Lysine acylation in cellular regulation, ageing and disease.

细胞调节、衰老和疾病中的赖氨酸酰化。

• 批准号: 389564084
• 负责人: Professor Dr. Michael Lammers
• 金额: --
• 依托单位: Institut für Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/389564084?language=en
• 关键词: Lysine; acylation; cellular; regulation; ageing

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.

通过定量质谱法在多种生物体的蛋白质组中发现了数千个赖氨酸乙酰化位点。不同带电或不带电赖氨酸酰化的鉴定使这种翻译后修饰变得更加复杂。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的位点。值得注意的是，迄今为止，只有不到 1% 的赖氨酸酰化事件得到了功能表征。我们采用合成生物学、生物物理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如何通过位点特异性赖氨酸乙酰化来调节。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是相对比率而不是化学计量。我们想要了解赖氨酸酰化如何通过双重系统和靶向方法影响蛋白质功能。使用老年小鼠的不同组织进行定量质谱分析，对携带胞质脱乙酰酶（HDAC6、Sirt2 和/或 Sirt5）基因缺失的细胞进行定量质谱分析，并结合胞质赖氨酸乙酰转移酶 (KAT) 的过表达研究，将表明赖氨酸乙酰化在多大程度上是一种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，我们将对其亚细胞定位、相互作用伙伴和底物进行分析。迄今为止，这些研究尚未系统地进行。然而，这些对于判断乙酰化事件的生物学影响至关重要。该提案的另一个重要方面是了解不同的赖氨酸酰化（例如棕榈酰化和琥珀酰化）如何在机制上发挥不同的调节作用。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整合到蛋白质中，以获得高纯度、均质的酰化蛋白质，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，它可以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及其脱乙酰化。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。该提案的另一个关键方面是对赖氨酸脱乙酰酶（Sirt1-3、Sirt5、HDAC6）全长底物复合物进行结构表征。到目前为止，脱乙酰酶的结构仅了解与酰化肽的复合物。结果将阐明脱乙酰酶底物特异性的分子决定因素，并将支持用于治疗方法的新型脱乙酰酶抑制剂的开发。