MOLECULAR DISSECTION OF A PROTEASOME ACTIVATOR

蛋白酶体激活剂的分子解剖

• 批准号: 6490196
• 负责人: MARTIN C RECHSTEINER
• 金额: $ 26.2万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6490196
• 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

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.

真核生物蛋白酶体是一种圆柱形多生育络合物，其中包含一个埋藏的中央腔室，其中细胞内蛋白被降解。 在1992年，我们发现了两个结合蛋白酶体末端的颗粒，并明显通过向中央腔室的通道打开通道来显着激活肽水解。这些激活剂之一是11S Reg，是一个包含alpha和beta亚基的七聚体环。 在过去的六年中，我们已经克隆并表达了编码Regalpha，Regbeta和同源激活剂Reggamma的cDNA。 我们表征了重组蛋白和各种富豪突变体。 现在，我们提出了其他诱变实验，以分配每个Reg同源物中区域的功能并产生显性阴性突变体。 我们的合作者克里斯·希尔（Chris Hill）解决了Regalpha Heptamer的晶体结构。 它是带有中央溶剂填充通道的圆锥形环。 Regalpha的“下部”表面结合蛋白酶体。 在每个亚基的“上部”表面上尚未分解的是39个残基，其中包含每个Reg同源物特有的序列。 在雷加达中，这种独特的氨基酸片由28个“交替”赖氨酸（K）和谷氨酸（E）残基组成。 我们称此类区域为“ Keke图案”，它们富含蛋白酶体亚基和伴侣。 使用酵母两个杂交筛选，GST-REG嵌合体和用放射性化的光电反应交联衍生化的REG同源物，我们将鉴定蛋白质结合了丽汉/β/β和reggamma Heptamers的“上部”表面。 有间接的证据表明，富豪/β七聚体在I类抗原表现中起作用。 我们将直接通过表达培养的小鼠细胞中的野生型或显性阴性的雷形和雷形突变体并测量OValbumin I类表位的表面表达来直接测试这一想法。由于Keke基序也富含在I类分子上呈现的肽的前体中，因此我们假设它们实际上可能促进了肽的表现。 我们将通过产生编码Keke或Non-Keke区域N末端的质粒来检验这一假设。 前体将在小鼠LKB细胞中表达，并使用定量单克隆抗体测定法测量I类蛋白肽络合物的表面表达。