Myristoylation is a post-translational protein modification which corresponds to the irreversible covalent linkage of a 14-carbon saturated fatty acid, the myristic acid, to the N-terminal glycine of an eukaryotic or viral protein. It is catalyzed by the enzyme N-myristoyltransferase (NMT).
This process is involved in directing and anchoring proteins to membranes and, as consequence, in cellular regulation, signal transduction, translocation, several viral induced pathological processes and apoptosis. Substrates of NMT include protein kinases such as the catalytic subunit of CAMP-dependent protein kinase (PK-A) and p60src, phosphatases such as calcineurin B, proteins involved in transmembrane signaling such as several guanine nucleotide-binding alpha subunits of heterotrimeric G proteins, the gag polyprotein precursors of a number of retroviruses (e.g. HIV-I and Moloney murine leukemia virus) as well as the capsid proteins of some papovaviruses and picornaviruses. Many of these proteins are involved in signalling cascades.
(The Biology and Enzymology of Protein N-Myristoylation, 2001)
(Protein N-Myristoylation, 1991)
The transfer of an acetyl group from one molecule to another is a fundamental biochemical process. Several unrelated classes of enzymes catalyze such a reaction, such as the GCN5-related N-acetyltransferases (GNAT), which catalyze the transfer of the acetyl group from acetyl coenzyme A (AcCoA, the “donor”) to a primary amine (the “acceptor”). In contrast to the acetyltransferases, members of the GNAT superfamily also catalyze the transfer of longer acyl groups from acyl-CoAs to proteins and small molecules.
In humans, myristoylation is catalyzed by two NMTs, NMT1 and NMT2: despite these enzymes not being specifically N-acetyltransferases, structure determinations of N-myristoyltransferase reveal a myristoyl-CoA binding domain with the same fold and mode of cofactor binding as those of the GNAT superfamily members. It seems likely that a common ancestral protein served as the precursor for both groups of enzymes.
NMT appears to be ubiquitous in eukaryotes and has been isolated and characterised in yeast and fungi.
(GCN5-RELATED N-ACETYLTRANSFERASES: A Structural Overview, 2000)
(Structure and functions of the GNAT superfamily of acetyltransferases, 2004)
Catalysis by NMT occurs via a sequential ordered Bi Bi mechanism. The enzyme first forms a Myristol-CoA – NMT binary complex with high selectivity for myristoyl-CoA. This complex influences interaction of NMT with peptide. A peptide substrate then binds to generate a Myristoyl-CoA-NMT-peptide ternary complex. Following the catalytic transfer of myristate from CoA to peptide substrate, free CoA is released first and the N-myristoylated protein second.
(N-Myristoyltransferase: A Novel Target, 2008)
The importance of N-Myristoylation
Myristoylation can occur during both co-translational protein synthesis and post-translationally, confers lipophilicity to protein molecules, and controls protein functions.
The myristoyl moiety can perform one or a combination of different roles in the cell:
- promote reversible membrane binding which, in conjunction with other interactions, localises a protein to a membrane,
- form an integral part of the protein tertiary structure to stabilise the conformation,
- form part of a protein–protein interaction site.
(Protein myristoylation in health and disease, 2009)
NMT1 is essential for growth and development, during which rapid cellular proliferation is required, in a variety of organisms. NMT1 is also reported to be elevated in many cancerous states, which also involve rapid cellular growth, albeit in an unwanted and uncontrolled manner. The delineation of myristoylation-dependent cellular functions is still in a state of infancy, and many of the roles of the myristoylated proteins remain to be established. For example, NMT1 plays a role in the development of cells of the leukocytic lineage, a phase of rapid growth and development.
(N-myristoyltransferase in the leukocytic development processes, 2011)
NMT appears to be over-expressed in colorectal tumours and adenocarcinomas, in ballbladder and oral squamous cell carcinomas and brain tumours. For this reason there are studies in progress on the myristoylation as a chemotherapeutic target for cancer.
In addition, several eukaryotic human pathogens such as pathogenic fungi and parasites require NMT for their survival and some viruses and bacteria use the host NMT enzyme to myristoylate their own proteins.
It is well established that the NMT substrate Src, a tyrosine kinase, has elevated activity in some human cancers and that this contributes to pathogenicity. A non-myristoylated mutant has a decreased rate of phosphorylation and suppressed kinase activity, and so is not as efficient at inducing events in proliferation.
On the other hand, NMT is important for tumor suppression too: Fus1, a tumour suppressor implicated in lung cancers, is a myristoylation target. Fus1, appears to promote apoptosis through the intrinsic apoptotic signalling pathway: non-myristoylated Fus1 loses its tumour suppressor activity and so myristoylation could play an important role in retarding lung cancer pathogenesis.
(Protein myristoylation in health and disease, 2009)
Another example of the importance of N-myristoylation in promoting apoptosis is his action on the protein BID.
The pro-apoptotic protein BID translocates from the cytosol to the mitochondrial outer membrane in response to death signals generated by Fas ligand (Fas-L) or tumor necrosis factor-alpha (TNF-α). It links proximal signals from death receptors to the core apoptotic pathway
Binding of Fas-L to a trimeric receptor (FAS/CD95) induces formation of a death-inducing signaling complex (DISC). This complex recruits, via the adaptor molecule Fas-associated death domain protein (FADD), multiple procaspase-8 molecules, resulting in autocatalytic cleavage of procaspase-8 and the formation of active caspase-8, which is followed by caspase-3-mediated apoptosis. Post-translational N-myristoylation of the pro-apoptotic molecule BID acts as an irreversible step in mitochondrial targeting of truncated BID (tBID) and is a critical event in the mitochondrial amplification loop between the death-inducing signaling complex (DISC) and caspase-3-mediated apoptosis.
(Protein N-myristoylation: Critical Role in Apoptosis and Salt Tolerance, 2000)
Myristoylation of human LanC-like protein 2 (LANCL2) is essential for the interaction with the plasma membrane and the increase in cellular sensitivity to adriamycin. 2006
Human LANCL2, also known as Testis-specific Adriamycin Sensitivity Protein (TASP), is a member of the highly conserved and widely distributed lanthionine synthetase component C-like (LANCL) protein family. Expression studies of tagged LANCL2 revealed the major localization to the plasma membrane, juxta-nuclear vesicles, and the nucleus, in contrast to the homologue LANCL1 that was mainly found in the cytosol and nucleus. We identified the unique N-terminus of LANCL2 to function as the membrane anchor and characterized the relevant N-terminal myristoylation and a basic phosphatidylinositol phosphate-binding site. Interestingly, the non-myristoylated protein was confined to the nucleus indicating that the myristoylation targets LANCL2 to the plasma membrane. Cholesterol depletion by methyl-beta-cyclodextrin caused the partial dissociation of overexpressed LANCL2 from the plasma membrane in vitro, whereas in vivo we observed an enhanced cell detachment from the matrix. We found that overexpressed LANCL2 interacts with the cortical actin cytoskeleton and therefore may play a role in cytoskeleton reorganization and in consequence to cell detachment. Moreover, we confirmed previous data that LANCL2 overexpression enhances the cellular sensitivity to the anticancer drug adriamycin and found that this sensitivity is dependent on the myristoylation and membrane association of LANCL2.