The <i>N</i>-myristoylome of <i>Trypanosoma cruzi</i>

Protein N-myristoylation is catalysed by N-myristoyltransferase (NMT), an essential and druggable target in Trypanosoma cruzi, the causative agent of Chagas’ disease. Here we have employed whole cell labelling with azidomyristic acid and click chemistry to identify N-myristoylated proteins in different life cycle stages of the parasite. Only minor differences in fluorescent-labelling were observed between the dividing forms (the insect epimastigote and mammalian amastigote stages) and the non-dividing trypomastigote stage. Using a combination of label-free and stable isotope labelling of cells in culture (SILAC) based proteomic strategies in the presence and absence of the NMT inhibitor DDD85646, we identified 56 proteins enriched in at least two out of the three experimental approaches. Of these, 6 were likely to be false positives, with the remaining 50 commencing with amino acids MG at the N-terminus in one or more of the T. cruzi genomes. Most of these are proteins of unknown function (32), with the remainder (18) implicated in a diverse range of critical cellular and metabolic functions such as intracellular transport, cell signalling and protein turnover. In summary, we have established that 0.43–0.46% of the proteome is N-myristoylated in T. cruzi approaching that of other eukaryotic organisms (0.5–1.7%)

Global profiling of co- and post-translationally N-myristoylated proteomes in human cells

Protein N-myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. Here, we report the global N-myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N-myristoyltransferase (NMT) inhibition. Global quantification of N-myristoylation during normal growth or apoptosis allowed the identification of >100 N-myristoylated proteins, >95% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N-myristoylation is determined for >70 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N-myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells

Targeting N-myristoylation for therapy of B-cell lymphomas

N-myristoyltransferases (NMTs) target many signaling proteins to membranes. Here the authors show an NMT inhibitor named PCLX-001 selectively kills lymphoma cells by shutting down their main survival signaling pathway and offers an additional treatment strategy for lymphoma patients

Bacterial entombment by intratubular mineralization following orthograde mineral trioxide aggregate obturation: A scanning electron microscopy study

The time domain entombment of bacteria by intratubular mineralization following orthograde canal obturation with mineral trioxide aggregate (MTA) was studied by scanning electron microscopy (SEM). Single-rooted human premolars (n=60) were instrumented to an apical size #50/0.06 using ProFile and treated as follows: Group 1 (n=10) was filled with phosphate buffered saline (PBS); Group 2 (n=10) was incubated with Enterococcus faecalis for 3 weeks, and then filled with PBS; Group 3 (n=20) was obturated orthograde with a paste of OrthoMTA (BioMTA, Seoul, Korea) and PBS; and Group 4 (n=20) was incubated with E. faecalis for 3 weeks and then obturated with OrthoMTA-PBS paste. Following their treatments, the coronal openings were sealed with PBS-soaked cotton and intermediate restorative material (IRM), and the roots were then stored in PBS for 1, 2, 4, 8 or 16 weeks. After each incubation period, the roots were split and their dentin/MTA interfaces examined in both longitudinal and horizontal directions by SEM. There appeared to be an increase in intratubular mineralization over time in the OrthoMTA-filled roots (Groups 3 and 4). Furthermore, there was a gradual entombment of bacteria within the dentinal tubules in the E. faecalis inoculated MTA-filled roots (Group 4). Therefore, the orthograde obturation of root canals with OrthoMTA mixed with PBS may create a favorable environment for bacterial entombment by intratubular mineralization