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Structural Basis for Regulation of Tyrosine Hydroxlyase by the Catecholamines
8
THEME A Catecholamine Biosynthesis and Storage
GTPCH promoters were enhanced by E2 over a wide range of concentrations with either ER subtype. Membrane-initiated estradiol signaling in regulation of TH and DBH promoter activity was demonstrated using membrane-impermeable estradiol conjugate (E2BSA). E2BSA elicited rapid phosphorylation of CREB and increased CRE-driven promoter activity. Over-expression of dominant negative forms of CREB, with mutations in DNA binding or phosphorylation site, prevented TH promoter response to E2BSA. Pre-treatment with protein kinase A (PKA) and MEK inhibitors reduced E2 dependent phosphorylation of CREB and ERK, and also decreased induction of TH promoter activity by E2 or E2BSA. CONCLUSIONS: The results in vivo demonstrate the importance of estrogens in regulation of basal and stress induced CA biosynthetic enzyme gene expression. Activation of ERα is especially important in modulation of the response to stress. The findings in vitro show that E2 induction of TH and DBH gene transcription with ERα involves membrane-initiated estradiol signaling. The role of membrane initiated ER signaling in regulation of CA gene expression by estrogens in vivo remains to be determined.
Structural Basis for Regulation of Tyrosine Hydroxlyase by the Catecholamines Gabrielle Briggs, Jesse Bulley, Peter R. Dunkley and Phillip W. Dickson University of Newcastle Australia
BACKGROUND: Tyrosine Hydroxylase (TH) performs the first and rate limiting step in the synthesis of catechoamines (CAs), which feedback to dramatically inhibit DOPA synthesis by irreversibly binding to the active site. Key residues in the N-terminal regulatory domain are required for this inhibition and are thought to interact with the CA-bound active site. During neuronal or chromaffin cell activation, phosphorylation of the N-terminal residue Ser40 relieves this inhibition. In addition, another CA binding site has lower affinity for the enzyme, is dissociable and is not abolished by phosphorylation. Both this site and the non-dissociable site inhibit TH by competing with the essential cofactor, tetrahydrobiopterin (BH4). In situ, the low affinity site regulates TH through binding and dissociating according to cytosolic CA levels, and operates in conjunction with the “on/off” regulation provided by high affinity CA binding and phosphorylation. While the functionality of these regulatory sites is understood, the structural basis underlying their functions is mostly unknown. This is largely due to the TH crystal structure being a CA-free form and lacking the 156 N-terminal residues containing those required for high affinity CA binding. OBJECTIVES: We aimed to identify residues involved in the CA binding sites via substitution of amino acids within and around the active site. In doing so, we would gain insight into how TH is
Structural Basis for Regulation of Tyrosine Hydroxlyase by the Catecholamines
9
regulated by CAs, in the absence of a complete crystal structure. A key area identified for the high affinity site was then used for computational screening of compounds and in vitro testing of hits for their effect on high affinity CA binding. METHODS AND RESULTS: 7 active site substitutions and 8 substitutions of residues around the outer regions of the active site were generated. Dopamine (DA) dependent inhibition of recombinant TH activity was assayed using the tritiated water release assay. The IC50s for DA inhibition through the low affinity site in active site mutants Y371F and E332D were 70-fold and 10-fold higher than wild-type respectively. Effects of CA bound to the high affinity site were assessed by measuring the competition of CA with the cofactor, BH4. High affinity bound DA produced a 10-fold increase in the Km for BH4 in wild-type TH. In Y371F and E332D, this inhibitory effect was absent. Substitutions made outside of the active site showed changes to the high affinity site, with A297L and D361N showing no increase in the Km for the cofactor upon high affinity CA binding. This region of TH was targeted for computational screening of a drug database using DOCK 6. 7 of the hits were tested in vitro. While DA inhibition was retained with these compounds, one was shown to increase TH activity up to 5-fold in the absence of DA. CONCLUSIONS: The common roles of E332 and Y371 in low and high affinity CA binding indicate that these sites overlap in the active site and use E332 and Y371to position CA in the BH4 binding site. Residues outside the active site seem to be involved in high affinity binding only and may constitute the interacting surface for the N-terminus in producing an irreversible binding of CA. Since low and high affinity CA binding occur simultaneously, they cannot occupy the same space, as these results suggest. However, TH is a dimer of dimers and CA binding stoichiometry has previously been measured as 1mol/mol TH dimer for each of the low and high affinity sites, suggesting that E332 and Y371 bind CA in both monomers of the dimer, while A297 and D361 have an additional role in producing the high affinity site in one monomer only. This model could be confirmed by testing for high and low affinity sites in TH monomers. The compound’s activation of TH activity suggests that this region may have roles outside that of high affinity CA inhibition.
THEME A Catecholamine Biosynthesis and Storage
GTPCH promoters were enhanced by E2 over a wide range of concentrations with either ER subtype. Membrane-initiated estradiol signaling in regulation of TH and DBH promoter activity was demonstrated using membrane-impermeable estradiol conjugate (E2BSA). E2BSA elicited rapid phosphorylation of CREB and increased CRE-driven promoter activity. Over-expression of dominant negative forms of CREB, with mutations in DNA binding or phosphorylation site, prevented TH promoter response to E2BSA. Pre-treatment with protein kinase A (PKA) and MEK inhibitors reduced E2 dependent phosphorylation of CREB and ERK, and also decreased induction of TH promoter activity by E2 or E2BSA. CONCLUSIONS: The results in vivo demonstrate the importance of estrogens in regulation of basal and stress induced CA biosynthetic enzyme gene expression. Activation of ERα is especially important in modulation of the response to stress. The findings in vitro show that E2 induction of TH and DBH gene transcription with ERα involves membrane-initiated estradiol signaling. The role of membrane initiated ER signaling in regulation of CA gene expression by estrogens in vivo remains to be determined.
Structural Basis for Regulation of Tyrosine Hydroxlyase by the Catecholamines Gabrielle Briggs, Jesse Bulley, Peter R. Dunkley and Phillip W. Dickson University of Newcastle Australia
BACKGROUND: Tyrosine Hydroxylase (TH) performs the first and rate limiting step in the synthesis of catechoamines (CAs), which feedback to dramatically inhibit DOPA synthesis by irreversibly binding to the active site. Key residues in the N-terminal regulatory domain are required for this inhibition and are thought to interact with the CA-bound active site. During neuronal or chromaffin cell activation, phosphorylation of the N-terminal residue Ser40 relieves this inhibition. In addition, another CA binding site has lower affinity for the enzyme, is dissociable and is not abolished by phosphorylation. Both this site and the non-dissociable site inhibit TH by competing with the essential cofactor, tetrahydrobiopterin (BH4). In situ, the low affinity site regulates TH through binding and dissociating according to cytosolic CA levels, and operates in conjunction with the “on/off” regulation provided by high affinity CA binding and phosphorylation. While the functionality of these regulatory sites is understood, the structural basis underlying their functions is mostly unknown. This is largely due to the TH crystal structure being a CA-free form and lacking the 156 N-terminal residues containing those required for high affinity CA binding. OBJECTIVES: We aimed to identify residues involved in the CA binding sites via substitution of amino acids within and around the active site. In doing so, we would gain insight into how TH is
Structural Basis for Regulation of Tyrosine Hydroxlyase by the Catecholamines
9
regulated by CAs, in the absence of a complete crystal structure. A key area identified for the high affinity site was then used for computational screening of compounds and in vitro testing of hits for their effect on high affinity CA binding. METHODS AND RESULTS: 7 active site substitutions and 8 substitutions of residues around the outer regions of the active site were generated. Dopamine (DA) dependent inhibition of recombinant TH activity was assayed using the tritiated water release assay. The IC50s for DA inhibition through the low affinity site in active site mutants Y371F and E332D were 70-fold and 10-fold higher than wild-type respectively. Effects of CA bound to the high affinity site were assessed by measuring the competition of CA with the cofactor, BH4. High affinity bound DA produced a 10-fold increase in the Km for BH4 in wild-type TH. In Y371F and E332D, this inhibitory effect was absent. Substitutions made outside of the active site showed changes to the high affinity site, with A297L and D361N showing no increase in the Km for the cofactor upon high affinity CA binding. This region of TH was targeted for computational screening of a drug database using DOCK 6. 7 of the hits were tested in vitro. While DA inhibition was retained with these compounds, one was shown to increase TH activity up to 5-fold in the absence of DA. CONCLUSIONS: The common roles of E332 and Y371 in low and high affinity CA binding indicate that these sites overlap in the active site and use E332 and Y371to position CA in the BH4 binding site. Residues outside the active site seem to be involved in high affinity binding only and may constitute the interacting surface for the N-terminus in producing an irreversible binding of CA. Since low and high affinity CA binding occur simultaneously, they cannot occupy the same space, as these results suggest. However, TH is a dimer of dimers and CA binding stoichiometry has previously been measured as 1mol/mol TH dimer for each of the low and high affinity sites, suggesting that E332 and Y371 bind CA in both monomers of the dimer, while A297 and D361 have an additional role in producing the high affinity site in one monomer only. This model could be confirmed by testing for high and low affinity sites in TH monomers. The compound’s activation of TH activity suggests that this region may have roles outside that of high affinity CA inhibition.