NEW, SALT-SENSITIVE COMPONENTS OF THE PROTEASOME

描述(由申请人提供)：芬利博士实验室以前的研究定义了酵母蛋白酶体调节颗粒的17个亚基。这些样品在常规提纯过程中暴露在高盐环境中。最近，芬利博士的实验室开发了一种蛋白酶体的亲和纯化方法。纯化的盐洗部分的蛋白质经质谱仪鉴定为真正的蛋白酶体相关蛋白(PAPs)。这些似乎是蛋白酶体的新的体内成分；其中一些似乎是近似化学计量的。新的成分是泛素蛋白连接酶(Hul5)，去泛素化酶(Ubp6)，稳定蛋白酶体核心颗粒和调节颗粒之间联系的蛋白质(Eru29)，以及与DNA修复有关的蛋白质(Bkn3)。这四个基因中的每一个都与已知蛋白酶体亚基的基因共同调控，并受到烷化剂MMS的强烈诱导。此外，已知的PAP蛋白在进化上都是保守的。芬利博士建议定义一整套主要的PAPs，并研究这些蛋白质在体内和体外蛋白酶体功能中的作用。负责蛋白酶体结合的序列将被映射到每个PAP中，并将确定蛋白酶体结合丧失的表型后果。将使用强大的新的质谱学方法对由PAP基因功能突变所稳定的蛋白酶体底物的谱进行全球评估。松散结合的辅因子对许多蛋白质复合体的功能至关重要，如聚合酶、核糖体、微管和核孔。因此，对蛋白酶体相关蛋白的系统研究可能会大大增强我们对这一重要蛋白质复合体的了解。具体目的如下：目的1.确定主要的蛋白酶体相关蛋白。目的2.用质谱仪在全球范围内寻找PAP突变体的降解缺陷。目的3.在蛋白酶体内定位特定相关蛋白的结合部位。目的4.定位蛋白酶体相关蛋白中介导其与蛋白酶体结合的序列。目的5.研究特定蛋白酶体相关蛋白的功能。

DESCRIPTION (provided by applicant): Previous studies from Dr. Finley's laboratory defined the 17 subunits of the yeast proteasome regulatory particle. These samples had been exposed to high salt during conventional purification. Recently an affinity-purification method for proteasomes was developed by Dr. Finley's laboratory. Proteins from the salt wash fraction of the purification were identified by mass spectrometry and subsequently shown to be bona fide proteasome-associated proteins (PAPs). These appear to be novel in vivo components of the proteasome; some appear to be approximately stoichiometric. The new components are a ubiquitin-protein ligase (Hul5), a deubiquitinating enzyme (Ubp6), a protein that stabilizes the association of the proteasome core particle and the regulatory particle (Ecru29), and a protein that has been implicated in DNA repair (Bkn3). Each of these four genes is co-regulated with genes for known proteasome subunits, and strongly induced by the alkylating agent MMS. In addition, the known PAP proteins are all evolutionarily conserved. Dr. Finley proposes to define the complete set of major PAPs, and to study the roles of these proteins in proteasome function in vivo and in vitro. Sequences responsible for proteasome binding will be mapped within each PAP, and the phenotypic consequences of the loss of proteasome association will be determined. The spectrum of proteasome substrates stabilized by loss of function mutations in PAP genes will be assessed globally using powerful new mass spectrometry methods. Loosely associated cofactors are critical for the functioning of many protein complexes, such as polymerases, ribosomes, microtubules, and nuclear pores. Therefore, systematic investigation of proteasome-associated proteins may significantly enhance our understanding of this important protein complex. Specific Aims are as follows: Aim 1. To define the major proteasome-associated proteins. Aim 2. To search globally for degradation defects in PAP mutants by mass spectrometry. Aim 3. To localize within the proteasome the binding sites for specific associated proteins. Aim 4. To map sequences within proteasome-associated proteins that mediate their binding to proteasomes. Aim 5. To study the functions of specific proteasome-associated proteins.