

DESCRIPTION (provided by applicant): Multiprotein machines control many complex biological processes, such as DNA replication, RNA processing, and protein degradation. The 26S proteasome is responsible for the majority of protein degradation in eukaryotes and is essential for numerous cellular activities. Aberrant proteasomal activity impacts many human diseases including cancer, neurodegenerative disorders, and diabetes. Like many multisubunit catalytic complexes, the proteasome forms ring-shaped structures through which a substrate is passed, allowing multiple enzymatic activities to act sequentially on the substrate. The proteasome consists of a cylindrical 20S proteasome core particle (CP) with a 19S regulatory particle (RP) on one or both ends. Each RP can be divided into lid and base subcomplexes. The CP has two outer heteroheptameric 1 rings that sandwich a pair of 2 rings, which house the central proteolytic chamber. The RP base includes six different ATPases that form a hexameric ring; the RP binds and unfolds substrates and drives them into the CP interior. How these large complexes are assembled in vivo is poorly understood. This is true for the CP and even more so for the ~19-subunit RP. Proteasome assembly appears to be an ordered process conserved across species. As proteasomes are highly abundant, assembly must occur with high fidelity to avoid excessive accumulation of nonproductive and potentially toxic off-pathway intermediates. Both CP and RP assembly depend on dedicated assembly chaperones. The long-range goal of this grant is to delineate the pathways of eukaryotic 26S proteasome biogenesis. The proteasome has emerged as an important target for anti-cancer treatment and other therapies. Interfering with its assembly will provide a new approach for pharmaceutical intervention. The proposed experiments use a combination of genetic, biochemical, cell biological and biophysical methods and are focused primarily on the model eukaryote Saccharomyces cerevisiae, which has a 26S proteasome very similar to the human complex. A major goal of this first renewal will be on deciphering the assembly-promoting mechanisms of a CP assembly factor (Aim 1) and four RP assembly chaperones (RACs) (Aim 2). Both sets of factors were discovered during the past cycle of this grant. Aim 3 is directed toward determining what intermediates of the RP lid form in vivo and whether these serve as assembly intermediates. Lid-base joining will be a key step to be analyzed. Finally, Aim 4 considers the question of where in the cell the proteasome assembles and how proteasomes relocalize within the cell in response to changes in growth conditions.