Yeast prions and Hsp70 chaperones

酵母朊病毒和 Hsp70 伴侣

• 批准号: 8553385
• 负责人: Daniel Masison
• 金额: $ 18.63万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553385
• 关键词: ATPase Domain; Affect; Am 80; Amyloid; Amyloidosis; Binding; Cell physiology; Cells; Cellular Stress; Data; Degradation Pathway; Disease; Genome; Goals; Homologous Gene; Human; Mediating; Molecular Chaperones; Mutation; Organism; Phenotype; Play; Point Mutation; Prions; Process; Protein Isoforms; Proteins; Regulation; Role; Saccharomyces cerevisiae; Specific qualifier value; Stress; Structure; Structure-Activity Relationship; Substrate Interaction; System; Work; Yeasts; base; cell growth; chaperone machinery; design; insight; protein degradation; protein folding; protein structure; small molecule; yeast prion

We are using a yeast system we constructed earlier that allows us to evaluate function of any Hsp70 isoform in yeast. We are using this system to investigate Hsp70 homologs within and across species with regard to cell growth and propagation of different prions. This very sensitive system gives us the unique ability to distinguish exquisite functional differences among nearly identical Hsp70 isoforms and provides a means to approach the problem of uncovering the underlying mechanisms. The constitutively expressed S. cerevisiae Hsp70 isoforms Ssa1 and Ssa2 are 98% identical and their stress-inducible counterparts Ssa3 and Ssa4 share 88% identity and are 80% identical to Ssa1/2. Using our system we have identified structural regions between Ssa1 and Ssa2 that underlie the differences in the way these isoforms function in both prion propagation and protein degradation. Having identified the Hsp70 regulatory ATPase domain as responsible for the differences, we then identified a specific residue of this domain (G83 or A83) as solely responsible. We show enzymatic activities of Ssa1p and Ssa2p to be nearly the same, suggesting the difference in phenotypes is probably conferred by differences in how this residue of Ssa1p and Ssa2p influences interaction with one or more of the many co-chaperones that regulate Hsp70. We further found that this same residue defined a functional distinction between Ssa1p and Ssa2p in a vacuolar-mediated protein degradation pathway. The data show how an exquisitely fine difference in structure can determine functions of nearly identical Hsp70s and imply that regulation of Hsp70 substrate interactions, rather than substrate binding per se, is the important parameter that determines functional distinctions. Our findings show that the ATPase domain is more than a regulatory domain in that it also specifies function, and provide insight into how nearly identical Hsp70s can behave differently not only in the processes that yeast prions need to grow and replicate, but also in important cellular processes. Our work emphasizes the importance of subtle differences in Hsp70 activity, and reveals that the smallest difference in structure of proteins whose functions overlap in many ways is all that is needed to direct them to perform specialized tasks in the cell. Continued work focuses on determining whether differences in functions of various Hsp70s are mediated by differences in the way they interact physically and functionally with co-chaperones that regulate Hsp70 activity. Additionally, our yeast system is being modified to study similar differences among human Hsp70s and several of their co-chaperone partners.

我们正在使用先前构建的酵母系统，该系统使我们能够评估酵母中任何HSP70同工型的功能。我们正在使用该系统来研究物种内部和整个物种中的HSP70同源物，以进行不同prions的细胞生长和传播。这个非常敏感的系统使我们具有独特的能力，可以区分几乎相同的HSP70同工型之间的精美功能差异，并提供了一种方法来解决揭示潜在机制的问题。组成型表达的酿酒酵母HSP70同工型SSA1和SSA2相同98％，其应力诱导型SSA3和SSA4具有88％的身份，与SSA1/2相同80％。 使用我们的系统，我们已经确定了SSA1和SSA2之间的结构区域，这些区域是这些同工型在prion传播和蛋白质降解中起作用的差异的基础。在确定HSP70调节ATPase域是负责差异的原因之后，我们将该域的特定残基（G83或A83）完全负责。我们显示SSA1P和SSA2P的酶促活性几乎相同，这表明表型的差异可能是由于SSA1P和SSA2P的残基如何影响与调节HSP70的许多共芯酮的一种或多种相互作用的差异所赋予的。我们进一步发现，相同的残基定义了在液泡介导的蛋白质降解途径中SSA1P和SSA2P之间的功能区别。该数据显示结构上的精细差异如何确定几乎相同的HSP70的功能，这意味着HSP70底物相互作用而不是底物结合本身的调节是决定功能区别的重要参数。我们的发现表明，ATPase域不仅仅是一个调节域，因为它也指定了功能，并且可以深入了解几乎相同的HSP70在酵母菌prions在酵母菌prions中的表现如何有所不同，而在酵母菌prions则需要成长和复制的过程中，而且在重要的细胞过程中。我们的工作强调了HSP70活动中细微差异的重要性，并揭示了蛋白质结构的最小差异以许多方式重叠的蛋白质结构是指向他们指导他们在单元格中执行专业任务所需的一切。 持续的工作着重于确定各种HSP70功能的差异是否是由它们与调节HSP70活性的副酮在物理和功能上相互作用的方式上介导的。此外，我们正在修改我们的酵母系统，以研究人类HSP70及其几个伴侣伙伴之间的类似差异。