Lysine acylation in cellular regulation, ageing and disease.

通过定量质谱分析，在不同生物体的蛋白质组中发现了数千个赖氨酸乙酰化位点。不同的带电或不带电赖氨酸酰化的鉴定使这种翻译后修饰的复杂性达到另一个水平。酰化研究领域的主要挑战之一是区分生物学相关和不相关位点。值得注意的是，到目前为止，这些赖氨酸酰化事件中只有不到1%被功能性表征。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来研究蛋白质功能是如何通过位点特异性赖氨酸乙酰化来调节的。在酰化研究领域的一个主要缺点是，最近的定量蛋白质组学研究产生的相对比例，而不是化学计量。我们希望了解赖氨酸酰化如何影响蛋白质功能的双重系统和有针对性的方法。使用来自老年小鼠的不同组织进行的定量质谱分析，结合细胞溶质赖氨酸乙酰转移酶（KAT）的过表达研究，将显示赖氨酸乙酰化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的库，我们将分析其亚细胞定位，其相互作用伙伴和底物。迄今为止，这些研究尚未系统地进行。然而，这些都是必不可少的判断乙酰化事件的生物学影响。本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，发挥不同的调节作用的机制。我们使用遗传密码扩展概念（GCEC），将不同的赖氨酸酰化位点特异性地掺入蛋白质中，以获得高纯度，均匀酰化的蛋白质，其产量足以进行生物物理研究，包括X射线晶体学。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而不是肽的影响。底物蛋白的结构特征影响赖氨酸乙酰化和脱乙酰化。此外，赖氨酸突变为谷氨酰胺通常是赖氨酸乙酰化的不良分子模拟物。该提议的另一个关键方面是在结构上修饰赖氨酸脱乙酰酶（Sirt1 - 3、Sirt5、HDAC6）全长底物复合物。到目前为止，已知的脱乙酰酶的结构只与酰化肽的复合物。结果将澄清脱乙酰酶底物特异性的分子决定因素，并将支持开发新的脱乙酰酶抑制剂的治疗方法。

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.