The Protein Partners of GTP Cyclohydrolase I in Rat Organs

GTP 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.A 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.GCH1 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

The interaction of GCH1 with GFRP in different organs.

<p>GCH1 and its interacting proteins were purified from brain, heart, liver and kidney, and analyzed by western blot against GCH1 antibody (A) and GFRP antibody (B). EL1 is the first eluate, EL2 is the second eluate. Eluates from IgG conjugated column were used as controls. Protein lysates from different organs were immunoblotted with GFRP antibody and HSP90. HSP90 was used as a loading control (C). HEK cells stably over-expressing GCH1 (GCH1-HEK) were immunoprecipitated with IgG or GCH1 antibody and immunoblotted against GFRP and GCH1. Straight cell lysate of GCH1-HEK cells (Lysate) was also loaded for comparison (the first lane). Liver homogenate was used as a positive control (D). HEK cells were transiently transfected with Flag-GCH1, HA-GFRP or pcDNA, and immunoprecipitated with Flag tag (E) or HA tag and immunoblotted with GCH1 and GFRP antibodies. In (E), cell lysates (the first lane) from HEK cells transfected with FLAG-GCH1 and HA-GFRP were used as positive controls for GFRP and GCH1 expression.</p

Purification of GCH1 and its interacting proteins.

<p>(A) Flow chart showing the procedure for purification and characterization of GCH1 complexes from rat organ (brain, heart, liver and kidney). (B) Protein samples from different steps of protein purification by IgG and GCH1 from rat liver were separated by SDS-PAGE, (B) stained with Gelcode Blue and (C) analyzed by Western blot analysis against GCH1 antibody. IgG and GCH1 purified samples from rat heart were (D) silver stained and (E) analyzed by Western blot analysis against GCH1. MK, molecular weight marker; Ig, IgG; GCH, GCH1; FL, flow-through; FLG, Flag tag, Flg-GCH is cell lysates from FLAG-GCH1 over-expressing HEK cells, used as positive control.</p

Inhibition of CDKS by roscovitine suppressed LPS-induced ·NO production through inhibiting NFκB activation and BH4 biosynthesis in macrophages

In inflammatory diseases, tissue damage is critically associated with nitric oxide (·NO) and cytokines, which are overproduced in response to cellular release of endotoxins. Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases (CDKs) on ·NO production in mouse macrophages. In RAW264.7 cells, we found that roscovitine abolished the production of ·NO induced by lipopolysaccharide (LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA and protein expression. Our data also showed that roscovitine attenuated LPS-induced phosphorylation of IκB kinase β (IKKβ), IκB, and p65 but enhanced the phosphorylation of ERK, p38, and c-Jun NH2-terminal kinase (JNK). In addition, roscovitine dose dependently inhibited LPS-induced expression of cyclooxygenase-2 (COX)-2, IL-1β, and IL-6 but not tumor necrosis factor (TNF)-α. Tetrahydrobiopterin (BH4), an essential cofactor for iNOS, is easily oxidized to 7,8-dihydrobiopterin (BH2). Roscovitine significantly inhibited LPS-induced BH4 biosynthesis and decreased BH4-to-BH2 ratio. Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase-1 (GCH-1), the rate-limiting enzyme for BH4 biosynthesis. Using other CDK inhibitors, we found that CDK1, CDK5, and CDK7, but not CDK2, significantly inhibited LPS-induced ·NO production in macrophages. Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited ·NO production, iNOS, and COX-2 upregulation, activation of NFκB, and induction of GCH-1 by LPS. Together, our data indicate that roscovitine abolishes LPS-induced ·NO production in macrophages by suppressing nuclear factor-κB activation and BH4 biosynthesis, which might be mediated by CDK1, CDK5, and CDK7. Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which roscovitine attenuates inflammation

Identified Proteins in GCH1 pull-down rat liver samples.

<p>Identified Proteins in GCH1 pull-down rat liver samples.</p

The interactions of GCH1 with its 6 protein partners identified by ESI/LC/MS were validated in rat liver by western blot analysis.

<p>The purified GCH1 complexes from rat liver were separated by SDS-PAGE and then immunoblotted with different antibodies as indicated. The blots were representatives of five independent biological repeats.</p

The interaction of rat liver GCH1-interacting proteins with GCH1 in brain, heart and kidney determined by western blot analysis.

<p>(A) The protein lysates from different rat organs were immunoblotted with antibodies indicated. (B) The GCH1 protein complexes purified from different organs were immunoblotted with the indicated antibodies.</p