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Mekuli R, Shoukat M, Dugat-Bony E, Bonnarme P, Landaud S, Swennen D, Hervé V. Iron-based microbial interactions: the role of iron metabolism in the cheese ecosystem. J Bacteriol 2025:e0053924. [PMID: 40237503 DOI: 10.1128/jb.00539-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/18/2025] Open
Abstract
Iron is involved in various microbial metabolisms and interactions and is an essential micronutrient for most microorganisms. This review focuses on the cheese ecosystem, in which iron is sparse (median concentration of 2.9 mg/kg based on a literature survey) and of limited bioavailability due to the presence of various metal-binding agents in the cheese matrix. Cheese microorganisms overcome this low bioavailability of iron by producing and/or importing ferric iron-specific chelators called siderophores. We introduce these siderophores and their specific transporters, which play a key role in ecological interactions and microbial metabolism. We discuss the impact of iron on all the major taxa (fungi, bacteria, and viruses) and functional groups (starters, ripening microorganisms, and pathogens) present and interacting in cheese, from the community to individual levels. We describe the ways in which cheese-ripening microorganisms use iron and the effects of iron limitation on major metabolic pathways, including the tricarboxylic acid (TCA) cycle and amino-acid biosynthesis. The cheese ecosystem is a relevant in situ model for improving our understanding of iron biochemistry and its putative role in microbe-microbe interactions. Yet, this review highlights critical gaps in our understanding of iron's role in cheese from fundamental ecological and biochemical perspectives to applied microbiology, with broader implications for the quality, safety, and organoleptic properties of cheese.
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Affiliation(s)
- Rina Mekuli
- Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, Palaiseau, France
| | - Mahtab Shoukat
- Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, Palaiseau, France
| | - Eric Dugat-Bony
- Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, Palaiseau, France
| | - Pascal Bonnarme
- Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, Palaiseau, France
| | - Sophie Landaud
- Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, Palaiseau, France
| | - Dominique Swennen
- Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, Palaiseau, France
| | - Vincent Hervé
- Université Paris-Saclay, INRAE, AgroParisTech, UMR SayFood, Palaiseau, France
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2
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Garg R, Zhu Z, Hernandez FG, Wang Y, David MS, Bruno VM, Culotta VC. A response to iron involving carbon metabolism in the opportunistic fungal pathogen Candida albicans. mSphere 2025:e0004025. [PMID: 40183578 DOI: 10.1128/msphere.00040-25] [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: 01/16/2025] [Accepted: 03/09/2025] [Indexed: 04/05/2025] Open
Abstract
Iron (Fe) is an essential micronutrient, and during infection, the host attempts to starve pathogens of this vital element through a process known as nutritional immunity. Successful pathogens have evolved means to evade this attack, an example being Candida albicans, the most prevalent human fungal pathogen. When Fe-starved, C. albicans induces multiple pathways for Fe uptake using the SEF1 trans-regulator, and we now describe a previously unrecognized effect of Fe on C. albicans metabolism that occurs independent of SEF1. Specifically, Fe limitation leads to inhibition of pyruvate dehydrogenase (PDH) connecting glycolysis to mitochondrial respiration. PDH inactivation involves loss of the LAT1 catalytic subunit harboring a lipoic acid co-factor. Protein lipoylation is a Fe-S dependent process, and lipoylated alpha-ketoglutarate dehydrogenase is also inhibited in Fe-starved C. albicans. SEF1 does not protect against PDH inactivation, and despite SEF1 induction of Fe import genes, cellular Fe levels drop dramatically during chronic Fe starvation. Such loss of LAT1 and lipoylation is also seen in Fe-starved bakers' yeast Saccharomyces cerevisiae. In both yeast species, glucose is diverted toward the pentose phosphate pathway (PPP) and PPP production of NADPH is increased in response to low Fe and PDH loss. Additionally, glucose consumption is lowered in Fe-starved C. albicans, and non-PDH alternatives to producing Ac-CoA are induced, including pyruvate bypass and fatty acid oxidation pathways. C. albicans can adapt well to the effects of micronutrient loss on cell metabolism. IMPORTANCE We describe a new response to Fe-starvation in a fungal pathogen involving carbon metabolism. Pyruvate dehydrogenase (PDH) that is central to glucose metabolism is inactivated at the post-translational level in Fe-starved cells. Nevertheless, the fungal pathogen can thrive by activating backup systems for metabolizing glucose. Methods that inhibit these compensatory pathways for carbon metabolism may prove beneficial in future anti-fungal strategies.
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Affiliation(s)
- Ritu Garg
- Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA
| | - Zhengkai Zhu
- Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA
| | - Francisco G Hernandez
- Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA
| | - Yiran Wang
- Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA
| | - Marika S David
- Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA
| | - Vincent M Bruno
- Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Valeria C Culotta
- Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA
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3
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Jospe-Kaufman M, Fridman M. Illuminating antifungal mode of action and resistance with fluorescent probes. Curr Opin Chem Biol 2025; 85:102570. [PMID: 39965367 DOI: 10.1016/j.cbpa.2025.102570] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2024] [Revised: 12/27/2024] [Accepted: 01/18/2025] [Indexed: 02/20/2025]
Abstract
The rise in fungal infections, driven by pathogens resistant to the limited scope of antifungal agents available, poses an increasing threat to global health and the economy. Addressing this challenge requires a thorough understanding of the mechanisms of antifungal agents and the development of advanced resistance diagnostic methods. This opinion manuscript highlights recent advancements in antifungal research, with a focus on chemical biology approaches, particularly the development of fluorescent probes derived from various antifungal agents. These probes reveal new aspects of antifungal activity and provide deeper insights into modes of action and resistance mechanisms. Live cell imaging of fungal pathogens labeled with these probes has uncovered novel strategies to enhance antifungal efficacy, understand virulence factors, and detect resistance. These unique small-molecule tools offer powerful new avenues for addressing the fungal infections crisis, harnessing chemical biology approaches to develop innovative solutions to the global challenges posed by fungi.
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Affiliation(s)
- Moriah Jospe-Kaufman
- School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 6997801, Israel
| | - Micha Fridman
- School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 6997801, Israel.
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Deng X, Li H, Wu A, He J, Mao X, Dai Z, Tian G, Cai J, Tang J, Luo Y. Composition, Influencing Factors, and Effects on Host Nutrient Metabolism of Fungi in Gastrointestinal Tract of Monogastric Animals. Animals (Basel) 2025; 15:710. [PMID: 40075993 PMCID: PMC11898470 DOI: 10.3390/ani15050710] [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: 01/07/2025] [Revised: 02/26/2025] [Accepted: 02/28/2025] [Indexed: 03/14/2025] Open
Abstract
Intestinal fungi, collectively referred to as mycobiota, constitute a small (0.01-2%) but crucial component of the overall intestinal microbiota. While fungi are far less abundant than bacteria in the gut, the volume of an average fungal cell is roughly 100-fold greater than that of an average bacterial cell. They play a vital role in nutrient metabolism and maintaining intestinal health. The composition and spatial organization of mycobiota vary across different animal species and are influenced by a multitude of factors, including age, diet, and the host's physiological state. At present, quantitative research on the composition of mycobiota in monogastric animals remains scarce, and investigations into the mechanisms underlying their metabolic functions are also relatively restricted. This review delves into the distribution characteristics of mycobiota, including Candida albicans, Saccharomyces cerevisiae, Kazachstania slooffiae, in monogastric animals, the factors influencing their composition, and the consequent impacts on host metabolism and health. The objective is to offer insights for a deeper understanding of the nutritional significance of intestinal fungi in monogastric animals and to explore the mechanisms by which they affect host health in relation to inflammatory bowel disease (IBD), diarrhea, and obesity. Through a systematic evaluation of their functional contributions, this review shifts our perception of intestinal fungi from overlooked commensals to key components in gut ecosystem dynamics, emphasizing their potential in personalized metabolic control regulation and the enhancement of disease prevention and treatment strategies.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | - Yuheng Luo
- Key Laboratory for Animal Disease-Resistance Nutrition of Ministry of Education of China, Key Laboratory for Animal Disease-Resistance Nutrition and Feed of Ministry of Agriculture of China, Key Laboratory of Animal Disease-Resistant Nutrition of Sichuan Province, Engineering Research Center of Animal Disease-Resistance Nutrition Biotechnology of Ministry of Education of China, Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China; (X.D.); (H.L.); (A.W.); (J.H.); (X.M.); (Z.D.); (G.T.); (J.C.); (J.T.)
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5
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Tanwar S, Kalra S, Bari VK. Insights into the role of sterol metabolism in antifungal drug resistance: a mini-review. Front Microbiol 2024; 15:1409085. [PMID: 39464401 PMCID: PMC11502366 DOI: 10.3389/fmicb.2024.1409085] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2024] [Accepted: 09/26/2024] [Indexed: 10/29/2024] Open
Abstract
Sterols are essential for eukaryotic cells and are crucial in cellular membranes' structure, function, fluidity, permeability, adaptability to environmental stressors, and host-pathogen interactions. Fungal sterol, such as ergosterol metabolism, involves several organelles, including the mitochondria, lipid droplets, endoplasmic reticulum, and peroxisomes that can be regulated mainly by feedback mechanisms and transcriptionally. The majority of sterol transport in yeast occurs via non-vesicular transport pathways mediated by lipid transfer proteins, which determine the quantity of sterol present in the cell membrane. Pathogenic fungi Candida, Aspergillus, and Cryptococcus species can cause a range of superficial to potentially fatal systemic and invasive infections that are more common in immunocompromised patients. There is a significant risk of morbidity and mortality from these infections, which are very difficult to cure. Several antifungal drugs with different modes of action have received clinical approval to treat fungal infections. Antifungal drugs targeting the ergosterol biosynthesis pathway are well-known for their antifungal activity; however, an imbalance in the regulation and transport of ergosterol could lead to resistance to antifungal therapy. This study summarizes how fungal sterol metabolism and regulation can modulate sterol-targeting antifungal drug resistance.
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6
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Hammond J, Das IM, Paenga R, Caddie M, Skinner D, Sheridan JP, Miller MR, Munkacsi AB. Multi-omic analysis reveals genes and proteins integral to bioactivity of Echinochrome A isolated from the waste stream of the sea urchin industry in Aotearoa New Zealand. Food Sci Nutr 2024; 12:4927-4943. [PMID: 39055184 PMCID: PMC11266889 DOI: 10.1002/fsn3.4140] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2023] [Revised: 03/07/2024] [Accepted: 03/16/2024] [Indexed: 07/27/2024] Open
Abstract
Evechinus chloroticus (commonly known as kina) is a sea urchin species endemic to New Zealand. Its roe is a culinary delicacy to the indigenous Māori and a globally exported food product. Echinochrome A (Ech A) is a bioactive compound isolated from the waste product of kina shells and spines; however, the molecular mechanisms of Ech A bioactivity are not well understood, partly due to Ech A never being studied using unbiased genome-wide analysis. To explore the high-value pharmaceutical potential of kina food waste, we obtained unbiased functional genomic and proteomic profiles of yeast cells treated with Echinochrome A. Abundance was measured for 4100 proteins every 30 min for four hours using fluorescent microscopy, resulting in the identification of 92 proteins with significant alterations in protein abundance caused by Ech A treatment that were over-represented with specific changes in DNA replication, repair and RNA binding after 30 min, followed by specific changes in the metabolism of metal ions (specifically iron and copper) from 60-240 min. Further analysis indicated that Ech A chelated iron, and that iron supplementation negated the growth inhibition caused by Ech A. Via a growth-based genome-wide analysis of 4800 gene deletion strains, 20 gene deletion strains were sensitive to Ech A in an iron-dependent manner. These genes were over-represented in the cellular response to oxidative stress, suggesting that Ech A suppressed growth inhibition caused by oxidative stress. Unexpectedly, genes integral to cardiolipin and inositol phosphate biosynthesis were required for Ech A bioactivity. Overall, these results identify genes, proteins, and cellular processes mediating the bioactivity of Ech A. Moreover, we demonstrate unbiased genomic and proteomic methodology that will be useful for characterizing bioactive compounds in food and food waste.
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Affiliation(s)
- Joseph Hammond
- School of Biological SciencesVictoria University of WellingtonWellingtonNew Zealand
| | | | - Ruihana Paenga
- Hikurangi Bioactives Limited PartnershipRuatōriaNew Zealand
| | - Manu Caddie
- Hikurangi Bioactives Limited PartnershipRuatōriaNew Zealand
| | - Damian Skinner
- Hikurangi Bioactives Limited PartnershipRuatōriaNew Zealand
| | - Jeffrey P. Sheridan
- School of Biological SciencesVictoria University of WellingtonWellingtonNew Zealand
| | | | - Andrew B. Munkacsi
- School of Biological SciencesVictoria University of WellingtonWellingtonNew Zealand
- Centre for BiodiscoveryVictoria University of WellingtonWellingtonNew Zealand
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7
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Calvanese M, D’Angelo C, Tutino ML, Lauro C. Whole-Cell Biosensor for Iron Monitoring as a Potential Tool for Safeguarding Biodiversity in Polar Marine Environments. Mar Drugs 2024; 22:299. [PMID: 39057408 PMCID: PMC11277574 DOI: 10.3390/md22070299] [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: 04/11/2024] [Revised: 06/05/2024] [Accepted: 06/24/2024] [Indexed: 07/28/2024] Open
Abstract
Iron is a key micronutrient essential for various essential biological processes. As a consequence, alteration in iron concentration in seawater can deeply influence marine biodiversity. In polar marine environments, where environmental conditions are characterized by low temperatures, the role of iron becomes particularly significant. While iron limitation can negatively influence primary production and nutrient cycling, excessive iron concentrations can lead to harmful algal blooms and oxygen depletion. Furthermore, the growth of certain phytoplankton species can be increased in high-iron-content environments, resulting in altered balance in the marine food web and reduced biodiversity. Although many chemical/physical methods are established for inorganic iron quantification, the determination of the bio-available iron in seawater samples is more suitably carried out using marine microorganisms as biosensors. Despite existing challenges, whole-cell biosensors offer other advantages, such as real-time detection, cost-effectiveness, and ease of manipulation, making them promising tools for monitoring environmental iron levels in polar marine ecosystems. In this review, we discuss fundamental biosensor designs and assemblies, arranging host features, transcription factors, reporter proteins, and detection methods. The progress in the genetic manipulation of iron-responsive regulatory and reporter modules is also addressed to the optimization of the biosensor performance, focusing on the improvement of sensitivity and specificity.
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Affiliation(s)
- Marzia Calvanese
- Department of Chemical Sciences, University of Naples “Federico II”, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Naples, Italy; (M.C.); (C.D.); (M.L.T.)
- Istituto Nazionale Biostrutture e Biosistemi (I.N.B.B), Viale Medaglie D’Oro 305, 00136 Roma, Italy
| | - Caterina D’Angelo
- Department of Chemical Sciences, University of Naples “Federico II”, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Naples, Italy; (M.C.); (C.D.); (M.L.T.)
| | - Maria Luisa Tutino
- Department of Chemical Sciences, University of Naples “Federico II”, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Naples, Italy; (M.C.); (C.D.); (M.L.T.)
- Istituto Nazionale Biostrutture e Biosistemi (I.N.B.B), Viale Medaglie D’Oro 305, 00136 Roma, Italy
| | - Concetta Lauro
- Department of Chemical Sciences, University of Naples “Federico II”, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Naples, Italy; (M.C.); (C.D.); (M.L.T.)
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8
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Liu D, Hu Z, Lu J, Yi C. Redox-Regulated Iron Metabolism and Ferroptosis in Ovarian Cancer: Molecular Insights and Therapeutic Opportunities. Antioxidants (Basel) 2024; 13:791. [PMID: 39061859 PMCID: PMC11274267 DOI: 10.3390/antiox13070791] [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: 05/01/2024] [Revised: 06/21/2024] [Accepted: 06/24/2024] [Indexed: 07/28/2024] Open
Abstract
Ovarian cancer (OC), known for its lethality and resistance to chemotherapy, is closely associated with iron metabolism and ferroptosis-an iron-dependent cell death process, distinct from both autophagy and apoptosis. Emerging evidence suggests that dysregulation of iron metabolism could play a crucial role in OC by inducing an imbalance in the redox system, which leads to ferroptosis, offering a novel therapeutic approach. This review examines how disruptions in iron metabolism, which affect redox balance, impact OC progression, focusing on its essential cellular functions and potential as a therapeutic target. It highlights the molecular interplay, including the role of non-coding RNAs (ncRNAs), between iron metabolism and ferroptosis, and explores their interactions with key immune cells such as macrophages and T cells, as well as inflammation within the tumor microenvironment. The review also discusses how glycolysis-related iron metabolism influences ferroptosis via reactive oxygen species. Targeting these pathways, especially through agents that modulate iron metabolism and ferroptosis, presents promising therapeutic prospects. The review emphasizes the need for deeper insights into iron metabolism and ferroptosis within the redox-regulated system to enhance OC therapy and advocates for continued research into these mechanisms as potential strategies to combat OC.
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Affiliation(s)
- Dan Liu
- Department of Obstetrics and Gynecology, The First Affiliated Hospital, Yangtze University, Jingzhou 434000, China; (D.L.); (Z.H.)
- Hubei Provincial Clinical Research Center for Personalized Diagnosis and Treatment of Cancer, Jingzhou 434000, China
| | - Zewen Hu
- Department of Obstetrics and Gynecology, The First Affiliated Hospital, Yangtze University, Jingzhou 434000, China; (D.L.); (Z.H.)
- Hubei Provincial Clinical Research Center for Personalized Diagnosis and Treatment of Cancer, Jingzhou 434000, China
| | - Jinzhi Lu
- Hubei Provincial Clinical Research Center for Personalized Diagnosis and Treatment of Cancer, Jingzhou 434000, China
- Department of Laboratory Medicine, The First Affiliated Hospital, Yangtze University, Jingzhou 434000, China
| | - Cunjian Yi
- Department of Obstetrics and Gynecology, The First Affiliated Hospital, Yangtze University, Jingzhou 434000, China; (D.L.); (Z.H.)
- Hubei Provincial Clinical Research Center for Personalized Diagnosis and Treatment of Cancer, Jingzhou 434000, China
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9
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Moyung K, Li Y, Hartemink AJ, MacAlpine DM. Genome-wide nucleosome and transcription factor responses to genetic perturbations reveal chromatin-mediated mechanisms of transcriptional regulation. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.05.24.595391. [PMID: 38826400 PMCID: PMC11142231 DOI: 10.1101/2024.05.24.595391] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/04/2024]
Abstract
Epigenetic mechanisms contribute to gene regulation by altering chromatin accessibility through changes in transcription factor (TF) and nucleosome occupancy throughout the genome. Despite numerous studies focusing on changes in gene expression, the intricate chromatin-mediated regulatory code remains largely unexplored on a comprehensive scale. We address this by employing a factor-agnostic, reverse-genetics approach that uses MNase-seq to capture genome-wide TF and nucleosome occupancies in response to the individual deletion of 201 transcriptional regulators in Saccharomyces cerevisiae, thereby assaying nearly one million mutant-gene interactions. We develop a principled approach to identify and quantify chromatin changes genome-wide, observing differences in TF and nucleosome occupancy that recapitulate well-established pathways identified by gene expression data. We also discover distinct chromatin signatures associated with the up- and downregulation of genes, and use these signatures to reveal regulatory mechanisms previously unexplored in expression-based studies. Finally, we demonstrate that chromatin features are predictive of transcriptional activity and leverage these features to reconstruct chromatin-based transcriptional regulatory networks. Overall, these results illustrate the power of an approach combining genetic perturbation with high-resolution epigenomic profiling; the latter enables a close examination of the interplay between TFs and nucleosomes genome-wide, providing a deeper, more mechanistic understanding of the complex relationship between chromatin organization and transcription.
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Affiliation(s)
- Kevin Moyung
- Program in Computational Biology and Bioinformatics, Duke University, Durham, NC 27708
- Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710
| | - Yulong Li
- Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710
- Department of Computer Science, Duke University, Durham, NC 27708
| | - Alexander J. Hartemink
- Program in Computational Biology and Bioinformatics, Duke University, Durham, NC 27708
- Department of Computer Science, Duke University, Durham, NC 27708
| | - David M. MacAlpine
- Program in Computational Biology and Bioinformatics, Duke University, Durham, NC 27708
- Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710
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10
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Yona A, Fridman M. Poacic Acid, a Plant-Derived Stilbenoid, Augments Cell Wall Chitin Production, but Its Antifungal Activity Is Hindered by This Polysaccharide and by Fungal Essential Metals. Biochemistry 2024; 63:1051-1065. [PMID: 38533731 PMCID: PMC11025111 DOI: 10.1021/acs.biochem.3c00595] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2023] [Revised: 03/17/2024] [Accepted: 03/18/2024] [Indexed: 03/28/2024]
Abstract
Climate and environmental changes have modified the habitats of fungal pathogens, inflicting devastating effects on livestock and crop production. Additionally, drug-resistant fungi are increasing worldwide, driving the urgent need to identify new molecular scaffolds for the development of antifungal agents for humans, animals, and plants. Poacic acid (PA), a plant-derived stilbenoid, was recently discovered to be a novel molecular scaffold that inhibits the growth of several fungi. Its antifungal activity has been associated with perturbation of the production/assembly of the fungal cell wall β-1,3-glucan, but its mode of action is not resolved. In this study, we investigated the antifungal activity of PA and its derivatives on a panel of yeast. PA had a fungistatic effect on S. cerevisiae and a fungicidal effect on plasma membrane-damaged Candida albicans mutants. Live cell fluorescence microscopy experiments revealed that PA increases chitin production and modifies its cell wall distribution. Chitin production and cell growth returned to normal after prolonged incubation. The antifungal activity of PA was reduced in the presence of exogenous chitin, suggesting that the potentiation of chitin production is a stress response that helps the yeast cell overcome the effect of this antifungal stilbenoid. Growth inhibition was also reduced by metal ions, indicating that PA affects the metal homeostasis. These findings suggest that PA has a complex antifungal mechanism of action that involves perturbation of the cell wall β-1,3-glucan production/assembly, chitin production, and metal homeostasis.
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Affiliation(s)
- Adi Yona
- School of Chemistry, Raymond
& Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 6997801, Israel
| | - Micha Fridman
- School of Chemistry, Raymond
& Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 6997801, Israel
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11
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Egebjerg JM, Szomek M, Thaysen K, Juhl AD, Kozakijevic S, Werner S, Pratsch C, Schneider G, Kapishnikov S, Ekman A, Röttger R, Wüstner D. Automated quantification of vacuole fusion and lipophagy in Saccharomyces cerevisiae from fluorescence and cryo-soft X-ray microscopy data using deep learning. Autophagy 2024; 20:902-922. [PMID: 37908116 PMCID: PMC11062380 DOI: 10.1080/15548627.2023.2270378] [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: 03/06/2023] [Revised: 09/12/2023] [Accepted: 10/02/2023] [Indexed: 11/02/2023] Open
Abstract
During starvation in the yeast Saccharomyces cerevisiae vacuolar vesicles fuse and lipid droplets (LDs) can become internalized into the vacuole in an autophagic process named lipophagy. There is a lack of tools to quantitatively assess starvation-induced vacuole fusion and lipophagy in intact cells with high resolution and throughput. Here, we combine soft X-ray tomography (SXT) with fluorescence microscopy and use a deep-learning computational approach to visualize and quantify these processes in yeast. We focus on yeast homologs of mammalian NPC1 (NPC intracellular cholesterol transporter 1; Ncr1 in yeast) and NPC2 proteins, whose dysfunction leads to Niemann Pick type C (NPC) disease in humans. We developed a convolutional neural network (CNN) model which classifies fully fused versus partially fused vacuoles based on fluorescence images of stained cells. This CNN, named Deep Yeast Fusion Network (DYFNet), revealed that cells lacking Ncr1 (ncr1∆ cells) or Npc2 (npc2∆ cells) have a reduced capacity for vacuole fusion. Using a second CNN model, we implemented a pipeline named LipoSeg to perform automated instance segmentation of LDs and vacuoles from high-resolution reconstructions of X-ray tomograms. From that, we obtained 3D renderings of LDs inside and outside of the vacuole in a fully automated manner and additionally measured droplet volume, number, and distribution. We find that ncr1∆ and npc2∆ cells could ingest LDs into vacuoles normally but showed compromised degradation of LDs and accumulation of lipid vesicles inside vacuoles. Our new method is versatile and allows for analysis of vacuole fusion, droplet size and lipophagy in intact cells.Abbreviations: BODIPY493/503: 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-Indacene; BPS: bathophenanthrolinedisulfonic acid disodium salt hydrate; CNN: convolutional neural network; DHE; dehydroergosterol; npc2∆, yeast deficient in Npc2; DSC, Dice similarity coefficient; EM, electron microscopy; EVs, extracellular vesicles; FIB-SEM, focused ion beam milling-scanning electron microscopy; FM 4-64, N-(3-triethylammoniumpropyl)-4-(6-[4-{diethylamino} phenyl] hexatrienyl)-pyridinium dibromide; LDs, lipid droplets; Ncr1, yeast homolog of human NPC1 protein; ncr1∆, yeast deficient in Ncr1; NPC, Niemann Pick type C; NPC2, Niemann Pick type C homolog; OD600, optical density at 600 nm; ReLU, rectifier linear unit; PPV, positive predictive value; NPV, negative predictive value; MCC, Matthews correlation coefficient; SXT, soft X-ray tomography; UV, ultraviolet; YPD, yeast extract peptone dextrose.
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Affiliation(s)
- Jacob Marcus Egebjerg
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark
- Department of Mathematics and Computer Science, University of Southern Denmark, Odense M, Denmark
| | - Maria Szomek
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark
| | - Katja Thaysen
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark
| | - Alice Dupont Juhl
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark
| | - Suzana Kozakijevic
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark
| | - Stephan Werner
- Department of X‑Ray Microscopy, Helmholtz-Zentrum Berlin, Germany and Humboldt-Universität zu Berlin, Institut für Physik, Berlin, Germany
| | - Christoph Pratsch
- Department of X‑Ray Microscopy, Helmholtz-Zentrum Berlin, Germany and Humboldt-Universität zu Berlin, Institut für Physik, Berlin, Germany
| | - Gerd Schneider
- Department of X‑Ray Microscopy, Helmholtz-Zentrum Berlin, Germany and Humboldt-Universität zu Berlin, Institut für Physik, Berlin, Germany
| | - Sergey Kapishnikov
- SiriusXT, 9A Holly Ave. Stillorgan Industrial Park, Blackrock, Co, Dublin, Ireland
| | - Axel Ekman
- Department of Biological and Environmental Science and Nanoscience Centre, University of Jyväskylä, Jyväskylä, Finland
| | - Richard Röttger
- Department of Mathematics and Computer Science, University of Southern Denmark, Odense M, Denmark
| | - Daniel Wüstner
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark
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12
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Carvalho LC, Oliveira ALS, Carsanba E, Lopes A, Leal T, Ribeiro M, Fernandes S, Pintado M, Oliveira C. Removal of phenolic compounds from sugarcane syrup and impact on Saccharomyces cerevisiae fermentation for β-farnesene production. Biotechnol J 2024; 19:e2300465. [PMID: 38403437 DOI: 10.1002/biot.202300465] [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: 09/05/2023] [Revised: 12/17/2023] [Accepted: 01/03/2024] [Indexed: 02/27/2024]
Abstract
This work aimed to study for the first time the effects of phenolic compounds from sugarcane syrup on Saccharomyces cerevisiae β-farnesene fermentation by removing them from this feedstock. Syrup purification was optimized through a central composite design using five types of activated charcoal: three contact times (1-24 h) and three adsorbent concentrations (10-150 g L-1 ). The optimal purification condition-charcoal pellets at 115 g L-1 and contact time of 12.5 h-led to 96.7% of phenolic compounds removal and 43.7% of syrup recovery. The effects of reducing phenolic content from approximately 7.0-0.3 mg L-1 in sugarcane syrup on yeast fermentation varied with the scale. An increase in biomolecule productivity was only observed in shake-flasks (11%) and in biomass productivity only in the 2 L bioreactor (12%). Thus, phenolic compounds from sugarcane syrup do not influence β-farnesene production at a large scale under the conditions tested.
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Affiliation(s)
- Luís Carlos Carvalho
- Amyris BioProducts Portugal, Unipessoal, Porto, Portugal
- Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal
| | - Ana L S Oliveira
- Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal
| | - Erdem Carsanba
- Amyris BioProducts Portugal, Unipessoal, Porto, Portugal
- Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal
| | - Ana Lopes
- Amyris BioProducts Portugal, Unipessoal, Porto, Portugal
- Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal
| | - Tânia Leal
- Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal
| | - Mónica Ribeiro
- Amyris BioProducts Portugal, Unipessoal, Porto, Portugal
- Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal
| | - Sara Fernandes
- Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal
| | - Manuela Pintado
- Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal
| | - Carla Oliveira
- Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal
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13
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Dong Z, Liu H, Wan D, Wu X, Yin Y. Ferrous-sucrose complex supplementation regulates maternal plasma metabolism and the fecal microbiota composition and improves neonatal immunity and placental glucose transportation by activating the EGF/PI3K/AKT signaling pathways in sows. Food Funct 2024; 15:906-916. [PMID: 38168829 DOI: 10.1039/d3fo03733a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2024]
Abstract
Pregnancy is a dynamic state involving rapid physiological changes in metabolism, affecting the health and development of the offspring. During pregnancy, the placenta constitutes a physical and immunological barrier to provide fetal nutrition through the maternal blood and prevent the exposure of the fetus to dangerous signals. Metabolic changes in the plasma, the fecal microbiota profile, and functional regulation in the placenta were studied in sows supplied with a ferrous-sucrose complex (FeSuc) from late gestation to parturition. The results revealed that maternal FeSuc supplementation enhanced arginine and proline metabolism, glutathione metabolism, with increased glutamic acid, beta-D-glucosamine, L-proline, 1-butylamine, and succinic acid and reduced sphingosine and chenodeoxycholic acid sulfate levels in the plasma. Moreover, significantly increased abundances of Christensenellaceae_R-7_group, Prevotellaceae_NK3B31_group, and Lachnospiraceae_NK4B4_group were detected in the feces of sows from the FeSuc group (P < 0.05). Spearman's correlation analysis indicated that Prevotellaceae_NK3B31_group abundances were positively correlated with glutamic acid, indoxyl sulfate, acetyl-DL-leucine, and beta-D-glucosamine, while Christensenellaceae_R-7_group was positively correlated with beta-D-glucosamine. Furthermore, maternal FeSuc supplementation significantly increased neonatal glucose (P < 0.01) and iron (P < 0.01) in the neonatal serum, significantly increased IL-10 and TGF-β1 levels in the neonatal liver (P < 0.01) and jejunum (P < 0.05), promoted the transcription of immune molecules in the placenta, and significantly increased the protein expressions of EGF (P < 0.05), PI3K (P < 0.01), p-PI3K (P < 0.001), p-AKT (P < 0.01), and glucose transporter 1 (GLUT1) (P < 0.001) in the placenta. The current study demonstrated that FeSuc supplementation regulated maternal metabolism processes by altering the fecal microbial composition and improved neonatal immunity and placental glucose transportation by activating the EGF/PI3K/AKT signaling pathways in sows.
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Affiliation(s)
- Zhenglin Dong
- Key Laboratory of Agro-Ecological Processes in Subtropical Region, Hunan Research Center of Livestock & Poultry Sciences, South-Central Experimental Station of Animal Nutrition and Feed Science in Ministry of Agriculture, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha, Hunan 410125, China.
| | - Hongwei Liu
- Key Laboratory of Agro-Ecological Processes in Subtropical Region, Hunan Research Center of Livestock & Poultry Sciences, South-Central Experimental Station of Animal Nutrition and Feed Science in Ministry of Agriculture, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha, Hunan 410125, China.
| | - Dan Wan
- Key Laboratory of Agro-Ecological Processes in Subtropical Region, Hunan Research Center of Livestock & Poultry Sciences, South-Central Experimental Station of Animal Nutrition and Feed Science in Ministry of Agriculture, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha, Hunan 410125, China.
| | - Xin Wu
- Key Laboratory of Agro-Ecological Processes in Subtropical Region, Hunan Research Center of Livestock & Poultry Sciences, South-Central Experimental Station of Animal Nutrition and Feed Science in Ministry of Agriculture, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha, Hunan 410125, China.
- Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
| | - Yulong Yin
- Key Laboratory of Agro-Ecological Processes in Subtropical Region, Hunan Research Center of Livestock & Poultry Sciences, South-Central Experimental Station of Animal Nutrition and Feed Science in Ministry of Agriculture, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha, Hunan 410125, China.
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14
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Pijuan J, Moreno DF, Yahya G, Moisa M, Ul Haq I, Krukiewicz K, Mosbah R, Metwally K, Cavalu S. Regulatory and pathogenic mechanisms in response to iron deficiency and excess in fungi. Microb Biotechnol 2023; 16:2053-2071. [PMID: 37804207 PMCID: PMC10616654 DOI: 10.1111/1751-7915.14346] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2023] [Revised: 09/14/2023] [Accepted: 09/19/2023] [Indexed: 10/09/2023] Open
Abstract
Iron is an essential element for all eukaryote organisms because of its redox properties, which are important for many biological processes such as DNA synthesis, mitochondrial respiration, oxygen transport, lipid, and carbon metabolism. For this reason, living organisms have developed different strategies and mechanisms to optimally regulate iron acquisition, transport, storage, and uptake in different environmental responses. Moreover, iron plays an essential role during microbial infections. Saccharomyces cerevisiae has been of key importance for decrypting iron homeostasis and regulation mechanisms in eukaryotes. Specifically, the transcription factors Aft1/Aft2 and Yap5 regulate the expression of genes to control iron metabolism in response to its deficiency or excess, adapting to the cell's iron requirements and its availability in the environment. We also review which iron-related virulence factors have the most common fungal human pathogens (Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans). These factors are essential for adaptation in different host niches during pathogenesis, including different fungal-specific iron-uptake mechanisms. While being necessary for virulence, they provide hope for developing novel antifungal treatments, which are currently scarce and usually toxic for patients. In this review, we provide a compilation of the current knowledge about the metabolic response to iron deficiency and excess in fungi.
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Affiliation(s)
- Jordi Pijuan
- Laboratory of Neurogenetics and Molecular MedicineInstitut de Recerca Sant Joan de DéuBarcelonaSpain
- Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIIIMadridSpain
| | - David F. Moreno
- Department of Molecular Cellular and Developmental BiologyYale UniversityNew HavenConnecticutUSA
- Systems Biology InstituteYale UniversityWest HavenConnecticutUSA
- Institut de Génétique et de Biologie Moléculaire et CellulaireIllkirchFrance
| | - Galal Yahya
- Department of Microbiology and Immunology, Faculty of PharmacyZagazig UniversityAl SharqiaEgypt
| | - Mihaela Moisa
- Faculty of Medicine and PharmacyUniversity of OradeaOradeaRomania
| | - Ihtisham Ul Haq
- Department of Physical Chemistry and Polymers TechnologySilesian University of TechnologyGliwicePoland
- Programa de Pós‐graduação em Inovação TecnológicaUniversidade Federal de Minas GeraisBelo HorizonteBrazil
| | - Katarzyna Krukiewicz
- Department of Physical Chemistry and Polymers TechnologySilesian University of TechnologyGliwicePoland
- Centre for Organic and Nanohybrid ElectronicsSilesian University of TechnologyGliwicePoland
| | - Rasha Mosbah
- Infection Control UnitHospitals of Zagazig UniversityZagazigEgypt
| | - Kamel Metwally
- Department of Medicinal Chemistry, Faculty of PharmacyUniversity of TabukTabukSaudi Arabia
- Department of Pharmaceutical Medicinal Chemistry, Faculty of PharmacyZagazig UniversityZagazigEgypt
| | - Simona Cavalu
- Faculty of Medicine and PharmacyUniversity of OradeaOradeaRomania
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15
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Jordá T, Rozès N, Martínez-Pastor MT, Puig S. The yeast mRNA-binding protein Cth2 post-transcriptionally modulates ergosterol biosynthesis in response to iron deficiency. BIOCHIMICA ET BIOPHYSICA ACTA. GENE REGULATORY MECHANISMS 2023; 1866:194959. [PMID: 37453649 DOI: 10.1016/j.bbagrm.2023.194959] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/26/2023] [Revised: 06/28/2023] [Accepted: 07/11/2023] [Indexed: 07/18/2023]
Abstract
Sterol synthesis is an iron-dependent metabolic pathway in eukaryotes. Consequently, fungal ergosterol biosynthesis (ERG) is down-regulated in response to iron deficiency. In this report, we show that, upon iron limitation or overexpression of the iron-regulated mRNA-binding protein Cth2, the yeast Saccharomyces cerevisiae down-regulates the three initial enzymatic steps of ergosterol synthesis (ERG1, ERG7 and ERG11). Mechanistically, we show that Cth2 protein limits the translation and promotes the decrease in the mRNA levels of these specific ERG genes, which contain consensus Cth2-binding sites defined as AU-rich elements (AREs). Thus, expression of CTH2 leads to the accumulation of initial sterol intermediates, such as squalene, and to the drop of ergosterol levels. Changes in CTH2 expression levels disturb the response of yeast cells to stresses related to membrane integrity such as high ethanol and sorbitol concentrations. Therefore, CTH2 should be considered as a critical regulatory factor of ergosterol biosynthesis during iron deficiency.
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Affiliation(s)
- Tania Jordá
- Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Paterna, Valencia, Spain.
| | - Nicolas Rozès
- Departament de Bioquímica i Biotecnologia, Facultat d'Enologia, Universitat Rovira i Virgili, Tarragona, Spain
| | - María Teresa Martínez-Pastor
- Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Paterna, Valencia, Spain; Departament de Bioquímica i Biologia Molecular, Facultat de Ciències Biològiques, Universitat de València, Burjassot, Valencia, Spain
| | - Sergi Puig
- Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Paterna, Valencia, Spain.
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16
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Hammond N, Snider J, Stagljar I, Mitchell K, Lagutin K, Jessulat M, Babu M, Teesdale-Spittle PH, Sheridan JP, Sturley SL, Munkacsi AB. Identification and characterization of protein interactions with the major Niemann-Pick type C disease protein in yeast reveals pathways of therapeutic potential. Genetics 2023; 225:iyad129. [PMID: 37440478 PMCID: PMC10471228 DOI: 10.1093/genetics/iyad129] [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: 01/12/2023] [Revised: 01/12/2023] [Accepted: 06/20/2023] [Indexed: 07/15/2023] Open
Abstract
Niemann-Pick type C (NP-C) disease is a rare lysosomal storage disease caused by mutations in NPC1 (95% cases) or NPC2 (5% cases). These proteins function together in cholesterol egress from the lysosome, whereby upon mutation, cholesterol and other lipids accumulate causing major pathologies. However, it is not fully understood how cholesterol is transported from NPC1 residing at the lysosomal membrane to the endoplasmic reticulum (ER) and plasma membrane. The yeast ortholog of NPC1, Niemann-Pick type C-related protein-1 (Ncr1), functions similarly to NPC1; when transfected into a mammalian cell lacking NPC1, Ncr1 rescues the diagnostic hallmarks of cholesterol and sphingolipid accumulation. Here, we aimed to identify and characterize protein-protein interactions (PPIs) with the yeast Ncr1 protein. A genome-wide split-ubiquitin membrane yeast two-hybrid (MYTH) protein interaction screen identified 11 ER membrane-localized, full-length proteins interacting with Ncr1 at the lysosomal/vacuolar membrane. These highlight the importance of ER-vacuole membrane interface and include PPIs with the Cyb5/Cbr1 electron transfer system, the ceramide synthase complex, and the Sec61/Sbh1 protein translocation complex. These PPIs were not detected in a sterol auxotrophy condition and thus depend on normal sterol metabolism. To provide biological context for the Ncr1-Cyb5 PPI, a yeast strain lacking this PPI (via gene deletions) exhibited altered levels of sterols and sphingolipids including increased levels of glucosylceramide that mimic NP-C disease. Overall, the results herein provide new physical and genetic interaction models to further use the yeast model of NP-C disease to better understand human NP-C disease.
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Affiliation(s)
- Natalie Hammond
- School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand
- Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand
| | - Jamie Snider
- Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada
| | - Igor Stagljar
- Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
- Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada
- Mediterranean Institute for Life Sciences, Meštrovićevo Šetalište 45, HR-21000 Split, Croatia
| | | | | | - Matthew Jessulat
- Department of Biochemistry, Research and Innovation Centre, University of Regina, Regina, Saskatchewan S4S 0A2, Canada
| | - Mohan Babu
- Department of Biochemistry, Research and Innovation Centre, University of Regina, Regina, Saskatchewan S4S 0A2, Canada
| | - Paul H Teesdale-Spittle
- School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand
- Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand
| | - Jeffrey P Sheridan
- School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand
- Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand
| | - Stephen L Sturley
- Department of Biology, Barnard College-Columbia University, New York, NY 10027, USA
| | - Andrew B Munkacsi
- School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand
- Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand
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17
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Johnston EJ, Tallis J, Cunningham-Oakes E, Moses T, Moore SJ, Hosking S, Rosser SJ. Yeast lacking the sterol C-5 desaturase Erg3 are tolerant to the anti-inflammatory triterpenoid saponin escin. Sci Rep 2023; 13:13617. [PMID: 37604855 PMCID: PMC10442444 DOI: 10.1038/s41598-023-40308-0] [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: 05/10/2023] [Accepted: 08/08/2023] [Indexed: 08/23/2023] Open
Abstract
Escin is a mixture of over 30 glycosylated triterpenoid (saponin) structures, extracted from the dried fruit of horse chestnuts. Escin is currently used as an anti-inflammatory, and has potential applications in the treatment of arthritis and cancer. Engineered yeast would enable production of specific bioactive components of escin at industrial scale, however many saponins have been shown to be toxic to yeast. Here we report that a Saccharomyces cerevisiae strain specifically lacking the sterol C-5 desaturase gene ERG3, exhibits striking enhanced tolerance to escin treatment. Transcriptome analyses, as well as pre-mixing of escin with sterols, support the hypothesis that escin interacts directly with ergosterol, but not as strongly with the altered sterols present in erg3Δ. A diverse range of saponins are of commercial interest, and this research highlights the value of screening lipidome mutants to identify appropriate hosts for engineering the industrial production of saponins.
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Affiliation(s)
- Emily J Johnston
- Centre for Engineering Biology, University of Edinburgh, Edinburgh, EH9 3BD, UK.
| | - Jess Tallis
- Centre for Engineering Biology, University of Edinburgh, Edinburgh, EH9 3BD, UK
| | - Edward Cunningham-Oakes
- Department of Infection Biology and Microbiomes, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool, L69 7ZB, UK
| | - Tessa Moses
- EdinOmics, RRID:SCR_021838, University of Edinburgh, Max Born Crescent, Edinburgh, EH9 3BF, UK
| | - Simon J Moore
- Genetic Science Division, Thermo Fisher Scientific, 7 Kingsland Grange, Warrington, Cheshire, WA1 4SR, UK
| | - Sarah Hosking
- Unilever R&D Port Sunlight, Quarry Road East, Bebington, Wirral, CH63 3JW, UK
| | - Susan J Rosser
- Centre for Engineering Biology, University of Edinburgh, Edinburgh, EH9 3BD, UK.
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18
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Elias D, Tóth Hervay N, Bujdos M, Gbelska Y. Essential Role of CgErg6p in Maintaining Oxidative Stress Tolerance and Iron Homeostasis in Candida glabrata. J Fungi (Basel) 2023; 9:jof9050579. [PMID: 37233290 DOI: 10.3390/jof9050579] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2023] [Revised: 05/12/2023] [Accepted: 05/16/2023] [Indexed: 05/27/2023] Open
Abstract
The human pathogenic fungus Candida glabrata is the second leading cause of candidemia, a life-threatening invasive mycosis. Clinical outcomes are complicated by reduced susceptibility of C. glabrata to azoles together with its ability to evolve stable resistance to both azoles and echinocandins following drug exposure. Compared to other Candida spp., C. glabrata displays robust oxidative stress resistance. In this study, we investigated the impact of CgERG6 gene deletion on the oxidative stress response in C. glabrata. CgERG6 gene encodes sterol-24-C-methyltransferase, which is involved in the final steps of ergosterol biosynthesis. Our previous results showed that the Cgerg6Δ mutant has a lower ergosterol content in its membranes. Here, we show that the Cgerg6Δ mutant displays increased susceptibility to oxidative stress inducing agents, such as menadione, hydrogen peroxide and diamide, accompanied with increased intracellular ROS production. The Cgerg6Δ mutant is not able to tolerate higher concentrations of iron in the growth media. We observed increased expression of transcription factors, CgYap1p, CgMsn4p and CgYap5p, together with increased expression of catalase encoding the CgCTA1 gene and vacuolar iron transporter CgCCC1 in the Cgerg6Δ mutant cells. However, it seems that the CgERG6 gene deletion does not influence the function of mitochondria.
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Affiliation(s)
- Daniel Elias
- Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovicova 6, 842 15 Bratislava, Slovakia
| | - Nora Tóth Hervay
- Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovicova 6, 842 15 Bratislava, Slovakia
| | - Marek Bujdos
- Faculty of Natural Sciences, Institute of Laboratory Research on Geomaterials, Comenius University in Bratislava, Ilkovicova 6, 842 15 Bratislava, Slovakia
| | - Yvetta Gbelska
- Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovicova 6, 842 15 Bratislava, Slovakia
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19
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Xu YS, Ma W, Li J, Huang PW, Sun XM, Huang H. Metal cofactor regulation combined with rational genetic engineering of Schizochytrium sp. for high-yield production of squalene. Biotechnol Bioeng 2023; 120:1026-1037. [PMID: 36522292 DOI: 10.1002/bit.28311] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2022] [Revised: 12/05/2022] [Accepted: 12/12/2022] [Indexed: 12/23/2022]
Abstract
The increasing market demand for squalene requires novel biotechnological production platforms. Schizochytrium sp. is an industrial oleaginous host with a high potential for squalene production due to its abundant native acetyl-CoA pool. We first found that iron starvation led to the accumulation of 1.5 g/L of squalene by Schizochytrium sp., which was 40-fold higher than in the control. Subsequent transcriptomic and lipidomic analyses showed that the high squalene titer is due to the diversion of precursors from lipid biosynthesis and increased triglycerides (TAG) content for squalene storage. Furthermore, we constructed the engineered acetyl-CoA C-acetyltransferase (ACAT)-overexpressing strain 18S::ACAT, which produced 2.79 g/L of squalene, representing an 86% increase over the original strain. Finally, a nitrogen-rich feeding strategy was developed to further increase the squalene titer of the engineered strain, which reached 10.78 g/L in fed-batch fermentation, a remarkable 161-fold increase over the control. To our best knowledge, this is the highest squalene yield in thraustochytrids reported to date.
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Affiliation(s)
- Ying-Shuang Xu
- School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China
| | - Wang Ma
- School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China
| | - Jin Li
- School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China
| | - Peng-Wei Huang
- School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China
| | - Xiao-Man Sun
- School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China
| | - He Huang
- School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China
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20
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Martins TS, Costa RS, Vilaça R, Lemos C, Teixeira V, Pereira C, Costa V. Iron Limitation Restores Autophagy and Increases Lifespan in the Yeast Model of Niemann-Pick Type C1. Int J Mol Sci 2023; 24:6221. [PMID: 37047194 PMCID: PMC10094029 DOI: 10.3390/ijms24076221] [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: 02/07/2023] [Revised: 03/21/2023] [Accepted: 03/22/2023] [Indexed: 03/29/2023] Open
Abstract
Niemann-Pick type C1 (NPC1) is an endolysosomal transmembrane protein involved in the export of cholesterol and sphingolipids to other cellular compartments such as the endoplasmic reticulum and plasma membrane. NPC1 loss of function is the major cause of NPC disease, a rare lysosomal storage disorder characterized by an abnormal accumulation of lipids in the late endosomal/lysosomal network, mitochondrial dysfunction, and impaired autophagy. NPC phenotypes are conserved in yeast lacking Ncr1, an orthologue of human NPC1, leading to premature aging. Herein, we performed a phosphoproteomic analysis to investigate the effect of Ncr1 loss on cellular functions mediated by the yeast lysosome-like vacuoles. Our results revealed changes in vacuolar membrane proteins that are associated mostly with vesicle biology (fusion, transport, organization), autophagy, and ion homeostasis, including iron, manganese, and calcium. Consistently, the cytoplasm to vacuole targeting (Cvt) pathway was increased in ncr1∆ cells and autophagy was compromised despite TORC1 inhibition. Moreover, ncr1∆ cells exhibited iron overload mediated by the low-iron sensing transcription factor Aft1. Iron deprivation restored the autophagic flux of ncr1∆ cells and increased its chronological lifespan and oxidative stress resistance. These results implicate iron overload on autophagy impairment, oxidative stress sensitivity, and cell death in the yeast model of NPC1.
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Affiliation(s)
- Telma S. Martins
- i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
- IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal
- Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal
| | - Rafaela S. Costa
- i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
- IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal
- Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal
| | - Rita Vilaça
- i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
- IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal
| | - Carolina Lemos
- i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
- IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal
- Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal
| | - Vitor Teixeira
- i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
- IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal
- Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal
| | - Clara Pereira
- i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
- IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal
| | - Vítor Costa
- i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
- IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal
- Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal
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21
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Zhang Y, Cai Y, Chen Z. Community-specific diffusion characteristics determine resistance of biofilms to oxidative stress. SCIENCE ADVANCES 2023; 9:eade2610. [PMID: 36961890 DOI: 10.1126/sciadv.ade2610] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/04/2022] [Accepted: 02/17/2023] [Indexed: 06/18/2023]
Abstract
Biofilms are multicellular communities with a spatial structure. Different from single-cell scale diffusion in planktonic systems, the diffusion distance becomes the dimension of multicellular clusters in a biofilm. Such differences in diffusion behavior affect the tolerance and response to exogenous stress. Here, we found that at the same doses of exogenous hydrogen peroxide (H2O2), planktonic Escherichia coli were completely killed within two hours, whereas the biofilm resumed growth in six hours by building a catalase barrier to block H2O2 penetration, despite the growth burden. Unexpectedly, when we changed the carbon source from glucose to glycerol, H2O2 instantly counterintuitively boosted biofilm growth due to supplemental oxygen, which was the growth-limiting factor. We further demonstrated that the energy metabolism modes determined the growth-limiting factor, which then determined the two patterns of biofilms resistances to H2O2.
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Affiliation(s)
- Yuzhen Zhang
- Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing 100084, China
- Tsinghua-Peking Center for Life Sciences, Beijing 100084, China
| | - Yumin Cai
- Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing 100084, China
| | - Zhaoyuan Chen
- Center for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing 100084, China
- Tsinghua-Peking Center for Life Sciences, Beijing 100084, China
- Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences, Ningbo 315020, China
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22
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Fridman M, Sakurai K. Deciphering the Biological Activities of Antifungal Agents with Chemical Probes. Angew Chem Int Ed Engl 2023; 62:e202211927. [PMID: 36628503 DOI: 10.1002/anie.202211927] [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: 08/12/2022] [Revised: 11/09/2022] [Accepted: 01/10/2023] [Indexed: 01/12/2023]
Abstract
The growing number of fungal infections caused by pathogens resistant to one or more classes of antifungal drugs emphasizes the threat that these microorganisms pose to animal and human health and global food security. Open questions remain regarding the mechanisms of action of the limited repertoire of antifungal agents, making it challenging to rationally develop more efficacious therapeutics. In recent years, the use of chemical biology approaches has resolved some of these questions and has provided new promising concepts to guide the design of antifungal agents. By focusing on examples from studies carried out in recent years, this minireview describes the key roles that probes based on antifungal agents and their derivatives have played in uncovering details about their activities, in detecting resistance, and in characterizing the interactions between these agents and their targets.
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Affiliation(s)
- Micha Fridman
- School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 6997801, Israel
| | - Kaori Sakurai
- Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 4-24-16, Naka-cho, Koganei-shi, Tokyo, 184-8588, Japan
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23
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Unveiling the Effect of NCgl0580 Gene Deletion on 5-Aminolevulinic Acid Biosynthesis in Corynebacterium glutamicum. FERMENTATION-BASEL 2023. [DOI: 10.3390/fermentation9030213] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/03/2023]
Abstract
5-Aminolevulinic acid (5-ALA) has recently received much attention for its wide applications in medicine and agriculture. In this study, we investigated the effect of NCgl0580 in Corynebacterium glutamicum on 5-ALA biosynthesis as well as its possible mechanism. It was found that the overexpression of NCgl0580 increased 5-ALA production by approximately 53.3%. Interestingly, the knockout of this gene led to an even more significant 2.49-fold increase in 5-ALA production. According to transcriptome analysis and functional validation of phenotype-related targets, the deletion of NCgl0580 brought about considerable changes in the transcript levels of genes involved in central carbon metabolism, leading to fluxes redistribution toward the 5-ALA precursor succinyl-CoA as well as ATP-binding cassette (ABC) transporters affecting 5-ALA biosynthesis. In particular, the positive effects of enhanced sugar transport (by overexpressing NCgl1445 and iolT1), glycolysis (by overexpressing pyk2), iron uptake (by overexpressing afuABC), and phosphate uptake (by overexpressing pstSCAB and ugpQ) on 5-ALA biosynthesis were demonstrated for the first time. Thus, the transcriptional mechanism underlying the effect of NCgl0580 deletion on 5-ALA biosynthesis was elucidated, providing new strategies to regulate the metabolic network of C. glutamicum to achieve a further increase in 5-ALA production.
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24
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Aft1 Nuclear Localization and Transcriptional Response to Iron Starvation Rely upon TORC2/Ypk1 Signaling and Sphingolipid Biosynthesis. Int J Mol Sci 2023; 24:ijms24032438. [PMID: 36768760 PMCID: PMC9916926 DOI: 10.3390/ijms24032438] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2023] [Revised: 01/20/2023] [Accepted: 01/24/2023] [Indexed: 01/28/2023] Open
Abstract
Iron scarcity provokes a cellular response consisting of the strong expression of high-affinity systems to optimize iron uptake and mobilization. Aft1 is a primary transcription factor involved in iron homeostasis and controls the expression of high-affinity iron uptake genes in Saccharomyces cerevisiae. Aft1 responds to iron deprivation by translocating from the cytoplasm to the nucleus. Here, we demonstrate that the AGC kinase Ypk1, as well as its upstream regulator TOR Complex 2 (TORC2), are required for proper Aft1 nuclear localization following iron deprivation. We exclude a role for TOR Complex 1 (TORC1) and its downstream effector Sch9, suggesting this response is specific for the TORC2 arm of the TOR pathway. Remarkably, we demonstrate that Aft1 nuclear localization and a robust transcriptional response to iron starvation also require biosynthesis of sphingolipids, including complex sphingolipids such as inositol phosphorylceramide (IPC) and upstream precursors, e.g., long-chain bases (LCBs) and ceramides. Furthermore, we observe the deficiency of Aft1 nuclear localization and impaired transcriptional response in the absence of iron when TORC2-Ypk1 is impaired is partially suppressed by exogenous addition of the LCB dihydrosphingosine (DHS). This latter result is consistent with prior studies linking sphingolipid biosynthesis to TORC2-Ypk1 signaling. Taken together, these results reveal a novel role for sphingolipids, controlled by TORC2-Ypk1, for proper localization and activity of Aft1 in response to iron scarcity.
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25
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Comparative Transcriptome Analysis Unravels the Response Mechanisms of Fusarium oxysporum f.sp. cubense to a Biocontrol Agent, Pseudomonas aeruginosa Gxun-2. Int J Mol Sci 2022; 23:ijms232315432. [PMID: 36499750 PMCID: PMC9735772 DOI: 10.3390/ijms232315432] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2022] [Revised: 11/30/2022] [Accepted: 12/01/2022] [Indexed: 12/12/2022] Open
Abstract
Banana Fusarium wilt, which is caused by Fusarium oxysporum f.sp. cubense Tropical Race 4 (FOC TR4), is one of the most serious fungal diseases in the banana-producing regions in east Asia. Pseudomonas aeruginosa Gxun-2 could significantly inhibit the growth of FOC TR4. Strain Gxun-2 strongly inhibited the mycelial growth of FOC TR4 on dual culture plates and caused hyphal wrinkles, ruptures, and deformities on in vitro cultures. Banana seedlings under pot experiment treatment with Gxun-2 in a greenhouse resulted in an 84.21% reduction in the disease. Comparative transcriptome analysis was applied to reveal the response and resistance of FOC TR4 to Gxun-2 stress. The RNA-seq analysis of FOC TR4 during dual-culture with P. aeruginosa Gxun-2 revealed 3075 differentially expressed genes (DEGs) compared with the control. Among the genes, 1158 genes were up-regulated, and 1917 genes were down-regulated. Further analysis of gene function and the pathway of DEGs revealed that genes related to the cell membrane, cell wall formation, peroxidase, ABC transporter, and autophagy were up-regulated, while down-regulated DEGs were enriched in the sphingolipid metabolism and chitinase. These results indicated that FOC TR4 upregulates a large number of genes in order to maintain cell functions. The results of qRT-PCR conducted on a subset of 13 genes were consistent with the results of RNA-seq data. Thus, this study serves as a valuable resource regarding the mechanisms of fungal pathogen resistance to biocontrol agents.
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26
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Ghio C, Soukup JM, Dailey LA, Ghio AJ, Schreinemachers DM, Koppes RA, Koppes AN. Lactate Production can Function to Increase Human Epithelial Cell Iron Concentration. Cell Mol Bioeng 2022; 15:571-585. [PMID: 36531860 PMCID: PMC9751240 DOI: 10.1007/s12195-022-00741-z] [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: 03/26/2022] [Accepted: 09/21/2022] [Indexed: 11/28/2022] Open
Abstract
Introduction Under conditions of limited iron availability, plants and microbes have evolved mechanisms to acquire iron. For example, metal deficiency stimulates reprogramming of carbon metabolism, increasing activity of enzymes involved in the Krebs cycle and the glycolytic pathway. Resultant carboxylates/hydroxycarboxylates then function as ligands to complex iron and facilitate solubilization and uptake, reversing the metal deficiency. Similarly, human intestinal epithelial cells may produce lactate, a hydroxycarboxylate, during absolute and functional iron deficiency to import metal to reverse limited availability. Methods Here we investigate (1) if lactate can increase cell metal import of epithelial cells in vitro, (2) if lactate dehydrogenase (LDH) activity in and lactate production by epithelial cells correspond to metal availability, and (3) if blood concentrations of LDH in a human cohort correlate with indices of iron homeostasis. Results Results show that exposures of human epithelial cells, Caco-2, to both sodium lactate and ferric ammonium citrate (FAC) increase metal import relative to FAC alone. Similarly, fumaric, isocitric, malic, and succinic acid coincubation with FAC increase iron import relative to FAC alone. Increased iron import following exposures to sodium lactate and FAC elevated both ferritin and metal associated with mitochondria. LDH did not change after exposure to deferoxamine but decreased with 24 h exposure to FAC. Lactate levels revealed decreased levels with FAC incubation. Review of the National Health and Nutrition Examination Survey demonstrated significant negative relationships between LDH concentrations and serum iron in human cohorts. Conclusions Therefore, we conclude that iron import in human epithelial cells can involve lactate, LDH activity can reflect the availability of this metal, and blood LDH concentrations can correlate with indices of iron homeostasis.
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Affiliation(s)
- Caroline Ghio
- Department of Chemical Engineering, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, MA 02115 USA
| | | | - Lisa A. Dailey
- US Environmental Protection Agency, Chapel Hill, NC 27514 USA
| | - Andrew J. Ghio
- US Environmental Protection Agency, Chapel Hill, NC 27514 USA
| | | | - Ryan A. Koppes
- Department of Chemical Engineering, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, MA 02115 USA
| | - Abigail N. Koppes
- Department of Chemical Engineering, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, MA 02115 USA
- Department of Biology, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, MA 02115 USA
- Department of Bioengineering, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, MA 02115 USA
- Northeastern University, 360 Huntington Ave., 332 Mugar Life Science Building, Boston, MA 02115 USA
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27
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Metal ion availability and homeostasis as drivers of metabolic evolution and enzyme function. Curr Opin Genet Dev 2022; 77:101987. [PMID: 36183585 DOI: 10.1016/j.gde.2022.101987] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2022] [Revised: 08/25/2022] [Accepted: 08/29/2022] [Indexed: 01/27/2023]
Abstract
Metal ions are potent catalysts and have been available for cellular biochemistry at all stages of evolution. Growing evidence suggests that metal catalysis was critical for the origin of the very first metabolic reactions. With approximately 80% of modern metabolic pathways being dependent on metal ions, metallocatalysis and homeostasis continue to be essential for intracellular metabolic networks and physiology. However, the genetic network that controls metal ion homeostasis and the impact of metal availability on metabolism is poorly understood. Here, we review recent work on gene and protein evolution relevant for better understanding metal ion biology and its role in metabolism. We highlight the importance of analysing the origin and evolution of enzyme catalysis in the context of catalytically relevant metal ions, summarise unanswered questions essential for developing a comprehensive understanding of metal ion homeostasis and advocate for the consideration of metal ion properties and availability in the design and directed evolution of novel enzymes and pathways.
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28
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Lindahl PA, Vali SW. Mössbauer-based molecular-level decomposition of the Saccharomyces cerevisiae ironome, and preliminary characterization of isolated nuclei. Metallomics 2022; 14:mfac080. [PMID: 36214417 PMCID: PMC9624242 DOI: 10.1093/mtomcs/mfac080] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2022] [Accepted: 09/23/2022] [Indexed: 11/25/2022]
Abstract
One hundred proteins in Saccharomyces cerevisiae are known to contain iron. These proteins are found mainly in mitochondria, cytosol, nuclei, endoplasmic reticula, and vacuoles. Cells also contain non-proteinaceous low-molecular-mass labile iron pools (LFePs). How each molecular iron species interacts on the cellular or systems' level is underdeveloped as doing so would require considering the entire iron content of the cell-the ironome. In this paper, Mössbauer (MB) spectroscopy was used to probe the ironome of yeast. MB spectra of whole cells and isolated organelles were predicted by summing the spectral contribution of each iron-containing species in the cell. Simulations required input from published proteomics and microscopy data, as well as from previous spectroscopic and redox characterization of individual iron-containing proteins. Composite simulations were compared to experimentally determined spectra. Simulated MB spectra of non-proteinaceous iron pools in the cell were assumed to account for major differences between simulated and experimental spectra of whole cells and isolated mitochondria and vacuoles. Nuclei were predicted to contain ∼30 μM iron, mostly in the form of [Fe4S4] clusters. This was experimentally confirmed by isolating nuclei from 57Fe-enriched cells and obtaining the first MB spectra of the organelle. This study provides the first semi-quantitative estimate of all concentrations of iron-containing proteins and non-proteinaceous species in yeast, as well as a novel approach to spectroscopically characterizing LFePs.
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Affiliation(s)
- Paul A Lindahl
- Department of Chemistry, Texas A&M University, College Station, TX, USA
- Department of Biochemistry and Biophysics, Texas A&M University, College Station TX, USA
| | - Shaik Waseem Vali
- Department of Chemistry, Texas A&M University, College Station, TX, USA
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29
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Jordá T, Barba-Aliaga M, Rozès N, Alepuz P, Martínez-Pastor MT, Puig S. Transcriptional regulation of ergosterol biosynthesis genes in response to iron deficiency. Environ Microbiol 2022; 24:5248-5260. [PMID: 36382795 DOI: 10.1111/1462-2920.16157] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2022] [Accepted: 08/01/2022] [Indexed: 01/07/2023]
Abstract
Iron participates as an essential cofactor in the biosynthesis of critical cellular components, including DNA, proteins and lipids. The ergosterol biosynthetic pathway, which is an important target of antifungal treatments, depends on iron in four enzymatic steps. Our results in the model yeast Saccharomyces cerevisiae show that the expression of ergosterol biosynthesis (ERG) genes is tightly modulated by iron availability probably through the iron-dependent variation of sterol and heme levels. Whereas the transcription factors Upc2 and Ecm22 are responsible for the activation of ERG genes upon iron deficiency, the heme-dependent factor Hap1 triggers their Tup1-mediated transcriptional repression. The combined regulation by both activating and repressing regulatory factors allows for the fine-tuning of ERG transcript levels along the progress of iron deficiency, avoiding the accumulation of toxic sterol intermediates and enabling efficient adaptation to rapidly changing conditions. The lack of these regulatory factors leads to changes in the yeast sterol profile upon iron-deficient conditions. Both environmental iron availability and specific regulatory factors should be considered in ergosterol antifungal treatments.
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Affiliation(s)
- Tania Jordá
- Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Paterna, Valencia, Spain
| | - Marina Barba-Aliaga
- Instituto de Biotecnología y Biomedicina (Biotecmed), Universitat de València, Burjassot, Valencia, Spain
- Departamento de Bioquímica y Biología Molecular, Universitat de València, Burjassot, Valencia, Spain
| | - Nicolas Rozès
- Departament de Bioquímica i Biotecnologia, Facultat d'Enologia, Universitat Rovira i Virgili, Tarragona, Spain
| | - Paula Alepuz
- Instituto de Biotecnología y Biomedicina (Biotecmed), Universitat de València, Burjassot, Valencia, Spain
- Departamento de Bioquímica y Biología Molecular, Universitat de València, Burjassot, Valencia, Spain
| | | | - Sergi Puig
- Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Paterna, Valencia, Spain
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30
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Jordá T, Martínez-Martín A, Martínez-Pastor MT, Puig S. Modulation of yeast Erg1 expression and terbinafine susceptibility by iron bioavailability. Microb Biotechnol 2022; 15:2705-2716. [PMID: 35837730 PMCID: PMC9618313 DOI: 10.1111/1751-7915.14102] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2022] [Revised: 06/01/2022] [Accepted: 06/03/2022] [Indexed: 11/28/2022] Open
Abstract
Ergosterol is a specific sterol component of yeast and fungal membranes. Its biosynthesis is one of the most effective targets for antifungal treatments. However, the emergent resistance to multiple sterol‐based antifungal drugs emphasizes the need for new therapeutic approaches. The allylamine terbinafine, which selectively inhibits squalene epoxidase Erg1 within the ergosterol biosynthetic pathway, is mainly used to treat dermatomycoses, whereas its effectiveness in other fungal infections is limited. Given that ergosterol biosynthesis depends on iron as an essential cofactor, in this report, we used the yeast Saccharomyces cerevisiae to investigate how iron bioavailability influences Erg1 expression and terbinafine susceptibility. We observed that both chemical and genetic depletion of iron decrease ERG1 expression, leading to an increase in terbinafine susceptibility. Deletion of either ROX1 transcriptional repressor or CTH1 and CTH2 post‐transcriptional repressors of ERG1 expression led to an increase in Erg1 protein levels and terbinafine resistance. On the contrary, overexpression of CTH2 led to the opposite effect, lowering Erg1 levels and increasing terbinafine susceptibility. Although strain‐specific particularities exist, opportunistic pathogenic strains of S. cerevisiae displayed a response similar to the laboratory strain. These data indicate that iron bioavailability and particular regulatory factors could be used to modulate susceptibility to terbinafine.
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Affiliation(s)
- Tania Jordá
- Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Paterna, Valencia, Spain
| | - Ana Martínez-Martín
- Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Paterna, Valencia, Spain
| | | | - Sergi Puig
- Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Paterna, Valencia, Spain
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31
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Were E, Schöne J, Viljoen A, Rasche F. De novo synthesis of ferrichrome by Fusarium oxysporum f. sp. cubense TR4 in response to iron starvation. Fungal Biol 2022; 126:521-527. [DOI: 10.1016/j.funbio.2022.05.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2022] [Revised: 05/03/2022] [Accepted: 05/24/2022] [Indexed: 11/04/2022]
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32
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Young DJ, Guydosh NR. Rebirth of the translational machinery: The importance of recycling ribosomes. Bioessays 2022; 44:e2100269. [PMID: 35147231 PMCID: PMC9270684 DOI: 10.1002/bies.202100269] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2021] [Revised: 01/26/2022] [Accepted: 01/27/2022] [Indexed: 11/10/2022]
Abstract
Translation of the genetic code occurs in a cycle where ribosomes engage mRNAs, synthesize protein, and then disengage in order to repeat the process again. The final part of this process-ribosome recycling, where ribosomes dissociate from mRNAs-involves a complex molecular choreography of specific protein factors to remove the large and small subunits of the ribosome in a coordinated fashion. Errors in this process can lead to the accumulation of ribosomes at stop codons or translation of downstream open reading frames (ORFs). Ribosome recycling is also critical when a ribosome stalls during the elongation phase of translation and must be rescued to allow continued translation of the mRNA. Here we discuss the molecular interactions that drive ribosome recycling, and their regulation in the cell. We also examine the consequences of inefficient recycling with regards to disease, and its functional roles in synthesis of novel peptides, regulation of gene expression, and control of mRNA-associated proteins. Alterations in ribosome recycling efficiency have the potential to impact many cellular functions but additional work is needed to understand how this regulatory power is utilized.
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Affiliation(s)
- David J Young
- Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Nicholas R Guydosh
- Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
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33
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Romero AM, García-Martínez J, Pérez-Ortín JE, Martínez-Pastor MT, Puig S. Changes in mRNA stability play an important role in the adaptation of yeast cells to iron deprivation. BIOCHIMICA ET BIOPHYSICA ACTA. GENE REGULATORY MECHANISMS 2022; 1865:194800. [PMID: 35218933 DOI: 10.1016/j.bbagrm.2022.194800] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/21/2021] [Revised: 02/09/2022] [Accepted: 02/10/2022] [Indexed: 06/14/2023]
Abstract
Eukaryotic cells rely on iron as an indispensable cofactor for multiple biological functions including mitochondrial respiration and protein synthesis. The budding yeast Saccharomyces cerevisiae utilizes both transcriptional and posttranscriptional mechanisms to couple mRNA levels to the requirements of iron deprivation. Thus, in response to iron deficiency, transcription factors Aft1 and Aft2 activate the expression of genes implicated in iron acquisition and mobilization, whereas two mRNA-binding proteins, Cth1 and Cth2, posttranscriptionally control iron metabolism. By using a genome-wide approach, we describe here a global stabilization of mRNAs, including transcripts encoding ribosomal proteins (RPs), when iron bioavailability diminishes. mRNA decay assays indicate that the mRNA-binding protein Pub1 contributes to RP transcript stabilization during adaptation to iron limitation. In fact, Pub1 becomes critical for growth and translational repression in low-iron conditions. Remarkably, we observe that pub1Δ cells also exhibit an increase in the transcription of RP genes that evidences the crosstalk between transcription and degradation mechanisms to maintain the appropriate mRNA balance under iron deficiency conditions.
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Affiliation(s)
- Antonia María Romero
- Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Agustín Escardino 7, E-46980 Paterna, Valencia, Spain.
| | - José García-Martínez
- Departamento de Genética, Universitat de València, Ave. Doctor Moliner 50, E-46100 Burjassot, Valencia, Spain; Instituto de Biotecnología y Biomedicina (BIOTECMED), Universitat de València, Ave. Doctor Moliner 50, E-46100 Burjassot, Valencia, Spain
| | - José Enrique Pérez-Ortín
- Instituto de Biotecnología y Biomedicina (BIOTECMED), Universitat de València, Ave. Doctor Moliner 50, E-46100 Burjassot, Valencia, Spain; Departamento de Bioquímica y Biología Molecular, Universitat de València, Ave. Doctor Moliner 50, E-46100 Burjassot, Valencia, Spain
| | - María Teresa Martínez-Pastor
- Departamento de Bioquímica y Biología Molecular, Universitat de València, Ave. Doctor Moliner 50, E-46100 Burjassot, Valencia, Spain
| | - Sergi Puig
- Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Agustín Escardino 7, E-46980 Paterna, Valencia, Spain.
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34
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Cordova LT, Palmer CM, Alper HS. Shifting the distribution: modulation of the lipid profile in Yarrowia lipolytica via iron content. Appl Microbiol Biotechnol 2022; 106:1571-1581. [PMID: 35099573 DOI: 10.1007/s00253-022-11800-w] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2021] [Revised: 01/14/2022] [Accepted: 01/21/2022] [Indexed: 11/02/2022]
Abstract
Microbial fermentation offers a sustainable source of fuels, commodity chemicals, and pharmaceuticals, yet strain performance is influenced greatly by the growth media selected. Specifically, trace metals (e.g., iron, copper, manganese, zinc, and others) are critical for proper growth and enzymatic function within microorganisms yet are non-standardized across media formulation. In this work, the effect of trace metal supplementation on the lipid production profile of Yarrowia lipolytica was explored using tube scale fermentation followed by biomass and lipid characterization. Addition of iron (II) to the chemically defined Yeast Synthetic Complete (YSC) medium increased final optical density nearly twofold and lipid production threefold, while addition of copper (II) had no impact. Additionally, dose-responsive changes in lipid distribution were observed, with the percent of oleic acid increasing and stearic acid decreasing as initial iron concentration increased. These changes were reversible with subsequent iron-selective chelation. Use of rich Yeast Peptone Dextrose (YPD) medium enabled further increases in the production of two specialty oleochemicals ultimately reaching 63 and 47% of the lipid pool as α-linolenic acid and cyclopropane fatty acid, respectively, compared to YSC medium. Selective removal of iron (II) natively present in YPD medium decreased this oleochemical production, ultimately aligning the lipid profile with that of non-supplemented YSC medium. These results provide further insight into the proposed mechanisms for iron regulation in yeasts especially as these productions strains contain a mutant allele of the iron regulator, mga2. The work presented here also suggests a non-genetic method for control of the lipid profile in Y. lipolytica for use in diverse applications. KEY POINTS: • Iron supplementation increases cell density and lipid titer in Yarrowia lipolytica. • Iron addition reversibly alters lipid portfolio increasing linolenic acid. • Removal of iron from YPD media provides a link to enhanced oleochemical production.
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Affiliation(s)
- Lauren T Cordova
- McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St. Stop C0400, Austin, TX, 78712, USA
| | - Claire M Palmer
- Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, TX, 78712, USA
| | - Hal S Alper
- McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St. Stop C0400, Austin, TX, 78712, USA. .,Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, TX, 78712, USA.
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35
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Hsieh EJ, Lin WD, Schmidt W. Genomically Hardwired Regulation of Gene Activity Orchestrates Cellular Iron Homeostasis in Arabidopsis. RNA Biol 2021; 19:143-161. [PMID: 35067184 PMCID: PMC8786333 DOI: 10.1080/15476286.2021.2024024] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2021] [Revised: 12/09/2021] [Accepted: 12/26/2021] [Indexed: 10/26/2022] Open
Abstract
Iron (Fe) is an essential micronutrient which plays pivotal roles as electron donor and catalyst across organisms. In plants, variable, often insufficient Fe supply necessitates mechanisms that constantly attune Fe uptake rates and recalibrate cellular Fe homoeostasis. Here, we show that short-term (0.5, 6, and 12 h) exposure of Arabidopsis thaliana plants to Fe deficiency triggered massive changes in gene activity governed by transcription and alternative splicing (AS), regulatory layers that were to a large extent mutually exclusive. Such preclusion was not observed for genes that are directly involved in the acquisition of Fe, which appears to be concordantly regulated by both expression and AS. Generally, genes with lower splice site strengths and higher intron numbers were more likely to be regulated by AS, no dependence on gene architecture was observed for transcriptionally controlled genes. Conspicuously, specific processes were associated with particular genomic features and biased towards either regulatory mode, suggesting that genomic hardwiring is functionally biased. Early changes in splicing patterns were, in many cases, congruent with later changes in transcript or protein abundance, thus contributing to the pronounced transcriptome-proteome discordance observed in plants.
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Affiliation(s)
- En-Jung Hsieh
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
| | - Wen-Dar Lin
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
| | - Wolfgang Schmidt
- Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
- Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan
- Genome and Systems Biology Degree Program, College of Life Science, National Taiwan University, Taipei, Taiwan
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36
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Wang T, Wang Z, de Fabritus L, Tao J, Saied EM, Lee HJ, Ramazanov BR, Jackson B, Burkhardt D, Parker M, Gleinich AS, Wang Z, Seo DE, Zhou T, Xu S, Alecu I, Azadi P, Arenz C, Hornemann T, Krishnaswamy S, van de Pavert SA, Kaech SM, Ivanova NB, Santori FR. 1-deoxysphingolipids bind to COUP-TF to modulate lymphatic and cardiac cell development. Dev Cell 2021; 56:3128-3145.e15. [PMID: 34762852 PMCID: PMC8628544 DOI: 10.1016/j.devcel.2021.10.018] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2020] [Revised: 08/30/2021] [Accepted: 10/19/2021] [Indexed: 12/12/2022]
Abstract
Identification of physiological modulators of nuclear hormone receptor (NHR) activity is paramount for understanding the link between metabolism and transcriptional networks that orchestrate development and cellular physiology. Using libraries of metabolic enzymes alongside their substrates and products, we identify 1-deoxysphingosines as modulators of the activity of NR2F1 and 2 (COUP-TFs), which are orphan NHRs that are critical for development of the nervous system, heart, veins, and lymphatic vessels. We show that these non-canonical alanine-based sphingolipids bind to the NR2F1/2 ligand-binding domains (LBDs) and modulate their transcriptional activity in cell-based assays at physiological concentrations. Furthermore, inhibition of sphingolipid biosynthesis phenocopies NR2F1/2 deficiency in endothelium and cardiomyocytes, and increases in 1-deoxysphingosine levels activate NR2F1/2-dependent differentiation programs. Our findings suggest that 1-deoxysphingosines are physiological regulators of NR2F1/2-mediated transcription.
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Affiliation(s)
- Ting Wang
- Department of Immunobiology, Yale University, New Haven, CT 06520, USA; Department of Hematology, Tianjin Medical University General Hospital, Tianjin 300052, China
| | - Zheng Wang
- Department of Genetics and Cell Biology, Basic Medical College, Qingdao University, Qingdao, Shandong 266071, China; Department of Reproductive Medicine, the Affiliated Hospital of Qingdao University, Qingdao, Shandong 266000, China
| | - Lauriane de Fabritus
- Aix-Marseille Universite, CNRS, INSERM, Centre d'Immunologie de Marseille-Luminy (CIML), 13288 Marseille Cedex 9, France
| | - Jinglian Tao
- Department of Hematology, Tianjin Medical University General Hospital, Tianjin 300052, China; Center for Molecular Medicine, Department of Genetics, University of Georgia, Athens, GA 30602, USA
| | - Essa M Saied
- Institut für Chemie, Humboldt Universität zu Berlin, Berlin 12489, Germany; Chemistry Department, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt
| | - Ho-Joon Lee
- Department of Genetics, Yale University, New Haven, CT 06520, USA; Center for Genome Analysis, Yale University, New Haven, CT 06510, USA
| | - Bulat R Ramazanov
- Department of Cell Biology, Yale University, New Haven, CT 06520, USA
| | - Benjamin Jackson
- Center for Molecular Medicine, Department of Genetics, University of Georgia, Athens, GA 30602, USA
| | - Daniel Burkhardt
- Department of Genetics, Yale University, New Haven, CT 06520, USA
| | - Mikhail Parker
- Center for Molecular Medicine, Department of Genetics, University of Georgia, Athens, GA 30602, USA
| | - Anne S Gleinich
- Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA
| | - Zhirui Wang
- Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA
| | - Dong Eun Seo
- Center for Molecular Medicine, Department of Genetics, University of Georgia, Athens, GA 30602, USA
| | - Ting Zhou
- Department of Immunobiology, Yale University, New Haven, CT 06520, USA
| | - Shihao Xu
- NOMIS Center for Immunobiology and Microbial Pathogenesis, the Salk Institute for Biological Studies, La Jolla, CA 92037, USA
| | - Irina Alecu
- Neural Regeneration Laboratory, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa K1H 8M5, Canada
| | - Parastoo Azadi
- Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA
| | - Christoph Arenz
- Institut für Chemie, Humboldt Universität zu Berlin, Berlin 12489, Germany
| | - Thorsten Hornemann
- Institute of Clinical Chemistry, University and University Hospital of Zurich, Zurich 8091, Switzerland
| | | | - Serge A van de Pavert
- Aix-Marseille Universite, CNRS, INSERM, Centre d'Immunologie de Marseille-Luminy (CIML), 13288 Marseille Cedex 9, France
| | - Susan M Kaech
- NOMIS Center for Immunobiology and Microbial Pathogenesis, the Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
| | - Natalia B Ivanova
- Center for Molecular Medicine, Department of Genetics, University of Georgia, Athens, GA 30602, USA.
| | - Fabio R Santori
- Department of Immunobiology, Yale University, New Haven, CT 06520, USA.
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Gu Y, Jiao X, Ye L, Yu H. Metabolic engineering strategies for de novo biosynthesis of sterols and steroids in yeast. BIORESOUR BIOPROCESS 2021; 8:110. [PMID: 38650187 PMCID: PMC10992410 DOI: 10.1186/s40643-021-00460-9] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2021] [Accepted: 10/16/2021] [Indexed: 12/17/2022] Open
Abstract
Steroidal compounds are of great interest in the pharmaceutical field, with steroidal drugs as the second largest category of medicine in the world. Advances in synthetic biology and metabolic engineering have enabled de novo biosynthesis of sterols and steroids in yeast, which is a green and safe production route for these valuable steroidal compounds. In this review, we summarize the metabolic engineering strategies developed and employed for improving the de novo biosynthesis of sterols and steroids in yeast based on the regulation mechanisms, and introduce the recent progresses in de novo synthesis of some typical sterols and steroids in yeast. The remaining challenges and future perspectives are also discussed.
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Affiliation(s)
- Yuehao Gu
- Key Laboratory of Biomass Chemical Engineering (Education Ministry), College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China
- Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China
| | - Xue Jiao
- Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China
| | - Lidan Ye
- Key Laboratory of Biomass Chemical Engineering (Education Ministry), College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China.
- Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China.
| | - Hongwei Yu
- Key Laboratory of Biomass Chemical Engineering (Education Ministry), College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China.
- Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China.
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38
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Jordá T, Rozès N, Puig S. Sterol Composition Modulates the Response of Saccharomyces cerevisiae to Iron Deficiency. J Fungi (Basel) 2021; 7:jof7110901. [PMID: 34829190 PMCID: PMC8620032 DOI: 10.3390/jof7110901] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2021] [Revised: 10/21/2021] [Accepted: 10/22/2021] [Indexed: 11/16/2022] Open
Abstract
Iron is a vital micronutrient that functions as an essential cofactor in multiple biological processes, including oxygen transport, cellular respiration, and metabolic pathways, such as sterol biosynthesis. However, its low bioavailability at physiological pH frequently leads to nutritional iron deficiency. The yeast Saccharomyces cerevisiae is extensively used to study iron and lipid metabolisms, as well as in multiple biotechnological applications. Despite iron being indispensable for yeast ergosterol biosynthesis and growth, little is known about their interconnections. Here, we used lipid composition analyses to determine that changes in the pattern of sterols impair the response to iron deprivation of yeast cells. Yeast mutants defective in ergosterol biosynthesis display defects in the transcriptional activation of the iron-acquisition machinery and growth defects in iron-depleted conditions. The transcriptional activation function of the iron-sensing Aft1 factor is interrupted due to its mislocalization to the vacuole. These data uncover novel links between iron and sterol metabolisms that need to be considered when producing yeast-derived foods or when treating fungal infections with drugs that target the ergosterol biosynthesis pathway.
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Affiliation(s)
- Tania Jordá
- Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), 46980 Valencia, Spain;
| | - Nicolas Rozès
- Departament de Bioquímica i Biotecnología, Facultat d’Enologia, Universitat Rovira i Virgili, 43007 Tarragona, Spain;
| | - Sergi Puig
- Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), 46980 Valencia, Spain;
- Correspondence:
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39
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Huang T, Suen D. Iron insufficiency in floral buds impairs pollen development by disrupting tapetum function. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2021; 108:244-267. [PMID: 34310779 PMCID: PMC9292431 DOI: 10.1111/tpj.15438] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/11/2020] [Revised: 06/25/2021] [Accepted: 07/20/2021] [Indexed: 06/13/2023]
Abstract
Reduction of crop yield due to iron (Fe) deficiency has always been a concern in agriculture. How Fe insufficiency in floral buds affects pollen development remains unexplored. Here, plants transferred to Fe-deficient medium at the reproductive stage had reduced floral Fe content and viable pollen and showed a defective pollen outer wall, all restored by supplying floral buds with Fe. A comparison of differentially expressed genes (DEGs) in Fe-deficient leaves, roots, and anthers suggested that changes in several cellular processes were unique to anthers, including increased lipid degradation. Co-expression analysis revealed that ABORTED MICROSPORES (AMS), DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION1, and BASIC HELIX-LOOP-HELIX 089/091/010 encode key upstream transcription factors of Fe deficiency-responsive DEGs involved in tapetum function and development, including tapetal ROS homeostasis, programmed cell death, and pollen outer wall formation-related lipid metabolism. Analysis of RESPIRATORY-BURST OXIDASE HOMOLOG E (RBOHE) gain- and loss-of-function under Fe deficiency indicated that RBOHE- and Fe-dependent regulation cooperatively control anther reactive oxygen species levels and pollen development. Since DEGs in Fe-deficient anthers were not significantly enriched in genes related to mitochondrial function, the changes in mitochondrial status under Fe deficiency, including respiration activity, density, and morphology, were probably because the Fe amount was insufficient to maintain proper mitochondrial protein function in anthers. To sum up, Fe deficiency in anthers may affect Fe-dependent protein function and impact upstream transcription factors and their downstream genes, resulting in extensively impaired tapetum function and pollen development.
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Affiliation(s)
- Tzu‐Hsiang Huang
- Agricultural Biotechnology Research CenterAcademia SinicaTaipei11529Taiwan
- Molecular and Biological Agricultural Sciences ProgramTaiwan International Graduate ProgramAcademia Sinica and National Chung‐Hsing UniversityTaipei11529Taiwan
- Graduate Institute of BiotechnologyNational Chung‐Hsing UniversityTaichung40227Taiwan
| | - Der‐Fen Suen
- Agricultural Biotechnology Research CenterAcademia SinicaTaipei11529Taiwan
- Molecular and Biological Agricultural Sciences ProgramTaiwan International Graduate ProgramAcademia Sinica and National Chung‐Hsing UniversityTaipei11529Taiwan
- Biotechnology CenterNational Chung‐Hsing UniversityTaichung40227Taiwan
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40
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Kang S, Seo H, Lee MG, Yun CW. Regulation of Copper Metabolism by Nitrogen Utilization in Saccharomyces cerevisiae. J Fungi (Basel) 2021; 7:jof7090756. [PMID: 34575794 PMCID: PMC8469692 DOI: 10.3390/jof7090756] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2021] [Revised: 09/03/2021] [Accepted: 09/10/2021] [Indexed: 11/17/2022] Open
Abstract
To understand the relationship between carbon or nitrogen utilization and iron homeostasis, we performed an iron uptake assay with several deletion mutants with partial defects in carbon or nitrogen metabolism. Among them, some deletion mutants defective in carbon metabolism partially and the MEP2 deletion mutant showed lower iron uptake activity than the wild type. Mep2 is known as a high-affinity ammonia transporter in Saccharomyces cerevisiae. Interestingly, we found that nitrogen starvation resulted in lower iron uptake activity than that of wild-type cells without downregulation of the genes involved in the high-affinity iron uptake system FET3/FTR1. However, the gene expression of FRE1 and CTR1 was downregulated by nitrogen starvation. The protein level of Ctr1 was also decreased by nitrogen starvation, and addition of copper to the nitrogen starvation medium partially restored iron uptake activity. However, the expression of MAC1, which is a copper-responsive transcriptional activator, was not downregulated by nitrogen starvation at the transcriptional level but was highly downregulated at the translational level. Mac1 was downregulated dramatically under nitrogen starvation, and treatment with MG132, which is an inhibitor of proteasome-dependent protein degradation, partially attenuated the downregulation of Mac1. Taken together, these results suggest that nitrogen starvation downregulates the high-affinity iron uptake system by degrading Mac1 in a proteasome-dependent manner and eventually downregulates copper metabolism.
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Affiliation(s)
| | | | | | - Cheol-Won Yun
- Correspondence: ; Tel.: +82-2-3290-3456; Fax: +82-2-927-9028
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41
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Abstract
Metal ions are essential to all living cells, as they can serve as cofactors of enzymes to drive catalysis of biochemical reactions. We present a constraint-based model of yeast that relates metabolism with metal ions via enzymes. The model is able to capture responses of metabolism and gene expression upon iron depletion, suggesting that yeast cells allocate iron resource in the way abiding to optimization principles. Interestingly, the model predicts up-regulation of several iron-containing enzymes that coincide with experiments, which raises the possibility that the decrease in activity due to limited iron could be compensated by elevated enzyme abundance. Moreover, the model paves the way for guiding biosynthesis of high-value compounds (e.g., p-coumaric acid) that relies on iron-containing enzymes. Metal ions are vital to metabolism, as they can act as cofactors on enzymes and thus modulate individual enzymatic reactions. Although many enzymes have been reported to interact with metal ions, the quantitative relationships between metal ions and metabolism are lacking. Here, we reconstructed a genome-scale metabolic model of the yeast Saccharomyces cerevisiae to account for proteome constraints and enzyme cofactors such as metal ions, named CofactorYeast. The model is able to estimate abundances of metal ions binding on enzymes in cells under various conditions, which are comparable to measured metal ion contents in biomass. In addition, the model predicts distinct metabolic flux distributions in response to reduced levels of various metal ions in the medium. Specifically, the model reproduces changes upon iron deficiency in metabolic and gene expression levels, which could be interpreted by optimization principles (i.e., yeast optimizes iron utilization based on metabolic network and enzyme kinetics rather than preferentially targeting iron to specific enzymes or pathways). At last, we show the potential of using the model for understanding cell factories that harbor heterologous iron-containing enzymes to synthesize high-value compounds such as p-coumaric acid. Overall, the model demonstrates the dependence of enzymes on metal ions and links metal ions to metabolism on a genome scale.
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An Internal Promoter Drives the Expression of a Truncated Form of CCC1 Capable of Protecting Yeast from Iron Toxicity. Microorganisms 2021; 9:microorganisms9061337. [PMID: 34203091 PMCID: PMC8235630 DOI: 10.3390/microorganisms9061337] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2021] [Revised: 06/11/2021] [Accepted: 06/15/2021] [Indexed: 11/17/2022] Open
Abstract
In yeast, iron storage and detoxification depend on the Ccc1 transporter that mediates iron accumulation in vacuoles. While deletion of the CCC1 gene renders cells unable to survive under iron overload conditions, the deletion of its previously identified regulators only partially affects survival, indicating that the mechanisms controlling iron storage and detoxification in yeast are still far from well understood. This work reveals that CCC1 is equipped with a complex transcriptional structure comprising several regulatory regions. One of these is located inside the coding sequence of the gene and drives the expression of a short transcript encoding an N-terminally truncated protein, designated as s-Ccc1. s-Ccc1, though less efficiently than Ccc1, is able to promote metal accumulation in the vacuole, protecting cells against iron toxicity. While the expression of the s-Ccc1 appears to be repressed in the normal genomic context, our current data clearly demonstrates that it is functional and has the capacity to play a role under iron overload conditions.
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Navarrete-Perea J, Guerra-Moreno A, Van Vranken J, Isasa M, Paulo JA, Gygi SP. Iron Deficiency and Recovery in Yeast: A Quantitative Proteomics Approach. J Proteome Res 2021; 20:2751-2761. [PMID: 33797912 DOI: 10.1021/acs.jproteome.1c00035] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Iron is an essential element for life, as it is critical for oxygen transport, cellular respiration, DNA synthesis, and metabolism. Disruptions in iron metabolism have been associated with several complex diseases like diabetes, cancer, infection susceptibility, neurodegeneration, and others; however, the molecular mechanisms linking iron metabolism with these diseases are not fully understood. A commonly used model to study iron deficiency (ID) is yeast, Saccharomyces cerevisiae. Here, we used quantitative (phospho)proteomics to explore the early (4 and 6 h) and late (12 h) response to ID. We showed that metabolic pathways like the Krebs cycle, amino acid, and ergosterol biosynthesis were affected by ID. In addition, during the late response, several proteins related to the ubiquitin-proteasome system and autophagy were upregulated. We also explored the proteomic changes during a recovery period after 12 h of ID. Several proteins recovered their steady-state levels, but some others, such as cytochromes, did not recover during the time tested. Additionally, we showed that autophagy is active during ID, and some of the degraded proteins during ID can be rescued using KO strains for several key autophagy genes. Our results highlight the complex proteome changes occurring during ID and recovery. This study constitutes a valuable data set for researchers interested in iron biology, offering a temporal proteomic data set for ID, as well as a compendium the proteomic changes associated with episodes of iron recovery.
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Affiliation(s)
- Jose Navarrete-Perea
- Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02155, United States
| | | | - Jonathan Van Vranken
- Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02155, United States
| | - Marta Isasa
- C4 Therapeutics, Cambridge, Massachusetts 02142, United States
| | - Joao A Paulo
- Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02155, United States
| | - Steven P Gygi
- Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02155, United States
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44
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Gasmi A, Peana M, Arshad M, Butnariu M, Menzel A, Bjørklund G. Krebs cycle: activators, inhibitors and their roles in the modulation of carcinogenesis. Arch Toxicol 2021; 95:1161-1178. [DOI: 10.1007/s00204-021-02974-9] [Citation(s) in RCA: 45] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2020] [Accepted: 01/04/2021] [Indexed: 12/12/2022]
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45
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Robinson JR, Isikhuemhen OS, Anike FN. Fungal-Metal Interactions: A Review of Toxicity and Homeostasis. J Fungi (Basel) 2021; 7:225. [PMID: 33803838 PMCID: PMC8003315 DOI: 10.3390/jof7030225] [Citation(s) in RCA: 56] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2021] [Revised: 03/15/2021] [Accepted: 03/17/2021] [Indexed: 12/18/2022] Open
Abstract
Metal nanoparticles used as antifungals have increased the occurrence of fungal-metal interactions. However, there is a lack of knowledge about how these interactions cause genomic and physiological changes, which can produce fungal superbugs. Despite interest in these interactions, there is limited understanding of resistance mechanisms in most fungi studied until now. We highlight the current knowledge of fungal homeostasis of zinc, copper, iron, manganese, and silver to comprehensively examine associated mechanisms of resistance. Such mechanisms have been widely studied in Saccharomyces cerevisiae, but limited reports exist in filamentous fungi, though they are frequently the subject of nanoparticle biosynthesis and targets of antifungal metals. In most cases, microarray analyses uncovered resistance mechanisms as a response to metal exposure. In yeast, metal resistance is mainly due to the down-regulation of metal ion importers, utilization of metallothionein and metallothionein-like structures, and ion sequestration to the vacuole. In contrast, metal resistance in filamentous fungi heavily relies upon cellular ion export. However, there are instances of resistance that utilized vacuole sequestration, ion metallothionein, and chelator binding, deleting a metal ion importer, and ion storage in hyphal cell walls. In general, resistance to zinc, copper, iron, and manganese is extensively reported in yeast and partially known in filamentous fungi; and silver resistance lacks comprehensive understanding in both.
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Affiliation(s)
| | - Omoanghe S. Isikhuemhen
- Department of Natural Resources and Environmental Design, North Carolina Agricultural and Technical State University, 1601 East Market Street, Greensboro, NC 27411, USA; (J.R.R.); (F.N.A.)
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Soczewka P, Tribouillard-Tanvier D, di Rago JP, Zoladek T, Kaminska J. Targeting Copper Homeostasis Improves Functioning of vps13Δ Yeast Mutant Cells, a Model of VPS13-Related Diseases. Int J Mol Sci 2021; 22:2248. [PMID: 33668157 PMCID: PMC7956333 DOI: 10.3390/ijms22052248] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2021] [Revised: 02/12/2021] [Accepted: 02/19/2021] [Indexed: 01/01/2023] Open
Abstract
Ion homeostasis is crucial for organism functioning, and its alterations may cause diseases. For example, copper insufficiency and overload are associated with Menkes and Wilson's diseases, respectively, and iron imbalance is observed in Parkinson's and Alzheimer's diseases. To better understand human diseases, Saccharomyces cerevisiae yeast are used as a model organism. In our studies, we used the vps13Δ yeast strain as a model of rare neurological diseases caused by mutations in VPS13A-D genes. In this work, we show that overexpression of genes encoding copper transporters, CTR1, CTR3, and CCC2, or the addition of copper salt to the medium, improved functioning of the vps13Δ mutant. We show that their mechanism of action, at least partially, depends on increasing iron content in the cells by the copper-dependent iron uptake system. Finally, we present that treatment with copper ionophores, disulfiram, elesclomol, and sodium pyrithione, also resulted in alleviation of the defects observed in vps13Δ cells. Our study points at copper and iron homeostasis as a potential therapeutic target for further investigation in higher eukaryotic models of VPS13-related diseases.
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Affiliation(s)
- Piotr Soczewka
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland;
| | - Déborah Tribouillard-Tanvier
- IBGC, UMR 5095, CNRS, Université de Bordeaux, F-33000 Bordeaux, France; (D.T.-T.); (J.-P.d.R.)
- Institut National de la Santé et de la Recherche Médicale (INSERM), F-33077 Bordeaux, France
| | - Jean-Paul di Rago
- IBGC, UMR 5095, CNRS, Université de Bordeaux, F-33000 Bordeaux, France; (D.T.-T.); (J.-P.d.R.)
| | - Teresa Zoladek
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland;
| | - Joanna Kaminska
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland;
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47
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Bulk autophagy induction and life extension is achieved when iron is the only limited nutrient in Saccharomyces cerevisiae. Biochem J 2021; 478:811-837. [DOI: 10.1042/bcj20200849] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2020] [Revised: 01/26/2021] [Accepted: 01/27/2021] [Indexed: 01/21/2023]
Abstract
We have investigated the effects that iron limitation provokes in Saccharomyces cerevisiae exponential cultures. We have demonstrated that one primary response is the induction of bulk autophagy mediated by TORC1. Coherently, Atg13 became dephosphorylated whereas Atg1 appeared phosphorylated. The signal of iron deprivation requires Tor2/Ypk1 activity and the inactivation of Tor1 leading to Atg13 dephosphorylation, thus triggering the autophagy process. Iron replenishment in its turn, reduces autophagy flux through the AMPK Snf1 and the subsequent activity of the iron-responsive transcription factor, Aft1. This signalling converges in Atg13 phosphorylation mediated by Tor1. Iron limitation promotes accumulation of trehalose and the increase in stress resistance leading to a quiescent state in cells. All these effects contribute to the extension of the chronological life, in a manner totally dependent on autophagy activation.
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48
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Smethurst DGJ, Kovalev N, McKenzie ER, Pestov DG, Shcherbik N. Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese. J Biol Chem 2020; 295:17200-17214. [PMID: 33040024 PMCID: PMC7863898 DOI: 10.1074/jbc.ra120.015025] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2020] [Revised: 10/05/2020] [Indexed: 02/05/2023] Open
Abstract
Protein biosynthesis is fundamental to cellular life and requires the efficient functioning of the translational machinery. At the center of this machinery is the ribosome, a ribonucleoprotein complex that depends heavily on Mg2+ for structure. Recent work has indicated that other metal cations can substitute for Mg2+, raising questions about the role different metals may play in the maintenance of the ribosome under oxidative stress conditions. Here, we assess ribosomal integrity following oxidative stress both in vitro and in cells to elucidate details of the interactions between Fe2+ and the ribosome and identify Mn2+ as a factor capable of attenuating oxidant-induced Fe2+-mediated degradation of rRNA. We report that Fe2+ promotes degradation of all rRNA species of the yeast ribosome and that it is bound directly to RNA molecules. Furthermore, we demonstrate that Mn2+ competes with Fe2+ for rRNA-binding sites and that protection of ribosomes from Fe2+-mediated rRNA hydrolysis correlates with the restoration of cell viability. Our data, therefore, suggest a relationship between these two transition metals in controlling ribosome stability under oxidative stress.
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Affiliation(s)
- Daniel G J Smethurst
- Department of Cell Biology and Neuroscience, Rowan University, School of Osteopathic Medicine, Stratford, New Jersey, USA
| | - Nikolay Kovalev
- Department of Cell Biology and Neuroscience, Rowan University, School of Osteopathic Medicine, Stratford, New Jersey, USA
| | - Erica R McKenzie
- Civil and Environmental Engineering Department, Temple University, Philadelphia, Pennsylvania, USA
| | - Dimitri G Pestov
- Department of Cell Biology and Neuroscience, Rowan University, School of Osteopathic Medicine, Stratford, New Jersey, USA
| | - Natalia Shcherbik
- Department of Cell Biology and Neuroscience, Rowan University, School of Osteopathic Medicine, Stratford, New Jersey, USA.
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49
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Hierarchical routing in carbon metabolism favors iron-scavenging strategy in iron-deficient soil Pseudomonas species. Proc Natl Acad Sci U S A 2020; 117:32358-32369. [PMID: 33273114 PMCID: PMC7768705 DOI: 10.1073/pnas.2016380117] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Siderophore secretion confers competitive advantage to pathogenic and beneficial bacteria in various nutritional environments, including human infections and rhizosphere microbiome. The siderophore biosynthesis must be sustained during a compromised carbon metabolism in Fe-deficient cells. Here we demonstrate that Fe-deficient Pseudomonas species overcome this paradox by coupling selectivity in carbon utilization with a hierarchy in metabolic pathways to favor carbon and energy fluxes for siderophore biosynthesis. A reprogrammed metabolism is predicted from genomics-based data obtained with several marine and soil bacterial systems in response to Fe deficiency, but metabolomics evidence is lacking. The present study offers an important roadmap for investigating the underlying metabolic connections between Fe or other metal nutrient availability and carbon utilization. High-affinity iron (Fe) scavenging compounds, or siderophores, are widely employed by soil bacteria to survive scarcity in bioavailable Fe. Siderophore biosynthesis relies on cellular carbon metabolism, despite reported decrease in both carbon uptake and Fe-containing metabolic proteins in Fe-deficient cells. Given this paradox, the metabolic network required to sustain the Fe-scavenging strategy is poorly understood. Here, through multiple 13C-metabolomics experiments with Fe-replete and Fe-limited cells, we uncover how soil Pseudomonas species reprogram their metabolic pathways to prioritize siderophore biosynthesis. Across the three species investigated (Pseudomonas putida KT2440, Pseudomonas protegens Pf-5, and Pseudomonas putida S12), siderophore secretion is higher during growth on gluconeogenic substrates than during growth on glycolytic substrates. In response to Fe limitation, we capture decreased flux toward the tricarboxylic acid (TCA) cycle during the metabolism of glycolytic substrates but, due to carbon recycling to the TCA cycle via enhanced anaplerosis, the metabolism of gluconeogenic substrates results in an increase in both siderophore secretion (up to threefold) and Fe extraction (up to sixfold) from soil minerals. During simultaneous feeding on the different substrate types, Fe deficiency triggers a hierarchy in substrate utilization, which is facilitated by changes in protein abundances for substrate uptake and initial catabolism. Rerouted metabolism further promotes favorable fluxes in the TCA cycle and the gluconeogenesis–anaplerosis nodes, despite decrease in several proteins in these pathways, to meet carbon and energy demands for siderophore precursors in accordance with increased proteins for siderophore biosynthesis. Hierarchical carbon metabolism thus serves as a critical survival strategy during the metal nutrient deficiency.
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50
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Cirigliano A, Amelina A, Biferali B, Macone A, Mozzetta C, Bianchi MM, Mori M, Botta B, Pick E, Negri R, Rinaldi T. Statins interfere with the attachment of S. cerevisiae mtDNA to the inner mitochondrial membrane. J Enzyme Inhib Med Chem 2020; 35:129-137. [PMID: 31694426 PMCID: PMC6844431 DOI: 10.1080/14756366.2019.1687461] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2019] [Revised: 09/25/2019] [Accepted: 09/26/2019] [Indexed: 12/16/2022] Open
Abstract
The 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme of the mevalonate pathway for the synthesis of cholesterol in mammals (ergosterol in fungi), is inhibited by statins, a class of cholesterol lowering drugs. Indeed, statins are in a wide medical use, yet statins treatment could induce side effects as hepatotoxicity and myopathy in patients. We used Saccharomyces cerevisiae as a model to investigate the effects of statins on mitochondria. We demonstrate that statins are active in S.cerevisiae by lowering the ergosterol content in cells and interfering with the attachment of mitochondrial DNA to the inner mitochondrial membrane. Experiments on murine myoblasts confirmed these results in mammals. We propose that the instability of mitochondrial DNA is an early indirect target of statins.
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Affiliation(s)
- Angela Cirigliano
- Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Rome, Italy
| | - Antonia Amelina
- Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Rome, Italy
| | - Beatrice Biferali
- Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Rome, Italy
- Institute of Molecular Biology and Pathology, CNR, Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Rome, Italy
| | - Alberto Macone
- Pasteur Institute-Cenci Bolognetti Foundation, Rome, Italy
- Institute of Molecular Biology and Pathology, CNR, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza University of Rome, Rome, Italy
| | - Chiara Mozzetta
- Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Rome, Italy
- Institute of Molecular Biology and Pathology, CNR, Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Rome, Italy
| | - Michele Maria Bianchi
- Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Rome, Italy
| | - Mattia Mori
- Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy
| | - Bruno Botta
- Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, Rome, Italy
| | - Elah Pick
- Department of Biology and Environment, Faculty of Natural Sciences, University of Haifa at Oranim, Tivon, Israel
| | - Rodolfo Negri
- Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Rome, Italy
- Institute of Molecular Biology and Pathology, CNR, Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Rome, Italy
| | - Teresa Rinaldi
- Department of Biology and Biotechnology “Charles Darwin”, Sapienza University of Rome, Rome, Italy
- Pasteur Institute-Cenci Bolognetti Foundation, Rome, Italy
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