Unravelling the regulation of beta-endorphin and other peptide hormones by N-terminal acetylation and deacetylation

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Obesity is one of several metabolic disorders, which is considered in our time as a major health issue in the EU and the Western world. Important regulators for metabolism, appetite, sexual behaviour and pain are the peptide hormones alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin (beta-END). Both hormones are regulated post-translationally by N-terminal acetylation (Nt-Ac). To date, six different N-terminal acetyltransferases (NatA-NatF) have been characterized in higher eukaryotes, responsible for co-translational acetylation of 80% of all proteins in humans. Nevertheless, the identity of the NAT (NatX) responsible for the modification of alpha-MSH and beta-END remains elusive. Identification of NatX would unravel the role and regulation of peptide hormones in obesity and several other important physiological processes that may be regulated by post-translational Nt-Ac. Furthermore, no mechanism of N-terminal deacetylation and no N-terminal deacetylase (NDAC) have been identified. Although, several indications render the possibility of the existence of NDAC(s), especially the fact that beta-END is stored in its biologically inactive form when acetylated, and thus has to be deacetylated to become active and be released. Further, recent findings demonstrate that Nt-Ac is dynamic in Saccharomyces cerevisiae upon alterations in growth. Hence, the major objectives of this project are to identify NatX as well as to reveal the existence of deacetylase activity in various lysates and to identify potential candidates for NDACs.For this purpose I will use recently developed unique bisubstrate analogues (acetyl coenzyme A chemically fused to peptides) or acetyl-peptides with photo-reactive crosslinkers to specifically bind and isolate NatX and potential NDACs. Further, I will use a global peptide library with metabolically labelled 100% N-terminal acetylated peptides and incubate it with various lysates that have a potentially high deacetylase activity. This will be followed by HPLC and mass spectrometry approaches with which I will analyse the peptides for a deacetylated moiety. By incubating these peptide analogues with the above mentioned lysates, I will be able to crosslink potential NDACs, isolate them and identify them by mass spectrometry. Altogether, these analyses hold the promise to significantly enhance our understanding of Nt-Ac and its regulatory function in so many important molecular and (patho-) physiological processes.