N-myristoylation: An Overview

N-myristoylation An OverviewN-myristoylationProtein N-myristoylation is the covalent attachment of myristate, a 14-carbon roly-poly pudding acid, onto the N-terminal genus Glycine residues of protein substrates. It is transferred co- or post-translationally to a subset of proteins from a thioester form, myristoyl-CoA, catalyzed by N-myristoyl transferases (NMTs). (insert genes expressing nmt1 and nmt2, NMT recognizes a general consensus sequence for myristoylation (Gly-X-X-X-(Ser/Thr/Cys)) containing a N-terminal glycine, 3 other amino acids and a serine, threonine or a cysteine in the fifth position.) While this process is often observed co-translationally on nascent shorter protein substrates, post-translation myristoylation ensues during apoptosis on N-terminal glycine residue exposed after caspase cleavage of protein substrates.1 An increase in proteins hydrophobicity conferred by this modification allows for weak protein-lipide and protein-protein interactions, as well as for m embrane targeting and die hard of proteins involved in signal transduction cascades.2NMTs served as therapeutic targets owing to their importance for the survival of human pathogens and their association with carcinogenesis.1 To globally profile NMT protein substrates, chemical proteomic approaches have been industrious where small tags on fatty acids such as an alkyne (YnMyr) or azide (AzMyr) (Fig. X) were developed to probe myristoylated proteins via metabolic labeling.3,4 This probe was successfully used in a high-confidence write of the co-translational myristoylome in human and zebra fish.5 Although YnMyr remains to be the probe of choice owing to its marginal background labeling6, it was demonstrated to label proteins with other known lipid-modifications such as N-myristoyl,7 S-palmitoyl8 and GPI-anchors9-compromising its specificity towards labeling of N-terminal myristoylated proteins. To circumvent the challenge of identifying the true NMT substrates, Tate et al. used an integrated chemical biology approach where selective inhibition of NMT with small-molecule inhibitors combined with YnMyr labeling and vicenary proteomics allowed for pen of much than 30 known and story protein candidates for N-myristoylation in blood-stage malaria poriferan.9 (describe that the presence of inhibitor abolished the labeling of the true substrates, which should non be enriched in control samples) This technology was likewise applied to globally profile the N-myristoylome of other human pathogens such as inLeishmania donovani,10 Trypanosoma brucei,11and freshly Trypanosoma cruzi.12Theprofile of a large set of N-myristoylated proteins with diverse cellular functions unravels the significance of this lipid modification in these parasites. Furthermore, this also validates NMT as a viable drug target in attenuating the virulence of these pathogens. Extending the selfsame(prenominal) approach to HeLa cancer cells enabled the identification of more than 100 of both co- and post-translationally modified N-myristoylated proteins, majority of which were identified at endogenous levels for the first age.13 Indeed, this robust technique proved to be powerful in lancinate on-target proteins from off-targets in a proteome-wide summary, resulting in the discovery of novel NMT protein substrates at high confidence.Although promising, the method described where NMT inhibitors were used may non be applicable to more complex systems where cell viability may be compromised, e.g. in the context of viral and bacterial infection. An alternative targeted approach tosimplify data analysis of enriched proteins employs isolating those that bear the N-terminal glycine requirement for N-myristoylation. This enabled the write of downregulated host N-myristoylated proteins upon infection with herpes simplex virus (HSV)14, as well as novel fatty-acylated proteins encoded by HSV. This same approach provided a more defined picture of the demyristoylating funct ion of the bacterial effector IpaJ upon host cell invasion of Shigella flexneri, which was determined to contribute to its virulence.15PalmitoylationProteins S-palmitoylation is the attachment of a 16-carbon long fatty acid (as palmitate-CoA) to cysteine residues, which was first discovered by radiolabeling of virus-infected cells with 3Hpalmitate.16 The formation of the thioester linkage is mediated by a family of protein acyl transferases (PATs) that bear a conserved Asp-His -His-Cys catalytic musical theme (DHHC-PATs), which can be removed by hydrolysis aided by acyl protein thioesterases (APTs).17 Owing to the reversibility of this modification, S-palmitoylation of proteins was thought to be alive(p)ally regulated ,whereby a subset of proteins are transiently palmitoylated in a certain time point/cellular activity. (insert something) S-palmitoylation has been demonstrated to be an essential mechanism for protein stability, activity, and proper cellular localization.18 Recent a dvances in identifying palmitoylated proteins revealed not only its key role in regulatory mechanisms but as well as in host invasion and virulence of pathogens.Large-scale proteomic pen of S-palmitoylated proteins employ metabolic labeling has been heavily dependent on employing the alkyne analogue of palmitic acid, 17-ODYA (Fig. X). This commercially available chemical dealer is suitable for these analyses as it has shown better specificity and has minimal background in labeling proteins that are ought to be acylated by shorter fatty alkyl chains.6 The subsequent click reaction with fluorophore- or biotin-azide then allows for in-gel fluorescence monitoring and biotin-pulldown strategy preliminary to LC-MS proteomic analysis of labeled proteins, respectively. In these studies, hundreds of palmitoylated proteins were identified with a wide range of functions, highlighting the importance of S-palmitoylation in a plethora of cellular mechanisms and pathways. For instance, the fir st report on using such strategy applied to mammalian cells identified around 125 candidate S-palmitoylated proteins at high confidence, including G proteins, receptors and uncharacterized hydrolases.19 Using the same strategy in dendritic immune cells (DC2.4) identified more than 150 predicted S-palmitoylated proteins and revealed that palmitoylation of interferon-induced transmembrane protein 3 (IFITM3)20 and Toll-like receptor 2 (TLR2)21 is essential for their antiviral activity. A more recent study on Cryptococcus neoformans revealed that a single PAT, Pfa4, palmitoylates the fungal proteins required for parasite integrity and virulence-palmitoylating 72 proteins identified in a global-scale approach.22A more quantitative approach to measure levels of palmitoylated proteins combines metabolic labeling with 17-ODYA and Stable Isotope Labeling with amino acids in Cultured Cells (SILAC). In virus-infected RPE-1 epithelial cells, selective repression was observed for host S-palmitoy lated proteins, including interferon signaling regulators and members of the tetraspanin family.14 A novel set of HSV-encoded proteins palmitoylated by the host machinery were selectively and significantly identified, further suggesting that HSV exploits the palmitoylation pathway which contributes to its virulence. As palmitoylation is a reversible process, the dynamic cycling of palmitoylated proteins in mouse T-cell hybridoma cells was investigated using this quantitative approach in combination with a pulse-chase technique.23 Through the use of a serine lipase-inhibitor as the chase, palmitoylated proteins that undergo fast turnovers were distinguished from those that are stably modified. This indicates that a subset of this dynamic palmitoylation event is regulated by serine hydrolases, validating the fundamental regulatory mechanism of depalmitoylation for proteins with rapid turnovers. It is important to note that in this study, only the insoluble protein fractions were analy zed, as the soluble proteins were not amenable to metabolic probe incorporation.19Given the dynamic nature of palmitoylation, the metabolic labeling strategy would allow labeling of only those that are palmitoylated at the time of probe treatment and were stably modified. An older approach, coined as acyl-biotin exchange (ABE), has the potential to capture the full complement of palmitoylated proteins. In this multistep procedure, the protein lysates are treated with hydroxylamine to selectively mystify the thioester bonds, followed by disulfide capture with thiol-containing biotin analogue, and subsequently enriched through a pulldown technique prior to LC-MS analysis. ABE was first utilized in tandem with semi-quantitative MudPit analysis on profiling the palmitome of Saccharomyces cerivisae.24 The 12 known and 35 new palmitoylated proteins identified presented the first evidence on the diverse specificities of PATs. The ABE method was further employed in profiling the palmitoylo me in rat neurons,25 human T cells,26 and recently in poplar tree cells,27 establishing its applicability to a wide range of biological systems.Both ABE and metabolic labeling approaches combined with SILAC revealed their large complementarity in profiling S-palmitoylated proteins in Plasmodium falciparum.28 A total of more than 400 palmitoylated proteins were identified where 202 proteins were enriched in both methods. As expected, metabolic labeling identified a lesser number of proteins, reflecting the less complexity in this approach. A pulse-chase labeling using ABE in a quantitative approach with 2-BMP as the parasite PAT inhibitor revealed the identification of a range of stably and dynamically palmitoylated proteins. Indeed, this study demonstrated the importance of palmitoylation in multiple parasite-specific processes, specifically in drug resistance, nonsexual stage development, host cell invasion, and protein export. Both methods were also employed in investigating the d ysregulation of palmitoylation in breast cancer cells by inducing Snail-overexpression- an event correlated with chemoresistance and metastasis.29 Results showed that some proteins were differentially expressed regardless of differential palmitoylation. Thus, Snail-overexpression compromises the dynamic palmitoylation of some proteins that may be involved in pathways that contribute to malignancy.Albeit most proteins are S -palmitoylated in their cysteine residues, others were reported to be O-palmitoylated30 and N-palmitoylated17, which are also labeled by 17-ODYA. To distinguish S-palmitoylated proteins from these other forms in Toxoplasma gondii, a method similar to ABE was employed which also takes advantage of the labilityof thioester bonds to hydrolysis.31 In this approach, the metabolic incorporation of 17-ODYA and enrichment is followed by hydroxylamine cleavage to profile S-palmitoylated proteins. This confirmed 282 hydroxylamine-sensitive proteins from 501 putative palmito ylated proteins enriched from the initial 17-ODYA labeling. This also revealed and validated that palmitoylation of AMA1, a protein essential for host-cell invasion, is not required on invasion but increases microneme secretion.Taken together, these studies presented underscore the utility of large-scale S-palmitome profiling in understanding the biological importance of this lipid modification. 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