Function and Assembly of Eukaryotic Proteasomes

描述（由申请人提供）：多蛋白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的大部分蛋白质降解，对许多细胞活动至关重要。异常的蛋白酶体活动影响许多人类疾病，包括癌症、神经退行性疾病和糖尿病。像许多多亚基催化配合物一样，蛋白酶体形成环状结构，底物通过该环状结构，从而允许多种酶活性依次作用于底物。该蛋白酶体由一个圆柱形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可分为盖子亚配合物和碱亚配合物。CP有两个外部的异七聚体1环夹在一对2环之间，这两个2环容纳了中心的蛋白水解室。RP碱基包括六种不同的atp酶，形成六聚体环；RP结合并展开底物并将它们驱入CP内部。这些大的复合体是如何在体内组装的，人们知之甚少。对于CP是如此，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，组装必须以高保真度进行，以避免非生产性和潜在毒性的旁路中间体的过度积累。CP和RP装配都依赖于专门的装配伴侣。这项资助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新的途径。本实验采用遗传学、生物化学、细胞生物学和生物物理学方法相结合的方法，主要集中在具有26S蛋白酶体与人类复合物非常相似的真核生物酿酒酵母（Saccharomyces cerevisiae）上。这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（目标2）的组装促进机制。这两组因素都是在这个拨款的上一个周期中发现的。目的3旨在确定RP盖在体内形成的中间体以及这些中间体是否作为组装中间体。盖底连接将是分析的关键步骤。最后，Aim 4考虑了蛋白酶体在细胞中的组装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. PUBLIC HEALTH RELEVANCE: Many human diseases result from an abnormally high or low level of some crucial protein that controls cell or tissue function. Some cancers, for instance, are caused by insufficient amounts of proteins called tumor suppressors. The proteasome is a huge enzyme complex that controls the levels of important proteins by degrading them; adjustment of proteasome assembly or activity can therefore have a profound impact on the course of many human disorders.