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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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bph13202-fig-0001: 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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bph13202-fig-0001: 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