1
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Der C, Courty PE, Recorbet G, Wipf D, Simon-Plas F, Gerbeau-Pissot P. Sterols, pleiotropic players in plant-microbe interactions. TRENDS IN PLANT SCIENCE 2024; 29:524-534. [PMID: 38565452 DOI: 10.1016/j.tplants.2024.03.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/17/2023] [Revised: 02/08/2024] [Accepted: 03/04/2024] [Indexed: 04/04/2024]
Abstract
Plant-microbe interactions (PMIs) are regulated through a wide range of mechanisms in which sterols from plants and microbes are involved in numerous ways, including recognition, transduction, communication, and/or exchanges between partners. Phytosterol equilibrium is regulated by PMIs through expression of genes involved in phytosterol biosynthesis, together with their accumulation. As such, PMI outcomes also include plasma membrane (PM) functionalization events, in which phytosterols have a central role, and activation of sterol-interacting proteins involved in cell signaling. In spite (or perhaps because) of such multifaceted abilities, an overall mechanism of sterol contribution is difficult to determine. However, promising approaches exploring sterol diversity, their contribution to PMI outcomes, and their localization would help us to decipher their crucial role in PMIs.
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Affiliation(s)
- Christophe Der
- Agroécologie, INRAE, Institut Agro, University of Bourgogne, Dijon, France
| | | | - Ghislaine Recorbet
- Agroécologie, INRAE, Institut Agro, University of Bourgogne, Dijon, France
| | - Daniel Wipf
- Agroécologie, INRAE, Institut Agro, University of Bourgogne, Dijon, France
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2
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Barata IS, Rueff J, Kranendonk M, Esteves F. Pleiotropy of Progesterone Receptor Membrane Component 1 in Modulation of Cytochrome P450 Activity. J Xenobiot 2024; 14:575-603. [PMID: 38804287 PMCID: PMC11130977 DOI: 10.3390/jox14020034] [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: 03/15/2024] [Revised: 04/26/2024] [Accepted: 04/29/2024] [Indexed: 05/29/2024] Open
Abstract
Progesterone receptor membrane component 1 (PGRMC1) is one of few proteins that have been recently described as direct modulators of the activity of human cytochrome P450 enzymes (CYP)s. These enzymes form a superfamily of membrane-bound hemoproteins that metabolize a wide variety of physiological, dietary, environmental, and pharmacological compounds. Modulation of CYP activity impacts the detoxification of xenobiotics as well as endogenous pathways such as steroid and fatty acid metabolism, thus playing a central role in homeostasis. This review is focused on nine main topics that include the most relevant aspects of past and current PGRMC1 research, focusing on its role in CYP-mediated drug metabolism. Firstly, a general overview of the main aspects of xenobiotic metabolism is presented (I), followed by an overview of the role of the CYP enzymatic complex (IIa), a section on human disorders associated with defects in CYP enzyme complex activity (IIb), and a brief account of cytochrome b5 (cyt b5)'s effect on CYP activity (IIc). Subsequently, we present a background overview of the history of the molecular characterization of PGRMC1 (III), regarding its structure, expression, and intracellular location (IIIa), and its heme-binding capability and dimerization (IIIb). The next section reflects the different effects PGRMC1 may have on CYP activity (IV), presenting a description of studies on the direct effects on CYP activity (IVa), and a summary of pathways in which PGRMC1's involvement may indirectly affect CYP activity (IVb). The last section of the review is focused on the current challenges of research on the effect of PGRMC1 on CYP activity (V), presenting some future perspectives of research in the field (VI).
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Affiliation(s)
- Isabel S. Barata
- Department of Pediatrics, Division of Endocrinology, Diabetology and Metabolism, University Children’s Hospital, University of Bern, 3010 Bern, Switzerland;
- Translational Hormone Research Program, Department of Biomedical Research, University of Bern, 3010 Bern, Switzerland
- Graduate School for Cellular and Biomedical Sciences, University of Bern, 3012 Bern, Switzerland
| | - José Rueff
- ToxOmics, NOVA Medical School, Faculdade de Ciências Médicas, NMS|FCM, Universidade NOVA de Lisboa, Campo Mártires da Pátria 130, 1169-056 Lisboa, Portugal;
| | - Michel Kranendonk
- ToxOmics, NOVA Medical School, Faculdade de Ciências Médicas, NMS|FCM, Universidade NOVA de Lisboa, Campo Mártires da Pátria 130, 1169-056 Lisboa, Portugal;
| | - Francisco Esteves
- ToxOmics, NOVA Medical School, Faculdade de Ciências Médicas, NMS|FCM, Universidade NOVA de Lisboa, Campo Mártires da Pátria 130, 1169-056 Lisboa, Portugal;
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3
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Xu D, Wang M, Zhang X, Mao H, Xu H, Zhang B, Zeng X, Li F. The Putative Cytochrome b5 Domain-Containing Protein CaDap1 Homologue Is Involved in Antifungal Drug Tolerance, Cell Wall Chitin Maintenance, and Virulence in Candida albicans. J Fungi (Basel) 2024; 10:316. [PMID: 38786671 PMCID: PMC11122062 DOI: 10.3390/jof10050316] [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: 03/14/2024] [Revised: 04/18/2024] [Accepted: 04/19/2024] [Indexed: 05/25/2024] Open
Abstract
Candida albicans (Ca), a prominent opportunistic fungal pathogen in humans, has garnered considerable attention due to its infectious properties. Herein, we have identified and characterized CaCDAP1 (Ca orf19.1034), a homolog of ScDAP1 found in Saccharomyces cerevisiae. CaCDAP1 encodes a 183-amino acid protein with a conserved cytochrome b5-like heme-binding domain. The deletion of CaDAP1 renders Ca cells susceptible to caspofungin and terbinafine. CaDAP1 deletion confers resistance to Congo Red and Calcofluor White, and sensitivity to sodium dodecyl sulfate. The deletion of CaDAP1 results in a 50% reduction in chitin content within the cell wall, the downregulation of phosphorylation levels in CaMkc1, and the upregulation of phosphorylation levels in CaCek1. Notably, CaDAP1 deletion results in the abnormal hyphal development of Ca cells and diminishes virulence in a mouse systemic infection model. Thus, CaDAP1 emerges as a critical regulator governing cellular responses to antifungal drugs, the synthesis of cell wall chitin, and virulence in Ca.
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Affiliation(s)
- Dayong Xu
- College of Life Sciences, Huaibei Normal University, Huaibei 235000, China; (M.W.); (X.Z.); (H.M.); (H.X.); (B.Z.); (X.Z.)
| | | | | | | | | | | | | | - Feng Li
- College of Life Sciences, Huaibei Normal University, Huaibei 235000, China; (M.W.); (X.Z.); (H.M.); (H.X.); (B.Z.); (X.Z.)
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4
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Badve P, Meier KK. Defining Requirements for Heme Binding in PGRMC1 and Identifying Key Elements that Influence Protein Dimerization. Biochemistry 2024; 63:926-938. [PMID: 38489495 DOI: 10.1021/acs.biochem.3c00718] [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: 03/17/2024]
Abstract
Progesterone receptor membrane component 1 (PGRMC1) binds heme via a surface-exposed site and displays some structural resemblance to cytochrome b5 despite their different functions. In the case of PGRMC1, it is the protein interaction with drug-metabolizing cytochrome P450s and the epidermal growth factor receptor that has garnered the most attention. These interactions are thought to result in a compromised ability to metabolize common chemotherapy agents and to enhance cancer cell proliferation. X-ray crystallography and immunoprecipitation data have suggested that heme-mediated PGRMC1 dimers are important for facilitating these interactions. However, more recent studies have called into question the requirement of heme binding for PGRMC1 dimerization. Our study employs spectroscopic and computational methods to probe and define heme binding and its impact on PGRMC1 dimerization. Fluorescence, electron paramagnetic resonance and circular dichroism spectroscopies confirm heme binding to apo-PGRMC1 and were used to demonstrate the stabilizing effect of heme on the wild-type protein. We also utilized variants (C129S and Y113F) to precisely define the contributions of disulfide bonds and direct heme coordination to PGRMC1 dimerization. Understanding the key factors involved in these processes has important implications for downstream protein-protein interactions that may influence the metabolism of chemotherapeutic agents. This work opens avenues for deeper exploration into the physiological significance of the truncated-PGRMC1 model and developing design principles for potential therapeutics to target PGRMC1 dimerization and downstream interactions.
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Affiliation(s)
- Prajakta Badve
- Department of Chemistry, University of Miami, Coral Gables, Florida 33146, United States
| | - Katlyn K Meier
- Department of Chemistry, University of Miami, Coral Gables, Florida 33146, United States
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5
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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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6
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Asady B, Sampels V, Romano JD, Levitskaya J, Lige B, Khare P, Le A, Coppens I. Function and regulation of a steroidogenic CYP450 enzyme in the mitochondrion of Toxoplasma gondii. PLoS Pathog 2023; 19:e1011566. [PMID: 37651449 PMCID: PMC10499268 DOI: 10.1371/journal.ppat.1011566] [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: 01/05/2023] [Revised: 09/13/2023] [Accepted: 07/19/2023] [Indexed: 09/02/2023] Open
Abstract
As an obligate intracellular parasite, Toxoplasma gondii must import essential nutrients from the host cell into the parasitophorous vacuole. We previously reported that the parasite scavenges cholesterol from host endocytic organelles for incorporation into membranes and storage as cholesteryl esters in lipid droplets. In this study, we have investigated whether Toxoplasma utilizes cholesterol as a precursor for the synthesis of metabolites, such as steroids. In mammalian cells, steroidogenesis occurs in mitochondria and involves membrane-bound type I cytochrome P450 oxidases that are activated through interaction with heme-binding proteins containing a cytochrome b5 domain, such as members of the membrane-associated progesterone receptor (MAPR) family. Our LC-MS targeted lipidomics detect selective classes of hormone steroids in Toxoplasma, with a predominance for anti-inflammatory hydroxypregnenolone species, deoxycorticosterone and dehydroepiandrosterone. The genome of Toxoplasma contains homologs encoding a single type I CYP450 enzyme (we named TgCYP450mt) and a single MAPR (we named TgMAPR). We showed that TgMAPR is a hemoprotein with conserved residues in a heme-binding cytochrome b5 domain. Both TgCYP450 and TgMAPR localize to the mitochondrion and show interactions in in situ proximity ligation assays. Genetic ablation of cyp450mt is not tolerated by Toxoplasma; we therefore engineered a conditional knockout strain and showed that iΔTgCYP450mt parasites exhibit growth impairment in cultured cells. Parasite strains deficient for mapr could be generated; however, ΔTgMAPR parasites suffer from poor global fitness, loss of plasma membrane integrity, aberrant mitochondrial cristae, and an abnormally long S-phase in their cell cycle. Compared to wild-type parasites, iΔTgCYP450mt and ΔTgMAPR lost virulence in mice and metabolomics studies reveal that both mutants have reduced levels of steroids. These observations point to a steroidogenic pathway operational in the mitochondrion of a protozoan that involves an evolutionary conserved TgCYP450mt enzyme and its binding partner TgMAPR.
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Affiliation(s)
- Beejan Asady
- Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, United States of America
| | - Vera Sampels
- Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, United States of America
| | - Julia D. Romano
- Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, United States of America
| | - Jelena Levitskaya
- Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, United States of America
| | - Bao Lige
- Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, United States of America
| | - Pratik Khare
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America
| | - Anne Le
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America
- Department of Pathology and Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
| | - Isabelle Coppens
- Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, United States of America
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7
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Turco G, Chang C, Wang RY, Kim G, Stoops EH, Richardson B, Sochat V, Rust J, Oughtred R, Thayer N, Kang F, Livstone MS, Heinicke S, Schroeder M, Dolinski KJ, Botstein D, Baryshnikova A. Global analysis of the yeast knockout phenome. SCIENCE ADVANCES 2023; 9:eadg5702. [PMID: 37235661 PMCID: PMC11326039 DOI: 10.1126/sciadv.adg5702] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/06/2023] [Accepted: 04/20/2023] [Indexed: 05/28/2023]
Abstract
Genome-wide phenotypic screens in the budding yeast Saccharomyces cerevisiae, enabled by its knockout collection, have produced the largest, richest, and most systematic phenotypic description of any organism. However, integrative analyses of this rich data source have been virtually impossible because of the lack of a central data repository and consistent metadata annotations. Here, we describe the aggregation, harmonization, and analysis of ~14,500 yeast knockout screens, which we call Yeast Phenome. Using this unique dataset, we characterized two unknown genes (YHR045W and YGL117W) and showed that tryptophan starvation is a by-product of many chemical treatments. Furthermore, we uncovered an exponential relationship between phenotypic similarity and intergenic distance, which suggests that gene positions in both yeast and human genomes are optimized for function.
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Affiliation(s)
- Gina Turco
- Calico Life Sciences LLC, South San Francisco, CA, USA
| | - Christie Chang
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA
| | | | - Griffin Kim
- Calico Life Sciences LLC, South San Francisco, CA, USA
| | | | - Brianna Richardson
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA
| | - Vanessa Sochat
- Lawrence Livermore National Laboratory, Livermore, CA, USA
| | - Jennifer Rust
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA
| | - Rose Oughtred
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA
| | | | - Fan Kang
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA
| | - Michael S Livstone
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA
| | - Sven Heinicke
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA
| | - Mark Schroeder
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA
| | - Kara J Dolinski
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA
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8
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Yang J, Park S, Kim HJ, Lee SJ, Jung WH. The Interkingdom Interaction with Staphylococcus Influences the Antifungal Susceptibility of the Cutaneous Fungus Malassezia. J Microbiol Biotechnol 2023; 33:180-187. [PMID: 36575858 PMCID: PMC9998211 DOI: 10.4014/jmb.2210.10039] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2022] [Revised: 12/10/2022] [Accepted: 12/12/2022] [Indexed: 12/29/2022]
Abstract
The skin is a dynamic ecosystem on which diverse microbes reside. The interkingdom interaction between microbial species in the skin microbiota is thought to influence the health and disease of the skin although the roles of the intra- and interkingdom interactions remain to be elucidated. In this context, the interactions between Malassezia and Staphylococcus, the most dominant microorganisms in the skin microbiota, have gained attention. This study investigated how the interaction between Malassezia and Staphylococcus affected the antifungal susceptibility of the fungus to the azole antifungal drug ketoconazole. The susceptibility was significantly decreased when Malassezia was co-cultured with Staphylococcus. We found that acidification of the environment by organic acids produced by Staphylococcus influenced the decrease of the ketoconazole susceptibility of M. restricta in the co-culturing condition. Furthermore, our data demonstrated that the significant increased ergosterol content and cell membrane and wall thickness of the M. restricta cells grown in the acidic environment may be the main cause of the altered azole susceptibility of the fungus. Overall, our study suggests that the interaction between Malassezia and Staphylococcus influences the antifungal susceptibility of the fungus and that pH has a critical role in the polymicrobial interaction in the skin environment.
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Affiliation(s)
- Juan Yang
- Department of Systems Biotechnology and Institute of Microbiomics, Chung-Ang University, Anseong 17546, Republic of Korea
| | - Sungmin Park
- Department of Systems Biotechnology and Institute of Microbiomics, Chung-Ang University, Anseong 17546, Republic of Korea
| | - Hyun Ju Kim
- Department of Systems Biotechnology and Institute of Microbiomics, Chung-Ang University, Anseong 17546, Republic of Korea
| | - Sang Jun Lee
- Department of Systems Biotechnology and Institute of Microbiomics, Chung-Ang University, Anseong 17546, Republic of Korea
| | - Won Hee Jung
- Department of Systems Biotechnology and Institute of Microbiomics, Chung-Ang University, Anseong 17546, Republic of Korea
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9
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McGuire MR, Espenshade PJ. PGRMC1: An enigmatic heme-binding protein. Pharmacol Ther 2023; 241:108326. [PMID: 36463977 PMCID: PMC9839567 DOI: 10.1016/j.pharmthera.2022.108326] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2022] [Revised: 11/22/2022] [Accepted: 11/28/2022] [Indexed: 12/03/2022]
Abstract
Progesterone Receptor Membrane Component 1 (PGRMC1) is a heme-binding protein that has been implicated in a wide range of cell and tissue functions, including cytochromes P450 activity, heme homeostasis, cancer, female reproduction, and protein quality control. Despite an extensive body of literature, a relative lack of mechanistic insight means that how PGRMC1 functions in these different aspects of biology is largely unknown. This review provides an overview of the PGRMC1 literature, highlighting what information is rigorously supported by experimental evidence and where additional investigation is warranted. The central role of PGRMC1 in supporting cytochrome P450 activity is discussed at length. Building on existing models of PGRMC1 function, a speculative model is proposed using the reviewed literature in which PGRMC1 functions as a heme chaperone to shuttle heme from its site of synthesis in the mitochondrion to other subcellular compartments. By spotlighting knowledge gaps, this review will motivate investigators to better understand this enigmatic protein.
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Affiliation(s)
- Meredith R McGuire
- Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Peter J Espenshade
- Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Oncology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Physiology 107B, Baltimore, MD 21205, USA.
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10
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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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11
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Zhao S, Hughes AL, Espenshade PJ. Fission yeast Dap1 heme iron-coordinating residue Y83 is required for cytochromes P450 function. MICROPUBLICATION BIOLOGY 2022; 2022:10.17912/micropub.biology.000631. [PMID: 36090151 PMCID: PMC9449707 DOI: 10.17912/micropub.biology.000631] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/10/2022] [Revised: 08/22/2022] [Accepted: 08/22/2022] [Indexed: 11/28/2022]
Abstract
Fission yeast Dap1 is a heme binding protein required for cytochromes P450 activity. Here, we tested whether Dap1 axial coordination of heme iron is required for its role in the function of the cytochrome P450 enzymes, Erg5 and Erg11. Two different dap1 mutants predicted to alter iron coordination failed to rescue growth on cobalt chloride containing medium which requires Erg5 and Erg11. In addition, deletion of dap1 + did not affect expression of Erg5 or Erg11. PGRMC1, a mammalian Dap1 homolog, does not require heme binding to bind and stabilize cytochromes P450. These experiments highlight important functional differences between these conserved proteins.
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Affiliation(s)
- Shan Zhao
- Johns Hopkins University School of Medicine, USA
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12
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Abstract
Progesterone receptor membrane component (PGRMC) proteins play important roles in tumor growth, progression, and chemoresistance, of which PGRMC1 is the best characterized. The ancestral member predates the evolution of metazoans, so it is perhaps not surprising that many of the purported actions of PGRMC proteins are rooted in fundamental metabolic processes such as proliferation, apoptosis, and DNA damage responses. Despite mediating some of the actions of progesterone (P4) and being fundamentally required for female fertility, PGRMC1 and PGRMC2 are broadly expressed in most tissues. As such, these proteins likely have both progesterone-dependent and progesterone-independent functions. It has been proposed that PGRMC1 acquired the ability to mediate P4 actions over evolutionary time through acquisition of its cytochrome b5-like heme/sterol-binding domain. Diverse reproductive and nonreproductive diseases associate with altered PGRMC1 expression, epigenetic regulation, or gene silencing mechanisms, some of which include polycystic ovarian disease, premature ovarian insufficiency, endometriosis, Alzheimer disease, and cancer. Although many studies have been completed using transformed cell lines in culture or in xenograft tumor approaches, recently developed transgenic model organisms are offering new insights in the physiological actions of PGRMC proteins, as well as pathophysiological and oncogenic consequences when PGRMC expression is altered. The purpose of this mini-review is to provide an overview of PGRMC proteins in cancer and to offer discussion of where this field must go to solidify PGRMC proteins as central contributors to the oncogenic process.
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Affiliation(s)
- James K Pru
- Correspondence: James K. Pru, PhD, Program in Reproductive Biology, Department of Animal Science, University of Wyoming, Laramie, WY, USA.
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13
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Zhang L, Ruan X, Gu M, Mueck AO. E2 + norethisterone promotes the PI3K-AKT pathway via PGRMC1 to induce breast cancer cell proliferation. Climacteric 2022; 25:467-475. [PMID: 35137666 DOI: 10.1080/13697137.2022.2029837] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
OBJECTIVE This study aimed to find evidence that progesterone receptor membrane component 1 (PGRMC1) promotes estradiol (E2) + norethisterone (NET)-induced breast cancer proliferation through activation of the phosphatidylinositol-3-kinase (PI3K)-AKT pathway. METHODS PGRMC1-mediated breast cancer cellular proliferation and phosphorylation of PGRMC1 were studied using wild-type (hemagglutinin [HA]-tagged) MCF-7 cells, which were stably transfected with expression vector containing HA (MCF-7-HA cells), PGRMC1 (MCF-7-PGRMC1 cells) and Ser181 point mutated PGRMC1 (MCF-7-PGRMC1-S181A cells). Bioinformatics, cell proliferation, western blot, isobaric tags for relative and absolute quantitation (iTRAQ)-based RNA sequencing, real-time quantitative polymerase chain reaction (RT-qPCR) and cell cycle in vitro assays were performed to indicate the function of PGRMC1 and its possible mechanisms in breast cancer. RESULTS NET + E2 elicited a significant proliferation in MCF-7-Vec at 10-6 M and 10-10 M, respectively. MCF-7-PGRMC1 did increase the phosphorylation of AKT or ERK, which can be blocked by treatment with casein kinase 2 (CK2) inhibitor quinalizarin or in MCF-7-PGRMC1-S181A cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the PI3K-AKT pathway is upregulated in MCF-7-PGRMC1 cells. Importantly, upregulation of the PI3K-AKT pathway mainly through promotion of cell cycle regulation strongly promoted cell proliferation in MCF-7-PGRMC1 cells. CONCLUSIONS CK2 is involved in phosphorylation of PGRMC1 at S181. The mechanism for the action of PGRMC1 for mediating proliferative progestogen effects obviously starts with promotion cell cycle regulation, and then activation of the PI3K-AKT pathway.
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Affiliation(s)
- L Zhang
- Department of Gynecological Endocrinology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China
| | - X Ruan
- Department of Gynecological Endocrinology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China.,Department of Women's Health, University Women's Hospital and Research Center for Women's Health, University of Tuebingen, Tuebingen, Germany
| | - M Gu
- Department of Gynecological Endocrinology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China
| | - A O Mueck
- Department of Gynecological Endocrinology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China.,Department of Women's Health, University Women's Hospital and Research Center for Women's Health, University of Tuebingen, Tuebingen, Germany
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14
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González AM, Venegas M, Barahona S, Gómez M, Gutiérrez MS, Sepúlveda D, Baeza M, Cifuentes V, Alcaíno J. Damage response protein 1 (Dap1) functions in the synthesis of carotenoids and sterols in Xanthophyllomyces dendrorhous. J Lipid Res 2022; 63:100175. [PMID: 35120994 PMCID: PMC8953664 DOI: 10.1016/j.jlr.2022.100175] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2021] [Revised: 01/22/2022] [Accepted: 01/24/2022] [Indexed: 11/25/2022] Open
Abstract
Cytochrome P450s (P450s) are heme-containing proteins involved in several cellular functions, including biosynthesis of steroidal hormones, detoxification of xenobiotic compounds, among others. Damage response protein 1 (Dap1) has been described as a positive regulator of P450s through protein-protein interactions in organisms such as Schizosaccharomyces pombe. Three P450s in the carotenogenic yeast Xanthophyllomyces dendrorhous have thus far been characterized: Cyp51 and Cyp61, which are involved in ergosterol biosynthesis, and CrtS (astaxanthin synthase), which is involved in biosynthesis of the carotenoid astaxanthin. In this work, we describe the X. dendrorhous DAP1 gene, deletion of which affected yeast pigmentation by decreasing the astaxanthin fraction and increasing the β-carotene (a substrate of CrtS) fraction, which is consistent with the known role of CrtS. We found that the proportion of ergosterol was also decreased in the Δdap1 mutant. However, even though the fractions of the end products of these two pathways (the synthesis of carotenoids and sterols) were decreased in the Δdap1 mutant, the transcript levels of genes from the P450 systems involved were higher than those in the wild-type strain. We demonstrate that Dap1 coimmunoprecipitates with these three P450s, suggesting that Dap1 interacts with these three proteins. We propose that Dap1 regulates the synthesis of astaxanthin and ergosterol in X. dendrorhous, probably by regulating the P450s involved in both biosynthetic pathways at the protein level. This work suggests a new role for Dap1 in the regulation of carotenoid biosynthesis in X. dendrorhous.
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Affiliation(s)
- Ana-María González
- Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
| | - Maximiliano Venegas
- Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
| | - Salvador Barahona
- Centro de Biotecnología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
| | - Melissa Gómez
- Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
| | - María-Soledad Gutiérrez
- Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
| | - Dionisia Sepúlveda
- Centro de Biotecnología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
| | - Marcelo Baeza
- Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile; Centro de Biotecnología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
| | - Víctor Cifuentes
- Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile; Centro de Biotecnología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
| | - Jennifer Alcaíno
- Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile; Centro de Biotecnología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
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15
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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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16
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Thieffry C, Van Wynendaele M, Aynaci A, Maja M, Dupuis C, Loriot A, Marbaix E, Henriet P. AG-205 Upregulates Enzymes Involved in Cholesterol Biosynthesis and Steroidogenesis in Human Endometrial Cells Independently of PGRMC1 and Related MAPR Proteins. Biomolecules 2021; 11:1472. [PMID: 34680104 PMCID: PMC8533447 DOI: 10.3390/biom11101472] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2021] [Revised: 10/01/2021] [Accepted: 10/04/2021] [Indexed: 12/27/2022] Open
Abstract
An inappropriate response to progestogens in the human endometrium can result in fertility issues and jeopardize progestin-based treatments against pathologies such as endometriosis. PGRMC1 can mediate progesterone response in the breast and ovaries but its endometrial functions remain unknown. AG-205 is an alleged PGRMC1 inhibitor but its specificity was recently questioned. We added AG-205 in the cultures of two endometrial cell lines and performed a transcriptomic comparison. AG-205 significantly increased expression of genes coding enzymes of the cholesterol biosynthetic pathway or of steroidogenesis. However, these observations were not reproduced with cells transfected with siRNA against PGRMC1 or its related proteins (MAPRs). Furthermore, AG-205 retained its ability to increase expression of selected target genes even when expression of PGRMC1 or all MAPRs was concomitantly downregulated, indicating that neither PGRMC1 nor any MAPR is required to mediate AG-205 effect. In conclusion, although AG-205 has attractive effects encouraging its use to develop therapeutic strategies, for instance against breast cancer, our study delivers two important warning messages. First, AG-205 is not specific for PGRMC1 or other MAPRs and its mechanisms of action remain unclear. Second, due to its effects on genes involved in steroidogenesis, its use may increase the risk for endometrial pathologies resulting from imbalanced hormones concentrations.
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Affiliation(s)
- Charlotte Thieffry
- CELL Unit, de Duve Institute and Université Catholique de Louvain, B-1200 Brussels, Belgium; (C.T.); (M.V.W.); (A.A.); (M.M.); (C.D.); (E.M.)
| | - Marie Van Wynendaele
- CELL Unit, de Duve Institute and Université Catholique de Louvain, B-1200 Brussels, Belgium; (C.T.); (M.V.W.); (A.A.); (M.M.); (C.D.); (E.M.)
| | - Asena Aynaci
- CELL Unit, de Duve Institute and Université Catholique de Louvain, B-1200 Brussels, Belgium; (C.T.); (M.V.W.); (A.A.); (M.M.); (C.D.); (E.M.)
| | - Mauriane Maja
- CELL Unit, de Duve Institute and Université Catholique de Louvain, B-1200 Brussels, Belgium; (C.T.); (M.V.W.); (A.A.); (M.M.); (C.D.); (E.M.)
| | - Caroline Dupuis
- CELL Unit, de Duve Institute and Université Catholique de Louvain, B-1200 Brussels, Belgium; (C.T.); (M.V.W.); (A.A.); (M.M.); (C.D.); (E.M.)
| | - Axelle Loriot
- GEPI Unit, de Duve Institute and Université Catholique de Louvain, B-1200 Brussels, Belgium;
| | - Etienne Marbaix
- CELL Unit, de Duve Institute and Université Catholique de Louvain, B-1200 Brussels, Belgium; (C.T.); (M.V.W.); (A.A.); (M.M.); (C.D.); (E.M.)
- Pathology Department, Cliniques Universitaires Saint-Luc, B-1200 Brussels, Belgium
| | - Patrick Henriet
- CELL Unit, de Duve Institute and Université Catholique de Louvain, B-1200 Brussels, Belgium; (C.T.); (M.V.W.); (A.A.); (M.M.); (C.D.); (E.M.)
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17
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Mestre-Fos S, Ito C, Moore CM, Reddi AR, Williams LD. Human ribosomal G-quadruplexes regulate heme bioavailability. J Biol Chem 2020; 295:14855-14865. [PMID: 32817343 PMCID: PMC7606673 DOI: 10.1074/jbc.ra120.014332] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2020] [Revised: 08/06/2020] [Indexed: 12/13/2022] Open
Abstract
The in vitro formation of stable G-quadruplexes (G4s) in human rRNA was recently reported. However, their formation in cells and their cellular roles were not resolved. Here, by taking a chemical biology approach that integrates results from immunofluorescence, G4 ligands, heme-affinity reagents, and a genetically encoded fluorescent heme sensor, we report that human ribosomes can form G4s in vivo that regulate heme bioavailability. Immunofluorescence experiments indicate that the vast majority of extra-nuclear G4s are associated with rRNA. Moreover, titrating human cells with a G4 ligand alters the ability of ribosomes to bind heme and disrupts cellular heme bioavailability as measured by a genetically encoded fluorescent heme sensor. Overall, these results suggest that ribosomes play a role in regulating heme homeostasis.
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Affiliation(s)
- Santi Mestre-Fos
- Center for the Origin of Life, Georgia Institute of Technology, Atlanta, Georgia, USA; School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA; Parker Petit Institute of Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia, USA
| | - Chieri Ito
- Center for the Origin of Life, Georgia Institute of Technology, Atlanta, Georgia, USA; School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA; Parker Petit Institute of Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia, USA
| | - Courtney M Moore
- School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA; Parker Petit Institute of Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia, USA
| | - Amit R Reddi
- School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA; Parker Petit Institute of Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia, USA; School of Biological Sciences, Georgia Institute of Technology, Atlanta, Georgia, USA.
| | - Loren Dean Williams
- Center for the Origin of Life, Georgia Institute of Technology, Atlanta, Georgia, USA; School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA; Parker Petit Institute of Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia, USA; School of Biological Sciences, Georgia Institute of Technology, Atlanta, Georgia, USA.
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18
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Regulation of Ergosterol Biosynthesis in Saccharomyces cerevisiae. Genes (Basel) 2020; 11:genes11070795. [PMID: 32679672 PMCID: PMC7397035 DOI: 10.3390/genes11070795] [Citation(s) in RCA: 222] [Impact Index Per Article: 44.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Revised: 07/06/2020] [Accepted: 07/13/2020] [Indexed: 12/14/2022] Open
Abstract
Ergosterol is an essential component of fungal cell membranes that determines the fluidity, permeability and activity of membrane-associated proteins. Ergosterol biosynthesis is a complex and highly energy-consuming pathway that involves the participation of many enzymes. Deficiencies in sterol biosynthesis cause pleiotropic defects that limit cellular proliferation and adaptation to stress. Thereby, fungal ergosterol levels are tightly controlled by the bioavailability of particular metabolites (e.g., sterols, oxygen and iron) and environmental conditions. The regulation of ergosterol synthesis is achieved by overlapping mechanisms that include transcriptional expression, feedback inhibition of enzymes and changes in their subcellular localization. In the budding yeast Saccharomyces cerevisiae, the sterol regulatory element (SRE)-binding proteins Upc2 and Ecm22, the heme-binding protein Hap1 and the repressor factors Rox1 and Mot3 coordinate ergosterol biosynthesis (ERG) gene expression. Here, we summarize the sterol biosynthesis, transport and detoxification systems of S. cerevisiae, as well as its adaptive response to sterol depletion, low oxygen, hyperosmotic stress and iron deficiency. Because of the large number of ERG genes and the crosstalk between different environmental signals and pathways, many aspects of ergosterol regulation are still unknown. The study of sterol metabolism and its regulation is highly relevant due to its wide applications in antifungal treatments, as well as in food and pharmaceutical industries.
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19
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Thejer BM, Adhikary PP, Teakel SL, Fang J, Weston PA, Gurusinghe S, Anwer AG, Gosnell M, Jazayeri JA, Ludescher M, Gray LA, Pawlak M, Wallace RH, Pant SD, Wong M, Fischer T, New EJ, Fehm TN, Neubauer H, Goldys EM, Quinn JC, Weston LA, Cahill MA. PGRMC1 effects on metabolism, genomic mutation and CpG methylation imply crucial roles in animal biology and disease. BMC Mol Cell Biol 2020; 21:26. [PMID: 32293262 PMCID: PMC7160964 DOI: 10.1186/s12860-020-00268-z] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2019] [Accepted: 03/20/2020] [Indexed: 01/08/2023] Open
Abstract
Background Progesterone receptor membrane component 1 (PGRMC1) is often elevated in cancers, and exists in alternative states of phosphorylation. A motif centered on PGRMC1 Y180 was evolutionarily acquired concurrently with the embryological gastrulation organizer that orchestrates vertebrate tissue differentiation. Results Here, we show that mutagenic manipulation of PGRMC1 phosphorylation alters cell metabolism, genomic stability, and CpG methylation. Each of several mutants elicited distinct patterns of genomic CpG methylation. Mutation of S57A/Y180/S181A led to increased net hypermethylation, reminiscent of embryonic stem cells. Pathways enrichment analysis suggested modulation of processes related to animal cell differentiation status and tissue identity, as well as cell cycle control and ATM/ATR DNA damage repair regulation. We detected different genomic mutation rates in culture. Conclusions A companion manuscript shows that these cell states dramatically affect protein abundances, cell and mitochondrial morphology, and glycolytic metabolism. We propose that PGRMC1 phosphorylation status modulates cellular plasticity mechanisms relevant to early embryological tissue differentiation.
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Affiliation(s)
- Bashar M Thejer
- School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia.,Department of Biology, College of Science, University of Wasit, Kut, Wasit, Iraq
| | - Partho P Adhikary
- School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia.,Present Address: Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, Canada
| | - Sarah L Teakel
- School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia
| | - Johnny Fang
- School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia
| | - Paul A Weston
- Graham Centre for Agricultural Innovation, Charles Sturt University, Boorooma Street, Wagga Wagga, NSW, 2678, Australia.,School of Agricultural and Wine Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, NSW, 2678, Australia
| | - Saliya Gurusinghe
- Graham Centre for Agricultural Innovation, Charles Sturt University, Boorooma Street, Wagga Wagga, NSW, 2678, Australia
| | - Ayad G Anwer
- ARC Centre of Excellence for Nanoscale BioPhotonics, Macquarie University, Sydney, NSW, 2109, Australia.,Present Address: The Graduate School of Biomedical Engineering, University of New South Wales, Sydney, Kensington, NSW, 2052, Australia
| | - Martin Gosnell
- ARC Centre of Excellence for Nanoscale BioPhotonics, Macquarie University, Sydney, NSW, 2109, Australia.,Quantitative (Biotechnology) Pty. Ltd., ABN 17 165 684 186, Australia
| | - Jalal A Jazayeri
- School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia
| | - Marina Ludescher
- Department of Gynecology and Obstetrics, University Women's Hospital of Dusseldorf, Dusseldorf, Germany
| | - Lesley-Ann Gray
- Australian Genome Research Facility Ltd., Victorian Comprehensive Cancer Centre, Melbourne, VIC, 3000, Australia
| | - Michael Pawlak
- NMI TT Pharmaservices, Protein Profiling, 72770 Reutlingen, Germany
| | - Robyn H Wallace
- School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia
| | - Sameer D Pant
- School of Animal and Veterinary Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, NSW, 2678, Australia
| | - Marie Wong
- Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Darlinghurst, NSW, 2010, Australia
| | - Tamas Fischer
- ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, 2601, Australia
| | - Elizabeth J New
- University of Sydney, School of Chemistry, Sydney, NSW, 2006, Australia
| | - Tanja N Fehm
- Department of Gynecology and Obstetrics, University Women's Hospital of Dusseldorf, Dusseldorf, Germany
| | - Hans Neubauer
- Department of Gynecology and Obstetrics, University Women's Hospital of Dusseldorf, Dusseldorf, Germany
| | - Ewa M Goldys
- ARC Centre of Excellence for Nanoscale BioPhotonics, Macquarie University, Sydney, NSW, 2109, Australia.,Present Address: The Graduate School of Biomedical Engineering, University of New South Wales, Sydney, Kensington, NSW, 2052, Australia
| | - Jane C Quinn
- Graham Centre for Agricultural Innovation, Charles Sturt University, Boorooma Street, Wagga Wagga, NSW, 2678, Australia.,Faculty of Science, Charles Sturt University, Boorooma Street, Wagga Wagga, NSW, 2678, Australia
| | - Leslie A Weston
- Graham Centre for Agricultural Innovation, Charles Sturt University, Boorooma Street, Wagga Wagga, NSW, 2678, Australia.,School of Agricultural and Wine Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, NSW, 2678, Australia
| | - Michael A Cahill
- School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2650, Australia. .,ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, 2601, Australia.
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20
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Hehenberger E, Eitel M, Fortunato SAV, Miller DJ, Keeling PJ, Cahill MA. Early eukaryotic origins and metazoan elaboration of MAPR family proteins. Mol Phylogenet Evol 2020; 148:106814. [PMID: 32278076 DOI: 10.1016/j.ympev.2020.106814] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2019] [Revised: 03/24/2020] [Accepted: 04/01/2020] [Indexed: 01/01/2023]
Abstract
The membrane-associated progesterone receptor (MAPR) family consists of heme-binding proteins containing a cytochrome b5 (cytb5) domain characterized by the presence of a MAPR-specific interhelical insert region (MIHIR) between helices 3 and 4 of the canonical cytb5-domain fold. Animals possess three MAPR genes (PGRMC-like, Neuferricin and Neudesin). Here we show that all three animal MAPR genes were already present in the common ancestor of the opisthokonts (comprising animals and fungi as well as related single-celled taxa). All three MAPR genes acquired extensions C-terminal to the cytb5 domain, either before or with the evolution of animals. The archetypical MAPR protein, progesterone receptor membrane component 1 (PGRMC1), contains phosphorylated tyrosines Y139 and Y180. The combination of Y139/Y180 appeared in the common ancestor of cnidarians and bilaterians, along with an early embryological organizer and synapsed neurons, and is strongly conserved in all bilaterian animals. A predicted protein interaction motif in the PGRMC1 MIHIR is potentially regulated by Y139 phosphorylation. A multilayered model of animal MAPR function acquisition includes some pre-metazoan functions (e.g., heme binding and cytochrome P450 interactions) and some acquired animal-specific functions that involve regulation of strongly conserved protein interaction motifs acquired by animals (Metazoa). This study provides a conceptual framework for future studies, against which especially PGRMC1's multiple functions can perhaps be stratified and functionally dissected.
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Affiliation(s)
- Elisabeth Hehenberger
- Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada
| | - Michael Eitel
- Department of Earth and Environmental Sciences, Paleontology and Geobiology, Ludwig-Maximilians-Universität München, Munich, Germany
| | - Sofia A V Fortunato
- ARC Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, QLD 4811, Australia
| | - David J Miller
- ARC Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, QLD 4811, Australia
| | - Patrick J Keeling
- Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada
| | - Michael A Cahill
- School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW 2678, Australia; ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, Canberra, ACT 2601, Australia.
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21
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Yang WJ, Wang HB, Wang WD, Bai PY, Lu HX, Sun CH, Liu ZS, Guan DK, Yang GW, Zhang GL. A network-based predictive gene expression signature for recurrence risks in stage II colorectal cancer. Cancer Med 2019; 9:179-193. [PMID: 31724326 PMCID: PMC6943157 DOI: 10.1002/cam4.2642] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2019] [Revised: 08/07/2019] [Accepted: 10/10/2019] [Indexed: 12/11/2022] Open
Abstract
The current criteria for defining the recurrence risks of stage II colorectal cancer (CRC) are not robust; therefore, we aimed to explore novel gene signatures to predict recurrence risks and to reveal the underlying mechanisms of stage II CRC. First, the gene expression profiles of 124 patients with stage II CRC from The Cancer Genome Atlas (TCGA) database were obtained to screen differentially expressed genes (DEGs). A total of 202 DEGs, including 128 upregulated and 74 downregulated, were identified in the recurrence group (n = 24) compared to the nonrecurrence group (n = 100). Furthermore, the top 5 DEGs (ZNF561, WFS1, SLC2A1, MFI2, and PTGR1) were identified by random forest variable hunting, and four (ZNF561, WFS1, SLC2A1, and PTGR1) were selected to create a four‐gene recurrent model (GRM), with an area under the curve (AUC) of 0.882 according to the receiver operating characteristic curve, and the robust diagnostic effectiveness of the GRM was further validated with another gene expression profiling dataset (GSE12032), with an AUC of 0.943. The diagnostic effectiveness of the GRM regarding recurrence was associated with poor disease‐free survival in all stages of CRC. In addition, gene ontology functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses revealed 18 enriched functions and 6 enriched pathways. Four genes, ABCG2, CACNA1F, CYP19A1, and TF, were identified as hub genes by the protein‐protein interaction network, which further validated that these genes were correlated with a poor pathologic stage and overall survival in all stages of CRC. In conclusion, the GRM can effectively classify stage II CRC into groups of high and low risks of recurrence, thereby making up for the prognostic value of the traditional clinicopathological risk factors defined by the National Comprehensive Cancer Network guidelines. The hub genes may be useful therapeutic targets for recurrence. Thus, the GRM and hub genes could offer clinical value in directing individualized and precision therapeutic regimens for stage II CRC patients.
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Affiliation(s)
- Wen-Jing Yang
- Department of Oncology, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, China
| | - Hai-Bo Wang
- Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing, China
| | - Wen-Da Wang
- Department of Anorectal Surgery, Shanxi Cancer Hospital, Taiyuan, China
| | - Peng-Yu Bai
- Department of Anorectal Surgery, Shanxi Cancer Hospital, Taiyuan, China
| | - Hong-Xia Lu
- Department of Gastroenterology, Shanxi Cancer Hospital, Taiyuan, China
| | - Chang-He Sun
- Department of Oncology, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, China
| | - Zi-Shen Liu
- Department of Oncology, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, China
| | - Ding-Kun Guan
- Department of Oncology, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, China
| | - Guo-Wang Yang
- Department of Oncology, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, China
| | - Gan-Lin Zhang
- Department of Oncology, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, China
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22
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Piel RB, Dailey HA, Medlock AE. The mitochondrial heme metabolon: Insights into the complex(ity) of heme synthesis and distribution. Mol Genet Metab 2019; 128:198-203. [PMID: 30709775 PMCID: PMC6640082 DOI: 10.1016/j.ymgme.2019.01.006] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/17/2018] [Revised: 12/04/2018] [Accepted: 01/10/2019] [Indexed: 10/27/2022]
Abstract
Heme is an essential cofactor in metazoans that is also toxic in its free state. Heme is synthesized by most metazoans and must be delivered to all cellular compartments for incorporation into a variety of hemoproteins. The heme biosynthesis enzymes have been proposed to exist in a metabolon, a protein complex consisting of interacting enzymes in a metabolic pathway. Metabolons enhance the function of enzymatic pathways by creating favorable microenvironments for pathway enzymes and intermediates, facilitating substrate transport, and providing a scaffold for interactions with other pathways, signaling molecules, or organelles. Herein we detail growing evidence for a mitochondrial heme metabolon and discuss its implications for the study of heme biosynthesis and cellular heme homeostasis.
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Affiliation(s)
- Robert B Piel
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, United States
| | - Harry A Dailey
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, United States; Biomedical and Health Sciences Institute, University of Georgia, Athens, GA 30602, United States; Department of Microbiology, University of Georgia, Athens, GA, 30602, United States
| | - Amy E Medlock
- Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, United States; Biomedical and Health Sciences Institute, University of Georgia, Athens, GA 30602, United States; Augusta University-University of Georgia, Medical Partnership, Athens, GA 30602, United States.
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23
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Sabbir MG. Progesterone induced Warburg effect in HEK293 cells is associated with post-translational modifications and proteasomal degradation of progesterone receptor membrane component 1. J Steroid Biochem Mol Biol 2019; 191:105376. [PMID: 31067491 DOI: 10.1016/j.jsbmb.2019.105376] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/13/2019] [Revised: 04/17/2019] [Accepted: 05/04/2019] [Indexed: 02/07/2023]
Abstract
Progesterone (P4) is a major steroid hormone that has important effects on metabolism. The progesterone receptor membrane component 1 (PGRMC1) is a non-canonical P4 binding protein. The biological functions affected by PGRMC1 include cholesterol/steroid biosynthesis and metabolism, iron homeostasis and heme trafficking, autophagy, regulation of cell cycle and proliferation, cell migration and invasion. PGRMC1 has been an attractive target for therapeutic intervention in cancer and neurodegenerative disorders due to its biological role in promoting cell survival. P4 has been used in a number of clinical applications and is considered neuroprotective. The involvement of PGRMC1 in P4-mediated regulation of cellular glucose metabolism is not well studied. PGRMC1 is a 21 kDa protein but complex post-translational modifications (PTMs) lead to the existence of several high molecular mass proteins whose molecular function, intracellular distribution, and physiological relevancies are not fully known. Therefore, in this study, P4-PGRMC1-mediated cellular glucose metabolism and PTMs of PGRMC1 were studied using wild-type and CRISPR/Cas9 mediated PGRMC1 knockout (KO) human embryonic kidney-derived (HEK293) cell lines. A 70 kDa (p70) and 100 kDa (p100) PGRMC1 proteins were identified that are predominantly associated with endoplasmic reticulum/mitochondria and nuclear fractions in the cells, respectively. Phosphorylation, acetylation, ubiquitination, and sumoylation of native PGRMC1 under serum starvation were identified which provided an explanation for the higher molecular masses. This study indicates that P4-PGRMC1 signaling caused a rapid increase in glycolysis in the presence of oxygen (aerobic glycolysis) and a corresponding decrease in cellular respiration, known as the Warburg effect. Further, it was demonstrated that the P4-induced increase in glycolysis is associated with rapid proteasomal degradation of the p70 and reduction of the nuclear p100 protein level. P4 treatment also caused significant alteration in the dynamics of PGRMC1 PTMs and its association with potential interacting proteins. Overall, this study provides a hitherto unknown aspect of P4-PGRMC1 mediated signaling that changes basic cellular metabolism in HEK293 cells.
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Affiliation(s)
- Mohammad Golam Sabbir
- Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, MB, R2H 2A6, Canada.
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24
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Wang Z, Qi Q, Lin Y, Guo Y, Liu Y, Wang Q. QTL analysis reveals genomic variants linked to high-temperature fermentation performance in the industrial yeast. BIOTECHNOLOGY FOR BIOFUELS 2019; 12:59. [PMID: 30923567 PMCID: PMC6423876 DOI: 10.1186/s13068-019-1398-7] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/30/2018] [Accepted: 03/08/2019] [Indexed: 05/26/2023]
Abstract
BACKGROUND High-temperature fermentation is desirable for the industrial production of ethanol, which requires thermotolerant yeast strains. However, yeast thermotolerance is a complicated quantitative trait. The understanding of genetic basis behind high-temperature fermentation performance is still limited. Quantitative trait locus (QTL) mapping by pooled-segregant whole genome sequencing has been proved to be a powerful and reliable approach to identify the loci, genes and single nucleotide polymorphism (SNP) variants linked to quantitative traits of yeast. RESULTS One superior thermotolerant industrial strain and one inferior thermosensitive natural strain with distinct high-temperature fermentation performances were screened from 124 Saccharomyces cerevisiae strains as parent strains for crossing and segregant isolation. Based on QTL mapping by pooled-segregant whole genome sequencing as well as the subsequent reciprocal hemizygosity analysis (RHA) and allele replacement analysis, we identified and validated total eight causative genes in four QTLs that linked to high-temperature fermentation of yeast. Interestingly, loss of heterozygosity in five of the eight causative genes including RXT2, ECM24, CSC1, IRA2 and AVO1 exhibited positive effects on high-temperature fermentation. Principal component analysis (PCA) of high-temperature fermentation data from all the RHA and allele replacement strains of those eight genes distinguished three superior parent alleles including VPS34, VID24 and DAP1 to be greatly beneficial to high-temperature fermentation in contrast to their inferior parent alleles. Strikingly, physiological impacts of the superior parent alleles of VPS34, VID24 and DAP1 converged on cell membrane by increasing trehalose accumulation or reducing membrane fluidity. CONCLUSIONS This work revealed eight novel causative genes and SNP variants closely associated with high-temperature fermentation performance. Among these genes, VPS34 and DAP1 would be good targets for improving high-temperature fermentation of the industrial yeast. It also showed that loss of heterozygosity of causative genes could contribute to the improvement of high-temperature fermentation capacities. Our findings would provide guides to develop more robust and thermotolerant strains for the industrial production of ethanol.
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Affiliation(s)
- Zhen Wang
- CAS Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
- University of Chinese Academy of Sciences, Beijing, 100049 China
| | - Qi Qi
- CAS Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
- University of Chinese Academy of Sciences, Beijing, 100049 China
| | - Yuping Lin
- CAS Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
| | - Yufeng Guo
- CAS Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
| | - Yanfang Liu
- CAS Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
- University of Chinese Academy of Sciences, Beijing, 100049 China
| | - Qinhong Wang
- CAS Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308 China
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25
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Bhattacharya S, Esquivel BD, White TC. Overexpression or Deletion of Ergosterol Biosynthesis Genes Alters Doubling Time, Response to Stress Agents, and Drug Susceptibility in Saccharomyces cerevisiae. mBio 2018; 9:e01291-18. [PMID: 30042199 PMCID: PMC6058291 DOI: 10.1128/mbio.01291-18] [Citation(s) in RCA: 134] [Impact Index Per Article: 19.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2018] [Accepted: 06/18/2018] [Indexed: 01/07/2023] Open
Abstract
Ergosterol (ERG) is a critical sterol in the cell membranes of fungi, and its biosynthesis is tightly regulated by 25 known enzymes along the ERG production pathway. The effects of changes in expression of each ERG biosynthesis enzyme in Saccharomyces cerevisiae were analyzed by the use of gene deletion or plasmid-borne overexpression constructs. The strains overexpressing the ERG pathway genes were examined for changes in doubling time and responses to a variety of stress agents. In addition, ERG gene overexpression strains and ERG gene deletion strains were tested for alterations in antifungal drug susceptibility. The data show that disruptions in ergosterol biosynthesis regulation can affect a diverse set of cellular processes and can cause numerous phenotypic effects. Some of the phenotypes observed include dramatic increases in doubling times, respiratory deficiencies on glycerol media, cell wall insufficiencies on Congo red media, and disrupted ion homeostasis under iron or calcium starvation conditions. Overexpression or deletion of specific enzymes in the ERG pathway causes altered susceptibilities to a variety of classes of antifungal ergosterol inhibitors, including fluconazole, fenpropimorph, lovastatin, nystatin, amphotericin B, and terbinafine. This analysis of the effect of perturbations to the ERG pathway caused by systematic overexpression of each of the ERG pathway genes contributes significantly to the understanding of the ergosterol biosynthetic pathway and its relationship to stress response and basic biological processes. The data indicate that precise regulation of ERG genes is essential for cellular homeostasis and identify several ERG genes that could be exploited in future antifungal development efforts.IMPORTANCE A common target of antifungal drug treatment is the fungal ergosterol biosynthesis pathway. This report helps to identify ergosterol biosynthesis enzymes that have not previously been appreciated as drug targets. The effects of overexpression of each of the 25 ERG genes in S. cerevisiae were analyzed in the presence of six stress agents that target essential cellular processes (cell wall biosynthesis, protein translation, respiration, osmotic/ionic stress, and iron and calcium homeostasis), as well as six antifungal inhibitors that target ergosterol biosynthesis. The importance of identifying cell perturbations caused by gene overexpression or deletion is emphasized by the prevalence of gene expression alterations in many pathogenic and drug-resistant clinical isolates. Genes whose altered expression causes the most extensive phenotypic alterations in the presence of stressors or inhibitors have the potential to be drug targets.
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Affiliation(s)
- Somanon Bhattacharya
- School of the Biological Sciences, University of Missouri, Kansas City, Kansas City, Missouri, USA
| | - Brooke D Esquivel
- School of the Biological Sciences, University of Missouri, Kansas City, Kansas City, Missouri, USA
| | - Theodore C White
- School of the Biological Sciences, University of Missouri, Kansas City, Kansas City, Missouri, USA
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26
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Song J, Zhang S, Lu L. Fungal cytochrome P450 protein Cyp51: What we can learn from its evolution, regulons and Cyp51-based azole resistance. FUNGAL BIOL REV 2018. [DOI: 10.1016/j.fbr.2018.05.001] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
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27
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Gou M, Ran X, Martin DW, Liu CJ. The scaffold proteins of lignin biosynthetic cytochrome P450 enzymes. NATURE PLANTS 2018; 4:299-310. [PMID: 29725099 DOI: 10.1038/s41477-018-0142-9] [Citation(s) in RCA: 98] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/13/2017] [Accepted: 03/28/2018] [Indexed: 05/18/2023]
Abstract
Lignin is a complex and irregular biopolymer of crosslinked phenylpropanoid units in plant secondary cell walls. Its biosynthesis requires three endoplasmic reticulum (ER)-resident cytochrome P450 monooxygenases, C4H, C3'H and F5H, to establish the structural characteristics of its monomeric precursors. These P450 enzymes were reported to associate with each other or potentially with other soluble monolignol biosynthetic enzymes to form an enzyme complex or a metabolon. However, the molecular basis governing such enzyme or pathway organization remains elusive. Here, we show that Arabidopsis membrane steroid-binding proteins (MSBPs) serve as a scaffold to physically organize monolignol P450 monooxygenases, thereby regulating the lignin biosynthetic process. We find that although C4H, C3'H and F5H are in spatial proximity to each other on the ER membrane in vivo, they do not appear to directly interact with each other. Instead, two MSBP proteins physically interact with all three P450 enzymes and, moreover, MSBPs themselves associate as homomers and heteromers on the ER membrane, thereby organizing P450 clusters. Downregulation of MSBP genes does not affect the transcription levels of monolignol biosynthetic P450 genes but substantially impairs the stability and activity of the MSBP-interacting P450 enzymes and, consequently, lignin deposition, and the accumulation of soluble phenolics in the monolignol branch but not in the flavonoid pathway. Our study suggests that MSBP proteins are essential structural components in the ER membrane that physically organize and stabilize the monolignol biosynthetic P450 enzyme complex, thereby specifically controlling phenylpropanoid-monolignol branch biosynthesis.
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Affiliation(s)
- Mingyue Gou
- Biology Department, Brookhaven National Laboratory, Upton, NY, USA
| | - Xiuzhi Ran
- Biology Department, Brookhaven National Laboratory, Upton, NY, USA
| | - Dwight W Martin
- Department of Medicine and the Proteomics Center, Stony Brook University, Stony Brook, NY, USA
| | - Chang-Jun Liu
- Biology Department, Brookhaven National Laboratory, Upton, NY, USA.
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28
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Tsai HW, Ho CL, Cheng SW, Lin YJ, Chen CC, Cheng PN, Yen CJ, Chang TT, Chiang PM, Chan SH, Ho CH, Chen SH, Wang YW, Chow NH, Lin JC. Progesterone receptor membrane component 1 as a potential prognostic biomarker for hepatocellular carcinoma. World J Gastroenterol 2018; 24:1152-1166. [PMID: 29563759 PMCID: PMC5850134 DOI: 10.3748/wjg.v24.i10.1152] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/11/2017] [Revised: 01/16/2018] [Accepted: 01/23/2018] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the clinicopathological significance of progesterone receptor membrane component 1 (PGRMC1) and PGRMC2 in hepatocellular carcinoma (HCC).
METHODS We performed immunohistochemical staining to evaluate the estrogen receptor (ER), progesterone receptor (PR), PGRMC1, and PGRMC2 in a clinical cohort consisting of 89 paired HCC and non-tumor liver samples. We also analyzed HCC data (n = 373) from The Cancer Genome Atlas (TCGA). We correlated the expression status of PGRMC1 and PGRMC2 with clinicopathological indicators and the clinical outcomes of the HCC patients. We knocked down or overexpressed PGRMC1 in HCC cell lines to evaluate its biological significance in HCC cell proliferation, differentiation, migration, and invasion.
RESULTS We found that few HCC cases expressed ER (5.6%) and PR (4.5%). In contrast, most HCC cases expressed PGRMC1 (89.9%) and PGRMC2 (100%). PGRMC1 and PGRMC2 exhibited significantly lower expression in tumor tissue than in non-tumor tissue (P < 0.001). Lower PGRMC1 expression in HCC was significantly associated with higher serum alpha-fetoprotein expression (P = 0.004), poorer tumor differentiation (P = 0.045) and liver capsule penetration (P = 0.038). Low PGRMC1 expression was an independent predictor for worse disease-free survival (P = 0.002, HR = 2.384, CI: 1.377-4.128) in our cases, as well as in the TCGA cohort (P < 0.001, HR = 2.857, CI: 1.781-4.584). The expression of PGRMC2 did not relate to patient outcome. PGRMC1 knockdown promoted a poorly differentiated phenotype and proliferation of HCC cells in vitro, while PGRMC1 overexpression caused the opposite effects.
CONCLUSION PGRMC1 is a non-classical hormonal receptor that negatively regulates hepatocarcinogenesis. PGRMC1 down-regulation is associated with progression of HCC and is a poor prognostic indicator.
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Affiliation(s)
- Hung-Wen Tsai
- Institute of Clinical Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
- Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
- Center of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
| | - Chung-Liang Ho
- Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
| | - Shu-Wen Cheng
- Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
| | - Yih-Jyh Lin
- Department of Surgery, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
| | - Chou-Cheng Chen
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
| | - Pin-Nan Cheng
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
| | - Chia-Jui Yen
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
| | - Ting-Tsung Chang
- Center of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
| | - Po-Min Chiang
- Institute of Clinical Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
| | - Shih-Huang Chan
- Department of Statistics, College of Management, National Cheng Kung University, Tainan 70403, Taiwan
| | - Cheng-Hsun Ho
- Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
- Research Center of Clinical Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
| | - Shu-Hui Chen
- Department of Chemistry, College of Sciences, National Cheng Kung University, Tainan 70403, Taiwan
| | - Yi-Wen Wang
- Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
| | - Nan-Haw Chow
- Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
| | - Jou-Chun Lin
- Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
- Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 70403, Taiwan
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29
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Identification of progesterone receptor membrane component-1 as an interaction partner and possible regulator of fatty acid 2-hydroxylase. Biochem J 2018; 475:853-871. [PMID: 29438993 DOI: 10.1042/bcj20170963] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2017] [Revised: 02/02/2018] [Accepted: 02/08/2018] [Indexed: 12/20/2022]
Abstract
The fatty acid 2-hydroxylase (FA2H) is essential for synthesis of 2-hydroxylated fatty acids in myelinating and other cells, and deficiency of this enzyme causes a complicated form of hereditary spastic paraplegia also known as fatty acid hydroxylase-associated neurodegeneration. Despite its important role in sphingolipid metabolism, regulation of FA2H and its interaction with other proteins involved in the same or other metabolic pathways is poorly understood. To identify potential interaction partners of the enzyme, quantitative mass spectrometry using stable isotope labeling of cells was combined with formaldehyde cross-linking and proximity biotinylation, respectively. Besides other enzymes involved in sphingolipid synthesis and intermembrane transfer of ceramide, and putative redox partners of FA2H, progesterone receptor membrane component-1 (PGRMC1) and PGRMC2 were identified as putative interaction partners. These two related heme-binding proteins are known to regulate several cytochrome P450 enzymes. Bimolecular fluorescence complementation experiments confirmed the interaction of FA2H with PGRMC1. Moreover, the PGRMC1 inhibitor AG-205 significantly reduced synthesis of hydroxylated ceramide and glucosylceramide in FA2H-expressing cells. This suggests that PGRMC1 may regulate FA2H activity, possibly through its heme chaperone activity.
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30
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Verma S, Shakya VPS, Idnurm A. Exploring and exploiting the connection between mitochondria and the virulence of human pathogenic fungi. Virulence 2018; 9:426-446. [PMID: 29261004 PMCID: PMC5955198 DOI: 10.1080/21505594.2017.1414133] [Citation(s) in RCA: 46] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2017] [Revised: 12/04/2017] [Accepted: 12/04/2017] [Indexed: 12/17/2022] Open
Abstract
Mitochondria are best known for their role in the production of ATP; however, recent research implicates other mitochondrial functions in the virulence of human pathogenic fungi. Inhibitors of mitochondrial succinate dehydrogenase or the electron transport chain are successfully used to combat plant pathogenic fungi, but similar inhibition of mitochondrial functions has not been pursued for applications in medical mycology. Advances in understanding mitochondrial function relevant to human pathogenic fungi are in four major directions: 1) the role of mitochondrial morphology in virulence, 2) mitochondrial genetics, with a focus on mitochondrial DNA recombination and mitochondrial inheritance 3) the role of mitochondria in drug resistance, and 4) the interaction of mitochondria with other organelles. Collectively, despite the similarities in mitochondrial functions between fungi and animals, this organelle is currently an under-explored potential target to treat medical mycoses. Future research could define and then exploit those mitochondrial components best suited as drug targets.
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Affiliation(s)
- Surbhi Verma
- Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah, USA
| | - Viplendra P. S. Shakya
- Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah, USA
| | - Alexander Idnurm
- School of BioSciences, University of Melbourne, Parkville, VIC, Australia
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31
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Ryu CS, Klein K, Zanger UM. Membrane Associated Progesterone Receptors: Promiscuous Proteins with Pleiotropic Functions - Focus on Interactions with Cytochromes P450. Front Pharmacol 2017; 8:159. [PMID: 28396637 PMCID: PMC5366339 DOI: 10.3389/fphar.2017.00159] [Citation(s) in RCA: 67] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2017] [Accepted: 03/13/2017] [Indexed: 12/22/2022] Open
Abstract
Membrane-associated progesterone receptors (MAPR) are a group of four rather small, partially homologous proteins, which share a similar non-covalent heme-binding domain that is related to cytochrome b5, a well-known functional interaction partner of microsomal cytochrome P450 (CYP) monooxygenase systems. Apart from their structural similarities the four proteins progesterone membrane component 1 (PGRMC1, also referred to as IZA, sigma-2 receptor, Dap1), PGRMC2, neudesin (NENF) and neuferricin (CYB5D2) display surprisingly divergent and multifunctional physiological properties related to cholesterol/steroid biosynthesis, drug metabolism and response, iron homeostasis, heme trafficking, energy metabolism, autophagy, apoptosis, cell cycle regulation, cell migration, neural functions, and tumorigenesis and cancer progression. The purpose of this mini-review is to briefly summarize the structural and functional properties of MAPRs with particular focus on their interactions with the CYP system. For PGRMC1, originally identified as a non-canonical progesterone-binding protein that mediates some immediate non-genomic actions of progesterone, available evidence indicates mainly activating interactions with steroidogenic CYPs including CYP11A1, CYP21A2, CYP17, CYP19, CYP51A1, and CYP61A1, while interactions with drug metabolizing CYPs including CYP2C2, CYP2C8, CYP2C9, CYP2E1, and CYP3A4 were either ineffective or slightly inhibitory. For the other MAPRs the evidence is so far less conclusive. We also point out that experimental limitations question some of the previous conclusions. Use of appropriate model systems should help to further clarify the true impact of these proteins on CYP-mediated metabolic pathways.
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Affiliation(s)
- Chang S Ryu
- Department of Molecular and Cell Biology, Dr. Margarete Fischer-Bosch-Institute of Clinical PharmacologyStuttgart, Germany; Eberhard-Karls-UniversityTübingen, Germany
| | - Kathrin Klein
- Department of Molecular and Cell Biology, Dr. Margarete Fischer-Bosch-Institute of Clinical PharmacologyStuttgart, Germany; Eberhard-Karls-UniversityTübingen, Germany
| | - Ulrich M Zanger
- Department of Molecular and Cell Biology, Dr. Margarete Fischer-Bosch-Institute of Clinical PharmacologyStuttgart, Germany; Eberhard-Karls-UniversityTübingen, Germany
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32
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Kim YJ, Lee N, Woo S, Ryu JC, Yum S. Transcriptomic change as evidence for cadmium-induced endocrine disruption in marine fish model of medaka, Oryzias javanicus. Mol Cell Toxicol 2017. [DOI: 10.1007/s13273-016-0045-7] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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33
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Song J, Zhai P, Lu L. Damage resistance protein (Dap) contributes to azole resistance in a sterol-regulatory-element-binding protein SrbA-dependent way. Appl Microbiol Biotechnol 2017; 101:3729-3741. [DOI: 10.1007/s00253-016-8072-9] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2016] [Revised: 11/23/2016] [Accepted: 12/07/2016] [Indexed: 02/06/2023]
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34
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Screening and Characterization of a Non-cyp51A Mutation in an Aspergillus fumigatus cox10 Strain Conferring Azole Resistance. Antimicrob Agents Chemother 2016; 61:AAC.02101-16. [PMID: 27799210 DOI: 10.1128/aac.02101-16] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2016] [Accepted: 10/18/2016] [Indexed: 12/14/2022] Open
Abstract
The rapid and global emergence of azole resistance in the human pathogen Aspergillus fumigatus has drawn attention. Thus, a thorough understanding of its mechanisms of drug resistance requires extensive exploration. In this study, we found that the loss of the putative calcium-dependent protein-encoding gene algA causes an increased frequency of azole-resistant A. fumigatus isolates. In contrast to previously identified azole-resistant isolates related to cyp51A mutations, only one isolate carries a point mutation in cyp51A (F219L mutation) among 105 independent stable azole-resistant isolates. Through next-generation sequencing (NGS), we successfully identified a new mutation (R243Q substitution) conferring azole resistance in the putative A. fumigatus farnesyltransferase Cox10 (AfCox10) (AFUB_065450). High-performance liquid chromatography (HPLC) analysis verified that the decreased absorption of itraconazole in related Afcox10 mutants is the primary reason for itraconazole resistance. Moreover, a complementation experiment by reengineering the mutation in a parental wild-type background strain demonstrated that both the F219L and R243Q mutations contribute to itraconazole resistance in an algA-independent manner. These data collectively suggest that the loss of algA results in an increased frequency of azole-resistant isolates with a non-cyp51A mutation. Our findings indicate that there are many unexplored non-cyp51A mutations conferring azole resistance in A. fumigatus and that algA defects make it possible to isolate drug-resistant alleles. In addition, our study suggests that genome-wide sequencing combined with alignment comparison analysis is an efficient approach to identify the contribution of single nucleotide polymorphism (SNP) diversity to drug resistance.
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35
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Piel RB, Shiferaw MT, Vashisht AA, Marcero JR, Praissman JL, Phillips JD, Wohlschlegel JA, Medlock AE. A Novel Role for Progesterone Receptor Membrane Component 1 (PGRMC1): A Partner and Regulator of Ferrochelatase. Biochemistry 2016; 55:5204-17. [PMID: 27599036 DOI: 10.1021/acs.biochem.6b00756] [Citation(s) in RCA: 84] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Heme is an iron-containing cofactor essential for multiple cellular processes and fundamental activities such as oxygen transport. To better understand the means by which heme synthesis is regulated during erythropoiesis, affinity purification coupled with mass spectrometry (MS) was performed to identify putative protein partners interacting with ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Both progesterone receptor membrane component 1 (PGRMC1) and progesterone receptor membrane component 2 (PGRMC2) were identified in these experiments. These interactions were validated by reciprocal affinity purification followed by MS analysis and immunoblotting. The interaction between PGRMC1 and FECH was confirmed in vitro and in HEK 293T cells, a non-erythroid cell line. When cells that are recognized models for erythroid differentiation were treated with a small molecule inhibitor of PGRMC1, AG-205, there was an observed decrease in the level of hemoglobinization relative to that of untreated cells. In vitro heme transfer experiments showed that purified PGRMC1 was able to donate heme to apo-cytochrome b5. In the presence of PGRMC1, in vitro measured FECH activity decreased in a dose-dependent manner. Interactions between FECH and PGRMC1 were strongest for the conformation of FECH associated with product release, suggesting that PGRMC1 may regulate FECH activity by controlling heme release. Overall, the data illustrate a role for PGRMC1 in regulating heme synthesis via interactions with FECH and suggest that PGRMC1 may be a heme chaperone or sensor.
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Affiliation(s)
- Robert B Piel
- Department of Biochemistry and Molecular Biology, Biomedical and Health Sciences Institute, AU/UGA Medical Partnership, University of Georgia , Athens, Georgia 30602, United States
| | - Mesafint T Shiferaw
- Department of Biochemistry and Molecular Biology, Biomedical and Health Sciences Institute, AU/UGA Medical Partnership, University of Georgia , Athens, Georgia 30602, United States
| | - Ajay A Vashisht
- Department of Biological Chemistry, University of California , Los Angeles, California 90095-1737, United States
| | - Jason R Marcero
- Department of Biochemistry and Molecular Biology, Biomedical and Health Sciences Institute, AU/UGA Medical Partnership, University of Georgia , Athens, Georgia 30602, United States
| | - Jeremy L Praissman
- Department of Biochemistry and Molecular Biology, Biomedical and Health Sciences Institute, AU/UGA Medical Partnership, University of Georgia , Athens, Georgia 30602, United States
| | - John D Phillips
- Hematology Division, University of Utah School of Medicine , Salt Lake City, Utah 84132, United States
| | - James A Wohlschlegel
- Department of Biological Chemistry, University of California , Los Angeles, California 90095-1737, United States
| | - Amy E Medlock
- Department of Biochemistry and Molecular Biology, Biomedical and Health Sciences Institute, AU/UGA Medical Partnership, University of Georgia , Athens, Georgia 30602, United States
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The Aspergillus fumigatus Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility. mBio 2016; 7:e01919-15. [PMID: 26908577 PMCID: PMC4791848 DOI: 10.1128/mbio.01919-15] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Ergosterol is a major and specific component of the fungal plasma membrane, and thus, the cytochrome P450 enzymes (Erg proteins) that catalyze ergosterol synthesis have been selected as valuable targets of azole antifungals. However, the opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in the cytochrome P450 enzyme Cyp51 (Erg11). In this study, we demonstrate that a cytochrome b5-like heme-binding damage resistance protein (Dap) family, comprised of DapA, DapB, and DapC, coordinately regulates the functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles. The expression of all three genes is induced in an azole concentration-dependent way, and the decreased susceptibility to azoles requires DapA stabilization of cytochrome P450 protein activity. In contrast, overexpression of DapB and DapC causes dysfunction of Erg5 and Erg11, resulting in abnormal accumulation of sterol intermediates and further accentuating the sensitivity of ΔdapA strains to azoles. The results of exogenous-hemin rescue and heme-binding-site mutagenesis experiments demonstrate that the heme binding of DapA contributes the decreased azole susceptibility, while DapB and -C are capable of reducing the activities of Erg5 and Erg11 through depletion of heme. In vivo data demonstrate that inactivated DapA combined with activated DapB yields an A. fumigatus mutant that is easily treatable with azoles in an immunocompromised mouse model of invasive pulmonary aspergillosis. Compared to the single Dap proteins found in Saccharomyces cerevisiae and Schizosaccharomyces pombe, we suggest that this complex Dap family regulatory system emerged during the evolution of fungi as an adaptive means to regulate ergosterol synthesis in response to environmental stimuli. Knowledge of the ergosterol biosynthesis route in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of antifungal-drug resistance mechanisms. In this study, we demonstrate that three cytochrome b5-like Dap proteins coordinately regulate the azole resistance and ergosterol biosynthesis catalyzed by cytochrome P450 proteins. Our new insights into the Dap regulatory system in fungal pathogens may have broad therapeutic ramifications beyond their usefulness for classic azole antifungals. Moreover, our elucidation of the molecular mechanism of Dap regulation of cytochrome P450 protein functionality through heme-binding activity may extend beyond the Kingdom Fungi with applicability toward Dap protein regulation of mammalian sterol synthesis.
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Hampton KK, Stewart R, Napier D, Claudio PP, Craven RJ. PGRMC1 Elevation in Multiple Cancers and Essential Role in Stem Cell Survival. ACTA ACUST UNITED AC 2016; 4:37-51. [PMID: 27867772 PMCID: PMC5113835 DOI: 10.4236/alc.2015.43006] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Cancer is one of the leading causes of death in America, and there is an urgent need for new therapeutic approaches. The progesterone receptor membrane component 1 (PGRMC1) is a cytoch-rome b5 related protein that binds heme and is associated with signaling, apoptotic suppression and autophagy. PGRMC1 is essential for tumor formation, invasion and metastasis, and is upregulated in breast, colon, lung and thyroid tumors. In the present study, we have analyzed PGRMC1 levels in over 600 tumor sections, including a larger cohort of lung tumors than in previous studies, and report the first clinical analysis of PGRMC1 levels in human oral cavity and ovarian tumors compared to corresponding nonmalignant tissues. PGRMC1 was highly expressed in lung and ovarian cancers and correlated with patient survival. PGRMC1 has been previously associated with drug resistance, a characteristic of cancer stem cells. The stem cell theory proposes that a subset of cancerous stem cells contribute to drug resistance and tumor maintenance, and PGRMC1 was detected in lung-tumor derived stem cells. Drug treatment with a PGRMC1 inhibitor, AG-205, triggered stem cell death whereas treatment with erlotinib and the ERK inhibitor, PD98059, did not, suggesting a specific role for PGRMC1 in cancer stem cell viability. Together, our data demonstrate PGRMC1 as a potential tumor biomarker across a variety of tumors, as well as a therapeutic target for cancer stem cells.
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Affiliation(s)
- Kaia K Hampton
- Department of Pharmacology and Nutritional Sciences, University of Kentucky, Lexington, KY, USA
| | - Rachel Stewart
- Department of Pathology and Laboratory Medicine, University of Kentucky, Lexington, KY, USA; Markey Cancer Center, University of Kentucky, Lexington, KY, USA
| | - Dana Napier
- Department of Pathology and Laboratory Medicine, University of Kentucky, Lexington, KY, USA; Markey Cancer Center, University of Kentucky, Lexington, KY, USA
| | - Pier Paolo Claudio
- Department of Biomolecular Sciences and National Center for Natural Products Research, School of Pharmacy, University of Mississippi, Oxford, MS, USA; Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS, USA
| | - Rolf J Craven
- Department of Pharmacology and Nutritional Sciences, University of Kentucky, Lexington, KY, USA; Markey Cancer Center, University of Kentucky, Lexington, KY, USA
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Clark NC, Friel AM, Pru CA, Zhang L, Shioda T, Rueda BR, Peluso JJ, Pru JK. Progesterone receptor membrane component 1 promotes survival of human breast cancer cells and the growth of xenograft tumors. Cancer Biol Ther 2016; 17:262-71. [PMID: 26785864 DOI: 10.1080/15384047.2016.1139240] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Triple negative breast cancers (TNBCs) are highly aggressive and grow in response to sex steroid hormones despite lacking expression of the classical estrogen (E2) and progesterone (P4) receptors. Since P4 receptor membrane component 1 (PGRMC1) is expressed in breast cancer tumors and is known to mediate P4-induced cell survival, this study was designed to determine the expression of PGRMC1 in TNBC tumors and the involvement of PGRMC1 in regulating proliferation and survival of TNBC cells in vitro and the growth of TNBC tumors in vivo. For the latter studies, the MDA-MB-231 (MDA) cell line derived from TNBC was used. These cells express PGRMC1 but lack expression of the classical P4 receptor. A lentiviral-based shRNA approach was used to generate a stably transfected PGRMC1-deplete MDA line for comparison to the PGRMC1-intact MDA line. The present studies demonstrate that PGRMC1: 1) is expressed in TNBC cells; 2) mediates the ability of P4 to suppress TNBC cell mitosis in vitro; 3) is required for P4 to reduce the apoptotic effects of doxorubicin in vitro; and 4) facilitates TNBC tumor formation and growth in vivo. Taken together, these findings indicate that PGRMC1 plays an important role in regulating the growth and survival of TNBC cells in vitro and ultimately in the formation and development of these tumors in vivo. Thus, PGRMC1 may be a therapeutic target for TNBCs.
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Affiliation(s)
- Nicole C Clark
- a Department of Animal Sciences , School of Molecular Biosciences, Center for Reproductive Biology, Washington State University , Pullman , WA , USA
| | - Anne M Friel
- b Vincent Center for Reproductive Biology and Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Massachusetts General Hospital, Harvard Medical School , Boston , MA , USA
| | - Cindy A Pru
- a Department of Animal Sciences , School of Molecular Biosciences, Center for Reproductive Biology, Washington State University , Pullman , WA , USA
| | - Ling Zhang
- b Vincent Center for Reproductive Biology and Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Massachusetts General Hospital, Harvard Medical School , Boston , MA , USA
| | - Toshi Shioda
- c Massachusetts General Hospital Cancer Center and Harvard Medical School , Charlestown , MA , USA
| | - Bo R Rueda
- b Vincent Center for Reproductive Biology and Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Massachusetts General Hospital, Harvard Medical School , Boston , MA , USA
| | - John J Peluso
- d Departments of Obstetrics and Gynecology and Cell Biology , University of Connecticut Health Center , Farmington , CT , USA
| | - James K Pru
- a Department of Animal Sciences , School of Molecular Biosciences, Center for Reproductive Biology, Washington State University , Pullman , WA , USA
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Li X, Rhee DK, Malhotra R, Mayeur C, Hurst LA, Ager E, Shelton G, Kramer Y, McCulloh D, Keefe D, Bloch KD, Bloch DB, Peterson RT. Progesterone receptor membrane component-1 regulates hepcidin biosynthesis. J Clin Invest 2015; 126:389-401. [PMID: 26657863 DOI: 10.1172/jci83831] [Citation(s) in RCA: 71] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2015] [Accepted: 11/05/2015] [Indexed: 01/19/2023] Open
Abstract
Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism.
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Xie Y, Shen YT, Kapoor A, Ojo D, Wei F, De Melo J, Lin X, Wong N, Yan J, Tao L, Major P, Tang D. CYB5D2 displays tumor suppression activities towards cervical cancer. Biochim Biophys Acta Mol Basis Dis 2015; 1862:556-565. [PMID: 26692170 DOI: 10.1016/j.bbadis.2015.12.013] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2015] [Revised: 12/08/2015] [Accepted: 12/11/2015] [Indexed: 01/15/2023]
Abstract
Cervical cancer is caused by infections with human papillomaviruses (HPV) and genetic alternations in the cervical epithelium. While the former is well studied, the latter remains unclear. We report here that CYB5D2/Neuferricin possesses tumor suppressing activity towards cervical tumorigenesis. Ectopic expression of CYB5D2 did not affect HeLa cell proliferation and the cell's ability to form xenograft tumors, but significantly inhibited HeLa cell invasion in vitro and the cell-produced lung metastasis in NOD/SCID mice. Knockdown of CYB5D2 enhanced HeLa cell invasion. Two mutations in CYB5D2, the substitutions of arginine (R) 7 with either proline (P) or glycine (G), were reported in colon cancer. Both CYB5D2(R7P) and CYB5D2(R7G) were incapable of inhibiting HeLa cell invasion. CYB5D2 binds heme, in which aspartate (D) 86 is required. While CYB5D2(D86G) is heme-binding defective, it inhibited HeLa cell invasion. On the other hand, CYB5D2(R7P) and CYB5D2(R7G) bound heme but did not inhibit HeLa cell invasion. Collectively, CYB5D2 inhibits HeLa cell invasion independently of its heme binding. Furthermore, immunohistochemistry examination of CYB5D2 expression in 20 normal cervical tissues and 40 cervical squamous cell carcinomas (SCC) revealed a CYB5D2 reduction in 87.5% (35/40) of SCC. Analysis of CYB5D2 gene expression and genomic alteration data available from Oncomeine™ detected significant reductions of CYB5D2 mRNA in 40 SCCs and CYB5D2 gene copy number in 107 SCCs. Collectively, we provide evidence that CYB5D2 is a candidate tumor suppressor of cervical tumorigenesis.
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Affiliation(s)
- Yanyun Xie
- Division of Nephrology, Department of Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, PR China; Division of Nephrology, Department of Medicine, McMaster University, Canada; Father Sean O'Sullivan Research Institute, Canada; The Hamilton Center for Kidney Research, St. Joseph's Hospital, Canada
| | - Yen Ting Shen
- Division of Nephrology, Department of Medicine, McMaster University, Canada; Father Sean O'Sullivan Research Institute, Canada; The Hamilton Center for Kidney Research, St. Joseph's Hospital, Canada
| | - Anil Kapoor
- Father Sean O'Sullivan Research Institute, Canada; Department of Surgery, McMaster University, Hamilton, Ontario, Canada
| | - Diane Ojo
- Division of Nephrology, Department of Medicine, McMaster University, Canada; Father Sean O'Sullivan Research Institute, Canada; The Hamilton Center for Kidney Research, St. Joseph's Hospital, Canada
| | - Fengxiang Wei
- The Genetics Laboratory, Institute of Women and Children's Health, Longgang District, Shenzhen, Guangdong, PR China; ZhunYi Medical University, Zhunyi, Guizhou, PR China
| | - Jason De Melo
- Division of Nephrology, Department of Medicine, McMaster University, Canada; Father Sean O'Sullivan Research Institute, Canada; The Hamilton Center for Kidney Research, St. Joseph's Hospital, Canada
| | - Xiaozeng Lin
- Division of Nephrology, Department of Medicine, McMaster University, Canada; Father Sean O'Sullivan Research Institute, Canada; The Hamilton Center for Kidney Research, St. Joseph's Hospital, Canada
| | - Nicholas Wong
- Division of Nephrology, Department of Medicine, McMaster University, Canada; Father Sean O'Sullivan Research Institute, Canada; The Hamilton Center for Kidney Research, St. Joseph's Hospital, Canada
| | - Judy Yan
- Division of Nephrology, Department of Medicine, McMaster University, Canada; Father Sean O'Sullivan Research Institute, Canada; The Hamilton Center for Kidney Research, St. Joseph's Hospital, Canada
| | - Lijian Tao
- Division of Nephrology, Department of Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, PR China
| | - Pierre Major
- Division of Medical Oncology, Department of Oncology, McMaster University, Hamilton, Ontario, Canada
| | - Damu Tang
- Division of Nephrology, Department of Medicine, McMaster University, Canada; Father Sean O'Sullivan Research Institute, Canada; The Hamilton Center for Kidney Research, St. Joseph's Hospital, Canada.
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Kong LA, Wu DQ, Huang WK, Peng H, Wang GF, Cui JK, Liu SM, Li ZG, Yang J, Peng DL. Large-scale identification of wheat genes resistant to cereal cyst nematode Heterodera avenae using comparative transcriptomic analysis. BMC Genomics 2015; 16:801. [PMID: 26475271 PMCID: PMC4609135 DOI: 10.1186/s12864-015-2037-8] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2015] [Accepted: 10/08/2015] [Indexed: 11/10/2022] Open
Abstract
Background Cereal cyst nematode Heterodera avenae, an important soil-borne pathogen in wheat, causes numerous annual yield losses worldwide, and use of resistant cultivars is the best strategy for control. However, target genes are not readily available for breeding resistant cultivars. Therefore, comparative transcriptomic analyses were performed to identify more applicable resistance genes for cultivar breeding. Methods The developing nematodes within roots were stained with acid fuchsin solution. Transcriptome assemblies and redundancy filteration were obtained by Trinity, TGI Clustering Tool and BLASTN, respectively. Gene Ontology annotation was yielded by Blast2GO program, and metabolic pathways of transcripts were analyzed by Path_finder. The ROS levels were determined by luminol-chemiluminescence assay. The transcriptional gene expression profiles were obtained by quantitative RT-PCR. Results The RNA-sequencing was performed using an incompatible wheat cultivar VP1620 and a compatible control cultivar WEN19 infected with H. avenae at 24 h, 3 d and 8 d. Infection assays showed that VP1620 failed to block penetration of H. avenae but disturbed the transition of developmental stages, leading to a significant reduction in cyst formation. Two types of expression profiles were established to predict candidate resistance genes after developing a novel strategy to generate clean RNA-seq data by removing the transcripts of H. avenae within the raw data before assembly. Using the uncoordinated expression profiles with transcript abundance as a standard, 424 candidate resistance genes were identified, including 302 overlapping genes and 122 VP1620-specific genes. Genes with similar expression patterns were further classified according to the scales of changed transcript abundances, and 182 genes were rescued as supplementary candidate resistance genes. Functional characterizations revealed that diverse defense-related pathways were responsible for wheat resistance against H. avenae. Moreover, phospholipase was involved in many defense-related pathways and localized in the connection position. Furthermore, strong bursts of reactive oxygen species (ROS) within VP1620 roots infected with H. avenae were induced at 24 h and 3 d, and eight ROS-producing genes were significantly upregulated, including three class III peroxidase and five lipoxygenase genes. Conclusions Large-scale identification of wheat resistance genes were processed by comparative transcriptomic analysis. Functional characterization showed that phospholipases associated with ROS production played vital roles in early defense responses to H. avenae via involvement in diverse defense-related pathways as a hub switch. This study is the first to investigate the early defense responses of wheat against H. avenae, not only provides applicable candidate resistance genes for breeding novel wheat cultivars, but also enables a better understanding of the defense mechanisms of wheat against H. avenae. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2037-8) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Ling-An Kong
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
| | - Du-Qing Wu
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
| | - Wen-Kun Huang
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
| | - Huan Peng
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
| | - Gao-Feng Wang
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
| | - Jiang-Kuan Cui
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
| | - Shi-Ming Liu
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
| | - Zhi-Gang Li
- State Key Laboratory of Agrobiotechnology and MOA Key Laboratory of Plant Pathology, China Agricultural University, Beijing, 100193, China.
| | - Jun Yang
- State Key Laboratory of Agrobiotechnology and MOA Key Laboratory of Plant Pathology, China Agricultural University, Beijing, 100193, China.
| | - De-Liang Peng
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
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Zhang C. Involvement of Iron-Containing Proteins in Genome Integrity in Arabidopsis Thaliana. Genome Integr 2015; 6:2. [PMID: 27330736 PMCID: PMC4911903 DOI: 10.4103/2041-9414.155953] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2015] [Accepted: 03/12/2015] [Indexed: 01/03/2023] Open
Abstract
The Arabidopsis genome encodes numerous iron-containing proteins such as iron-sulfur (Fe-S) cluster proteins and hemoproteins. These proteins generally utilize iron as a cofactor, and they perform critical roles in photosynthesis, genome stability, electron transfer, and oxidation-reduction reactions. Plants have evolved sophisticated mechanisms to maintain iron homeostasis for the assembly of functional iron-containing proteins, thereby ensuring genome stability, cell development, and plant growth. Over the past few years, our understanding of iron-containing proteins and their functions involved in genome stability has expanded enormously. In this review, I provide the current perspectives on iron homeostasis in Arabidopsis, followed by a summary of iron-containing protein functions involved in genome stability maintenance and a discussion of their possible molecular mechanisms.
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Affiliation(s)
- Caiguo Zhang
- Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado, Aurora, Colorado, USA
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Kaluka D, Batabyal D, Chiang BY, Poulos TL, Yeh SR. Spectroscopic and mutagenesis studies of human PGRMC1. Biochemistry 2015; 54:1638-47. [PMID: 25675345 DOI: 10.1021/bi501177e] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Progesterone receptor membrane component 1 (PGRMC1) is a 25 kDa protein with an N-terminal transmembrane domain and a putative C-terminal cytochrome b5 domain. Heme-binding activity of PGRMC1 has been shown in various homologues of PGRMC1. Although the general definition of PGRMC1 is as a progesterone receptor, progesterone-binding activity has not been directly demonstrated in any of the purified PGRMC1 proteins fully loaded with heme. Here, we show that the human homologue of PGRMC1 (hPGRMC1) binds heme in a five-coordinate (5C) high-spin (HS) configuration, with an axial tyrosinate ligand, likely Y95. The negatively charged tyrosinate ligand leads to a relatively low redox potential of approximately -331 mV. The Y95C or Y95F mutation dramatically reduces the ability of the protein to bind heme, supporting the assignment of the axial heme ligand to Y95. On the other hand, the Y95H mutation retains ∼90% of the heme-binding activity. The heme in Y95H is also 5CHS, but it has a hydroxide axial ligand, conceivably stabilized by the engineered-in H95 via an H-bond; CO binding to the distal ligand-binding site leads to an exchange of the axial ligand to a histidine, possibly H95. We show that progesterone binds to hPGRMC1 and introduces spectral changes that manifest conformational changes to the heme. Our data offer the first direct evidence supporting progesterone-binding activity of PGRMC1.
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Affiliation(s)
- Daniel Kaluka
- Department of Physiology and Biophysics, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States
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Mir SUR, Schwarze SR, Jin L, Zhang J, Friend W, Miriyala S, St Clair D, Craven RJ. Progesterone receptor membrane component 1/Sigma-2 receptor associates with MAP1LC3B and promotes autophagy. Autophagy 2014; 9:1566-78. [DOI: 10.4161/auto.25889] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
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Bruce A, Rybak AP. CYB5D2 requires heme-binding to regulate HeLa cell growth and confer survival from chemotherapeutic agents. PLoS One 2014; 9:e86435. [PMID: 24466094 PMCID: PMC3899279 DOI: 10.1371/journal.pone.0086435] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2013] [Accepted: 12/10/2013] [Indexed: 12/19/2022] Open
Abstract
The cytochrome b5 domain containing 2 (CYB5D2; Neuferricin) protein has been reported to bind heme, however, the critical residues responsible for heme-binding are undefined. Furthermore, the relationship between heme-binding and CYB5D2-mediated intracellular functions remains unknown. Previous studies examining heme-binding in two cytochrome b5 heme-binding domain-containing proteins, damage-associated protein 1 (Dap1; Saccharomyces cerevisiae) and human progesterone receptor membrane component 1 (PGRMC1), have revealed that conserved tyrosine (Y) 73, Y79, aspartic acid (D) 86, and Y127 residues present in human CYB5D2 may be involved in heme-binding. CYB5D2 binds to type b heme, however, only the substitution of glycine (G) at D86 (D86G) within its cytochrome b5 heme-binding (cyt-b5) domain abolished its heme-binding ability. Both CYB5D2 and CYB5D2(D86G) localize to the endoplasmic reticulum. Ectopic CYB5D2 expression inhibited cell proliferation and anchorage-independent colony growth of HeLa cells. Conversely, CYB5D2 knockdown and ectopic CYB5D2(D86G) expression increased cell proliferation and colony growth. As PGRMC1 has been reported to regulate the expression and activities of cytochrome P450 proteins (CYPs), we examined the role of CYB5D2 in regulating the activities of CYPs involved in sterol synthesis (CYP51A1) and drug metabolism (CYP3A4). CYB5D2 co-localizes with cytochrome P450 reductase (CYPOR), while CYB5D2 knockdown reduced lanosterol demethylase (CYP51A1) levels and rendered HeLa cells sensitive to mevalonate. Additionally, knockdown of CYB5D2 reduced CYP3A4 activity. Lastly, CYB5D2 expression conferred HeLa cell survival from chemotherapeutic agents (paclitaxel, cisplatin and doxorubicin), with its ability to promote survival being dependent on its heme-binding ability. Taken together, this study provides evidence that heme-binding is critical for CYB5D2 in regulating HeLa cell growth and survival, with endogenous CYB5D2 being required to modulate CYP activities.
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Affiliation(s)
- Anthony Bruce
- Medical Sciences Program, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada
- Father Sean O’Sullivan Research Institute, Hamilton, Ontario, Canada
- Hamilton Centre for Kidney Research (HCKR), St. Joseph’s Hospital, Hamilton, Ontario, Canada
- * E-mail: (AB); (APR)
| | - Adrian P. Rybak
- Medical Sciences Program, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada
- Father Sean O’Sullivan Research Institute, Hamilton, Ontario, Canada
- Hamilton Centre for Kidney Research (HCKR), St. Joseph’s Hospital, Hamilton, Ontario, Canada
- * E-mail: (AB); (APR)
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Ciesielska K, Li B, Groeneboer S, Van Bogaert I, Lin YC, Soetaert W, Van de Peer Y, Devreese B. SILAC-Based Proteome Analysis of Starmerella bombicola Sophorolipid Production. J Proteome Res 2013; 12:4376-92. [DOI: 10.1021/pr400392a] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Affiliation(s)
- Katarzyna Ciesielska
- Laboratory
for Protein Biochemistry and Biomolecular Engineering, Department
of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat
35, 9000 Ghent, Belgium
| | - Bing Li
- VIB
Department of Plant Systems Biology and Department of Plant Biotechnology
and Bioinformatics, Ghent University, Technologiepark 927 B-9052, 9000 Ghent, Belgium
| | - Sara Groeneboer
- Laboratory
for Protein Biochemistry and Biomolecular Engineering, Department
of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat
35, 9000 Ghent, Belgium
| | - Inge Van Bogaert
- Laboratory
of Industrial Biotechnology and Biocatalysis, Ghent University, Coupure
Links 653, 9000 Ghent, Belgium
| | | | - Wim Soetaert
- Laboratory
of Industrial Biotechnology and Biocatalysis, Ghent University, Coupure
Links 653, 9000 Ghent, Belgium
| | - Yves Van de Peer
- VIB
Department of Plant Systems Biology and Department of Plant Biotechnology
and Bioinformatics, Ghent University, Technologiepark 927 B-9052, 9000 Ghent, Belgium
| | - Bart Devreese
- Laboratory
for Protein Biochemistry and Biomolecular Engineering, Department
of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat
35, 9000 Ghent, Belgium
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Hosogaya N, Miyazaki T, Nagi M, Tanabe K, Minematsu A, Nagayoshi Y, Yamauchi S, Nakamura S, Imamura Y, Izumikawa K, Kakeya H, Yanagihara K, Miyazaki Y, Kugiyama K, Kohno S. The heme-binding protein Dap1 links iron homeostasis to azole resistance via the P450 protein Erg11 in Candida glabrata. FEMS Yeast Res 2013; 13:411-21. [PMID: 23496820 DOI: 10.1111/1567-1364.12043] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2013] [Revised: 03/07/2013] [Accepted: 03/08/2013] [Indexed: 11/29/2022] Open
Abstract
The pathogenic fungus Candida glabrata is relatively resistant to azole antifungals, which target lanosterol 14α-demethylase (Erg11p) in the ergosterol biosynthesis pathway. Our study revealed that C. glabrata exhibits increased azole susceptibility under low-iron conditions. To investigate the molecular basis of this phenomenon, we generated a strain lacking the heme (iron protoporphyrin IX)-binding protein Dap1 in C. glabrata. The Δdap1 mutant displayed growth defects under iron-limited conditions, decreased azole tolerance, decreased production of ergosterol, and increased accumulation of 14α-methylated sterols lanosterol and squalene. All the Δdap1 phenotypes were complemented by wild-type DAP1, but not by DAP1(D91G) , in which a heme-binding site is mutated. Furthermore, azole tolerance of the Δdap1 mutant was rescued by exogenous ergosterol but not by iron supplementation alone. These results suggest that heme binding by Dap1 is crucial for Erg11 activity and ergosterol biosynthesis, thereby being required for azole tolerance. A Dap1-GFP fusion protein predominantly localized to vacuolar membranes and endosomes, and the Δdap1 cells exhibited aberrant vacuole morphologies, suggesting that Dap1 is also involved in the regulation of vacuole structures that could be important for iron storage. Our study demonstrates that Dap1 mediates a functional link between iron homeostasis and azole resistance in C. glabrata.
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Affiliation(s)
- Naoki Hosogaya
- Department of Molecular Microbiology and Immunology, Nagasaki University School of Medicine, Nagasaki, Japan
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48
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Hornick JR, Spitzer D, Goedegebuure P, Mach RH, Hawkins WG. Therapeutic targeting of pancreatic cancer utilizing sigma-2 ligands. Surgery 2012; 152:S152-6. [PMID: 22763259 DOI: 10.1016/j.surg.2012.05.014] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2012] [Accepted: 05/11/2012] [Indexed: 01/05/2023]
Abstract
One major barrier in the development of pancreas cancer therapeutics is the selective delivery of the drugs to their cellular targets. We have previously developed several sigma-2 ligands and reported the discovery of a component of the receptor for these ligands. Several sigma-2 ligands have been shown to trigger apoptosis in pancreas cancer cells. More importantly, sigma-2 ligands are internalized rapidly by the cancer cells and are capable of delivering other small-molecule therapeutics. Here we review sigma-2 ligands and conjugates as a potential novel therapy suitable for investigation in patients with pancreatic cancer.
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Affiliation(s)
- John R Hornick
- Department of Surgery, Harvard Medical School, Boston, MA, USA
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Kao AL, Lin YH, Chen RPY, Huang YY, Chen CC, Yang CC. E3-independent ubiquitination of AtMAPR/MSBP1. PHYTOCHEMISTRY 2012; 78:7-19. [PMID: 22513011 DOI: 10.1016/j.phytochem.2012.03.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/01/2010] [Revised: 03/01/2012] [Accepted: 03/16/2012] [Indexed: 05/31/2023]
Abstract
AtMAPR5/MSBP1 and its homologs can be ubiquitinated in the absence of E3 ligase in in vitro ubiquitination assays. Ubiquitinated AtMAPR3, AtMAPR5/MSBP1, and AtMAPR2 were identified using LC-MS/MS. Analysis of trypsin-released signature peptides showed that this E3-independent ubiquitination of AtMAPR3, AtMAPR5/MSBP1, and AtMAPR2 was dominated by mono-ubiquitination at multiple sites. Unlike AtUBC8-type E2s, AtUBC36 was not able to transfer ubiquitin to AtMAPR2. The truncated mutants AtMAPR2Δ1-10, AtMAPR2Δ1-30, and AtMAPR2_1-73 could also be ubiquitinated. The presence of a ubiquitin-binding domain (UBD) allows proteins to be ubiquitinated independently of E3 ligases. However, AtMAPRs do not contain any known UBD. In vitro ubiquitination of AtMAPR2 observed in this study will be further studied in biochemical and physiological aspects.
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Affiliation(s)
- Ai-Ling Kao
- Institute of Microbiology and Biochemistry, National Taiwan University, Taiwan
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50
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An integrated approach for identification and target validation of antifungal compounds active against Erg11p. Antimicrob Agents Chemother 2012; 56:4233-40. [PMID: 22615293 DOI: 10.1128/aac.06332-11] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Systemic life-threatening fungal infections represent a significant unmet medical need. Cell-based, phenotypic screening can be an effective means of discovering potential novel antifungal compounds, but it does not address target identification, normally required for compound optimization by medicinal chemistry. Here, we demonstrate a combination of screening, genetic, and biochemical approaches to identify and characterize novel antifungal compounds. We isolated a set of novel non-azole antifungal compounds for which no target or mechanism of action is known, using a screen for inhibition of Saccharomyces cerevisiae proliferation. Haploinsufficiency profiling of these compounds in S. cerevisiae suggests that they target Erg11p, a cytochrome P450 family member, which is the target of azoles. Consistent with this, metabolic profiling in S. cerevisiae revealed a buildup of the metabolic intermediates prior to Erg11p activity, following compound treatment. Further, human cytochrome P450 is also inhibited in in vitro assays by these compounds. We modeled the Erg11p protein based on the human CYP51 crystal structure, and in silico docking of these compounds suggests that they interact with the heme center in a manner similar to that of azoles. Consistent with these docking observations, Candida strains carrying azole-resistant alleles of ERG11 are also resistant to the compounds in this study. Thus, we have identified non-azole Erg11p inhibitors, using a systematic approach for ligand and target characterization.
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