Untargeted metabolite profiling reveals that nitric oxide bioynthesis is an endogenous modulator of carotenoid biosynthesis in Deinococcus radiodurans and is required for extreme ionizing radiation resistance.

Deinococcus radiodurans (Drad) is the most radioresistant organism known. Although mechanisms that underlie the extreme radioresistance of Drad are incompletely defined, resistance to UV irradiation-induced killing was found to be greatly attenuated in an NO synthase (NOS) knockout strain of Drad (Δnos). We now show that endogenous NO production is also critical for protection of Drad against γ-irradiation (3000 Gy), a result of accelerated growth recovery, not protection against killing. NO-donor treatment rescued radiosensitization in Δnos Drad but did not influence radiosensitivity in wild type Drad. To discover molecular mechanisms by which endogenous NO confers radioresistance, metabolite profiling studies were performed. Untargeted LC-MS-based metabolite profiling in Drad quantified relative abundances of 1425 molecules and levels of 294 of these were altered by >5-fold (p < 0.01). Unexpectedly, these studies identified a dramatic perturbation in carotenoid biosynthetic intermediates in Δnos Drad, including a reciprocal switch in the pathway end-products from deoxydeinoxanthin to deinoxanthin. NO supplementation rescued these nos deletion-associated changes in carotenoid biosynthesis, and fully-restored radioresistance to wildtype levels. Because carotenoids were shown to be important contributors to radioprotection in Drad, our findings suggest that endogenously-produced NO serves to maintain a spectrum of carotenoids critical for Drad's ability to withstand radiation insult.

A requirement for Gch1 and tetrahydrobiopterin in embryonic development.

INTRODUCTION: GTP cyclohydrolase I (GTPCH) catalyses the first and rate-limiting reaction in the synthesis of the enzymatic cofactor, tetrahydrobiopterin (BH4). Loss of function mutations in the GCH1 gene lead to congenital neurological diseases such as DOPA-responsive dystonia and hyperphenylalaninemia. However, little is known about how GTPCH and BH4 affects embryonic development in utero, and in particular whether metabolic replacement or supplementation in pregnancy is sufficient to rescue genetic GTPCH deficiency in the developing embryo. METHODS AND RESULTS: Gch1 deficient mice were generated by the insertion of loxP sites flanking exons 2-3 of the Gch1 gene. Gch1(fl/fl) mice were bred with Sox2cre mice to generate mice with global Gch1 deficiency. Genetic ablation of Gch1 caused embryonic lethality by E13.5. Despite loss of Gch1 mRNA and GTPCH enzymatic activity, whole embryo BH4 levels were maintained until E11.5, indicating sufficient maternal transfer of BH4 to reach this stage of development. After E11.5, Gch1(-/-) embryos were deficient in BH4, but an unbiased metabolomic screen indicated that the lethality was not due to a gross disturbance in metabolic profile. Embryonic lethality in Gch1(-/-) embryos was not caused by structural abnormalities, but was associated with significant bradycardia at E11.5. Embryonic lethality was not rescued by maternal supplementation of BH4, but was partially rescued, up to E15.5, by maternal supplementation of BH4 and l-DOPA. CONCLUSION: These findings demonstrate a requirement for Gch1 in embryonic development and have important implications for the understanding of pathogenesis and treatment of genetic BH4 deficiencies, as well as the identification of new potential roles for BH4.

Untargeted metabolite profiling of murine embryos to reveal metabolic perturbations associated with neural tube closure defects.

BACKGROUND: Neural tube closure defects (NTDs) are among the most common congenital malformation in human, typically presenting in liveborns as spina bifida. At least 240 gene mutations in mouse are known to increase the risk of NTD. There is a growing appreciation that environmental factors significantly contribute to NTD expression, and that NTDs likely arise from complex gene-environment interactions. Because maternal folic acid supplementation reduces human NTD risk in some populations by 60 to 70%, it is likely that NTD predisposition is often associated with a defect in folate-dependent one-carbon metabolism. A comprehensive, untargeted metabolic survey of NTD-associated changes in embryo metabolism would provide a valuable test of this assumption. We sought to establish a metabolic profiling platform that is capable of broadly assessing metabolic aberrations associated with NTD-promoting gene mutations in early-stage mouse embryos. METHODS: A liquid chromatography/mass spectrometry-based untargeted metabolite profiling platform was used to broadly identify significant differences in small molecule levels (50-1000 Da) in NTD-affected embryonic day (E) 9.5 mouse embryos (Lrp6(-) (/) (-) ) versus unaffected (Lrp6(+/+) ) control embryos. RESULTS: Results provide proof-of-principal feasibility for the broad survey of the metabolome of individual E9.5 mouse embryos and identification of metabolic changes associated with NTDs and gene mutations. Levels of 30 different metabolites were altered in association with Lrp6 gene deletion. Some metabolites link to folate-dependent one-carbon transfer reactions, as anticipated, while others await structure elucidation and pathway integration. CONCLUSION: Whole-embryo metabolomics offers the potential to identify metabolic changes in genetically determined NTD-prone embryos.

KCNQ1, KCNE2, and Na+-coupled solute transporters form reciprocally regulating complexes that affect neuronal excitability.

Na(+)-coupled solute transport is crucial for the uptake of nutrients and metabolic precursors, such as myo-inositol, an important osmolyte and precursor for various cell signaling molecules. We found that various solute transporters and potassium channel subunits formed complexes and reciprocally regulated each other in vitro and in vivo. Global metabolite profiling revealed that mice lacking KCNE2, a K(+) channel ß subunit, showed a reduction in myo-inositol concentration in cerebrospinal fluid (CSF) but not in serum. Increased behavioral responsiveness to stress and seizure susceptibility in Kcne2(-/-) mice were alleviated by injections of myo-inositol. Suspecting a defect in myo-inositol transport, we found that KCNE2 and KCNQ1, a voltage-gated potassium channel α subunit, colocalized and coimmunoprecipitated with SMIT1, a Na(+)-coupled myo-inositol transporter, in the choroid plexus epithelium. Heterologous coexpression demonstrated that myo-inositol transport by SMIT1 was augmented by coexpression of KCNQ1 but was inhibited by coexpression of both KCNQ1 and KCNE2, which form a constitutively active, heteromeric K(+) channel. SMIT1 and the related transporter SMIT2 were also inhibited by a constitutively active mutant form of KCNQ1. The activities of KCNQ1 and KCNQ1-KCNE2 were augmented by SMIT1 and the glucose transporter SGLT1 but were suppressed by SMIT2. Channel-transporter signaling complexes may be a widespread mechanism to facilitate solute transport and electrochemical crosstalk.

LRP6 exerts non-canonical effects on Wnt signaling during neural tube closure.

Low-density lipoprotein receptor related protein 6 (Lrp6) mutational effects on neurulation were examined using gain (Crooked tail, Lrp6(Cd)) and loss (Lrp6(-)) of function mouse lines. Two features often associated with canonical Wnt signaling, dorsal-ventral patterning and proliferation, were no different from wild-type (WT) in the Lrp6(Cd/Cd) neural tube. Lrp6(-/-) embryos showed reduced proliferation and subtle patterning changes in the neural folds. Cell polarity defects in both Lrp6(Cd/Cd) and Lrp6(-/-) cranial folds were indicated by cell shape, centrosome displacement and failure of F-actin and GTP-RhoA accumulation at the apical surface. Mouse embryonic fibroblasts (MEFs) derived from Lrp6(Cd/Cd) or Lrp6(-/-) embryos exhibited elevated and decreased RhoA basal activity levels, respectively. While ligand-independent activation of canonical Wnt signaling, bypassing Lrp-Frizzled receptors, did not activate RhoA, non-canonical Wnt5a stimulation of RhoA activity was impaired in Lrp6(-/-) MEFs. RhoA inhibition exacerbated NTDs in cultured Lrp6 knockout embryos compared with WT littermates. In contrast, a ROCK inhibitor rescued Lrp6(Cd/Cd) embryos from NTDs. Lrp6 co-immunoprecipitated with Disheveled-associated activator of morphogenesis 1 (DAAM1), a formin promoting GEF activity in Wnt signaling. Biochemical and cell biological data revealed intracellular accumulation of Lrp6(Cd) protein where interaction with DAAM1 could account for observed elevated RhoA activity. Conversely, null mutation that eliminates Lrp6 interaction with DAAM1 led to lower basal RhoA activity in Lrp6(-/-) embryos. These results indicate that Lrp6 mediates not only canonical Wnt signaling, but can also modulate non-canonical pathways involving RhoA-dependent mechanisms to impact neurulation, possibly through intracellular complexes with DAAM1.

Protein nitrotryptophan: formation, significance and identification.

Reactive nitrogen species are formed during a variety of disease states and have been shown to modify several amino acids on proteins. To date, the majority of research in this area has focused on the nitration of tyrosine residues to form 3-nitrotyrosine. However, emerging evidence suggests that another modification, nitration of tryptophan residues, to form nitrotryptophan (NO(2)-Trp), may also play a significant role in the biology of nitrosative stress. This review takes an in-depth look at NO(2)-Trp, presenting the current research about its formation, prevalence and biological significance, as well as the methods used to identify NO(2)-Trp-modified proteins. Although more research is needed to understand the full biological role of NO(2)-Trp, the data presented herein suggest a contribution to nitrosative stress-induced cell dysregulation and perhaps even in physiological cell processes.