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Cystatin C Amyloid Mechanism, Interaction with Amyloid-Beta, and Regulatory Implications for Alzheimer's Disease

机译：胱抑素C淀粉样蛋白的机制，与淀粉样蛋白β的相互作用，以及对阿尔茨海默氏病的调控意义

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摘要

Alzheimer's disease (AD) is the most common form of dementia, affecting over five million Americans today. There are currently no approved drugs which are shown to effectively modify the progression of the disease. Hundreds of clinical trials attempting drug intervention have failed over the past decade, a testament to the complexity of AD pathology. In the absence of effective treatment, the number of Americans suffering from the disease is expected to rise to around 16 million by 2050.;In the amyloid cascade hypothesis, soluble amyloid-beta (Abeta) spontaneously aggregates into highly beta-sheet-rich oligomers and fibrils. Fibrils associate and precipitate as amyloid plaque on brain tissue and within blood vessel walls. Soluble oligomers and fibrils are thought to be neurotoxic, initiating a series of events leading to formation of neurofibrillary tangles, loss of synapse activity, and neuronal cell death. Intervention strategies are currently focused on reducing Abeta generation, enhancing Abeta clearance, or binding soluble Abeta to prevent aggregation.;Cystatin C (CysC) is a 13.4 kDa protease inhibitor which is highly secreted into the cerebrospinal fluid (CSF). CysC performs many housekeeping functions within the CSF, including regulating tissue degradation and cell death. CysC has also been reported to bind Abeta and inhibit aggregation. In vivo studies have found that CysC not only co-deposits with Abeta in AD plaques, but polymorphisms in the CST3 gene encoding CysC are correlated to risk of developing late-onset AD. In vitro binding studies between CysC and Abeta lack mechanistic detail, and little has been reported about the nature of this interaction.;In addition to its anti-amyloidogenic role in binding Abeta, a specific mutation of CysC (L68Q) is known to cause Hereditary Cystatin C Amyloid Angiopathy (HCCAA), another amyloid disease affecting the vasculature of the central nervous system. Fibrillation was proposed by others to be a consequence of CysC dimerization via a domain-swapping mechanism. We hypothesized that CysC amyloid propensity is related to its strong interaction with Abeta. We investigated the CysC fibril-forming mechanism in order to further understand this interaction.;We first developed a protocol for producing and purifying recombinant human CysC in E. coli, using an intein-based affinity tag for ease of purification. The self-cleavable chemistry of the intein tag allowed a simple, one-step preparation procedure to isolate full-length CysC. Upon purification, we found CysC was a mixture of monomers and oligomers. We separated and further characterized the oligomers. The oligomers were not domain-swapped, which is the first reported instance of oligomerization without domain-swapping in CysC. We also found that non-swapped oligomeric CysC had pre-amyloid characteristics and inhibited Abeta aggregation much more potently than monomeric CysC.;We next investigated the CysC fibrillation mechanism. The propagated domain-swapping (PDS) hypothesis, wherein CysC associates into fibrils via an open-ended domain-swapping fashion, has been used to explain CysC fibrillation for nearly 20 years. We produced a single-point mutant of CysC, V57N, that retains native folded structure but does not undergo domain-swapping. V57N formed fibrils faster than wild-type, contradicting the PDS hypothesis. Furthermore, non-swapped oligomers were resistant to fibril formation. We conclude that CysC fibrillation likely initiates from the monomeric state and is related to the stability of the native protein fold, and does not fibrillize via propagated domain-swapping.;CysC and Abeta interaction in vivo is likely modified by other proteins, notably cathepsin B (CatB). CatB is a cysteine-type protease typically found in acidic cellular compartments, but which can be transferred to the CSF through secretory pathways or by apoptosis. Because CatB retains some of its activity in the neutral pH of CSF, CysC has the important job of specifically and potently inhibiting leaked CatB. CatB is known to degrade soluble Abeta into non-aggregating fragments, forming a natural Abeta-clearance mechanism.;We modeled the CysC/CatB/Abeta interaction by considering binding, degradation, and aggregation reactions. We performed in vitro experiments to measure critical equilibrium and kinetic rate constants. We found that our model could accurately predict the aggregation behavior of the ternary system in vitro. We developed a dimensionless parameter to describe the ratio of characteristic degradation and aggregation timescales, and found that the steady-state model solution was controlled by this dimensionless parameter.;Researchers studying disease-modifying therapies in animal models can easily calculate this dimensionless parameter and predict aggregation behavior, simply by knowledge of typical CysC/CatB/Abeta levels in the model. This model can be extended to consider more protein interactions relevant to AD pathology. Additionally, it can be adapted to include an Abeta generation step, in which CatB is also known to play a role.
机译：阿尔茨海默氏病（AD）是痴呆症的最常见形式，目前已影响超过500万美国人。目前尚无批准的药物能有效改变疾病的进展。在过去十年中，数以百计的尝试药物干预的临床试验都以失败告终，这证明了AD病理学的复杂性。在缺乏有效治疗的情况下，到2050年，罹患该疾病的美国人人数预计将增加到1600万左右。在淀粉样蛋白级联假设中，可溶性淀粉样β（Abeta）自发聚集为高度富含β折叠的低聚物。和原纤维。原纤维在淀粉样蛋白斑块上结合并沉淀在脑组织上和血管壁内。可溶性低聚物和原纤维被认为具有神经毒性，引发一系列事件，导致神经原纤维缠结的形成，突触活性的丧失和神经元细胞的死亡。干预策略目前集中在减少Abeta的产生，增强Abeta的清除或结合可溶性Abe​​ta以防止聚集。胱抑素C（CysC）是一种13.4 kDa的蛋白酶抑制剂，高度分泌到脑脊液（CSF）中。 CysC在CSF中执行许多管家功能，包括调节组织降解和细胞死亡。也有报道称CysC结合Abeta并抑制聚集。体内研究发现，CysC不仅与AD斑块中的Abeta共同沉积，而且编码CysC的CST3基因的多态性与发生迟发性AD的风险有关。 CysC和Abeta之间的体外结合研究缺乏机理细节，关于这种相互作用的性质几乎没有报道。;除其在结合Abeta中具有抗淀粉样蛋白作用外，已知CysC（L68Q）的特定突变可导致遗传性胱抑素C淀粉样血管病（HCCAA），另一种影响中枢神经系统脉管系统的淀粉样疾病。其他人提出原纤化是通过域交换机制使CysC二聚化的结果。我们假设CysC淀粉样蛋白的倾向与其与Abeta的强相互作用有关。为了进一步了解这种相互作用，我们研究了CysC原纤维形成的机制。我们首先开发了一种在大肠杆菌中生产和纯化重组人CysC的方案，使用基于内含蛋白的亲和标签进行纯化。内含子标签的可自我切割的化学性质使得可以通过简单的一步制备程序来分离全长的CysC。纯化后，我们发现CysC是单体和低聚物的混合物。我们分离并进一步表征了低聚物。寡聚体不进行域交换，这是在CysC中首次报道的不进行域交换的低聚反应。我们还发现，未交换的寡聚CysC具有淀粉样前特性，并且比单体CysC更有效地抑制Abeta聚集。我们接下来研究了CysC的原纤化机制。 CysC通过开放域结构域交换方式缔合为原纤维的传播结构域交换（PDS）假设已被用于解释CysC的原纤维形成近20年。我们生产了CysC，V57N的单点突变体，该突变体保留了天然的折叠结构，但不进行域交换。 V57N形成原纤维的速度比野生型更快，这与PDS假设相反。此外，未交换的低聚物对原纤维形成有抵抗力。我们得出结论，CysC的原纤维形成可能是从单体状态开始的，并且与天然蛋白质折叠的稳定性有关，并且不通过传播的结构域交换来原纤维形成。；体内的CysC和Abeta相互作用可能被其他蛋白质（尤其是组织蛋白酶B）修饰（CatB）。 CatB是半胱氨酸型蛋白酶，通常在酸性细胞区室中发现，但可以通过分泌途径或凋亡将其转移至CSF。由于CatB在CSF的中性pH中保留了其某些活性，因此CysC在特异性和有效地抑制泄漏的CatB方面起着重要的作用。已知CatB可将可溶性Abe​​ta降解为非聚集片段，形成天然的Abeta清除机制。我们通过考虑结合，降解和聚集反应对CysC / CatB / Abeta相互作用进行建模。我们进行了体外实验，以测量临界平衡和动力学速率常数。我们发现我们的模型可以准确地预测三元系统在体外的聚集行为。我们开发了一个无量纲的参数来描述特征降解和聚集时间尺度的比率，发现稳态模型解决方案受该无量纲的参数控制。研究动物模型中的疾病改变疗法的研究人员可以轻松地计算出该无量纲的参数并进行预测聚集行为，只需了解模型中典型的CysC / CatB / Abeta水平即可。可以扩展该模型以考虑与AD病理相关的更多蛋白质相互作用。另外，它可以适用于包括Abeta生成步骤，其中CatB也起着重要作用。

著录项

  • 作者

    Perlenfein, Tyler John.;

  • 作者单位

    The University of Wisconsin - Madison.;

  • 授予单位 The University of Wisconsin - Madison.;
  • 学科 Bioengineering.
  • 学位 Ph.D.
  • 年度 2017
  • 页码 231 p.
  • 总页数 231
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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