DHFR Human

What is human DHFR and what is its primary function?

Human Dihydrofolate Reductase (DHFR) is a 21-23 kDa member of the dihydrofolate reductase family of enzymes. It plays a critical role in folate metabolism by catalyzing the reduction of dihydrofolate to tetrahydrofolate (THF), a crucial intermediate used in the synthesis of purines and thymidylic acid necessary for DNA and RNA synthesis. DHFR is ubiquitously expressed as a monomer and is classified as a housekeeping gene due to its critical function across multiple cellular pathways. The human DHFR protein consists of 187 amino acids, with its functional DHFR domain spanning residues 4-185 .

How does DHFR expression vary across different developmental stages?

While DHFR is generally classified as a housekeeping gene that is expressed in all cycling cells proportionally to cell growth, recent single-cell gene expression analyses have revealed that DHFR expression is dynamically regulated during development. For instance, in the developing neocortex, DHFR mRNA is highly expressed in PAX6+ apical progenitors at early developmental stages and is downregulated in these progenitors at later stages. Expression is also lower in intermediate progenitors (TBR2+) and differentiating neurons . Western blot analyses confirm that DHFR protein reaches peak expression at embryonic day 12.5 (E12.5) in the developing mouse head .

What are the known structural features of human DHFR?

Human DHFR is a monomeric protein with a clearly defined DHFR domain (amino acids 4-185). Research has identified its mRNA binding motif, which involves specific amino acid residues including Cysteine 6, Leucine 22, Glutamic acid 30, and Serine 118 . Within the cell, DHFR exists in two distinct pools: one where DHFR is bound to its own RNA (functioning as a transcriptional repressor) and another where DHFR is bound to NADPH . These structural features are crucial for both its enzymatic function and regulatory roles.

How does DHFR regulate its own expression at the translational level?

DHFR exhibits a fascinating mechanism of translational autoregulation by binding to its own mRNA. In this process, the DHFR protein can recognize and bind to specific regions of its own mRNA transcript, thereby inhibiting translation. This represents a negative feedback loop that allows precise control of DHFR levels within the cell. Recent research has shown that the newly discovered DHFRL1 enzyme is also capable of the same translational autoregulation by binding to its own mRNA. Interestingly, each enzyme (DHFR and DHFRL1) is capable of replacing the other in this regulatory function , suggesting evolutionary conservation of this important regulatory mechanism.

How do the kinetic properties of DHFR and DHFRL1 differ?

Comparative enzymatic analyses have revealed important differences in the kinetic properties of DHFR and DHFRL1:

These differences suggest that DHFRL1, with its lower affinity for dihydrofolate, may be optimized for the specific conditions within mitochondria, providing a specialized dihydrofolate reductase activity in this cellular compartment .

What are the recommended immunofluorescence protocols for detecting DHFR in cell cultures?

For optimal detection of DHFR in cultured cells using immunofluorescence, researchers should implement the following protocol:

• Fix cells using immersion fixation (typically 4% paraformaldehyde for 15-20 minutes)

• Use a specific antibody such as Sheep Anti-Human Dihydrofolate Reductase/DHFR Antigen Affinity-purified Polyclonal Antibody at an optimized concentration (e.g., 10 μg/mL)

• Incubate for 3 hours at room temperature

• Use appropriate species-specific secondary antibodies (e.g., Northern-Lights™ 557-conjugated Anti-Sheep IgG)

• Counterstain nuclei with DAPI

• Analyze using confocal microscopy with appropriate filters

This protocol has been successfully employed to visualize DHFR in MCF-7 human breast cancer cell lines, revealing specific staining localized to the cytoplasm . For detection of DHFRL1, additional optimization may be necessary, with particular attention to mitochondrial markers for colocalization studies.

How can DHFR activity be accurately measured in tissue samples?

Measuring DHFR activity in tissue samples requires careful consideration of assay conditions. A recommended approach involves:

• Prepare tissue extracts under conditions that preserve enzymatic activity (typically involving protease inhibitors and maintaining samples at 4°C)

• Quantify DHFR activity using a spectrophotometric assay that monitors the oxidation of NADPH (which exhibits decreased absorbance at 340 nm) in the presence of dihydrofolate

• Include appropriate controls to account for background NADPH oxidation

• Normalize activity to protein concentration

• Compare to wild-type or standard reference samples

This methodology has been successfully applied to measure DHFR activity in embryonic tissue samples, revealing that DHFR activity is halved in Dhfr+/Δ heterozygous embryos compared to wild-type . For accurate results, it is essential to optimize assay conditions for the specific tissue being studied.

What approaches can be used to differentiate between DHFR and DHFRL1 in experimental systems?

Distinguishing between DHFR and DHFRL1 in experimental systems presents technical challenges due to their structural similarities. Researchers should consider a multi-pronged approach:

• Antibody-based methods: Use specific antibodies capable of distinguishing between the two proteins, though this may require custom antibody development

• Subcellular fractionation: Exploit the differential localization (DHFRL1 is predominantly mitochondrial) through careful subcellular fractionation

• Gene-specific knockdown: Employ siRNA or CRISPR targeting specific sequences unique to each gene

• Mass spectrometry: Use high-resolution mass spectrometry with careful attention to peptides that differ between the two proteins

• Recombinant expression systems: Express tagged versions of each protein to study their individual properties

• Kinetic assays: Leverage the different Km values for dihydrofolate to help distinguish their activities

Researchers should be aware that earlier studies on DHFR may have unknowingly captured combined effects of both enzymes, potentially necessitating reinterpretation of previous results .

How does DHFR activity influence neurogenic transitions in cortical development?

DHFR plays a critical role in neurogenic transitions during cortical development. Research with Dhfr+/Δ haploinsufficient mouse embryos has shown that reduced DHFR activity leads to:

• Initial developmental delay followed by accelerated indirect neurogenesis

• Increased production of TBR2+ intermediate progenitors at the expense of PAX6+ apical progenitors

• Decreased generation of CTIP2+ early-born neurons

• Increased production of SATB2+ late-born neurons

These findings suggest that DHFR activity is essential for maintaining the proper balance between direct neurogenesis (generating CTIP2+ neurons) and indirect neurogenesis (generating SATB2+ neurons from TBR2+ progenitors) . The temporal sequence of neurogenesis appears to be accelerated when DHFR activity is reduced, indicating that DHFR plays a regulatory role in the timing of neurogenic transitions.

What metabolic pathways link DHFR deficiency to altered histone methylation in neural development?

DHFR deficiency impacts neural development through alterations in one-carbon metabolism and subsequent epigenetic modifications. The pathway proceeds as follows:

• Reduced DHFR activity decreases the conversion of dihydrofolate to tetrahydrofolate (THF)

• Reduced THF availability impacts the methionine cycle, resulting in decreased levels of S-adenosyl methionine (SAM)

• SAM is the primary methyl donor for histone methyltransferases

• Decreased SAM levels lead to reduced histone H3K4 trimethylation (H3K4me3)

• Changes in H3K4me3 marks affect genes specific to neuronal subtypes

Genome-wide analyses have revealed that DHFR haploinsufficiency results in altered histone methylation patterns at genes critical for neuronal subtype specification . This indicates that DHFR's role in one-carbon metabolism extends beyond DNA synthesis to include epigenetic regulation, providing a mechanistic link between folate metabolism and neuronal differentiation.

How do human neural organoid models complement mouse models in studying DHFR's role in neurogenesis?

Human neural organoid (HNO) models offer complementary insights to mouse models when studying DHFR's role in neurogenesis:

• HNOs treated with DHFR inhibitors (e.g., methotrexate/MTX) at early developmental stages show depletion of PAX6+ apical progenitors and overproduction of TBR2+ intermediate progenitors

• This results in accelerated generation of both CTIP2+ early-born neurons and SATB2+ late-born neurons

• These phenotypes parallel observations in Dhfr+/Δ mouse models, strengthening the evidence that DHFR inhibition directly impacts neural progenitor behavior

Using both mouse models and human neural organoids strengthens the translational relevance of findings and helps distinguish direct effects of DHFR inhibition in neural progenitors from potential systemic effects in whole-animal models . This combined approach is particularly valuable given that DHFR mutations in humans are associated with severe neurological disorders.

What computational approaches are being used to design novel hDHFR inhibitors?

Modern computational approaches for designing novel hDHFR inhibitors incorporate deep learning methodologies. One such workflow includes:

• An artificial neural network trained on molecules from the ChEMBL database with experimental DHFR inhibition data

• Conditional generative adversarial networks (cGAN) to generate candidate molecules with predicted high inhibitory activity

• Molecular docking simulations to verify binding of candidate molecules to the DHFR active site

This integrated approach allows researchers to efficiently explore chemical space and identify drug-like compounds with DHFR inhibition comparable to currently used inhibitors . These computational methods accelerate the discovery of novel DHFR inhibitors with potential applications as anti-cancer, anti-malarial, and antibacterial agents.

How can researchers assess the specificity of DHFR inhibitors against DHFR versus DHFRL1?

Developing inhibitors specific to either DHFR or DHFRL1 requires careful assessment of differential binding and inhibition. Researchers should implement a systematic approach:

• Express and purify recombinant DHFR and DHFRL1 proteins

• Perform enzyme kinetic assays with candidate inhibitors to determine IC50 values for each enzyme

• Conduct structural analysis (X-ray crystallography or molecular modeling) to identify binding mode differences

• Assess cellular localization of inhibitors to determine if they preferentially accumulate in cytosol (targeting DHFR) or mitochondria (targeting DHFRL1)

• Evaluate phenotypic effects in cellular models with selective knockdown of either DHFR or DHFRL1

The discovery of DHFRL1 necessitates reassessment of existing DHFR inhibitors, as compounds previously thought to target only DHFR may affect both enzymes. This has significant implications for drug development and understanding of mechanism of action for both existing and novel inhibitors .

What methodological considerations are important when evaluating DHFR inhibitors in cellular systems?

When evaluating DHFR inhibitors in cellular systems, researchers should consider several methodological factors:

• Cell type selection: Different cell types may express varying levels of DHFR and DHFRL1, potentially affecting inhibitor efficacy

• Proliferation status: As DHFR expression correlates with cell cycle, the proliferation rate of test cells will impact results

• Metabolic state: Cellular metabolic status affects folate pathway activity and should be standardized

• Transport mechanisms: Consider cell-specific uptake mechanisms for inhibitors

• Resistance mechanisms: Evaluate potential upregulation of DHFR or DHFRL1 in response to inhibitor treatment

• Downstream effects: Monitor not only DHFR activity but also downstream metabolites (THF, SAM) and processes (DNA synthesis, histone methylation)

• Specificity controls: Include experiments with DHFR or DHFRL1 knockdown to confirm target specificity

These considerations are essential for accurate evaluation of inhibitor efficacy and specificity, particularly in light of the newly recognized role of DHFRL1 .