Molecular Chaperones and Protein Degradation

分子伴侣和蛋白质降解

基本信息

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
项目类别：
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

项目摘要

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项重要功能是选择性地破坏错误折叠或受损的蛋白质。蛋白质在各种人类疾病和衰老过程中积累，我们的首要目标是进一步了解。泛素蛋白酶体途径的功能以及分子伴侣在此过程中的参与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，其中 19S 蛋白酶体是由 26S 蛋白酶体催化的。调节颗粒包含六种作为分子伴侣的 ATP 酶，以了解它们是如何发挥作用的。 ATP酶展开并将底物转移到核心20S蛋白酶体中，我们也在研究更简单的，同源 ATP 酶复合物 PAN，可激活古细菌中 20S 蛋白酶体的蛋白水解作用。我们最近发现，在 ATP 结合后，这些区域中存在一个保守的 C 端序列（HbYX 基序） ATPase 对接至 20S 外环的口袋中，就像“钥匙锁”一样触发其门控的打开一个主要目标是进一步阐明开门和底物进入的分子事件。在真核生物 26S 中，有 6 个 ATP 酶和 7 个 20S ¿ -亚基不同，门的开口仅限于两个 ATP 酶，我们希望了解哪些 C 末端对接至哪些 20S 口袋以及是否对接。其他含有 HbYX 基序的蛋白酶体调节因子（BLM10、PI131）和 p97/cdc48 激活 20S 蛋白酶体也类似。我们已将蛋白酶体降解分解为一系列需要 ATP 的步骤，并希望能够阐明我们将进一步探讨真核生物 26S 是否利用了额外的多步反应序列。我们发现六个 ATP 酶亚基相互作用。通过负协同作用并在 PAN 中鉴定出两个底物结合域，其功能是我们希望阐明这些在蛋白质降解中的功能意义。合作相互作用和相关的构象变化。我们最近发现酵母中温度升高和氧自由基会导致特定的降解新合成的蛋白质，而不影响大多数细胞蛋白质的分解，我们希望进一步阐明这一点。不同泛素化酶和分子伴侣在其降解中的作用并鉴定新合成的蛋白质对这种损伤最敏感。热休克、氧化应激、接近冰点的温度），酵母产生大量的“化学物质” 海藻糖，它可以增强细胞活力。我们最近发现，海藻糖也是细胞活力所必需的。有效的蛋白质降解，即使在正常细胞中也是如此。这种意想不到的新保护功能的机制。海藻糖的含量将被研究。