Molecular Chaperones and Protein Degradation

分子伴侣和蛋白质降解

• 批准号: 7999992
• 负责人: ALFRED L GOLDBERG
• 金额: $ 6.27万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-29 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7999992
• 关键词: 26S proteasome; ATP Hydrolysis; ATP phosphohydrolase; Affect; Aging; Aging-Related Process; Archaea; Binding; Binding Proteins; Biochemical; Biological Assay; C-terminal; Cell Survival; Cells; Chemicals; Complex; Degradation Pathway; Disaccharides; Docking; Enzymes; Event; Free Radicals; Freezing; Goals; Grant; Heat-Shock Response; Hydrolysis; Inherited; Lead; Learning; Mammalian Cell; Maps; Molecular; Molecular Chaperones; Mutagenesis; Mutation; Neurodegenerative Disorders; Normal Cell; Nucleotides; Oxidants; Oxidative Stress; Pathway interactions; Process; Proteins; Proteolysis; Reaction; Reactive Oxygen Species; Role; Series; Superoxides; Temperature; Testing; Trehalose; Ubiquitin; Ubiquitination; X ray diffraction analysis; X-Ray Diffraction; Yeasts; abstracting; base; cell injury; crosslink; free radical oxygen; human disease; in vivo; multicatalytic endopeptidase complex; particle; protein degradation; protein misfolding; reconstitution

ABSTRACT One important function of intracellular protein degradation is to selectively destroy misfolded or damaged proteins, as accumulate in various human diseases and during aging. Our primary goal is to understand further the functioning of the ubiquitin proteasome pathway and the involvement of molecular chaperons in this process. Most protein breakdown in mammalian cells is catalyzed by the 26S proteasome, whose 19S regulatory particle contains six ATPases which function as molecular chaperones. To understand how these ATPases unfold and translocate substrates into the core 20S proteasome, we are also studying the simpler, homologous ATPase complex, PAN, which activates proteolysis by 20S proteasomes in archaea. We recently discovered that upon ATP-binding, a conserved C-terminal sequence (HbYX motif) in these ATPases docks into pockets in the 20S's outer ring and like a "key-in-a-lock" triggers opening of its gated channel for substrate entry. A major goal will be to elucidate further the molecular events for gate-opening and its structural basis. In the eukaryotic 26S, the six ATPases and seven 20S ¿-subunits differ, and gate opening is restricted to two ATPases. We hope to learn which C-termini dock into which 20S pockets and whether other proteasome regulators (BLM10, PI131) and p97/cdc48 that contain the HbYX motif activate 20S proteasomes similarly. We have dissociated proteasomal degradation into a series of ATP-requiring steps, and hope to elucidate further this multistep reaction sequence. We shall explore whether the eukaryotic 26S utilizes additional reaction steps in degrading ubiquitin-conjugated proteins. We've found that the six ATPase subunits interact through negative cooperativity and identified in PAN two substrate-binding domains whose function is nucleotide-dependent. We hope to clarify the functional significance in protein degradation of these cooperative interactions and the associated conformational changes. We recently found that increased temperatures and oxygen radicals in yeast cause the degradation specifically of newly synthesized proteins, without affecting breakdown of most cell proteins. We hope to clarify further the roles of different ubiquitination enzymes and molecular chaperones in their degradation and to identify the newly synthesized proteins most sensitive to such damage. In harsh conditions that damage cell proteins (e.g. heat-shock, oxidative stress, near-freezing temperatures), yeast produce large amounts of the "chemical chaperone", trehalose, which enhances cell viability. We recently found that trehalose is also required for efficient protein degradation, even in normal cells. The mechanisms of this unexpected new protective function of trehalose will be investigated.

抽象的 细胞内蛋白质降解的一个重要功能是选择性破坏错误折叠或损坏 蛋白质，随着各种人类疾病和衰老期间积累。我们的主要目标是进一步了解 泛素蛋白酶体途径的功能和分子伴侣的参与 过程。哺乳动物细胞中的大多数蛋白质分解是由26S蛋白酶体催化的，其19s 调节粒子包含六个ATPases，它们充当分子伴侣。了解这些如何 ATPases展开并将底物转移到核心20S蛋白酶体中，我们还研究了更简单的， 同源ATPase复合物PAN，该复合物在古细菌中激活20S蛋白酶体的蛋白水解。 我们最近发现，在ATP结合时，这些是一个配置的C末端序列（HBYX基序） ATPASE码头码头登上20年代外环的口袋，就像“锁定锁”触发了门的开口 基材进入的频道。一个主要目标是阐明进一步的分子事件，以进行门开放和 它的结构基础。在真核生物26s中，六个ATPase和7个20S„ - subunits不同，门开口 仅限于两个ATPases。我们希望了解哪个C-termini码头有20秒的口袋以及是否 其他蛋白酶体调节剂（BLM10，PI131）和P97/CDC48包含HBYX基序激活20S 蛋白酶体类似。 我们已将蛋白酶体降解分解为一系列ATP的步骤，并希望阐明 此外，该多步反应序列。我们将探讨26S是否利用其他 降解泛素偶联蛋白的反应步骤。我们发现六个ATPase亚基相互作用 通过负协调，并在pan两个底物结合域中确定，其功能为 核苷酸依赖性。我们希望阐明这些蛋白质降解的功能意义 合作互动和相关的构象变化。 我们最近发现，酵母中的温度和氧自由基的升高会导致降解特别降解 新合成的蛋白质，而不会影响大多数细胞蛋白的分解。我们希望进一步澄清 不同泛素化酶和分子伴侣在降解中的作用，并确定 新合成的蛋白质对这种损伤最敏感。在损害细胞蛋白的危险条件下（例如 热震，氧化应激，接近冻结的温度），酵母产生大量的“化学品 伴侣，海藻糖，可以增强细胞活力。我们最近发现，海藻糖也需要 有效的蛋白质降解，即使在正常细胞中也是如此。这种意外的新保护功能的机制 将调查海藻糖的。