An expanded genetic code for native state, live labeling of prions
An expanded genetic code for native state, live labeling of prions
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天然状态的扩展遗传密码，朊病毒的实时标记
DOI：
10.1016/j.bpj.2023.11.2530
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发表时间：
2024
影响因子：
3.4
通讯作者：
Trainer T
中科院分区：
文献类型：
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作者：
Trainer T
朊病毒是一种可传播的蛋白质物质，会导致致命的神经退行性疾病。朊病毒通过一种未确定的机制传播，涉及天然细胞朊病毒蛋白（PrP C）结构转变为感染性朊病毒聚集体。用于监测朊病毒的荧光标记方法在很大程度上依赖于基于抗体的方法，这种方法避免了 PrPC 向朊病毒的结构转化，并且通常需要细胞的固定和透化。另一种方法是 PrP C 的荧光融合蛋白，仅允许受损的结构转换并改变转换材料的生化特性。当涉及到了解朊病毒生物学的纳米级时，这些标记方法产生的假象变得难以忍受。开发一种荧光标记系统至关重要，该系统可以在活体、天然条件下以对其生化特性影响最小的方式标记朊病毒。遗传密码扩展 (GCE) 利用生物正交翻译组件，通过琥珀终止密码子抑制将非天然氨基酸 (UAA) 定点整合到蛋白质中。通过使用 GCE 将与基于四嗪的点击化学相容的 UAA 掺入小鼠神经母细胞瘤细胞系和原代小鼠皮质海马神经元的 PrP C 中，我们生产了仅具有单个氨基酸修饰的荧光标记的 PrP C (PrPamb)。 PrPamb 能够转化为朊病毒，复制其相关的生化特征，同时能够在活细胞的天然条件下结合荧光标记。使用该系统，可以在原始状态下以高分辨率研究朊病毒。它非常适合与低温相关光学和电子显微镜 (cryo-CLEM) 结合使用，以查明朊病毒感染的微环境，同时通过标记过程最大限度地减少对它们的干扰。
Prions are transmissible proteinaceous agents that cause invariably fatal neurodegenerative disease. Prions propagate through an undetermined mechanism involving the structural conversion of the native cellular prion protein (PrP C) into infectious prion aggregates. Fluorescent labelling methods for monitoring prions rely heavily on antibody-based approaches, which obviate the structural conversion of PrP C into prions and often require fixation and permeabilisation of cells. The alternative, fluorescent fusion proteins of PrP C, only allow compromised structural conversion and change the biochemical characteristics of converted material. Artefacts produced by these labelling methods become intolerable when it comes to understanding the nanoscale of prion biology. It is vital to develop a fluorescent labelling system that can label prions under live, native conditions in a way that minimally affects their biochemical characteristics. Genetic code expansion (GCE) exploits bio-orthogonal translational components to site-specifically incorporate unnatural amino acids (UAAs) into proteins by amber stop codon suppression. By using GCE to incorporate a UAA compatible with tetrazine-based click chemistry into PrP C in a mouse neuroblastoma cell line and primary mouse cortico-hippocampal neurons, we have produced fluorescently labelled PrP C (PrPamb) with only a single amino acid modification. PrPamb is capable of converting into prions, replicating their relevant biochemical characteristics, while being able to bind a fluorescent label under native conditions in live cells. Using this system, prions can be investigated at high resolutions in their native state. It is ideal for use in combination with cryogenic correlative light and electron microscopy (cryo-CLEM), to pinpoint microenvironments of prion infection while minimally disturbing them via the labelling process.