Yeast prions and Hsp104 chaperone

酵母朊病毒和 Hsp104 伴侣

• 批准号: 8939502
• 负责人: Daniel Masison
• 金额: $ 33.61万
• 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/8939502
• 关键词: Affect; Amyloid; Cell Count; Cell physiology; Cells; Chimeric Proteins; Data; Dependency; Event; Fiber; Goals; Heterogeneity; Kinetics; Learning; Life; Mediating; Modeling; Molecular; Molecular Chaperones; Monitor; Pattern; Play; Population; Prions; Process; Protein Structure Initiative; Proteins; Proteolysis; Role; Seeds; Stress; System; Therapeutic; Work; Yeasts; base; in vivo; insight; monomer; overexpression; particle; prion seeds; protein aggregate; segregation; sup35; yeast prion

Hsp104 is a protein chaperone that helps cells recover from stress by resolubilizing proteins from aggregates. This disaggregation activity requires assistance of Hsp40 and Hsp70 and is necessary for fragmentation of prion fibers that underlies replication of amyloid-based yeast prions. Depleting or inactivating Hsp104 arrests prion replication, which causes yeast prions to be lost as cells divide because the number of cells eventually becomes greater than the number of infectious prion particles, or seeds. Elevating expression of Hsp104 under conditions where it is not normally induced also leads to prion loss. The mechanism of this "curing" is unknown, but differences in curing kinetics, dependency on other factors, and the inadequacy of Hsp104 disaggregation activity alone suggest it is unrelated to that of curing by Hsp104 inactivation. To assess more definitively how overexpressing Hsp104 cures cells of prions, we monitored the fate of prion particles (fluorescent foci) in live cells using a Sup35-GFP fusion protein after overexpressing Hsp104. We found a large heterogeneity in the distribution of fluorescent foci in cells of partially cured populations and distinct kinetics of prion loss that were incompatible with the mechanism being inhibition of fragmentation. We also found no differences in segregation pattern of prion seeds, ruling out asymmetric segregation of prion seeds as a possible mechanism. Our data support a mechanism by which prions are removed in a two-step process of trimming by Hsp104 to reduce their size followed by elimination, probably by proteolysis. Expressing Hsp104 is being explored as a therapeutic approach in mammalian amyloid models. Our work is providing insight into the mechanisms underlying functions of this chaperone system that might reveal unanticipated limitations or benefits of such an approach.

HSP104是一种蛋白质伴侣，可以通过从聚集体中分辨出蛋白质来帮助细胞从胁迫中恢复。这种分解活性需要HSP40和HSP70的帮助，对于基于淀粉样蛋白酵母菌prions复制的prion纤维的分裂是必需的。耗尽或灭活的HSP104会阻止prion复制，这会导致酵母菌群体随着细胞的分裂而丢失，因为细胞的数量最终变得大于感染性prion虫的数量或种子。在通常不会诱导的条件下，HSP104的表达升高也导致prion虫丧失。这种“固化”的机制尚不清楚，但是固化动力学，对其他因素的依赖以及HSP104分解活性不足的差异表明它与HSP104失活的固化无关。 为了更明确地评估过度表达HSP104如何治愈王室的细胞，我们使用SUP35-GFP融合蛋白过表达HSP104后，使用SUP35-GFP融合蛋白监测了活细胞中的prion颗粒（荧光焦点）的命运。我们发现，部分治愈群体细胞中荧光灶的分布有很大的异质性，而prion骨损失的独特动力学与机制抑制碎片的机制不相容。我们还发现，王室种子的隔离模式没有差异，排除了prion种子的不对称分离为可能的机制。我们的数据支持一种机制，通过HSP104的两步进行修剪过程，通过该机制减少了大小，然后消除了蛋白质水解。 在哺乳动物淀粉样蛋白模型中，正在探索表达HSP104的治疗方法。我们的工作正在洞悉该伴侣系统的基本功能的机制，这些机制可能揭示了这种方法的意外限制或好处。