NEW, SALT-SENSITIVE COMPONENTS OF THE PROTEASOME

描述（由申请人提供）：Finley博士实验室的先前研究确定了酵母蛋白酶体调节颗粒的17个亚基。这些样品在常规纯化过程中暴露于高盐。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。来自纯化的盐洗涤部分的蛋白质通过质谱法鉴定，随后显示为真正的蛋白酶体相关蛋白（PAP）。这些似乎是新的蛋白酶体的体内成分;有些似乎是近似化学计量的。新的组分是一种泛素-蛋白质连接酶（Hul 5），一种去泛素化酶（Ubp 6），一种稳定蛋白酶体核心颗粒和调节颗粒（Ecru 29）结合的蛋白质，以及一种与DNA修复有关的蛋白质（Bkn 3）。这四个基因中的每一个都与已知的蛋白酶体亚基的基因共同调节，并且由烷化剂MMS强烈诱导。此外，已知的PAP蛋白都是进化保守的。Finley博士建议定义一套完整的主要PAP，并研究这些蛋白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在每个PAP中进行定位，并确定蛋白酶体结合丧失的表型后果。将使用强大的新质谱方法在全球范围内评估PAP基因功能突变丧失所稳定的蛋白酶体底物谱。松散相关的辅因子对于许多蛋白质复合物的功能至关重要，例如聚合酶、核糖体、微管和核孔。因此，蛋白酶体相关蛋白的系统研究可能会显着提高我们对这一重要的蛋白质复合物的理解。具体目标如下：目标1。确定主要的蛋白酶体相关蛋白。目标二。通过质谱法在全球范围内搜索PAP突变体中的降解缺陷。目标3：在蛋白酶体内定位特定相关蛋白的结合位点。目标4。绘制介导蛋白酶体结合的蛋白酶体相关蛋白的序列。目标5。研究特异性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.