权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

MOLECULAR DISSECTION OF A PROTEASOME ACTIVATOR

蛋白酶体激活剂的分子解剖

基本信息

批准号：
6627253
负责人：
MARTIN C RECHSTEINER
金额：
$ 26.98万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-01-01 至 2004-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6627253
关键词：
MHC class I antigen antigen presentation cell component structure /function molecular chaperones proteasome protein binding recombinant proteins site directed mutagenesis tissue /cell culture yeast two hybrid system

项目摘要

The eukaryotic proteasome is a cylindrical multisubunit complex that contains a buried central chamber in which intracellular proteins are degraded. In 1992, we discovered two particles that bind the ends of the proteasome and markedly activate peptide hydrolysis presumably by opening channels to the central chamber. One of these activators, the 11S REG, is a heptameric ring containing alpha and beta subunits. Over the past six years, we have cloned and expressed cDNAs encoding REGalpha, REGbeta and a homologous activator, REGgamma. We have characterized the recombinant proteins and a variety of REGalpha, mutants. We now propose additional mutagenesis experiments to assign functions for regions within each REG homolog and to generate dominant-negative mutants. Our collaborator, Chris Hill, has solved the crystal structure of the REGalpha heptamer. It is a conical ring with a central, solvent-filled channel. The "lower" surface of the REGalpha, ring binds the proteasome. Unresolved on the "upper" surface of each subunit are 39 residues that encompass sequences unique to each REG homolog. In REGalpha this unique stretch of amino acids consists of 28 "alternating" lysine (K) and glutamate (E) residues. We call such regions "KEKE motifs", and they are enriched in proteasome subunits and in chaperones. Using yeast two hybrid screens, GST-REG chimeras and REG homologs derivatized with radioiodinated photoreactive crosslinkers, we will identify proteins that bind the "upper" surfaces of the REGalpha/beta and the REGgamma heptamers. There is circumstantial evidence that REGalpha/beta heptamers play a role in Class I antigen presentation. We will test this idea directly by over expressing wild-type or dominant negative REGalpha and REGbeta mutants in cultured mouse cells and measuring the surface expression of an ovalbumin Class I epitope. Because KEKE motifs are also enriched in precursors to peptides presented on Class I molecules, we hypothesize that they may actually promote presentation of peptides. We will test this hypothesis by producing plasmids that encode KEKE or non-KEKE regions N-terminal to the ovalbumin epitope. The precursors will be expressed in mouse LKb cells, and the surface expression of Class I-ovalbumin peptide complexes will be measured using a quantitative monoclonal antibody assay.

真核蛋白酶体是一种圆柱形多亚基复合物，包含一个埋藏的中央室，细胞内蛋白质在其中被降解。 1992 年，我们发现了两种颗粒，它们结合了蛋白酶体的末端，并可能通过打开通往中央室的通道来显着激活肽水解。其中一种激活剂 11S REG 是包含 α 和 β 亚基的七聚环。在过去的六年里，我们克隆并表达了编码 REGalpha、REGbeta 和同源激活剂 REGgamma 的 cDNA。我们已经表征了重组蛋白和各种 REGalpha 突变体。我们现在提出额外的诱变实验来为每个 REG 同源物内的区域分配功能并生成显性失活突变体。我们的合作者 Chris Hill 解析了 REGalpha 七聚体的晶体结构。它是一个锥形环，具有一个中心充满溶剂的通道。 REGalpha 环的“下”表面与蛋白酶体结合。每个亚基的“上”表面上未解析的是 39 个残基，其中包含每个 REG 同源物所独有的序列。在 REGalpha 中，这段独特的氨基酸由 28 个“交替”赖氨酸 (K) 和谷氨酸 (E) 残基组成。我们将这些区域称为“KEKE 基序”，它们富含蛋白酶体亚基和伴侣。使用酵母两种杂交筛选、GST-REG嵌合体和用放射性碘标记光反应交联剂衍生的REG同源物，我们将鉴定结合REGα/β和REGγ七聚体“上”表面的蛋白质。有间接证据表明 REGα/β 七聚体在 I 类抗原呈递中发挥作用。我们将通过在培养的小鼠细胞中过度表达野生型或显性失活 REGα 和 REGβ 突变体并测量卵清蛋白 I 类表位的表面表达来直接测试这一想法。由于 KEKE 基序也富含 I 类分子上呈递的肽的前体，因此我们假设它们实际上可能促进肽的呈递。我们将通过产生编码卵清蛋白表位 N 端 KEKE 或非 KEKE 区域的质粒来检验这一假设。前体将在小鼠 LKb 细胞中表达，并且将使用定量单克隆抗体测定来测量 I 类卵清蛋白肽复合物的表面表达。