Abstract
N-Myristoyltransferase (NMT) covalently attaches a C14 fatty acid to the N-terminal glycine of proteins and has been proposed as a therapeutic target in cancer. We have recently shown that selective NMT inhibition leads to dose-responsive loss of N-myristoylation on more than 100 protein targets in cells, and cytotoxicity in cancer cells. N-myristoylation lies upstream of multiple pro-proliferative and oncogenic pathways, but to date the complex substrate specificity of NMT has limited determination of which diseases are most likely to respond to a selective NMT inhibitor. We describe here the phenotype of NMT inhibition in HeLa cells and show that cells die through apoptosis following or concurrent with accumulation in the G1 phase. We used quantitative proteomics to map protein expression changes for more than 2700 proteins in response to treatment with an NMT inhibitor in HeLa cells and observed down-regulation of proteins involved in cell cycle regulation and up-regulation of proteins involved in the endoplasmic reticulum stress and unfolded protein response, with similar results in breast (MCF-7, MDA-MB-231) and colon (HCT116) cancer cell lines. This study describes the cellular response to NMT inhibition at the proteome level and provides a starting point for selective targeting of specific diseases with NMT inhibitors, potentially in combination with other targeted agents.
Highlights
N-myristoylation is the irreversible attachment of a C14 fatty acid to the N-terminal glycine of a protein, catalyzed by myristoyl CoA: protein N-myristoyltransferase (NMT).[1,2] the majority of N-myristoylation occurs cotranslationally, we recently identified at least 30 proteins that are myristoylated post-translationally during apoptosis, where cleavage of proteins by caspases reveals a new N-terminal glycine.[3]
The effect of NMT inhibition on cell proliferation and apoptosis was evaluated in HeLa cells treated with various concentrations of inhibitor 1 or with vehicle (DMSO) for 1, 3, or 7 days. 0.2 μM inhibitor 1 corresponds to the EC50 value measured by a standard metabolic activity (MTS) assay.[3]
Selective NMT inhibition is characterized by a progressive onset of cytotoxicity, and we hypothesized that this is due to the time required to turn over existing N-myristoylated proteins in cells, concurrent with evolution of the proteome toward a predominance of nonmyristoylated NMT substrates
Summary
N-myristoylation is the irreversible attachment of a C14 fatty acid to the N-terminal glycine of a protein, catalyzed by myristoyl CoA: protein N-myristoyltransferase (NMT).[1,2] the majority of N-myristoylation occurs cotranslationally, we recently identified at least 30 proteins that are myristoylated post-translationally during apoptosis, where cleavage of proteins by caspases reveals a new N-terminal glycine.[3]. HeLa cells maintain a “plateau” of residual metabolic activity (approximately 25% of untreated levels) following 3 days of inhibition even in the presence of concentrations of inhibitor 1 (from 1 μM to 10 μM) that deliver complete inhibition of N-myristoylation in cells (Supporting Information Figure 1). We applied quantitative proteomics to study proteome level effects of NMT inhibition on HeLa cells, characterize the cytotoxic phenotype, and identify top-level pathways that are modulated by NMT inhibition These data provide a starting point for future studies to decipher the mode of action of NMT inhibitors in specific disease contexts and for validation of human NMT as a therapeutic target through identification of sensitive disease subtypes or novel drug combinations
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