Upgrading the mammalian translation system to probe multi-site post-translational modifications

蛋白质是驱动生物学基础过程的分子机器。蛋白质的功能经常受到各种可逆化学修饰的调节，如赖氨酸残基的乙酰化。虽然已经在各种人类蛋白质上鉴定出许多这样的乙酰化，但它们在生物学中的作用仍然不清楚。许多人类蛋白质在多个不同的位点被乙酰化，但这些个体修饰如何促进调控仍然知之甚少。目前无法产生在一组选定位点均匀乙酰化的靶蛋白是一个主要挑战。这一限制将通过建立一种新技术来克服。这项技术将允许在人类细胞合成过程中直接修饰靶蛋白。这将使系统阐明多位点乙酰化如何调节蛋白质功能。将开发一个跨学科的夏季研究计划，以培训和激励来自大波士顿地区的高中生。在过去的二十年中，分析技术的快速发展已经鉴定出许多在多个位点乙酰化的人类蛋白质。然而，在均质乙酰化状态下生产重组蛋白的艰巨挑战一直是一个主要的障碍。遗传密码扩增技术提供了一种克服这种限制的强有力的策略，通过使用工程化的无义抑制氨酰-tRNA合成酶/tRNA对，允许N（N）-乙酰基赖氨酸（AcK）共翻译位点特异性掺入重组蛋白中。然而，目前在哺乳动物细胞中每个蛋白质掺入一个以上的AcK残基是具有挑战性的。为了解决这一限制，将对限制AcK掺入效率的哺乳动物翻译系统的组分进行系统工程改造。将开发一种新的定向进化平台，用于根据这些组件在活哺乳动物细胞中的性能对它们进行工程改造。增强的AcK掺入系统将被用来询问HMGCS 2的功能是如何通过三个不同的赖氨酸残基的乙酰化来调节的。外联工作包括针对当地高中生的暑期研究方案。这个项目叫做“你进化出一种蛋白质！(YEP!)”强调了利用实验室进化来设计具有所需特性的蛋白质。参与者将从发射绿色光的荧光蛋白开始，创造出具有改变颜色的工程变体。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

Proteins are molecular machines that drive the processes underlying biology. The function of proteins are frequently regulated by a wide-variety of reversible chemical modifications, such as acetylation of lysine residues. While numerous such acetylations have been identified on various human proteins, their role in biology remains unclear. Many human proteins are acetylated at multiple different sites, but how these individual modifications contribute to regulation remains poorly understood. The current inability to generate target proteins homogeneously acetylated at a set of chosen sites is a major challenge. This limitation will be overcome by establishing a novel technology. This technology will allow direct modification of a target protein during its synthesis in human cells. This will enable systematic elucidation of how multi-site acetylation regulates protein function. A cross-disciplinary summer research program will be developed to train and motivate high-school students from the greater Boston area. In the last two decades, rapid advances in analytical techniques have identified numerous human proteins that are acetylated at multiple sites. However, the daunting challenge of producing recombinant proteins in a homogeneous state of acetylation been a major roadblock. The genetic code expansion technology offers a powerful strategy to overcome this limitation, by allowing co-translational site-specific incorporation of N (epsilon)-acetyllysine (AcK) into recombinant proteins using an engineered nonsense suppressing aminoacyl-tRNA synthetase/tRNA pair. However, it is currently challenging to incorporate more than one AcK residue per protein in mammalian cells. To address this limitation, systematic engineering of the components of the mammalian translation system that limit the incorporation efficiency of AcK, will be carried out. A novel directed evolution platform for engineering these components based on their performance in live mammalian cells will be developed. The enhanced AcK-incorporation system will be used to interrogate how the function of HMGCS2 is regulated by the acetylation of three different lysine residues. Outreach efforts include a summer research program targeted to local high school students. This program called "You Evolve a Protein!(YEP!)" highlights the use of laboratory evolution to engineer proteins with desired properties. Starting from a fluorescent protein that emits green light, the participant will create engineered variants with altered color.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.