Mechanisms of ATP-dependent proteolytic enzymes

ATP依赖性蛋白水解酶的机制

• 批准号: 6745460
• 负责人: ALFRED L GOLDBERG
• 金额: $ 1.79万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6745460
• 关键词: Archaea; Escherichia coli; X ray crystallography; active sites; adenosinetriphosphatase; bioenergetics; chemical chain length; chemical cleavage; chemical kinetics; electron microscopy; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; exopeptidase; fluorescent dye /probe; high performance liquid chromatography; interferon gamma; nuclear factor kappa beta; proteasome; proteolysis; tomography

DESCRIPTION (Verbatim from the applicant's abstract): A major goal of this research is to increase our understanding of the 26S proteasome, the ATP-dependent complex, which preferentially degrades ubiquitin-conjugated proteins and catalyzes most of the protein degradation in animal cells. Unlike conventional proteases, this enzyme and its simpler homologs in prokaryotes are large ATP-hydrolyzing complexes that degrade polypeptides processively to small peptide. Recently, we discovered that the different active sites of the core 20S proteasome are allosterically regulated by peptide substrates and thus appear to function in a cyclical manner during protein breakdown (the "bite-chew model). In coming years, we hope to elucidate the structural basis for these allosteric effects to determine whether the proteasome digests proteins in a preferred direction and to clarify how products are released by these particles. To understand the role of ATP hydrolysis, we are using as a model system the simpler proteasomes from archaea, where we recently discovered a proteasome ATPase complex, PAN, which homologous to the six ATPases in the 26S proteasome. We hope to clarify PAN's structure and how stimulates unfolding and translocation of substrates into the lumen of the 20S particle. Proteasomes degrade proteins to peptides that range from 3-22 residues. Most are quickly hydrolyzed in the cytosol to amino acids, but in mammals, some escape this fate and serve as precursors for the 8- to 9-residue peptides that are presented to the immune system on MHC-class I molecules. We have developed new methods to determine to what extent the 26S proteasomes and the alternative forms induced by gamma-interferon (immunoproteasomes and PA28-containing complexes) directly generate the antigenic peptides or yield larger precursors that are trimmed by aminopeptidases to the MHC-presented epitopes. We also hope to learn more about this trimming process and about the competing pathway by which most proteasome products are digested to amino acids.

描述（申请人摘要的逐字记录）：此项目的主要目标 研究的目的是增加我们对 26S 蛋白酶体的了解 ATP 依赖性复合物，优先降解泛素结合物 蛋白质并催化动物细胞中的大部分蛋白质降解。不像 传统的蛋白酶，这种酶及其在原核生物中的更简单的同系物是 大的 ATP 水解复合物将多肽逐渐降解为小分子 肽。最近，我们发现核心的不同活性位点 20S 蛋白酶体受肽底物变构调节，因此 似乎在蛋白质分解过程中以循环方式发挥作用（ “咬咀模型）。在未来几年，我们希望阐明其结构基础 对于这些变构效应来确定蛋白酶体是否消化 蛋白质的首选方向，并阐明产品是如何释放的 这些颗粒。为了了解 ATP 水解的作用，我们使用 模型系统来自我们最近发现的古细菌的更简单的蛋白酶体 蛋白酶体 ATP 酶复合物 PAN，与细胞中的六种 ATP 酶同源 26S蛋白酶体。我们希望阐明 PAN 的结构以及如何刺激展开 以及底物易位到20S颗粒的内腔中。蛋白酶体 将蛋白质降解为 3-22 个残基的肽。大多数都很快 在细胞质中水解成氨基酸，但在哺乳动物中，有些逃脱了这种命运 并作为 8 至 9 残基肽的前体，呈递给 免疫系统对 MHC I 类分子的影响。我们开发了新方法 确定 26S 蛋白酶体和替代形式诱导的程度 直接通过 γ-干扰素（免疫蛋白酶体和含 PA28 的复合物） 产生抗原肽或产生更大的前体，并被修剪 氨肽酶作用于 MHC 呈递的表位。我们也希望能够了解更多 这个修剪过程以及大多数蛋白酶体的竞争途径 产品被消化成氨基酸。