Quantitative proteomics suggests metabolic reprogramming during ETHE1 deficiency

Deficiency of mitochondrial sulfur dioxygenase (ETHE1) causes the severe metabolic disorder ethylmalonic encephalopathy, which is characterized by early‐onset encephalopathy and defective cytochrome C oxidase because of hydrogen sulfide accumulation. Although the severe systemic consequences of the disorder are becoming clear, the molecular effects are not well defined. Therefore, for further elucidating the effects of ETHE1‐deficiency, we performed a large scale quantitative proteomics study on liver tissue from ETHE1‐deficient mice. Our results demonstrated a clear link between ETHE1‐deficiency and redox active proteins, as reflected by downregulation of several proteins related to oxidation‐reduction, such as different dehydrogenases and cytochrome P450 (CYP450) members. Furthermore, the protein data indicated impact of the ETHE1‐deficiency on metabolic reprogramming through upregulation of glycolytic enzymes and by altering several heterogeneous ribonucleoproteins, indicating novel link between ETHE1 and gene expression regulation. We also found increase in total protein acetylation level, pointing out the link between ETHE1 and acetylation, which is likely controlled by both redox state and cellular metabolites. These findings are relevant for understanding the complexity of the disease and may shed light on important functions influenced by ETHE1 deficiency and by the concomitant increase in the gaseous mediator hydrogen sulfide. All MS data have been deposited in the ProteomeXchange with the dataset identifiers PXD002741 ( http://proteomecentral.proteomexchange.org/dataset/PXD002741 ) and PXD002742 ( http://proteomecentral.proteomexchange.org/dataset/PXD002741 ).     

Flavoprotein-linked substrate oxidation in preparations of hamster brown adipocytes: A discrimination between internally and externally oxidized substrates

Noradrenaline-stimulated oxidative metabolism in isolated hamster brown fat cells is very reproducible between different cell preparations, 565 ± 81 (S.D.) nmol O/min per 10⁶ cells (n = 25).In contrast, the oxygen consumption rate induced by the addition of succinate or sn-glycerol 3-phosphate strongly varies between different cell preparation, although these substances have been reported to be potent substrates for isolated hamster brown fat cells.By filtration and by successive washings we demonstrate that the flavoprotein-linked substrate oxidation is mainly dependent on extracellular succinate and sn-glycerol 3-phosphate-oxidizing enzymes. These enzymes originate from damaged and broken cells and are present in different amounts in different cell preparations.In discriminating between intra- and extracellular succinate oxidation 5,5′-dithiobis(2-nitrobenzoate) is used as an inhibitor of the extracellular portion. This application of 5,5′-dithiobis(2-nitrobenzoate) ougth to be useful also in other cell or tissue preparations.Added succinate can, however, be oxidized by the intact brown adipocyte but a very low rate, probably as a result of a limited transport rate over the membrane(s). In the presence of noradrenaline, added succinated can potentiate the noradrenaline-inducible oxygen consumption by catalytically increasing the oxidative capacity of the citric acid cycle.Our conclusions is that the only effectors which significantly increase oxidative metabolism in intact isolated hamster brown fat cells are catecholamines and free fatty acids. Provided the cells are uncoupled, also pyruvate can function as substrate for these cells.     

OMERACT: An international initiative to improve outcome measurement in rheumatology

Background and objective

Cerebral microembolism detected by transcranial Doppler (TCD) occurs systematically during cardiac catheterization, but its clinical relevance, remains unknown. Studies suggest that asymptomatic embolic cerebral infarction detectable by diffusion-weighted (DW) MRI might exist after percutaneous cardiac interventions with a frequency as high as 15 to 22% of cases. We have set up, for the first time, a prospective multicenter trial to assess the rate of silent cerebral infarction after cardiac catheterization and to compare the impact of the arterial access site, comparing radial and femoral access, on this phenomenon.

Study design

This prospective study will be performed in patients with severe aortic valve stenosis. To assess the occurrence of cerebral infarction, all patients will undergo cerebral DW-MRI and neurological assessment within 24 hours before, and 48 hours after cardiac catheterization and retrograde catheterization of the aortic valve. Randomization for the access site will be performed before coronary angiography. A subgroup will be monitored by transcranial power M-mode Doppler during cardiac catheterization to observe cerebral blood flow and track emboli. Neuropsychological tests will also be recorded in a subgroup of patients before and after the interventional procedures to assess the impact of silent brain injury on potential cognitive decline. The primary end-point of the study is a direct comparison of ischemic cerebral lesions as detected by serial cerebral DW-MRI between patients explored by radial access and patients explored by femoral access. Secondary end-points include comparison of neuropsychological test performance and number of microembolism signals observed in the two groups.

Implications

Using serial DW-MRI, silent cerebral infarction rate will be defined and the potential influence of vascular access site will be evaluated. Silent cerebral infarction might be a major concern during cardiac catheterization and its potential relationship to cognitive decline needs to be assessed.

Study registration

The SCIPION study is registered through National Institutes of Health-sponsored clinical trials registry and has been assigned the Identifier: NCT 00329979.     

Ablation of mouse phosphomannose isomerase (Mpi) causes mannose 6-phosphate accumulation, toxicity, and embryonic lethality

MPI encodes phosphomannose isomerase, which interconverts fructose 6-phosphate and mannose 6-phosphate (Man-6-P), used for glycoconjugate biosynthesis. MPI mutations in humans impair protein glycosylation causing congenital disorder of glycosylation Ib (CDG-Ib), but oral mannose supplements normalize glycosylation. To establish a mannose-responsive mouse model for CDG-Ib, we ablated Mpi and provided dams with mannose to rescue the anticipated defective glycosylation. Surprisingly, although glycosylation was normal, Mpi(-/-) embryos died around E11.5. Mannose supplementation even hastened their death, suggesting that man-nose was toxic. Mpi(-/-) embryos showed growth retardation and placental hyperplasia. More than 90% of Mpi(-/-) embryos failed to form yolk sac vasculature, and 35% failed chorioallantoic fusion. We generated primary embryonic fibroblasts to investigate the mechanisms leading to embryonic lethality and found that mannose caused a concentration- and time-dependent accumulation of Man 6-P in Mpi(-/-) fibroblasts. In parallel, ATP decreased by more than 70% after 24 h compared with Mpi(+/+) controls. In cell lysates, Man-6-P inhibited hexokinase (70%), phosphoglucose isomerase (65%), and glucose-6-phosphate dehydrogenase (85%), but not phosphofructokinase. Incubating intact Mpi(-/-) fibroblasts with 2-[(3)H]deoxyglucose confirmed mannose-dependent hexokinase inhibition. Our results in vitro suggest that mannose toxicity in Mpi(-/-) embryos is caused by Man-6-P accumulation, which inhibits glucose metabolism and depletes intracellular ATP. This was confirmed in E10.5 Mpi(-/-) embryos where Man-6-P increased more than 10 times, and ATP decreased by 50% compared with Mpi(+/+) littermates. Because Mpi ablation is embryonic lethal, a murine CDG-Ib model will require hypomorphic Mpi alleles.     

Ethylene-dependent and -independent pathways controlling floral abscission are revealed to converge using promoter::reporter gene constructs in the ida abscission mutant

The process of floral organ abscission in Arabidopsis thaliana can be modulated by ethylene and involves numerous genes contributing to cell separation. One gene that is absolutely required for abscission is INFLORESCENCE DEFICIENT IN ABSCISSION, IDA, as the ida mutant is completely blocked in abscission. To elucidate the genetic pathways regulating floral abscission, molecular markers expressed in the floral abscission zone have been studied in an ida mutant background. Using plants with promoter-reporter gene constructs including promoters of a novel FLORAL ABSCISSION ASSOCIATED gene (FAA) encoding a putative single-stranded binding protein (BASIL), chitinase (CHIT::GUS) and cellulase (BAC::GUS), it is shown that IDA acts in the last steps of the abscission process. These markers, as well as HAESA, encoding a receptor-like kinase, were unaffected in their temporal expression patterns in ida compared with wild-type plants; thus showing that different regulatory pathways are active in the abscission process. In contrast to BASIL, CHIT::GUS and BAC::GUS showed, however, much weaker induction of expression in an ida background, consistent with a reduction in pathogen-associated responses and a lack of total dissolution of cell walls in the mutant. IDA, encoding a putative secreted peptide ligand, and HAESA appeared to have identical patterns of expression in floral abscission zones. Lastly, to address the role of ethylene, IDA::GUS expression in the wild type and the ethylene-insensitive mutant etr1-1 was compared. Similar temporal patterns, yet restricted spatial expression patterns were observed in etr1-1, suggesting that the pathways regulated by IDA and by ethylene act in parallel, but are, to some degree, interdependent.     

Megalin is a receptor for apolipoprotein M, and kidney-specific megalin-deficiency confers urinary excretion of apolipoprotein M

Apolipoprotein (apo) M is a novel apolipoprotein belonging to the lipocalin protein superfamily, i.e. proteins binding small lipophilic compounds. Like other apolipoproteins, it is expressed in hepatocytes and secreted into plasma where it associates with high-density lipoprotein particles. In addition, apoM is expressed at high levels in the kidney tubule cells. In this study, we show that the multiligand receptor megalin, which is expressed in kidney proximal tubule cells, is a receptor for apoM and mediates its uptake in the kidney. To examine apoM binding to megalin, a recombinant apoM was expressed in Escherichia coli and used in surface plasmon resonance and cell culture studies. The results showed apoM binding to immobilized megalin [dissociation constant (Kd) approximately 0.3-1 microm] and that the apoM was endocytosed by cultured rat yolk sac cells in a megalin-dependent manner. To examine the importance of apoM binding by megalin in vivo, we analyzed mice with a tissue-specific deficiency of megalin in the kidney. Megalin deficiency was associated with pronounced urinary excretion of apoM, whereas apoM was not detected in normal mouse, human, or rat urine. Gel filtration analysis showed that the urinary apoM-containing particles were small and devoid of apoA-I. The results suggest that apoM binds to megalin and that megalin-mediated endocytosis in kidney proximal tubules prevents apoM excretion in the urine.     

Late type apoptosis and apoptosis free lethal effect of quercetin in human melanoma

No satisfactory treatment is currently available for metastatic malignant melanoma. Recently, the flavonoid quercetin was suggested as a potential treatment due to its anti-tumorogenic properties. Some of these properties appeared to correspond to those published for UVB irradiation. To determine quercetin's long-term effects, type of apoptosis, and shared properties with UVB, we exposed Mel-Juso, M14, and G361 human melanoma cell-lines to a large range of quercetin or UVB doses, 20-400 microm and 25-1000 mJ/cm2 respectively. Apoptosis was measured for 4 consecutive d by flow cytometry and cell viability was studied by colony-forming assay. Quercetin decreased cell viability level in a dose-dependent manner to almost zero at 100 microm. Up to this concentration, it did not induce significant apoptosis nor did it decrease the survival-fractions below 90% during a 4 d follow-up. The data suggest that Quercetin is lethal to melanoma cells at concentrations that do not activate apoptosis during the first 4 d post-exposure and that quercetin's effects extend beyond the period of direct contact. Both quercetin and UVB induced late-type apoptosis at the upper range of the tested doses, but they do not appear to share all the pathways that they activate. Finally, this paper provides novel data showing that quercetin causes two different lethal effects on human melanoma cells, suggesting the activation of at least two different dose-depended mechanisms.     

Ecological Characterisation of the Colonic Microbiota in Arctic and Sub-Arctic Seals

Dominant colonic bacteria in wild hooded (n = 9), harbour (n = 1) and grey (n = 1) seals were identified using 16S rRNA gene clone libraries (313 clones), revealing 52.7% Bacteroidetes, 41.5% Firmicutes, 4.5% Proteobacteria and 1.0% Fusobacteria. Thirty (77%) of the 39 phylotypes identified were novel, showing <97% sequence similarity to their nearest cultivated relatives. Mean colonic bacterial cell density, determined by real-time PCR, was high (12.8 log₁₀ cells/g wet wt) for the hooded seals, while the number of methanogenic Archea was low (4.0 log₁₀ cells/g wet wt). The level of ampicillin (amp^r) and tetracycline-resistant (tet^r) isolates was investigated by cultivation. Aerobic amp^r isolates were only detected in colon contents from four hooded seals, whereas aerobic tet^r isolates were found in seven of the nine hooded seals. These data provide novel insight to the gut microbiota of Arctic and sub-Arctic seals living in the wild.     

Mammalian ALKBH8 Possesses tRNA Methyltransferase Activity Required for the Biogenesis of Multiple Wobble Uridine Modifications Implicated in Translational Decoding

Uridines in the wobble position of tRNA are almost invariably modified. Modifications can increase the efficiency of codon reading, but they also prevent mistranslation by limiting wobbling. In mammals, several tRNAs have 5-methoxycarbonylmethyluridine (mcm⁵U) or derivatives thereof in the wobble position. Through analysis of tRNA from Alkbh8^−/− mice, we show here that ALKBH8 is a tRNA methyltransferase required for the final step in the biogenesis of mcm⁵U. We also demonstrate that the interaction of ALKBH8 with a small accessory protein, TRM112, is required to form a functional tRNA methyltransferase. Furthermore, prior ALKBH8-mediated methylation is a prerequisite for the thiolation and 2′-O-ribose methylation that form 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U) and 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm⁵Um), respectively. Despite the complete loss of all of these uridine modifications, Alkbh8^−/− mice appear normal. However, the selenocysteine-specific tRNA (tRNA^Sec) is aberrantly modified in the Alkbh8^−/− mice, and for the selenoprotein Gpx1, we indeed observed reduced recoding of the UGA stop codon to selenocysteine.tRNAs are frequently modified at the wobble uridine, a feature that is believed to either promote or restrict wobbling depending on the type of modification. In the case of eukaryotes, the functions of wobble uridine modifications have been studied in the greatest detail in Saccharomyces cerevisiae. Here, the modifications 5-methoxycarbonylmethyluridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), and 5-carbamoylmethyluridine (ncm⁵U) or its 2′-O-ribose-methylated form, ncm⁵Um, are found in 11 out of 13 wobble uridine-containing tRNAs (22). mcm⁵U and mcm⁵s²U are mostly found in “split” codon boxes, where the pyrimidine- and purine-ending codons encode different amino acids, while ncm⁵U is found in “family” codon boxes, where all four codons encode a single amino acid. Early reports based on in vitro experiments suggested that wobble nucleosides, such as mcm⁵U, ncm⁵U, and their derivatives, may restrict wobbling (17, 37, 45), but the results of a recent comprehensive study performed in vivo in S. cerevisiae show that such modifications can improve the reading both of the cognate, A-ending codons and of the wobble, G-ending codons (22). This may suggest that the primary role of these modified nucleosides is to improve translational efficiency rather than to restrict wobbling.The characterization of wobble uridine modifications in higher eukaryotes is very limited, and little is known about the enzymes that introduce them. In mammals, mcm⁵s²U has been found in the wobble position of tRNA^Glu(UUC), tRNA^Lys(UUU), and tRNA^Arg(UCU) (40). Unlike yeast, mammals possess a specialized tRNA that is responsible for recoding the UGA stop codon to insert the 21st amino acid, selenocysteine (Sec). The mammalian tRNA^Sec population consists of two subpopulations containing either mcm⁵U or the ribose-methylated derivative mcm⁵Um in the wobble position. Interestingly, ribose methylation of mcm⁵U in tRNA^Sec appears to have a role in regulating selenoprotein synthesis, as the expression of some selenoproteins, such as glutathione peroxidase 1 (Gpx1), appears to be promoted by mcm⁵Um-containing tRNA^Sec (5, 7, 9, 32).Some years ago, the Escherichia coli AlkB protein was found to be a 2-oxoglutarate- and iron-dependent dioxygenase capable of demethylating the lesions 1-methyladenosine and 3-methylcytosine in DNA (13, 42). Multicellular organisms generally possess several different AlkB homologues (ALKBH), and bioinformatics analysis has identified eight different mammalian ALKBH proteins, denoted ALKBH1 to ALKBH8 in humans and Alkbh1 to Alkbh8 in mice, as well as the somewhat-less-related, obesity-associated FTO protein (2, 16, 30). Among the ALKBH proteins of unknown function, ALKBH8 is the only one containing additional annotated protein domains. Here, the AlkB domain is localized between an N-terminal RNA recognition motif (RRM) and a C-terminal methyltransferase (MT) domain. Interestingly, the MT domain has sequence homology to the S. cerevisiae tRNA methyltransferase Trm9, which has been shown to catalyze the methyl esterification of modified wobble uridine (U34) residues of tRNA^Arg and tRNA^Glu, resulting in the formation of mcm⁵U and mcm⁵s²U, respectively (23, 43). Until recently, human ALKBH8 was incorrectly annotated in the protein sequence database, and another human protein, KIAA1456, has been designated the human Trm9 homologue (3, 23).We have generated for this study Alkbh8-targeted mice that lack exons critical for both the MT and AlkB activities of Alkbh8. The mice did not display any overt phenotype, but tRNA from these mice was completely devoid of mcm⁵U, mcm⁵s²U, and mcm⁵Um, and the relevant tRNA isoacceptors instead contained the acid form 5-carboxymethyluridine (cm⁵U) and/or the amide forms ncm⁵U/ncm⁵s²U. Furthermore, we show that recombinant ALKBH8 and TRM112 form a heterodimeric complex capable of catalyzing the methyl esterification of cm⁵U and cm⁵s²U to mcm⁵U and mcm⁵s²U, respectively. In agreement with the involvement of mcm⁵Um in selenoprotein synthesis, we observed a reduced level of Gpx1 in the Alkbh8^−/− mice, and tRNA^Sec from these mice showed a reduced ability to decode the UGA stop codon to Sec.