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Vieira CSP, Segundo MA, Araújo AN. Cytochrome P450 electrochemical biosensors transforming in vitro metabolism testing - Opportunities and challenges. Bioelectrochemistry 2025; 163:108913. [PMID: 39854934 DOI: 10.1016/j.bioelechem.2025.108913] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2024] [Revised: 01/06/2025] [Accepted: 01/18/2025] [Indexed: 01/27/2025]
Abstract
The ability of the living world to flourish in the face of constant exposure to dangerous chemicals depends on the management ability of a widespread group of enzymes known as heme-thiolate monooxygenases or cytochrome P450 superfamily. About three-quarters of all reactions determining the metabolism of endogenous compounds, of those carried in foods, of taken drugs, or even of synthetic chemicals discarded into the environment depend on their catalytic performance. The chromatographic and (photo)luminometric methods routinely used as predictive and analytical tools in laboratories have significant drawbacks ranging from limited shelf-life of reagents, use of synthetic substrates, laborious and tedious procedures for highly sensitive detection. In this review, alternative electrochemical biosensors using the cytochrome P450 enzymes as bio-element are emphasized in their main aspects as well regarding their implementation and usefulness. Despite the various schemes proposed for the implementation, reports on real applications are scant for several reasons, including low reaction rates, broad substrate specificity, uncoupling reactions occurrence, and the need for expensive electron transfer partners to promote electron transfer. Finally, the prospect for future developments is introduced, focusing on integrating miniaturized systems with electrochemical techniques, alongside optimizing enzyme immobilization methods and electrode modifications to improve enzymatic stability and enhance sensor reliability. This progress represents a crucial step towards the creation of portable biosensors that mimic human physiological responses, supporting the precision medicine approach.
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Affiliation(s)
- Carina S P Vieira
- LAQV-REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal
| | - Marcela A Segundo
- LAQV-REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal
| | - Alberto N Araújo
- LAQV-REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal.
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2
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Harlington AC, Das T, Shearwin KE, Bell SG, Whelan F. Structural insights into S-lignin O-demethylation via a rare class of heme peroxygenase enzymes. Nat Commun 2025; 16:1815. [PMID: 39979323 PMCID: PMC11842817 DOI: 10.1038/s41467-025-57129-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2024] [Accepted: 02/05/2025] [Indexed: 02/22/2025] Open
Abstract
The O-demethylation of lignin aromatics is a rate-limiting step in their bioconversion to higher-value compounds. A recently discovered cytochrome P450, SyoA, demethylates the S-lignin aromatic syringol. In this work, we solve high-resolution X-ray crystal structures of substrate-free and substrate-bound SyoA and evaluate demethylation of para-substituted S-lignin aromatics via monooxygenase and peroxide shunt pathways. We find that SyoA demethylates S-lignin aromatics exclusively using the peroxide shunt pathway. The atomic-resolution structures reveal the position of non-canonical residues in the I-helix (Gln252, Glu253). Mutagenesis of this amide-acid pair in SyoA shows they are critical for catalytic activity. This work expands the enzymatic toolkit for improving the capacity to funnel lignin derived aromatics towards higher value compounds and defines the chemistry within the active site of the enzyme that enables peroxygenase activity. These insights provide a framework for engineering peroxygenase activity in other heme enzymes to generate easier to use biocatalysts.
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Affiliation(s)
- Alix C Harlington
- Department of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia
| | - Tuhin Das
- Department of Chemistry, University of Adelaide, Adelaide, SA, Australia
| | - Keith E Shearwin
- Department of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia
| | - Stephen G Bell
- Department of Chemistry, University of Adelaide, Adelaide, SA, Australia.
| | - Fiona Whelan
- Department of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia.
- Adelaide Microscopy, University of Adelaide, Adelaide, SA, Australia.
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3
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Üremiş N, Üremiş MM. Oxidative/Nitrosative Stress, Apoptosis, and Redox Signaling: Key Players in Neurodegenerative Diseases. J Biochem Mol Toxicol 2025; 39:e70133. [PMID: 39799559 PMCID: PMC11725306 DOI: 10.1002/jbt.70133] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2024] [Revised: 12/12/2024] [Accepted: 12/29/2024] [Indexed: 01/15/2025]
Abstract
Neurodegenerative diseases are significant health concerns that have a profound impact on the quality and duration of life for millions of individuals. These diseases are characterized by pathological changes in various brain regions, specific genetic mutations associated with the disease, deposits of abnormal proteins, and the degeneration of neurological cells. As neurodegenerative disorders vary in their epidemiological characteristics and vulnerability of neurons, treatment of these diseases is usually aimed at slowing disease progression. The heterogeneity of genetic and environmental factors involved in the process of neurodegeneration makes current treatment methods inadequate. However, the existence of common molecular mechanisms in the pathogenesis of these diseases may allow the development of new targeted therapeutic strategies. Oxidative and nitrosative stress damages membrane components by accumulating ROS and RNS and disrupting redox balance. This process results in the induction of apoptosis, which is important in the pathogenesis of neurodegenerative diseases through oxidative stress. Studies conducted using postmortem human samples, animal models, and cell cultures have demonstrated that oxidative stress, nitrosative stress, and apoptosis are crucial factors in the development of diseases such as Alzheimer's, Parkinson's, Multiple Sclerosis, amyotrophic lateral sclerosis, and Huntington's disease. The excessive production of reactive oxygen and nitrogen species, elevated levels of free radicals, heightened mitochondrial stress, disturbances in energy metabolism, and the oxidation and nitrosylation of cellular macromolecules are recognized as triggers for neuronal cell death. Challenges in managing and treating neurodegenerative diseases require a better understanding of this field at the molecular level. Therefore, this review elaborates on the molecular mechanisms by which oxidative and nitrosative stress are involved in neuronal apoptosis.
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Affiliation(s)
- Nuray Üremiş
- Department of Medical BiochemistryFaculty of Medicine, Kahramanmaraş Sütçü İmam UniversityKahramanmaraşTurkey
| | - Muhammed Mehdi Üremiş
- Department of Medical BiochemistryFaculty of Medicine, Kahramanmaraş Sütçü İmam UniversityKahramanmaraşTurkey
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4
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Medina D, Omanakuttan B, Nguyen R, Alwarsh E, Walgama C. Electrochemical Probing of Human Liver Subcellular S9 Fractions for Drug Metabolite Synthesis. Metabolites 2024; 14:429. [PMID: 39195525 DOI: 10.3390/metabo14080429] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2024] [Revised: 07/24/2024] [Accepted: 07/30/2024] [Indexed: 08/29/2024] Open
Abstract
Human liver subcellular fractions, including liver microsomes (HLM), liver cytosol fractions, and S9 fractions, are extensively utilized in in vitro assays to predict liver metabolism. The S9 fractions are supernatants of human liver homogenates that contain both microsomes and cytosol, which include most cytochrome P450 (CYP) enzymes and soluble phase II enzymes such as glucuronosyltransferases and sulfotransferases. This study reports on the direct electrochemistry and biocatalytic features of redox-active enzymes in S9 fractions for the first time. We investigated the electrochemical properties of S9 films by immobilizing them onto a high-purity graphite (HPG) electrode and performing cyclic voltammetry under anaerobic (Ar-saturated) and aerobic (O2-saturated) conditions. The heterogeneous electron transfer rate between the S9 film and the HPG electrode was found to be 14 ± 3 s-1, with a formal potential of -0.451 V vs. Ag/AgCl reference electrode, which confirmed the electrochemical activation of the FAD/FMN cofactor containing CYP450-reductase (CPR) as the electron receiver from the electrode. The S9 films have also demonstrated catalytic oxygen reduction under aerobic conditions, identical to HLM films attached to similar electrodes. Additionally, we investigated CYP activity in the S9 biofilm for phase I metabolism using diclofenac hydroxylation as a probe reaction and identified metabolic products using liquid chromatography-mass spectrometry (LC-MS). Investigating the feasibility of utilizing liver S9 fractions in such electrochemical assays offers significant advantages for pharmacological and toxicological evaluations of new drugs in development while providing valuable insights for the development of efficient biosensor and bioreactor platforms.
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Affiliation(s)
- Daphne Medina
- Department of Physical & Applied Sciences, University of Houston-Clear Lake, 2700 Bay Area Boulevard, Houston, TX 77058, USA
| | - Bhavana Omanakuttan
- Department of Physical & Applied Sciences, University of Houston-Clear Lake, 2700 Bay Area Boulevard, Houston, TX 77058, USA
| | - Ricky Nguyen
- Department of Physical & Applied Sciences, University of Houston-Clear Lake, 2700 Bay Area Boulevard, Houston, TX 77058, USA
| | - Eman Alwarsh
- Department of Physical & Applied Sciences, University of Houston-Clear Lake, 2700 Bay Area Boulevard, Houston, TX 77058, USA
| | - Charuksha Walgama
- Department of Physical & Applied Sciences, University of Houston-Clear Lake, 2700 Bay Area Boulevard, Houston, TX 77058, USA
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5
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Font-Farre M, Brown D, Toth R, Mahadevan C, Brazier-Hicks M, Morimoto K, Kaschani F, Sinclair J, Dale R, Hall S, Morris M, Kaiser M, Wright AT, Burton J, van der Hoorn RAL. Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta. Sci Rep 2024; 14:10091. [PMID: 38698065 PMCID: PMC11066006 DOI: 10.1038/s41598-024-60333-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2023] [Accepted: 04/22/2024] [Indexed: 05/05/2024] Open
Abstract
Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.
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Affiliation(s)
- Maria Font-Farre
- The Plant Chemetics Laboratory, Department of Biology, University of Oxford, Oxford, UK
| | - Daniel Brown
- Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UK
| | - Reka Toth
- Department of Biology, University of Oxford, Oxford, UK
| | | | | | - Kyoko Morimoto
- The Plant Chemetics Laboratory, Department of Biology, University of Oxford, Oxford, UK
| | - Farnusch Kaschani
- ZMB Chemical Biology, Faculty of Biology, University of Duisburg-Essen, Essen, Germany
| | - John Sinclair
- Bioscience, Syngenta, Jealotts Hill International Research Centre, Bracknell, UK
| | - Richard Dale
- Bioscience, Syngenta, Jealotts Hill International Research Centre, Bracknell, UK
| | - Samantha Hall
- Bioscience, Syngenta, Jealotts Hill International Research Centre, Bracknell, UK
| | - Melloney Morris
- Bioscience, Syngenta, Jealotts Hill International Research Centre, Bracknell, UK
| | - Markus Kaiser
- ZMB Chemical Biology, Faculty of Biology, University of Duisburg-Essen, Essen, Germany
| | | | - Jonathan Burton
- Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UK
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6
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Giuriato D, Catucci G, Correddu D, Nardo GD, Gilardi G. CYP116B5-SOX: An artificial peroxygenase for drug metabolites production and bioremediation. Biotechnol J 2024; 19:e2300664. [PMID: 38719620 DOI: 10.1002/biot.202300664] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2023] [Revised: 04/02/2024] [Accepted: 04/05/2024] [Indexed: 06/06/2024]
Abstract
CYP116B5 is a class VII P450 in which the heme domain is linked to a FMN and 2Fe2S-binding reductase. Our laboratory has proved that the CYP116B5 heme domain (CYP116B5-hd) is capable of catalyzing the oxidation of substrates using H2O2. Recently, the Molecular Lego approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides H2O2 in-situ by the sarcosine oxidation. In this work, the chimeric self-sufficient fusion enzyme CYP116B5-SOX was heterologously expressed, purified, and characterized for its functionality by absorbance and fluorescence spectroscopy. Differential scanning calorimetry (DSC) experiments revealed a TM of 48.4 ± 0.04 and 58.3 ± 0.02°C and a enthalpy value of 175,500 ± 1850 and 120,500 ± 1350 cal mol-1 for the CYP116B5 and SOX domains respectively. The fusion enzyme showed an outstanding chemical stability in presence of up to 200 mM sarcosine or 5 mM H2O2 (4.4 ± 0.8 and 11.0 ± 2.6% heme leakage respectively). Thanks to the in-situ H2O2 generation, an improved kcat/KM for the p-nitrophenol conversion was observed (kcat of 20.1 ± 0.6 min-1 and KM of 0.23 ± 0.03 mM), corresponding to 4 times the kcat/KM of the CYP116B5-hd. The aim of this work is the development of an engineered biocatalyst to be exploited in bioremediation. In order to tackle this challenge, an E. coli strain expressing CYP116B5-SOX was employed to exploit this biocatalyst for the oxidation of the wastewater contaminating-drug tamoxifen. Data show a 12-fold increase in tamoxifen N-oxide production-herein detected for the first time as CYP116B5 metabolite-compared to the direct H2O2 supply, equal to the 25% of the total drug conversion.
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Affiliation(s)
- Daniele Giuriato
- Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy
| | - Gianluca Catucci
- Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy
| | - Danilo Correddu
- Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy
| | - Giovanna Di Nardo
- Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy
| | - Gianfranco Gilardi
- Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy
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7
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Du J, Yin H, Li J, Zhang W, Ding G, Zhou D, Sun Y, Shen B. Transcription factor B-H2 regulates CYP9J34 expression conveying deltamethrin resistance in Culex pipiens pallens. PEST MANAGEMENT SCIENCE 2024; 80:1991-2000. [PMID: 38092527 DOI: 10.1002/ps.7934] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/17/2023] [Revised: 12/08/2023] [Accepted: 12/14/2023] [Indexed: 01/19/2024]
Abstract
BACKGROUND Mosquitoes are vectors of various diseases, posing significant health threats worldwide. Chemical pesticides, particularly pyrethroids like deltamethrin, are commonly used for mosquito control, but the emergence of resistant mosquito populations has become a concern. In the deltamethrin-resistant (DR) strain of Culex pipiens pallens, the highly expressed cytochrome P450 9 J34 (CYP9J34) gene is believed to play a role in resistance, yet the underlying mechanism remains unclear. RESULTS Quantitative polymerase chain reaction with reverse transcription (qRT-PCR) analysis revealed that the expression of CYP9J34 was 14.6-fold higher in DR strains than in deltamethrin-susceptible (DS) strains. The recombinant production of CYP9J34 protein of Cx. pipiens pallens showed that the protein could directly metabolize deltamethrin, yielding the major metabolite 4'-OH deltamethrin. Through dual luciferase reporter assays and RNA interference, the transcription factor homeobox protein B-H2-like (B-H2) was identified to modulate the expression of the CYP9J34 gene, contributing to mosquito resistance to deltamethrin. CONCLUSIONS Our findings demonstrate that the CYP9J34 protein could directly degrade deltamethrin, and the transcription factor B-H2 could regulate CYP9J34 expression, influencing the resistance of mosquitoes to deltamethrin. © 2023 Society of Chemical Industry.
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Affiliation(s)
- Jiajia Du
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, China
| | - Haitao Yin
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, China
| | - Jinze Li
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, China
| | - Wenxing Zhang
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, China
| | - Guangshuo Ding
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, China
| | - Dan Zhou
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, China
| | - Yan Sun
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, China
| | - Bo Shen
- Department of Pathogen Biology, Nanjing Medical University, Nanjing, China
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8
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Wang H, Fan Q, Liang Q, Wu Y, Ye Z, Wu H, Sun Q, Tang H, Liu Y, Liu Q, Chen Y. Human CYP1A1-activated aneugenicity of aflatoxin B1 in mammalian cells and its combined effect with benzo(a)pyrene. Chem Biol Interact 2024; 392:110923. [PMID: 38382706 DOI: 10.1016/j.cbi.2024.110923] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2023] [Revised: 01/31/2024] [Accepted: 02/18/2024] [Indexed: 02/23/2024]
Abstract
Aflatoxin B1 (AFB1) is the most toxic mycotoxin and a proven human carcinogen that requires metabolic activation, known by cytochrome P450 (CYP) 1A2 and 3A4. Previous evidence showed that AFB1 is activated by human recombinant CYP1A1 expressed in budding yeast. Yet, the toxicity, in particular the genotoxicity of the reactive metabolites formed from AFB1 remains unclear. Humans could be exposed to both AFB1 and benzo(a)pyrene (BaP) simultaneously, thus we were interested in their combined genotoxic effects subsequent to metabolic activation by CYP1A1. In this study, molecular docking of AFB1 to human CYP1A1 indicated that AFB1 is valid as a substrate. In the incubations with AFB1 in human CYP1A1-expressed microsomes, AFM1 as a marking metabolite of AFB1 was detected. Moreover, AFB1 induced micronucleus formation in a Chinese hamster V79-derived cell line and in a human lung epithelial BEAS-2B cell line, both expressing recombinant human CYP1A1, V79-hCYP1A1 and 2B-hCYP1A1 cells, respectively. Immunofluorescence of centromere protein B stained micronuclei was dominant in AFB1-treated BEAS-2B cells exposed to AFB1, suggesting an aneugenic effect. Moreover, AFB1 elevated the levels of ROS, 8-OHdG, AFB1-DNA adduct, and DNA breaks in 2B-hCYP1A1 cells, compared with those in the parental BEAS-2B cells. Meanwhile, AFB1 increased CYP1A1, RAD51, and γ-H2AX protein levels in 2B-hCYP1A1 cells, which were attenuated by the CYP1A1 inhibitor bergamottin. Co-exposure of AFB1 with BaP increased 8-OHdG, RAD51, and γ-H2AX levels (indicating DNA damage). In conclusion, AFB1 could be activated by human CYP1A1 for potent aneugenicity, which may be further enhanced by co-exposure to BaP.
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Affiliation(s)
- Huanhuan Wang
- Dongguan Key Laboratory of Environmental Medicine, The First Dongguan Affiliated Hospital, School of Public Health, Guangdong Medical University, Dongguan, 523808, China
| | - Qin Fan
- Dongguan Key Laboratory of Environmental Medicine, The First Dongguan Affiliated Hospital, School of Public Health, Guangdong Medical University, Dongguan, 523808, China
| | - Qian Liang
- Dongguan Key Laboratory of Environmental Medicine, The First Dongguan Affiliated Hospital, School of Public Health, Guangdong Medical University, Dongguan, 523808, China
| | - Yao Wu
- Dongguan Key Laboratory of Environmental Medicine, The First Dongguan Affiliated Hospital, School of Public Health, Guangdong Medical University, Dongguan, 523808, China
| | - Zhongming Ye
- Dongguan Key Laboratory of Environmental Medicine, The First Dongguan Affiliated Hospital, School of Public Health, Guangdong Medical University, Dongguan, 523808, China
| | - Haipeng Wu
- Dongguan Key Laboratory of Environmental Medicine, The First Dongguan Affiliated Hospital, School of Public Health, Guangdong Medical University, Dongguan, 523808, China
| | - Qian Sun
- Dongguan Key Laboratory of Environmental Medicine, The First Dongguan Affiliated Hospital, School of Public Health, Guangdong Medical University, Dongguan, 523808, China
| | - Huanwen Tang
- Dongguan Key Laboratory of Environmental Medicine, The First Dongguan Affiliated Hospital, School of Public Health, Guangdong Medical University, Dongguan, 523808, China
| | - Yungang Liu
- Department of Toxicology, School of Public Health (Guangdong Provincial Key Laboratory of Tropical Disease Research), Southern Medical University, Guangzhou, 510515, China
| | - Qizhan Liu
- Dongguan Key Laboratory of Environmental Medicine, The First Dongguan Affiliated Hospital, School of Public Health, Guangdong Medical University, Dongguan, 523808, China; Center for Global Health, The Key Laboratory of Modern Toxicology, Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, 211166, Jiangsu, China.
| | - Yuting Chen
- Dongguan Key Laboratory of Environmental Medicine, The First Dongguan Affiliated Hospital, School of Public Health, Guangdong Medical University, Dongguan, 523808, China.
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9
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Knauer JF, Schulz C, Zemella A, Wüstenhagen DA, Walter RM, Küpper JH, Kubick S. Synthesis of mono Cytochrome P450 in a modified CHO-CPR cell-free protein production platform. Sci Rep 2024; 14:1271. [PMID: 38218994 PMCID: PMC10787779 DOI: 10.1038/s41598-024-51781-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2023] [Accepted: 01/09/2024] [Indexed: 01/15/2024] Open
Abstract
Cytochromes P450 (CYPs) are a group of monooxygenases that can be found in almost all kinds of organisms. For CYPs to receive electrons from co-substrate NADPH, the activity of NADPH-Cytochrome-P450-oxidoreductase (CPR) is required as well. In humans, CYPs are an integral part of liver-based phase-1 biotransformation, which is essential for the metabolization of multiple xenobiotics and drugs. Consequently, CYPs are important players during drug development and therefore these enzymes are implemented in diverse screening applications. For these applications it is usually advantageous to use mono CYP microsomes containing only the CYP of interest. The generation of mono-CYP containing mammalian cells and vesicles is difficult since endogenous CYPs are present in many cell types that contain the necessary co-factors. By obtaining translationally active lysates from a modified CHO-CPR cell line, it is now possible to generate mono CYPs in a cell-free protein synthesis process in a straightforward manner. As a proof of principle, the synthesis of active human CYPs from three different CYP450 gene families (CYP1A2, CYP2B6 and CYP3A4), which are of outstanding interest in industry and academia was demonstrated. Luciferase based activity assays confirm the activity of the produced CYPs and enable the individual adaptation of the synthesis process for efficient cell-free enzyme production. Furthermore, they allow for substrate and inhibitor screenings not only for wild-type CYPs but also for mutants and further CYP isoforms and variants. As an example, the turnover of selected CYP substrates by cell-free synthesized CYPs was demonstrated via an indirect luciferase assay-based screening setup.
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Affiliation(s)
- Jan Felix Knauer
- Fraunhofer Project Group PZ-Syn of the Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany
- Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany
- Freie Universität Berlin, Institute of Chemistry and Biochemistry - Biochemistry, Berlin, Germany
| | - Christian Schulz
- Fraunhofer Project Group PZ-Syn of the Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany
- Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany
| | - Anne Zemella
- Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany.
| | - Doreen A Wüstenhagen
- Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany
| | - Ruben Magnus Walter
- Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany
- Institute of Biotechnology, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355, Berlin, Germany
| | - Jan-Heiner Küpper
- Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany
| | - Stefan Kubick
- Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany
- Freie Universität Berlin, Institute of Chemistry and Biochemistry - Biochemistry, Berlin, Germany
- Faculty of Health Sciences, Joint Faculty of the Brandenburg University of Technology Cottbus -Senftenberg, the Brandenburg Medical School Theodor Fontane and the University of Potsdam, Potsdam, Germany
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10
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Dolz M, Monterrey DT, Beltrán-Nogal A, Menés-Rubio A, Keser M, González-Pérez D, de Santos PG, Viña-González J, Alcalde M. The colors of peroxygenase activity: Colorimetric high-throughput screening assays for directed evolution. Methods Enzymol 2023; 693:73-109. [PMID: 37977739 DOI: 10.1016/bs.mie.2023.09.006] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2023]
Abstract
Fungal unspecific peroxygenases (UPOs) are arising as versatile biocatalysts for C-H oxyfunctionalization reactions. In recent years, several directed evolution studies have been conducted to design improved UPO variants. An essential part of this protein engineering strategy is the design of reliable colorimetric high-throughput screening (HTS) assays for mutant library exploration. Here, we present a palette of 12 colorimetric HTS assays along with their step-by-step protocols, which have been validated for directed UPO evolution campaigns. This array of colorimetric assays will pave the way for the discovery and design of new UPO variants.
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Affiliation(s)
- Mikel Dolz
- Department of Biocatalysis, Institute of Catalysis, CSIC, C/ Marie Curie 2, Cantoblanco, Madrid, Spain
| | - Dianelis T Monterrey
- Department of Biocatalysis, Institute of Catalysis, CSIC, C/ Marie Curie 2, Cantoblanco, Madrid, Spain
| | - Alejandro Beltrán-Nogal
- Department of Biocatalysis, Institute of Catalysis, CSIC, C/ Marie Curie 2, Cantoblanco, Madrid, Spain
| | - Andrea Menés-Rubio
- Department of Biocatalysis, Institute of Catalysis, CSIC, C/ Marie Curie 2, Cantoblanco, Madrid, Spain
| | - Merve Keser
- Department of Biocatalysis, Institute of Catalysis, CSIC, C/ Marie Curie 2, Cantoblanco, Madrid, Spain
| | - David González-Pérez
- Department of Biocatalysis, Institute of Catalysis, CSIC, C/ Marie Curie 2, Cantoblanco, Madrid, Spain
| | | | - Javier Viña-González
- EvoEnzyme S.L., C/ Faraday 7. Parque Científico de Madrid, Cantoblanco, Madrid, Spain
| | - Miguel Alcalde
- Department of Biocatalysis, Institute of Catalysis, CSIC, C/ Marie Curie 2, Cantoblanco, Madrid, Spain.
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11
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Mohammadpour P, Safaei E, Mazarei E, Zeinalipour-Yazdi CD. TEMPO and a co-reductant mediated aerobic epoxidation of olefins using a new magnetically recoverable iron(III) bis(phenol)diamine complex: experimental and computational studies. Phys Chem Chem Phys 2023; 25:26588-26603. [PMID: 37753780 DOI: 10.1039/d3cp02254d] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/28/2023]
Abstract
A magnetically recoverable catalyst of an iron(III) bis(phenol) diamine complex immobilized onto amine functionalized silica-coated magnetic nanoparticles has been synthesized. The catalyst was characterized using FESEM, TEM and XRD which confirmed the nano structure of the catalyst. The physicochemical techniques of ICP, FT-IR, XPS, EDS and TGA proved the loading of the ligand and metal complex on silica-coated magnetic nanoparticles. Using the prepared heterogeneous catalyst, aerobic epoxidation reactions of different alkenes have been investigated in the presence of SO32- as a reducing agent. Moreover, using TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) to discover the mechanism of the aerobic epoxidation of olefins, a new TEMPO-assisted route has been explored. Both of the reaction pathways led to a moderate to high percentage yield of epoxides in water at room temperature. For further understanding mechanistic aspects, density functional theory (DFT) computational studies have been performed. The DFT calculations confirm the suggested mechanism for the title reaction and show the electron density in the vicinity of Fe(II) in the presence of TEMPO as a co-catalyst was more than that in the presence of SO32-.
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Affiliation(s)
- Pegah Mohammadpour
- Department of Chemistry, College of Sciences, Shiraz University, Shiraz, 7194684795, Iran.
| | - Elham Safaei
- Department of Chemistry, College of Sciences, Shiraz University, Shiraz, 7194684795, Iran.
| | - Elham Mazarei
- Theoretical Chemistry, Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany
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12
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Panda SK, Peng V, Sudan R, Ulezko Antonova A, Di Luccia B, Ohara TE, Fachi JL, Grajales-Reyes GE, Jaeger N, Trsan T, Gilfillan S, Cella M, Colonna M. Repression of the aryl-hydrocarbon receptor prevents oxidative stress and ferroptosis of intestinal intraepithelial lymphocytes. Immunity 2023; 56:797-812.e4. [PMID: 36801011 PMCID: PMC10101911 DOI: 10.1016/j.immuni.2023.01.023] [Citation(s) in RCA: 72] [Impact Index Per Article: 36.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2022] [Revised: 12/05/2022] [Accepted: 01/18/2023] [Indexed: 02/18/2023]
Abstract
The aryl-hydrocarbon receptor (AHR) is a ligand-activated transcription factor that buoys intestinal immune responses. AHR induces its own negative regulator, the AHR repressor (AHRR). Here, we show that AHRR is vital to sustaining intestinal intraepithelial lymphocytes (IELs). AHRR deficiency reduced IEL representation in a cell-intrinsic fashion. Single-cell RNA sequencing revealed an oxidative stress profile in Ahrr-/- IELs. AHRR deficiency unleashed AHR-induced expression of CYP1A1, a monooxygenase that generates reactive oxygen species, increasing redox imbalance, lipid peroxidation, and ferroptosis in Ahrr-/- IELs. Dietary supplementation with selenium or vitamin E to restore redox homeostasis rescued Ahrr-/- IELs. Loss of IELs in Ahrr-/- mice caused susceptibility to Clostridium difficile infection and dextran sodium-sulfate-induced colitis. Inflamed tissue of inflammatory bowel disease patients showed reduced Ahrr expression that may contribute to disease. We conclude that AHR signaling must be tightly regulated to prevent oxidative stress and ferroptosis of IELs and to preserve intestinal immune responses.
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Affiliation(s)
- Santosh K Panda
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Vincent Peng
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Raki Sudan
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Alina Ulezko Antonova
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Blanda Di Luccia
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Takahiro E Ohara
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Jose Luis Fachi
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Gary E Grajales-Reyes
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Natalia Jaeger
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Tihana Trsan
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Susan Gilfillan
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Marina Cella
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Marco Colonna
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.
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13
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Hlavica P. Key regulators in the architecture of substrate access/egress channels in mammalian cytochromes P450 governing flexibility in substrate oxyfunctionalization. J Inorg Biochem 2023; 241:112150. [PMID: 36731371 DOI: 10.1016/j.jinorgbio.2023.112150] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2022] [Revised: 01/21/2023] [Accepted: 01/22/2023] [Indexed: 01/31/2023]
Abstract
Cytochrome P450s (CYP) represent a superfamily of b-type hemoproteins catalyzing oxifunctionalization of a vast array of endogenous and exogenous compounds. The present review focuses on assessment of the topology of prospective determinants in substrate entry and product release channels of mammalian P450s, steering the conformational dynamics of substrate accessibility and productive ligand orientation toward the iron-oxene core. Based on a generalized, CYP3A4-related construct, the sum of critical elements from diverse target enzymes was found to cluster within the known substrate recognition sites. The majority of prevalent substrate access/egress tunnels revealed to be of fairly balanced functional importance. The hydrophobicity profile of the candidates revealed to be the most salient feature in functional interaction throughout the conduits, while bulkiness of the residues imposes steric restrictions on substrate traveling. Thus, small amino acids such as prolines and glycines serve as hinges, driving conformational flexibility in ligand passage. Similarly, bottlenecks in the tunnel architecture, being narrowest encounter points within the CYP3A4 model, have a vital function in substrate selectivity along with clusters of aromatic amino acids acting as gatekeepers. In addition, peripheral patches in conduits may house determinants modulating allosteric cooperativity between remote and central domains in the P450 structure. Remarkably, the bulk critical residues lining tunnels in the various isozymes reside in helices B'/C and F/G inclusive of their interhelical turns as well as in helix I. This suggests these regions to represent hotspots for targeted genetic engineering to tailor more sophisticated mammalian P450s exploitable in industrial, biotechnological and medicinal areas.
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Affiliation(s)
- Peter Hlavica
- Walther-Straub Institut fuer Pharmakologie und Toxikologie, Goethestrasse 33, D80336 Muenchen, Germany.
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14
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Guan C, Zhao G, Sun C, Zhang M, Liu S, Jiang Z, Li W, Peng Y, Zheng J. Metabolic Activation of Pesticide Isoprocarb Mediated by CYP3A4 and the Possible Correlation with Its Cytotoxicity. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2023; 71:2390-2398. [PMID: 36706223 DOI: 10.1021/acs.jafc.2c07206] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/18/2023]
Abstract
Isoprocarb (IPC), one of the most important carbamate pesticides, is used to control pests, such as rice planthoppers in crops. Studies have found that IPC induced hepatotoxicity in poultry chicken. However, the mechanisms of IPC-induced hepatotoxicity are unclear. The objectives of this study were to characterize reactive metabolites of IPC in vitro and in vivo, to identify cytochrome P450 enzymes for metabolic activation, and to define a possible correlation between the metabolic activation and cytotoxicity of IPC. In GSH- or NAC-supplemented microsomal incubations, one GSH conjugate (M6) and two NAC conjugates (M7 and M8) were detected after exposure to IPC. The corresponding GSH conjugate and NAC conjugates were found in the liver homogenates and urine of mice after IPC administration. IPC was found to be metabolized to a quinone intermediate reactive to GSH in vitro and in vivo. IPC was found to induce marked cytotoxicity in cultured mouse primary hepatocytes. Ketoconazole, a selective CYP3A4/5 enzyme inhibitor, attenuated the susceptibility of hepatocytes to IPC cytotoxicity.
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Affiliation(s)
- Chunjing Guan
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
| | - Guode Zhao
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
| | - Chen Sun
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
| | - Mingyu Zhang
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
| | - Siyu Liu
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
| | - Ziying Jiang
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
| | - Weiwei Li
- State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou 550025, P. R. China
| | - Ying Peng
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
| | - Jiang Zheng
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
- State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou 550025, P. R. China
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15
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Tran AD, Cho K, Han O. Rice peroxygenase catalyzes lipoxygenase-dependent regiospecific epoxidation of lipid peroxides in the response to abiotic stressors. Bioorg Chem 2023; 131:106285. [PMID: 36450198 DOI: 10.1016/j.bioorg.2022.106285] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2022] [Revised: 11/15/2022] [Accepted: 11/17/2022] [Indexed: 11/25/2022]
Abstract
The peroxygenase pathway plays pivotal roles in plant responses to oxidative stress and other environmental stressors. Analysis of a network of co-expressed stress-regulated rice genes demonstrated that expression of OsPXG9 is negatively correlated with expression of genes involved in jasmonic acid biosynthesis. DNA sequence analysis and structure/function studies reveal that OsPXG9 is a caleosin-like peroxygenase with amphipathic α-helices that localizes to lipid droplets in rice cells. Enzymatic studies demonstrate that 12-epoxidation is slightly more favorable with 9(S)-hydroperoxyoctadecatrienoic acid than with 9(S)-hydroperoxyoctadecadienoic acid as substrate. The products of 12-epoxidation are labile, and the epoxide ring is hydrolytically cleaved into corresponding trihydroxy compounds. On the other hand, OsPXG9 catalyzed 15-epoxidation of 13(S)-hydroperoxyoctadecatrienoic acid generates a relatively stable epoxide product. Therefore, the regiospecific 12- or 15-epoxidation catalyzed by OsPXG9 strongly depends on activation of the 9- or 13- peroxygenase reaction pathways, with their respective preferred substrates. The relative abundance of products in the 9-PXG and 13-PXG pathways suggest that the 12-epoxidation involves intramolecular oxygen transfer while the 15-epoxidation can proceed via intramolecular or intermolecular oxygen transfer. Expression of OsPXG9 is up-regulated by abiotic stimuli such as drought and salt stress, but it is down-regulated by biotic stimuli such as flagellin 22 and salicylic acid. The results suggest that the primary function of OsPXG9 is to modulate the level of lipid peroxides to facilitate effective defense responses to abiotic and biotic stressors.
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Affiliation(s)
- Anh Duc Tran
- Department of Molecular Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 61186, Republic of Korea
| | - Kyoungwon Cho
- Department of Molecular Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 61186, Republic of Korea
| | - Oksoo Han
- Department of Molecular Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 61186, Republic of Korea.
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16
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Wei K, Zhang H, Yang S, Cui Y, Zhang B, Liu J, Tang L, Tan Y, Liu S, Chen S, Yuan W, Luo X, Chen C, Li F, Liu J, Chen J, Xu P, Lv J, Tang K, Zhang Y, Ma J, Huang B. Chemo-drugs in cell microparticles reset antitumor activity of macrophages by activating lysosomal P450 and nuclear hnRNPA2B1. Signal Transduct Target Ther 2023; 8:22. [PMID: 36658134 PMCID: PMC9852455 DOI: 10.1038/s41392-022-01212-7] [Citation(s) in RCA: 23] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2022] [Revised: 09/01/2022] [Accepted: 09/28/2022] [Indexed: 01/21/2023] Open
Abstract
Macrophages in tumors (tumor-associated macrophages, TAMs), a major population within most tumors, play key homeostatic functions by stimulating angiogenesis, enhancing tumor cell growth, and suppressing antitumor immunity. Resetting TAMs by simple, efficacious and safe approach(s) is highly desirable to enhance antitumor immunity and attenuate tumor cell malignancy. Previously, we used tumor cell-derived microparticles to package chemotherapeutic drugs (drug-MPs), which resulted in a significant treatment outcome in human malignant pleural effusions via neutrophil recruitments, implicating that drug-MPs might reset TAMs, considering the inhibitory effects of M2 macrophages on neutrophil recruitment and activation. Here, we show that drug-MPs can function as an antitumor immunomodulator by resetting TAMs with M1 phenotype and IFN-β release. Mechanistically, drug molecules in tumor MPs activate macrophage lysosomal P450 monooxygenases, resulting in superoxide anion formation, which further amplifies lysosomal ROS production and pH value by activating lysosomal NOX2. Consequently, lysosomal Ca2+ signaling is activated, thus polarizing macrophages towards M1. Meanwhile, the drug molecules are delivered from lysosomes into the nucleus where they activate DNA sensor hnRNPA2B1 for IFN-β production. This lysosomal-nuclear machinery fully arouses the antitumor activity of macrophages by targeting both lysosomal pH and the nuclear innate immunity. These findings highlight that drug-MPs can act as a new immunotherapeutic approach by revitalizing antitumor activity of macrophages. This mechanistic elucidation can be translated to treat malignant ascites by drug-MPs combined with PD-1 blockade.
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Affiliation(s)
- Keke Wei
- Department of Immunology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China
| | - Huafeng Zhang
- Department of Pathology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China
| | - Shuaishuai Yang
- Department of Immunology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China
| | - Yuxiao Cui
- Department of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China
| | - Bingxia Zhang
- Cardiovascular Surgery, Union Hospital, Huazhong University of Science & Technology, Wuhan, 430071, China
| | - Jincheng Liu
- Department of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China
| | - Liang Tang
- Department of Immunology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China
| | - Yaoyao Tan
- Department of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China
| | - Simin Liu
- Department of Immunology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China
| | - Shiqi Chen
- Cardiovascular Surgery, Union Hospital, Huazhong University of Science & Technology, Wuhan, 430071, China
| | - Wu Yuan
- Department of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China
| | - Xiao Luo
- Department of Pathology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China
| | - Chen Chen
- Department of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China
| | - Fei Li
- Department of Immunology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China
| | - Junwei Liu
- Cardiovascular Surgery, Union Hospital, Huazhong University of Science & Technology, Wuhan, 430071, China
| | - Jie Chen
- Department of Immunology & National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College, Beijing, 100005, China
| | - Pingwei Xu
- Translational Medicine Laboratory, First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China
| | - Jiadi Lv
- Department of Immunology & National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College, Beijing, 100005, China
| | - Ke Tang
- Department of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China
| | - Yi Zhang
- Biotherapy Center and Cancer Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China
| | - Jingwei Ma
- Department of Immunology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China.
| | - Bo Huang
- Department of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430030, China.
- Department of Immunology & National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College, Beijing, 100005, China.
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17
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Mani S, Dubey R, Lai IC, Babu MA, Tyagi S, Swargiary G, Mody D, Singh M, Agarwal S, Iqbal D, Kumar S, Hamed M, Sachdeva P, Almutary AG, Albadrani HM, Ojha S, Singh SK, Jha NK. Oxidative Stress and Natural Antioxidants: Back and Forth in the Neurological Mechanisms of Alzheimer's Disease. J Alzheimers Dis 2023; 96:877-912. [PMID: 37927255 DOI: 10.3233/jad-220700] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2023]
Abstract
Alzheimer's disease (AD) is characterized by the progressive degeneration of neuronal cells. With the increase in aged population, there is a prevalence of irreversible neurodegenerative changes, causing a significant mental, social, and economic burden globally. The factors contributing to AD are multidimensional, highly complex, and not completely understood. However, it is widely known that aging, neuroinflammation, and excessive production of reactive oxygen species (ROS), along with other free radicals, substantially contribute to oxidative stress and cell death, which are inextricably linked. While oxidative stress is undeniably important in AD, limiting free radicals and ROS levels is an intriguing and potential strategy for deferring the process of neurodegeneration and alleviating associated symptoms. Therapeutic compounds from natural sources have recently become increasingly accepted and have been effectively studied for AD treatment. These phytocompounds are widely available and a multitude of holistic therapeutic efficiencies for treating AD owing to their antioxidant, anti-inflammatory, and biological activities. Some of these compounds also function by stimulating cholinergic neurotransmission, facilitating the suppression of beta-site amyloid precursor protein-cleaving enzyme 1, α-synuclein, and monoamine oxidase proteins, and deterring the occurrence of AD. Additionally, various phenolic, flavonoid, and terpenoid phytocompounds have been extensively described as potential palliative agents for AD progression. Preclinical studies have shown their involvement in modulating the cellular redox balance and minimizing ROS formation, displaying them as antioxidant agents with neuroprotective abilities. This review emphasizes the mechanistic role of natural products in the treatment of AD and discusses the various pathological hypotheses proposed for AD.
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Affiliation(s)
- Shalini Mani
- Centre for Emerging Diseases, Department of Biotechnology, Jaypee Institute of Information Technology, Noida, UP, India
| | - Rajni Dubey
- Division of Cardiology, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan
| | - I-Chun Lai
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
- Division of Radiation Oncology, Department of Oncology, Taipei Veterans General Hospital, Taipei, Taiwan
- The Ph.D. Program for Translational Medicine, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei, Taiwan
| | - M Arockia Babu
- Institute of Pharmaceutical Research, GLA University, Mathura, India
| | - Sakshi Tyagi
- Centre for Emerging Diseases, Department of Biotechnology, Jaypee Institute of Information Technology, Noida, UP, India
| | - Geeta Swargiary
- Centre for Emerging Diseases, Department of Biotechnology, Jaypee Institute of Information Technology, Noida, UP, India
| | - Deepansh Mody
- Centre for Emerging Diseases, Department of Biotechnology, Jaypee Institute of Information Technology, Noida, UP, India
| | - Manisha Singh
- Centre for Emerging Diseases, Department of Biotechnology, Jaypee Institute of Information Technology, Noida, UP, India
| | - Shriya Agarwal
- Department of Molecular Sciences, Macquarie University, Sydney, Australia
| | - Danish Iqbal
- Department of Health Information Management, College of Applied Medical Sciences, Buraydah Private Colleges, Buraydah, Saudi Arabia
| | - Sanjay Kumar
- Department of Life Sciences, School of Basic Sciences and Research (SBSR), Sharda University, Greater Noida, Uttar Pradesh, India
| | - Munerah Hamed
- Department of Pathology, Faculty of Medicine, Umm Al-Qura University, Makkah, Saudi Arabia
| | | | - Abdulmajeed G Almutary
- Department of Biomedical Sciences, College of Health Sciences, Abu Dhabi University, Abu Dhabi, United Arab Emirates
| | - Hind Muteb Albadrani
- Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Imam Abdulrahman Bin Faisal University, Dammam, Eastern Province, Kingdom of Saudi Arabia
| | - Shreesh Ojha
- Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, Abu Dhabi, United Arab Emirates
| | | | - Niraj Kumar Jha
- Department of Biotechnology, School of Engineering & Technology (SET), Sharda University, Greater Noida, Uttar Pradesh, India
- School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India
- Department of Biotechnology, School of Applied & Life Sciences (SALS), Uttaranchal University, Dehradun, Uttarakhand, India
- Department of Biotechnology Engineering and Food Technology, Chandigarh University, Mohali, India
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18
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Owumi SE, Arunsi UO, Oyewumi OM, Altayyar A. Accidental lead in contaminated pipe-borne water and dietary furan intake perturbs rats' hepatorenal function altering oxidative, inflammatory, and apoptotic balance. BMC Pharmacol Toxicol 2022; 23:76. [PMID: 36180958 PMCID: PMC9526313 DOI: 10.1186/s40360-022-00615-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2022] [Accepted: 09/15/2022] [Indexed: 12/03/2022] Open
Abstract
Inadvertent exposure to furan and Pb is associated with hepatorenal abnormalities in humans and animals. It is perceived that these two chemical species may work in synergy to orchestrate liver and kidney damage. Against this background, we investigated the combined effect of furan and incremental lead (Pb) exposure on hepatorenal dysfunction. Wistar rats (n = 30; 150 g) were treated for 28 days accordingly: Control; FUR (8 mg/kg), PbAc (100 µg/L), FUR + PbAc1 (8 mg/kg FUR + 1 µg/L PbAc); FUR + PbAc1 (8 mg/kg FUR + 10 µg/L PbAc), and FUR + PbAc1 (8 mg/kg FUR + 100 µg/L PbAc). Biomarkers of hepatorenal function, oxidative stress, inflammation, DNA damage, and apoptosis were examined. Furan and incrementally Pb exposure increased the levels of hepatorenal biomarkers and oxidative and pro-inflammatory mediators, including lipid peroxidation, reactive oxygen and nitrogen species, and interleukin-1 beta. Increased DNA damage, caspases- 9 and -3, and atypical histoarchitecture of the hepatorenal tissues exemplified furan and Pb treatment-related perturbations. Furthermore, the levels of antioxidants and IL-10 were also suppressed. Furan and Pb dose-dependently exacerbated hepatorenal derangements by altering the redox and inflammatory rheostats, worsened DNA damage, and related apoptotic onset that may potentiate hepatorenal disorders in humans and animals. The findings validate the synergistic effect of furan and Pb in the pathophysiology of kidney and liver disorders.
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Affiliation(s)
- Solomon E Owumi
- ChangeLab-Changing Life Cancer Research and Molecular Biology Laboratories, Department of Biochemistry, Room NB302 Faculty of Basic Medical Sciences, University of Ibadan, Ibadan, Oyo, 200004, Nigeria.
| | - Uche O Arunsi
- Department of Cancer Immunology and Biotechnology, School of Medicine, University of Nottingham, Nottingham, NG7 2RD, UK
| | - Omolola M Oyewumi
- ChangeLab-Changing Life Cancer Research and Molecular Biology Laboratories, Department of Biochemistry, Room NB302 Faculty of Basic Medical Sciences, University of Ibadan, Ibadan, Oyo, 200004, Nigeria
| | - Ahmad Altayyar
- Department of Cancer Immunology and Biotechnology, School of Medicine, University of Nottingham, Nottingham, NG7 2RD, UK
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19
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Enzymology on an Electrode and in a Nanopore: Analysis Algorithms, Enzyme Kinetics, and Perspectives. BIONANOSCIENCE 2022. [DOI: 10.1007/s12668-022-01037-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/14/2022]
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20
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Wen J, Aili A, Yan YX, Lai Y, Niu S, He S, Zhang X, Zhang G, Li J. OIT3 serves as a novel biomarker of hepatocellular carcinoma by mediating ferroptosis via regulating the arachidonic acid metabolism. Front Oncol 2022; 12:977348. [PMID: 36132142 PMCID: PMC9483180 DOI: 10.3389/fonc.2022.977348] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2022] [Accepted: 08/18/2022] [Indexed: 11/21/2022] Open
Abstract
Background Oncoprotein-Induced Transcript 3 Protein (OIT3) was identified as a liver-specific gene with abnormal expression in hepatocellular carcinoma (HCC). Herein, we aimed to examine the function and specific mechanism of OIT3 in HCC. Methods Bioinformatic analyses and tissue microarray via immunohistochemistry were used to validate the expression of OIT3 in HCC. The biofunctions of OIT3 in HCC were determined in vitro and in vivo. The mechanism was confirmed by RNA-Sequence and Western blotting. The uni- and multivariate analyses were used to identify the independent predictors for HCC. Results Low expression of OIT3 was observed in HCC and predicted a poor clinical outcome. Ectopic expression of OIT3 could inhibit the proliferation, migration, and invasion abilities of HCC cells. Mechanistically, OIT3 upregulated the expression of ALOX15 and CYP4F3, thus inducing arachidonic acid increase, ROS accumulation, and lipid peroxidation, and eventually causing ferroptosis. OIT3 was validated as a prognostic predictor for HCC patients. Conclusions Our findings revealed a novel role of OIT3 in the process of tumorigenesis of HCC. OIT3 inhibited reproliferation, migration, and invasion of HCC cells by triggering ferroptosis, which indicates that OIT3 could serve as a potential biomarker in HCC.
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Affiliation(s)
- Jie Wen
- Department of Interventional Oncology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China and Department of Medical Oncology and Radiation Sickness, Peking University Third Hospital, Beijing, China
- *Correspondence: Jie Wen, ; Jiaping Li,
| | - Abudureyimujiang Aili
- Department of Medical Oncology and Radiation Sickness, Peking University Third Hospital, Beijing, China
| | - Yao Xue Yan
- Department of Dermatology, Peking University People’s Hospital, Beijing, China
| | - YuLin Lai
- Deparment of Radiotherapy, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Shaoqing Niu
- Deparment of Radiotherapy, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Shasha He
- Deparment of Radiotherapy, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Xiaokai Zhang
- Department of Interventional Oncology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Guixiong Zhang
- Department of Interventional Oncology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Jiaping Li
- Department of Interventional Oncology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
- *Correspondence: Jie Wen, ; Jiaping Li,
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Wang B, Zhang X, Fang W, Rovira C, Shaik S. How Do Metalloproteins Tame the Fenton Reaction and Utilize •OH Radicals in Constructive Manners? Acc Chem Res 2022; 55:2280-2290. [PMID: 35926175 DOI: 10.1021/acs.accounts.2c00304] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
This Account describes the manner whereby nature controls the Fenton-type reaction of O-O homolysis of hydrogen peroxide and harnesses it to carry out various useful oxidative transformations in metalloenzymes. H2O2 acts as the cosubstrate for the heme-dependent peroxidases, P450BM3, P450SPα, P450BSβ, and the P450 decarboxylase OleT, as well as the nonheme enzymes HppE and the copper-dependent lytic polysaccharide monooxygenases (LPMOs). Whereas heme peroxidases use the Poulos-Kraut heterolytic mechanism for H2O2 activation, some heme enzymes prefer the alternative Fenton-type mechanism, which produces •OH radical intermediates. The fate of the •OH radical is controlled by the protein environment, using tight H-bonding networks around H2O2. The so-generated •OH radical is constrained by the surrounding H-bonding interactions, the orientation of which is targeted to perform H-abstraction from the Fe(III)-OH group and thereby leading to the formation of the active species, called Compound I (Cpd I), Por+•Fe(IV)═O, which performs oxidation of the substrate. Alternatively, for the nonheme HppE enzyme, the O-O homolysis catalyzed by the resting state Fe(II) generates an Fe(III)-OH species that effectively constrains the •OH radical species by a tight H-bonding network. The so-formed H-bonded •OH radical acts directly as the oxidant, since it is oriented to perform H-abstraction from the C-H bond of the substrate (S)-2-HPP. The Fenton-type H2O2 activation is strongly suggested by computations to occur also in copper-dependent LPMOs and pMMO. In LPMOs, the Cu(I)-catalyzed O-O homolysis of the H2O2 cosubstrate generates an •OH radical that abstracts a hydrogen atom from Cu(II)-OH and forms thereby the active species of the enzyme, Cu(II)-O•. Such Fenton-type O-O activation can be shared by both the O2-dependent activations of LPMOs and pMMOs, in which the O2 cosubstrate may be reduced to H2O2 by external reductants. Our studies show that, generally, the H2O2 activation is highly dependent on the protein environment, as well as on the presence/absence of substrates. Since H2O2 is a highly flexible and hydrophilic molecule, the absence of suitable substrates may lead to unproductive binding or even to the release of H2O2 from the active site, as has been suggested in P450cam and LPMOs, whereas the presence of the substrate seems to play a role in steering a Fenton-type H2O2 activation. In the absence of a substrate, the hydrophilic active site of P450BM3 disfavors the binding and activation of H2O2 and protects thereby the enzyme from the damage by the Fenton reaction. Due to the distinct coordination and reaction environment, the Fenton-type H2O2 activation mechanism by enzymes differs from the reaction in synthetic systems. In nonenzymatic reactions, the H-bonding networks are quite dynamic and flexible and the reactivity of H2O2 is not strategically constrained as in the enzymatic environment. As such, our Account describes the controlled Fenton-type mechanism in metalloenzymes, and the role of the protein environment in constraining the •OH radical against oxidative damage, while directing it to perform useful oxidative transformations.
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Affiliation(s)
- Binju Wang
- State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People's Republic of China
| | - Xuan Zhang
- State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People's Republic of China
| | - Wenhan Fang
- State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People's Republic of China
| | - Carme Rovira
- Departament de Química Inorgànica i Orgànica & IQTCUB, Universitat de Barcelona, 08028 Barcelona, Spain.,Institució Catalana de Recerca i Estudis Avançats (ICREA), 08020 Barcelona, Spain
| | - Sason Shaik
- Institute of Chemistry, The Hebrew University of Jerusalem, Givat Ram Campus, 91904 Jerusalem, Israel
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22
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Hou D, Hu F, Mao Y, Yan L, Zhang Y, Zheng Z, Wu A, Forouzanfar T, Pathak JL, Wu G. Cationic antimicrobial peptide NRC-03 induces oral squamous cell carcinoma cell apoptosis via CypD-mPTP axis-mediated mitochondrial oxidative stress. Redox Biol 2022; 54:102355. [PMID: 35660629 PMCID: PMC9511698 DOI: 10.1016/j.redox.2022.102355] [Citation(s) in RCA: 30] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2022] [Revised: 05/14/2022] [Accepted: 05/24/2022] [Indexed: 02/07/2023] Open
Abstract
Pleurocidin-family cationic antimicrobial peptide NRC-03 exhibits potent and selective cytotoxicity towards cancer cells. However, the anticancer effect of NRC-03 in oral squamous cell carcinoma (OSCC) and the molecular mechanism of NRC-03 induced cancer cell death is still unclear. This study focused to investigate mitochondrial oxidative stress-mediated altered mitochondrial function involved in NRC-03-induced apoptosis of OSCC cells. NRC-03 entered the OSCC cells more easily than that of normal cells and bound to mitochondria as well as the nucleus, causing cell membrane blebbing, mitochondria swelling, and DNA fragmentation. NRC-03 induced high oxygen consumption, reactive oxygen species (ROS) release, mitochondrial dysfunction, and apoptosis in OSCC cells. Non-specific antioxidant N-acetyl-l-cysteine (NAC), or mitochondria-specific antioxidant mitoquinone (MitoQ) alleviated NRC-03-induced apoptosis and mitochondrial dysfunction indicated that NRC-03 exerts a cytotoxic effect in cancer cells via inducing cellular and mitochondrial oxidative stress. Moreover, the expression of cyclophilin D (CypD), the key component of mitochondrial permeability transition pore (mPTP), was upregulated in NRC-03-treated cancer cells. Blockade of CypD by siRNA-mediated depletion or pharmacological inhibitor cyclosporine A (CsA) significantly suppressed NRC-03-induced mitochondrial oxidative stress, mitochondrial dysfunction, and apoptosis. NRC-03 also activated MAPK/ERK and NF-κB pathways. Importantly, intratumoral administration of NRC-03 inhibited the growth of CAL-27 cells-derived tumors on xenografted animal models. Taken together, our study indicates that NRC-03 induces apoptosis in OSCC cells via the CypD-mPTP axis mediated mitochondrial oxidative stress.
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Affiliation(s)
- Dan Hou
- Affiliated Stomatology Hospital of Guangzhou Medical University, Guangdong Engineering Research Center of Oral Restoration and Reconstruction, Guangzhou Key Laboratory of Basic and Applied Research of Oral Regenerative Medicine, Guangzhou, Guangdong, 510182, China; Department of Oral and Maxillofacial Surgery/Oral Pathology, Amsterdam UMC/VUmc and Academic Centre for Dentistry Amsterdam (ACTA), Vrije Universiteit Amsterdam, Amsterdam Movement Science, Amsterdam, 1081 HZ, the Netherlands
| | - Fengjun Hu
- Institute of Information Technology, Zhejiang Shuren University, Hangzhou, Zhejiang, 310000, China
| | - Yixin Mao
- Department of Prosthodontics, School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, 325027, China; Institute of Stomatology, School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, 325027, China; Laboratory for Myology, Department of Human Movement Sciences, Faculty of Behavioural and Movement Sciences, Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, 1081 HZ, Netherlands
| | - Liang Yan
- Department of Medical Biochemistry and Molecular Biology, School of Medicine, Jinan University, Guangzhou, 510632, China
| | - Yuhui Zhang
- Affiliated Stomatology Hospital of Guangzhou Medical University, Guangdong Engineering Research Center of Oral Restoration and Reconstruction, Guangzhou Key Laboratory of Basic and Applied Research of Oral Regenerative Medicine, Guangzhou, Guangdong, 510182, China
| | - Zhichao Zheng
- Affiliated Stomatology Hospital of Guangzhou Medical University, Guangdong Engineering Research Center of Oral Restoration and Reconstruction, Guangzhou Key Laboratory of Basic and Applied Research of Oral Regenerative Medicine, Guangzhou, Guangdong, 510182, China
| | - Antong Wu
- Affiliated Stomatology Hospital of Guangzhou Medical University, Guangdong Engineering Research Center of Oral Restoration and Reconstruction, Guangzhou Key Laboratory of Basic and Applied Research of Oral Regenerative Medicine, Guangzhou, Guangdong, 510182, China
| | - Tymour Forouzanfar
- Department of Oral and Maxillofacial Surgery/Oral Pathology, Amsterdam UMC/VUmc and Academic Centre for Dentistry Amsterdam (ACTA), Vrije Universiteit Amsterdam, Amsterdam Movement Science, Amsterdam, 1081 HZ, the Netherlands
| | - Janak L Pathak
- Affiliated Stomatology Hospital of Guangzhou Medical University, Guangdong Engineering Research Center of Oral Restoration and Reconstruction, Guangzhou Key Laboratory of Basic and Applied Research of Oral Regenerative Medicine, Guangzhou, Guangdong, 510182, China.
| | - Gang Wu
- Department of Oral and Maxillofacial Surgery/Oral Pathology, Amsterdam UMC/VUmc and Academic Centre for Dentistry Amsterdam (ACTA), Vrije Universiteit Amsterdam, Amsterdam Movement Science, Amsterdam, 1081 HZ, the Netherlands; Department of Oral Cell Biology, Academic Centre of Dentistry Amsterdam (ACTA), University van Amsterdam and Vrije Universiteit Amsterdam, Amsterdam, 1081LA, Netherlands.
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Abstract
Here, the choice of the first coordination shell of the metal center is analyzed from the perspective of charge maintenance in a binary enzyme-substrate complex and an O2-bound ternary complex in the nonheme iron oxygenases. Comparing homogentisate 1,2-dioxygenase and gentisate dioxygenase highlights the significance of charge maintenance after substrate binding as an important factor that drives the reaction coordinate. We then extend the charge analysis to several common types of nonheme iron oxygenases containing either a 2-His-1-carboxylate facial triad or a 3-His or 4-His ligand motif, including extradiol and intradiol ring-cleavage dioxygenases, thiol dioxygenases, α-ketoglutarate-dependent oxygenases, and carotenoid cleavage oxygenases. After forming the productive enzyme-substrate complex, the overall charge of the iron complex at the 0, +1, or +2 state is maintained in the remaining catalytic steps. Hence, maintaining a constant charge is crucial to promote the reaction of the iron center beginning from the formation of the Michaelis or ternary complex. The charge compensation to the iron ion is tuned not only by protein-derived carboxylate ligands but also by substrates. Overall, these analyses indicate that charge maintenance at the iron center is significant when all the necessary components form a productive complex. This charge maintenance concept may apply to most oxygen-activating metalloenzymes systems that do not draw electrons and protons step-by-step from a separate reactant, such as NADH, via a reductase. The charge maintenance perception may also be useful in proposing catalytic pathways or designing prototypical reactions using artificial or engineered enzymes for biotechnological applications.
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Affiliation(s)
- Ephrahime S. Traore
- Department of Chemistry, The University of Texas at San Antonio, San Antonio, Texas 78249, United States
| | - Aimin Liu
- Department of Chemistry, The University of Texas at San Antonio, San Antonio, Texas 78249, United States
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25
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Zhang M, Hu Y, Li W, Sun C, Guan C, Peng Y, Zheng J. In Vitro and In Vivo Metabolic Activation and Hepatotoxicity of Environmental Pollutant 2,6-Dimethylphenol. Chem Res Toxicol 2022; 35:1036-1044. [PMID: 35583464 DOI: 10.1021/acs.chemrestox.2c00048] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
2,6-Dimethylphenol (2,6-DMP) is an environmental pollutant found in industrial wastewater. Exposure to 2,6-DMP is of increasing concern as it endangered reportedly some aquatic animals. In this study, we investigated the metabolic activation and hepatotoxicity of 2,6-DMP. 2,6-DMP was metabolized to an o-quinone methide intermediate in vitro and in vivo. The electrophilic metabolite was reactive to the sulfhydryl groups of glutathione, N-acetyl cysteine, and cysteine. NADPH was required for the formation of the reactive metabolite. The quinone methide intermediate reacted with cysteine residues to form hepatic protein adduction. A single dose of 2,6-DMP induced marked elevation of serum ALT and AST in mice. Both the protein adduction and hepatotoxicity of 2,6-DMP showed dose dependency.
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Affiliation(s)
- Mingyu Zhang
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
| | - Yaodong Hu
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
| | - Wei Li
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
| | - Chen Sun
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
| | - Chunjing Guan
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
| | - Ying Peng
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China
| | - Jiang Zheng
- Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, P. R. China.,State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou 550025, P. R. China.,Key Laboratory of Environmental Pollution, Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang, Guizhou 550025, P. R. China
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26
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Superoxide Radicals in the Execution of Cell Death. Antioxidants (Basel) 2022; 11:antiox11030501. [PMID: 35326151 PMCID: PMC8944419 DOI: 10.3390/antiox11030501] [Citation(s) in RCA: 126] [Impact Index Per Article: 42.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Revised: 03/01/2022] [Accepted: 03/01/2022] [Indexed: 12/24/2022] Open
Abstract
Superoxide is a primary oxygen radical that is produced when an oxygen molecule receives one electron. Superoxide dismutase (SOD) plays a primary role in the cellular defense against an oxidative insult by ROS. However, the resulting hydrogen peroxide is still reactive and, in the presence of free ferrous iron, may produce hydroxyl radicals and exacerbate diseases. Polyunsaturated fatty acids are the preferred target of hydroxyl radicals. Ferroptosis, a type of necrotic cell death induced by lipid peroxides in the presence of free iron, has attracted considerable interest because of its role in the pathogenesis of many diseases. Radical electrons, namely those released from mitochondrial electron transfer complexes, and those produced by enzymatic reactions, such as lipoxygenases, appear to cause lipid peroxidation. While GPX4 is the most potent anti-ferroptotic enzyme that is known to reduce lipid peroxides to alcohols, other antioxidative enzymes are also indirectly involved in protection against ferroptosis. Moreover, several low molecular weight compounds that include α-tocopherol, ascorbate, and nitric oxide also efficiently neutralize radical electrons, thereby suppressing ferroptosis. The removal of radical electrons in the early stages is of primary importance in protecting against ferroptosis and other diseases that are related to oxidative stress.
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27
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Akhtar M, Wright JN. A review of 18O labelling Studies to probe the mechanism of aromatase (CYP191A). J Steroid Biochem Mol Biol 2022; 216:106010. [PMID: 34757095 DOI: 10.1016/j.jsbmb.2021.106010] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/27/2021] [Revised: 10/12/2021] [Accepted: 10/18/2021] [Indexed: 11/25/2022]
Abstract
Our previous studies, using precursors for two classes of estrogens, estrone and estriol, have highlighted the following facets of aromatase. The overall reaction, converting androgens into estrogens, occurs in three steps, each requiring NADPH and O2. In Step 1, a 19-hydroxy intermediate is produced, which in Step 2, is converted into a 19-oxo derivative via a gem -diol intermediate with the stereospecific loss of HRe. In Step 3, a scission of the C-10-C-19 bond occurs releasing C-19 as formic acid (HCOOH) and incorporating an atom of oxygen from O2, The other oxygen atom of formic acid is derived from the hydroxyl group introduced in Step 1. These experiments were performed using the classical placental microsomal system. Our findings were confirmed and extended by (the late) Caspi's group. However, incorporation of oxygen in Step 3, has been challenged in a subsequent study using a soluble reconstituted system. The latter authors have implied the superiority of their system over the microsomal preparation. However, several assumptions under pinning their own work were derived from the use of placental microsomes. Furthermore, the authors have not considered that when a previous work is challenged it needs to be repeated under the conditions described in the original publication.
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Affiliation(s)
- Muhammad Akhtar
- School of Biological Sciences, University of the Punjab, New Campus, Lahore 54590, Pakistan; School of Biological Sciences, University of Southampton, Building 85, SO17 1BJ, UK.
| | - J Neville Wright
- School of Biological Sciences, University of Southampton, Building 85, SO17 1BJ, UK
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28
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Shumyantseva VV, Bulko TV, Gnedenko OV, Yablokov EO, Usanov SA, Ivanov AS. [Adrenodoxins and their role in the cytochrome P450 systems]. BIOMEDITSINSKAIA KHIMIIA 2022; 68:47-54. [PMID: 35221296 DOI: 10.18097/pbmc20226801047] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
The role of partner proteins in the formation of functional complexes in cytochrome P450 systems was investigated by means of optical biosensor technique. Kinetic constants and equilibrium dissociation constants of complexes of cytochrome CYP11A1 (P450scc) with wild-type adrenodoxin (Adx WT) and mutant forms of adrenodoxin R106D and D109R were determined using an optical biosensor. Wild-type adrenodoxin (Kd = (1.23±0.09)⋅10⁻⁶ M) and mutant D109R (Kd = (2.37±0.09)⋅10⁻⁸ M) formed complexes with cytochrome P450scc. For the R106D mutant, no complex formation was detected. To investigate the possibility of the participation of adrenodoxins and their mutant variants in the process of electron transfer as electron donors in mitochondrial cytochrome P450 systems, the electrochemical properties of these iron-sulfur proteins Adx WT and mutant forms of adrenodoxins were studied. Adx WT, mutant forms R106D and D109R have redox potentials E1/2 significantly more negative than cytochromes P450 (-579±10 mV, -590±15 mV, and -528±10 mV, respectively). These results suggest that Adx WT and mutant forms may be electron donors in the cytochrome P450 systems.
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Affiliation(s)
- V V Shumyantseva
- Institute of Biomedical Chemistry, Moscow, Russia; Pirogov Russian National Research Medical University, Moscow, Russia
| | - T V Bulko
- Institute of Biomedical Chemistry, Moscow, Russia
| | - O V Gnedenko
- Institute of Biomedical Chemistry, Moscow, Russia
| | - E O Yablokov
- Institute of Biomedical Chemistry, Moscow, Russia
| | - S A Usanov
- Institute of Bioorganic Chemistry, Minsk, Belarus
| | - A S Ivanov
- Institute of Biomedical Chemistry, Moscow, Russia
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29
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Cheng T, Lam AK, Gopalan V. Diet derived polycyclic aromatic hydrocarbons and its pathogenic roles in colorectal carcinogenesis. Crit Rev Oncol Hematol 2021; 168:103522. [PMID: 34748942 DOI: 10.1016/j.critrevonc.2021.103522] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2021] [Revised: 11/02/2021] [Accepted: 11/03/2021] [Indexed: 12/11/2022] Open
Abstract
Polycyclic aromatic hydrocarbon (PAHs) are molecules that contaminate meat products during the high-temperature cooking of meat. This study reviewed the pathogenic roles of meat derived polycyclic aromatic hydrocarbons in the carcinogenesis of colorectal cancer (CRC). Ingested PAHs undergo xenobiotic metabolism resulting in the activation of genotoxic metabolites that can induce DNA damage in the colorectum. Genetic polymorphisms in PAH xenobiotic enzymes are linked to the risk of CRC and suggest a role for PAH-meat ingestion in carcinogenesis of colorectal malignancies. Furthermore, PAH specific DNA adducts have been identified in colorectal cancer tissue and linked to high meat intake. DNA adduct resolution is mediated by the nucleotide excision repair, and polymorphisms within genes of this repair pathway and high meat intake are associated with increased CRC risk. In the literature, there is evidence from metabolic enzyme gene variants, DNA repair genes, PAH metabolites, and epidemiological studies suggesting PAH involvement in CRC.
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Affiliation(s)
- Tracie Cheng
- Cancer Molecular Pathology, School of Medicine & Dentistry, Griffith University, Gold Coast, Queensland 4222, Australia
| | - Alfred K Lam
- Cancer Molecular Pathology, School of Medicine & Dentistry, Griffith University, Gold Coast, Queensland 4222, Australia
| | - Vinod Gopalan
- Cancer Molecular Pathology, School of Medicine & Dentistry, Griffith University, Gold Coast, Queensland 4222, Australia.
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30
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Oliw EH. Fatty acid dioxygenase-cytochrome P450 fusion enzymes of filamentous fungal pathogens. Fungal Genet Biol 2021; 157:103623. [PMID: 34520871 DOI: 10.1016/j.fgb.2021.103623] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2021] [Accepted: 09/07/2021] [Indexed: 11/27/2022]
Abstract
Oxylipins designate oxygenated unsaturated C18 fatty acids. Many filamentous fungi pathogens contain dioxygenases (DOX) in oxylipin biosynthesis with homology to human cyclooxygenases. They contain a DOX domain, which is often fused to a functional cytochrome P450 at the C-terminal end. A Tyr radical in the DOX domain initiates dioxygenation of linoleic acid by hydrogen abstraction with formation of 8-, 9-, or 10-hydroperoxy metabolites. The P450 domains can catalyze heterolytic cleavage of 8- and 10-hydroperoxides with oxidation of the heme thiolate iron for hydroxylation at C-5, C-7, C-9, or C-11 and for epoxidation of the 12Z double bond; thus displaying linoleate diol synthase (LDS) and epoxy alcohol synthase (EAS) activities. LSD activities are present in the rice blast pathogen Magnaporthe oryzae, Botrytis cinerea causing grey mold and the black scurf pathogen Rhizoctonia solani. 10R-DOX-EAS has been found in M. oryzae and Fusarium oxysporum. The P450 domains may also catalyze homolytic cleavage of 8- and 9-hydroperoxy fatty acids and dehydration to produce epoxides with an adjacent double bond, i.e., allene oxides, thus displaying 8- and 9-DOX-allene oxide synthases (AOS). F. oxysporum, F. graminearum, and R. solani express 9S-DOX-AOS and Zymoseptoria tritici 8S-and 9R-DOX-AOS. Homologues are present in endemic human-pathogenic fungi with extensive studies in Aspergillus fumigatus, A. flavus (also a plant pathogen) as well as the genetic model A. nidulans. 8R-and 10R-DOX appear to bind fatty acids "headfirst" in the active site, whereas 9S-DOX binds them "tail first" in analogy with cyclooxygenases. The biological relevance of 8R-DOX-5,8-LDS (also designated PpoA) was first discovered in relation to sporulation of A. nidulans and recently for development and programmed hyphal branching of A. fumigatus. Gene deletion DOX-AOS homologues in F. verticillioides, A. flavus, and A. nidulans alters, inter alia, mycotoxin production, sporulation, and gene expression.
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Affiliation(s)
- Ernst H Oliw
- Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Box 591, SE-751 24 Uppsala, Sweden.
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Oliw EH. WITHDRAWN: Fatty acid dioxygenase-cytochrome P450 fusion enzymes of the top 10 fungal pathogens in molecular plant pathology and human-pathogenic fungi. Fungal Genet Biol 2021:103603. [PMID: 34214670 DOI: 10.1016/j.fgb.2021.103603] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2020] [Revised: 02/21/2021] [Accepted: 06/11/2021] [Indexed: 11/22/2022]
Abstract
This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal
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Affiliation(s)
- Ernst H Oliw
- Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Box 591, SE-751 24 Uppsala, Sweden.
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33
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Donoso RA, Ruiz D, Gárate-Castro C, Villegas P, González-Pastor JE, de Lorenzo V, González B, Pérez-Pantoja D. Identification of a self-sufficient cytochrome P450 monooxygenase from Cupriavidus pinatubonensis JMP134 involved in 2-hydroxyphenylacetic acid catabolism, via homogentisate pathway. Microb Biotechnol 2021; 14:1944-1960. [PMID: 34156761 PMCID: PMC8449657 DOI: 10.1111/1751-7915.13865] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2020] [Revised: 05/24/2021] [Accepted: 05/26/2021] [Indexed: 11/28/2022] Open
Abstract
The self-sufficient cytochrome P450 RhF and its homologues belonging to the CYP116B subfamily have attracted considerable attention due to the potential for biotechnological applications based in their ability to catalyse an array of challenging oxidative reactions without requiring additional protein partners. In this work, we showed for the first time that a CYP116B self-sufficient cytochrome P450 encoded by the ohpA gene harboured by Cupriavidus pinatubonensis JMP134, a β-proteobacterium model for biodegradative pathways, catalyses the conversion of 2-hydroxyphenylacetic acid (2-HPA) into homogentisate. Mutational analysis and HPLC metabolite detection in strain JMP134 showed that 2-HPA is degraded through the well-known homogentisate pathway requiring a 2-HPA 5-hydroxylase activity provided by OhpA, which was additionally supported by heterologous expression and enzyme assays. The ohpA gene belongs to an operon including also ohpT, coding for a substrate-binding subunit of a putative transporter, whose expression is driven by an inducible promoter responsive to 2-HPA in presence of a predicted OhpR transcriptional regulator. OhpA homologues can be found in several genera belonging to Actinobacteria and α-, β- and γ-proteobacteria lineages indicating a widespread distribution of 2-HPA catabolism via homogentisate route. These results provide first time evidence for the natural function of members of the CYP116B self-sufficient oxygenases and represent a significant input to support novel kinetic and structural studies to develop cytochrome P450-based biocatalytic processes.
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Affiliation(s)
- Raúl A Donoso
- Programa Institucional de Fomento a la Investigación, Desarrollo e Innovación (PIDi), Universidad Tecnológica Metropolitana, Santiago, Chile.,Center of Applied Ecology and Sustainability (CAPES), Santiago, Chile
| | - Daniela Ruiz
- Center of Applied Ecology and Sustainability (CAPES), Santiago, Chile.,Facultad de Ingeniería y Ciencias, Universidad Adolfo Ibáñez, Santiago, Chile
| | - Carla Gárate-Castro
- Programa Institucional de Fomento a la Investigación, Desarrollo e Innovación (PIDi), Universidad Tecnológica Metropolitana, Santiago, Chile.,Center of Applied Ecology and Sustainability (CAPES), Santiago, Chile
| | - Pamela Villegas
- Programa Institucional de Fomento a la Investigación, Desarrollo e Innovación (PIDi), Universidad Tecnológica Metropolitana, Santiago, Chile
| | - José Eduardo González-Pastor
- Laboratory of Molecular Adaptation, Department of Molecular Evolution, Centro de Astrobiología (CSIC-INTA), Madrid, Spain
| | - Víctor de Lorenzo
- Systems and Synthetic Biology Department, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid, Spain
| | - Bernardo González
- Center of Applied Ecology and Sustainability (CAPES), Santiago, Chile.,Facultad de Ingeniería y Ciencias, Universidad Adolfo Ibáñez, Santiago, Chile
| | - Danilo Pérez-Pantoja
- Programa Institucional de Fomento a la Investigación, Desarrollo e Innovación (PIDi), Universidad Tecnológica Metropolitana, Santiago, Chile
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Faponle AS, Roy A, Adelegan AA, Gauld JW. Molecular Dynamics Simulations of a Cytochrome P450 from Tepidiphilus thermophilus (P450-TT) Reveal How Its Substrate-Binding Channel Opens. Molecules 2021; 26:molecules26123614. [PMID: 34204747 PMCID: PMC8231624 DOI: 10.3390/molecules26123614] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2021] [Revised: 06/10/2021] [Accepted: 06/10/2021] [Indexed: 01/08/2023] Open
Abstract
Cytochrome P450s (P450) are important enzymes in biology with useful biochemical reactions in, for instance, drug and xenobiotics metabolisms, biotechnology, and health. Recently, the crystal structure of a new member of the CYP116B family has been resolved. This enzyme is a cytochrome P450 (CYP116B46) from Tepidiphilus thermophilus (P450-TT) and has potential for the oxy-functionalization of organic molecules such as fatty acids, terpenes, steroids, and statins. However, it was thought that the opening to its hitherto identified substrate channel was too small to allow organic molecules to enter. To investigate this, we performed molecular dynamics simulations on the enzyme. The results suggest that the crystal structure is not relaxed, possibly due to crystal packing effects, and that its tunnel structure is constrained. In addition, the simulations revealed two key amino acid residues at the mouth of the channel; a glutamyl and an arginyl. The glutamyl’s side chain tightens and relaxes the opening to the channel in conjunction with the arginyl’s, though the latter’s side chain is less dramatically changed after the initial relaxation of its conformations. Additionally, it was observed that the effect of increased temperature did not considerably affect the dynamics of the enzyme fold, including the relative solvent accessibility of the amino acid residues that make up the substrate channel wall even as compared to the changes that occurred at room temperature. Interestingly, the substrate channel became distinguishable as a prominent tunnel that is likely to accommodate small- to medium-sized organic molecules for bioconversions. That is, P450-TT has the ability to pass appropriate organic substrates to its active site through its elaborate substrate channel, and notably, is able to control or gate any molecules at the opening to this channel.
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Affiliation(s)
- Abayomi S. Faponle
- Department of Biochemistry, Faculty of Basic Medical Sciences, Sagamu Campus, Olabisi Onabanjo University, Ago-Iwoye, Nigeria; (A.S.F.); (A.A.A.)
| | - Anupom Roy
- Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON N9B3P4, Canada;
| | - Ayodeji A. Adelegan
- Department of Biochemistry, Faculty of Basic Medical Sciences, Sagamu Campus, Olabisi Onabanjo University, Ago-Iwoye, Nigeria; (A.S.F.); (A.A.A.)
| | - James W. Gauld
- Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON N9B3P4, Canada;
- Correspondence: ; Tel.: +1-519-253-3000 (ext. 3992); Fax: +1-519-973-7098
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Eleftheriadis T, Pissas G, Golfinopoulos S, Liakopoulos V, Stefanidis I. Role of indoleamine 2,3-dioxygenase in ischemia-reperfusion injury of renal tubular epithelial cells. Mol Med Rep 2021; 23:472. [PMID: 33899121 PMCID: PMC8097759 DOI: 10.3892/mmr.2021.12111] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2020] [Accepted: 03/18/2021] [Indexed: 01/22/2023] Open
Abstract
The present study evaluated indoleamine 2,3‑dioxygenase 1 (IDO) kinetics and how it affects cell survival during the two distinct phases of ischemia‑reperfusion (I‑R) injury. Primary renal proximal tubular epithelial cells (RPTECs) were cultured under anoxia or reoxygenation with or without the IDO inhibitor 1‑DL‑methyltryptophan, the aryl‑hydrocarbon receptor (AhR) inhibitor CH223191 or the ferroptosis inhibitor α‑tocopherol. Using cell imaging, colorimetric assays, PCR and western blotting, it was demonstrated that IDO was upregulated and induced apoptosis during anoxia. The related molecular pathway entails tryptophan degradation, general control non‑derepressible‑2 kinase (GCN2K) activation, increased level of phosphorylated eukaryotic translation initiation factor 2α, activating transcription factor (ATF)4, ATF3, C/EBP homologous protein, phosphorylated p53, p53, Bax, death receptor‑5 and eventually activated cleaved caspase‑3. Reoxygenation also upregulated IDO, which, in this case, induced ferroptosis. The related molecular pathway encompasses kynurenine production, AhR activation, cytochrome p450 enzymes increase, reactive oxygen species generation and eventually ferroptosis. In conclusion, in RPTECs, both anoxia and reoxygenation upregulated IDO, which in turn induced GCN2K‑mediated apoptosis and AhR‑mediated ferroptosis. Since both phases of I‑R injury share IDO upregulation as a common point, its inhibition may prove a useful therapeutic strategy for preventing or attenuating I‑R injury.
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Affiliation(s)
- Theodoros Eleftheriadis
- Department of Nephrology, Faculty of Medicine, University of Thessaly, 41110 Larissa, Greece
| | - Georgios Pissas
- Department of Nephrology, Faculty of Medicine, University of Thessaly, 41110 Larissa, Greece
| | - Spyridon Golfinopoulos
- Department of Nephrology, Faculty of Medicine, University of Thessaly, 41110 Larissa, Greece
| | - Vassilios Liakopoulos
- Department of Nephrology, Faculty of Medicine, University of Thessaly, 41110 Larissa, Greece
| | - Ioannis Stefanidis
- Department of Nephrology, Faculty of Medicine, University of Thessaly, 41110 Larissa, Greece
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Eleftheriadis T, Pissas G, Filippidis G, Liakopoulos V, Stefanidis I. Reoxygenation induces reactive oxygen species production and ferroptosis in renal tubular epithelial cells by activating aryl hydrocarbon receptor. Mol Med Rep 2021; 23:41. [PMID: 33179104 PMCID: PMC7684866 DOI: 10.3892/mmr.2020.11679] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2020] [Accepted: 10/08/2020] [Indexed: 12/13/2022] Open
Abstract
During the reperfusion phase of ischemia‑reperfusion injury, reactive oxygen species (ROS) production aggravates the course of many diseases, including acute kidney injury. Among the various enzymes implicated in ROS production are the enzymes of the cytochromes P450 superfamily (CYPs). Since arylhydrocarbon receptor (AhR) controls the expression of certain CYPs, the involvement of this pathway was evaluated in reperfusion injury. Because AhR may interact with the nuclear factor erythroid 2‑related factor 2 (Nrf2) and the hypoxia‑inducible factor‑1α (HIF‑1α), whether such an interaction takes place and affects reperfusion injury was also assessed. Proximal renal proximal tubular epithelial cells were subjected to anoxia and subsequent reoxygenation. At the onset of reoxygenation, the AhR inhibitor CH223191, the HIF‑1α activator roxadustat, or the ferroptosis inhibitor α‑tocopherol were used. The activity of AhR, Nrf2, HIF‑1α, and their transcriptional targets were assessed with western blotting. ROS production, lipid peroxidation and cell death were measured with colorimetric assays or cell imaging. Reoxygenation induced ROS production, lipid peroxidation and cell ferroptosis, whereas CH223191 prevented all. Roxadustat did not affect the above parameters. Reoxygenation activated AhR and increased CYP1A1, while CH223191 prevented both. Reoxygenation with or without CH223191 did not alter Nrf2 or HIF‑1α activity. Thus, AhR is activated during reoxygenation and induces ROS production, lipid peroxidation and ferroptotic cell death. These detrimental effects may be mediated by AhR‑induced CYP overexpression, while the Nrf2 or the HIF‑1α pathways remain unaffected. Accordingly, the AhR pathway may represent a promising therapeutic target for the prevention of reperfusion injury.
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Affiliation(s)
- Theodoros Eleftheriadis
- Department of Nephrology, Faculty of Medicine, University of Thessaly, 41110 Larissa, Greece
| | - Georgios Pissas
- Department of Nephrology, Faculty of Medicine, University of Thessaly, 41110 Larissa, Greece
| | - Georgios Filippidis
- Department of Nephrology, Faculty of Medicine, University of Thessaly, 41110 Larissa, Greece
| | - Vassilios Liakopoulos
- Department of Nephrology, Faculty of Medicine, University of Thessaly, 41110 Larissa, Greece
| | - Ioannis Stefanidis
- Department of Nephrology, Faculty of Medicine, University of Thessaly, 41110 Larissa, Greece
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Chi ZC. Research status and prgoress of nonalcoholic fatty pancreatic disease. Shijie Huaren Xiaohua Zazhi 2020; 28:933-950. [DOI: 10.11569/wcjd.v28.i19.933] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Nonalcoholic fatty pancreatic disease (NAFPD) is a disease characterized by an increase in pancreatic fat accumulation. It is a component of the metabolic syndrome and often coexists with nonalcoholic fatty liver disease. Once the diagnosis is established, it is closely related to acute and chronic pancreatitis, type 2 diabetes mellitus, pancreatic fibrosis, and pancreatic cancer. In recent years, it has been confirmed that NAFPD is closely related to cardiovascular disease, liver fibrosis, and liver cancer. The prevalence of NAFPD ranges between 11% and 69%, and increases with age. It is worth noting that the prevalence in obese children is twice as high as that in non-obese children. The high prevalence rate and complexity of the disease have aroused people's high attention. Therefore, to improve the understanding of NAFPD, fully understand the clinical significance of NAFPD, and further study its pathogenesis, diagnosis, and treatment require the collaboration and joint efforts of multiple disciplines, including hepatopathy, gastroenterology, endocrine metabolism, cardiovascular disease, imaging, pathology, and others. In this paper, we review the clinical significance, pathogenesis, and imaging diagnosis of NAFPD and propose our personal understanding of the key points in future research.
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Affiliation(s)
- Zhao-Chun Chi
- Department of Gastroenterology, Qingdao Municipal Hospital, Qingdao 266011, Shandong Province, China
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Casnati A, Lanzi M, Cera G. Recent Advances in Asymmetric Iron Catalysis. Molecules 2020; 25:E3889. [PMID: 32858925 PMCID: PMC7503417 DOI: 10.3390/molecules25173889] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2020] [Revised: 08/24/2020] [Accepted: 08/25/2020] [Indexed: 11/16/2022] Open
Abstract
Asymmetric transition-metal catalysis represents a fascinating challenge in the field of organic chemistry research. Since seminal advances in the late 60s, which were finally recognized by the Nobel Prize to Noyori, Sharpless and Knowles in 2001, the scientific community explored several approaches to emulate nature in producing chiral organic molecules. In a scenario that has been for a long time dominated by the use of late-transition metals (TM) catalysts, the use of 3d-TMs and particularly iron has found, recently, a widespread application. Indeed, the low toxicity and the earth-abundancy of iron, along with its chemical versatility, allowed for the development of unprecedented and more sustainable catalytic transformations. While several competent reviews tried to provide a complete picture of the astounding advances achieved in this area, within this review we aimed to survey the latest achievements and new concepts brought in the field of enantioselective iron-catalyzed transformations.
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Affiliation(s)
- Alessandra Casnati
- Laboratoire des Systèmes Complexes en Synthèse et Catalyse, Institut de Science et d’Ingénierie Supramoléculaires, Université de Strasbourg &CNRS, 8 Allèe Gaspard Monge, BP 70028, F-67083 Strasbourg, France;
| | - Matteo Lanzi
- Laboratoire de Chemie Moléculaire (UMR CNRS 7509), Université de Strasbourg, ECPM 25 Rue Becquerel, 67087 Strasbourg, France;
| | - Gianpiero Cera
- Dipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale, Università di Parma, Parco Area delle Scienze 17/A, I-43124 Parma, Italy
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Jacoby C, Ferlaino S, Bezold D, Jessen H, Müller M, Boll M. ATP-dependent hydroxylation of an unactivated primary carbon with water. Nat Commun 2020; 11:3906. [PMID: 32764563 PMCID: PMC7411048 DOI: 10.1038/s41467-020-17675-7] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2020] [Accepted: 07/09/2020] [Indexed: 11/09/2022] Open
Abstract
Enzymatic hydroxylation of unactivated primary carbons is generally associated with the use of molecular oxygen as co-substrate for monooxygenases. However, in anaerobic cholesterol-degrading bacteria such as Sterolibacterium denitrificans the primary carbon of the isoprenoid side chain is oxidised to a carboxylate in the absence of oxygen. Here, we identify an enzymatic reaction sequence comprising two molybdenum-dependent hydroxylases and one ATP-dependent dehydratase that accomplish the hydroxylation of unactivated primary C26 methyl group of cholesterol with water: (i) hydroxylation of C25 to a tertiary alcohol, (ii) ATP-dependent dehydration to an alkene via a phosphorylated intermediate, (iii) hydroxylation of C26 to an allylic alcohol that is subsequently oxidised to the carboxylate. The three-step enzymatic reaction cascade divides the high activation energy barrier of primary C–H bond cleavage into three biologically feasible steps. This finding expands our knowledge of biological C–H activations beyond canonical oxygenase-dependent reactions. Monooxygenases catalyse the hydroxylation of C-H bonds using oxygen as a co-substrate, which, in turn, is unavailable for anaerobic bacteria. Here, the authors report a three-step reaction cascade involving two hydroxylases and one dehydratase which hydroxylate the C26 methyl group of cholesterol with water as a co-substrate.
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Affiliation(s)
- Christian Jacoby
- Microbiology, Faculty of Biology, Albert-Ludwigs-Universität Freiburg, Schänzlestr. 1, 79104, Freiburg, Germany
| | - Sascha Ferlaino
- Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, 79104, Freiburg, Germany
| | - Dominik Bezold
- Institute of Organic Chemistry, Albert-Ludwigs-Universität Freiburg, Albertstrasse 21, 79104, Freiburg, Germany
| | - Henning Jessen
- Institute of Organic Chemistry, Albert-Ludwigs-Universität Freiburg, Albertstrasse 21, 79104, Freiburg, Germany
| | - Michael Müller
- Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, 79104, Freiburg, Germany
| | - Matthias Boll
- Microbiology, Faculty of Biology, Albert-Ludwigs-Universität Freiburg, Schänzlestr. 1, 79104, Freiburg, Germany.
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Mykles DL, Chang ES. Hormonal control of the crustacean molting gland: Insights from transcriptomics and proteomics. Gen Comp Endocrinol 2020; 294:113493. [PMID: 32339519 DOI: 10.1016/j.ygcen.2020.113493] [Citation(s) in RCA: 40] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/08/2020] [Accepted: 04/21/2020] [Indexed: 01/17/2023]
Abstract
Endocrine control of molting in decapod crustaceans involves the eyestalk neurosecretory center (X-organ/sinus gland complex), regenerating limbs, and a pair of Y-organs (YOs), as molting is induced by eyestalk ablation or multiple leg autotomy and suspended in early premolt by limb bud autotomy. Molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), produced in the X-organ/sinus gland complex, inhibit the YO. The YO transitions through four physiological states over the molt cycle: basal in intermolt; activated in early premolt; committed in mid- and late premolt; and repressed in postmolt. We assembled the first comprehensive YO transcriptome over the molt cycle in the land crab, Gecarcinus lateralis, showing that as many as 23 signaling pathways may interact in controlling ecdysteroidogenesis. A proposed model of the MIH/cyclic nucleotide pathway, which maintains the basal YO, consists of cAMP/Ca2+ triggering and nitric oxide (NO)/cGMP summation phases. Mechanistic target of rapamycin (mTOR) signaling is required for YO activation in early premolt and affects the mRNA levels of thousands of genes. Transforming Growth Factor-β (TGFβ)/Activin signaling is required for YO commitment in mid-premolt and high ecdysteroid titers at the end of premolt may trigger YO repression. The G. lateralis YO expresses 99 G protein-coupled receptors, three of which are putative receptors for MIH/CHH. Proteomic analysis shows the importance of radical oxygen species scavenging, cytoskeleton, vesicular secretion, immune response, and protein homeostasis and turnover proteins associated with YO function over the molt cycle. In addition to eyestalk ganglia, MIH mRNA and protein are present in brain, optic nerve, ventral nerve cord, and thoracic ganglion, suggesting that they are secondary sources of MIH. Down-regulation of mTOR signaling genes, in particular Ras homolog enriched in brain or Rheb, compensates for the effects of elevated temperature in the YO, heart, and eyestalk ganglia in juvenile Metacarcinus magister. Rheb expression increases in the activated and committed YO. These data suggest that mTOR plays a central role in mediating molt regulation by physiological and environmental factors.
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Affiliation(s)
- Donald L Mykles
- Department of Biology, Colorado State University, Fort Collins, CO 80523, USA; University of California-Davis Bodega Marine Laboratory, Bodega Bay, CA 94923, USA
| | - Ernest S Chang
- University of California-Davis Bodega Marine Laboratory, Bodega Bay, CA 94923, USA
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Strohmaier SJ, De Voss JJ, Jurva U, Andersson S, Gillam EMJ. Oxygen Surrogate Systems for Supporting Human Drug-Metabolizing Cytochrome P450 Enzymes. Drug Metab Dispos 2020; 48:432-437. [PMID: 32238418 DOI: 10.1124/dmd.120.090555] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2020] [Accepted: 03/12/2020] [Indexed: 01/13/2023] Open
Abstract
Oxygen surrogates (OSs) have been used to support cytochrome P450 (P450) enzymes for diverse purposes in drug metabolism research, including reaction phenotyping, mechanistic and inhibition studies, studies of redox partner interactions, and to avoid the need for NADPH or a redox partner. They also have been used in engineering P450s for more cost-effective, NADPH-independent biocatalysis. However, despite their broad application, little is known of the preference of individual P450s for different OSs or the substrate dependence of OS-supported activity. Furthermore, the biocatalytic potential of OSs other than cumene hydroperoxide (CuOOH) and hydrogen peroxide (H2O2) is yet to be explored. Here, we investigated the ability of the major human drug-metabolizing P450s, namely CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP1A2, to use the following OSs: H2O2, tert-butyl hydroperoxide (tert-BuOOH), CuOOH, (diacetoxyiodo)benzene, and bis(trifluoroacetoxy)iodobenzene. Overall, CuOOH and tert-BuOOH were found to be the most effective at supporting these P450s. However, the ability of P450s to be supported by OSs effectively was also found to be highly dependent on the substrate used. This suggests that the choice of OS should be tailored to both the P450 and the substrate under investigation, underscoring the need to employ screening methods that reflect the activity toward the substrate of interest to the end application. SIGNIFICANCE STATEMENT: Cytochrome P450 (P450) enzymes can be supported by different oxygen surrogates (OSs), avoiding the need for a redox partner and costly NADPH. However, few data exist comparing relative activity with different OSs and substrates. This study shows that the choice of OS used to support the major drug-metabolizing P450s influences their relative activity and regioselectivity in a substrate-specific fashion and provides a model for the more efficient use of P450s for metabolite biosynthesis.
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Affiliation(s)
- Silja J Strohmaier
- School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia (S.J.S., J.J.D.V., E.M.J.G.); and DMPK, Early Cardiovascular, Renal and Metabolism (U.J.) and Discovery Sciences (S.A.), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
| | - James J De Voss
- School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia (S.J.S., J.J.D.V., E.M.J.G.); and DMPK, Early Cardiovascular, Renal and Metabolism (U.J.) and Discovery Sciences (S.A.), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
| | - Ulrik Jurva
- School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia (S.J.S., J.J.D.V., E.M.J.G.); and DMPK, Early Cardiovascular, Renal and Metabolism (U.J.) and Discovery Sciences (S.A.), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
| | - Shalini Andersson
- School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia (S.J.S., J.J.D.V., E.M.J.G.); and DMPK, Early Cardiovascular, Renal and Metabolism (U.J.) and Discovery Sciences (S.A.), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
| | - Elizabeth M J Gillam
- School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia (S.J.S., J.J.D.V., E.M.J.G.); and DMPK, Early Cardiovascular, Renal and Metabolism (U.J.) and Discovery Sciences (S.A.), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
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Kuzikov A, Masamrekh R, Ershov P, Mezentsev Y, Ivanov A, Gilep A, Usanov S, Shumyantseva V. Interaction of Isatin with Cytochrome P450 Isoenzymes: Investigation by Means of Spectral and Electrochemical Methods The role of Isatin in Cytochromes P450 Ligand-Protein Binding Events. BIONANOSCIENCE 2020. [DOI: 10.1007/s12668-019-00707-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
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Bhunia S, Rana A, Dey SG, Ivancich A, Dey A. A designed second-sphere hydrogen-bond interaction that critically influences the O-O bond activation for heterolytic cleavage in ferric iron-porphyrin complexes. Chem Sci 2020; 11:2681-2695. [PMID: 34084327 PMCID: PMC8157560 DOI: 10.1039/c9sc04388h] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2019] [Accepted: 01/26/2020] [Indexed: 12/18/2022] Open
Abstract
Heme hydroperoxidases catalyze the oxidation of substrates by H2O2. The catalytic cycle involves the formation of a highly oxidizing species known as Compound I, resulting from the two-electron oxidation of the ferric heme in the active site of the resting enzyme. This high-valent intermediate is formed upon facile heterolysis of the O-O bond in the initial FeIII-OOH complex. Heterolysis is assisted by the histidine and arginine residues present in the heme distal cavity. This chemistry has not been successfully modeled in synthetic systems up to now. In this work, we have used a series of iron(iii) porphyrin complexes (FeIIIL2(Br), FeIIIL3(Br) and FeIIIMPh(Br)) with covalently attached pendent basic groups (pyridine and primary amine) mimicking the histidine and arginine residues in the distal-pocket of natural heme enzymes. The presence of pendent basic groups, capable of 2nd sphere hydrogen bonding interactions, leads to almost 1000-fold enhancement in the rate of Compound I formation from peracids relative to analogous complexes without these residues. The short-lived Compound I intermediate formed at cryogenic temperatures could be detected using UV-vis electronic absorption spectroscopy and also trapped to be unequivocally identified by 9 GHz EPR spectroscopy at 4 K. The broad (2000 G) and axial EPR spectrum of an exchange-coupled oxoferryl-porphyrin radical species, [FeIV[double bond, length as m-dash]O Por˙+] with g eff ⊥ = 3.80 and g eff ‖ = 1.99, was observed upon a reaction of the FeIIIL3(Br) porphyrin complex with m-CPBA. The characterization of the reactivity of the FeIII porphyrin complexes with a substrate in the presence of an oxidant like m-CPBA by UV-vis electronic absorption spectroscopy showed that they are capable of oxidizing two equivalents of inorganic and organic substrate(s) like ferrocene, 2,4,6-tritertiary butyl phenol and o-phenylenediamine. These oxidations are catalytic with a turnover number (TON) as high as 350. Density Functional Theory (DFT) calculations show that the mechanism of O-O bond activation by 2nd sphere hydrogen bonding interaction from these pendent basic groups, which are protonated by a peracid, involves polarization of the O-O σ-bond, leading to lowering of the O-O σ*-orbital allowing enhanced back bonding from the iron center. These results demonstrate how inclusion of 2nd sphere hydrogen bonding interaction can play a critical role in O-O bond heterolysis.
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Affiliation(s)
- Sarmistha Bhunia
- Department of Inorganic Chemistry, Indian Association for the Cultivation of Science Kolkata 700032 India
| | - Atanu Rana
- Department of Inorganic Chemistry, Indian Association for the Cultivation of Science Kolkata 700032 India
| | - Somdatta Ghosh Dey
- Department of Inorganic Chemistry, Indian Association for the Cultivation of Science Kolkata 700032 India
| | - Anabella Ivancich
- CNRS, Aix-Marseille Univ, Laboratoire de Bioénergétique et Ingénierie des Protéines (UMR 7281), IMM FR3479 Marseille France
| | - Abhishek Dey
- Department of Inorganic Chemistry, Indian Association for the Cultivation of Science Kolkata 700032 India
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Fungal Peroxygenases: A Phylogenetically Old Superfamily of Heme Enzymes with Promiscuity for Oxygen Transfer Reactions. ACTA ACUST UNITED AC 2020. [DOI: 10.1007/978-3-030-29541-7_14] [Citation(s) in RCA: 51] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
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Tischler D, Kumpf A, Eggerichs D, Heine T. Styrene monooxygenases, indole monooxygenases and related flavoproteins applied in bioremediation and biocatalysis. FLAVIN-DEPENDENT ENZYMES: MECHANISMS, STRUCTURES AND APPLICATIONS 2020; 47:399-425. [DOI: 10.1016/bs.enz.2020.05.011] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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ROS Generation and Antioxidant Defense Systems in Normal and Malignant Cells. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2019; 2019:6175804. [PMID: 31467634 PMCID: PMC6701375 DOI: 10.1155/2019/6175804] [Citation(s) in RCA: 511] [Impact Index Per Article: 85.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/05/2019] [Accepted: 06/24/2019] [Indexed: 02/08/2023]
Abstract
Reactive oxygen species (ROS) are by-products of normal cell activity. They are produced in many cellular compartments and play a major role in signaling pathways. Overproduction of ROS is associated with the development of various human diseases (including cancer, cardiovascular, neurodegenerative, and metabolic disorders), inflammation, and aging. Tumors continuously generate ROS at increased levels that have a dual role in their development. Oxidative stress can promote tumor initiation, progression, and resistance to therapy through DNA damage, leading to the accumulation of mutations and genome instability, as well as reprogramming cell metabolism and signaling. On the contrary, elevated ROS levels can induce tumor cell death. This review covers the current data on the mechanisms of ROS generation and existing antioxidant systems balancing the redox state in mammalian cells that can also be related to tumors.
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Chylenski P, Bissaro B, Sørlie M, Røhr ÅK, Várnai A, Horn SJ, Eijsink VG. Lytic Polysaccharide Monooxygenases in Enzymatic Processing of Lignocellulosic Biomass. ACS Catal 2019. [DOI: 10.1021/acscatal.9b00246] [Citation(s) in RCA: 122] [Impact Index Per Article: 20.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Affiliation(s)
- Piotr Chylenski
- Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, N-1432 Ås, Norway
| | - Bastien Bissaro
- Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, N-1432 Ås, Norway
| | - Morten Sørlie
- Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, N-1432 Ås, Norway
| | - Åsmund K. Røhr
- Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, N-1432 Ås, Norway
| | - Anikó Várnai
- Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, N-1432 Ås, Norway
| | - Svein J. Horn
- Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, N-1432 Ås, Norway
| | - Vincent G.H. Eijsink
- Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, N-1432 Ås, Norway
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Rothe ML, Li J, Garibay E, Moore BS, McKinnie SMK. Synthesis, bioactivity, and enzymatic modification of antibacterial thiotetromycin derivatives. Org Biomol Chem 2019; 17:3416-3423. [PMID: 30869693 PMCID: PMC6437001 DOI: 10.1039/c8ob03109f] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Thiotetronate-containing natural products, including thiolactomycin, thiotetromycin, and thiotetroamide, are potent, broad-spectrum antibacterial compounds that target fatty acid synthesis in bacteria. Natural modifications at the C-5 dialkyl position in this molecular series result in pronounced bioactivity differences. The C-5 acetamide-containing thiotetroamide, which is the more potent antibacterial agent in this family, is biosynthesized from the C-5 ethyl analogue thiotetromycin via a unique two-enzyme process involving the cytochrome P450-amidotransferase enzyme pair TtmP-TtmN. Herein we synthesized a focused library of 17 novel thiotetromycin derivatives differing at the 5-position alkyl substituent to investigate their biological activities and their reactivity towards the hydroxylase TtmP. Although we observed marginal anti-tuberculosis activity, select thiotetromycin analogues showed antibacterial activity against an Escherichia coli ΔtolC strain with IC50 values in a range of 1.9-36 μg mL-1. Additional screening efforts highlighted select thiotetronate analogues as inhibitors of the cancer-associated enzyme nicotinamide N-methyltransferase (NNMT), with a unique scaffold compared to previously identified NNMT inhibitors. In vitro assays further showed that the TtmP P450 was capable of resolving racemic substrate mixtures and had modest promiscuity to hydroxylate derivatives with variable alkyl chains; however triple oxidation to a carboxylic acid remained specific for the natural thiotetromycin substrate. The tendency of TtmP to accept a range of unnatural substrates for hydroxylation makes it an interesting target for P450 engineering towards broader applications.
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Affiliation(s)
- Marlene L. Rothe
- Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, 92093, USA
| | - Jie Li
- Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, 92093, USA
- Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC,29208, USA
| | - Ernesto Garibay
- Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, 92093, USA
| | - Bradley S. Moore
- Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, 92093, USA
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, 92093, USA
| | - Shaun M. K. McKinnie
- Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, 92093, USA
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Chang HC, Mondal B, Fang H, Neese F, Bill E, Ye S. Electron Paramagnetic Resonance Signature of Tetragonal Low Spin Iron(V)-Nitrido and -Oxo Complexes Derived from the Electronic Structure Analysis of Heme and Non-Heme Archetypes. J Am Chem Soc 2019; 141:2421-2434. [PMID: 30620571 PMCID: PMC6728100 DOI: 10.1021/jacs.8b11429] [Citation(s) in RCA: 48] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
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Iron(V)-nitrido and -oxo complexes
have been proposed as key intermediates
in a diverse array of chemical transformations. Herein we present
a detailed electronic-structure analysis of [FeV(N)(TPP)]
(1, TPP2– = tetraphenylporphyrinato),
and [FeV(N)(cyclam-ac)]+ (2, cyclam-ac
= 1,4,8,11-tetraazacyclotetradecane-1-acetato) using electron paramagnetic
resonance (EPR) and 57Fe Mössbauer spectroscopy
coupled with wave function based complete active-space self-consistent
field (CASSCF) calculations. The findings were compared with all other
well-characterized genuine iron(V)-nitrido and -oxo complexes, [FeV(N)(MePy2tacn)](PF6)2 (3, MePy2tacn = methyl-N′,N″-bis(2-picolyl)-1,4,7-triazacyclononane), [FeV(N){PhB(t-BuIm)3}]+ (4, PhB(tBuIm)3– = phenyltris(3-tert-butylimidazol-2-ylidene)borate),
and [FeV(O)(TAML)]− (5,
TAML4– = tetraamido macrocyclic ligand). Our results
revealed that complex 1 is an authenticated iron(V)-nitrido
species and contrasts with its oxo congener, compound I, which contains
a ferryl unit interacting with a porphyrin radical. More importantly,
tetragonal iron(V)-nitrido and -oxo complexes 1–3 and 5 all possess an orbitally nearly doubly
degenerate S = 1/2 ground state. Consequently, analogous
near-axial EPR spectra with g|| < g⊥ ≤ 2 were measured for them,
and their g|| and g⊥ values were found to obey a simple relation of g⊥2 + (2 – g∥)2 = 4. However, the bonding situation for trigonal iron(V)-nitrido
complex 4 is completely different as evidenced by its
distinct EPR spectrum with g|| < 2
< g⊥. Further in-depth analyses
suggested that tetragonal low spin iron(V)-nitrido and -oxo complexes
feature electronic structures akin to those found for complexes 1–3 and 5. Therefore, the
characteristic EPR signals determined for 1–3 and 5 can be used as a spectroscopic marker
to identify such highly reactive intermediates in catalytic processes.
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Affiliation(s)
- Hao-Ching Chang
- Max-Planck-Institut für Kohlenforschung , Kaiser-Wilhelm-Platz 1 , D-45470 Mülheim an der Ruhr , Germany
| | - Bhaskar Mondal
- Max-Planck-Institut für Chemische Energiekonversion , Stiftstr. 34-36 , D-45470 Mülheim an der Ruhr , Germany
| | - Huayi Fang
- Max-Planck-Institut für Chemische Energiekonversion , Stiftstr. 34-36 , D-45470 Mülheim an der Ruhr , Germany
| | - Frank Neese
- Max-Planck-Institut für Kohlenforschung , Kaiser-Wilhelm-Platz 1 , D-45470 Mülheim an der Ruhr , Germany
| | - Eckhard Bill
- Max-Planck-Institut für Chemische Energiekonversion , Stiftstr. 34-36 , D-45470 Mülheim an der Ruhr , Germany
| | - Shengfa Ye
- Max-Planck-Institut für Kohlenforschung , Kaiser-Wilhelm-Platz 1 , D-45470 Mülheim an der Ruhr , Germany
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