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，所有生命领域的蛋白质都是使用相同的20种典型的生物合成方法合成的。 氨基酸结构单元。然而，蛋白质可及的化学和功能空间极大地 通过各种不同的翻译后修饰（PTM）在生命系统中扩展。这些 PTM在我们生物学的各个方面都发挥着重要作用。近年来，已知的PTM目录在 由于基于质谱的蛋白质组学的进步，我们的蛋白质组以惊人的速度扩展 和相关技术。然而，绝大多数这些新的功能性后果 已鉴定的PTM的特征仍然很差。在这个深刻的知识差距的核心，一个至关重要的 我们生物学的一个方面在于，很难在修饰的同质状态下产生真核蛋白质 用于探测它们的性质如何在体外或体内被PTM调节。对于通过以下方式确定的大多数PTM， MS-蛋白质组学，确切的生物化学起源是未知的或具有挑战性的重建， 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 通过使修饰的残基能够共翻译位点特异性地掺入到几乎任何 任何蛋白质的位置。然而，尽管其巨大的潜力，该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，包括有限的结构多样性， 非典型氨基酸（ncAA）可以被遗传编码，它们的掺入效率差， 等在过去的五年里，我们的团队通过开发 创新的解决方案，以克服这些长期存在的挑战，包括：A）新的平台，遗传 B）基于哺乳动物细胞的定向进化系统，以改善 该机制的性能，和C）有效递送ncAA掺入的新型病毒载体 机器到各种各样的哺乳动物细胞和组织。这些进步开启了令人兴奋的 有机会使用这种强大的技术来系统地破译各种PTM中观察到的作用， 人类蛋白质组为此，在未来五年，我们建议开发新的GCE平台， 新的ncAA结构类别，使用它们在真核生物中遗传编码以前无法获得的PTM， 通过定向进化优化它们的效率，并利用它们来破译PTM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PTM（例如，与阅读器/擦除器蛋白），通过位点特异性结合两个ncAA：一个模拟 感兴趣的PTM和另一个含有光交联剂。最后，通过克服长期存在的挑战， 我们将极大地推进GCE技术在哺乳动物细胞中的应用范围， 其广泛而深刻的影响远远超出了本提案的范围。

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.