New Reagents for Protein Modification

近年来，半胱氨酸氧化引起了越来越多的关注，因为越来越多的证据表明，半胱氨酸氧化在几个关键和复杂的细胞过程中起着关键作用，特别是氧化应激反应机制和氧化还原细胞信号通路。据估计，人类蛋白质组中多达12%的半胱氨酸在细胞和组织中以氧化形式存在。令人惊讶的是，仅半胱氨酸亚磺酸（Cys-SO2H，“CSA”）就被认为占人类蛋白质组中可接近（即不埋在蛋白质核心）半胱氨酸的约5%。虽然近年来越来越多的证据表明CSA作为一种调节修饰，但其生物形成和活性的全部范围仍然知之甚少。一个主要的障碍是缺乏将CSA化学纳入纯化蛋白的方法，以实现准确的生化研究。事实上，目前制备CSA的方法已经被证明是非常费力的，并且依赖于用过氧化氢或过氧酸等氧化剂处理含有感兴趣肽/蛋白（POI）的半胱氨酸硫醇（Cys-SH）。这种方法效率特别低，而且会产生氧化poi的复杂混合物，进一步阻碍了生化和生物物理分析的纯化和准确表征。为了解决这一关键问题，我们的目标是开发新的方法，通过调节半胱氨酸硫醇的亲核性，从而调节其对氧化的反应性，从而允许将CSA掺入重组蛋白中进行功能研究。这项工作计划将涉及两个主要方面：首先，它将建立设计和合成路线，以capp -氧化- uncapping一锅序列的新型半胱氨酸化学选择性试剂，以及将半胱氨酸有效转化为短肽中的CSA的生物相容性方案。其次，我们将利用这些新试剂/方案，在概念验证实验中将CSA引入一系列重组、治疗相关的蛋白质中。我们研究的一个重要重点将是初步阐明CSA对蛋白质结构、稳定性和分子识别特性的影响。总之，这些方法的成功发展将为研究CSA在人类生理和疾病中的功能和相关性提供有价值的新工具。最后，它还将为开发新一代的合成蛋白质/肽编码新的活性和功能开辟令人兴奋的途径，用于基础科学和治疗应用。

Cysteine oxidation has attracted increasing interest recently, due to cumulative evidence of its key role in several critical and complex cellular processes, notably oxidative stress response mechanisms and redox cellular signalling pathways. It is estimated that up to 12% of all cysteines in the human proteome is in an oxidised form in cells and tissues. Astonishingly, cysteine sulfinic acid (Cys-SO2H, "CSA") alone is thought to account for ~5% of accessible (i.e. not buried in the protein core) cysteines in the human proteome. While increasing evidence in recent years have suggested that CSA acts as a regulatory modification, the full scope of its biological formation and activity remains poorly understood.A major obstacle is the lack of methods to incorporate CSA chemically within purified proteins to enable accurate biochemical studies. Indeed, current methods for the preparation of CSA have proven extremely laborious and have relied on treating a cysteine thiol (Cys-SH) containing peptide/protein of interest (POI) with oxidising agents such as hydrogen peroxide or peroxyacids. Such methods are particularly inefficient and produce complex mixtures of oxidised POIs, further hampering purification and accurate characterisation in biochemical and biophysical assays.To address this critical caveat, we aim to develop new methods allowing the incorporation of CSA within recombinant proteins for functional studies, by modulating the nucleophilicity hence reactivity of the cysteine thiol towards oxidation. This programme of work will involve two main aspects: First, it will establish the design and synthetic routes towards novel cysteine chemoselective reagents in a CAPPING-OXIDATION-UNCAPPING one-pot sequence, along with biocompatible protocols for the efficient conversion of cysteine to CSA in short peptides. Second, we will then take advantage of these new reagents/protocols for the controlled introduction of CSA in an array of recombinant, therapeutically relevant proteins in proof-of-concept experiments.An important focus of our investigation will be directed at shedding initial light on the influence of CSA on the structure, stability and molecular recognition properties of proteins. Overall, the successful development of these methodologies will provide the community with a valuable new tools to investigate the function and relevance of CSA in human physiology and disease. Finally, it will also open exciting avenues for the development of new generations of synthetic proteins/peptides encoding new activities and functions, for both fundamental science and therapeutic applications.