S-Myristoylation: Functions, Regulation, and Experimental Insights

What is S-Myristoylation?

S-myristoylation is a vital and distinct post-translational modification entailing the covalent binding of a myristoyl group, a 14-carbon saturated fatty acid, to specific proteins. This process forms an amide linkage between the N-terminal glycine of the target protein and the myristoyl group, resulting in the creation of a thioester bond.

S-myristoylation is a reversible modification that significantly impacts protein behavior and function within cells. It plays a critical role in determining protein localization, membrane association, and interactions with other cellular constituents. This modification serves as a fundamental regulatory mechanism in cell biology, influencing a wide array of cellular processes and signaling pathways. A comprehensive understanding of S-myristoylation is essential for grasping its significance in maintaining cellular balance and its implications for various biological functions.

Schematic representation of N-myristoylation of proteins.

The Process of S-Myristoylation

1. Protein Synthesis: The process of S-myristoylation initiates with the synthesis of the target protein. This synthesis unfolds within the cell's ribosomes, where the genetic information encoded in mRNA orchestrates the assembly of a polypeptide chain. Once the polypeptide chain forms, it may undergo additional modifications to transform into a functional protein.
2. N-terminal Glycine Recognition: To undergo S-myristoylation, a protein must feature a specific glycine residue at its N-terminus. This glycine acts as the attachment site for the myristoyl group. Typically, the N-terminal glycine is part of a distinct motif, which the enzymes participating in myristoylation recognize.
3. Myristoyl-CoA Synthesis: Within the cell's cytoplasm, myristic acid, a 14-carbon fatty acid, undergoes activation by binding to coenzyme A (CoA), forming myristoyl-CoA. This activation step is executed by an enzyme called myristoyl-CoA synthetase. Myristoyl-CoA serves as the donor molecule for the subsequent attachment of the myristoyl group.
4. N-Myristoyltransferase (NMT) Catalysis: The pivotal enzyme in S-myristoylation is N-Myristoyltransferase (NMT). NMT recognizes the N-terminal glycine of the target protein and catalyzes the transfer of the myristoyl group from myristoyl-CoA to this glycine residue. This transfer occurs through the formation of a covalent amide bond between the myristoyl group and the glycine.
5. Membrane Association: Once the myristoyl group attaches to the N-terminal glycine, the protein becomes myristoylated. The myristoyl group serves as a hydrophobic tail, rendering the protein more lipophilic. This hydrophobic tail facilitates the association of the myristoylated protein with the lipid bilayer of cell membranes. The interaction between the myristoyl group and the lipid bilayer secures the protein to the cell membrane.

The association of myristoylated proteins with the cell membrane assumes critical importance in ensuring their proper functionality. Numerous signaling proteins, kinases, and G-proteins, for instance, rely on S-myristoylation for their localization to the plasma membrane. This localization is indispensable for their interactions with other proteins and receptors, ensuring they are in the correct position to carry out their cellular roles.

S-myristoylation can be reversible in certain instances, allowing specific enzymes to remove the myristoyl group. This reversibility is pivotal for the precise control of protein activity and localization in cellular processes.

Biological Significance of S-Myristoylation

Membrane Association and Targeting

Anchoring to Membranes: S-myristoylation plays a central role in anchoring proteins to cellular membranes. The attachment of a myristoyl group enables proteins to interact with the hydrophobic lipid bilayer, thereby determining their spatial distribution within the cell.

Subcellular Localization: The precise localization of proteins within cells is governed by S-myristoylation. This modification ensures that proteins are correctly positioned at the membrane, where they are needed for specific functions.

Cellular Signaling Pathways

Regulation of Signaling Events: S-myristoylation is intricately linked with cellular signaling pathways. It often targets key components of these pathways, allowing for precise spatial and temporal control of signaling events. In doing so, it influences the initiation, propagation, and termination of intracellular signaling cascades, ensuring the specificity and fidelity of cellular responses to extracellular cues.

Role in Protein-Protein Interactions

Facilitating Protein Interactions: S-myristoylation significantly impacts protein-protein interactions. Myristoylated proteins can act as scaffolds or anchors, bringing other proteins into close proximity, thus facilitating specific interactions. These interactions are essential for the formation of multi-protein complexes vital for diverse cellular functions, including cell adhesion, cell motility, and intracellular transport.

Localization and Compartmentalization

Determination of Protein Localization: S-myristoylation serves as a pivotal determinant of protein localization within cells. It ensures that proteins are in the right place at the right time, aiding them in fulfilling their intended functions. This is particularly critical for proteins that need to translocate between various cellular compartments, such as the nucleus, cytoplasm, and organelles.

Disease Implications

Cancer and Dysregulation: Dysregulated S-myristoylation has been associated with various diseases, including cancer. When oncoproteins undergo aberrant S-myristoylation, it can contribute to the uncontrolled growth of cancer cells and tumor formation. Understanding the biological significance of S-myristoylation is crucial for identifying potential therapeutic targets and interventions for these diseases.

Regulatory Mechanisms

Fine-Tuned Control: The precise regulation of S-myristoylation is essential for maintaining cellular homeostasis. The modification can be dynamically controlled through phosphorylation events, interactions with regulatory proteins, or other post-translational modifications. Unraveling these regulatory mechanisms provides valuable insights into the fine-tuned control of cellular processes.

Regulation and Control of S-Myristoylation

Phosphorylation as a Regulatory Mechanism

• Phosphorylation of Myristoylated Proteins: Protein kinases can phosphorylate myristoylated proteins, influencing their interaction with cellular membranes and other proteins. This phosphorylation can either enhance or inhibit the association of myristoylated proteins with membranes, depending on the specific phosphorylation events and the kinases involved.
• Kinase-Myristoylation Crosstalk: The interplay between protein kinases and S-myristoylation is highly complex. myristoylation can impact the localization and activity of kinases, while kinase activity can reciprocally influence S-myristoylation. For instance, the phosphorylation of a myristoylated protein can lead to its relocalization within the cell or affect its interactions with other proteins.

Dynamic Nature of S-myristoylation

• Reversibility: The reversible nature of S-myristoylation is a noteworthy feature. Myristoyl groups can be enzymatically removed from proteins by specific enzymes, allowing proteins to adjust their subcellular localization and function in response to changing conditions.
• Adaptability to Cellular Demands: Cells can fine-tune the myristoylation status of proteins to adapt to variations in environmental cues. For instance, during cellular responses to extracellular signals, S-myristoylation can be rapidly added or removed from specific proteins, leading to alterations in their subcellular localization and function.

Diseases and S-myristoylation

• Cancer Implications: Dysregulated S-myristoylation, especially in oncoproteins, can contribute to cancer progression. Aberrant myristoylation can lead to the hyperactivity of proteins involved in cell cycle regulation, apoptosis, or signaling pathways, resulting in uncontrolled cell growth and tumor formation.
• Neurodegenerative Disorders: In some neurodegenerative diseases, altered S-myristoylation of specific proteins can disrupt cellular homeostasis, potentially influencing protein aggregation and toxicity in neurons. These disruptions may contribute to the pathogenesis of neurodegenerative disorders.

Therapeutic Implications

• Targeting Dysregulated S-myristoylation: Given the involvement of S-myristoylation in various diseases, there is growing interest in targeting this modification for therapeutic interventions. Small molecules and chemical probes are being developed to modulate S-myristoylation in the context of disease.
• Precision Medicine: Precision medicine approaches may capitalize on our understanding of S-myristoylation, potentially leading to the development of targeted therapies tailored to individual patients based on their unique genetic and molecular profiles.

Experimental Techniques for S-Myristoylation Analysis

The study of S-myristoylation relies on a diverse array of experimental techniques that enable the identification, quantification, and functional analysis of myristoylated proteins. These techniques contribute to our understanding of how S-Myristoylation impacts various cellular processes.

Mass Spectrometry for S-Myristoylation Detection

• Myristoylated Protein Identification: Mass spectrometry (MS) is a pivotal technique for identifying S-myristoylated proteins. It involves enzymatic cleavage of the myristoyl group from the modified proteins followed by MS analysis. This technique allows for the precise identification of myristoylated protein substrates.
• Quantification and Stoichiometry: MS can be utilized to quantify the extent of S-myristoylation on specific proteins, providing insights into the stoichiometry of the modification. Quantitative proteomic approaches, such as SILAC (Stable Isotope Labeling by Amino acids in Cell culture), can be combined with MS for accurate quantification.
• High-Resolution Mass Spectrometry: Recent advancements in high-resolution mass spectrometry have significantly improved the sensitivity and accuracy of S-myristoylation detection. This technology permits the identification of low-abundance myristoylated proteins and the precise determination of the exact site of myristoylation.

In Vitro and In Vivo Assays

• In Vitro Assays: Researchers often employ in vitro assays to study the enzymatic machinery involved in S-myristoylation. These assays typically include recombinant myristoyltransferases, substrate proteins, and myristoyl-CoA. They help decipher the enzymatic kinetics and substrate specificity of S-myristoylation reactions.
• In Vivo Assays: In vivo assays are crucial for understanding the physiological relevance of S-myristoylation. Transfection or gene knockdown techniques are used to manipulate the myristoylation status of specific proteins in living cells or model organisms. Subsequently, the impact on protein localization and function can be assessed.
• Imaging Techniques: Fluorescence microscopy and other imaging techniques are often employed in conjunction with in vivo assays to visualize the impact of S-myristoylation on protein localization in real-time. Techniques like confocal microscopy and super-resolution microscopy provide detailed subcellular localization information.

Genetic and Chemical Manipulations

• Genetic Approaches: Genetic manipulation, including the generation of knockout or knock-in models, is a powerful strategy for studying the in vivo role of S-Myristoylated proteins. CRISPR/Cas9 technology allows for precise genome editing to create models that mimic the loss or gain of S-myristoylation.
• Chemical Probes and Inhibitors: Small molecules and chemical inhibitors targeting the S-myristoylation process are under development. These compounds can be used to modulate the myristoylation status of proteins, providing insights into the functional consequences of altered myristoylation.

Crosslinking and Protein-Protein Interaction Studies

• Crosslinking Agents: Crosslinking reagents can be utilized to stabilize transient protein-protein interactions facilitated by S-myristoylation. These agents help capture and identify interacting partners.
• Co-Immunoprecipitation (Co-IP): Co-IP assays are routinely used to confirm protein-protein interactions mediated by S-myristoylation. These experiments involve the immunoprecipitation of a myristoylated protein followed by the identification of associated partners using mass spectrometry or immunoblotting.

Subcellular Fractionation

• Subcellular Fractionation Techniques: Subcellular fractionation is essential for studying the localization of S-myristoylated proteins within specific cellular compartments. These methods entail the separation of organelles and membrane fractions, followed by the analysis of myristoylated proteins in each fraction. This provides insights into the subcellular distribution of modified proteins.

The use of these experimental techniques contributes to our comprehensive understanding of the S-myristoylation process. It aids in the identification of myristoylated proteins, quantification of their modification, and the exploration of their functional roles in various cellular processes.

Reference

1. Udenwobele, Daniel Ikenna, et al. "Myristoylation: an important protein modification in the immune response." Frontiers in immunology 8 (2017): 751.

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