GCHFR Human

What is the basic structure of human GCHFR protein?

Human GCHFR is an 84 amino acid enzyme inhibitor with a molecular mass of approximately 11-12 kDa in its monomeric form. The protein typically exists as a 20 kDa homodimer in cellular environments. The full amino acid sequence is well-characterized, with the recombinant form containing 107 amino acids (including the His-tag) when produced in E. coli expression systems . X-ray crystallography and biophysical studies have revealed that GCHFR forms a specific tertiary structure that enables its regulatory function. When studying GCHFR structure in experimental settings, researchers should consider using reducing conditions during western blot analysis, as demonstrated in validated protocols that successfully detect the protein at approximately 11 kDa .

What is the primary biological function of GCHFR?

GCHFR binds to and regulates the activity of GTP cyclohydrolase I (GTPCH1), which is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin (BH4) . This regulation represents a critical feedback mechanism in the BH4 synthesis pathway. BH4 serves as an essential cofactor for nitric oxide synthases and aromatic amino acid hydroxylases, thus GCHFR indirectly influences nitric oxide production and neurotransmitter synthesis . Researchers investigating GCHFR function should design experiments that account for its dynamic interaction with GTPCH1 and consider measuring downstream products like BH4 levels and activity of BH4-dependent enzymes to fully characterize phenotypic effects of GCHFR modulation.

What is the tissue expression pattern of human GCHFR?

Human GCHFR demonstrates widespread tissue distribution, with expression detected in multiple cell types and organs. According to the Human Protein Atlas data, GCHFR is found in the nucleus and cytoplasm of endothelial cells, keratinocytes, and melanocytes . The protein is expressed in diverse tissues including brain regions (cerebral cortex, cerebellum, hippocampal formation, amygdala), liver, kidney, lung, heart, skeletal muscle, and reproductive organs . When designing tissue-specific studies, researchers should consider this broad expression pattern and implement appropriate controls to account for baseline GCHFR levels in different tissues.

How do experimental conditions affect GCHFR expression in cell culture models?

GCHFR expression in cell culture can be modulated by several factors including cell density, oxygen tension, inflammatory cytokines, and phenylalanine levels. Researchers should standardize culture conditions when comparing GCHFR expression across experimental groups. When designing in vitro studies, hepatocellular carcinoma cell lines such as HepG2 and Hep3B have been validated for detectable GCHFR expression and can serve as positive controls for expression studies . Methodologically, western blot analysis using validated antibodies (such as Mouse Anti-Human GCHFR Monoclonal Antibody) can reliably detect GCHFR at approximately 11 kDa under reducing conditions .

What are the optimal methods for detecting GCHFR protein in experimental samples?

Several validated techniques are available for GCHFR detection:

• Western Blot: The most common method utilizes reducing conditions with specific antibodies such as the Mouse Anti-Human GCHFR Monoclonal Antibody (Clone #846011). GCHFR appears at approximately 11 kDa .

• Simple Western™: This automated capillary-based immunoassay has been validated for GCHFR detection in hepatocellular carcinoma cell lines, requiring approximately 0.5 mg/mL of lysate concentration for optimal results .

• Immunohistochemistry: Can be used for tissue localization studies, though careful antibody validation is essential.

For all detection methods, researchers should include appropriate positive controls (such as Hep3B or HepG2 cell lysates) and negative controls to ensure specificity. Antibody dilution optimization is critical, with 1 μg/mL being a recommended starting concentration for western blot applications .

What approaches are recommended for studying GCHFR-GTPCH1 interactions?

The investigation of GCHFR-GTPCH1 interactions requires specialized techniques:

• Co-immunoprecipitation: Can be used to demonstrate physical interaction between GCHFR and GTPCH1 in cell lysates.

• Bioluminescence Resonance Energy Transfer (BRET) or Fluorescence Resonance Energy Transfer (FRET): These techniques allow real-time monitoring of protein-protein interactions in living cells.

• Surface Plasmon Resonance (SPR): Provides quantitative binding kinetics data using purified recombinant proteins.

When designing these experiments, researchers should consider that GCHFR-GTPCH1 interactions are modulated by tetrahydrobiopterin levels and phenylalanine concentration, which should be controlled or measured in the experimental system . Additionally, the homodimeric nature of GCHFR should be taken into account when interpreting interaction data .

How is GCHFR activity regulated at the molecular level?

GCHFR activity is subject to complex regulation involving:

• Feedback inhibition: Tetrahydrobiopterin binds to GCHFR and enhances its inhibitory effect on GTPCH1, creating a negative feedback loop that maintains appropriate BH4 levels .

• Allosteric activation: Phenylalanine can bind to GCHFR and counteract the inhibitory effect on GTPCH1, stimulating BH4 synthesis when phenylalanine levels are elevated .

• Post-translational modifications: Phosphorylation events may alter GCHFR's regulatory capacity.

For experimental investigation of these regulatory mechanisms, researchers should employ enzyme activity assays with purified components and implement systems where BH4 and phenylalanine concentrations can be precisely controlled. Mutations in key binding residues can be introduced to dissect the specific molecular interactions involved in regulation.

What signaling pathways influence GCHFR expression and function?

GCHFR expression and function are influenced by several signaling pathways:

• Inflammatory cytokine signaling: Pro-inflammatory cytokines can modulate GCHFR expression levels, potentially altering BH4 synthesis in inflammatory conditions.

• Nitric oxide (NO) signaling: As GCHFR indirectly regulates NO production through BH4 availability, there may be feedback mechanisms from NO signaling pathways.

• Metabolic regulation: Amino acid metabolism, particularly phenylalanine homeostasis, interacts with GCHFR function.

When designing studies to investigate these pathways, researchers should consider employing cytokine treatments, NO donors/inhibitors, or metabolic perturbations in cell culture systems, followed by assessment of GCHFR expression and activity. Time-course experiments are recommended to capture the dynamic nature of these regulatory events.

What is known about GCHFR's role in human diseases?

While direct GCHFR mutations are not commonly reported as primary disease causes, its regulatory role in BH4 biosynthesis connects it to several pathological conditions:

• Phenylketonuria (PKU): As GCHFR regulates phenylalanine metabolism in the liver, it may influence PKU phenotypes or response to treatment .

• Neurodegenerative diseases: Through its impact on BH4 availability and subsequent effects on neurotransmitter synthesis, GCHFR may be relevant in neurological disorders.

• Vascular pathologies: Via regulation of nitric oxide production, GCHFR might influence endothelial function and vascular health.

Researchers investigating these associations should consider genetic association studies, analysis of GCHFR expression in disease tissues, and functional assays comparing normal and disease states. Case-control designs with adequately powered sample sizes are necessary for meaningful clinical correlations.

How does GCHFR function differ in disease states compared to normal physiology?

In pathological conditions, GCHFR function may be altered in several ways:

• Expression level changes: Altered GCHFR expression has been observed in certain disease states, potentially disrupting the balance of BH4 regulation.

• Regulatory coupling: The sensitivity of GCHFR to its modulators (BH4, phenylalanine) may be altered in disease contexts.

• Protein-protein interactions: Changes in GCHFR-GTPCH1 binding dynamics could affect downstream metabolic pathways.

When investigating these differences, researchers should employ comparative studies between normal and disease tissues or models, using techniques such as quantitative PCR, western blotting, and activity assays. Analysis of protein complexes using co-immunoprecipitation or proximity ligation assays can reveal altered interaction patterns in disease states.

What gene editing approaches are most effective for GCHFR functional studies?

Several gene editing strategies have proven effective for GCHFR functional studies:

• CRISPR-Cas9 knockout: Complete elimination of GCHFR expression allows assessment of its necessity in BH4 regulation. Guide RNA selection should target early exons to ensure functional knockout.

• Knock-in mutations: Introduction of specific mutations can help dissect the importance of particular residues in GCHFR function.

• Conditional knockout systems: Tissue-specific or inducible GCHFR deletion can circumvent potential developmental effects of constitutive knockout.

When implementing these approaches, researchers should verify editing efficiency through sequencing and confirm functional consequences through protein expression analysis. Phenotypic characterization should include measurement of GTPCH1 activity, BH4 levels, and downstream functional readouts such as NO production or neurotransmitter synthesis.

What are emerging techniques for studying GCHFR's dynamic regulation in living systems?

Cutting-edge approaches for investigating GCHFR dynamics include:

• Optogenetic control: Light-inducible protein interaction systems can be adapted to modulate GCHFR-GTPCH1 interactions with temporal precision.

• Biosensors: Genetically encoded fluorescent biosensors for BH4 or related metabolites can provide real-time readouts of pathway activity.

• Single-molecule imaging: Advanced microscopy techniques can reveal the dynamics of individual GCHFR molecules and their interactions.

• Metabolic flux analysis: Isotope labeling combined with mass spectrometry can track the impact of GCHFR modulation on metabolic pathways.

These methodologies require specialized equipment and expertise but offer unprecedented insights into GCHFR biology. Researchers should consider collaborations with laboratories specialized in these techniques when designing advanced functional studies.

What expression systems are optimal for producing recombinant human GCHFR?

E. coli represents the most commonly used and validated expression system for human GCHFR production . When using bacterial expression systems, researchers should consider the following:

• Construct design: Addition of an N-terminal His-tag (23 amino acids) has been successfully employed for purification purposes, resulting in a recombinant protein of 107 amino acids (versus 84 for the native protein) .

• Expression conditions: Optimization of induction parameters (temperature, IPTG concentration, duration) is critical for maximizing protein yield while maintaining proper folding.

• Solubility considerations: GCHFR is generally expressed as a soluble protein, but inclusion body formation can occur under certain conditions.

For functional studies requiring post-translational modifications, mammalian or insect cell expression systems may be considered as alternatives, though with typically lower yields.

What storage conditions maintain GCHFR stability for long-term experimental use?

For maintaining GCHFR stability, validated storage conditions include:

• Short-term storage (2-4 weeks): 4°C in an appropriate buffer (e.g., 20mM Tris-HCl buffer pH 8.0, containing 0.15M NaCl, 40% glycerol and 1mM DTT) .

• Long-term storage: -20°C to -70°C for periods up to 12 months .

• Stability enhancers: Addition of carrier proteins (0.1% HSA or BSA) is recommended for long-term storage to prevent protein adhesion to container surfaces and maintain activity .

• Freeze-thaw considerations: Multiple freeze-thaw cycles should be avoided as they can lead to protein denaturation and activity loss .

Researchers should validate protein activity after storage using functional assays to ensure that experimental results aren't compromised by protein degradation.