Intracellular processing of vitamin B12 by MMACHC (CblC).

Vitamin B12 (cobalamin, Cbl, B12) is a water-soluble micronutrient synthesized exclusively by a group of microorganisms. Human beings are unable to make B12 and thus obtain the vitamin via intake of animal products, fermented plant-based foods or supplements. Vitamin B12 obtained from the diet comprises three major chemical forms, namely hydroxocobalamin (HOCbl), methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl). The most common form of B12 present in supplements is cyanocobalamin (CNCbl). Yet, these chemical forms cannot be utilized directly as they come, but instead, they undergo chemical processing by the MMACHC protein, also known as CblC. Processing of dietary B12 by CblC involves removal of the upper-axial ligand (beta-ligand) yielding the one-electron reduced intermediate cob(II)alamin. Newly formed cob(II)alamin undergoes trafficking and delivery to the two B12-dependent enzymes, cytosolic methionine synthase (MS) and mitochondrial methylmalonyl-CoA mutase (MUT). The catalytic cycles of MS and MUT incorporate cob(II)alamin as a precursor to regenerate the coenzyme forms MeCbl and AdoCbl, respectively. Mutations and epimutations in the MMACHC gene result in cblC disease, the most common inborn error of B12 metabolism, which manifests with combined homocystinuria and methylmalonic aciduria. Elevation of metabolites homocysteine and methylmalonic acid occurs because the lack of an active CblC blocks formation of the indispensable precursor cob(II)alamin that is necessary to activate MS and MUT. Thus, in patients with cblC disease, vitamin B12 is absorbed and present in circulation in normal to high concentrations, yet, cells are unable to make use of it. Mutations in seemingly unrelated genes that modify MMACHC gene expression also result in clinical phenotypes that resemble cblC disease. We review current knowledge on structural and functional aspects of intracellular processing of vitamin B12 by the versatile protein CblC, its partners and possible regulators.

Thiolatocobalamins repair the activity of pathogenic variants of the human cobalamin processing enzyme CblC.

Thiolatocobalamins are a class of cobalamins comprised of naturally occurring and synthetic ligands. Glutathionylcobalamin (GSCbl) occurs naturally in mammalian cells, and also as an intermediate in the glutathione-dependent dealkylation of methylcobalamin (MeCbl) to form cob(I)alamin by pure recombinant CblC from C. elegans. Glutathione-driven deglutathionylation of GSCbl was demonstrated both in mammalian as well as in C. elegans CblC. Dethiolation is orders of magnitude faster than dealkylation of Co-C bonded cobalamins, which motivated us to investigate two synthetic thiolatocobalamins as substrates to repair the enzymatic activity of pathogenic CblC variants in humans. We report the synthesis and kinetic characterization of cysteaminylcobalamin (CyaCbl) and 2-mercaptopropionylglycinocobalamin (MpgCbl). Both CyaCbl and MpgCbl were obtained in high purity (90-95%) and yield (78-85%). UV-visible spectral properties agreed with those reported for other thiolatocobalamins with absorbance maxima observed at 372 nm and 532 nm. Both CyaCbl and MpgCbl bound to wild type human recombinant CblC inducing spectral blue-shifts characteristic of the respective base-on to base-off transitions. Addition of excess glutathione (GSH) resulted in rapid elimination of the ß-ligand to give aquacobalamin (H2OCbl) as the reaction product under aerobic conditions. Further, CyaCbl and MpgCbl underwent spontaneous dethiolation thereby repairing the loss of activity of pathogenic variants of human CblC, namely R161G and R161Q. We posit that thiolatocobalamins could be exploited therapeutically for the treatment of inborn errors of metabolism that impair processing of dietary and supplemental cobalamin forms. While these disorders are targets for newborn screening in some countries, there is currently no effective treatment available to patients.

Proteomics of vitamin B12 processing.

The causes of cobalamin (B12, Cbl) deficiency are multifactorial. Whether nutritional due to poor dietary intake, or functional due to impairments in absorption or intracellular processing and trafficking events, the major symptoms of Cbl deficiency include megaloblastic anemia, neurological deterioration and in extreme cases, failure to thrive and death. The common biomarkers of Cbl deficiency (hyperhomocysteinemia and methylmalonic acidemia) are extremely valuable diagnostic indicators of the condition, but little is known about the changes that occur at the protein level. A mechanistic explanation bridging the physiological changes associated with functional B12 deficiency with its intracellular processers and carriers is lacking. In this article, we will cover the effects of B12 deficiency in a cblC-disrupted background (also referred to as MMACHC) as a model of functional Cbl deficiency. As will be shown, major protein changes involve the cytoskeleton, the neurological system as well as signaling and detoxification pathways. Supplementation of cultured MMACHC-mutant cells with hydroxocobalamin (HOCbl) failed to restore these variants to the normal phenotype, suggesting that a defective Cbl processing pathway produces irreversible changes at the protein level.

Both selenium deficiency and modest selenium supplementation lead to myocardial fibrosis in mice via effects on redox-methylation balance.

SCOPE: Selenium has complex effects in vivo on multiple homeostatic mechanisms such as redox balance, methylation balance, and epigenesis, via its interaction with the methionine-homocysteine cycle. In this study, we examined the hypothesis that selenium status would modulate both redox and methylation balance and thereby modulate myocardial structure and function. METHODS AND RESULTS: We examined the effects of selenium-deficient (<0.025 mg/kg), control (0.15 mg/kg), and selenium-supplemented (0.5 mg/kg) diets on myocardial histology, biochemistry and function in adult C57/BL6 mice. Selenium deficiency led to reactive myocardial fibrosis and systolic dysfunction accompanied by increased myocardial oxidant stress. Selenium supplementation significantly reduced methylation potential, DNA methyltransferase activity and DNA methylation. In mice fed the supplemented diet, inspite of lower oxidant stress, myocardial matrix gene expression was significantly altered resulting in reactive myocardial fibrosis and diastolic dysfunction in the absence of myocardial hypertrophy. CONCLUSION: Our results indicate that both selenium deficiency and modest selenium supplementation leads to a similar phenotype of abnormal myocardial matrix remodeling and dysfunction in the normal heart. The crucial role selenium plays in maintaining the balance between redox and methylation pathways needs to be taken into account while optimizing selenium status for prevention and treatment of heart failure.

The MMACHC proteome: hallmarks of functional cobalamin deficiency in humans.

Cobalamin (Cbl, B(12)) is an essential micronutrient required to fulfill the enzymatic reactions of cytosolic methylcobalamin-dependent methionine synthase and mitochondrial adenosylcobalamin-dependent methylmalonyl-CoA mutase. Mutations in the MMACHC gene (cblC complementation group) disrupt processing of the upper-axial ligand of newly internalized cobalamins, leading to functional deficiency of the vitamin. Patients with cblC disease present with both hyperhomocysteinemia and methylmalonic acidemia, cognitive dysfunction, and megaloblastic anemia. In the present study we show that cultured skin fibroblasts from cblC patients export increased levels of both homocysteine and methylmalonic acid compared to control skin fibroblasts, and that they also have decreased levels of total intracellular folates. This is consistent with the clinical phenotype of functional cobalamin deficiency in vivo. The protein changes that accompany human functional Cbl deficiency are unknown. The proteome of control and cblC fibroblasts was quantitatively examined by two dimensional difference in-gel electrophoresis (2D-DIGE) and liquid chromatography-electrospray ionization-mass spectrometry (LC/ESI/MS). Major changes were observed in the expression levels of proteins involved in cytoskeleton organization and assembly, the neurological system and cell signaling. Pathway analysis of the differentially expressed proteins demonstrated strong associations with neurological disorders, muscular and skeletal disorders, and cardiovascular diseases in the cblC mutant cell lines. Supplementation of the cell cultures with hydroxocobalamin did not restore the cblC proteome to the patterns of expression observed in control cells. These results concur with the observed phenotype of patients with the cblC disorder and their sometimes poor response to treatment with hydroxocobalamin. Our findings could be valuable for designing alternative therapies to alleviate the clinical manifestation of the cblC disorder, as some of the protein changes detected in our study are common hallmarks of known pathologies such as Alzheimer's and Parkinson's diseases as well as muscular dystrophies.

Taurine supplementation prevents ethanol-induced decrease in serum adiponectin and reduces hepatic steatosis in rats.

UNLABELLED: Chronic ethanol feeding decreases expression of adiponectin by adipocytes and circulating adiponectin. Adiponectin treatment during chronic ethanol feeding prevents liver injury in mice. Chronic ethanol feeding also increases oxidative and endoplasmic reticulum (ER) stress in adipose tissue. Here we tested the hypothesis that supplemental taurine, an amino acid that functions as a chemical chaperone/osmolyte and enhances cellular antioxidant activity, would prevent ethanol-induced decreases in adiponectin expression and attenuate liver injury. Serum adiponectin concentrations decreased as early as 4 to 7 days after feeding rats a 36% ethanol diet. This rapid decrease was associated with increased oxidative, but not ER, stress in subcutaneous adipose tissue. Taurine prevented ethanol-induced oxidative stress and increased inflammatory cytokine expression in adipose tissue. Ethanol feeding also rapidly decreased expression of transcription factors regulating adiponectin expression (CCAAT/enhancer binding protein alpha; peroxisome proliferator-activated receptor alpha/gamma) in subcutaneous adipose tissue. Taurine prevented the ethanol-induced decrease in CCAAT/enhancer binding protein alpha and peroxisome proliferator-activated receptor alpha, normalizing adiponectin messenger (m)RNA and serum adiponectin concentrations. In the liver, taurine prevented ethanol-induced oxidative stress and attenuated tumor necrosis factor alpha expression and steatosis, at least in part, by increasing expression of genes involved in fatty acid oxidation. CONCLUSION: In subcutaneous adipose tissue, taurine decreased ethanol-induced oxidative stress and cytokine expression, as well as normalized expression of adiponectin mRNA. Taurine prevented ethanol-induced decreases in serum adiponectin; normalized adiponectin was associated with a reduction in hepatic oxidative stress, tumor necrosis factor alpha expression, and steatosis. Taken together, these data demonstrate that taurine has important protective effects against ethanol-induced tissue injury in both adipose and liver tissue.

Exogenous thioredoxin prevents ethanol-induced oxidative damage and apoptosis in mouse liver.

UNLABELLED: Ethanol-induced liver injury is characterized by increased formation of reactive oxygen species (ROS) and inflammatory cytokines, resulting in the development of hepatic steatosis, injury, and cell death by necrosis and apoptosis. Thioredoxin (Trx), a potent antioxidant and antiinflammatory molecule with antiapoptotic properties, protects animals from a number of inflammatory diseases. However, the effects of ethanol on Trx or its role in ethanol-induced liver injury are not known. Female C57BL/6 mice were allowed ad libitum access to a Lieber-deCarli ethanol diet with 5.4% of calories as ethanol for 2 days to acclimate them to the diet, followed by 2 days with 32.4% of calories as ethanol or pair-fed control diet. Hepatic Trx-1 was decreased by ethanol feeding; daily supplementation with recombinant human Trx (rhTrx) prevented this ethanol-induced decrease. Therefore, we tested the hypothesis that administration of rhTrx during ethanol exposure would attenuate ethanol-induced oxidative stress, inflammatory cytokine production, and apoptosis. Mice were treated with a daily intraperitoneal injection of either 5 g/kg of rhTrx or phosphate-buffered saline (PBS). CONCLUSION: Ethanol feeding increased accumulation of hepatic 4-hydroxynonenal protein adducts, expression of hepatic tumor necrosis factor alpha, and resulted in hepatic steatosis and increased plasma aspartate aminotransferase and alanine aminotransferase. In ethanol-fed mice, treatment with rhTrx reduced 4-hydroxynonenal adduct accumulation, inflammatory cytokine expression, decreased hepatic triglyceride, and improved liver enzyme profiles. Ethanol feeding also increased transferase-mediated dUTP-biotin nick-end labeling-positive cells, caspase-3 activity, and cytokeratin-18 staining in the liver. rhTrx treatment prevented these increases. In summary, rhTrx attenuated ethanol-induced increases in markers of oxidative stress, inflammatory cytokine expression, and apoptosis.

Hyperhomocysteinemia induced by methionine supplementation does not independently cause atherosclerosis in C57BL/6J mice.

A causal relationship between diet-induced hyperhomocysteinemia (HHcy) and accelerated atherosclerosis has been established in apolipoprotein E-deficient (apoE(-/-)) mice. However, it is not known whether the proatherogenic effect of HHcy in apoE(-/-) mice is independent of hyperlipidemia and/or deficiency of apoE. In this study, a comprehensive dietary approach using C57BL/6J mice was used to investigate whether HHcy is an independent risk factor for accelerated atherosclerosis or dependent on additional dietary factors that increase plasma lipids and/or inflammation. C57BL/6J mice at 4 wk of age were divided into 6 dietary groups: chow diet (C), chow diet + methionine (C+M), western-type diet (W), western-type diet + methionine (W+M), atherogenic diet (A), or atherogenic diet + methionine (A+M). After 2, 10, 20, or 40 wk on the diets, mice were sacrificed, and the levels of total plasma homocysteine, cysteine, and glutathione, as well as total plasma cholesterol and triglycerides were analyzed. Aortic root sections were examined for atherosclerotic lesions. HHcy was induced in all groups supplemented with methionine, compared to diet-matched control groups. Plasma total cholesterol was significantly increased in mice fed the W or A diet. However, the W diet increased LDL/IDL and HDL levels, while the A diet significantly elevated plasma VLDL and LDL/IDL levels without increasing HDL. No differences in plasma total cholesterol levels or lipid profiles were observed between methionine-supplemented groups and the diet-matched control groups. Early atherosclerotic lesions containing macrophage foam cells were only observed in mice fed the A or A + M diet. Furthermore, lesion size was significantly larger in the A + M group compared to the A group at 10 and 20 wk; however, mature lesions were never observed even after 40 wk on these diets. The presence of lymphocytes, increased hyaluronan staining, and the expression of endoplasmic reticulum (ER) stress markers were also increased in atherosclerotic lesions from the A + M group. Taken together, these results suggest that HHcy does not independently cause atherosclerosis in C57BL/6J mice even in the presence of increased total plasma lipids induced by the W diet. However, HHcy can accelerate atherosclerotic lesion development under dietary conditions that increase plasma VLDL levels and/or inflammation.

TDAG51 is induced by homocysteine, promotes detachment-mediated programmed cell death, and contributes to the cevelopment of atherosclerosis in hyperhomocysteinemia.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and accelerates atherosclerosis in apoE-/- mice. Despite the observations that homocysteine causes endoplasmic reticulum (ER) stress and programmed cell death (PCD) in cultured human vascular endothelial cells, the cellular factors responsible for this effect and their relevance to atherogenesis have not been completely elucidated. We report here that homocysteine induces the expression of T-cell death-associated gene 51 (TDAG51), a member of the pleckstrin homology-related domain family, in cultured human vascular endothelial cells. This effect was observed for other ER stress-inducing agents, including dithiothreitol and tunicamycin. TDAG51 expression was attenuated in homozygous A/A mutant eukaryotic translation initiation factor 2 alpha mouse embryonic fibroblasts treated with homocysteine or tunicamycin, suggesting that ER stress-induced phosphorylation of eukaryotic translation initiation factor 2 alpha is required for TDAG51 transcriptional activation. Transient overexpression of TDAG51 elicited significant changes in cell morphology, decreased cell adhesion, and promoted detachment-mediated PCD. In support of these in vitro findings, TDAG51 expression was increased and correlated with PCD in the atherosclerotic lesions from apoE-/- mice fed hyperhomocysteinemic diets, compared with mice fed a control diet. Collectively, these findings provide evidence that TDAG51 is induced by homocysteine, promotes detachment-mediated PCD, and contributes to the development of atherosclerosis observed in hyperhomocysteinemia.

Usefulness of plasma vitamin B(6), B(12), folate, homocysteine, and creatinine in predicting outcomes in heart transplant recipients.

Atherothrombotic complications are frequently seen in patients undergoing heart transplantation. These patients have high plasma total homocysteine concentrations associated with lower folate and vitamin B(6) levels. The relation between these metabolic abnormalities and the development of vascular complications, however, remains unclear. Fasting plasma total homocysteine, folate, vitamin B(12), vitamin B(6), and creatinine were measured in 160 cardiac transplant recipients who were followed for a mean duration of 28 +/- 9 months after blood draw (mean 59 +/- 28 months after transplant). Cardiovascular events and causes of mortality were determined and Cox proportional-hazards regression analysis was used to identify the independent predictors for cardiovascular events and mortality. Twenty-five patients developed cardiovascular events and 17 died (11 cardiovascular deaths). Mean +/- SD total homocysteine value was 18.4 +/- 8.5 (range 4.3 to 63.5 micromol/L). Hyperhomocysteinemia (> or =15 micromol/L) was seen in 99 patients (62%). Levels were no different in patients with or without cardiovascular complications/death (16.8 +/- 6.2 vs 18.9 +/- 9 micromol/L, p = 0.4). However, vitamin B(6) deficiency was seen in 21% of recipients with and in 9% without cardiovascular complications/death (p = 0.05). The relative risk for cardiovascular events, including cardiovascular death, increased 2.7 times (confidence interval 1.2 to 5.9) for B(6) levels < or =20 nmol/L compared with those with normal B(6) levels (p = 0.02). Thus, hyperhomocysteinemia is common in transplant recipients but may have no causal role in the atherothrombotic vascular complications of transplantation. Deficiency of vitamin B(6), however, may predict adverse outcomes, suggesting a possible role for supplementation with this vitamin.