Site-specific N-terminal labelling of proteins in vitro and in vivo using N-myristoyl transferase and bioorthogonal ligation chemistry.
ABSTRACT N-Myristoyl transferase-mediated modification with azide-bearing substrates is introduced as a highly selective and practical method for in vitro and in vivo N-terminal labelling of a recombinant protein using bioorthogonal ligation chemistry.
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ABSTRACT: Protein prenyltransferases catalyze the attachment of C15 (farnesyl) and C20 (geranylgeranyl) groups to proteins at specific sequences localized at or near the C-termini of specific proteins. Determination of the specific protein prenyltransferase substrates affected by the inhibition of these enzymes is critical for enhancing knowledge of the mechanism of such potential drugs. Here, we investigate the utility of alkyne-containing isoprenoid analogs for chemical proteomics experiments by showing that these compounds readily penetrate mammalian cells in culture and become incorporated into proteins that are normally prenylated. Derivatization via Cu(I) catalyzed click reaction with a fluorescent azide reagent allows the proteins to be visualized and their relative levels to be analyzed. Simultaneous treatment of cells with these probes and inhibitors of prenylation reveals decreases in the levels of some but not all of the labeled proteins. Two-dimensional electrophoretic separation of these labeled proteins followed by mass spectrometric analysis allowed several labeled proteins to be unambiguously identified. Docking experiments and density functional theory calculations suggest that the substrate specificity of protein farnesyl transferase may vary depending on whether azide- or alkyne-based isoprenoid analogs is employed. These results demonstrate the utility of alkyne-containing analogs for chemical proteomic applications.Chemical Biology & Drug Design 10/2010; 76(6):460-71. · 2.28 Impact Factor
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ABSTRACT: Myristoylation corresponds to the irreversible covalent linkage of the 14-carbon saturated fatty acid, myristic acid, to the N-terminal glycine of many eukaryotic and viral proteins. It is catalyzed by N-myristoyltransferase. Typically, the myristate moiety participates in protein subcellular localization by facilitating protein-membrane interactions as well as protein-protein interactions. Myristoylated proteins are crucial components of a wide variety of functions, which include many signalling pathways, oncogenesis or viral replication. Initially, myristoylation was described as a co-translational reaction that occurs after the removal of the initiator methionine residue. However, it is now well established that myristoylation can also occur post-translationally in apoptotic cells. Indeed, during apoptosis hundreds of proteins are cleaved by caspases and in many cases this cleavage exposes an N-terminal glycine within a cryptic myristoylation consensus sequence, which can be myristoylated. The principal objective of this review is to provide an overview on the implication of myristoylation in health and disease with a special emphasis on post-translational myristoylation. In addition, new advancements in the detection and identification of myristoylated proteins are also briefly reviewed.Biochimie 11/2010; 93(1):18-31. · 3.02 Impact Factor