Probing the biochemical mechanism of amyloid disease

我们试图了解甲状腺素运载蛋白（TTR）聚集的过程，以及聚集的结构 导致TTR淀粉样疾病中有丝分裂后组织的功能障碍并最终死亡。 了解这种结构-蛋白毒性关系将使新的治疗药物的开发成为可能。 策略，以及建立量化治疗反应的早期诊断生物标志物。的突变 在一个TTR等位基因中产生包含突变型和野生型（WT）TTR亚基的不稳定的异源四聚体 从肝脏分泌，其表现出更快的限速四聚体解离和单体错误折叠， 提供一系列聚集结构，包括与常染色体显性遗传家族性淀粉样纤维相关的淀粉样纤维， TTR淀粉样心肌病还有散发性WT TTR心肌病，影响多达10%的 老年男性人口。在一项评估tafamlitazone（一种结合并 稳定天然TTR四聚体，防止新合成的TTR的错误折叠和聚集）， 治疗TTR心肌病，心脏中的淀粉样蛋白负荷在治疗的时间尺度上没有可检测到的变化。 他伐他汀的临床反应，使得心脏的淀粉样蛋白浸润不太可能是 mortality.在具体目标1中，我们将使用心脏病理学重新审视人类心脏的病理学。 方法来探测存在的非天然TTR结构、宿主分子与 其中聚集体是共定位的，辨别聚集体是在心脏细胞内部还是外部， 表征免疫细胞如巨噬细胞的浸润，并探测是否存在任何可辨别的 自主神经系统缺陷另一个关键问题是为什么WT TTR会聚合？我们将测试 一个较少的群体的动力学不太稳定的假设，交替折叠TTR四聚体形式 在非最佳细胞折叠期间或在失败的溶酶体降解期间产生，提供TTR 不稳定并导致聚集的四聚体。在具体目标2中，我们将使用抗体和肽 从多发性神经病患者血浆中分离非天然TTR的探针，以表征 与着陆器合作，用原子力显微镜和低温电子显微镜观察天然结构 实验室，以测试不同的聚集体结构驱动组织向性的假设。我们将比较 来自V30M血浆（纯多发性神经病）的聚集体与来自纯多发性神经病的聚集体的结构 心肌病，以评估其对DRG神经元与心肌细胞的相对细胞毒性，以测试以下观点： 这些疾病的组织嗜性是基于聚集体结构的。与科埃略集团，我们将继续 通过研究V30M携带者发展为多发性神经病来提高多发性神经病的早期诊断 使用无偏倚血浆蛋白质组学的患者。蛋白质组学也将被用来试图理解为什么 的TTR多发性神经病患者对任何批准的治疗均无反应，主要集中在炎症和 免疫调节分子和细胞可能成为多发性神经病的驱动因素。

We seek to understand how the process of transthyretin (TTR) aggregation, and which aggregate structures lead to the dysfunction and ultimately the death of post-mitotic tissue in the TTR amyloid diseases. Understanding this structure-proteotoxicity relationship would enable the development of novel therapeutic strategies, and the establishment of early diagnostic biomarkers that quantify response to therapy. A mutation in one TTR allele results in destabilized heterotetramers comprising mutant and wild type (WT) TTR subunits being secreted from the liver, which exhibit faster rate-limiting tetramer dissociation and monomer misfolding, affording a spectrum of aggregate structures, including amyloid fibrils linked to autosomal dominant familial TTR amyloid cardiomyopathy. There is also sporadic WT TTR cardiomyopathy, affecting as much as 10% of the elderly male population. In a clinical trial assessing the potential of tafamidis (a drug that binds to and stabilizes the native TTR tetramer preventing misfolding and aggregation of newly synthesized TTR) for treating TTR cardiomyopathy, the amyloid load in the heart did not detectably change on the timescale of the tafamidis clinical response, rendering it unlikely that amyloid infiltration of the heart is the main driver of mortality. In Specific Aim 1, we will take a fresh look at the pathology of the human heart using the CLARITY method to probe the 3D relationships between the non-native TTR structures present, the host molecules to which the aggregates are colocalized, discern whether aggregates are inside or outside heart cells, characterize infiltration by immune cells such as macrophages, and probe whether there are any discernable autonomic nervous system deficiencies. Another key question is why does WT TTR aggregate? We will test the hypothesis that a minor population of a less kinetically stable, alternatively folded TTR tetramer forms during non-optimal cellular folding or is produced during failed lysosomal degradation, affording a TTR tetramer that is unstable and leads to aggregation. In Specific Aim 2, we will use antibodies and peptide probes to isolate non-native TTR from polyneuropathy patient plasma to characterize the circulating non- native structure(s) by atomic force microscopy and cryo-electron microscopy in collaboration with the Lander Lab, to test the hypothesis that distinct aggregate structures drive tissue tropism. We will compare the structure of aggregates from V30M plasma (a pure polyneuropathy) vs aggregates from a pure cardiomyopathy to assess their relative cytotoxicities to DRG neurons vs cardiomyocytes, to test the idea that the tissue tropism of these diseases is aggregate structure-based. With the Coelho group, we will continue to improve early polyneuropathy diagnosis by studying V30M carriers as they progress to polyneuropathy patients using unbiased plasma proteomics. Proteomics will also be used to attempt to understand why ≈ 30% of TTR polyneuropathy patients do not respond to any approved therapies, focusing on inflammatory and immune-modulatory molecules and cells that may become drivers of polyneuropathy.