GO:0018377 protein myristoylation: Protein Lipidation Pathway, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0018377 protein myristoylation is the covalent attachment of a myristoyl group to a protein, typically on an N-terminal glycine after removal of the initiator methionine.
• The reaction is catalyzed by N-myristoyltransferases (NMT1 and NMT2 in humans) using myristoyl-CoA as the acyl donor.
• Myristoylation is co-translational for many proteins but can also occur post-translationally during apoptosis, and it drives membrane targeting and protein-protein interactions.
• Quality control of N-myristoylation is mediated by a glycine-specific N-degron pathway, linking myristoylation status to protein stability.
• Myristoylation is critical in innate immunity, immune signaling, and host-pathogen interactions, and is being explored for antiviral and anticancer drug development.
• Dysregulated myristoylation contributes to cancer, neurodegeneration, and viral replication, making NMTs and myristoylated proteins attractive therapeutic targets.
Description
Protein myristoylation (GO:0018377) is a lipid modification in which a 14-carbon saturated fatty acid, myristate, is covalently attached to a protein. This modification is one of the most studied forms of protein lipidation and is essential for membrane association, protein-protein interactions, and signal transduction in eukaryotic cells. The reaction is typically catalyzed by N-myristoyltransferase (NMT) enzymes, which transfer myristate from myristoyl-CoA to the N-terminal glycine of a substrate protein after the initiator methionine has been removed. Because myristoylation is irreversible and often co-translational, it serves as a stable anchor that directs proteins to specific cellular membranes and signaling complexes. Researchers study protein myristoylation to understand how cells control protein localization, immune responses, and viral replication, and to develop therapies that target this modification in cancer and infectious diseases. The importance of this process is underscored by its evolutionary conservation and by the severe phenotypes observed when NMT activity or substrate recognition is disrupted.
protein myristoylation At A Glance
| GO ID | GO:0018377 |
|---|---|
| GO term | protein myristoylation |
| Ontology | biological_process |
| Synonym | protein amino acid myristoylation |
| Definition | The covalent attachment of a myristoyl group to a protein. |
| Major function | Covalent lipid modification that targets proteins to membranes and regulates protein-protein interactions, signaling, and stability. |
| Enzymes involved | N-myristoyltransferases (NMT1, NMT2) transfer myristate from myristoyl-CoA to substrate proteins. |
| Substrate motif | Typically an N-terminal glycine after methionine removal, though internal sites exist. |
| Biological impact | Essential for innate immunity, immune signaling, viral replication, and cellular homeostasis. |
What Is GO:0018377?
According to the Gene Ontology, GO:0018377 protein myristoylation is defined as the covalent attachment of a myristoyl group to a protein. This biological process typically involves the addition of myristate (a 14-carbon fatty acid) to an N-terminal glycine residue, although internal myristoylation sites have also been reported. The modification is catalyzed by N-myristoyltransferase enzymes and uses myristoyl-CoA as the donor substrate. Protein myristoylation is distinct from other lipid modifications such as palmitoylation and prenylation in its enzymatic machinery, substrate specificity, and biological consequences.
Why Is protein myristoylation Important in Cell Biology?
Protein myristoylation is a fundamental regulatory modification that controls the localization and function of hundreds of proteins in eukaryotic cells. By anchoring proteins to membranes, myristoylation enables key signaling events in immunity, cell growth, and apoptosis. Disruption of myristoylation leads to mislocalization of critical proteins and has been linked to cancer, neurodegenerative disorders, and viral pathogenesis. Because N-myristoyltransferases are druggable enzymes, myristoylation is an active area for therapeutic development against infectious diseases and cancer.
• Myristoylation is required for the membrane targeting of many signaling proteins, including Src-family kinases and G proteins.
• It plays a central role in innate immune signaling by modifying proteins such as Myd88 and TRAM.
• N-myristoylation is essential for the replication of several viruses, including HIV-1 and parvoviruses.
• Dysregulated myristoylation contributes to cancer progression through effects on cell proliferation and survival.
• The glycine-specific N-degron pathway links myristoylation status to protein quality control and degradation.
• Myristoylation is involved in arachidonic acid metabolism and macrophage responses to interferon gamma.
• NMT inhibitors are being explored as anticancer and antiviral therapeutics.
• Myristoylation regulates protein-lipid and protein-protein interactions in diverse cellular contexts.
• It is a co-translational modification for many proteins, coupling translation and membrane targeting.
• Post-translational myristoylation occurs during apoptosis and can expose cryptic glycine residues.
What Happens During protein myristoylation?
Substrate recognition and N-terminal processing
In simple terms: First, the cell exposes a glycine at the start of the protein so myristate can be attached.
Most myristoylated proteins begin with a methionine that is removed by methionine aminopeptidases, exposing an N-terminal glycine. This glycine serves as the acceptor site for myristate. The recognition of this N-terminal glycine is a key determinant of substrate specificity, and the glycine-specific N-degron pathway monitors this exposed residue for quality control. In some cases, post-translational myristoylation can occur after caspase cleavage exposes an internal glycine, as seen during apoptosis.
Catalysis by N-myristoyltransferases
In simple terms: An enzyme called NMT grabs myristate and attaches it to the protein.
N-myristoyltransferases (NMT1 and NMT2 in humans) catalyze the transfer of myristate from myristoyl-CoA to the N-terminal glycine of the substrate protein. The reaction proceeds via a ping-pong bi-bi mechanism, forming a covalent myristoyl-enzyme intermediate before transferring the acyl group to the protein. NMTs are highly conserved and essential for viability in many organisms, highlighting the importance of this modification.
Membrane targeting and protein interactions
In simple terms: Once myristate is attached, the protein can stick to membranes and find its partners.
The myristoyl group inserts into lipid bilayers, providing a weak but stable membrane anchor that is often reinforced by additional modifications or electrostatic interactions. This membrane association is critical for the function of many signaling proteins, including Src-family kinases, G proteins, and ARF GTPases. Myristoylation also promotes specific protein-protein interactions by creating a hydrophobic surface that can be recognized by other proteins.
Quality control and degradation
In simple terms: If myristoylation fails, the cell can tag the protein for destruction.
The glycine-specific N-degron pathway recognizes unmyristoylated N-terminal glycines and targets these proteins for ubiquitin-mediated degradation. This quality control mechanism ensures that only properly myristoylated proteins accumulate, preventing mislocalized or dysfunctional proteins from interfering with cellular processes. This pathway links myristoylation directly to protein stability and turnover.
Biological outcomes in immunity and disease
In simple terms: Myristoylation helps immune cells respond to threats and can be hijacked by viruses.
Myristoylation is essential for innate immune signaling, as it targets proteins such as Myd88 and TRAM to membranes where they assemble signaling complexes. It also plays a role in the nuclear entry of viruses like the parvovirus Minute Virus of Mice. In macrophages, myristoylation is involved in arachidonic acid metabolism and responses to interferon gamma. These diverse roles make myristoylation a central node in host-pathogen interactions and immune regulation.
Key Genes Involved in GO:0018377 protein myristoylation
The following genes and proteins are central to protein myristoylation, including the enzymes that catalyze the reaction and the substrates that are modified.
| Gene | Major Role | Research Relevance |
|---|---|---|
| NMT1 | Catalyzes myristoyl transfer from myristoyl-CoA to protein substrates | Target for anticancer and antiviral drug development; knockout studies show essential roles in cell viability |
| NMT2 | N-myristoyltransferase paralog with overlapping and distinct substrate specificity | Potential redundancy with NMT1; studied in cancer and immune signaling |
| SRC | Myristoylated tyrosine kinase involved in cell growth and cancer | Model for studying membrane targeting and oncogenic signaling |
| LYN | Src-family kinase myristoylated for membrane localization | Key regulator of immune cell signaling; knockout models available |
| MYD88 | Myristoylated adaptor protein in innate immune signaling | Critical for TLR/IL-1R signaling; myristoylation required for function |
| TRAM | Myristoylated adaptor in TLR4 signaling | Studied for its role in innate immunity and inflammation |
| GNAI1 | Myristoylated G protein alpha subunit | Model for studying G protein-coupled receptor signaling |
| ARF1 | Myristoylated small GTPase regulating vesicle trafficking | Essential for Golgi function; knockout is lethal |
| ARF6 | Myristoylated GTPase involved in endocytosis and actin remodeling | Studied in cancer metastasis and membrane trafficking |
| CAPN1 | Calpain-1, post-translationally myristoylated during apoptosis | Model for post-translational myristoylation and cell death |
| BID | Pro-apoptotic protein with post-translational myristoylation | Studied in apoptosis and cancer therapy |
| MARCKS | Myristoylated protein kinase C substrate | Model for membrane-cytoskeleton interactions |
| Hck | Src-family kinase myristoylated in myeloid cells | Studied in immune cell activation and leukemia |
| Fyn | Myristoylated kinase involved in T-cell signaling | Model for immune synapse formation |
| Yes1 | Myristoylated Src-family kinase | Studied in cancer and cell adhesion |
| GNAO1 | Myristoylated G protein alpha subunit in neurons | Linked to neurodevelopmental disorders |
| NOS3 | Endothelial nitric oxide synthase, myristoylated for membrane localization | Studied in cardiovascular disease |
| PPM1B | Protein phosphatase myristoylated for membrane targeting | Model for studying myristoylation-dependent phosphatase function |
How Is protein myristoylation Regulated?
Protein myristoylation is regulated at multiple levels, including the availability of myristoyl-CoA, the expression and activity of N-myristoyltransferases, and the accessibility of substrate N-terminal glycines. NMT activity can be modulated by cellular metabolic state, and myristoyl-CoA levels fluctuate with fatty acid metabolism. The glycine-specific N-degron pathway provides a quality control layer that degrades unmyristoylated proteins, indirectly regulating the abundance of myristoylated proteins. Additionally, post-translational myristoylation during apoptosis is triggered by caspase cleavage, which exposes cryptic glycine residues. In immune cells, myristoylation of signaling proteins is dynamically regulated in response to interferon gamma and other stimuli.
protein myristoylation and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| NMT1 | Cancer, viral infections | Knockout and point-mutation cell lines to study NMT1 dependency |
| NMT2 | Cancer, immune signaling | Knockout models to assess redundancy with NMT1 |
| MYD88 | Innate immune disorders | Myristoylation-site point mutant knock-in to block membrane targeting |
| SRC | Cancer | Overexpression of myristoylated vs. non-myristoylated Src |
| ARF6 | Cancer metastasis, trafficking | Knockout and knock-in of myristoylation-deficient ARF6 |
Cancer
Dysregulated myristoylation contributes to cancer through the mislocalization and hyperactivation of oncogenic signaling proteins such as Src-family kinases. NMT1 is overexpressed in several cancers and is considered a therapeutic target. Inhibitors of N-myristoyltransferase have shown anticancer activity in preclinical models.
Viral infections
Many viruses exploit myristoylation for their replication. For example, the parvovirus Minute Virus of Mice requires myristoylation for nuclear entry. HIV-1 Nef and other viral proteins are myristoylated to interact with host membranes. Targeting myristoylation is a promising antiviral strategy.
Immune disorders and inflammation
Myristoylation is essential for innate immune signaling, and its disruption impairs responses to pathogens. Myristoylated adaptors like Myd88 and TRAM are critical for TLR signaling, and their dysfunction is linked to inflammatory diseases. Macrophage myristoylation is involved in arachidonic acid metabolism and interferon gamma responses.
Neurodegeneration
Myristoylation of neuronal proteins such as GNAO1 and Fyn is important for neuronal signaling, and defects in this process have been implicated in neurodevelopmental and neurodegenerative conditions. However, direct evidence linking myristoylation to neurodegeneration is still emerging.
From protein myristoylation-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of NMT1 affect cell viability? | NMT1 knockout cell line (e.g., HAP1 or HeLa) |
| Is myristoylation required for Myd88 signaling? | Myd88 G2A point-mutation knock-in cells |
| How does myristoylation affect Src localization? | Knock-in of tagged Src with or without myristoylation site |
| What proteins are myristoylated in a cell? | Overexpression of tagged NMT1 followed by proteomics |
| Does myristoylation regulate viral entry? | Knockout of host NMTs in virus infection models |
| Can NMT inhibitors alter immune responses? | Overexpression of NMT1 or NMT2 in immune cells |
How to Study the protein myristoylation Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Click chemistry proteomics | Global profile of myristoylated proteins | Identifying new myristoylation substrates |
| Metabolic labeling with azido-myristate | Dynamic changes in myristoylation | Imaging and tracking myristoylated proteins |
| NMT activity assay | Enzymatic transfer of myristate | Inhibitor screening and kinetics |
| Site-directed mutagenesis (G2A) | Requirement of myristoylation for function | Studying substrate-specific effects |
| Knockout/knockdown of NMTs | Loss-of-function phenotypes | Assessing essentiality in cancer and infection |
| Immunoprecipitation and Western blot | Protein expression and modification status | Validating myristoylation of candidate proteins |
| Fluorescence microscopy | Subcellular localization | Determining membrane targeting |
| Viral infection assays | Viral replication and entry | Testing myristoylation dependence |
Proteomic identification of myristoylated proteins
Myristoylome profiling using click chemistry or acyl-biotin exchange coupled with mass spectrometry allows global identification of myristoylated proteins. These methods often use alkynyl-myristate analogs that can be conjugated to tags for enrichment and detection.
Metabolic labeling and imaging
Metabolic labeling with azido-myristate followed by click chemistry and fluorescent tagging enables visualization of myristoylated proteins in cells. This approach is useful for studying dynamic changes in myristoylation during signaling or infection.
Genetic and pharmacological perturbation
Knockout or knockdown of NMT1 and NMT2, combined with NMT inhibitors, helps determine the functional consequences of myristoylation loss. Point mutations that remove the myristoylation site (e.g., Gly-to-Ala) are commonly used to study substrate-specific effects.
Biochemical assays for NMT activity
In vitro NMT activity assays using recombinant enzymes and peptide substrates measure the transfer of myristate from myristoyl-CoA. These assays are used for inhibitor screening and kinetic studies.
How CRISPR Can Be Used to Study GO:0018377 protein myristoylation
Knockout
CRISPR knockout of NMT1, NMT2, or specific myristoylated substrate genes allows researchers to study loss-of-function phenotypes, including effects on cell viability, signaling, and viral replication. Knockout cell lines are valuable for validating drug targets and understanding redundancy between NMT paralogs.
Point Mutation
Introducing point mutations that abolish myristoylation, such as Gly-to-Ala at the N-terminus, is a powerful way to dissect the specific contribution of myristoylation to protein function without deleting the entire gene. These models are particularly useful for studying membrane targeting and protein-protein interactions.
Knock-in
Knock-in of tagged or fluorescently labeled myristoylated proteins enables real-time tracking of localization and dynamics in live cells. Knock-in of disease-associated mutations can also model human disorders linked to myristoylation defects.
Overexpression
Overexpression of NMT1, NMT2, or myristoylated substrates is used to study gain-of-function effects, including oncogenic transformation and immune activation. Overexpression models are also valuable for biochemical purification and interaction studies.
How EDITGENE Supports protein myristoylation Research
Researchers studying protein myristoylation-related genes often need to determine whether a candidate gene is causally involved in a specific biological process or disease. CRISPR-based models provide a robust way to test causality by precisely manipulating the genome. EDITGENE offers a comprehensive suite of services to support these studies, from knockout to knock-in and library screening.
Contact EDITGENE today to design your custom CRISPR model for protein myristoylation research.
Frequently Asked Questions About protein myristoylation
What is protein myristoylation?
Protein myristoylation is the covalent attachment of a myristoyl group to a protein, typically on an N-terminal glycine, catalyzed by N-myristoyltransferases.
What genes are involved in protein myristoylation?
Key genes include NMT1 and NMT2, which encode the enzymes that catalyze the reaction, as well as substrate genes such as SRC, MYD88, and ARF1.
What is the GO term for protein myristoylation?
The Gene Ontology term is GO:0018377, defined as the covalent attachment of a myristoyl group to a protein.
How does myristoylation affect protein function?
Myristoylation targets proteins to membranes and promotes protein-protein interactions, which are essential for signaling, immunity, and viral replication.
What enzymes catalyze protein myristoylation?
N-myristoyltransferases (NMT1 and NMT2 in humans) catalyze the transfer of myristate from myristoyl-CoA to substrate proteins.
Is myristoylation reversible?
Myristoylation is generally considered irreversible, unlike palmitoylation, and serves as a stable membrane anchor.
What diseases are linked to myristoylation?
Myristoylation is linked to cancer, viral infections, immune disorders, and potentially neurodegeneration.
How can I study myristoylation in the lab?
Common methods include click chemistry proteomics, metabolic labeling, NMT activity assays, and CRISPR knockout of NMT genes.
What is the role of myristoylation in immunity?
Myristoylation is required for innate immune signaling by targeting adaptors like Myd88 and TRAM to membranes.
Can myristoylation be targeted therapeutically?
Yes, NMT inhibitors are being developed as anticancer and antiviral agents.
Conclusion
Protein myristoylation (GO:0018377) is a fundamental lipid modification that controls protein localization, signaling, and stability. Its roles in immunity, cancer, and viral infection make it a high-priority research area. CRISPR-based models and advanced proteomic methods are accelerating our understanding of this process and its therapeutic potential.
References
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- 3. Udenwobele DI et al.. 2017. Myristoylation: An Important Protein Modification in the Immune Response.. Front Immunol 8:751 PMID: 28713376
- 4. Giglione C et al.. 2022. Mapping the myristoylome through a complete understanding of protein myristoylation biochemistry.. Prog Lipid Res 85:101139 PMID: 34793862
- 5. Aderem AA. 1988. Protein myristoylation as an intermediate step during signal transduction in macrophages: its role in arachidonic acid metabolism and in responses to interferon gamma.. J Cell Sci Suppl 9:151-67 PMID: 3151694
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- 8. Yang S et al.. 2022. Protein Myristoylation Plays a Role in the Nuclear Entry of the Parvovirus Minute Virus of Mice.. J Virol 96(17):e0111822 PMID: 35950857
