Protein Methylation in Cellular Function

What Is Protein Methylation?

Protein methylation is a critical post-translational modification (PTM) that regulates a wide array of cellular processes, including gene expression, protein–protein interactions, and signal transduction. This modification involves the enzymatic transfer of a methyl group from S-adenosylmethionine (SAM) to specific amino acid residues, predominantly arginine and lysine. The dynamic nature of protein methylation, mediated by methyltransferases and demethylases, underscores its role in cellular homeostasis and disease pathogenesis. This article provides a comprehensive analysis of protein methylation, covering its biochemical mechanisms, enzymatic regulation, types, biological implications, and state-of-the-art analytical techniques used for its investigation.

What Enzymes Are Involved in Protein Methylation?

The methylation process is catalyzed by specific enzymes known as methyltransferases, which utilize SAM as a methyl donor. Demethylases can reverse methylation, making this modification a dynamic regulatory mechanism.

Protein Arginine Methyltransferases (PRMTs)

PRMTs catalyze the methylation of arginine residues, yielding three distinct forms:

• Monomethylarginine (MMA)
• Asymmetric dimethylarginine (ADMA)
• Symmetric dimethylarginine (SDMA)

PRMTs are classified into three types:

• Type I PRMTs: Generate ADMA (e.g., PRMT1, PRMT3, PRMT4).
• Type II PRMTs: Generate SDMA (e.g., PRMT5).
• Type III PRMTs: Generate MMA (e.g., PRMT7).

Protein Lysine Methyltransferases (PKMTs)

PKMTs catalyze the methylation of lysine residues and often contain a conserved SET domain (Su(var)3-9, Enhancer of zeste, and Trithorax). Examples include:

• SUV39H1: Methylates H3K9, facilitating chromatin compaction.
• MLL (Mixed Lineage Leukemia): Methylates H3K4, promoting transcriptional activation.

Other Methyltransferases

• N-terminal methyltransferases (NTMTs): Irreversibly modify protein α-amino groups, influencing stability and interactions.
• Prenylcysteine methyltransferases: Modify proteins involved in membrane signaling.
• Protein phosphatase methyltransferases: Regulate the activity of phosphatases such as PP2A.

Protein Demethylases

Specialized demethylases reverse methylation:

• Lysine demethylases (KDMs): Including the LSD1 (FAD-dependent) and JmjC domain-containing (Fe²⁺-dependent) families.
• Arginine demethylases: Including JMJD6, which can reverse arginine methylation in histones.

Common Types of Protein Methylation

Arginine Methylation in Proteins

Arginine residues, enriched within glycine-arginine-rich (GAR) motifs, undergo methylation via the action of distinct classes of PRMTs.

• Type I PRMTs (e.g., PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, and PRMT8) catalyze the formation of asymmetric dimethylarginine by transferring two methyl groups to a single terminal nitrogen.
• Type II PRMTs (e.g., PRMT5 and PRMT9) produce symmetric dimethylarginine through methylation of both terminal nitrogens.
• Type III PRMTs, such as PRMT7, exclusively generate monomethylarginine.

This modification is integral to processes such as RNA splicing, nucleic acid binding, and signal transduction, with implications for the modulation of chromatin structure and transcriptional activity.

Lysine Methylation in Proteins

Lysine methylation is predominantly mediated by lysine methyltransferases, which frequently contain a conserved SET domain. Lysine residues may be mono-, di-, or trimethylated, with the degree of methylation serving as an epigenetic signal:

• Histone Modulation: Trimethylation at histone H3 lysine 4 (H3K4me3) is typically associated with transcriptional activation, whereas methylation at lysine 9 (H3K9me3) correlates with gene repression and heterochromatin formation.
• Non-Histone Proteins: Methylation of lysine residues on proteins such as p53 influences protein stability and transcriptional regulation, underscoring the broader functional spectrum of this modification.

Figure 1. Mechanism of lysine (K) and arginine (R) methylation. (Adhikary, et al., 2019)

N-terminal and Prenylcysteine Methylation

N-terminal Methylation

Proteins bearing a specific N-terminal consensus motif undergo α-amino methylation by NTMTs. The methylation of the amino terminus, which is irreversible in nature, imparts a permanent positive charge that can affect protein-protein interactions and cellular localization. Notable examples include the methylation of centromere-associated proteins CENP-A and CENP-B.

Prenylcysteine Methylation

Proteins featuring a C-terminal CAAX motif are subject to a series of post-translational modifications, including prenylation, proteolytic cleavage, and subsequent methylation of the exposed prenylcysteine group. This modification is critical for the proper targeting of proteins, such as Ras and nuclear lamins, to the plasma membrane, thus influencing signal transduction and cellular compartmentalization.

C-terminal Methylation

C-terminal methylation is exemplified by the modification of the catalytic subunit of protein phosphatase 2A (PP2A). Here, the reversible methylation of the terminal leucine carboxyl group modulates the assembly of the phosphatase holoenzyme by enhancing the binding affinity for regulatory subunits. This dynamic process is facilitated by a unique protein phosphatase methyltransferase and its corresponding methylesterase, providing a mechanism for the fine-tuning of phosphatase activity in response to extracellular signals.

Mechanisms of Protein Methylation

• Substrate Recognition: Enzymes recognize specific amino acid sequences or motifs (e.g., GAR motifs in arginine methylation or the SET domain-specific sites in lysine methylation) that confer selectivity for the modification.
• Catalytic Activity: The catalytic domains, such as the SET domain in lysine methyltransferases or the Rossmann fold in PRMTs, facilitate the transfer reaction. The subsequent addition of methyl groups can alter the steric and electronic properties of the target residue, thereby modulating its interaction with other biomolecules.
• Reversibility: In many cases, the methylation process is reversible, with specific demethylases excising methyl groups. This reversibility allows for dynamic regulation of protein function, akin to phosphorylation-dephosphorylation cycles observed in other PTMs.

Analytical Techniques for Protein Methylation Research

Mass Spectrometry and Proteomics Approaches

Mass spectrometry (MS) is the cornerstone of methyl-proteomics, enabling the precise qualitative and quantification of methylated peptides.

• Sample Preparation and Digestion: Proteins are enzymatically digested into peptides, which are then separated via high-performance liquid chromatography (HPLC).
• Mass Analysis: Advanced MS techniques, such as tandem mass spectrometry (MS/MS), facilitate the detection of characteristic mass shifts indicative of methyl group addition.
• Database Matching: Bioinformatics tools are employed to compare the acquired spectra against protein databases, confirming the identity and specific methylation sites.

Antibody-Based Detection and Immunoprecipitation Methods

Antibody-based methodologies are instrumental in the selective enrichment and detection of methylated proteins.

• Immunoprecipitation (IP): Utilization of antibodies raised against specific methylated epitopes allows for the targeted isolation of methylated proteins from complex mixtures.
• Western Blotting: Post-IP, Western blotting is employed to visualize and confirm the presence of methylated proteins, utilizing secondary antibodies conjugated to detection enzymes.
• Co-Immunoprecipitation (Co-IP): This technique facilitates the study of protein-protein interactions influenced by methylation, providing insights into the formation and regulation of multi-protein complexes.

Functional Assays and Binding Studies

Functional assays are indispensable for elucidating the biological consequences of protein methylation.

• In Vitro Enzyme Assays: These assays assess the impact of methylation on enzyme activity, often revealing alterations in catalytic efficiency or substrate affinity.
• Binding Studies: Techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) quantify the binding interactions between methylated proteins and their partners.
• Cellular Assays: Manipulating methylation levels within cellular models allows for observing downstream effects on signal transduction pathways, cell cycle progression, and other vital processes.

Figure 2. Detection of lysine methylation. The scheme is divided into candidate-based (top part) and high-throughput (bottom part) experimental platforms. (Levy D. 2019)

Biological Significance of Protein Methylation

Epigenetic Regulation: Histone methylation modulates chromatin accessibility, influencing transcriptional activity. For instance, H3K4me3 is linked to gene activation, while H3K9me3 correlates with gene repression.

Signal Transduction: Methylation of non-histone proteins, including transcription factors and kinases, governs signaling cascades.

Protein-Protein Interactions: Methylated residues serve as docking sites for protein complexes, regulating molecular assembly and stability.

Cellular Differentiation and Development: Lysine and arginine methylation orchestrate gene expression patterns essential for cell fate determination.

Protein Methylation and Disease

Cancer: Aberrant histone methylation patterns are hallmarks of tumorigenesis. Overexpression of methyltransferases such as EZH2 is frequently observed in cancers, contributing to epigenetic silencing of tumor suppressor genes.

Neurodegenerative Disorders: Defective arginine methylation of RNA-binding proteins can lead to neuronal dysfunction, as observed in amyotrophic lateral sclerosis (ALS).

Cardiovascular Diseases: Methylation of signaling molecules involved in vascular biology influences endothelial function and atherosclerosis development.

Case Study

Methylation of the Retinoblastoma Tumor Suppressor by SMYD2*

Journal: Journal of Biological Chemistry

Published: 2010

DOI: 10.1074/jbc.M110.137612

Background

RB (retinoblastoma tumor suppressor) is a key regulator of the cell cycle, whose activity is modulated by various post-translational modifications (PTMs) such as phosphorylation, acetylation, and ubiquitination. Prior studies indicated that non-histone proteins, including transcription factors like p53, could be methylated, suggesting that methylation might also regulate RB function.

Purpose

The study aimed to determine whether RB is methylated, identify the specific methylation site, and elucidate the functional consequences of this modification, particularly its role in regulating RB's interactions and activity in cellular processes.

Methods

• In Vitro Assays & Mass Spectrometry: Recombinant proteins were used in methyltransferase assays with SMYD2, followed by mass spectrometry to detect methylation on RB, pinpointing lysine 860 as the target.
• Cellular Experiments: To confirm cell methylation, specific antibodies recognizing monomethylated RB at lysine 860 were used in immunoprecipitation and immunoblotting. Knockdown experiments with shRNA against SMYD2 further validated its role.
• Binding Studies: Peptide pull-down assays, co-immunoprecipitation, and isothermal titration calorimetry (ITC) were conducted to assess the interaction between methylated RB and the methyl-binding protein L3MBTL1.

Results

• SMYD2 was shown to monomethylate RB at lysine 860 both in vitro and in cells.
• The methylation level of RB at K860 was regulated during cell cycle progression, differentiation, and in response to DNA damage.
• Methylation at K860 creates a binding site for L3MBTL1, and mutations at this site significantly reduce this interaction, implicating methylation in modulating RB's functional associations.

Figure 3. Mass spectrometry analysis of RB methylation by SMYD2 in vitro.

Figure 4. Methylation of human RB by SMYD2 at lysine 860 in vivo.

Conclusion

The study demonstrates that SMYD2-mediated monomethylation of RB at lysine 860 is a novel regulatory PTM. This modification plays a crucial role in modulating RB function by facilitating its interaction with L3MBTL1, thereby contributing to an "RB code" that integrates multiple PTMs to finely regulate cell cycle progression and cellular responses to various stimuli.

References

• Adhikary, et al. "The Role of Protein Lysine Methylation in the Regulation of Protein Function: Looking Beyond the Histone Code." The DNA, RNA, and Histone Methylomes. Cham: Springer International Publishing, 2019. 453-477. DOI: 10.1007/978-3-030-14792-1_18
• Levy D. Lysine methylation signaling of non-histone proteins in the nucleus. Cellular and Molecular Life Sciences, 2019, 76: 2873-2883. DOI: 10.1007/s00018-019-03142-0