MTHFD2 Human

What is MTHFD2 and what are its primary enzymatic functions?

MTHFD2 is a bifunctional mitochondrial enzyme with 5,10-methenyl-THF (CH+-THF) cyclohydrolase and 5,10-methylene-THF (CH2-THF) dehydrogenase activities . This 37kDa protein (350 amino acids) is primarily localized to the mitochondria and plays a crucial role in mitochondrial folate one-carbon metabolism .
Its enzymatic functions include:

• Converting 5,10-methylene-THF to 5,10-methenyl-THF in the presence of NAD+ or NADP+

• Catalyzing the cyclohydrolase reaction that converts 5,10-methenyl-THF to 10-formyl-THF
  Methodologically, enzyme activity assays using purified recombinant MTHFD2 with varying concentrations of substrates and cofactors remain the gold standard for characterizing its catalytic properties. Researchers should consider using both forward and reverse reaction assays to fully understand its kinetic parameters.

How does MTHFD2 expression differ between developing, normal adult, and cancer tissues?

MTHFD2 exhibits distinct expression patterns across different tissue states:

• Developing tissues: Highly expressed during embryogenesis, particularly in early developmental stages, with expression beginning to taper off during later stages .

• Normal adult tissues: Generally low or undetectable levels in most differentiated adult tissues .

• Cancer tissues: Significantly upregulated in various cancer types and transformed cells .

• Inflammatory conditions: Consistently overexpressed in multiple inflammatory and autoimmune diseases including ulcerative colitis, Crohn's disease, Celiac's disease, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, psoriatic arthritis, Sjogren's syndrome, and multiple sclerosis .
  To study expression patterns, researchers should employ multiple complementary techniques including RT-qPCR, Western blotting, immunohistochemistry, and RNA-seq. Careful selection of antibodies with confirmed specificity is essential, as is the inclusion of appropriate positive and negative controls.

What is the cofactor specificity of MTHFD2 and how does it differ from MTHFD2L?

MTHFD2 was initially characterized as strictly NAD+-dependent, but recent studies have revealed more complex cofactor utilization:

• MTHFD2 can use both NAD+ and NADP+ as cofactors at physiologically relevant substrate concentrations .

• This dual redox cofactor specificity is similar to that of MTHFD2L, which shares 60-65% amino acid sequence identity with MTHFD2 .

• The relative use of NAD+ versus NADP+ has significant implications for the direction of one-carbon unit flux in mitochondria by affecting the equilibrium between 5,10-CH2-THF and 10-CHO-THF .
  For experimental determination of cofactor specificity, researchers should conduct enzyme kinetics studies using purified enzyme with varying concentrations of both NAD+ and NADP+ under physiologically relevant conditions. Isothermal titration calorimetry and spectrophotometric assays can provide quantitative measurements of binding affinity and reaction rates with different cofactors.

How does MTHFD2 regulate T cell differentiation and function in inflammatory conditions?

MTHFD2 serves as a critical metabolic checkpoint controlling effector and regulatory T cell differentiation:

• In activated T cells, MTHFD2 regulates de novo purine synthesis and signaling to promote proliferation and inflammatory cytokine production .

• In pathogenic T helper-17 (Th17) cells, MTHFD2 prevents aberrant upregulation of the transcription factor FoxP3 and inappropriate gain of suppressive capacity .

• MTHFD2 deficiency promotes regulatory T (Treg) cell differentiation .

• MTHFD2 inhibition leads to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased mTORC1 signaling .

• MTHFD2 also regulates DNA and histone methylation in Th17 cells, suggesting epigenetic mechanisms of action .
  Methodologically, researchers investigating MTHFD2's role in T cell biology should:

• Use both genetic (CRISPR/Cas9) and pharmacological approaches to modulate MTHFD2 activity

• Incorporate metabolic profiling (LC-MS) to measure purine metabolites and other relevant metabolic intermediates

• Assess changes in mTORC1 signaling through phosphorylation status of downstream targets

• Measure epigenetic modifications using techniques such as ChIP-seq and bisulfite sequencing

What non-canonical functions does MTHFD2 perform beyond its metabolic enzyme activity?

MTHFD2 exhibits several notable non-enzymatic "moonlighting" functions:

• DNA damage repair: MTHFD2 unexpectedly promotes non-homologous end joining (NHEJ) in response to DNA damage by forming a complex with PARP3 to enhance its ribosylation .

• PARP3 interaction: The introduction of a PARP3-binding but enzymatically inactive MTHFD2 mutant (D155A) is sufficient to prevent DNA damage, separating its enzymatic and non-enzymatic functions .

• Nuclear localization: Despite being primarily a mitochondrial protein, MTHFD2 can localize to the nucleus where it may directly interact with DNA repair machinery .
  To investigate these non-canonical functions, researchers should:

• Use co-immunoprecipitation and proximity ligation assays to confirm protein-protein interactions

• Employ subcellular fractionation and immunofluorescence microscopy to track MTHFD2 localization under different conditions

• Design structure-function studies using point mutations that specifically disrupt enzymatic activity versus protein-protein interactions

• Utilize DNA damage assays (comet assay, γH2AX foci formation) to assess functional outcomes

What is the relationship between p53 and MTHFD2 in cancer development?

The p53-MTHFD2 axis represents a critical regulatory mechanism in cancer metabolism:

• p53 transcriptionally suppresses MTHFD2 expression by binding to the MTHFD2 gene .

• p53 loss or mutation leads to upregulation of MTHFD2, resulting in increased folate metabolism, de novo purine synthesis, and enhanced tumor growth both in vivo and in vitro .

• MTHFD2 depletion strongly restrains proliferation in p53-deficient cells specifically and sensitizes these cells to chemotherapeutic agents .

• MTHFD2 depletion in p53-deficient cells preferably induces apoptosis and cell proliferative arrest .

• Conversely, in p53 wild-type cells, MTHFD2 downregulation can induce AICAR-mediated AMPK-p53-p21 activation, which may actually protect these cells .
  Experimental approaches to study this relationship should include:

• Chromatin immunoprecipitation (ChIP) to confirm p53 binding to the MTHFD2 promoter

• Luciferase reporter assays to quantify transcriptional regulation

• Isogenic cell line pairs (p53+/+ vs. p53-/-) to assess differential effects of MTHFD2 modulation

• In vivo xenograft models with varying p53 status to validate therapeutic implications

How does MTHFD2 expression correlate with clinical outcomes in different cancer types?

MTHFD2 has significant prognostic implications across multiple cancer types:

• Analyze large-scale cancer genomics databases (TCGA, ICGC) stratified by cancer type, stage, and molecular subtypes

• Perform multivariate analyses to control for confounding factors

• Validate findings using tissue microarrays and independent patient cohorts

• Correlate MTHFD2 expression with other molecular markers to identify potential synergistic biomarkers

What are the most effective experimental approaches for modulating MTHFD2 activity in research models?

Researchers have several options for investigating MTHFD2 function through modulation:

• Genetic approaches:

  • CRISPR/Cas9-mediated knockout or knockdown (complete gene deletion or conditional systems)

  • siRNA or shRNA for transient or stable knockdown

  • Overexpression systems using wild-type or mutant MTHFD2 constructs (e.g., D155A mutant that retains PARP3 binding but lacks enzymatic activity)

• Pharmacological approaches:

  • Small molecule inhibitors targeting MTHFD2 enzymatic activity

  • Metabolic perturbations of related pathways (e.g., folate antagonists)

• Combination approaches:

  • Rescue experiments with metabolic intermediates

  • Dual targeting of MTHFD2 and compensatory pathways
    When designing MTHFD2 modulation experiments, researchers should consider:

• The timing of intervention (e.g., before or after T cell activation)

• The specificity of the approach (potential off-target effects)

• The completeness of inhibition (partial vs. complete)

• The cellular context (different cell types may respond differently)

How can researchers distinguish between metabolic and non-metabolic functions of MTHFD2?

Separating MTHFD2's dual roles requires specialized experimental designs:

• Structure-function analysis:

  • Generate enzymatically inactive but structurally intact MTHFD2 mutants (e.g., D155A)

  • Compare phenotypes between wild-type, complete knockout, and catalytically inactive mutants

• Metabolic rescue experiments:

  • Supplement with metabolic products (purines, one-carbon units) to bypass MTHFD2's enzymatic function

  • If phenotypes persist despite metabolic rescue, this suggests non-metabolic roles

• Subcellular localization studies:

  • Generate constructs with altered localization signals (mitochondrial vs. nuclear)

  • Assess which functions correlate with specific localizations

• Interactome analysis:

  • Identify protein interaction partners using techniques like BioID or IP-MS

  • Map interactions to specific functional domains of MTHFD2
    These approaches should be complemented with comprehensive metabolomics and functional readouts to delineate which phenotypes result from metabolic versus non-metabolic activities.

What is the therapeutic potential of targeting MTHFD2 in inflammatory and autoimmune diseases?

MTHFD2 represents a promising target for inflammatory conditions:

• MTHFD2 deficiency reduces disease severity in multiple in vivo inflammatory disease models .

• MTHFD2 is consistently overexpressed across multiple inflammatory and autoimmune diseases including ulcerative colitis, Crohn's disease, and multiple sclerosis .

• MTHFD2 expression is significantly elevated in recently diagnosed MS patients compared to healthy donors or MS patients undergoing therapy with disease remission .

• MTHFD2 inhibition can promote regulatory T cell differentiation while impairing inflammatory T cell function .
  Therapeutic development strategies should include:

• High-throughput screening for selective MTHFD2 inhibitors

• Testing in relevant animal models of inflammatory disease

• Evaluation of effects on different immune cell subsets

• Assessment of potential combination therapies with existing immunomodulatory drugs

• Development of biomarkers to identify patients most likely to respond to MTHFD2-targeted therapy

What are the current challenges in developing specific MTHFD2 inhibitors for cancer therapy?

Development of MTHFD2-targeted therapeutics faces several key challenges:

• Selectivity: Achieving specificity over related enzymes like MTHFD2L and MTHFD1

• Dual cofactor usage: Designing inhibitors effective against both NAD+ and NADP+-utilizing forms of the enzyme

• Mitochondrial targeting: Ensuring sufficient drug penetration into mitochondria

• Differential effects: Managing the opposing effects in p53-wild-type versus p53-deficient cancers

• Resistance mechanisms: Identifying and addressing potential compensatory pathways
  Researchers should approach these challenges through:

• Structure-based drug design leveraging crystallographic data

• Phenotypic screening in cancer models with defined p53 status

• Development of mitochondria-targeted drug delivery systems

• Combination strategies to address resistance mechanisms

• Identification of biomarkers to select appropriate patients for MTHFD2-targeted therapy

How might the temporal expression differences between MTHFD2 and MTHFD2L inform developmental biology research?

The distinct temporal expression patterns of MTHFD2 and MTHFD2L during development present intriguing research opportunities:

• MTHFD2 is expressed more abundantly during early developmental stages and begins to taper off, with little or no expression in most adult tissues .

• MTHFD2L expression is low in early developmental stages but begins to increase at embryonic day 10.5 and remains elevated through birth and into adulthood .

• This suggests a developmental switch from MTHFD2 to MTHFD2L to support one-carbon metabolism at different stages .
  Future research directions should include:

• Precise mapping of the spatiotemporal expression patterns of both enzymes during embryogenesis

• Investigation of the regulatory mechanisms controlling this developmental switch

• Functional studies using conditional knockout models with stage-specific deletion

• Exploration of potential developmental defects resulting from altered expression timing

• Assessment of the interplay between these enzymes and developmental signaling pathways

How does MTHFD2 integrate with broader cellular stress response networks?

MTHFD2 appears to function at the intersection of multiple cellular stress response pathways:

• DNA damage response: MTHFD2 directly participates in NHEJ repair through PARP3 interaction .

• Metabolic stress: MTHFD2 influences AMPK-p53-p21 signaling when depleted in p53-competent cells .

• Oxidative stress: MTHFD2 inhibition may lead to increased oxidative stress in cancer cells .

• Immune activation: MTHFD2 expression is rapidly induced following T cell activation .
  Future investigations should focus on:

• Global interactome and phosphoproteome analysis of MTHFD2 under various stress conditions

• Integrated multi-omics approaches to map MTHFD2-dependent responses to different stressors

• Investigation of potential post-translational modifications regulating MTHFD2 function

• Exploration of MTHFD2's role in cellular adaptation to microenvironmental stresses This comprehensive research agenda will help elucidate MTHFD2's position within cellular stress response networks and potentially identify novel therapeutic opportunities for conditions characterized by dysregulated stress responses.