| 1996 |
GFRP (GCHFR) was purified to homogeneity from rat liver as a homodimer of 9.5-kDa subunits with a native molecular mass of ~20 kDa, co-purifying with GTP cyclohydrolase I (GTPCHI) in a complex that binds GTP-agarose. GFRP mediates tetrahydrobiopterin (BH4)-dependent inhibition of GTPCHI activity, and this inhibition is specifically reversed by L-phenylalanine. Recombinant GFRP expressed as a thioredoxin fusion protein in E. coli retained BH4-dependent GTPCHI inhibitory activity, confirming its direct regulatory function. |
Co-purification via GTP-affinity chromatography, gel filtration, anion-exchange HPLC; cDNA cloning and recombinant expression; in vitro enzymatic inhibition assay |
The Journal of biological chemistry |
High |
8702680
|
| 2002 |
Crystal structure of the stimulatory GTPCHI·GFRP complex reveals that the GTPCHI decamer is sandwiched by two GFRP homopentamers. Each GFRP pentamer forms a five-membered ring with beta-propeller-like symmetry. Five phenylalanine molecules are buried at each GFRP–GTPCHI interface, enhancing protein–protein binding and locking GTPCHI in the active state to stimulate enzyme activity. |
X-ray crystallography of the reconstituted phenylalanine-induced stimulatory complex |
Proceedings of the National Academy of Sciences of the United States of America |
High |
11818540
|
| 2001 |
Crystal structure of rat GFRP alone at 2.6 Å resolution shows the protein forms a pentamer, establishing the oligomeric state of the regulatory subunit and providing a structural model for how it interacts with the GTPCHI decamer. Kinetic studies confirmed that both rat and human GFRP produce similar regulatory effects on human GTPCHI. |
X-ray crystallography (2.6 Å); kinetic enzyme assays with human GCH1 |
Journal of molecular biology |
High |
11580249
|
| 2004 |
Crystal structure of the biopterin-induced inhibitory GTPCHI·GFRP complex shows five biopterin molecules at each GTPCHI–GFRP interface. Biopterin binding induces large conformational changes ('induced fit') in GTPCHI peptide segments forming the active site, resulting in enzyme inhibition. Comparison with the stimulatory complex reveals that the same interfaces are used for both stimulation (phenylalanine) and inhibition (biopterin), but with opposite structural outcomes. Dystonia-associated mutations in GTPCHI map to regions that may disrupt GFRP-mediated regulation. |
X-ray crystallography of biopterin-bound inhibitory complex; structural comparison with stimulatory complex; mapping of disease mutations onto structure |
The Journal of biological chemistry |
High |
15448133
|
| 2010 |
The N-terminal 45-amino-acid peptide of rat GTPCHI functions as an autoinhibitory element and is required for GFRP binding in the absence of small-molecule effectors. Deletion of this peptide (Δ45-GTPCH) abolishes GFRP complex formation and phenocopies GFRP-stimulated wild-type GTPCHI (increased Vmax, decreased KmGTP, increased Hill coefficient). GFRP binding to wild-type GTPCHI in the absence of any effector provides allosteric stimulation (20% increase in Vmax, 50% decrease in KmGTP). Expression of Δ45-GTPCH in HEK-293 cells produced 3-fold greater BH4 accumulation than equivalent wild-type GTPCHI. |
Recombinant protein expression and purification of truncation mutants; in vitro kinetic enzyme assays; GFRP binding assays; cellular BH4 measurement in HEK-293 cells |
The Journal of biological chemistry |
High |
21163945
|
| 2005 |
In human aortic endothelial cells (HAECs) and adult rat cardiomyocytes, LPS treatment decreased GFRP mRNA without changing GTPCHI protein levels, and this disruption of the GTPCHI:GFRP complex correlated with enhanced de novo biopterin synthesis. Conversely, hydrogen peroxide increased both GTPCHI and GFRP mRNA in HAECs while depleting BH4, suggesting GFRP overrides GTPCHI upregulation to inhibit enzyme activity. Transient transfection of GFRP in cells depleted biopterin levels, directly demonstrating GFRP's inhibitory role on cellular BH4 production. |
Western blot, qRT-PCR, transient transfection with GFRP cDNA, biopterin measurement by HPLC |
Free radical biology & medicine |
Medium |
15649650
|
| 2013 |
Transgenic mice ubiquitously overexpressing Gfrp showed increased GFRP–GTPCHI interaction (confirmed by co-immunoprecipitation), reduced BH4 levels, reduced glutathione (GSH), and altered mitochondrial bioenergetics compared to control littermates, establishing that GFRP overexpression in vivo suppresses BH4 biosynthesis and increases oxidative stress. |
Cre-Lox transgenic mouse model; co-immunoprecipitation; BH4 and GSH quantification; mitochondrial bioenergetics profiling |
Antioxidants & redox signaling |
Medium |
23521531
|
| 2020 |
High-resolution X-ray crystal structures of ligand-free and ligand-bound human GFRP and human GCH1–GFRP complexes reveal that effector-induced conformational changes in GCH1 or GFRP create highly complementary surfaces, driving picomolar-affinity complex formation with extremely slow koff values. In the absence of BH4 or phenylalanine, the GCH1–GFRP complex rapidly disintegrates. Binding of the substrate analogue 7-deaza-GTP to active and inhibited GCH1–GFRP complexes indicates a dissociation rate-controlled mechanism of non-competitive inhibition. The disordered GCH1 N-terminus was found NOT to impact complex formation or enzymatic activity in human GCH1 (contrasting with prior findings in rat GTPCHI). |
X-ray crystallography; SPR kinetics (koff measurement); enzymatic assays; comparison of full-length vs. N-terminally truncated GCH1 |
Journal of structural biology |
High |
33387654
|
| 2018 |
L-phenylalanine administration in spontaneously hypertensive rats restored vascular BH4 levels and improved vascular function through activation of the GCH1–GFRP complex, demonstrating that pharmacological engagement of GFRP-mediated feed-forward activation is achievable in a disease model. |
In vivo rodent model (spontaneously hypertensive rats); L-phenylalanine administration; vascular function assays; BH4 measurement |
JACC. Basic to translational science |
Medium |
29963647
|