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

Prominin 1 is crucial for the early development of photoreceptor outer segments

Abstract Prominin 1 (PROM1) is a pentaspan transmembrane glycoprotein localized on the nascent photoreceptor discs. Mutations in PROM1 are linked to various retinal diseases. In this study, we assessed the role of PROM1 in photoreceptor biology and physiology using the PROM1 knockout murine model (rd19). Our study found that PROM1 is essential for vision and photoreceptor development. We found an early reduction in photoreceptor response beginning at post-natal day 12 (P12) before eye opening in the absence of PROM1 with no apparent loss in photoreceptor cells. However, at this stage, we observed an increased glial cell activation, indicative of cell damage. Contrary to our expectations, dark rearing did not mitigate photoreceptor degeneration or vision loss in PROM1 knockout mice. In addition to physiological defects seen in PROM1 knockout mice, ultrastructural analysis revealed malformed outer segments characterized by whorl-like continuous membranes instead of stacked disks. In parallel to the reduced rod response at P12, proteomics revealed a significant reduction in the levels of protocadherin, a known interactor of PROM1, and rod photoreceptor outer segment proteins, including rhodopsin. Overall, our results underscore the indispensable role of PROM1 in photoreceptor development and maintenance of healthy vision

Cross talk between NADPH oxidase and autophagy in pulmonary artery endothelial cells with intrauterine persistent pulmonary hypertension

Autophagy is a process for cells to degrade proteins or entire organelles to maintain a balance in the synthesis, degradation, and subsequent recycling of cellular products. Increased reactive oxygen species formation is known to induce autophagy. We previously reported that increased NADPH oxidase (NOX) activity in pulmonary artery endothelial cells (PAEC) from fetal lambs with persistent pulmonary hypertension (PPHN) contributes to impaired angiogenesis in PPHN-PAEC compared with normal PAEC. We hypothesized that increased NOX activity in PPHN-PAEC is associated with increased autophagy, which, in turn, contributes to impaired angiogenesis in PPHN-PAEC. In the present study, we detected increased autophagy in PPHN-PAEC as shown by increased ratio of the microtubule-associated protein 1 light chain (LC3)-II to LC3-I and increased percentage of green fluorescent protein-LC3 punctate positive cells. Inhibiting autophagy by 3-methyladenine, chloroquine, and beclin-1 knockdown in PPHN-PAEC has led to decreased autophagy and increased in vitro angiogenesis. Inhibition of autophagy also decreased the association between gp91phox and p47phox, NOX activity, and superoxide generation. A nonspecific antioxidant N-acetylcysteine and a NOX inhibitor apocynin decreased autophagy in PPHN-PAEC. In conclusion, autophagy may contribute to impaired angiogenesis in PPHN-PAEC through increasing NOX activity. Our results suggest that, in PPHN-PAEC, a positive feedback relationship between autophagy and NOX activity may regulate angiogenesis. </jats:p

RANS-Based Modelling of Turbulent Flow in Submarine Pipe Bends: Effect of Computational Mesh and Turbulence Modelling

Pipe bend is a critical integral component, widely used in slurry pipeline systems involving various engineering applications, including natural gas hydrate production. The aim of this study is to assess the capability of RANS-based CFD models to capture the main features of the turbulent single-phase flow in pipe bends, in view of the future investigation of the hydrate slurry flow in the same geometry. This is different from the available literature in which only a few accounted for the effects of a combination of computational mesh, turbulence model, and near-wall treatment approach. In this study, three types of mesh configuration were adopted to carry out the computations, namely unstructured mesh and two structured meshes with a uniform and nonuniform inflation layer, respectively. To explore the influence of the turbulence model, standard k-ε, low-Reynolds k-ε, and nonlinear eddy viscosity turbulence model were selected to close RANS equations. Pressure coefficient, mean axial velocity, turbulence intensity, secondary flow velocity, and magnitude of secondary flow were regarded as the critical variables to make a comprehensive sensitivity analysis. Predicted results suggest that turbulent kinetic energy is the most sensitive variable to the computational mesh while others tend to stabilize. The largest difference of turbulence kinetic energy was around 26% between unstructured mesh and structured mesh with a nonuniform inflation layer. Additionally, a fully resolved boundary layer can reduce the sensitivity of mesh on turbulent kinetic energy, especially for a nonlinear turbulence model. However, the large gradient and peak value of turbulence intensity near the inner wall of the bend was not captured by the case with a fully resolved boundary layer, compared with that of the wall function used. Furthermore, it has been confirmed that the same rule was detected also for different curvature ratios, Reynolds numbers, and dimensionless wall distance y+.</jats:p

The distribution of GCH1 in mitochondria.

<p>Cytosolic and mitochondrial fractions were extracted from liver, heart and kidney and probed for GCH1, COX-1 and HSP90 by western blot analysis (A). The liver mitochondrial fraction was immunoprecipitated with IgG or GCH1 and immunoblotted with GCH1 antibody. IP, Immunoprecipitation, IB, western blot analysis (B). BH4 levels from liver and liver mitochondria were assayed by HPLC and normalized by protein concentrations (C) (N = 4). *P<0.05 vs. Liver BH4.</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

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