Proteome-wide identification and quantification of S-glutathionylation targets in mouse liver
Protein S-glutathionylation is a reversible post-translational modification regulating sulfhydryl homeostasis. However, little is known about the proteins and pathways regulated by S-glutathionylation in whole organisms and current approaches lack the sensitivity to examine this modification under basal conditions. We now report the quantification and identification of S-glutathionylated proteins from animal tissue, using a highly sensitive methodology combining high-accuracy proteomics with Tandem Mass Tagging to provide precise, extensive coverage of S-glutathionylated targets in mouse liver. Critically, we show signific...
Source: BJ Energy - April 20, 2015 Category: Biochemistry Authors: D J McGarry, W Chen, P Chakravarty, D J Lamont, C Roland Wolf, C J Henderson Tags: BJ Energy Source Type: research
Bioenergetic Programming Of Macrophages By The Apolipoprotein A-I Mimetic Peptide 4F
The apolipoprotein (apo)A-I mimetic peptide 4F favors the differentiation of human monocytes to an alternatively-activated M2 phenotype. The goal of the current study was to test whether the 4F-mediated differentiation of monocyte-derived macrophages (MDMs) requires the induction of an oxidative metabolic program. 4F treatment induced several genes in MDMs that play an important role in lipid metabolism, including peroxisome proliferator-activated receptor γ (PPARγ) and CD36. Addition of 4F was associated with a significant increase in fatty acid (FA) uptake and oxidation compared to vehicle treatment. Mitoch...
Source: BJ Energy - March 5, 2015 Category: Biochemistry Authors: G Datta, P A Kramer, M S Johnson, H Sawada, L E Smythies, D K Crossman, B Chacko, S W Ballinger, D G Westbrook, P Mayakonda, G M Anantharamaiah, V M Darley-Usmar, C Roger White Tags: BJ Energy Source Type: research
DJ1 represses glycolysis and cell proliferation by transcriptionally up-regulating pink1
DJ1 is a multifunctional protein whose mutations cause autosomal recessive early-onset Parkinson disease (PD). DJ1 loss of function disrupts mitochondrial function, but the signaling pathway whereby it interferes with energy metabolism is unknown. Here, we found that mouse embryonic fibroblasts obtained from DJ1-null (dj1-/-) mice showed higher glycolytic rate than those from wild type DJ1 (dj1+/+). This effect could be counteracted by the expression of the full-length cDNA encoding the wild type DJ1, but not its DJ1-L166P mutant form associated with PD. Loss of DJ1 increased hypoxia-inducible factor-1α...
Source: BJ Energy - February 11, 2015 Category: Biochemistry Authors: R Requejo-Aguilar, I Lopez-Fabuel, D Jimenez-blasco, E Fernandez, A Almeida, J P Bolaños Tags: BJ Energy Source Type: research
Evolutionarily Divergent, Na{+}-Regulated H{+}-Transporting Membrane-Bound Pyrophosphatases
Membrane-bound PPases (mPPases) of various types consume PPi to drive active H+ or Na+ transport across membranes. H+-transporting PPases are divided into phylogenetically distinct K+-independent and K+-dependent subfamilies. Here we describe a group of 46 bacterial proteins and one archaeal protein that are only distantly related to known mPPases (23–34% sequence identity). Despite this evolutionary divergence, these proteins contain the full set of 12 polar residues that interact with PPi, the nucleophilic water, and five cofactor Mg2+ ions found in “canonicalȁ...
Source: BJ Energy - February 9, 2015 Category: Biochemistry Authors: H H Luoto, E Nordbo, A M Malinen, A A Baykov, R Lahti Tags: BJ Energy Source Type: research
Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy consuming redox circuit
Cellular proteins rely on reversible redox reactions to establish and maintain biological structure and function. How redox catabolic (NAD+:NADH) and anabolic (NADP+:NADPH) processes integrate during metabolism to maintain cellular redox homeostasis however is unknown. The present work identifies a continuously cycling, mitochondrial membrane potential-dependent redox circuit between the pyruvate dehydrogenase complex (PDHC) and nicotinamide nucleotide transhydrogenase (NNT). PDHC is shown to produce H2O2 in relation to reducing pressure within the complex. The H2O2 produced however is effectively masked by a...
Source: BJ Energy - February 3, 2015 Category: Biochemistry Authors: K H Fisher-Wellman, C Lin, T E Ryan, L R Reese, L A. Gilliam, B L. Cathey, D S. Lark, C D. Smith, D M. Muoio, P Darrell Neufer Tags: BJ Energy Source Type: research
High Glucose Induces Mitochondrial Dysfunction Independent of Protein O-GlcNAcylation
In this study, we tested whether high glucose-induced increases in O-GlcNAc modifications directly regulate mitochondrial function in isolated cardiomyocytes. Augmentation of O-GlcNAcylation with high glucose (33 mM) was associated with diminished basal and maximal cardiomyocyte respiration, a decreased mitochondrial reserve capacity, and lower Complex II-dependent respiration (p<0.05); however, pharmacological or genetic modulation of O-GlcNAc modifications under normal or high glucose conditions showed few significant effects on mitochondrial respiration, suggesting that O-GlcNAc does not play a major role in regulati...
Source: BJ Energy - January 27, 2015 Category: Biochemistry Authors: S Dassanayaka, R D Readnower, J K Salabei, B W Long, A L Aird, Y Zheng, S Muthusamy, H T Facundo, B G Hill, S P Jones Tags: BJ Metabolism Source Type: research
Bioenergetic differences between MCF-7 and T47D breast cancer cells and their regulation by estradiol and tamoxifen
Estrogen receptor α (ERα+) breast tumors rely on mitochondria (mt) to generate ATP. The goal of this study was to determine how estradiol (E2) and 4-hydroxytamoxifen (4-OHT) affect cellular bioenergetic function in MCF-7 and T47D ERα+ breast cancer cells in serum-replete versus dextran-coated charcoal (DCC)-stripped fetal bovine serum (FBS)-containing medium (‘serum-starved’). Serum-starvation reduced oxygen consumption rate (OCR), extracellular acidification rate (ECAR), ATP-linked OCR, and maximum mt capacity, reflecting lower ATP demand and mt respiration. Cellular respir...
Source: BJ Energy - October 3, 2014 Category: Biochemistry Authors: B N Radde, M M Ivanova, H Xuan Mai, J K Salabei, B G Hill, C M Klinge Tags: BJ Metabolism Source Type: research
The disease-associated mutation of the mitochondrial sulphydryl oxidase Erv1 impairs cofactor binding during its catalytic reaction
In this study, we use yeast Erv1 as a model to provide clear evidence for a progressive functional defect in the catalytic activity of the corresponding Erv1 R182H mutant. We show that the FAD cofactor was released from Erv1 R182H during its catalytic cycle, which led to the inactivation of the enzyme. We also characterised the effects of the mutation on the folding and stability of Erv1, and tested our in vitro findings in vivo using a yeast genetic approach. The results of this study allows us to provide a model for the functional defect in Erv1 R182H, which could potentially be extended to human ALR R194H, and provides ...
Source: BJ Energy - October 1, 2014 Category: Biochemistry Authors: E Ceh-Pavia, S Ang, M P Spiller, H Lu Tags: BJ Biomolecules Source Type: research
Structural characterisation of Spinach Ribulose-1,5-Bisphosphate carboxylase/oxygenase activase isoforms reveals hexameric assemblies with increased thermal stability
Most plants contain two isoforms of Rubisco activase, a chloroplast protein that maintains the activity of Rubisco during photosynthesis. The longer (α-) Rubisco activase isoform has previously been shown to regulate the activity of Rubisco in response to both the ADP:ATP ratio and redox potential via thioredoxin-f. We have characterised the arrangement of the different spinach (Spinacia oleracea) isoforms in solution, and show how the presence of nucleotides changes the oligomeric state. While the shorter (β-) isoform from both tobacco (Nicotiana tabacum) and spinach tend to form a range of oligomers in solu...
Source: BJ Energy - September 23, 2014 Category: Biochemistry Authors: J R Keown, F Grant Pearce Tags: BJ Plant Source Type: research
Comparisons of subunit 5A and 5B isozymes of yeast cytochrome c oxidase
Subunit 5 of Saccharomyces cerevisiae CcO is essential for assembly and has two isoforms, 5A and 5B. 5A is expressed under normoxic conditions while 5B is expressed at very low oxygen tensions. As a consequence, COX5A-deleted strains (Δcox5A) have no or only low levels of CcO under normoxic conditions rendering them respiratory-deficient. Previous studies reported that respiratory growth could be restored by combining Δcox5A with mutations of ROX1 that encodes a repressor of COX5B expression. In these mutants 5B isozyme expression level was 30-50 % of wild type (5A isozyme) and exhibited a maximum catalytic a...
Source: BJ Energy - September 22, 2014 Category: Biochemistry Authors: R Dodia, B Meunier, C W.M. Kay, P R Rich Tags: BJ Energy Source Type: research
Cytochrome P450 System Proteins Reside in Different Regions of the Endoplasmic Reticulum
Cytochrome P450 function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher levels of unsaturated fatty acids. The goal of this study was to determine if the P450 system proteins localize to different regions of the...
Source: BJ Energy - September 19, 2014 Category: Biochemistry Authors: J Park, J Robert Reed, L M Brignac-Huber, W L. Backes Tags: BJ Energy Source Type: research
Investigating the contribution of the active site environment to the slow reaction of hypoxia-inducible factor prolyl hydroxylase domain 2 with oxygen
Prolyl hydroxylase domain 2 (PHD2) catalyses the post-translational hydroxylation of the Hypoxia Inducible Factor (HIF), a modification that regulates the hypoxic response in humans. PHD2 is an FeII/2-oxoglutarate (2OG) oxygenase; its catalysis is proposed to provide a link between cellular HIF levels and changes in O2 availability. Transient kinetic studies have shown that PHD2 reacts slowly with O2 compared to some other studied 2OG oxygenases, a property which may be related to its hypoxia sensing role. PHD2 forms a stable complex with FeII and 2OG; crystallographic and kinetic analyses indicate that an FeII-coordinated...
Source: BJ Energy - August 14, 2014 Category: Biochemistry Authors: H Tarhonskaya, R Chowdhury, I K. H. Leung, N D. Loik, J S. O. McCullagh, T D.W. Claridge, C J. Schofield, E Flashman Tags: BJ Biomolecules Source Type: research
Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria
We report that biguanides inhibit complex I by inhibiting ubiquinone reduction (but not competitively) and, independently, stimulate reactive oxygen species production by the complex I flavin. Biguanides also inhibit mitochondrial ATP synthase, and two of them inhibit only ATP hydrolysis, not synthesis. Thus, we identify biguanides as a new class of complex I and ATP synthase inhibitor. By comparing biguanide effects on isolated complex I and cultured cells, we distinguish three anti-diabetic and potentially anti-neoplastic biguanides (metformin, buformin and phenformin) from two anti-malarial biguanides (cycloguanil and p...
Source: BJ Energy - July 14, 2014 Category: Biochemistry Authors: H R Bridges, A J Y Jones, M N Pollak, J Hirst Tags: BJ Energy Source Type: research
A small molecule that induces reactive oxygen species via cellular glutathione depletion
Induction of excessive levels of reactive oxygen species (ROS) by small molecule compounds has been considered as a potentially effective therapeutic strategy against cancer cells, which are often subjected to chronic oxidative stress. However, to elucidate the mechanisms of action of bioactive compounds is generally a time-consuming process. We recently identified NPD926, a small molecule that induces rapid cell death in cancer cells. By a combination of two comprehensive and complementary approaches, proteomic profiling and affinity purification, combined with the subsequent biochemical assays, we elucidated the mechanis...
Source: BJ Energy - July 11, 2014 Category: Biochemistry Authors: T Kawamura, Y Kondoh, M Muroi, M Kawatani, H Osada Tags: BJ ChemBio Source Type: research
Elucidation of the preferred routes of C8-vinyl reduction in chlorophyll and bacteriochlorophyll biosynthesis
Most of the chlorophylls and bacteriochlorophylls utilised for light harvesting by phototrophic organisms carry an ethyl group at the C8 position of the molecule, the product of a C8-vinyl reductase acting on a chlorophyll/bacteriochlorophyll biosynthetic precursor. Two unrelated classes of C8-vinyl reductase are known to exist, BciA and BciB, found in the purple phototroph Rhodobacter sphaeroides and the cyanobacterium Synechocystis sp. PCC6803, respectively. We constructed strains of each bacterium with the native C8-vinyl reductase swapped for the other class of the enzyme, and combined these replacements with a series ...
Source: BJ Energy - June 19, 2014 Category: Biochemistry Authors: D P. Canniffe, J W. Chidgey, C Neil Hunter Tags: BJ Energy Source Type: research
