A genetically encoded toolset to decipher the biology of post-translational modifications in the mammalian proteome

摘要 除了极少数例外，生命中所有领域的蛋白质都是用相同的二十种典型的 氨基酸积木。然而，蛋白质可获得的化学和功能空间极大地 通过各种不同的翻译后修饰(PTM)在生活系统中扩展。这些 PTM在我们生物学的各个方面都扮演着重要的角色。近年来，已知的PTM目录中 我们的蛋白质组以疯狂的速度扩张，这要归功于基于质谱学的蛋白质组学的进步。 以及相关技术。然而，绝大多数这些新的功能后果 已确定的PTM的特征仍然很差。这一深刻的知识鸿沟的核心是一个极其重要的 我们生物学的一个方面在于难以在均一修饰状态下生产真核蛋白质 用于探索PTM在体外或体内如何调节它们的性质。对于通过以下方式确定的大多数PTM MS-蛋白质组学，准确的生化来源要么未知，要么具有挑战性，如果没有 额外的多效性后果。遗传密码扩展(GCE)技术提供了一个令人兴奋的解决方案 通过使修饰残基能够与几乎任何 任何蛋白质的位置。然而，尽管它具有巨大的潜力，但这项技术在真核生物中的范围 系统仍然受到几个技术挑战的限制，包括有限的 可遗传编码的非规范氨基酸(NCAA)，其整合效率较低， 等。在过去的五年里，我们团队通过开发出 克服这些长期挑战的创新解决方案，包括：a)新的转基因平台 编码以前不可访问的NCAA，B)基于哺乳动物细胞的定向进化系统，以改进 这一机制的性能，以及C)有效地传递NCAA整合的新型病毒载体 机械连接到种类繁多的哺乳动物细胞和组织。这些进步开启了激动人心的 有机会利用这一强大的技术来系统地破译各种PTM在 人类蛋白质组。为此，我们建议在未来五年内开发新的GCE平台，以获取 新的NCAA结构类，用它们在基因上编码以前在真核生物中无法获得的PTM， 通过定向进化优化它们的效率，并用它们来破译PTMS的后果。 此外，我们将开发技术，系统地探索新的蛋白质-蛋白质相互作用触发 通过PTMS(例如，带有读取器/擦除蛋白)，按位置特定地结合两个NCAA：一个模拟 另一种含有光致交联剂。最后，通过克服长期存在的挑战， 我们将大幅推进GCE技术在哺乳动物细胞中的应用范围，这将 产生广泛而深刻的影响，远远超出这项提案的范围。

Abstract With rare exceptions, proteins in all domains of life are biosynthesized using the same twenty canonical amino acid building blocks. However, the chemical and functional space accessible to proteins are greatly expanded in living systems by a wide variety of different post-translational modifications (PTMs). These PTMs play important roles in all aspects of our biology. In recent years, the catalog of known PTMs within our proteome have expanded at a furious pace, thanks to advances in mass-spectrometry based proteomics and related technologies. However, functional consequences of the overwhelming majority of these newly identified PTMs remain poorly characterized. At the core of this deep knowledge-gap on a critically important facet of our biology lies the difficulty of producing eukaryotic proteins in a homogeneous state of modification for probing how their properties are modulated by a PTM in vitro or in vivo. For most PTMs identified through MS-proteomics, the exact biochemical origin is either unknown or challenging to reconstitute without additional pleiotropic consequences. Genetic code expansion (GCE) technology provides an exciting solution for this problem by enabling co-translational site-specific incorporation of a modified residue into virtually any site of any protein. However, despite its enormous potential, the scope of this technology in eukaryotic systems remains limited by several technical challenges, including the restricted structural diversity of noncanonical amino acids (ncAAs) that can be genetically encoded, poor efficiency of their incorporation, etc. Over the last five years, our group has greatly expanded the scope of this technology by developing innovative solutions to overcome these longstanding challenges, including: A) new platforms for genetically encoding previously inaccessible ncAAs, B) a mammalian cell-based directed evolution system to improve the performance of this machinery, and C) novel viral vectors that efficiently deliver the ncAA incorporation machinery to wide variety of mammalian cells and tissues. These advances have opened the exciting opportunity to use this powerful technology to systematically decipher the role of various PTMs observed in the human proteome. To this end, in the next five years, we propose to develop new GCE platforms to access new structural classes of ncAAs, use them to genetically encode previously inaccessible PTMs in eukaryotes, optimize their efficiency through directed evolution, and use them to decipher the consequences of PTMs. Furthermore, we will develop technology to systematically explore new protein-protein interactions triggered by PTMs (e.g., with reader/eraser proteins), by site-specifically incorporating two ncAAs: one modeling the PTM of interest and another harboring a photo-crosslinker. Finally, by overcoming longstanding challenges, we will dramatically advance the scope of the GCE technology for application in mammalian cells, which will have broad and deep impact far beyond the scope of this proposal.