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.

通过定量质谱法在不同生物体的蛋白质组中发现了数千种赖氨酸乙酰化位点。鉴定多种带电或未充电的赖氨酸酰基化的鉴定在这种翻译后修饰上增加了另一个复杂性。酰基化研究领域的主要挑战之一是区分生物学相关和无关紧要的位点。值得注意的是，到目前为止，这些赖氨酸酰化事件的少于1％的功能表征。我们使用合成的生物学，生物物理和细胞生物学方法进行结构和功能上如何通过位点特异性赖氨酸乙酰化调节蛋白质功能。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了相对比率，而不是石化的比率。我们想了解赖氨酸酰化如何在双重系统性和靶向方法中影响蛋白质功能。使用来自年龄小鼠的各种组织，携带胞质脱乙酰基酶遗传缺失的细胞（HDAC6，SIRT2和/或SIRT5）的细胞进行的定量质谱法，以及胞质赖氨酸碱性乙酰乙酰基转移酶（KATS）的过表达研究，将显示出源自溶质膜乙酰化的蛋白酶量化的蛋白质。我们建立了一个包含所有人类Kats的库，我们将分析有关其亚细胞定位，其相互作用伙伴和底物的图书馆。到目前为止，这些研究尚未进行。但是，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基化（例如棕榈酰化和琥珀酰化）如何发挥机械上不同的调节作用。我们使用遗传密码扩展概念（GCEC）将不同的赖氨酸酰基化掺入蛋白质中，以获得足以进行包括X射线晶体学（X射线晶体学）的生物物理研究的产量，以获得高度纯净的同质酰化蛋白。这种实验方法的一个主要好处是，它允许研究赖氨酸酰化在原生蛋白中而不是在肽中的影响。底物蛋白的结构特征会影响赖氨酸乙酰化及其脱染色。此外，赖氨酸对谷氨酰胺的突变通常是赖氨酸乙酰化的不良分子模仿。该建议的另一个关键方面是结构表征赖氨酸脱乙酰基酶（SIRT1-3，SIRT5，HDAC6）的全长底物复合物。到目前为止，脱乙酰基酶的结构仅因酰化肽的络合物而闻名。结果将阐明脱乙酰基酶 - 基底特异性的分子决定因素，并将支持新型脱乙酰基酶抑制剂的发展，以进行治疗方法。