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Validating the GTP-cyclohydrolase 1-feedback regulatory complex as a therapeutic target using biophysical and in vivo approaches.

Hussein D, Starr A, Heikal L, McNeill E, Channon KM, Brown PR, Sutton BJ, McDonnell JM, Nandi M - Br. J. Pharmacol. (2015)

Bottom Line: Therefore, orally bioavailable pharmacological activators of endogenous BH4 biosynthesis hold significant therapeutic potential.We investigated the effects of L-phe on the biophysical interactions of GCH1 and GFRP and its potential to alter BH4 levels in vivo.In vivo, L-phe challenge induced a sustained elevation of aortic BH4 , an effect absent in GCH1(fl/fl)-Tie2Cre mice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical Science, Faculty of Life Sciences & Medicine, King's College London, London, UK.

No MeSH data available.


Related in: MedlinePlus

Expression and activity of human recombinant native/full length GCH1 (F-GCH1), truncated GCH1 (T-GCH1) and GTP cyclohydrolase 1 (GCH1) feedback regulatory protein (GFRP). (A) F-GCH1 expression in uninduced (lane 1) and IPTG induced (lane 2) cells. T-GCH1 expression from IPTG induced (lanes 3 and 4) cells. GFRP expression in uninduced (lane 5) and IPTG (isopropyl β-D-1-thiogalactopyranoside) induced (lanes 6 and 7) cells. Products were resolved on a 4–15% SDS-PAGE gradient gel. (B) Elution of purified proteins from dual expression cultures: His-T-GCH1 and GFRP bands are observed at ∼25 and ∼10 kDa, respectively; His-F-GCH1–GFRP bands observed at ∼28 and ∼10 kDa respectively. (C) (i): Immunoreactive bands for GFRP at ∼10 kDa were observed using a commercially available GFRP antibody; (ii) immunoreactive bands for both GFRP and T-GCH1 using a commercially available His-tag antibody were observed at ∼10 and ∼24 kDa, respectively; (iii) immunoreactive band at ∼28 kDa for F-GCH1 using an N-terminal GCH1 antibody; no immunoreactive bands for T-GCH1. (D) Native 5% PAGE gels run prior to surface plasmon resonance (SPR) analysis. Predominant bands highlighted for F-GCH1, T-GCH1 and GFRP as indicated, from two independent experiments. (E) Activity of purified F-GCH1 and T-GCH1 was quantified by HPLC detecting neopterin production; mouse liver homogenate and empty vector control were used for comparison (n = 6, mean ± SEM). *P < 0.05, ***P < 0.001, significantly different from control. (F) GCH1 activity measured by microplate assay in the presence of 250 nM GCH1, 1 μM GFRP and 100 μM GTP (n = 10, mean ± SEM). ***P < 0.01, significantly different from F-GCH1 without GFRP).
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fig01: Expression and activity of human recombinant native/full length GCH1 (F-GCH1), truncated GCH1 (T-GCH1) and GTP cyclohydrolase 1 (GCH1) feedback regulatory protein (GFRP). (A) F-GCH1 expression in uninduced (lane 1) and IPTG induced (lane 2) cells. T-GCH1 expression from IPTG induced (lanes 3 and 4) cells. GFRP expression in uninduced (lane 5) and IPTG (isopropyl β-D-1-thiogalactopyranoside) induced (lanes 6 and 7) cells. Products were resolved on a 4–15% SDS-PAGE gradient gel. (B) Elution of purified proteins from dual expression cultures: His-T-GCH1 and GFRP bands are observed at ∼25 and ∼10 kDa, respectively; His-F-GCH1–GFRP bands observed at ∼28 and ∼10 kDa respectively. (C) (i): Immunoreactive bands for GFRP at ∼10 kDa were observed using a commercially available GFRP antibody; (ii) immunoreactive bands for both GFRP and T-GCH1 using a commercially available His-tag antibody were observed at ∼10 and ∼24 kDa, respectively; (iii) immunoreactive band at ∼28 kDa for F-GCH1 using an N-terminal GCH1 antibody; no immunoreactive bands for T-GCH1. (D) Native 5% PAGE gels run prior to surface plasmon resonance (SPR) analysis. Predominant bands highlighted for F-GCH1, T-GCH1 and GFRP as indicated, from two independent experiments. (E) Activity of purified F-GCH1 and T-GCH1 was quantified by HPLC detecting neopterin production; mouse liver homogenate and empty vector control were used for comparison (n = 6, mean ± SEM). *P < 0.05, ***P < 0.001, significantly different from control. (F) GCH1 activity measured by microplate assay in the presence of 250 nM GCH1, 1 μM GFRP and 100 μM GTP (n = 10, mean ± SEM). ***P < 0.01, significantly different from F-GCH1 without GFRP).

Mentions: Soluble human recombinant T-GCH1, F-GCH1 and GFRP proteins were successfully expressed individually in BL21 (GCH1) or Rosetta (GFRP) cells (Figure 1A). In dual expression cultures, metal affinity purification of His6T-GCH1 or His6F-GCH1 revealed that GFRP was able to bind to both forms of GCH1 and could be co-isolated (Figure 1B).


Validating the GTP-cyclohydrolase 1-feedback regulatory complex as a therapeutic target using biophysical and in vivo approaches.

Hussein D, Starr A, Heikal L, McNeill E, Channon KM, Brown PR, Sutton BJ, McDonnell JM, Nandi M - Br. J. Pharmacol. (2015)

Expression and activity of human recombinant native/full length GCH1 (F-GCH1), truncated GCH1 (T-GCH1) and GTP cyclohydrolase 1 (GCH1) feedback regulatory protein (GFRP). (A) F-GCH1 expression in uninduced (lane 1) and IPTG induced (lane 2) cells. T-GCH1 expression from IPTG induced (lanes 3 and 4) cells. GFRP expression in uninduced (lane 5) and IPTG (isopropyl β-D-1-thiogalactopyranoside) induced (lanes 6 and 7) cells. Products were resolved on a 4–15% SDS-PAGE gradient gel. (B) Elution of purified proteins from dual expression cultures: His-T-GCH1 and GFRP bands are observed at ∼25 and ∼10 kDa, respectively; His-F-GCH1–GFRP bands observed at ∼28 and ∼10 kDa respectively. (C) (i): Immunoreactive bands for GFRP at ∼10 kDa were observed using a commercially available GFRP antibody; (ii) immunoreactive bands for both GFRP and T-GCH1 using a commercially available His-tag antibody were observed at ∼10 and ∼24 kDa, respectively; (iii) immunoreactive band at ∼28 kDa for F-GCH1 using an N-terminal GCH1 antibody; no immunoreactive bands for T-GCH1. (D) Native 5% PAGE gels run prior to surface plasmon resonance (SPR) analysis. Predominant bands highlighted for F-GCH1, T-GCH1 and GFRP as indicated, from two independent experiments. (E) Activity of purified F-GCH1 and T-GCH1 was quantified by HPLC detecting neopterin production; mouse liver homogenate and empty vector control were used for comparison (n = 6, mean ± SEM). *P < 0.05, ***P < 0.001, significantly different from control. (F) GCH1 activity measured by microplate assay in the presence of 250 nM GCH1, 1 μM GFRP and 100 μM GTP (n = 10, mean ± SEM). ***P < 0.01, significantly different from F-GCH1 without GFRP).
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Related In: Results  -  Collection

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fig01: Expression and activity of human recombinant native/full length GCH1 (F-GCH1), truncated GCH1 (T-GCH1) and GTP cyclohydrolase 1 (GCH1) feedback regulatory protein (GFRP). (A) F-GCH1 expression in uninduced (lane 1) and IPTG induced (lane 2) cells. T-GCH1 expression from IPTG induced (lanes 3 and 4) cells. GFRP expression in uninduced (lane 5) and IPTG (isopropyl β-D-1-thiogalactopyranoside) induced (lanes 6 and 7) cells. Products were resolved on a 4–15% SDS-PAGE gradient gel. (B) Elution of purified proteins from dual expression cultures: His-T-GCH1 and GFRP bands are observed at ∼25 and ∼10 kDa, respectively; His-F-GCH1–GFRP bands observed at ∼28 and ∼10 kDa respectively. (C) (i): Immunoreactive bands for GFRP at ∼10 kDa were observed using a commercially available GFRP antibody; (ii) immunoreactive bands for both GFRP and T-GCH1 using a commercially available His-tag antibody were observed at ∼10 and ∼24 kDa, respectively; (iii) immunoreactive band at ∼28 kDa for F-GCH1 using an N-terminal GCH1 antibody; no immunoreactive bands for T-GCH1. (D) Native 5% PAGE gels run prior to surface plasmon resonance (SPR) analysis. Predominant bands highlighted for F-GCH1, T-GCH1 and GFRP as indicated, from two independent experiments. (E) Activity of purified F-GCH1 and T-GCH1 was quantified by HPLC detecting neopterin production; mouse liver homogenate and empty vector control were used for comparison (n = 6, mean ± SEM). *P < 0.05, ***P < 0.001, significantly different from control. (F) GCH1 activity measured by microplate assay in the presence of 250 nM GCH1, 1 μM GFRP and 100 μM GTP (n = 10, mean ± SEM). ***P < 0.01, significantly different from F-GCH1 without GFRP).
Mentions: Soluble human recombinant T-GCH1, F-GCH1 and GFRP proteins were successfully expressed individually in BL21 (GCH1) or Rosetta (GFRP) cells (Figure 1A). In dual expression cultures, metal affinity purification of His6T-GCH1 or His6F-GCH1 revealed that GFRP was able to bind to both forms of GCH1 and could be co-isolated (Figure 1B).

Bottom Line: Therefore, orally bioavailable pharmacological activators of endogenous BH4 biosynthesis hold significant therapeutic potential.We investigated the effects of L-phe on the biophysical interactions of GCH1 and GFRP and its potential to alter BH4 levels in vivo.In vivo, L-phe challenge induced a sustained elevation of aortic BH4 , an effect absent in GCH1(fl/fl)-Tie2Cre mice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical Science, Faculty of Life Sciences & Medicine, King's College London, London, UK.

No MeSH data available.


Related in: MedlinePlus