Lysine acylation in cellular regulation, ageing and disease.

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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.