Lysine acylation in cellular regulation, ageing and disease.

定量质谱法在多种生物的蛋白质组中发现了数以千计的赖氨酸乙酰化位点。各种带电荷或不带电荷的赖氨酸酰化的鉴定使这种翻译后修饰变得更加复杂。在酰化研究领域的主要挑战之一是区分生物相关和不相关的位点。值得注意的是，到目前为止，只有不到1%的赖氨酸酰化事件具有功能特征。我们使用合成生物学、生物物理学和细胞生物学相结合的方法，从结构和功能上研究蛋白质功能是如何被位点特异性赖氨酸乙酰化调节的。酰化研究领域的一个主要缺陷是，最近的定量蛋白质组学研究得出的是相对比率，而不是化学计量学。我们想要了解赖氨酸酰化如何在双系统和靶向方法中影响蛋白质功能。使用携带遗传缺失的细胞质去乙酰化酶（HDAC6， Sirt2和/或Sirt5）的老年小鼠的不同组织进行定量质谱分析，结合细胞质赖氨酸乙酰转移酶（KATs）的过表达研究，将显示赖氨酸乙酰化在多大程度上是蛋白质功能的全局调节剂。我们建立了一个包含所有人类kat的文库，我们将分析其亚细胞定位，其相互作用伙伴和底物。到目前为止，还没有系统地进行过这些研究。然而，这些对于判断乙酰化事件的生物学影响是必不可少的。该建议的另一个重要方面是了解不同的赖氨酸酰化，如棕榈酰化和琥珀酰化，如何发挥不同的机制调节作用。我们使用遗传密码扩展概念（GCEC）特异性地将不同的赖氨酸酰化结合到蛋白质中，以获得高纯度，均质酰化的蛋白质，其产量足以进行包括x射线晶体学在内的生物物理研究。这种实验方法的一个主要好处是，它允许研究赖氨酸酰化对天然折叠蛋白质的影响，而不是对肽的影响。底物蛋白的结构特征影响赖氨酸乙酰化和去乙酰化。此外，赖氨酸向谷氨酰胺的突变往往是赖氨酸乙酰化的不良分子模拟物。该建议的另一个关键方面是在结构上表征赖氨酸去乙酰化酶（Sirt1-3, Sirt5, HDAC6）全长底物复合物。到目前为止，去乙酰化酶的结构只知道与酰基化肽的复合物。该结果将阐明脱乙酰酶-底物特异性的分子决定因素，并将支持开发用于治疗方法的新型脱乙酰酶抑制剂。

Thousands of lysine acetylation sites have been found in the proteome of diverse organisms by quantitative mass-spectrometry. The identification of diverse charged or uncharged lysine acylations puts another level of complexity on this post-translational modification. One of the major challenges in the acylation research field is to distinguish between biologically relevant and irrelevant sites. Notably, less than 1% of these lysine acylation events were functionally characterized so far. We use a combined synthetic biological, biophysical and cell biological approach to structurally and functionally investigate how protein function is regulated by site-specific lysine acetylation. One major drawback in the acylation research field is that recent quantitative proteomics studies yielded relative ratios rather than stoichiometries. We want to understand how lysine acylation affects protein function in a dual systemic and a targeted approach. Quantitative mass spectrometry performed using diverse tissues from aged mice, of the cells carrying genetic deletions of cytosolic deacetylases (HDAC6, Sirt2 and/or Sirt5) in combination with overexpression studies of cytosolic lysine acetyltransferases (KATs) will show to which extend lysine acetylation is a global regulator of protein function. We established a library encompassing all human KATs, which we will analyse concerning its subcellular localisation, its interaction partners and substrates. These studies have sytematically not been performed so far. However, these are essential to judge the biological impact of an acetylation event.Another important aspect of this proposal is to understand how different lysine acylations, such as palmitoylation and succinylation, exert mechanistically different regulatory roles. We use the genetic code expansion concept (GCEC) to site-specifically incorporate different lysine acylations into proteins to obtain highly pure, homogenously acylated proteins in yields sufficient to conduct biophysical studies including X-ray crystallography. A major benefit of this experimental approach is, that it allows to study the impact of lysine acylation in natively-folded proteins rather than in peptides. Structural features in substrate proteins affect lysine acetylation and its deactylation. Moreover, mutation of Lysine to Glutamin is often a bad molecular mimic for lysine acetylation. Another key aspect of this proposal is to structurally characterise lysine deacetylase (Sirt1-3, Sirt5, HDAC6) full-length substrate complexes. So far, structures of deacetylases are only known for complexes with acylated peptides. The results will clarify the molecular determinants of deacetylase-substrate specificity and will support the development of novel deacetylase inhibitors for therapeutic approaches.