Monitoring Early-Stage Protein Aggregation by an Aggregation Induced Emission Fluorogen
Monitoring Early-Stage Protein Aggregation by an Aggregation Induced Emission Fluorogen
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DOI：
10.1021/acs.analchem.7b02090
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发表时间：
2017-09-05
影响因子：
7.4
通讯作者：
Carver, John A.
中科院分区：
文献类型：
--
作者：
Kumar, Manjeet;Hong, Yuning;Carver, John A.
高度有序的蛋白质聚集体，称为淀粉样纤维，与广泛的疾病有关，其中许多是神经退行性疾病，例如阿尔茨海默病和帕金森病。从可溶性的功能性蛋白质到不溶性淀粉样蛋白原纤维的转变是通过一个复杂的过程发生的，该过程涉及高度动态的早期聚集体或前原纤维物质的初始生成。淀粉样蛋白探针，例如硫磺素T和刚果红，已经被用作检测溶液和组织切片中淀粉样蛋白原纤维的金标准数十年。然而，这些公认的染料不能检测到蛋白质聚集早期阶段形成的前原纤维物质的存在。已提出前纤维种类在淀粉样纤维的细胞毒性和神经退行性疾病的发病机制中起关键作用。在此，我们报告了一种新的荧光染料（双（三苯基膦）四苯乙烯（TPE-TPP））与聚集诱导的发射特性，用于监测淀粉样纤维的聚集过程。TPE-TPP荧光强度的增加仅在有序的蛋白质聚集，如淀粉样原纤维形成时观察到，而在稳定的熔融球状态或无定形聚集物质时观察不到。重要的是，TPE-TPP可以检测在原纤维形成过程中早期形成的预原纤维物质的存在。TPE-TPP表现出一个独特的光谱位移的存在下prefibrillar物种，表明这些中间体的独特结构特征。使用荧光偏振，这反映了发射实体的流动性，在原纤化过程中由各种蛋白质进行的特定寡聚途径可以辨别。此外，我们证明了TPE-TPP监测淀粉样蛋白原纤维聚集的广泛适用性，包括在不同的条件下，如在酸性pH值和升高的温度下，或在淀粉样蛋白抑制剂的存在下。
Highly ordered protein aggregates, termed amyloid fibrils, are associated with a broad range of diseases, many of which are neurodegenerative, for example, Alzheimers and Parkinsons. The transition from soluble, functional protein into insoluble amyloid fibril occurs via a complex process involving the initial generation of highly dynamic early stage aggregates or prefibrillar species. Amyloid probes, for example, thioflavin T and Congo red, have been used for decades as the gold standard for detecting amyloid fibrils in solution and tissue sections. However, these well-established dyes do not detect the presence of prefibrillar species formed during the early stages of protein aggregation. Prefibillar species have been proposed to play a key role in the cytotoxicity of amyloid fibrils and the pathogenesis of neurodegenerative diseases. Herein, we report a novel fluorescent dye (bis(triphenylphosphonium) tetraphenylethene (TPE-TPP)) with aggregation-induced emission characteristics for monitoring the aggregation process of amyloid fibrils. An increase in TPE-TPP fluorescence intensity is observed only with ordered protein aggregation, such as amyloid fibril formation, and not with stable molten globules states or amorphously aggregating species. Importantly, TPE-TPP can detect the presence of prefibrillar species formed early during fibril formation. TPE-TPP exhibits a distinctive spectral shift in the presence of prefibrillar species, indicating a unique structural feature of these intermediates. Using fluorescence polarization, which reflects the mobility of the emitting entity, the specific oligomeric pathways undertaken by various proteins during fibrillation could be discerned. Furthermore, we demonstrate the broad applicability of TPE-TPP to monitor amyloid fibril aggregation, including under diverse conditions such as at acidic pH and elevated temperature, or in the presence of amyloid inhibitors.