Aggregation, Inhibition, Degradation: The Cystatin C-Beta-Amyloid-Cathepsin B System

阿尔茨海默氏症或帕金森氏症等退行性神经疾病是由蛋白质异常聚集成称为“淀粉样蛋白”的沉积物引起的。淀粉样蛋白可以通过将蛋白质分解成无毒片段的酶来防止，或者通过附着在蛋白质上并防止结块的分子来防止。在这个项目中，将研究三种大脑蛋白质：(1)β-淀粉样蛋白(BA)，与阿尔茨海默病相关的沉积形成的蛋白质；(2)组织蛋白酶B，一种分解BA的酶；以及(3)胱抑素C，它可以形成淀粉样沉积，防止组织蛋白酶B降解BA，并附着到BA上，防止聚集。将探索这三种蛋白质之间的相互作用，提供关于健康和疾病脑细胞中蛋白质聚集和降解的新知识，并可能为这些疾病的更有效治疗策略提供一些线索。将为来自代表性不足群体的学生提供研究机会，并将修改本科工程学教材，以纳入更多与蛋白质折叠疾病相关的例子。淀粉样纤维是具有交叉β-折叠结构和纤维形态等物理化学性质的蛋白质聚集体。由于单个淀粉样蛋白在神经退行性疾病中的重要性，许多研究都集中在它们的聚集上。然而，蛋白质聚集不是在孤立的情况下发生的，而是在相互竞争的复杂生物环境中发生的。由胱抑素C、β-淀粉样蛋白和组织蛋白B组成的三联体构成了一个蛋白质聚集、结合和降解相互交织的网络。胱抑素C是脑脊液的一种成分，在脑脊液中它是组织蛋白酶B等蛋白酶的抑制剂。β-淀粉样蛋白是一种生物学功能未知的多肽，在阿尔茨海默病中聚集成淀粉样蛋白纤维。组织蛋白酶B降解β-淀粉样蛋白，但被胱抑素C抑制。此外，胱抑素C与β-淀粉样蛋白的结合抑制了β-淀粉样蛋白纤维的形成。因此，胱抑素C有两个相反的作用：隔离β-淀粉样蛋白和防止纤维形成，同时抑制组织蛋白酶B介导的β-淀粉样蛋白的蛋白分解。本项目的目标是(1)表征胱抑素C二聚体、寡聚体和纤维的结构和形成，(2)测量胱抑素C对β-淀粉样蛋白聚集的影响，以及(3)确定胱抑素C-β-淀粉样蛋白相互作用在组织蛋白酶B蛋白分解活性调节中的作用。这些实验数据将被用来建立一个“淀粉样蛋白调控网络”的数学模型。对这种网络的识别和对其相互作用的探索，将为健康和疾病组织中蛋白质的错误折叠、聚集和降解开辟新的研究路线。

Degenerative neurological disorders such as Alzheimer's or Parkinson's disease are caused by abnormal clumping of proteins into deposits called "amyloid". Amyloid can be prevented by enzymes that chop up the protein into nontoxic fragments, or by molecules that attach to the protein and prevent clumping. In this project, three brain proteins will be studied: (1) beta-amyloid (BA), the protein that forms deposits related to Alzheimer's disease, (2) cathepsin B, an enzyme that chops up BA, and (3) cystatin C, which can form amyloid deposits, prevents cathepsin B from degrading BA, and attaches to BA and prevents clumping. The interactions among these three proteins will be explored, providing new knowledge on protein clumping and degradation in healthy and diseased brain cells, and potentially providing some clues as to more effective treatment strategies for these diseases. Research opportunities will be provided to students from underrepresented groups, and an undergraduate engineering text will be revised to incorporate more examples related to protein folding diseases.Amyloid fibrils are protein aggregates that share physicochemical properties such as cross-beta-sheet structure and fibrillar morphology. Much research has focused on aggregation of individual amyloidogenic proteins because of their importance in neurodegenerative disorders. However, protein aggregation occurs not in isolation but in a complex biological milieu of competing interactions. The triad of cystatin C, beta-amyloid, and cathepsin B constitute a network in which protein aggregation, binding, and degradation are intertwined. Cystatin C is a constituent of cerebrospinal fluid, where it serves as an inhibitor of proteases such as cathepsin B. Cystatin C amyloid deposits are found in patients with cerebral amyloid angiopathy. Beta-amyloid is a peptide of unknown biological function that aggregates into amyloid fibrils in Alzheimer's disease. Cathepsin B degrades beta-amyloid but is inhibited by cystatin C. Furthermore, binding of cystatin C to beta-amyloid inhibits beta-amyloid fibrillogenesis. Thus, cystatin C has two opposing roles: sequestering beta-amyloid and preventing fibril formation, while inhibiting cathepsin B-mediated proteolysis of beta-amyloid. The objectives of this project are to (1) characterize the structure and formation of cystatin C dimers, oligomers, and fibrils, (2) measure the effect of cystatin C on beta-amyloid aggregation, and (3) determine the role of cystatin C- beta-amyloid interactions in regulation of cathepsin B proteolytic activity. The experimental data will be used to build a mathematical model of an "amyloid regulatory network". Identification of such a network, and exploration of its interactions, will open up new lines of inquiry in protein misfolding, aggregation, and degradation in healthy and diseased tissues.