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Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a five-subunit enzyme complex responsible for the carbonyl branch of the Wood–Ljungdahl (WL) pathway, considered one of the most ancient metabolisms for anaerobic carbon fixation, but its origin and evolutionary history have been unclear. While traditionally associated with methanogens and acetogens, the presence of CODH/ACS homologs has been reported in a large number of uncultured anaerobic lineages. Here, we have carried out an exhaustive phylogenomic study of CODH/ACS in over 6,400 archaeal and bacterial genomes. The identification of complete and likely functional CODH/ACS complexes in these genomes significantly expands its distribution in microbial lineages. The CODH/ACS complex displays astounding conservation and vertical inheritance over geological times. Rare intradomain and interdomain transfer events might tie into important functional transitions, including the acquisition of CODH/ACS in some archaeal methanogens not known to fix carbon, the tinkering of the complex in a clade of model bacterial acetogens, or emergence of archaeal–bacterial hybrid complexes. Once these transfers were clearly identified, our results allowed us to infer the presence of a CODH/ACS complex with at least four subunits in the last universal common ancestor (LUCA). Different scenarios on the possible role of ancestral CODH/ACS are discussed. Despite common assumptions, all are equally compatible with an autotrophic, mixotrophic, or heterotrophic LUCA. Functional characterization of CODH/ACS from a larger spectrum of bacterial and archaeal lineages and detailed evolutionary analysis of the WL methyl branch will help resolve this issue.

Regulation of mRNA translation elongation impacts nascent protein synthesis and integrity and plays a critical role in disease establishment. Here, we investigate features linking regulation of codon-dependent translation elongation to protein expression and homeostasis. Using knockdown models of enzymes that catalyze the mcm 5 s 2 wobble uridine tRNA modification (U 34 -enzymes), we show that gene codon content is necessary but not sufficient to predict protein fate. While translation defects upon perturbation of U 34 -enzymes are strictly dependent on codon content, the consequences on protein output are determined by other features. Specific hydrophilic motifs cause protein aggregation and degradation upon codon-dependent translation elongation defects. Accordingly, the combination of codon content and the presence of hydrophilic motifs define the proteome whose maintenance relies on U 34 -tRNA modification. Together, these results uncover the mechanism linking wobble tRNA modification to mRNA translation and aggregation to maintain proteome homeostasis. Wobble uridine (U 34 ) tRNA modifications are important for the decoding of AA-ending codons. Here the authors show that while the U 34 -codon content of mRNAs are predictive of changes in ribosome translation elongation, the resulting outcome in protein expression also relies on specific hydrophilic motifs-dependent protein aggregation and clearance.

Many cellular proteins are post-translationally modified by the addition of a single ubiquitin or a polyubiquitin chain 1 . Among these are receptor tyrosine kinases (RTKs), which undergo ligand-dependent ubiquitination 2 . The ubiquitination of RTKs has become recognized as an important signal for their endocytosis and degradation in the lysosome 3 ; however, it is not clear whether ubiquitination itself is sufficient for this process or simply participates in its regulation. The issue is further complicated by the fact that RTKs are thought to be polyubiquitinated — a modification that is linked to protein degradation by the proteasome 4 . By contrast, monoubiquitination has been associated with diverse proteasome-independent cellular functions including intracellular protein movement 5 . Here we show that the epidermal growth factor and platelet-derived growth factor receptors are not polyubiquitinated but rather are monoubiquitinated at multiple sites after their ligand-induced activation. By using different biochemical and molecular genetics approaches, we show that a single ubiquitin is sufficient for both receptor internalization and degradation. Thus, monoubiquitination is the principal signal responsible for the movement of RTKs from the plasma membrane to the lysosome.

Aims/hypothesis The aim of the study was to examine the effects of pioglitazone (PIO), a peroxisome proliferator-activated receptor (PPAR)-γ agonist, and fenofibrate (FENO), a PPAR-α agonist, as monotherapy and in combination on glucose and lipid metabolism. Subjects and methods Fifteen type 2 diabetic patients received FENO (n = 8) or PIO (n = 7) for 3 months, followed by the addition of the other agent for 3 months in an open-label study. Subjects received a 4 h hyperinsulinaemic-euglycaemic clamp and a hepatic fat content measurement at 0, 3 and 6 months. Results Following PIO, fasting plasma glucose (FPG) (p < 0.05) and HbA₁c (p < 0.01) decreased, while plasma adiponectin (AD) (5.5 ± 0.9 to 13.8 ± 3.5 μg/ml [SEM], p < 0.03) and the rate of insulin-stimulated total-body glucose disposal (R d) (23.8 ± 3.8 to 40.5 ± 4.4 μmol kg-¹ min-¹, p < 0.005) increased. After FENO, FPG, HbA₁c, AD and R d did not change. PIO reduced fasting NEFA (784 ± 53 to 546 ± 43 μmol/l, p < 0.05), triacylglycerol (2.12 ± 0.28 to 1.61 ± 0.22 mmol/l, p < 0.05) and hepatic fat content (20.4 ± 4.8 to 10.2 ± 2.5%, p < 0.02). Following FENO, fasting NEFA and hepatic fat content did not change, while triacylglycerol decreased (2.20 ± 0.14 to 1.59 ± 0.13 mmol/l, p < 0.01). Addition of FENO to PIO had no effect on R d, FPG, HbA₁c, NEFA, hepatic fat content or AD, but triacylglycerol decreased (1.61 ± 0.22 to 1.00 ± 0.15 mmol/l, p < 0.05). Addition of PIO to FENO increased R d (24.9 ± 4.4 to 36.1 ± 2.2 μmol kg-¹ min-¹, p < 0.005) and AD (4.1 ± 0.8 to 13.1 ± 2.5 μg/ml, p < 0.005) and reduced FPG (p < 0.05), HbA₁c (p < 0.05), NEFA (p < 0.01), hepatic fat content (18.3 ± 3.1 to 13.5 ± 2.1%, p < 0.03) and triacylglycerol (1.59 ± 0.13 to 0.96 ± 0.9 mmol/l, p < 0.01). Muscle adenosine 5'-monophosphate-activated protein kinase (AMPK) activity did not change following FENO; following the addition of PIO, muscle AMPK activity increased significantly (phosphorylated AMPK:total AMPK ratio 1.2 ± 0.2 to 2.2 ± 0.3, p < 0.01). Conclusions/interpretation We conclude that PPAR-α therapy has no effect on NEFA or glucose metabolism and that addition of a PPAR-α agonist to a PPAR-γ agent causes a further decrease in plasma triacylglycerol, but has no effect on NEFA or glucose metabolism.

Background UDP-GlcNAc 2-epimerase/ManNAc 6-kinase (GNE) is a bifunctional enzyme responsible for the first committed steps in the synthesis of sialic acid, a common terminal monosaccharide in both protein and lipid glycosylation. GNE mutations are responsible for a rare autosomal recessive neuromuscular disorder, GNE myopathy (also called hereditary inclusion body myopathy). The connection between the impairment of sialic acid synthesis and muscle pathology in GNE myopathy remains poorly understood. Methods Glycosphingolipid (GSL) analysis was performed by HPLC in multiple models of GNE myopathy, including patients’ fibroblasts and plasma, control fibroblasts with inhibited GNE epimerase activity through a novel imino sugar, and tissues of Gne M712T/M712T knock-in mice. Results Not only neutral GSLs, but also sialylated GSLs, were significantly increased compared to controls in all tested models of GNE myopathy. Treatment of GNE myopathy fibroblasts with N-acetylmannosamine (ManNAc), a sialic acid precursor downstream of GNE epimerase activity, ameliorated the increased total GSL concentrations. Conclusion GNE myopathy models have increased total GSL concentrations. ManNAc supplementation results in decrease of GSL levels, linking abnormal increase of total GSLs in GNE myopathy to defects in the sialic acid biosynthetic pathway. These data advocate for further exploring GSL concentrations as an informative biomarker, not only for GNE myopathy, but also for other disorders of sialic acid metabolism.

5-Aminoimidazole-4-Carboxamide 1-β- d -Ribofuranoside Acutely Stimulates Skeletal Muscle 2-Deoxyglucose Uptake in Healthy Men Daniel J. Cuthbertson 1 , John A. Babraj 2 , Kirsteen J.W. Mustard 3 , Mhairi C. Towler 4 , Kevin A. Green 3 , Henning Wackerhage 4 , Graeme P. Leese 1 , Keith Baar 3 , Michaela Thomason-Hughes 3 , Calum Sutherland 5 , D. Grahame Hardie 3 and Michael J. Rennie 6 1 Department of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, Scotland 2 School of Life Sciences, Heriot Watt University, Edinburgh, Scotland 3 Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee, Scotland 4 College of Life Sciences and Medicine, University of Aberdeen, Aberdeen, Scotland 5 Department of Pharmacology and Neurosciences, University of Dundee, Dundee, Scotland 6 School of Biomedical Sciences, Graduate Entry Medical School, University of Nottingham, Derby City Hospital, Derby, U.K Address correspondence and reprint requests to Dr. Daniel Cuthbertson, Department of Medicine, Ninewells Hospital and Medical School, Dundee, Scotland DD1 9SY. E-mail: d.j.r.cuthbertson{at}dundee.ac.uk Abstract Activation of AMP-activated protein kinase (AMPK) in rodent muscle by exercise, metformin, 5-aminoimidazole-4-carboxamide 1-β- d -ribofuranoside (AICAR), and adiponectin increases glucose uptake. The aim of this study was to determine whether AICAR stimulates muscle glucose uptake in humans. We studied 29 healthy men (aged 26 ± 8 years, BMI 25 ± 4 kg/m 2 [mean ± SD]). Rates of muscle 2-deoxyglucose (2DG) uptake were determined by measuring accumulation of total muscle 2DG (2DG and 2DG-6-phosphate) during a primed, continuous 2DG infusion. The effects of AICAR and exercise on muscle AMPK activity/phosphorylation and 2DG uptake were determined. Whole-body glucose disposal was compared before and during AICAR with the euglycemic-hyperinsulinemic clamp. Muscle 2DG uptake was linear over 9 h ( R 2 = 0.88 ± 0.09). After 3 h, 2DG uptake increased 2.1 ± 0.8- and 4.7 ± 1.7-fold in response to AICAR or bicycle exercise, respectively. AMPK α 1 and α 2 activity or AMPK phosphorylation was unchanged after 20 min or 3 h of AICAR, but AMPK phosphorylation significantly increased immediately and 3 h after bicycle exercise. AICAR significantly increased phosphorylation of extracellular signal–regulated kinase 1/2, but phosphorylation of β-acetyl-CoA carboxylase, glycogen synthase, and protein kinase B or insulin receptor substrate-1 level was unchanged. Mean whole-body glucose disposal increased by 7% with AICAR from 9.3 ± 0.6 to 10 ± 0.6 mg · kg −1 · min −1 ( P < 0.05). In healthy people, AICAR acutely stimulates muscle 2DG uptake with a minor effect on whole-body glucose disposal. 2DG, 2-deoxyglucose 2DG6P, 2-deoxyglucose 6-phosphate ACC, acetyl-CoA carboxylase AICAR, 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside AMPK, AMP-activated protein kinase aPKC, atypical protein kinase C ERK, extracellular signal–regulated kinase FDG, fluorodeoxyglucose GS, glycogen synthase GSK, glycogen synthase kinase IRS-1, insulin receptor substrate-1 PKB, protein kinase B ZMP, 5-aminoimidazole-4-carboxamide ribonucleoside Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 18 May 2007. DOI: 10.2337/db06-1716. D.J.C. and J.A.B. contributed equally to this work. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted May 11, 2007. Received December 8, 2006. DIABETES

Dominant Negative Pathogenesis by Mutant Proinsulin in the Akita Diabetic Mouse Tetsuro Izumi 1 , Hiromi Yokota-Hashimoto 1 , Shengli Zhao 1 , Jie Wang 1 , Philippe A. Halban 2 and Toshiyuki Takeuchi 1 1 Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan 2 Louis-Jeantet Research Laboratories, University Medical Centre, Geneva, Switzerland Abstract Autosomal dominant diabetes in the Akita mouse is caused by mutation of the insulin 2 gene, whose product replaces a cysteine residue that is engaged in the formation of an intramolecular disulfide bond. These heterozygous mice exhibit severe insulin deficiency despite coexpression of normal insulin molecules derived from three other wild-type alleles of the insulin 1 and 2 genes. Although the results of our previous study suggested that the mutant proinsulin 2 is misfolded and blocked in the transport from the endoplasmic reticulum to the Golgi apparatus, its dominant negative nature has not been fully characterized. In the present study, we investigated the possible pathogenic mechanisms induced by the mutant proinsulin 2. There is no evidence that the mutant proinsulin 2 attenuates the overall protein synthesis rate or promotes the formation of aberrant disulfide bonds. The trafficking of constitutively secreted alkaline phosphatase, however, is significantly decreased in the islets of Akita mice, indicating that the function of early secretory pathways is nonspecifically impaired. Morphological analysis has revealed that secretory pathway organelle architecture is progressively devastated in the β-cells of Akita mice. These findings suggest that the organelle dysfunction resulting from the intracellular accumulation of misfolded proinsulin 2 is primarily responsible for the defect of coexisting wild-type insulin secretion in Akita β-cells. Footnotes Address correspondence and reprint requests to Dr. Tetsuro Izumi, Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan. E-mail: tizumi{at}showa.gunma-u.ac.jp . Received for publication 1 August 2002 and accepted in revised form 5 November 2002. Current address for J.W. is Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637. ER, endoplasmic reticulum; SEAP, secretory alkaline phosphatase; TUNEL, transferase-mediated dUTP nick-end labeling. DIABETES

The mitochondrial electron transport chain (ETC) is necessary for tumour growth 1 – 6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies 7 – 9 . Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria 10 , 11 . However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP—that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)—an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX) 12 , which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis ( Lb NOX) 13 targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity. Oxidation of ubiquinol by the mitochondrial electron transfer chain drives tumour growth by maintaining the function of the oxidative Krebs cycle and de novo pyrimidine synthesis.

Certain adenosine residues within mammalian RNAs undergo reversible N 6 methylation. Two methyltransferase enzymes, METTL3 and METTL14, as well as the splicing factor WTAP are identified as core components of the multiprotein complex that deposits RNA N 6 -methyladenosine (m 6 A) in nuclear RNAs. N 6 -methyladenosine (m 6 A) is the most prevalent and reversible internal modification in mammalian messenger and noncoding RNAs. We report here that human methyltransferase-like 14 (METTL14) catalyzes m 6 A RNA methylation. Together with METTL3, the only previously known m 6 A methyltransferase, these two proteins form a stable heterodimer core complex of METTL3–METTL14 that functions in cellular m 6 A deposition on mammalian nuclear RNAs. WTAP, a mammalian splicing factor, can interact with this complex and affect this methylation.

Nutrition in CAPD: Serum bicarbonate and the ubiquitin-proteasome system in muscle. Metabolic acidosis in chronic renal failure (CRF) induces loss of lean body mass while elimination of acidosis during a one year trial improved anthropometric indices in continuous ambulatory peritoneal dialysis (CAPD) patients. In rats with CRF, the mechanisms causing loss of lean body mass have been linked to acidosis-induced destruction of the essential, branched-chain amino acids (BCAA) and activation of the ubiquitin-proteasome system that degrades muscle protein; the latter response includes increased transcription of the ubiquitin gene. Our aim was to determine if increasing the serum bicarbonate (HCO3) concentration of CAPD patients would improve their nutritional status, increase plasma BCAA levels, and reduce ubiquitin mRNA in their muscle as an index of suppressed activity of the ubiquitin-proteasome system. Eight, stable, long-term CAPD patients underwent vastus lateralis muscle biopsy before being randomized to continue 35 mmol/L lactate dialysate or convert to a 40 mmol/L lactate dialysate. After four weeks, measurements were repeated. Serum HCO3 increased in all patients and final values did not differ statistically between the two groups so results for all patients were combined. Weight and body mass index increased significantly as did plasma BCAA. Muscle levels of ubiquitin mRNA decreased significantly; serum tumor necrosis factor-α (TNF-α) also decreased. Our results indicate that even a small correction of serum HCO3 improves nutritional status, and provide evidence for down-regulation of BCAA degradation and muscle proteolysis via the ubiquitin-proteasome system. Whether acidosis and inflammatory cytokines (such as, TNF-α) interact to impair nutrition is unknown.