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央小室，在这个小室中细胞内的蛋白质被降解。1992年，我们发现了两个颗粒，它们结合在蛋白酶体的末端，可能通过打开通往中央腔室的通道来显著激活多肽水解。其中一个激活剂，11S Reg，是一个包含α和β亚基的七聚环。在过去的六年里，我们已经克隆和表达了编码REGalpha、REGbeta和同源激活剂REGGamma的cDNA。我们已经鉴定了重组蛋白和各种REGalpha，突变体。我们现在建议进行额外的突变实验，为每个REG同源物中的区域分配功能，并产生显性负突变。我们的合作者克里斯·希尔已经解决了REGalpha七聚体的晶体结构。它是一个圆锥形的环，中间有一个充满溶剂的通道。REGalpha环的“下”表面结合了蛋白酶体。在每个亚基的“上”表面有39个残基未被分解，这些残基包含每个REG同源基因所特有的序列。在REGalpha中，这段独特的氨基酸由28个“交替”的赖氨酸(K)和谷氨酸(E)残基组成。我们将这些区域称为“可可基序”，它们富含蛋白酶体亚基和伴侣蛋白。使用酵母双杂交筛选，GST-REG嵌合体和用放射性碘标记的光反应交联剂衍生的REG同系物，我们将识别结合REGalpha/β和REGGamma七聚体“上”表面的蛋白质。有间接证据表明，REGalpha/β七聚体在I类抗原递呈中发挥作用。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REGalpha和REGbeta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类分子上呈现的多肽的前体，我们假设它们实际上可能促进多肽的呈现。我们将通过产生编码卵清蛋白表位的KEKE或非KEKE区域N-末端的质粒来检验这一假设。这些前体将在小鼠LKB细胞中表达，并将使用定量单抗分析来测量I类卵清蛋白多肽复合体的表面表达。

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.