Acetyl:Succinate CoA-transferase in ProcyclicTrypanosoma brucei

Acetyl:succinate CoA-transferase (ASCT) is an acetate-producing enzyme shared by hydrogenosomes, mitochondria of trypanosomatids, and anaerobically functioning mitochondria. The gene encoding ASCT in the protozoan parasiteTrypanosoma bruceiwas identified as a new member of the CoA transferase family. Its assignment to ASCT activity was confirmed by 1) a quantitative correlation of protein expression and activity upon RNA interference-mediated repression, 2) the absence of activity in homozygous Δasct/Δasctknock out cells, 3) mitochondrial colocalization of protein and activity, 4) increased activity and acetate excretion upon transgenic overexpression, and 5) depletion of ASCT activity from lysates upon immunoprecipitation. Genetic ablation of ASCT produced a severe growth phenotype, increased glucose consumption, and excretion of β-hydroxybutyrate and pyruvate, indicating accumulation of acetyl-CoA. Analysis of the excreted end products of13C-enriched and14C-labeled glucose metabolism showed that acetate excretion was only slightly reduced. Adaptation to ASCT deficiency, however, was an infrequent event at the population level, indicating the importance of this enzyme. These studies show that ASCT is indeed involved in acetate production, but is not essential, as apparently it is not the only enzyme that produces acetate inT. brucei.

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Acetate:Succinate CoA-transferase in the Hydrogenosomes of Trichomonas vaginalis

Journal of Biological Chemistry ◽

10.1074/jbc.m702528200 ◽

2007 ◽

Vol 283 (3) ◽

pp. 1411-1418 ◽

Cited By ~ 45

Author(s):

Koen W. A. van Grinsven ◽

Silke Rosnowsky ◽

Susanne W. H. van Weelden ◽

Simone Pütz ◽

Mark van der Giezen ◽

...

Keyword(s):

Trichomonas Vaginalis ◽

Enzymatic Reaction ◽

Protozoan Parasite ◽

Transferase Activity ◽

Acetate Production ◽

Acetyl Coa ◽

Gene Encoding ◽

Homologous Overexpression ◽

Coa Transferase ◽

Coa Hydrolase

Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.

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Na+/K+-ATPase α isoform deficiency results in distinct spreading depolarization phenotypes

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x19833757 ◽

2019 ◽

Vol 40 (3) ◽

pp. 622-638 ◽

Cited By ~ 5

Author(s):

Clemens Reiffurth ◽

Mesbah Alam ◽

Mahdi Zahedi-Khorasani ◽

Sebastian Major ◽

Jens P Dreier

Keyword(s):

Familial Hemiplegic Migraine ◽

Gene Encoding ◽

Experimental Conditions ◽

High Potassium ◽

Knock Out ◽

Genetic Ablation ◽

Spreading Depolarization ◽

Underlying Mechanisms ◽

Knock Out Mice

Compromised Na+/K+-ATPase function is associated with the occurrence of spreading depolarization (SD). Mutations in ATP1A2, the gene encoding the α2 isoform of the Na+/K+-ATPase, were identified in patients with familial hemiplegic migraine type 2 (FHM2), a Mendelian model disease for SD. This suggests a distinct role for the α2 isoform in modulating SD susceptibility and raises questions about underlying mechanisms including the roles of other Na+/K+-ATPase α isoforms. Here, we investigated the effects of genetic ablation and pharmacological inhibition of α1, α2, and α3 on SD using heterozygous knock-out mice. We found that only α2 heterozygous mice displayed higher SD susceptibility when challenged with prolonged extracellular high potassium concentration ([K+]o), a pronounced post SD oligemia and higher SD speed in-vivo. By contrast, under physiological [K+]o, α2 heterozygous mice showed similar SD susceptibility compared to wild-type littermates. Deficiency of α3 resulted in increased resistance against electrically induced SD in-vivo, whereas α1 deficiency did not affect SD. The results support important roles of the α2 isoform in SD. Moreover, they suggest that specific experimental conditions can be necessary to reveal an inherent SD phenotype by driving a (meta-) stable system into decompensation, reminiscent of the episodic nature of SDs in various diseases.

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Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering

Applied and Environmental Microbiology ◽

10.1128/aem.01628-18 ◽

2018 ◽

Vol 84 (23) ◽

Cited By ~ 2

Author(s):

Efstratios Nikolaivits ◽

Maria Dimarogona ◽

Ioanna Karagiannaki ◽

Angelina Chalima ◽

Ayelet Fishman ◽

...

Keyword(s):

Substrate Specificity ◽

Fruits And Vegetables ◽

Homology Model ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Gene Encoding ◽

Content Type ◽

Polyphenol Oxidases ◽

Increased Activity ◽

Work Knowledge

ABSTRACTPolyphenol oxidases (PPOs) have been mostly associated with the undesirable postharvest browning in fruits and vegetables and have implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical, and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome ofThermothelomyces thermophilaand expressed inPichia pastoris. The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/liter, and it appeared to be a glycosylated and N-terminally processed protein.TtPPO showed broad substrate specificity, as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols, and methoxyphenols. Its optimum temperature was 65°C, with a half-life of 18.3 h at 50°C, while its optimum pH was 7.5. The homology model ofTtPPO was constructed, and site-directed mutagenesis was performed in order to increase its activity on mono- and dichlorophenols (di-CPs). The G292N/Y296V variant ofTtPPO 5.3-fold increased activity on 3,5-dichlorophenol (3,5-diCP) compared to the wild type.IMPORTANCEA novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

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Quantification and Isolation ofBacillus subtilisSpores using Cell Sorting and automated Gating

10.1101/528257 ◽

2019 ◽

Author(s):

Marianna Karava ◽

Felix Bracharz ◽

Johannes Kabisch

Keyword(s):

Bacillus Subtilis ◽

Cell Sorting ◽

Fluorescent Dyes ◽

Model Organism ◽

Gaussian Mixture ◽

Spore Formation ◽

Gene Encoding ◽

Knock Out ◽

Optimal Separation ◽

Genome Modifications

AbstractThe Gram-positive bacteriumBacillus subtilisis able to form endospores which have a variety of biotechnological applications. Due to this ability,B. subtilisis as well a model organism for cellular differentiation processes. Sporulating cultures ofBacillus subtilisform sub-populations which include vegetative cells, spore forming cells and spores. In order to readily and rapidly quantify spore formation we employed flow cytometric and fluorescence activated cell sorting techniques in combination with nucleic acid fluorescent staining in order to investigate the distribution of sporulating cultures on a single cell level. Moreover we tested different fluorescent dyes as well as different conditions in order to develop a method for optimal separation of distinct populations during sporulation. Automated gating procedures using k-means clustering and thresholding by gaussian mixture modeling were employed to avoid subjective gating and allow for the simultaneous measurement of controls. We utilized the presented method for monitoring sporulation over time in strains harboring different genome modifications. We identified the different subpopulations formed during sporulation by employing sorting and microscopy. Finally, we employed the technique to show that a double knock-out mutant of the phosphatase gene encoding Spo0E and of the spore killing factor SkfA results in faster spore formation.

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Abstract 13469: Drp1 Accumulates in Mitochondria and Plays a Protective Role in the Heart in Response to Pressure Overload

Circulation ◽

10.1161/circ.130.suppl_2.13469 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Akihiro Shirakabe ◽

Yoshiyuki Ikeda ◽

Peiyong Zai ◽

Junichi Sadoshima

Keyword(s):

Diastolic Pressure ◽

Pressure Overload ◽

Protective Role ◽

Left Ventricular ◽

Multiple Time ◽

Lung Weight ◽

Knock Out ◽

Multiple Time Points ◽

Increased Activity ◽

Adaptive Role

Dynamin-related protein 1 (Drp1) plays an essential role in maintaining the quality control of mitochondria through mitochondrial (Mt) fission and mitophagy. We investigated how Mt function, autophagy and Drp1 are regulated in the heart during pressure overload (PO) and whether endogenous Drp1 plays an important role in regulating cardiac function. Mice were subjected to transverse aortic constriction (TAC) at multiple time points between 6 hours and 30 days. Left ventricular (LV) weight/tibial length (LVW/TL) was significantly elevated at Day 7 (TAC vs sham; 5.92 ± 0.27 vs 4.22 ± 0.12, p<0.05). Ejection fraction (EF) was maintained at Day 7, but gradually decreased thereafter (at 30 days; 65±9 vs 83±9 %, p<0.05). LC3-II was decreased (-45.7%, p<0.05) while p62 accumulated (1.17 fold, p<0.05) significantly at Day 7. Both Mt ATP content (-65.6%, p<0.05) and production (-90.3%, p<0.05) were reduced significantly at Days 7 and 14, respectively, and thereafter. Mt mass, evaluated by electron microscopy, was also reduced (-19.9%, p<0.05) at Day 7. Drp1 accumulated in mitochondria at Day 7, and S616 phosphorylation of Drp1, associated with increased activity, was increased at Day 7. Thus, PO suppresses autophagy and induces Mt dysfunction by Day 7, at which time Drp1 accumulates in mitochondria and Mt mass is decreased. To examine the functional significance of endogenous Drp1 during PO, cardiac-specific heterozygous Drp1 knock out (Drp-hetCKO) mice were subjected to TAC. At Day 7, decreases in EF (61± 2 vs 84 ± 7%, p<0.05) and increases in LVW/TL (7.22 ± 0.26 vs 5.86 ± 0.65, p<0.05) and lung weight/TL (12.01 ± 1.10 vs 6.31 ± 1.19, p<0.05) were exacerbated in Drp-hetCKO compared to in control mice. LV end diastolic pressure was significantly higher (22.0 ± 2.8 vs 5.7 ± 2.9 mmHg, p<0.05) and myocardial fibrosis (14.1 ± 2.5 vs 6.2 ± 4.3 %, p<0.05) was greater in Drp-hetCKO than in control mice. Mt mass was also significantly greater in Drp-hetCKO than in control mice (relative Mt mass, 1.21 ± 0.46 vs 1.00 ± 0.02, p<0.05). These results suggest that PO inhibits autophagy and induces mitochondrial dysfunction by Day7, which coincides with Mt accumulation of Drp1. Drp1 plays an adaptive role in this condition, mediating decreases in Mt mass and protecting the heart from dysfunction.

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Abstract 304: Global Transcriptional Effects of Vitamin A Deficiency on the Postnatal Heart

Circulation Research ◽

10.1161/res.111.suppl_1.a304 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Cristi L Galindo ◽

Truc-Linh Tran ◽

Xuyang Peng ◽

Douglas B Sawyer ◽

Mary Asson-Batres

Keyword(s):

Vitamin A ◽

Natriuretic Peptide ◽

Heart Development ◽

Ventricular Remodeling ◽

Vitamin A Deficiency ◽

Healing Process ◽

Mutant Mice ◽

Wound Healing Process ◽

Gene Encoding ◽

Knock Out

Vitamin A (VA) is the chemical precursor of retinoic acid (RA), which is critical for embryonic development and for growth, immunity, metabolism, and cell differentiation in postnatal regenerating systems such as skin, sensory organs, and stem cell niches in the brain. VA is also essential for embryonic heart development, and we hypothesized that Vitamin A might exert an effect on the postnatal heart similar to what is observed for other tissues. Here, we report the global transcriptional profiles of wild-type (WT) mice fed a VA sufficient diet (VAS) compared with retinyl acyl transferase (LRAT) knock-out mice fed either a VAS or VA deficient (VAD) diet. Knockout of the LRAT gene alone was sufficient to induce differential expression of 576 genes relative to WT. Feeding LRAT mutant mice a VAD diet resulted in a change in the relative expression levels of 257 genes relative to LRAT mutant mice fed a VAS diet. As expected, we observed transcriptional alterations related to Vitamin A metabolism, including an increase in the gene encoding cellular retinoid binding protein 7 and down-regulation of the retinol metabolic enzymes Cy1a2 and Cyp2a4. Importantly, several cardiac genes not previously known to require VA were perturbed, including the gene encoding B-type natriuretic peptide, which was down-regulated in mutant mice irrespective of diet, and A-type natriuretic peptide, which was decreased only in mice fed the VAD diet. There was also a striking effect of VAD on genes important for immune responses, which could have an impact on the wound healing process subsequent to injury of the heart. This is consistent with recent evidence that showed that Vitamin A deficiency influences post-infarct ventricular remodeling in rats. In summary, this is the first microarray study of Vitamin A deficiency in the postnatal heart, which suggests mechanisms by which Vitamin A depletion may alter myocardial maintenance and repair after injury.

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Restraint Stress in Mice Alters Set of 25 miRNAs Which Regulate Stress- and Depression-Related mRNAs

International Journal of Molecular Sciences ◽

10.3390/ijms21249469 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9469

Author(s):

Joanna Solich ◽

Maciej Kuśmider ◽

Agata Faron-Górecka ◽

Paulina Pabian ◽

Marta Dziedzicka-Wasylewska

Keyword(s):

Machine Learning ◽

Restraint Stress ◽

Molecular Level ◽

Norepinephrine Transporter ◽

Mouse Serum ◽

Control Group ◽

Gene Encoding ◽

Knock Out ◽

Differentially Expressed Mirnas ◽

Mirnas Expression

In the present study, we aim to identify the effect of restrain stress (RS) on the expression of miRNAs in mouse serum. We used three genotypes of animals (mice with knock-out of the gene-encoding norepinephrine transporter, NET-KO; C57BL/6J, and SWR/J) which had previously been shown to display different sensitivity to RS, and focused on miRNAs which were altered by RS in the serum of all three genotypes. An analysis of miRNAs expression allowed for the identification of a set of 25 differentially expressed miRNAs; 10 were down-regulated compared to an appropriate control group of animals, while 15 were up-regulated. The application of DIANA-miRPath v. 3.0 allowed for the identification of selected pathways (KEGG) and Gene Ontology (GO) categories that were significantly controlled by these miRNAs, while miRWalk v. 3.0—the platform that used the machine learning based algorithm, TaRPmiR—was used to find their targets. The results indicate that 25 miRNAs, identified as altered upon RS in three genotypes of mice, are responsible for regulation of mRNA-encoding proteins that are key for the main hypotheses of depression; therefore, they may help to understand the link between stress and depression at the molecular level.

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Ribokinase fromLeishmania donovani: purification, characterization and X-ray crystallographic analysis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18000109 ◽

2018 ◽

Vol 74 (2) ◽

pp. 99-104 ◽

Cited By ~ 1

Author(s):

Santhosh Gatreddi ◽

Sayanna Are ◽

Insaf Ahmed Qureshi

Keyword(s):

Functional Characterization ◽

Three Dimensional ◽

Protozoan Parasite ◽

Crystallographic Analysis ◽

Size Exclusion ◽

Dimensional Structure ◽

Diffusion Method ◽

Gene Encoding ◽

Cell Parameters ◽

Α Helix

Leishmaniais an auxotrophic protozoan parasite which acquires D-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating D-ribose using ATP to produce D-ribose-5-phosphate. To understand its structure and function, the gene encoding RK fromL. donovaniwas cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α-helix in the secondary-structure content, and thermal unfolding revealed theTmto be 317.2 K. Kinetic parameters were obtained by functional characterization ofL. donovaniRK, and theKmvalues for ribose and ATP were found to be 296 ± 36 and 116 ± 9.0 µM, respectively. Crystals obtained by the hanging-drop vapour-diffusion method diffracted to 1.95 Å resolution and belonged to the hexagonal space groupP61, with unit-cell parametersa=b= 100.25,c= 126.77 Å. Analysis of the crystal content indicated the presence of two protomers in the asymmetric unit, with a Matthews coefficient (VM) of 2.45 Å3 Da−1and 49.8% solvent content. Further study revealed that human counterpart of this protein could be used as a template to determine the first three-dimensional structure of the RK from trypanosomatid parasites.

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Genome-scale CRISPR screens are efficient in non-homologous end-joining deficient cells

Scientific Reports ◽

10.1038/s41598-019-52078-9 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Joana Ferreira da Silva ◽

Sejla Salic ◽

Marc Wiedner ◽

Paul Datlinger ◽

Patrick Essletzbichler ◽

...

Keyword(s):

Genome Editing ◽

Large Scale ◽

Dependent Manner ◽

Dna Double Strand Breaks ◽

End Joining ◽

Knock Out ◽

Genetic Ablation ◽

Alternative End Joining ◽

Non Homologous End Joining ◽

Genome Scale

Abstract The mutagenic repair of Cas9 generated breaks is thought to predominantly rely on non-homologous end-joining (NHEJ), leading to insertions and deletions within DNA that culminate in gene knock-out (KO). In this study, by taking focused as well as genome-wide approaches, we show that this pathway is dispensable for the repair of such lesions. Genetic ablation of NHEJ is fully compensated for by alternative end joining (alt-EJ), in a POLQ-dependent manner, resulting in a distinct repair signature with larger deletions that may be exploited for large-scale genome editing. Moreover, we show that cells deficient for both NHEJ and alt-EJ were still able to repair CRISPR-mediated DNA double-strand breaks, highlighting how little is yet known about the mechanisms of CRISPR-based genome editing.

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Thrombasthenic mice generated by replacement of the integrin αIIb gene: demonstration that transcriptional activation of this megakaryocytic locus precedes lineage commitment

Blood ◽

10.1182/blood.v96.4.1399 ◽

2000 ◽

Vol 96 (4) ◽

pp. 1399-1408 ◽

Cited By ~ 66

Author(s):

Diana Tronik-Le Roux ◽

Valérie Roullot ◽

Christel Poujol ◽

Thierry Kortulewski ◽

Paquita Nurden ◽

...

Keyword(s):

Transcriptional Activation ◽

Fibrin Clot ◽

Lineage Commitment ◽

Gene Encoding ◽

Α Subunit ◽

Knock Out ◽

Toxic Gene ◽

Specific Locus ◽

Deficient Mice

Abstract To analyze the transcriptional activity of the gene encoding the α subunit of the platelet integrin αIIbβ3during the hematopoietic differentiation, mice were produced in which the herpes virus thymidine kinase (tk) was introduced in this megakaryocytic specific locus using homologous recombination technology. This provided a convenient manner in which to induce the eradication of particular hematopoietic cells expressing the targeted gene. Results of progenitor cell cultures and long-term bone marrow (BM) assays showed that the growth of a subset of stem cells was reduced in the presence of the antiherpetic drug ganciclovir, demonstrating that the activation of the toxic gene occurs before the commitment to the megakaryocytic lineage. Furthermore theknock-in of the tk gene into the αIIb locus resulted in the knock-out of the αIIb gene in homozygous mice. Cultures of BM cells of these animals, combined with ultrastructural analysis, established that the αIIbglycoprotein is dispensable for lineage commitment and megakaryocytic maturation. Platelets collected from αIIb-deficient mice failed to bind fibrinogen, to aggregate, and to retract a fibrin clot. Moreover, platelet α-granules did not contain fibrinogen. Consistent with these characteristics, the mice displayed bleeding disorders similar to those in humans with Glanzmann thrombasthenia.

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