In mammalian cells, N-terminal (Nt) acetylation is one of the most abundant protein modifications. It is catalysed by N-terminal acetyltransferases (NATs) and mostly occurs co-translationally. However, in contrast to the defined co-translational NATs, post-translational NATs which have crucial regulatory roles are mostly unexplored. Distinct peptide hormones regulate appetite, metabolism, sexual behaviour and pain, and their biological activity is critically modulated by post-translational Nt-acetylation. However, the identity of the NAT responsible for this modification, ‘HormNat’, is unknown, thus the molecular and physiological ramifications of this regulatory circuit remain elusive. Another example is actin, a key regulator of cell motility and cell division. The actin N-terminus is crucial for actin function and in mammals actin is modified by an unknown post-translational NAT, ‘ActNat’. Hence, the objectives of this project are to identify these human NATs acting post-translationally, and to investigate their molecular mechanisms, regulation and impact. We will identify the novel NATs by a combination of classical and newly developed in-house tools like in vitro acetylation assays, unique bisubstrate analogues, Nt-acetylation specific antibodies, and targeted mass spectrometry. Interestingly, Nt-acetylation is considered irreversible, but there is reason to believe that specific substrates are Nt-deacetylated. Elucidation of post-translational NATs and the reversible nature of Nt-acetylation would represent a new era in the field of protein and peptide regulation and identify key cellular and organismal switches.