Abstract
ObjectiveGTP cyclohydrolase I (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin biosynthesis and has been shown to be a promising therapeutic target in ischemic heart disease, hypertension, atherosclerosis and diabetes. The endogenous GCH1-interacting partners have not been identified. Here, we determined endogenous GCH1-interacting proteins in rat.Methods and ResultsA pulldown and proteomics approach were used to identify GCH1 interacting proteins in rat liver, brain, heart and kidney. We demonstrated that GCH1 interacts with at least 17 proteins including GTP cyclohydrolase I feedback regulatory protein (GFRP) in rat liver by affinity purification followed by proteomics and validated six protein partners in liver, brain, heart and kidney by immunoblotting. GCH1 interacts with GFRP and very long-chain specific acyl-CoA dehydrogenase in the liver, tubulin beta-2A chain in the liver and brain, DnaJ homolog subfamily A member 1 and fatty aldehyde dehydrogenase in the liver, heart and kidney and eukaryotic translation initiation factor 3 subunit I (EIF3I) in all organs tested. Furthermore, GCH1 associates with mitochondrial proteins and GCH1 itself locates in mitochondria.ConclusionGCH1 interacts with proteins in an organ dependant manner and EIF3I might be a general regulator of GCH1. Our finding indicates GCH1 might have broader functions beyond tetrahydrobiopterin biosynthesis.
Highlights
Tetrahydrobiopterin (BH4) is an essential cofactor for phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, and nitric oxide synthases (NOS) and alkylglycerol monooxygenase [1]
GCH1 interacts with proteins in an organ dependant manner and eukaryotic translation initiation factor 3 subunit I (EIF3I) might be a general regulator of GCH1
Our finding indicates GCH1 might have broader functions beyond tetrahydrobiopterin biosynthesis
Summary
Tetrahydrobiopterin (BH4) is an essential cofactor for phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, and nitric oxide synthases (NOS) and alkylglycerol monooxygenase [1]. The normal BH4 level is required for the degradation of phenylalanine, the biosynthesis of catecholamine, serotonin, and the balance of nitric oxide and superoxide [2]. GCH1 has been linked to hypertension, atherosclerosis, diabetes, cardiac hypertrophy, and myocardial ischemia [2] and has become a potential therapeutic target in cardiovascular disease. We have found that GCH1 confers the increased resistance to myocardial ischemia in Brown Norway rats compared to Dahl S rats [8]. Over-expression of GCH1 restores ischemic preconditioning during hyperglycemia [9], protects against acute cardiac allograft rejection [10], attenuates blood pressure progression in salt-sensitive low-renin hypertension [11], and reduces endothelial dysfunction and atherosclerosis in ApoE-knockout mice [12]. The understanding of molecular mechanisms of the protective functions by GCH1 remains very limited
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