For lipid synthesis, energy production via ␤ -oxidation, or for protein fatty acylation to occur, long-chain fatty acids (LCFAs) must be activated by conversion to their CoA derivatives (LCFA-CoAs) by fatty acyl-CoA synthetase (FAS ). Protein fatty acylation is one of many types of posttranslational modifi cations of proteins by lipids, which also includes isoprenoids, glycosylphosphatidylinositols, and cholesterol. Typically, lipids covalently attached to proteins serve as hydrophobic membrane anchors ( 1-6 ).Protein fatty acylation is mainly divided into two categories: N-myristoylation and S-acylation. The corresponding reactions are catalyzed by N-myristoyl transferases (NMT1 and NMT2) and two families of protein acyltransferases (PATs) referred to as zinc fi nger, Asp-His-His-Cys PATs Abstract Progress in understanding the biology of protein fatty acylation has been impeded by the lack of rapid direct detection and identifi cation methods. We fi rst report that a synthetic -alkynyl-palmitate analog can be readily and specifi cally incorporated into GAPDH or mitochondrial 3-hydroxyl-3-methylglutaryl-CoA synthase in vitro and reacted with an azido-biotin probe or the fl uorogenic probe 3-azido-7-hydroxycoumarin using click chemistry for rapid detection by Western blotting or fl at bed fl uorescence scanning. The acylated cysteine residues were confi rmed by MS. Second, -alkynyl-palmitate is preferentially incorporated into transiently expressed H-or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with -alkynyl-myristate or -alkynyl-stearate, via an alkali sensitive thioester bond. Third, -alkynyl-myristate is specifi cally incorporated into endogenous co-and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of -alkynylpalmitate and -alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with -alkynyl-palmitate does not affect membrane association of N-Ras. Furthermore, the palmitoylation of endogenous proteins including H-and N-Ras could be easily detected using -alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The -alkynylmyristate and -palmitate analogs used with click chemistry

Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, is critical for membrane targeting and cell signaling. Because cancer cells often have increased N-myristoyltransferase (NMT) expression, NMTs were proposed as anti-cancer targets. To systematically investigate this, we performed robotic cancer cell line screens and discovered a marked sensitivity of hematological cancer cell lines, including B-cell lymphomas, to the potent pan-NMT inhibitor PCLX-001. PCLX-001 treatment impacts the global myristoylation of lymphoma cell proteins and inhibits early B-cell receptor (BCR) signaling events critical for survival. In addition to abrogating myristoylation of Src family kinases, PCLX-001 also promotes their degradation and, unexpectedly, that of numerous non-myristoylated BCR effectors including c-Myc, NFκB and P-ERK, leading to cancer cell death in vitro and in xenograft models. Because some treated lymphoma patients experience relapse and die, targeting B-cell lymphomas with a NMT inhibitor potentially provides an additional much needed treatment option for lymphoma.

Myristoylation occurs cotranslationally on nascent proteins and post-translationally during apoptosis after caspase cleavages expose cryptic myristoylation sites. We demonstrate a drastic change in the myristoylated protein proteome in apoptotic cells, likely as more substrates are revealed by caspases. We show for the first time that both N-myristoyltransferases (NMTs) 1 and 2 are cleaved during apoptosis and that the caspase-3- or -8-mediated cleavage of NMT1 at Asp-72 precedes the cleavage of NMT2 by caspase-3 mainly at Asp-25. The cleavage of NMTs did not significantly affect their activity in apoptotic cells until the 8 h time point. However, the cleavage of the predominantly membrane bound NMT1 (64%) removed a polybasic domain stretch and led to a cytosolic relocalization (>55%), whereas predominantly cytosolic NMT2 (62%) relocalized to membranes when cleaved (>80%) after the removal of a negatively charged domain. The interplay between caspases and NMTs during apoptosis is of particular interest since caspases may not only control the rates of substrate production but also their myristoylation rate by regulating the location and perhaps the specificity of NMTs. Since apoptosis is often suppressed in cancer, the reduced caspase activity seen in cancer cells might also explain the higher NMT levels observed in many cancers.

Myristoylation, the addition of a 14-carbon fatty acid to the N-terminal glycine of a protein, is key to protein-membrane and protein-protein interactions. Typically, myristoylation occurs cotranslationally; however, post-translational myristoylation of caspase-cleaved proteins is now emerging as a well-established protein modification and as a novel regulator of apoptosis. To identify additional post-translationally myristoylated proteins, we engineered a plasmid vector encoding for a caspase-cleavable reporter protein named tandem reporter assay for myristoylation of proteins post-translationally (TRAMPP). pTRAMPP consists of tdTomato-DEVD-"test myristoylation sequence"-enhanced green fluorescent protein (EGFP). After induction of apoptosis, the reporter protein is cleaved by caspases, which frees a new N-terminal glycine residue attached to EGFP that can be myristoylated. We used pTRAMPP in appropriately transfected cells to identify 7 post-translationally myristoylated proteins. First, we confirmed the post-translational myristoylation of two previously identified putative substrates, cytoplasmic dynein intermediate chain 2A and PKCε (ctPKCε), and identified 5 more caspase-cleaved potential substrates for myristoylation that include the antiapoptotic regulator of apoptosis, Mcl-1, and the causative agent of Huntington's disease, huntingtin protein. Further investigation revealed that post-translationally myristoylated ctPKCε localized to membranes and increased Erk signaling and degradation of the proapoptotic protein Bim, which prevented a significant loss of mitochondrial potential of 17% over nonmyristoylated ctPKCε in HeLa cells in the presence of apoptotic stimuli. Taken together, these findings suggest a possible antiapoptotic role for post-translationally myristoylated caspase-cleaved ctPKCε.

Background: GDC-0980 is a selective small molecule inhibitor of class I PI3K and mTOR pathway with a potent anti-proliferative activity. Objective: We set out to evaluate the efficacy of GDC-0980, in pre-clinical studies, against pediatric leukemia cells. Methods: The anti-neoplastic activity of GDC-0980 was evaluated in vitro using five different pediatric leukemia cells. Results: Our data show that GDC-0980 significantly inhibited the proliferation of leukemia cell lines, KOPN8 (IC50, 532 nM), SEM (IC50,720 nM), MOLM-13 (IC50,346 nM), MV4;11 (IC50,199 nM), and TIB-202 (IC50, 848 nM), compared to normal control cells (1.23 µM). This antiproliferative activity was associated with activation of cellular apoptotic mechanism characterized by a decrease in Bcl-2 protein phosphorylation and enhanced PARP cleavage. Western blot analyses of GDC-0980 treated cells also showed decreased phosphorylation levels of mTOR, Akt and S6, but not ERK1/2. Notably, FLT3 phosphorylation was decreased in Molm-13 and MV4;11 cells following the application of GDC-0980. We further examined cellular viability of GDC-0980-treated primary leukemia cells isolated from pediatric leukemia patients. This study revealed a potential therapeutic effect of GDC-0980 on two ALL patients (IC50’s, 1.23 and 0.625 µM, respectively). Drug combination analyses of GDC-0980 demonstrated a synergistic activity with the MEK inhibitor Cobimetinib (MV4-11; 11, CI, 0.25, SEM, CI, 0.32, and TIB-202, CI, 0.55) and the targeted FLT3 inhibitor, Crenolanib (MV4-11; 11, CI, 0.25, SEM, CI, 0.7, and TIB-202, CI, 0.42). Conclusion: These findings provide initial proof-of-concept data and rationale for further investigation of GDC-0980 in selected subgroups of pediatric leukemia patients.