scholarly journals Is reduced ferredoxin the physiological electron donor for MetVF-type methylenetetrahydrofolate reductases in acetogenesis? A hypothesis

2021 ◽  
Author(s):  
Christian Öppinger ◽  
Florian Kremp ◽  
Volker Müller
Keyword(s):  
Electron Donor ◽  
Methanol Metabolism ◽  
Clostridium Ljungdahlii ◽  
Apparent Homogeneity ◽  
Key Enzyme ◽  
Clostridium Aceticum ◽  
Thermoanaerobacter Kivui ◽  
Acid Labile

AbstractThe methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.

scholarly journals USP29-mediated HIF1α stabilization is associated with Sorafenib resistance of hepatocellular carcinoma cells by upregulating glycolysis

Oncogenesis ◽  
2021 ◽  
Vol 10 (7) ◽  
Author(s):  
Ruize Gao ◽  
David Buechel ◽  
Ravi K. R. Kalathur ◽  
Marco F. Morini ◽  
Mairene Coto-Llerena ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Transcriptional Activity ◽  
Kinase Inhibitor ◽  
Aerobic Glycolysis ◽  
Hypoxia Inducible Factor ◽  
Aberrant Expression ◽  
Key Enzyme ◽  
Hcc Cells

AbstractUnderstanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients.

Characterization of the Metamitron Deaminating Enzyme Activity from Sugar Beet ( Beta vulgaris L.) Leaves

1979 ◽  
Vol 34 (11) ◽  
pp. 948-950 ◽  
Author(s):  
Carl Fedtke ◽  
Robert R. Schmidt
Keyword(s):  
Cytochrome C ◽  
Sugar Beet ◽  
Electron Donor ◽  
Nitrogen Atmosphere ◽  
Enzyme Preparations ◽  
Reducing Conditions ◽  
Trade Name ◽  
Membrane Bound

Abstract The enzymatic activity from sugar beet leaves which is responsible for the detoxification of the herbicide metamitron (4-amino-4,5-dihydro-3-methyl-6-phenyl-1, 2, 4-triazin-5-one, trade name Goltix®) has been characterized in vitro. The detoxification occurs by rapid deamination in vivo as well as in vitro. However, the deamination in vitro is only maximal under reducing conditions, i. e. with an electron donor and in a nitrogen atmosphere. The electron donor may be cystein, glutathione, dithionite or ascorbate. The enzymatic deamination further requires the addition of cytochrome c and a “supernatant factor”, which may be replaced by FMN, FAD or DCPIP. However, in the presence of FMN or DCPIP cytochrome c is not essential but only stimulatory. The partic­ulate as well as the soluble metamitron deaminating enzyme preparations obtained take up oxygen when supplied with cysteine and FMN. The particulate enzyme appears in the peroxysome-fraction. It is therefore suggested, that the enzymatic deamination of metamitron in sugar beet leaves is mediated by a proxisomal membrane bound electron transport system which alternatively may reduce oxygen or metamitron (deaminating).

scholarly journals Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16

2015 ◽  
Vol 82 (3) ◽  
pp. 910-921 ◽  
Author(s):  
Leonie Wenning ◽  
Nadine Stöveken ◽  
Jan Hendrik Wübbeler ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Metal Chelate ◽  
Sulfinic Acid ◽  
Cysteine Sulfinic Acid ◽  
Content Type ◽  
Apparent Homogeneity ◽  
Metal Cofactor ◽  
Metal Chelate Affinity Chromatography

ABSTRACTCysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. InRalstonia eutrophaH16, two Cdo homologues (CdoA and CdoB) have been identified previously.In vivostudies showed thatEscherichia colicells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing anin vitrocharacterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos fromR. eutrophaH16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3′-thiodipropionic acid (TDP) and 3,3′-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis inR. eutrophaH16, deletion ofcdoAmight enable increased synthesis of PTEs.

scholarly journals Branched-chain amino acid aminotransferase 2 regulates ferroptotic cell death in cancer cells

2020 ◽  
Author(s):  
Kang Wang ◽  
Zhengyang Zhang ◽  
Hsiang-i Tsai ◽  
Yanfang Liu ◽  
Jie Gao ◽  
...  
Keyword(s):  
Cell Death ◽  
Amino Acid ◽  
Cancer Cells ◽  
Ectopic Expression ◽  
Branched Chain Amino Acid ◽  
Pancreatic Cancer Cells ◽  
Key Enzyme ◽  
Branched Chain

Abstract Ferroptosis, a form of iron-dependent cell death driven by cellular metabolism and iron-dependent lipid peroxidation, has been implicated as a tumor-suppressor function for cancer therapy. Recent advance revealed that the sensitivity to ferroptosis is tightly linked to numerous biological processes, including metabolism of amino acid and the biosynthesis of glutathione. Here, by using a high-throughput CRISPR/Cas9-based genetic screen in HepG2 hepatocellular carcinoma cells to search for metabolic proteins inhibiting ferroptosis, we identified a branched-chain amino acid aminotransferase 2 (BCAT2) as a novel suppressor of ferroptosis. Mechanistically, ferroptosis inducers (erastin, sorafenib, and sulfasalazine) activated AMPK/SREBP1 signaling pathway through iron-dependent ferritinophagy, which in turn inhibited BCAT2 transcription. We further confirmed that BCAT2 as the key enzyme mediating the metabolism of sulfur amino acid, regulated intracellular glutamate level, whose activation by ectopic expression specifically antagonize system Xc– inhibition and protected liver and pancreatic cancer cells from ferroptosis in vitro and in vivo. On the contrary, direct inhibition of BCAT2 by RNA interference, or indirect inhibition by blocking system Xc– activity, triggers ferroptosis. Finally, our results demonstrate the synergistic effect of sorafenib and sulfasalazine in downregulating BCAT2 expression and dictating ferroptotic death, where BCAT2 can also be used to predict the responsiveness of cancer cells to ferroptosis-inducing therapies. Collectively, these findings identify a novel role of BCAT2 in ferroptosis, suggesting a potential therapeutic strategy for overcoming sorafenib resistance.

scholarly journals Heme Oxygenase-1, a Key Enzyme for the Cytoprotective Actions of Halophenols by Upregulating Nrf2 Expression via Activating Erk1/2 and PI3K/Akt in EA.hy926 Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Xiu E. Feng ◽  
Tai Gang Liang ◽  
Jie Gao ◽  
De Peng Kong ◽  
Rui Ge ◽  
...  
Keyword(s):  
Heme Oxygenase ◽  
Antioxidant Activities ◽  
Heme Oxygenase 1 ◽  
Protective Effects ◽  
Key Enzyme ◽  
Ea.Hy926 Cells ◽  
Anti Inflammatory ◽  
Mechanistic Basis

Increasing evidence has demonstrated that heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, exhibiting cytoprotective, antioxidant, and anti-inflammatory abilities. Previously, we prepared a series of novel active halophenols possessing strong antioxidant activities in vitro and in vivo. In the present study, we demonstrated that these halophenols exhibited significant protective effects against H2O2-induced injury in EA.hy926 cells by inhibition of apoptosis and ROS and TNF-αproduction, as well as induction of the upregulation of HO-1, the magnitude of which correlated with their cytoprotective actions. Further experiments which aimed to determine the mechanistic basis of these actions indicated that the halophenols induced the activation of Nrf2, Erk1/2, and PI3K/Akt without obvious effects on the phosphorylation of p38, JNK, or the expression of PKC-δ. This was validated with the use of PD98059 and Wortmannin, specific inhibitors of Erk1/2 and PI3K, respectively. Overall, our study is the first to demonstrate that the cytoprotective actions of halophenols involve their antiapoptotic, antioxidant, and anti-inflammatory abilities, which are mediated by the upregulation of Nrf2-dependent HO-1 expression and reductions in ROS and TNF-αgeneration via the activation of Erk1/2 and PI3K/Akt in EA.hy926 cells. HO-1 may thus be an important potential target for further research into the cytoprotective actions of halophenols.

scholarly journals Uranium Reduction by Desulfovibrio desulfuricans Strain G20 and a Cytochrome c3 Mutant

2002 ◽  
Vol 68 (6) ◽  
pp. 3129-3132 ◽  
Author(s):  
Rayford B. Payne ◽  
Darren M. Gentry ◽  
Barbara J. Rapp-Giles ◽  
Laurence Casalot ◽  
Judy D. Wall
Keyword(s):  
Electron Donor ◽  
Desulfovibrio Desulfuricans ◽  
Desulfovibrio Vulgaris ◽  
In Vitro Experiments ◽  
Wild Type ◽  
Cytochrome C3 ◽  
Uranium Reduction

ABSTRACT Previous in vitro experiments with Desulfovibrio vulgaris strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome c 3 could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome c 3 mutant of D. desulfuricans strain G20 was assayed and found to be able to reduce U(VI) with lactate or pyruvate as the electron donor at rates about one-half of those of the wild type. With electrons from hydrogen, the rate was more severely impaired. Cytochrome c 3 appears to be a part of the in vivo electron pathway to U(VI), but additional pathways from organic donors can apparently bypass this protein.

scholarly journals Isolation of a glycoprotein responsible for the enhanced concanavalin A agglutinability of erythrocytes in Yoshida-ascites-sarcoma-bearing rats: the mechanism of paraneoplastic syndromes

1993 ◽  
Vol 292 (1) ◽  
pp. 163-170 ◽  
Author(s):  
R D Kalraiya ◽  
A Sanjay ◽  
N G Mehta
Keyword(s):  
Molecular Mass ◽  
Concanavalin A ◽  
Paraneoplastic Syndromes ◽  
Host Cells ◽  
Ascites Fluid ◽  
Con A ◽  
Tumour Bearing ◽  
Apparent Homogeneity

As a model for the development of paraneoplastic syndromes, we have studied the mechanism by which erythrocytes in the circulation of rats bearing intraperitoneal Yoshida ascites sarcoma acquire higher agglutinability with concanavalin A (Con A). The in vitro incubation of erythrocytes from normal animals with the cell-free ascites fluid or the plasma of tumour-bearing animals is able to confer an enhanced agglutinability on the cells. Fractionation of the ascites fluid has yielded three subfractions that are active in vitro. Two of these, occurring in small amounts, are a particulate fraction rich in plasma-membrane markers and a soluble fraction containing protein of molecular mass equal to or less than 50 kDa. These two are, however, unable to affect the agglutinability of erythrocytes in vivo, i.e. when injected intraperitoneally into normal rats. The third, and major, fraction consists of proteins of molecular mass equal to or greater than 680 kDa, and is able to modify the erythrocyte agglutinability in vivo. From this fraction, by using a combination of Con A affinity chromatography, gel filtration, (NH4)2SO4 fractionation and DEAE-Sephadex chromatography, an active protein has been purified to apparent homogeneity. It yields a subunit of 310 kDa in the presence of SDS and further breaks down into a polypeptide of 170 kDa when reduced with 2-mercaptoethanol. It has a pI of 5.35. The protein is rich in Glx, and appears to contain hybrid-type N-linked oligosaccharides. The protein is also present in the blood plasma of tumour-bearing, but not normal, rats. The radioiodinated protein binds to the erythrocyte surface adding about 7400 molecules/cell. The study unequivocally demonstrates that a protein from the tumour fluid can appear in the circulation, interact with host cells that are not in contact with the tumour and modify their properties.

scholarly journals Carrier-Mediated Delivery of 9-(2-Phosphonylmethoxyethyl)Adenine to Parenchymal Liver Cells: a Novel Therapeutic Approach for Hepatitis B

2000 ◽  
Vol 44 (3) ◽  
pp. 477-483 ◽  
Author(s):  
Remco L. A. de Vrueh ◽  
Erik T. Rump ◽  
Erika van de Bilt ◽  
Richard van Veghel ◽  
Jan Balzarini ◽  
...  
Keyword(s):  
Hepatitis B ◽  
Lithocholic Acid ◽  
Hepatic Uptake ◽  
Liver Cells ◽  
Asialoglycoprotein Receptor ◽  
Parenchymal Liver ◽  
Lipophilic Prodrug ◽  
Acid Labile

ABSTRACT Our aim is to selectively deliver 9-(2-phosphonylmethoxyethyl)adenine (PMEA) to parenchymal liver cells, the primary site of hepatitis B virus (HBV) infection. Selective delivery is necessary because PMEA, which is effective against HBV in vitro, is hardly taken up by the liver in vivo. Lactosylated reconstituted high-density lipoprotein (LacNeoHDL), a lipid particle that is specifically internalized by parenchymal liver cells via the asialoglycoprotein receptor, was used as the carrier. PMEA could be incorporated into the lipid moiety of LacNeoHDL by attaching, via an acid-labile bond, lithocholic acid-3α-oleate to the drug. The uptake of the lipophilic prodrug (PMEA-LO) by the liver was substantially increased after incorporation into LacNeoHDL. Thirty minutes after injection of [3H]PMEA-LO-loaded LacNeoHDL into rats, the liver contained 68.9% ± 7.7% of the dose (free [3H]PMEA, <5%). Concomitantly, the uptake by the kidney was reduced to <2% of the dose (free [3H]PMEA, >45%). The hepatic uptake of PMEA-LO-loaded LacNeoHDL occurred mainly by parenchymal cells (88.5% ± 8.2% of the hepatic uptake). Moreover, asialofetuin inhibited the liver association by >75%, indicating uptake via the asialoglycoprotein receptor. The acid-labile linkage in PMEA-LO, designed to release PMEA during lysosomal processing of the prodrug-loaded carrier, was stable at physiological pH but was hydrolyzed at lysosomal pH (half-life, 60 to 70 min). Finally, subcellular fractionation indicates that the released PMEA is translocated to the cytosol, where it is converted into its active diphosphorylated metabolite. In conclusion, lipophilic modification and incorporation of PMEA into LacNeoHDL improves the biological fate of the drug and may lead to an enhanced therapeutic efficacy against chronic hepatitis B.

scholarly journals Deposition of Doxorubicin in Rats following Administration of Three Newly Synthesized Doxorubicin Conjugates

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Menglei Huan ◽  
Shuang Tian ◽  
Han Cui ◽  
Bangle Zhang ◽  
Dan Su ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Antitumor Activity ◽  
Tumor Targeting ◽  
Circulatory System ◽  
Systemic Toxicity ◽  
Chemical Structures ◽  
Low Levels ◽  
Acid Labile

We previously reported the synthesis of three DOX conjugates that represented different targeting vehicles and showed them to have antitumor activity bothin vitroandin vivo. However, the relationships between the pharmacokinetics of these DOX conjugates and their chemical structures were not characterized. In the current study, free DOX derived from each of the conjugates was found at low levels in the rat circulatory system, with conjugated DOX being the major form. The two polyethylene glycol (PEG) conjugates slowly released DOX, andt1/2βfor total DOX from DOX-LNA, PEG-ami-DOX, and PEG-hyd-DOX was 5.79, 10.22, and 15.18 h, respectively. All three conjugates also deposited less DOX into normal organs than did an equivalent dose of free DOX, and theCmaxvalue of free DOX released by DOX- LNA, PEG-ami-DOX, and PEG-hyd-DOX was 32.5, 9.5, and 4.7 μg/g, respectively. Among the conjugates, the compound with an acid-labile bond between PEG and DOX exhibited the lowest free DOX deposition in healthy tissues, which should decrease the systemic toxicity of free DOX while allowing for tumor targeting by PEG.

Sign in / Sign up

Export Citation Format

Share Document