MOLECULAR DISSECTION OF A PROTEASOME ACTIVATOR

真核蛋白酶体是一个圆柱形的多亚基复合体，它包含一个埋藏的中心腔室，细胞内蛋白质在其中被降解。1992年，我们发现了两个结合蛋白酶体末端的粒子，它们可能通过打开通往中心腔室的通道来显著激活肽水解。其中一种激活剂，11S REG，是一个七聚体环，包含α和β亚基。在过去的六年里，我们克隆并表达了编码REGalpha、REGbeta和同源激活子REGgamma的cdna。我们已经描述了重组蛋白和各种regα突变体。我们现在提出额外的诱变实验，为每个REG同源物内的区域分配功能，并产生显性阴性突变体。我们的合作者，克里斯·希尔，已经解决了regα七聚体的晶体结构。它是一个圆锥形环，中心有一个充满溶剂的通道。regα环的“下”表面与蛋白酶体结合。在每个亚基的“上”表面未解析的是39个残基，这些残基包含每个REG同源物独有的序列。在REGalpha中，这种独特的氨基酸延伸由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”，它们在蛋白酶体亚基和伴侣中富集。利用酵母的两个杂交筛选，GST-REG嵌合体和用放射性碘化光反应交联剂衍生的REG同源物，我们将识别结合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类抗原呈递中起作用。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regα和regβ突变体，并测量卵清蛋白I类表位的表面表达，直接验证这一想法。由于KEKE基序在I类分子上呈递肽的前体中也丰富，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KEKE或非KEKE区域n端到卵白蛋白表位的质粒来验证这一假设。前体将在小鼠LKb细胞中表达，i类卵清蛋白肽复合物的表面表达将使用定量单克隆抗体测定法进行测量。

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.