MOLECULAR DISSECTION OF A PROTEASOME ACTIVATOR

真核生物蛋白酶体是一个圆柱形的多亚基复合体，包含一个埋藏的中心腔室，细胞内的蛋白质在其中降解。 在1992年，我们发现了两种结合蛋白酶体末端的颗粒，它们可能通过打开通向中央腔室的通道而显著激活肽水解。这些激活剂之一，11 S REG，是含有α和β亚基的七聚体环。 在过去的六年中，我们已经克隆和表达编码REGalpha，REGbeta和同源激活剂，REGgamma的cDNA。 我们已经表征了重组蛋白和多种REG α突变体。 我们现在提出额外的诱变实验，以分配功能的区域内每个REG同源物，并产生显性负突变体。 我们的合作者Chris Hill已经解决了REG alpha七聚体的晶体结构。 它是一个锥形环，具有一个中心的、充满溶剂的通道。 REG α环的“下”表面结合蛋白酶体。 在每个亚基的“上”表面上未分辨的是39个残基，其包含每个REG同源物特有的序列。 在REGalpha中，这种独特的氨基酸序列由28个“交替”赖氨酸（K）和谷氨酸（E）残基组成。 我们称这些区域为“KEKE基序”，它们富含蛋白酶体亚基和分子伴侣。 使用酵母双杂交筛选，GST-REG嵌合体和REG同系物与放射性碘标记的光反应性交联剂衍生，我们将确定结合的REG α/β和REGgamma七聚体的“上”表面的蛋白质。 有间接证据表明REG α/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负性REG α和REG β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。因为KEKE基序也富含在I类分子上呈递的肽的前体，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白蛋白表位N-末端的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。

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.