Extending the limits of cryo-EM to better understand TTR misfolding and aggregation

应用程序ID：9981223 授权编号：1 R21 AG 067594 -01 PI姓名：Lander，Gabriel 扩展cryo-EM的限制以更好地理解TTR错误折叠和聚集 项目摘要 天然折叠的蛋白质转化为非功能性聚集体与广泛的年龄相关的退行性疾病相关，包括阿尔茨海默病（AD）。令人极为关切的是，随着我国预期寿命的增加，这些疾病的流行率将越来越高。因此，开发预防或逆转退行性疾病中涉及的蛋白质错误折叠事件的治疗方法已成为许多研究工作的焦点。蛋白质甲状腺素运载蛋白（TTR），甲状腺素和holoretinol载体蛋白输出到脑脊液（CSF）和血清，是一个这样的蛋白质，表现出增加的倾向，采用一个非天然的折叠和形成不溶性的聚集体随着年龄的增长。该过程可以在TTR蛋白的天然野生型形式中发生，并且负责野生型TTR淀粉样变性（也称为老年系统性淀粉样变性），其引起限制性心肌病。值得注意的是，有证据表明TTR与Aβ肽相互作用，从而防止Aβ原纤维形成和聚集。研究表明，AD的CSF 与年龄匹配的非AD个体相比，患者CSF中TTR的浓度显著更低，这支持TTR的神经保护作用。我们认为年龄相关的TTR错误折叠和聚集破坏了TTR阻止Aβ原纤维形成和随后AD发病的能力。为了更好地了解天然折叠的野生型TTR蛋白如何变得易于发生错误折叠事件，我们寻求资金来开发结构方法来研究TTR的天然和异常形式。关于这种蛋白质的不稳定和非天然寡聚化的详细结构信息将深刻影响我们对TTR错误折叠和纤维形成的理解，并可能导致开发更有效的TTR稳定药物，可以恢复AD患者TTR的神经保护特性。第一个目标，我们将推动的大小和分辨率的限制，可通过冷冻电子显微镜检查的高分辨率的三维结构的不稳定TTR四聚体，并比较它们的四聚体是稳定的小分子配体，揭示如何纳入错误折叠的亚基或不正确的纳入天然折叠的亚基影响TTR稳定性在原子水平。第二个目标将定义TTR聚集体的原子精度的架构，以阐明组装途径以及不同的寡聚状态如何差异地有助于各种不同的发病机制。值得注意的是， 由于野生型蛋白质的天然折叠途径的扰动可能是AD的细胞毒性驱动因素，因此这项工作的发现可能具有超出TTR淀粉样变性的深远影响。

Appl ID: 9981223 Grant Number : 1R21 AG067594-01 PI Name : Lander, Gabriel Grant Title : Extending the limits of cryo-EM to better understand TTR misfolding and aggregation PROJECT SUMMARY The conversion of natively folded proteins into non-functional aggregates is associated with a wide range of age-related degenerative diseases, including Alzheimer’s Disease (AD). Of great concern is the prediction that these diseases will become more prevalent in prevalence as our nation’s life expectancy increases. The development of therapeutics to prevent or reverse the protein misfolding events implicated in degenerative diseases has thus become the focus of many investigative efforts. The protein transthyretin (TTR), a thyroxine and holoretinol carrier protein exported to cerebrospinal fluid (CSF) and serum, is one such protein that demonstrates increased propensity to adopt a non-native fold and form insoluble aggregates with age. This process can occur in the native, wild type form of the TTR protein, and is responsible for wild type TTR amyloidosis (also known as senile systemic amyloidosis), which causes restrictive cardiomyopathy. Notably, there is evidence that TTR interacts with the Aβ peptide, thereby preventing Aβ fibril formation and aggregation. Studies demonstrating that the CSF of AD patients contain substantially lower concentrations of TTR than in the CSF of age-matched non-AD individuals supports a neuroprotective role of TTR. We posit that age-related TTR misfolding and aggregation abolishes the capacity of TTR to prevent Aβ fibril formation and the subsequent onset of AD. In order to better understand how a natively folded wild type TTR protein becomes predisposed to misfolding events, we seek funding to develop structural approaches to study both the native and aberrant forms of TTR. Detailed structural information regarding the destabilization and non-native oligomerization of this protein will profoundly impact our understanding of TTR misfolding and fibrillogenesis, and could lead to the development of more potent TTR stabilizing drugs that could restore neuroprotective properties of TTR in AD patients. The first aim we will push the limits of size and resolution attainable by cryo-electron microscopy to examine the high-resolution three-dimensional structures of destabilized TTR tetramers and compare them to tetramers that are stabilized by small molecule ligands, revealing how the incorporation of misfolded subunits or the improper incorporation of natively folded subunits impact TTR stability at an atomic level. The second aim will define the architecture of TTR aggregates with atomic precision in order to shed light on the assembly pathways and how different oligomeric states differentially contribute to a variety of distinct pathogeneses. Notably, since perturbations of the native folding pathway of wild type proteins are the likely cytotoxic drivers of AD, the findings of this work will likely have far-reaching impact beyond TTR amyloidoses.