Yeast prions and Hsp70 co-chaperones

酵母朊病毒和 Hsp70 共伴侣

• 批准号: 8741356
• 负责人: Daniel Masison
• 金额: $ 29.04万
• 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/8741356
• 关键词: Affect; Alleles; Amino Acids; Amyloid; Binding; Cell physiology; Cells; Client; Complex; Escherichia coli; Goals; Growth; Guanine Nucleotide Exchange Factors; Homologous Gene; Link; Mediating; Molecular Chaperones; Mutation; Nucleotides; Pathway interactions; Phosphotransferases; Physiological; Prions; Process; Protein Structure Initiative; Proteins; Reaction; Role; Signaling Protein; Specificity; Substrate Interaction; Temperature; Tertiary Protein Structure; Work; Yeasts; insight; mutant; overexpression; protein folding; transcription factor; yeast prion

Earlier we showed that altering the expression of wild type Hsp40s (J-proteins), nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain proteins can either enhance or impair propagation of different prions in both wild type and Hsp70 mutant cells. We also identified several mutant alleles of Hsp40s and TPR proteins that impair or enhance propagation of different prions. Our further work pointed to the fact that many, if not all, of the observed effects that these co-chaperones have on prions are mediated by the various ways they can regulate activities of Hsp70. Hsp70 has a major role in cooperating with Hsp90 in a protein folding pathway that is important for maturation of many "client" proteins in the cell, among which are many signaling proteins and transcription factors.The Hsp70 TPR co-chaperone Sti1 interacts with both Hsp70 and Hsp90 to facilitate the transfer of substrates between them. We showed earlier that the two TPR regions of Sti1 can act separately to regulate the functions of Hsp70 and Hsp90 independently. However, we provided the first direct evidence that the client folding pathway requires a pysical bridging of Hsp70 and Hsp90 by an intact Sti1 protein. We also found that Sti1 has a critical role in the curing of PSI+ prions when Hsp104 is overexpressed and that Sti1 links Hsp90 to this curing process. Work by another group on the E. coli Hsp90 homolog HtpG identified single amino acid alterations in HtpG that impair its function. Further study suggested the residues identified participate in the binding of HtpG to client substrates. To gain more insight into physiological role and degree of functional conservation of these residues we assessed the effects of making homologous alterations in the yeast Hsp90 (Hsp82). We found the mutations had effects in various cellular functions including folding of different clients, such as transcription factors and kinases, and ability to support growth at optimal and non-optimal temperature. Effects of the mutations ranged from only slight to abolishing Hsp90's ability to support growth. Although the results with mutations in Hsp82 confirm the importance of the identified residues for Hsp90 function, they did not provide direct evidence that the same residues are as critical for substrate interactions with Hsp82 as they are with HtpG. Since Hsp82 function is known to be regulated by more co-chaperones than HtpG, this difference could be related to a more complex situation for binding of substrates by Hsp82. Continuing our work on these mutations we found that none of them affected propagation of PSI+ prions, but some of them did interfere with the ability of overexpressed Hsp104 to cure cells of PSI+ prions. These results are consistent with our assertion over the years that the way Hsp104 functions in prion replication differs from the way it acts in curing cells of prions. They also are in line with our earlier work that uncovered a role for Hsp90 in the mechanism of this Hsp104 curing, and imply that continued work with these mutants will provide insight into that mechanism.

早些时候，我们表明，改变野生型HSP40（J蛋白），核苷酸交换因子（NEFS）和四肽重复（TPR）结构蛋白的表达可以增强或损害野生型和HSP70突变细胞中不同PRION的传播。我们还确定了HSP40S和TPR蛋白的几个突变等位基因，它们会损害或增强不同prions的传播。我们的进一步工作表明，这些共同伴侣对病毒的观察到的影响是许多（即使不是全部）的事实，它们是通过调节HSP70活动的各种方式来介导的。 HSP70在与HSP90合作中在蛋白质折叠途径中合作具有重要作用，这对于细胞中许多“客户”蛋白的成熟很重要，其中许多信号蛋白和转录因子包括HSP70 TPR CO-CHAPERONE STI1与HSP70和HSP90相互作用，可与HSP90相互作用，以促进它们之间的转移之间的转移。我们之前表明，STI1的两个TPR区域可以单独起作用以独立调节HSP70和HSP90的功能。但是，我们提供了第一个直接的证据，表明客户折叠途径需要通过完整的STI1蛋白将HSP70和HSP90进行刺激性桥接。我们还发现，当HSP104过表达时，STI1在PSI+ Prions的固化中具有关键作用，并且STI1将HSP90与此固化过程联系起来。 另一组在大肠杆菌HSP90同源物HTPG上的工作确定了HTPG中损害其功能的单个氨基酸的改变。进一步的研究表明，鉴定出的残基参与HTPG与客户底物的结合。为了进一步了解这些残基的生理作用和功能保护程度，我们评估了在酵母HSP90（HSP82）中进行同源改变的影响。我们发现这些突变在各种细胞功能中具有影响，包括不同客户的折叠，例如转录因子和激酶，以及在最佳和非最佳温度下支持生长的能力。突变的影响范围从仅轻微到消除HSP90支持生长的能力。尽管HSP82突变的结果证实了已鉴定的残基对HSP90功能的重要性，但它们没有提供直接的证据，表明相同的残基对于与HSP82的底物相互作用与与HTPG的相互作用至关重要。由于已知HSP82函数比HTPG更受副酮调节，因此这种差异可能与HSP82对底物结合的更复杂情况有关。继续我们对这些突变的工作，我们发现它们都没有影响PSI+ Prions的传播，但是其中一些确实干扰了过表达HSP104治愈PSI+ Prions细胞的能力。这些结果与我们多年来的断言一致，即Hsp104在prion复制中的作用与蛋固化细胞的作用方式不同。它们还与我们较早的工作一致，该工作发现了HSP90在这种HSP104固化机制中的作用，这意味着与这些突变体的继续工作将提供对该机制的见解。