Chaperone-amyloid interactions

伴侣-淀粉样蛋白相互作用

• 批准号: 10008653
• 负责人: Daniel Masison
• 金额: $ 31.16万
• 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/10008653
• 关键词: Actins; Affect; Amyloid; Binding; Cell division; Cells; Cytoplasm; Defect; Goals; Heat-Shock Proteins 70; Human; Huntington Disease; In Vitro; Mediating; Molecular; Molecular Chaperones; Monitor; Pathology; Phenotype; PrP; Prions; Process; Property; Protein Family; Protein Structure Initiative; Proteins; Quality Control; Reaction; Reagent; Structure; System; Tissues; Toxic effect; Work; Yeasts; fibrous protein; in vivo; interest; member; polyglutamine; protein aggregate; response; segregation; yeast prion

The URE3 prion of yeast propagates in vivo as an amyloid form of the Ure2 protein. Altering function or abundance of many different chaperones and co-chaperones, or disrupting various other protein quality control (PQC) processes can disrupt URE3 propagation in a variety of ways and to varying degrees. Central among these chaperones is Hsp70, whose activity is regulated by by many co-chaperones. Sis1 and Ydj1 are essential members of the Hsp40 J-protein family of Hsp70 co-chaperones. We are investigating interactions of these and other PQC factors with each other and with amyloid-forming proteins, such as Ure2, to understand mechanisms of how cellular PQC systems have these effects on amyloid in vivo. We earlier developed and used used a reagent to monitor aggregation status of Ure2 amyloid in URE3 cells as cells are being cured by altering various PQC factors. We confirmed that some of these PQC factors cured cells of the prion by collecting disperse prion aggregates into larger structures that are less likely to be transferred during cell division. We also used this system to show human Hsp70 co-chaperone DnaJB6 (related to yeast Sis1) cures cells of URE3 prions differently by causing more gradual dispersion and solubilization of Ure2 amyloid aggregates. We continued this work with an aim to uncover molecular mechanisms of how DnaJB6 acts to mitigate toxicity of amyloidogenic polyglutamine (polyQ), whose aggregagtion is associated with tissue pathology in Huntington's disease. We found DnaJB6 bound to polyQ aggregates and sequestered them into large cytoplasmic foci in a reaction that depended on Hsp70 and actin. Such patial segregation of aggregates is recognized in the field as a broad cellular response that reduces toxicity of small disperse aggregates. Earlier we also showed that normally non-toxic PSI prions become lethal in cells with defects in Sis1, which implies that Sis1 protects cells from the toxic effects of PSI prions. Another aspect of our work focuses on how Sis1 mediates this protection. By assessing functions of various altered forms of DnaJB6, however, we showed ability of DnaJB6 to protect cells from toxicity required its ability to bind polyQ, but not to spatially segregate poly(Q) aggregates. Moreover, some versions of DnaJB6 that drove formation of large polyQ foci did not protect cells from toxicity. By separating ability to drive spatial segregation of aggergates from ability to protect cells from toxicity, these findings show that something in addition to spatial segregation can be necessary to provide protection and raise questions of when and how toxicity of protein aggregates is neutralized in such PQC processes.

酵母菌的URE3 pr在体内传播为URE2蛋白的淀粉样蛋白形式。改变许多不同的伴侣和共伴侣的功能或丰度，或破坏其他各种蛋白质质量控​​制（PQC）过程，可以以多种方式和不同程度的方式破坏URE3的传播。这些伴侣中的中心是HSP70，其活性受许多共同伴侣的调节。 SIS1和YDJ1是HSP70共同伴侣HSP40 J蛋白家族的重要成员。我们正在研究这些和其他PQC因子的相互作用，以及与淀粉样蛋白（例如URE2）的相互作用，以了解细胞PQC系统如何对体内淀粉样蛋白产生这些影响的机制。 我们早些时候开发并使用了一种试剂来监测URE2淀粉样细胞中URE2淀粉样蛋白的聚集状态，因为细胞正在通过改变各种PQC因子来治愈。我们证实，通过将分散的prion聚集物收集到较大的结构中，这些PQC因素中的一些因素固化的细胞较少而在细胞分裂期间转移。 We also used this system to show human Hsp70 co-chaperone DnaJB6 (related to yeast Sis1) cures cells of URE3 prions differently by causing more gradual dispersion and solubilization of Ure2 amyloid aggregates. 我们继续进行这项工作，目的是揭示DNAJB6如何降低淀粉样蛋白酶生成聚谷氨酰胺（PolyQ）的毒性的分子机制，淀粉样蛋白酶生成的聚集作用与亨廷顿病中的组织病理有关。我们发现DNAJB6与Polyq聚集体结合，并将其隔离为大细胞质灶，这取决于HSP70和肌动蛋白。这种骨料的这种patial隔离在该领域被认为是一种较大的细胞反应，可降低小分散骨料的毒性。 早些时候，我们还表明，在SIS1缺陷的细胞中，通常无毒的PSI Prions致死，这意味着SIS1保护细胞免受PSI Prions的毒性作用。我们工作的另一个方面集中于SIS1如何介导这种保护。 然而，通过评估DNAJB6各种改变形式的功能，我们显示了DNAJB6保护细胞免受毒性的能力，需要其结合PolyQ的能力，但不能在空间上分离poly（q）聚集体。此外，某些驱动大型PolyQ焦点形成的DNAJB6的某些版本不能保护细胞免受毒性的影响。通过将Aggergates的空间隔离与保护细胞免受毒性的能力的分离，这些发现表明，除空间隔离外，还需要在这种PQC过程中中和蛋白质聚集物的何时以及如何中和蛋白质聚集物的毒性以及如何中和。