Chaperone-amyloid interactions

伴侣-淀粉样蛋白相互作用

• 批准号: 7733990
• 负责人: Daniel Masison
• 金额: $ 9.44万
• 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/7733990
• 关键词: Affect; Amyloid; Amyloid fibers; Binding; Biochemical Genetics; Cells; Fiber; Fluorescence; Fractionation; Goals; Immunoblotting; In Vitro; Kinetics; Label; Methods; Molecular; Molecular Chaperones; Monitor; Neurofibrillary Tangles; Phenotype; Polymers; Prions; Property; Proteins; Reaction; Structure; System; Testing; Yeasts; amyloid formation; base; in vivo; interest; mutant

To date our genetic and biochemical studies indicate that our original Hsp70 mutant impairs prions either by promoting self-association of amyloid fibers into large aggregates or by interfering with the disentangling of amyloid fibers from such higher-order aggregates. Unlike other in vitro systems used for studying interaction of chaperones with amyloid, the system we are developing allows testing the ability of chaperones or chaotropic agents to disentangle aggregated amyloid fibers. The basis of the system is a version of a multiply tagged yeast prion domain that can be differentially labeled by fluorescent and other moieties. Fibers are labeled by either or both probes, and those containing both can be labeled either sequentially or simultaneously. Fluorescence can be used in conjunction with physical methods such as fractionation and immunoblotting to test the extent to which chaperones bind to or break fibers. Our system will also allow monitoring the action of chaperones in the disentangling of polymers from aggregates. Ultimately, products generated from in vitro reactions will be used for infecting cells to test our predictions of how chaperone interaction with amyloid affects prion infectivity. In addition to studying effect of chaperones on amyloid formed from wild type prion protein, we are interested in studying how alterations in primary structure of prion proteins can affect chaperone interactions. To this end we have been isolating mutants of prion proteins that interfere with propagation of prions formed of wild type protein or that form prions with weakened phenotypes when the wild type protein is absent.

迄今为止，我们的遗传和生化研究表明，我们的原始HSP70突变体通过促进淀粉样纤维的自我关联或通过干扰从此类高阶聚集体中淀粉样蛋白纤维的解散来损害王子。与用于研究伴侣与淀粉样蛋白相互作用的其他体外系统不同，我们正在开发的系统允许测试伴侣或交际剂脱离骨料聚集的淀粉样淀粉样蛋白纤维的能力。该系统的基础是一个倍数标记的酵母菌prion域的版本，可以用荧光和其他部分进行差异标记。纤维用两种探针都标记，并且两个探针都可以同时或同时标记。荧光可与物理方法（例如分馏和免疫印迹）结合使用，以测试伴侣与纤维结合或断裂纤维的程度。我们的系统还将允许监测伴侣在与聚合物的分离中的作用。最终，由体外反应产生的产物将用于感染细胞，以测试我们对伴侣与淀粉样蛋白如何影响prion感染性的预测。 除了研究伴侣对由野生型prion蛋白形成的淀粉样蛋白的淀粉样蛋白的作用外，我们也有兴趣研究prion蛋白主要结构的变化如何影响伴侣的相互作用。为此，我们一直在隔离干扰由野生型蛋白质形成的prions的prion蛋白的突变体，或者在不存在野生型蛋白质时形成弱的表型蛋白。