VCPKMT Human

What is VCPKMT and what is its primary function in human cells?

VCPKMT (VCP lysine methyltransferase, also known as METTL21D) is a member of a novel human protein methyltransferase family that specifically trimethylates lysine 315 (K315) in Valosin-containing protein (VCP/p97). This S-adenosyl methionine (SAM)-dependent enzyme is predominantly localized in the cytoplasm and is expressed ubiquitously across human tissues. VCPKMT's primary function is to catalyze the trimethylation of VCP, which then assembles into fully methylated homohexameric complexes .

The enzyme belongs to a family that includes calmodulin-lysine methyltransferase and eight other previously uncharacterized proteins, collectively forming a distinct methyltransferase family . While the physiological significance of VCPKMT-mediated methylation is still being elucidated, it has been implicated in potential roles in cancer metastasis and cell migration .

What methodologies can reliably detect and quantify VCPKMT expression in human tissues?

Detection and quantification of VCPKMT in human tissues can be accomplished through several complementary techniques:

• Immunoblotting and immunofluorescence: Using validated VCPKMT antisera for protein detection in tissue extracts or fixed cells. This approach has been successfully employed to demonstrate cytoplasmic localization of VCPKMT .

• qRT-PCR: For quantifying VCPKMT transcript levels across different tissues, revealing its ubiquitous expression pattern .

• Mass spectrometry: For both detecting VCPKMT protein and analyzing its post-translational modifications. This technique has been crucial in confirming the absence of VCP methylation in VCPKMT knockout models .

• In situ hybridization: For spatial localization of VCPKMT transcripts in tissue sections, particularly useful for heterogeneous tissues.

For optimal results, researchers should validate antibody specificity using appropriate controls, such as VCPKMT knockout cell lines generated through zinc-finger nucleases or CRISPR-Cas9 technology .

How specific is VCPKMT for VCP, and what experimental evidence supports this specificity?

VCPKMT exhibits remarkably high substrate specificity for VCP. This specificity has been demonstrated through multiple lines of experimental evidence:

• In vitro methylation assays: When recombinant VCPKMT and radioactive AdoMet were incubated with protein extracts from wild-type and VCPKMT-knockout mouse tissues (liver, brain, or testis), radiolabeled methyl groups were incorporated only into VCP when using extracts from knockout mice. This suggests VCP is the sole substrate, as it was already fully methylated in wild-type extracts .

• Protein interaction studies: Two independent studies identified VCP as a binding partner of VCPKMT using different approaches - yeast two-hybrid screening and tandem affinity purification .

• Knockout validation: Analysis of tissues and cells from VCPKMT knockout mice demonstrated complete absence of VCP K315 methylation, with no other apparent methylation defects, further supporting VCP as the primary physiological substrate .

The only other detectable methylation in these assays appeared to be auto-methylation of VCPKMT itself, observed as a band at approximately 22 kDa .

What is the structural basis for VCPKMT recognition of VCP, and how does it access K315 in the assembled VCP hexamer?

The structural mechanism by which VCPKMT recognizes VCP and accesses K315 involves a complex interaction with the VCP hexamer structure:

• VCP monomerization: Evidence suggests that VCPKMT preferentially methylates VCP monomers rather than assembled hexamers. The protein TUG (Tether containing UBX domain for GLUT4) has been shown to disassemble VCP hexamers into monomers, which are then more efficiently methylated by VCPKMT .

• Spatial considerations: K315 is located near the Walker B motif of the D1 ATPase domain of VCP. This positioning may have functional significance for how methylation impacts VCP's enzymatic activity .

• Recognition elements: While the specific structural recognition elements haven't been fully characterized in the provided search results, the high specificity of VCPKMT for VCP suggests precise molecular recognition mechanisms.

Research using structural biology approaches such as crystallography or cryo-EM would be valuable to further elucidate the details of this interaction.

How does VCPKMT-mediated methylation affect VCP's ATPase activity and cellular functions?

The impact of VCPKMT-mediated methylation on VCP's ATPase activity remains somewhat contradictory in the literature:

This discrepancy highlights the complexity of studying VCP regulation in different experimental contexts. The K315 methylation site's proximity to the Walker B motif of the D1 ATPase domain suggests a potential functional significance, but the precise mechanisms remain to be fully elucidated.

The methylation status may impact VCP's diverse cellular functions, which include:

• Endoplasmic reticulum-associated degradation

• Membrane fusion

• Protein degradation and quality control

• Transcription factor regulation

• Cell cycle regulation

• DNA damage response

What phenotypic consequences have been observed in VCPKMT knockout models?

Surprisingly, despite the complete loss of VCP K315 trimethylation, VCPKMT knockout models have displayed minimal phenotypic consequences under standard laboratory conditions:

• Mouse models: VCPKMT knockout mice were viable, fertile, and showed no obvious pathological phenotypes. Their body weight, lifespan, and acute endurance capacity were comparable to wild-type controls. Distribution of genotypes followed expected Mendelian ratios, indicating no defects in embryonic development .

• Cellular studies: Mouse embryonic fibroblasts (MEFs) derived from VCPKMT knockout mice showed proliferation rates and growth characteristics comparable to wild-type cells .

• Human cell lines: Some VCPKMT-deficient human cell lines showed reduced migration and invasive potential, as well as slower cell proliferation in certain cases, though this was not consistently observed across all studies .

These findings suggest that while VCPKMT-mediated methylation of VCP may be dispensable for basic development and survival under unstressed conditions, it might play more subtle regulatory roles or become important under specific stress conditions or in particular disease contexts .

What is the evidence linking VCPKMT to cancer progression and metastasis?

Several lines of evidence connect VCPKMT to cancer progression and metastasis:

• Promotion of metastasis: VCPKMT was identified as a promoter of tumor metastasis in experimental models. When VCPKMT-overexpressing cells were injected into mice, enhanced metastasis formation in lymph nodes was observed, although primary tumor size was reduced .

• Cell migration and invasion: VCPKMT-deficient human cell lines demonstrated reduced migration and invasive potential in vitro, suggesting a role for VCPKMT in cellular processes relevant to metastasis .

• Proliferation effects: VCPKMT-deficient cells showed reduced proliferation in some studies, although this effect wasn't consistent across all experimental systems .

The molecular mechanisms underlying these observations remain incompletely understood. They could involve VCPKMT-mediated regulation of VCP's roles in protein degradation, stress response, or other cellular pathways relevant to cancer progression. Further research is needed to elucidate the precise pathways through which VCPKMT influences metastatic potential.

Is there any relationship between VCPKMT function and VCP-associated neurodegenerative diseases?

The relationship between VCPKMT and VCP-associated neurodegenerative diseases appears complex:

• VCP mutations and disease: Mutations in VCP have been linked to several neurodegenerative disorders, including inclusion body myopathy with frontotemporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). These conditions collectively represent multisystem proteinopathy (VCP-MSP) .

• K315 methylation site: Importantly, no pathogenic mutations have been reported at the K315 methylation site that is targeted by VCPKMT. Most disease-causing VCP mutations are located in the N-terminal domain, which is predominantly responsible for protein interactions .

• Pathogenic mechanisms: The pathogenesis of VCP-associated disorders is typically attributed to altered ATPase activity or disrupted protein interactions rather than methylation status .

Further studies examining VCPKMT activity and VCP methylation status in patients with VCP-associated diseases would be valuable to clarify any potential relationships.

What are the most effective methods for measuring VCPKMT enzymatic activity in vitro?

Several complementary approaches have proven effective for measuring VCPKMT enzymatic activity:

• Radioactive methylation assays: Using S-adenosyl-[methyl-³H]methionine (³H-SAM) or S-adenosyl-[methyl-¹⁴C]methionine as methyl donors, followed by SDS-PAGE and fluorography or scintillation counting. This approach allows visualization and quantification of methylated substrates .

• Methylation-specific antibody detection: Anti-trimethyl lysine antibodies or antibodies specifically raised against VCP-K315me3 can be used in Western blotting to detect the methylation product .

• Mass spectrometry: For precise identification and quantification of methylation states on specific lysine residues. This technique has been crucial in confirming VCPKMT's activity toward K315 in VCP .

For optimal results when studying VCPKMT specificity, researchers can use:

• Recombinant VCPKMT as the enzyme source

• Protein extracts from VCPKMT knockout tissues/cells as substrate sources (ensuring VCP is unmethylated)

• Controls including wild-type extracts (where VCP is already methylated)

Additionally, researchers can incorporate TUG protein or its C-terminal fragment to potentially enhance VCPKMT activity by promoting VCP hexamer disassembly .

What strategies can be employed to generate and validate VCPKMT-deficient cellular and animal models?

Several proven strategies exist for generating and validating VCPKMT-deficient models:

• Genetic knockout approaches:

  • Zinc-finger nucleases: Successfully used to generate human VCPKMT knockout cell lines

  • CRISPR-Cas9: For targeted disruption of the VCPKMT gene

  • Homologous recombination: Used to create conditional knockout mice by introducing loxP sites flanking exons 1-4 of the VCPKMT gene

• Validation methods:

  • Genomic PCR: To confirm deletion of targeted exons

  • RT-PCR and immunoblotting: To verify absence of VCPKMT transcript and protein

  • Immunofluorescence: To confirm loss of VCPKMT protein expression in cellular models

  • Mass spectrometry: To demonstrate absence of VCP K315 methylation

  • In vitro methylation assays: Using extracts from putative knockout models as substrate sources, with wild-type samples as controls

• Functional validation:

  • Analysis of VCP methylation status: Complete absence of K315 methylation should be observed in knockout models

  • Phenotypic characterization: Assessment of growth, development, and cellular functions to determine consequences of VCPKMT deficiency

The thorough validation approaches used in published VCPKMT knockout studies provide excellent templates for researchers developing new model systems.

How might the interaction between VCPKMT, VCP, and TUG regulate glucose transporter trafficking?

The interaction between VCPKMT, VCP, and TUG (Tether containing UBX domain for GLUT4 protein) suggests a potential regulatory mechanism for glucose transporter trafficking:

• TUG's dual functions: TUG retains GLUT4 transporters in the cytoplasm under basal conditions. Upon insulin stimulation, TUG undergoes cleavage, facilitating GLUT4 translocation to the cell membrane and muscle sarcolemma .

• TUG-VCPKMT-VCP interplay: TUG has been identified as an interaction partner of VCPKMT and can disassemble VCP hexamers into monomers. This disassembly appears to enhance VCPKMT-mediated methylation of VCP, as monomers are preferentially methylated .

• Regulatory hypothesis: It has been speculated that TUG and VCPKMT may regulate ubiquitin-dependent sorting of GLUT4 via VCP methylation, potentially affecting glucose homeostasis .

This model suggests a molecular pathway where:

• TUG promotes VCP monomerization

• VCPKMT methylates monomeric VCP

• Methylated VCP potentially influences GLUT4 trafficking

What are the most promising approaches for identifying additional substrates or interacting partners of VCPKMT?

Despite the apparent high specificity of VCPKMT for VCP, researchers interested in identifying additional substrates or interacting partners might employ these approaches:

• Proximity-based labeling: BioID or APEX2 fusion proteins to identify proteins in close proximity to VCPKMT in living cells.

• Affinity purification-mass spectrometry: Using tagged VCPKMT to pull down interacting proteins, followed by identification via mass spectrometry. This approach has already identified TUG as a VCPKMT interactor .

• Methylome analysis: Comparing global protein methylation patterns between wild-type and VCPKMT-deficient cells using advanced mass spectrometry techniques and methylation-specific enrichment strategies.

• Candidate-based approaches: Testing potential substrates from the same cellular compartments or pathways as VCP, particularly those with structural similarities in the region surrounding K315.

• Synthetic peptide arrays: Screening libraries of peptides representing potential methylation sites to assess VCPKMT substrate specificity in vitro.

• Genetic screens: Using CRISPR-based screens to identify genes that display synthetic interactions with VCPKMT, potentially revealing functional relationships.

These complementary approaches might reveal context-specific or lower-affinity substrates that weren't detected in previous studies, although current evidence strongly suggests VCP is the primary physiological substrate of VCPKMT .