ATP Synthase Α-subunit Research Articles

From cyanobacteria and cyanophages to chloroplasts: the fate of the genomes of oxyphototrophs and the genes encoding photosystem II proteins.

Chloroplasts are the result of endosymbiosis of cyanobacterial organisms with proto-eukaryotes. The psbA, psbD and psbO genes are present in all oxyphototrophs and encode the D1/D2 proteins of photosystem II (PSII) and PsbO, respectively. PsbO is a peripheral protein that stabilizes the O2-evolving complex in PSII. Of these genes, psbA and psbD remained in the chloroplastic genome, while psbO was transferred to the nucleus. The genomes of selected cyanobacteria, chloroplasts and cyanophages carrying psbA and psbD, respectively, were analysed. The highest density of genes and coding sequences (CDSs) was estimated for the genomes of cyanophages, cyanobacteria and chloroplasts. The synonymous mutation rate (rS) of psbA and psbD in chloroplasts remained almost unchanged and is lower than that of psbO. The results indicate that the decreasing genome size in chloroplasts is more similar to the genome reduction observed in contemporary endosymbiotic organisms than in streamlined genomes of free-living cyanobacteria. The rS of atpA, which encodes the α-subunit of ATP synthase in chloroplasts, suggests that psbA and psbD, and to a lesser extent psbO, are ancient and conservative and arose early in the evolution of oxygenic photosynthesis. The role of cyanophages in the evolution of oxyphototrophs and chloroplastic genomes is discussed.

Molecular Bulkiness ofa Single Amino Acid inthe F1 α-Subunit Determines the Robustness ofCyanobacterial ATP Synthase.

Cyanobacteria are promising photosynthetic organisms owing to their ease of genetic manipulation. Among them, Synechococcus elongatus UTEX 2973 exhibits faster growth, higher biomass production efficiency and more robust stress tolerance compared with S. elongatus PCC 7942. This is due to specific genetic differences, including four single-nucleotide polymorphisms (SNPs) in three genes. One of these SNPs alters an amino acid at position 252 of the FoF1 ATP synthase α-subunit from Tyr to Cys (αY252C) in S. elongatus 7942. This change has been shown to significantly affect growth rate and stress tolerance, specifically in S. elongatus. Furthermore, experimental substitutions with several other amino acids have been shown to alter the ATP synthesis rate in the cell. In the present study, we introduced identical amino acid substitutions into Synechocystis sp. PCC 6803 at position 252 to elucidate the amino acid's significance and generality across cyanobacteria. We investigated the resulting impact on growth, intracellular enzyme complex levels, intracellular ATP levels and enzyme activity. The results showed that the αY252C substitution decreased growth rate and high-light tolerance. This indicates that a specific bulkiness of this amino acid's side chain is important for maintaining cell growth. Additionally, a remarkable decrease in the membrane-bound enzyme complex level was observed. However, the αY252C substitution did not affect enzyme activity or intracellular ATP levels. Although the mechanism of growth suppression remains unknown, the amino acid at position 252 is expected to play an important role in enzyme complex formation.

Photosynthesis-related genes induce resistance against soybean mosaic virus: Evidence for involvement of the RNA silencing pathway.

Increasing lines of evidence indicate that chloroplast‐related genes are involved in plant–virus interactions. However, the involvement of photosynthesis‐related genes in plant immunity is largely unexplored. Analysis of RNA‐Seq data from the soybean cultivar L29, which carries the Rsv3 resistance gene, showed that several chloroplast‐related genes were strongly induced in response to infection with an avirulent strain of soybean mosaic virus (SMV), G5H, but were weakly induced in response to a virulent strain, G7H. For further analysis, we selected the PSaC gene from the photosystem I and the ATP‐synthase α‐subunit (ATPsyn‐α) gene whose encoded protein is part of the ATP‐synthase complex. Overexpression of either gene within the G7H genome reduced virus levels in the susceptible cultivar Lee74 (rsv3‐null). This result was confirmed by transiently expressing both genes in Nicotiana benthamiana followed by G7H infection. Both proteins localized in the chloroplast envelope as well as in the nucleus and cytoplasm. Because the chloroplast is the initial biosynthesis site of defence‐related hormones, we determined whether hormone‐related genes are involved in the ATPsyn‐α‐ and PSaC‐mediated defence. Interestingly, genes involved in the biosynthesis of several hormones were up‐regulated in plants infected with SMV‐G7H expressing ATPsyn‐α. However, only jasmonic and salicylic acid biosynthesis genes were up‐regulated following infection with the SMV‐G7H expressing PSaC. Both chimeras induced the expression of several antiviral RNA silencing genes, which indicate that such resistance may be partially achieved through the RNA silencing pathway. These findings highlight the role of photosynthesis‐related genes in regulating resistance to viruses.

Repeated Aconitine Treatment Induced the Remodeling of Mitochondrial Function via AMPK-OPA1-ATP5A1 Pathway.

Aconitine is attracting increasing attention for its unique positive inotropic effect on the cardiovascular system, but underlying molecular mechanisms are still not fully understood. The cardiotonic effect always requires abundant energy supplement, which is mainly related to mitochondrial function. And OPA1 has been documented to play a critical role in mitochondrial morphology and energy metabolism in cardiomyocytes. Hence, this study was designed to investigate the potential role of OPA1-mediated regulation of energy metabolism in the positive inotropic effect caused by repeated aconitine treatment and the possible mechanism involved. Our results showed that repeated treatment with low-doses (0–10 μM) of aconitine for 7 days did not induce detectable cytotoxicity and enhanced myocardial contraction in Neonatal Rat Ventricular Myocytes (NRVMs). Also, we first identified that no more than 5 μM of aconitine triggered an obvious perturbation of mitochondrial homeostasis in cardiomyocytes by accelerating mitochondrial fusion, biogenesis, and Parkin-mediated mitophagy, followed by the increase in mitochondrial function and the cellular ATP content, both of which were identified to be related to the upregulation of ATP synthase α-subunit (ATP5A1). Besides, with compound C (CC), an inhibitor of AMPK, could reverse aconitine-increased the content of phosphor-AMPK, OPA1, and ATP5A1, and the following mitochondrial function. In conclusion, this study first demonstrated that repeated aconitine treatment could cause the remodeling of mitochondrial function via the AMPK–OPA1–ATP5A1 pathway and provide a possible explanation for the energy metabolism associated with cardiotonic effect induced by medicinal plants containing aconitine.

Overexpression of native IF1 downregulates glucose-stimulated insulin secretion by pancreatic INS-1E cells

We have previously reported that transient knock-down of ATPase inhibitory factor 1 (IF1) by siRNA upregulates ATP levels and subsequently augments insulin secretion in model pancreatic β-cells INS-1E. Here we investigated how long-term IF1-overexpression impacts pancreatic β-cell bioenergetics and insulin secretion. We generated INS-1E cell line stably overexpressing native IF1. We revealed that IF1 overexpression leads to a substantial decrease in ATP levels and reduced glucose-stimulated insulin secretion. A decrease in total cellular ATP content was also reflected in decreased free ATP cytosolic and mitochondrial levels, as monitored with ATeam biosensor. Consistently, cellular respiration of IF1-overexpressing cells was decreased. 3D structured illumination microscopy (SIM) revealed a higher amount of insulin granules with higher volume in IF1-overexpressing cells. Similar effects occurred when cells were incubated at low glucose concentrations. Noteworthy, activation of PKA by dibutyryl cAMP entirely abolished the inhibitory effect of IF1 overexpression on ATP production and insulin secretion. Mitochondrial network morphology and cristae ultrastructure in INS-1E overexpressing IF1 remained mostly unchanged. Finally, we show that INS-1E cells decrease their IF1 protein levels relative to ATP synthase α-subunit in response to increased glucose. In conclusion, IF1 actively downregulates INS-1E cellular metabolism and reduces their ability to secrete insulin.

Intron-based phylogeny revealed a substrate role in the origin of the gastropod species flock from Lake Baikal

A nuclear marker, an intron of ATP synthase α-subunit gene, used to determine phylogenetic relationships between 22 species of the family Baicaliidae (Caenogastropoda: Rissooidea) indicated two genetic groups of these gastropods in Lake Baikal. The obtained groups partially coincide with the clusters revealed by the mitochondrial COI gene and do not fully coincide with the modern taxonomy of the family. Thirteen species of the first group inhabit different sediments (silty sand, sand, stones, and rocks), eight out of the nine species from the second group are psammobionts. Both genetic groups include eurybathic and stenobathic species widely distributed in the North, Middle and Southern Basins of the lake, as well as species, whose habitats are limited to one or two basins. Phylogenetic analysis suggests that the main way of baicaliids speciation was the repeated occupation of the same habitats by different ancestral forms. Sculpture of the shells (ribs, carina and/or periostracal hairs/plates) also most likely appeared independently in each group due to similar adaptation.

Nitric Oxide Donor NOC-18-Induced Changes of Mitochondrial Phosphoproteome in Rat Cardiac Ischemia Model.

Background and objective: Nitric oxide (NO) is known to exert cardioprotective effects against heart ischemic damage and may be involved in ischemic pre- and postconditioning. NO-triggered cardioprotective mechanisms are not well understood but may involve regulation of mitochondrial permeability transition pore (mPTP). In this study, we aimed to identify differentially phosphorylated mitochondrial proteins possibly involved in the NO/protein kinase G (PKG)/mPTP signaling pathway that can increase the resistance of cardiomyocytes to ischemic damage. Materials and methods: Isolated hearts from Wistar rats were perfused with NO donor NOC-18 prior to induction of stop–flow ischemia. To quantify and characterize the phosphoproteins, mitochondrial proteins were resolved and analyzed by two-dimensional gel electrophoresis followed by Pro-Q Diamond phosphoprotein gel staining, excision, trypsin digestions, and mass spectrometry. Quantitative proteomic analysis coupled with liquid chromatography–tandem mass spectrometry was also performed. Results: Mitochondrial protein phosphorylation patterns in NOC-18-pretreated ischemic hearts versus ischemic hearts were compared. Pretreatment of hearts with NOC-18 caused changes in mitochondrial phosphoproteome after ischemia which involved modifications of 10 mitochondrial membrane-bound and 10 matrix proteins. Among them, α-subunit of ATP synthase and adenine nucleotide (ADP/ATP) translocase 1, both of which are considered as potential structural components of mPTP, were identified. We also found that treatment of isolated non-ischemic mitochondria with recombinant PKG did not cause the same protein phosphorylation as pretreatment of hearts with NOC-18. Conclusions: Our study suggests that pretreatment of hearts with NOC-18 causes changes in mitochondrial phosphoproteome after ischemia which involves modifications of certain proteins thought to be involved in the regulation of mPTP opening and intracellular redox state. These proteins may be potential targets for pharmacological preconditioning of the heart.

An unexpected benefit from E. coli: how enterobactin benefits host health.

Iron plays many critical roles in human biology, such as aiding the transport of oxygen and mediating redox reactions. Iron is essential for life, yet little is known about how iron is taken up into mitochondria to impact the labile iron pool. Iron deficiency is one of the most prevalent human nutrient-deficiency diseases in the world and is a major cause of anemia that affects >25% of the world’s population, but unfortunately the current treatment (oral iron supplementation) is inefficient and has many side effects. A greater understanding of iron uptake, and discovery of molecules that aid in this process, may lead to more effective treatments for iron deficiency. In this study, we uncovered a unique and surprising role for an Escherichia coli-produced siderophore enterobactin (Ent) that facilitates iron uptake by the host, observed in both C. elegans and mammalian cells. Although siderophores are well-known Fe+3 scavengers, this activity has previously been described to only benefit iron acquisition by bacteria, not the host. This unexpected function is dependent on the binding of Ent to the host’s ATP synthase α-subunit but is independent of other subunits of the ATP synthase. This finding marks a major shift regarding the role of this siderophore in the “iron tug-of-war” paradigm, which is often used to describe the fight between the bacteria and the host for this essential micronutrient. Instead, this study presents E. coli as a commensal “friend” that provides a molecule that supports the host’s iron homeostasis. This work reveals a novel, beneficial role of a bacteria-generated molecule in aiding the host’s iron homeostasis, and points to surprising new benefits from commensal bacteria.

Enterococcus saigonensis sp. nov., isolated from retail chicken meat and liver.

Two Gram-stain-positive strains, VE80T and VE116, which were resistant to vancomycin, were isolated from retail chicken meat and liver in Ho Chi Minh, Vietnam, respectively. These strains were characterized by sequence analyses of 16S rRNA, RNA polymerase α-subunit (rpoA), ATP synthase α-subunit (atpA), and phenylalanyl-tRNA synthase α-subunit (pheS) genes, determination of DNA G+C content, cellular fatty acid methyl ester analysis, DNA-DNA hybridization, and conventional morphological and biochemical tests. Strains VE80T and VE116 had 99.6 % 16S rRNA gene sequence similarity with Enterococcus canintestini LMG 13590T, and 99.1 % 16S rRNA gene sequence similarity with Enterococcus dispar ATCC 51266T. However, the two isolates could be clearly differentiated from these reference strains by the low sequence similarities (86.1-86.8 %) of the atpA gene, low DNA-DNA relatedness (<22.8 %), and differences in the production of acid from melezitose and methyl α-d-glucoside. Based on the results obtained in the present study, these two isolates are considered to represent a novel species of the genus Enterococcus, for which the name Enterococcus saigonensis sp. nov., is proposed. The type strain is VE80T (=JCM 31193T=CCUG 68827T).

Genetic and morphological diversification in gastropods of the Baicaliidae family

The combined (“total evidence”) phylogenetic inference was based on the comparison of the intronic nucleotide sequences of the ATP synthase α-subunit gene of 11 gastropod species belonging to the rapidly evolving Baikalian endemic family Baicaliidae, the morphological description of these species, and the previously published sequences of the mitochondrial gene coding the first subunit of cytochrome c oxidase. The phylogeny based on the intron sequences agrees well with the morphology. It is shown that sister species usually have similar substrate preferences. Certain discrepancy in the phylogenetic tree topology and the taxonomy based on morphological traits is probably due to the rapid morphological evolution of mollusks of the family Baicaliidae.

HAI-178 antibody-conjugated fluorescent magnetic nanoparticles for targeted imaging and simultaneous therapy of gastric cancer

The successful development of safe and highly effective nanoprobes for targeted imaging and simultaneous therapy of in vivo gastric cancer is a great challenge. Herein we reported for the first time that anti-α-subunit of ATP synthase antibody, HAI-178 monoclonal antibody-conjugated fluorescent magnetic nanoparticles, was successfully used for targeted imaging and simultaneous therapy of in vivo gastric cancer. A total of 172 specimens of gastric cancer tissues were collected, and the expression of α-subunit of ATP synthase in gastric cancer tissues was investigated by immunohistochemistry method. Fluorescent magnetic nanoparticles were prepared and conjugated with HAI-178 monoclonal antibody, and the resultant HAI-178 antibody-conjugated fluorescent magnetic nanoparticles (HAI-178-FMNPs) were co-incubated with gastric cancer MGC803 cells and gastric mucous GES-1 cells. Gastric cancer-bearing nude mice models were established, were injected with prepared HAI-178-FMNPs via tail vein, and were imaged by magnetic resonance imaging and small animal fluorescent imaging system. The results showed that the α-subunit of ATP synthase exhibited high expression in 94.7% of the gastric cancer tissues. The prepared HAI-178-FMNPs could target actively MGC803 cells, realized fluorescent imaging and magnetic resonance imaging of in vivo gastric cancer, and actively inhibited growth of gastric cancer cells. In conclusion, HAI-178 antibody-conjugated fluorescent magnetic nanoparticles have a great potential in applications such as targeted imaging and simultaneous therapy of in vivo early gastric cancer cells in the near future.

L-methionase: a therapeutic enzyme to treat malignancies.

Cancer is an increasing cause of mortality and morbidity throughout the world. L-methionase has potential application against many types of cancers. L-Methionase is an intracellular enzyme in bacterial species, an extracellular enzyme in fungi, and absent in mammals. L-Methionase producing bacterial strain(s) can be isolated by 5,5′-dithio-bis-(2-nitrobenzoic acid) as a screening dye. L-Methionine plays an important role in tumour cells. These cells become methionine dependent and eventually follow apoptosis due to methionine limitation in cancer cells. L-Methionine also plays an indispensable role in gene activation and inactivation due to hypermethylation and/or hypomethylation. Membrane transporters such as GLUT1 and ion channels like Na2+, Ca2+, K+, and Cl− become overexpressed. Further, the α-subunit of ATP synthase plays a role in cancer cells growth and development by providing them enhanced nutritional requirements. Currently, selenomethionine is also used as a prodrug in cancer therapy along with enzyme methionase that converts prodrug into active toxic chemical(s) that causes death of cancerous cells/tissue. More recently, fusion protein (FP) consisting of L-methionase linked to annexin-V has been used in cancer therapy. The fusion proteins have advantage that they have specificity only for cancer cells and do not harm the normal cells.

Analysis of Proteins Associated with Ozone Stress Response in Soybean Cultivars

Ozone stress induces leaf injuries and yield losses in soybean. To identify the proteins associated with the stress response, ozone tolerant and sensitive cultivars were analyzed by 2D-PAGE. After 11 days of ozone stress unifoliate leaves of tolerant soybean cv. Enrei developed 15% and sensitive cv. Nakasennari developed 52% leaf injury symptoms. Analysis of proteins in the unifoliate leaves identified six proteins in tolerant cv. Enrei and three proteins in sensitive cv. Nakasennari, responded significantly to ozone stress. The significantly responded proteins identified in this study were ATP synthase α-subunit, ATP synthase β-subunit, phosphoglycerate kinase, aldo/ketoreductase, rubiscoactivase and glutamine synthetase. To understand that the differences in response of ATP synthase proteins were ultimately associated with extracellular ATP signaling response, ozone sensitive cv. Nakasennari was treated with 1 mM ATP. Unifoliate leaves of ATP treated plants have significantly reduced injury symptoms (20%) under ozone stress compare to control plants (47%). This confirms that extracellular ATP signaling in ATP synthase dependent manner is playing a pivotal role in inducing ozone stress tolerance and preventing injuries in soybean cultivars.

Metabolic Labeling of Caenorhabditis elegans Primary Embryonic Cells with Azido-Sugars as a Tool for Glycoprotein Discovery

Glycobiology research with Caenorhabditis elegans (C. elegans) has benefitted from the numerous genetic and cell biology tools available in this system. However, the lack of a cell line and the relative inaccessibility of C. elegans somatic cells in vivo have limited the biochemical approaches available in this model. Here we report that C. elegans primary embryonic cells in culture incorporate azido-sugar analogs of N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc), and that the labeled glycoproteins can be analyzed by mass spectrometry. By using this metabolic labeling approach, we have identified a set of novel C. elegans glycoprotein candidates, which include several mitochondrially-annotated proteins. This observation was unexpected given that mitochondrial glycoproteins have only rarely been reported, and it suggests that glycosylation of mitochondrially-annotated proteins might occur more frequently than previously thought. Using independent experimental strategies, we validated a subset of our glycoprotein candidates. These include a mitochondrial, atypical glycoprotein (ATP synthase α-subunit), a predicted glycoprotein (aspartyl protease, ASP-4), and a protein family with established glycosylation in other species (actin). Additionally, we observed a glycosylated isoform of ATP synthase α-subunit in bovine heart tissue and a primate cell line (COS-7). Overall, our finding that C. elegans primary embryonic cells are amenable to metabolic labeling demonstrates that biochemical studies in C. elegans are feasible, which opens the door to labeling C. elegans cells with other radioactive or azido-substrates and should enable the identification of additional post-translationally modified targets and analysis of the genes required for their modification using C. elegans mutant libraries.

Hypoxia Affects Mitochondrial Protein Expression in Rat Skeletal Muscle

Hypoxia affects mammalian mitochondrial function, as well as mitochondria-based energy metabolism. The detail mechanism has not been fully understood. In this study, we detected protein expression levels in mitochondrial fractions of Wistar rats exposed to hypobaric hypoxia by use of proteomic methods. Adult male Wistar rats were randomized into an hypoxic (4,500 m, 30 days) group and a normoxic control group (sea level). Gastrocnemius muscles mitochondria were extracted and purified. Mitochondrial oxygen consumption was measured with a Clark oxygen electrode; mitochondrial transmembrane potential was detected with Rhodamine 123 as a fluoresce probe. Using 2-DE and MALDI-TOF MS analysis, we identified eight mitochondrial protein spots that were differentially expressed in the hypoxic group compared with the normoxic control. These proteins included Chain A of F1-ATPase, voltage dependent anion channel 1 (VDAC), hydroxyacyl Coenzyme A dehydrogenase α-subunit, mitochondrial F1 complex γ-subunit, androgen-regulated protein and tripartite motif protein 50. Two of the spots, VDAC and ATP synthase α-subunit, were confirmed by Western blotting analysis. Oxygen consumption during State 3 respiration, as well as the respiratory control ratio (RCR) was significantly higher in the control than that in the hypoxic group; mitochondrial transmembrane potential was significantly higher in hypoxic group than that in the control. With successful use of multiple proteomic analysis techniques, we demonstrates that 30 days hypoxia exposure has effects on the expression of mitochondrial proteins involved in ATP production and lipid metabolism, decrease the stability of mitochondrial membrane, and affect the mitochondrial electron transport chain.

β-amyloid Peptide Binds and Regulates Ectopic ATP Synthase α-Chain on Neural Surface

ABSTRACTAccumulation of the amyloid β protein (Aβ) in the brain is an important step in the pathogenesis of Alzheimer's disease. Many molecules could bind with Aβ, among which some molecules mediate Aβ neuronal toxicity. Thus, it is of interest to study the binding proteins of Aβ, and the functions that might be affected by Aβ. In the present study, we observed that accumulation of α-subunit of ATP synthase is associated with aggregates of Aβ proteins in amyloid plaques of amyloid precursor protein/presennillin-1 transgenic mice, and identified the α-subunit of ATP synthase as one of the Aβ binding proteins on the plasma membrane of neural cells by Western blot and mass spectrometry. In order to evaluate the consequences of the interaction between Aβ and surface α-subunit of ATP synthase, the extracellular ATP generation was analyzed, which showed that aggregated Aβ partially inhibited the extracellular generation of ATP, but was unable to significantly induce a decrease in cell surface ATP synthase α on neurons. These results suggest that the cell surface ATP synthase α is a binding protein for Aβ on neural cells, the functional inhibition of surface ATP synthase might be involved in machinery of brain malfunction in Aβ-mediated pathogenesis of Alzheimer's disease.

ATP synthase ecto-α-subunit: a novel therapeutic target for breast cancer

BackgroundTreatment failure for breast cancer is frequently due to lymph node metastasis and invasion to neighboring organs. The aim of the present study was to investigate invasion- and metastasis-related genes in breast cancer cells in vitro and in vivo. Identification of new targets will facilitate the developmental pace of new techniques in screening and early diagnosis. Improved abilities to predict progression and metastasis, therapeutic response and toxicity will help to increase survival of breast cancer patients.MethodsDifferential protein expression in two breast cancer cell lines, one with high and the other with low metastatic potential, was analyzed using two-dimensional liquid phase chromatographic fractionation (Proteome Lab PF 2D system) followed by matrix-assisted laser desorption/time-of-flight mass spectrometry (MALDI-TOF/MS).ResultsUp regulation of α-subunit of ATP synthase was identified in high metastatic cells compared with low metastatic cells. Immunohistochemical analysis of 168 human breast cancer specimens on tissue microarrays revealed a high frequency of ATP synthase α-subunit expression in breast cancer (94.6%) compared to normal (21.2%) and atypical hyperplasia (23%) breast tissues. Levels of ATP synthase expression levels strongly correlated with large tumor size, poor tumor differentiation and advanced tumor stages (P < 0.05). ATP synthase α-subunit over-expression was detected on the surface of a highly invasive breast cancer cell line. An antibody against the ATP synthase α-subunit inhibited proliferation, migration and invasion in these breast cancer cells but not that of a non-tumor derived breast cell line.ConclusionsOver-expression of ATP synthase α-subunit may be involved in the progression and metastasis of breast cancer, perhaps representing a potential biomarker for diagnosis, prognosis and a therapeutic target for breast cancer. This finding of this study will help us to better understand the molecular mechanism of tumor metastasis and to improve the screening, diagnosis, as well as prognosis and/or prediction of responses to therapy for breast cancer.

In This Issue

HomeCirculation ResearchVol. 109, No. 7In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Originally published16 Sep 2011https://doi.org/10.1161/RES.0b013e31823377bfCirculation Research. 2011;109:715Exosomes and Paracrine Effects of CD34+ Cells (p 724)Exosomes are responsible for the angiogenic effects of CD34+ stem cells, report Losordo and colleagues.Download figureDownload PowerPointAlthough stem cell transplantation improves the function of injured hearts, the mechanism of their salubrious effects remains mysterious. Too few of these cells are incorporated into the injured tissue to account for the remarkable functional and physiological benefits that are observed. To account for this discrepancy, some investigators have suggested that stem cells secrete trophic factors that promote the growth of new blood vessels or new cells within the injured tissue (paracrine hypothesis). In an effort to explain the paracrine effect of stem cells, Losordo et al collected exosomes secreted by CD34+ stem cells, shown previously to reduce angina and lower the rates of amputation in patients with critical limb ischemia. They report that these exosomes were just as potent as the CD34+ cells in promoting the growth of endothelial cells in culture and in stimulating angiogenesis in vivo. Exosomes from non-CD34+ cells were ineffective. How stem cell–derived exosomes stimulate angiogenesis remains a mystery, but the authors suggest that the role of angiogenic microRNAs (miR-126 and miR-130), sequestered within these vesicles, is one mechanism to explore in future studies.Peroxiredoxin 2 Ameliorates Atherosclerosis (p 739)Park et al uncover a unique role of peroxiredoxin 2 in preventing atherosclerotic lesion formation.Download figureDownload PowerPointAtherosclerotic lesions preferentially develop in areas where blood vessels are branched or curved. Turbulent blood flow in these areas stimulates the production of reactive oxygen species (ROS), which promote leukocyte adhesion to the vessel wall and stimulate the development of atherosclerotic plaques. Park et al show that vascular areas exposed to turbulent flow express high levels of the hydrogen peroxide–removing enzyme peroxiredoxin 2 (Prdx2) and that deletion of this enzyme increases vascular adhesion and the formation of atherosclerotic lesions in apoE-null mice. They report that loss of Prdx2 increases infiltration of immune cells into plaques and that the absence of this enzyme, either in vascular or immune cells, is equally atherogenic. Significantly, deletion of other peroxide-removing enzymes such as glutathione peroxidase and catalase was less harmful than Prdx2 deficiency, suggesting that Prdx-2 has a unique role in removing peroxides from sites at which they can cause the most damage. Such specificity of action may also explain the limited success of attempts to inhibit atherosclerosis by global antioxidant interventions.Redox Regulation of ATP Synthase (p 750)Van Eyk and associates identify a mitochondrial redox sensor in failing hearts.Download figureDownload PowerPointBecause of conduction defects, heart failure results in discoordinate contraction of the myocardium. Resynchronization of contraction by biventricular stimulation improves heart function and enhances long-term survival of patients with heart failure. Cardiac resynchronization therapy (CRT) also improves the energetic efficiency of the heart, but the molecular and cellular basis for this improvement remains unknown. In their previous work, Van Eyk et al had found that heart failure in dogs results in partial inhibition of mitochondrial ATP synthase and that this activity could be restored by CRT. They also found that CRT affects several mitochondrial proteins involved in energy production and redox regulation. They now link these two phenomena together, demonstrating that dyssynchronous heart failure results in the formation of disulfide bonds between the α- and the γ-subunits of ATP synthase as well as S-glutathiolation of the α-subunit. These changes were reversed by CRT, which induced S-nitrosation of the protein. The authors suggest that a uniquely reactive cysteine residue (Cys-294), located within the α-subunit of ATP synthase, functions as a redox switch that is able to sense the redox state and adjust energy production accordingly.Written by Aruni Bhatnagar. Previous Back to top Next FiguresReferencesRelatedDetails September 16, 2011Vol 109, Issue 7 Advertisement Article InformationMetrics © 2011 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e31823377bf Originally publishedSeptember 16, 2011 PDF download Advertisement

A targeted mass spectrometry-based approach for the identification and characterization of proteins containing α-aminoadipic and γ-glutamic semialdehyde residues

The site-specific identification of α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) residues in proteins is reported. Semialdehydic protein modifications result from the metal-catalyzed oxidation of Lys or Arg and Pro residues, respectively. Most of the analytical methods for the analysis of protein carbonylation measure change to the global level of carbonylation and fail to provide details regarding protein identity, site, and chemical nature of the carbonylation. In this work, we used a targeted approach, which combines chemical labeling, enrichment, and tandem mass spectrometric analysis, for the site-specific identification of AAS and GGS sites in proteins. The approach is applied to in vitro oxidized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and an untreated biological sample, namely cardiac mitochondrial proteins. The analysis of GAPDH resulted in the site-specific identification of two AAA and four GGS residues. Computational evaluation of the identified AAS and GGS sites in GAPDH indicated that these sites are located in flexible regions, show high solvent accessibility values, and are in proximity with possible metal ion binding sites. The targeted proteomic analysis of semialdehydic modifications in cardiac mitochondria yielded nine AAS modification sites which were unambiguously assigned to distinct lysine residues in the following proteins: ATP/ATP translocase isoforms 1 and 2, ubiquinol cytochrome-c reductase core protein 2, and ATP synthase α-subunit.

Subjects With Early-Onset Type 2 Diabetes Show Defective Activation of the Skeletal Muscle PGC-1α/Mitofusin-2 Regulatory Pathway in Response to Physical Activity

OBJECTIVEType 2 diabetes is associated with insulin resistance and skeletal muscle mitochondrial dysfunction. We have found that subjects with early-onset type 2 diabetes show incapacity to increase Vo2max in response to chronic exercise. This suggests a defect in muscle mitochondrial response to exercise. Here, we have explored the nature of the mechanisms involved.RESEARCH DESIGN AND METHODSMuscle biopsies were collected from young type 2 diabetic subjects and obese control subjects before and after acute or chronic exercise protocols, and the expression of genes and/or proteins relevant to mitochondrial function was measured. In particular, the regulatory pathway peroxisome proliferator–activated receptor γ coactivator (PGC)-1α/mitofusin-2 (Mfn2) was analyzed.RESULTSAt baseline, subjects with diabetes showed reduced expression (by 26%) of the mitochondrial fusion protein Mfn2 and a 39% reduction of the α-subunit of ATP synthase. Porin expression was unchanged, consistent with normal mitochondrial mass. Chronic exercise led to a 2.8-fold increase in Mfn2, as well as increases in porin, and the α-subunit of ATP synthase in muscle from control subjects. However, Mfn2 was unchanged after chronic exercise in individuals with diabetes, whereas porin and α-subunit of ATP synthase were increased. Acute exercise caused a fourfold increase in PGC-1α expression in muscle from control subjects but not in subjects with diabetes.CONCLUSIONSOur results demonstrate alterations in the regulatory pathway that controls PGC-1α expression and induction of Mfn2 in muscle from patients with early-onset type 2 diabetes. Patients with early-onset type 2 diabetes display abnormalities in the exercise-dependent pathway that regulates the expression of PGC-1α and Mfn2.