| Popis: |
N-terminal modifications may constitute the first modifications any protein acquires and can modulate protein function by altering protein stability and 3D structure, promoting or interfering with protein-protein interactions and regulating membrane targeting, localisation or secretion. Here, two interlinked N-terminal protein modifications have been studied: initiator methionine (iMet) excision and co-translational N-myristoylation. N-myristoylation involves de attachment of myristic acid, a 14-carbon saturated fatty acid, onto exposed N-terminal glycines of protein substrates. In humans, this reaction is catalysed by N-myristoyltransferases 1 and 2. The two NMT isoforms share a high degree of sequence and structural similarity, which has hindered the development of isoform-specific inhibitors to date and prevented the dissection of isoform- specific substrate pools. Using CRISPR/Cas9 in combination with a previously described metabolic labelling approach and whole proteome profiling, NMT1, and not NMT2, was defined as the main enzyme responsible for N-myristoylation of proteins in the cancer cell. In addition, a novel method was designed to accurately assess on-target activity of NMT inhibitors and the fate of N-myristoylated substrates across the whole proteome upon NMT inhibition. This new approach relies on post-lysis labelling of exposed N-terminal glycines by S. aureus sortase A (SrtA) and no longer relying on metabolic labelling, it can be applied to any type of biological sample. Methionine aminopeptidases (MetAPs) catalyse initiator methionine (iMet) removal from nascent proteins and are essential to maintain healthy proteome dynamics by priming other N-terminal modifications such as N-acetylation or N-myristoylation and modulating protein localisation and stability. MetAP2 has been explored by pharmaceutical companies for decades for the treatment of cancer and obesity. However, the links between MetAP2 inhibition and phenotypic effects are still poorly understood. Here, a novel chemical proteomics workflow is proposed to elucidate the substrates of MetAP2 systematically and uncover the missing link between MetAP2 inhibition and phenotype. This new strategy is based on metabolic labelling of cells with the methionine analogue azidohomoalanine (AHA) and in combination with specific pharmacological inhibition of MetAP2, allowed identification of >70 substrates of MetAP2, 94% of which were unknown until reported here. Together, this work provides fundamental insights into the biological role and importance of N-myristoylation and iMet excision in cancer and shapes the path for future steps in the use of NMT and MetAP2 inhibitors for the treatment of human disease. Open Access |
