Dissecting the Structure, Thermodynamic Stability, and Aggregation Properties of the A25T Transthyretin (A25T-TTR) Variant Involved in Leptomeningeal Amyloidosis: Identifying Protein Partners That Co-Aggregate during A25T-TTR Fibrillogenesis in Cerebrospinal Fluid
Dissecting the Structure, Thermodynamic Stability, and Aggregation Properties of the A25T Transthyretin (A25T-TTR) Variant Involved in Leptomeningeal Amyloidosis: Identifying Protein Partners That Co-Aggregate during A25T-TTR Fibrillogenesis in Cerebrospinal Fluid
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DOI：
10.1021/bi201365r
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发表时间：
2011-12-27
期刊：
影响因子：
2.9
通讯作者：
Palhano, Fernando L.
中科院分区：
文献类型：
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作者：
Azevedo, Estefania P. C.;Pereira, Humberto M.;Palhano, Fernando L.
软脑膜和蛛网膜下血管中甲状腺素运载蛋白（TTR）的无定形聚集体和纤维沉积是软脑膜淀粉样变性（LA）的特征，LA是目前无法治疗的脑血管病。在此，我们报告的X-射线结构的A25 T同源四聚体TTR，一个自然的突变体中描述的患者LA。A25 T-TTR的结构与野生型TTR（wt-TTR）的结构难以区分，这表明A25 T-TTR和wt-TTR之间的淀粉样蛋白生成差异不能归因于总体结构差异。使用压力诱导解离的四聚体，我们表明，A25 T-TTR是3千卡/摩尔不稳定比L55 P-TTR，最积极的TTR突变体描述的日期。在37 ° C（pH 7.3）下孵育15天后，A25 T-TTR形成成熟的淀粉样蛋白原纤维。为了模拟TTR聚集的环境，我们研究了脑脊液（CSF）中的聚集。与L55 P-TTR不同，A25 T-TTR在CSF中快速形成淀粉样蛋白聚集体，其中包含几种蛋白质伴侣。利用蛋白质组学方法，我们鉴定了19种与A25 T-TTR淀粉样纤维共纯化的蛋白质。我们证实了先前在TTR淀粉样变性患者的活检组织中发现的与TTR聚集体相关的蛋白质的存在，如丛生蛋白、载脂蛋白E和补体蛋白。此外，我们发现了新的蛋白质，如凝血蛋白。总之，我们的结果揭示了TTR聚集的体外特征
Deposition of amorphous aggregates and fibrils of transthyretin (TTR) in leptomeninges and subarachnoid vessels is a characteristic of leptomeningeal amyloidosis (LA), a currently untreatable cerebral angiopathy. Herein, we report the X-ray structure of the A25T homotetramer of TTR, a natural mutant described in a patient with LA. The structure of A25T-TTR is indistinguishable from that of wild-type TTR (wt-TTR), indicating that the difference in amyloidogenicity between A25T-TTR and wt-TTR cannot be ascribed to gross structural differences. Using pressure-induced dissociation of the tetramer, we show that A25T-TTR is 3 kcal/mol less stable than L55P-TTR, the most aggressive mutant of TTR described to date. After incubation for 15 days at 37 degrees C (pH 7.3), A25T-TTR forms mature amyloid fibrils. To mimic the environment in which TTR aggregates, we investigated aggregation in cerebrospinal fluid (CSF). Unlike L55P-TTR, A25T-TTR rapidly forms amyloid aggregates in CSF that incorporated several protein partners. Utilizing a proteomics methodology, we identified 19 proteins that copurified with A25T-TTR amyloid fibrils. We confirmed the presence of proteins previously identified to be associated with TTR aggregates in biopsies of TTR amyloidosis patients, such as clusterin, apolipoprotein E, and complement proteins. Moreover, we identified novel proteins, such as blood coagulation proteins. Overall, our results revealed the in vitro characterization of TTR aggregation