Conformational targeting of fibrillar polyglutamine proteins in live cells escalates aggregation and cytotoxicity.
Conformational targeting of fibrillar polyglutamine proteins in live cells escalates aggregation and cytotoxicity.
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DOI：
10.1371/journal.pone.0005727
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发表时间：
2009-05-28
期刊：
影响因子：
3.7
通讯作者：
Messer A
中科院分区：
文献类型：
--
作者：
Kvam E;Nannenga BL;Wang MS;Jia Z;Sierks MR;Messer A
作为帕金森病、亨廷顿病和马查多-约瑟夫病基础的易于错误折叠和聚集的蛋白质，即α-突触核蛋白、亨廷顿蛋白和共济失调蛋白-3，在发病过程中采用多种细胞内构象，包括球状中间体和不溶性淀粉样蛋白样原纤维。这种构象多样性使淀粉样蛋白相关的细胞内功能障碍和神经变性的研究复杂化。为此，重组单链Fv抗体（scFv）是引人注目的分子工具，其可以针对特定蛋白质构象进行选择，并在细胞内作为胞内抗体表达，用于研究和治疗目的。使用原子力显微镜（AFM）和活细胞荧光显微镜，我们报告了针对α-突触核蛋白的纤维形式选择的人scFv靶向错误折叠的聚谷氨酰胺蛋白的同构构象。当在纹状体细胞的细胞质中表达时，这种构象特异性胞内抗体与错误折叠的共济失调蛋白-3和亨廷顿蛋白的病理片段的细胞内聚集体共定位，并且增强了与疾病相关的多聚谷氨酰胺蛋白的聚集倾向。使用这种intrabody作为一种工具，用于调节淀粉样蛋白生成的动力学，我们表明，不断升级的聚集体形成的病理性亨廷顿蛋白片段是不是在纹状体细胞的细胞保护，而是加剧氧化应激和细胞死亡，通过流式细胞术检测。相反，细胞保护是通过使用先前描述的与亨廷顿蛋白的淀粉样蛋白N末端结合的胞内抗体抑制聚集来实现的。类似的细胞毒性结果观察到以下的正常或聚谷氨酰胺扩增的人共济失调蛋白-3，其中部分聚集通过非聚谷氨酰胺结构域的构象靶向。这些发现证实了聚集速率调节多聚谷氨酰胺介导的细胞内功能障碍，并警告说，专门加速聚集的分子作为多聚谷氨酰胺疾病的治疗方法可能是有害的。此外，我们的研究结果引入了一种新的基于抗体的工具，由于其对纤维状构象的一般特异性及其在细胞内发挥作用的能力，为各种人类淀粉样蛋白疾病提供了广泛的研究潜力。
Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely α-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs) are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes. Using atomic force microscopy (AFM) and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of α-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains. These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to specifically hasten aggregation may be detrimental as therapies for polyglutamine disorders. Moreover, our findings introduce a novel antibody-based tool that, as a consequence of its general specificity for fibrillar conformations and its ability to function intracellularly, offers broad research potential for a variety of human amyloid diseases.
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影响因子：
2.9
作者：
Conway, KA;Harper, JD;Lansbury, PT
通讯作者：
Lansbury, PT
影响因子：
5.3
作者：
Cong, Shu-Yan;Pepers, Barry A.;Dorsman, Josephine C.
通讯作者：
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影响因子：
5.4
作者：
Firdaus, Wance J. J.;Wyttenbach, Andreas;Arrigo, Andre-Patrick
通讯作者：
Arrigo, Andre-Patrick
DOI：
10.1073/pnas.0502698102
发表时间：
2005-08-23
影响因子：
11.1
作者：
Charvin, D;Vanhoutte, P;Caboche, J
通讯作者：
Caboche, J
影响因子：
2.4
作者：
Barkhordarian, Hedieh;Emadi, Sharareh;Sierks, Michael R.
通讯作者：
Sierks, Michael R.