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Schultz CR, Aleiwi B, Zhou XE, Suino-Powell K, Melcher K, Almeida NMS, Wilson AK, Ellsworth EL, Bachmann AS. Design, Synthesis, and Biological Activity of Novel Ornithine Decarboxylase (ODC) Inhibitors. J Med Chem 2025; 68:5760-5773. [PMID: 40035393 PMCID: PMC11912471 DOI: 10.1021/acs.jmedchem.4c03120] [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: 12/18/2024] [Revised: 01/31/2025] [Accepted: 02/24/2025] [Indexed: 03/05/2025]
Abstract
We here describe the design, synthesis, and biological activity of novel ornithine decarboxylase (ODC) inhibitors that show significantly higher potency in vitro than α-difluoromethylornithine (DFMO), a U.S. Food and Drug Administration (FDA) approved drug. We report two X-ray structures of ODC complexed with new ODC inhibitors, computational docking, molecular dynamics, and binding free energy calculations to validate the experimental models. The X-ray structures reveal that covalent adducts with pyridoxal phosphate (PLP) are formed in the active site of the human ODC enzyme, as verified by their preparation and enzymatic testing. Finally, we verified that the cellular activity of endogenous ODC was inhibited, and polyamine levels were reduced. Given that ODC is a clinically validated target, combined with the fact that DFMO is currently the only ODC inhibitor in clinical use for several indications, the further development of more potent ODC inhibitors with superior activity and physical properties is warranted.
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Affiliation(s)
- Chad R Schultz
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, Michigan 49503, United States
- International Center for Polyamine Disorders, Grand Rapids, Michigan 49503, United States
| | - Bilal Aleiwi
- Department of Pharmacology and Toxicology, College of Human Medicine, East Lansing, Michigan 48824, United States
| | - X Edward Zhou
- Department of Structural Biology, Van Andel Institute, Grand Rapids, Michigan 49503, United States
| | - Kelly Suino-Powell
- Department of Structural Biology, Van Andel Institute, Grand Rapids, Michigan 49503, United States
| | - Karsten Melcher
- Department of Structural Biology, Van Andel Institute, Grand Rapids, Michigan 49503, United States
| | - Nuno M S Almeida
- Department of Chemistry, College of Natural Science, Michigan State University, East Lansing, Michigan 48824, United States
| | - Angela K Wilson
- Department of Chemistry, College of Natural Science, Michigan State University, East Lansing, Michigan 48824, United States
| | - Edmund L Ellsworth
- Department of Pharmacology and Toxicology, College of Human Medicine, East Lansing, Michigan 48824, United States
| | - André S Bachmann
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, Michigan 49503, United States
- International Center for Polyamine Disorders, Grand Rapids, Michigan 49503, United States
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2
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Liu H, Tian X, Wen J, Liu J, Huo Y, Yuan K, Guo J, Wang X, Yang M, Jiang A, Cao Q, Jiang J. Ame-miR-1-3p of bee venom reduced cell viability through the AZIN1/OAZ1-ODC1-polyamines pathway and enhanced the defense ability of honeybee (Apis mellifera L.). INSECT MOLECULAR BIOLOGY 2024; 33:312-322. [PMID: 38767730 DOI: 10.1111/imb.12899] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/07/2023] [Accepted: 01/08/2024] [Indexed: 05/22/2024]
Abstract
Bee venom serves as an essential defensive weapon for bees and also finds application as a medicinal drug. MicroRNAs (miRNAs) serve as critical regulators and have been demonstrated to perform a variety of biological functions. However, the presence of miRNAs in bee venom needs to be confirmed. Therefore, we conducted small RNA sequencing and identified 158 known miRNAs, 15 conserved miRNAs and 4 novel miRNAs. It is noteworthy that ame-miR-1-3p, the most abundant among them, accounted for over a quarter of all miRNA reads. To validate the function of ame-miR-1-3p, we screened 28 candidate target genes using transcriptome sequencing and three target gene prediction software (miRanda, PITA and TargetScan) for ame-miR-1-3p. Subsequently, we employed real-time quantitative reverse transcription PCR (qRT-PCR), Western blot and other technologies to confirm that ame-miR-1-3p inhibits the relative expression of antizyme inhibitor 1 (AZIN1) by targeting the 3' untranslated region (UTR) of AZIN1. This, in turn, caused ODC antizyme 1 (OAZ1) to bind to ornithine decarboxylase 1 (ODC1) and mark ODC1 for proteolytic destruction. The reduction in functional ODC1 ultimately resulted in a decrease in polyamine biosynthesis. Furthermore, we determined that ame-miR-1-3p accelerates cell death through the AZIN1/OAZ1-ODC1-polyamines pathway. Our studies demonstrate that ame-miR-1-3p diminishes cell viability and it may collaborate with sPLA2 to enhance the defence capabilities of honeybees (Apis mellifera L.). Collectively, these data further elucidate the defence mechanism of bee venom and expand the potential applications of bee venom in medical treatment.
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Affiliation(s)
- Haifeng Liu
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
| | - Xue Tian
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
| | - Jie Wen
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
| | - Jie Liu
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
| | - Yunfei Huo
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
| | - Kangqi Yuan
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
| | - Jiazhong Guo
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
| | - Xun Wang
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
| | - Mingxian Yang
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
| | - Anan Jiang
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
| | - Quanquan Cao
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
| | - Jun Jiang
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
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3
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Sahu PN, Sen A. Preventing Cancer by Inhibiting Ornithine Decarboxylase: A Comparative Perspective on Synthetic vs. Natural Drugs. Chem Biodivers 2024; 21:e202302067. [PMID: 38404009 DOI: 10.1002/cbdv.202302067] [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: 12/20/2023] [Revised: 02/22/2024] [Accepted: 02/24/2024] [Indexed: 02/27/2024]
Abstract
This perspective delves into the investigation of synthetic and naturally occurring inhibitors, their patterns of inhibition, and the effectiveness of newly utilized natural compounds as inhibitors targeting the Ornithine decarboxylase enzyme. This enzyme is known to target the MYC oncogene, thereby establishing a connection between polyamine metabolism and oncogenesis in both normal and cancerous cells. ODC activation and heightened polyamine activity are associated with tumor development in numerous cancers and fluctuations in ODC protein levels exert a profound influence on cellular activity for inhibition or suppressing tumor cells. This perspective outlines efforts to develop novel drugs, evaluate natural compounds, and identify promising inhibitors to address gaps in cancer prevention, highlighting the potential of newly designed synthetic moieties and natural flavonoids as alternatives. It also discusses natural compounds with potential as enhanced inhibitors.
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Affiliation(s)
- Preeti Nanda Sahu
- Department of Chemistry, (CMDD Lab) GITAM (Deemed to be), University, Rushikonda, Visakhapatnam, 530045, India
| | - Anik Sen
- Department of Chemistry, (CMDD Lab) GITAM (Deemed to be), University, Rushikonda, Visakhapatnam, 530045, India
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4
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Cruz-Pulido YE, Mounce BC. Good cop, bad cop: Polyamines play both sides in host immunity and viral replication. Semin Cell Dev Biol 2023; 146:70-79. [PMID: 36604249 PMCID: PMC10101871 DOI: 10.1016/j.semcdb.2022.12.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2022] [Revised: 12/12/2022] [Accepted: 12/13/2022] [Indexed: 01/05/2023]
Abstract
Viruses rely on host cells for energy and synthesis machinery required for genome replication and particle assembly. Due to the dependence of viruses on host cells, viruses have evolved multiple mechanisms by which they can induce metabolic changes in the host cell to suit their specific requirements. The host immune response also involves metabolic changes to be able to react to viral insult. Polyamines are small ubiquitously expressed polycations, and their metabolism is critical for viral replication and an adequate host immune response. This is due to the variety of functions that polyamines have, ranging from condensing DNA to enhancing the translation of polyproline-containing proteins through the hypusination of eIF5A. Here, we review the diverse mechanisms by which viruses exploit polyamines, as well as the mechanisms by which immune cells utilize polyamines for their functions. Furthermore, we highlight potential avenues for further study of the host-virus interface.
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Affiliation(s)
- Yazmin E Cruz-Pulido
- Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, IL, USA
| | - Bryan C Mounce
- Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, IL, USA; Infectious Disease and Immunology Research Institute, Loyola University Chicago Stritch School of Medicine, Maywood, IL, USA.
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5
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Xing Y, Nakahama T, Wu Y, Inoue M, Kim JI, Todo H, Shibuya T, Kato Y, Kawahara Y. RNA editing of AZIN1 coding sites is catalyzed by ADAR1 p150 after splicing. J Biol Chem 2023; 299:104840. [PMID: 37209819 PMCID: PMC10404624 DOI: 10.1016/j.jbc.2023.104840] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Revised: 05/09/2023] [Accepted: 05/12/2023] [Indexed: 05/22/2023] Open
Abstract
Adenosine-to-inosine RNA editing is catalyzed by nuclear adenosine deaminase acting on RNA 1 (ADAR1) p110 and ADAR2, and cytoplasmic ADAR1 p150 in mammals, all of which recognize dsRNAs as targets. RNA editing occurs in some coding regions, which alters protein functions by exchanging amino acid sequences, and is therefore physiologically significant. In general, such coding sites are edited by ADAR1 p110 and ADAR2 before splicing, given that the corresponding exon forms a dsRNA structure with an adjacent intron. We previously found that RNA editing at two coding sites of antizyme inhibitor 1 (AZIN1) is sustained in Adar1 p110/Aadr2 double KO mice. However, the molecular mechanisms underlying RNA editing of AZIN1 remain unknown. Here, we showed that Azin1 editing levels were increased upon type I interferon treatment, which activated Adar1 p150 transcription, in mouse Raw 264.7 cells. Azin1 RNA editing was observed in mature mRNA but not precursor mRNA. Furthermore, we revealed that the two coding sites were editable only by ADAR1 p150 in both mouse Raw 264.7 and human embryonic kidney 293T cells. This unique editing was achieved by forming a dsRNA structure with a downstream exon after splicing, and the intervening intron suppressed RNA editing. Therefore, deletion of a nuclear export signal from ADAR1 p150, shifting its localization to the nucleus, decreased Azin1 editing levels. Finally, we demonstrated that Azin1 RNA editing was completely absent in Adar1 p150 KO mice. Thus, these findings indicate that RNA editing of AZIN1 coding sites is exceptionally catalyzed by ADAR1 p150 after splicing.
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Affiliation(s)
- Yanfang Xing
- Department of RNA Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan
| | - Taisuke Nakahama
- Department of RNA Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan; Integrated Frontier Research for Medical Science Division and RNA Frontier Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, Suita, Osaka, Japan; Center for Infectious Disease Education and Research (CiDER), Osaka University, Suita, Osaka, Japan.
| | - Yuke Wu
- Department of RNA Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
| | - Maal Inoue
- Department of RNA Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan
| | - Jung In Kim
- Department of RNA Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan
| | - Hiroyuki Todo
- Department of RNA Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan
| | - Toshiharu Shibuya
- Department of RNA Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan
| | - Yuki Kato
- Department of RNA Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan; Integrated Frontier Research for Medical Science Division and RNA Frontier Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, Suita, Osaka, Japan
| | - Yukio Kawahara
- Department of RNA Biology and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan; Integrated Frontier Research for Medical Science Division and RNA Frontier Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, Suita, Osaka, Japan; Center for Infectious Disease Education and Research (CiDER), Osaka University, Suita, Osaka, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan; Genome Editing Research and Development Center, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.
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Gan WL, Ng L, Ng BYL, Chen L. Recent Advances in Adenosine-to-Inosine RNA Editing in Cancer. Cancer Treat Res 2023; 190:143-179. [PMID: 38113001 DOI: 10.1007/978-3-031-45654-1_5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2023]
Abstract
RNA epigenetics, or epitranscriptome, is a growing group of RNA modifications historically classified into two categories: RNA editing and RNA modification. RNA editing is usually understood as post-transcriptional RNA processing (except capping, splicing and polyadenylation) that changes the RNA nucleotide sequence encoded by the genome. This processing can be achieved through the insertion or deletion of nucleotides or deamination of nucleobases, generating either standard nucleotides such as uridine (U) or the rare nucleotide inosine (I). Adenosine-to-inosine (A-to-I) RNA editing is the most prevalent type of RNA modification in mammals and is catalyzed by adenosine deaminase acting on the RNA (ADAR) family of enzymes that recognize double-stranded RNAs (dsRNAs). Inosine mimics guanosine (G) in base pairing with cytidine (C), thereby A-to-I RNA editing alters dsRNA secondary structure. Inosine is also recognized as guanosine by the splicing and translation machineries, resulting in mRNA alternative splicing and protein recoding. Therefore, A-to-I RNA editing is an important mechanism that causes and regulates "RNA mutations" in both normal physiology and diseases including cancer. In this chapter, we reviewed current paradigms and developments in the field of A-to-I RNA editing in the context of cancer.
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Affiliation(s)
- Wei Liang Gan
- Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, Singapore, 117599, Singapore
| | - Larry Ng
- Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, Singapore, 117599, Singapore
| | - Bryan Y L Ng
- Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, Singapore, 117599, Singapore
| | - Leilei Chen
- Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, Singapore, 117599, Singapore.
- Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117594, Singapore.
- NUS Centre for Cancer Research, Yong Loo Lin School of Medicine, National University Singapore, Singapore, Singapore.
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7
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Lian J, Liang Y, Zhang H, Lan M, Ye Z, Lin B, Qiu X, Zeng J. The role of polyamine metabolism in remodeling immune responses and blocking therapy within the tumor immune microenvironment. Front Immunol 2022; 13:912279. [PMID: 36119047 PMCID: PMC9479087 DOI: 10.3389/fimmu.2022.912279] [Citation(s) in RCA: 28] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2022] [Accepted: 08/15/2022] [Indexed: 11/13/2022] Open
Abstract
The study of metabolism provides important information for understanding the biological basis of cancer cells and the defects of cancer treatment. Disorders of polyamine metabolism is a common metabolic change in cancer. With the deepening of understanding of polyamine metabolism, including molecular functions and changes in cancer, polyamine metabolism as a new anti-cancer strategy has become the focus of attention. There are many kinds of polyamine biosynthesis inhibitors and transport inhibitors, but not many drugs have been put into clinical application. Recent evidence shows that polyamine metabolism plays essential roles in remodeling the tumor immune microenvironment (TIME), particularly treatment of DFMO, an inhibitor of ODC, alters the immune cell population in the tumor microenvironment. Tumor immunosuppression is a major problem in cancer treatment. More and more studies have shown that the immunosuppressive effect of polyamines can help cancer cells to evade immune surveillance and promote tumor development and progression. Therefore, targeting polyamine metabolic pathways is expected to become a new avenue for immunotherapy for cancer.
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Affiliation(s)
- Jiachun Lian
- Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, The First Dongguan Affiliated Hospital, Guangdong Medical University, Dongguan, China
- Institute of Laboratory Medicine, School of Medical Technology, Guangdong Medical University, Dongguan, China
| | - Yanfang Liang
- Department of Pathology, Dongguan Hospital Affiliated to Jinan University, Binhaiwan Central Hospital of Dongguan, Dongguan, China
| | - Hailiang Zhang
- Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, The First Dongguan Affiliated Hospital, Guangdong Medical University, Dongguan, China
- Institute of Laboratory Medicine, School of Medical Technology, Guangdong Medical University, Dongguan, China
| | - Minsheng Lan
- Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, The First Dongguan Affiliated Hospital, Guangdong Medical University, Dongguan, China
| | - Ziyu Ye
- Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, The First Dongguan Affiliated Hospital, Guangdong Medical University, Dongguan, China
- Department of Pathology, Dongguan Hospital Affiliated to Jinan University, Binhaiwan Central Hospital of Dongguan, Dongguan, China
- Dongguan Metabolite Analysis Engineering Technology Center of Cells for Medical Use, Guangdong Xinghai Institute of Cell, Dongguan, China
| | - Bihua Lin
- Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, The First Dongguan Affiliated Hospital, Guangdong Medical University, Dongguan, China
- Key Laboratory of Medical Bioactive Molecular Research for Department of Education of Guangdong Province, Collaborative Innovation Center for Antitumor Active Substance Research and Development, Zhanjiang, China
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Guangdong Medical University, Zhanjiang, China
| | - Xianxiu Qiu
- Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, The First Dongguan Affiliated Hospital, Guangdong Medical University, Dongguan, China
- Key Laboratory of Medical Bioactive Molecular Research for Department of Education of Guangdong Province, Collaborative Innovation Center for Antitumor Active Substance Research and Development, Zhanjiang, China
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Guangdong Medical University, Zhanjiang, China
| | - Jincheng Zeng
- Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, The First Dongguan Affiliated Hospital, Guangdong Medical University, Dongguan, China
- Dongguan Metabolite Analysis Engineering Technology Center of Cells for Medical Use, Guangdong Xinghai Institute of Cell, Dongguan, China
- Key Laboratory of Medical Bioactive Molecular Research for Department of Education of Guangdong Province, Collaborative Innovation Center for Antitumor Active Substance Research and Development, Zhanjiang, China
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Guangdong Medical University, Zhanjiang, China
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8
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Tabbaa M, Ruz Gomez T, Campelj DG, Gregorevic P, Hayes A, Goodman CA. The regulation of polyamine pathway proteins in models of skeletal muscle hypertrophy and atrophy: a potential role for mTORC1. Am J Physiol Cell Physiol 2021; 320:C987-C999. [PMID: 33881936 DOI: 10.1152/ajpcell.00078.2021] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Polyamines have been shown to be absolutely required for protein synthesis and cell growth. The serine/threonine kinase, the mechanistic target of rapamycin complex 1 (mTORC1), also plays a fundamental role in the regulation of protein turnover and cell size, including in skeletal muscle, where mTORC1 is sufficient to increase protein synthesis and muscle fiber size, and is necessary for mechanical overload-induced muscle hypertrophy. Recent evidence suggests that mTORC1 may regulate the polyamine metabolic pathway, however, there is currently no evidence in skeletal muscle. This study examined changes in polyamine pathway proteins during muscle hypertrophy induced by mechanical overload (7 days), with and without the mTORC1 inhibitor, rapamycin, and during muscle atrophy induced by food deprivation (48 h) and denervation (7 days) in mice. Mechanical overload induced an increase in mTORC1 signaling, protein synthesis and muscle mass, and these were associated with rapamycin-sensitive increases in adenosylmethione decarboxylase 1 (Amd1), spermidine synthase (SpdSyn), and c-Myc. Food deprivation decreased mTORC1 signaling, protein synthesis, and muscle mass, accompanied by a decrease in spermidine/spermine acetyltransferase 1 (Sat1). Denervation, resulted increased mTORC1 signaling and protein synthesis, and decreased muscle mass, which was associated with an increase in SpdSyn, spermine synthase (SpmSyn), and c-Myc. Combined, these data show that polyamine pathway enzymes are differentially regulated in models of altered mechanical and metabolic stress, and that Amd1 and SpdSyn are, in part, regulated in a mTORC1-dependent manner. Furthermore, these data suggest that polyamines may play a role in the adaptive response to stressors in skeletal muscle.
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Affiliation(s)
- Michael Tabbaa
- Institute for Health and Sport (iHeS), Victoria University, Melbourne, Victoria, Australia.,Australian Institute for Musculoskeletal Science (AIMSS), Victoria University, St Albans, Victoria, Australia
| | - Tania Ruz Gomez
- Institute for Health and Sport (iHeS), Victoria University, Melbourne, Victoria, Australia.,Australian Institute for Musculoskeletal Science (AIMSS), Victoria University, St Albans, Victoria, Australia
| | - Dean G Campelj
- Institute for Health and Sport (iHeS), Victoria University, Melbourne, Victoria, Australia.,Australian Institute for Musculoskeletal Science (AIMSS), Victoria University, St Albans, Victoria, Australia
| | - Paul Gregorevic
- Centre for Muscle Research (CMR), Department of Physiology, The University of Melbourne, Victoria, Australia.,Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia.,Department of Neurology, The University of Washington School of Medicine, Seattle, Washington
| | - Alan Hayes
- Institute for Health and Sport (iHeS), Victoria University, Melbourne, Victoria, Australia.,Australian Institute for Musculoskeletal Science (AIMSS), Victoria University, St Albans, Victoria, Australia.,Department of Medicine - Western Health, Melbourne Medical School, The University of Melbourne, Melbourne, Victoria, Australia
| | - Craig A Goodman
- Institute for Health and Sport (iHeS), Victoria University, Melbourne, Victoria, Australia.,Australian Institute for Musculoskeletal Science (AIMSS), Victoria University, St Albans, Victoria, Australia.,Centre for Muscle Research (CMR), Department of Physiology, The University of Melbourne, Victoria, Australia
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9
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Prokop JW, Bupp CP, Frisch A, Bilinovich SM, Campbell DB, Vogt D, Schultz CR, Uhl KL, VanSickle E, Rajasekaran S, Bachmann AS. Emerging Role of ODC1 in Neurodevelopmental Disorders and Brain Development. Genes (Basel) 2021; 12:genes12040470. [PMID: 33806076 PMCID: PMC8064465 DOI: 10.3390/genes12040470] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2021] [Revised: 03/15/2021] [Accepted: 03/22/2021] [Indexed: 01/18/2023] Open
Abstract
Ornithine decarboxylase 1 (ODC1 gene) has been linked through gain-of-function variants to a rare disease featuring developmental delay, alopecia, macrocephaly, and structural brain anomalies. ODC1 has been linked to additional diseases like cancer, with growing evidence for neurological contributions to schizophrenia, mood disorders, anxiety, epilepsy, learning, and suicidal behavior. The evidence of ODC1 connection to neural disorders highlights the need for a systematic analysis of ODC1 genotype-to-phenotype associations. An analysis of variants from ClinVar, Geno2MP, TOPMed, gnomAD, and COSMIC revealed an intellectual disability and seizure connected loss-of-function variant, ODC G84R (rs138359527, NC_000002.12:g.10444500C > T). The missense variant is found in ~1% of South Asian individuals and results in 2.5-fold decrease in enzyme function. Expression quantitative trait loci (eQTLs) reveal multiple functionally annotated, non-coding variants regulating ODC1 that associate with psychiatric/neurological phenotypes. Further dissection of RNA-Seq during fetal brain development and within cerebral organoids showed an association of ODC1 expression with cell proliferation of neural progenitor cells, suggesting gain-of-function variants with neural over-proliferation and loss-of-function variants with neural depletion. The linkage from the expression data of ODC1 in early neural progenitor proliferation to phenotypes of neurodevelopmental delay and to the connection of polyamine metabolites in brain function establish ODC1 as a bona fide neurodevelopmental disorder gene.
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Affiliation(s)
- Jeremy W. Prokop
- Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI 49503, USA; (C.P.B.); (A.F.); (S.M.B.); (D.B.C.); (D.V.); (C.R.S.); (K.L.U.); (S.R.)
- Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824, USA
- Center for Research in Autism, Intellectual, and Other Neurodevelopmental Disabilities, Michigan State University, East Lansing, MI 48824, USA
- Correspondence: (J.W.P.); (A.S.B.)
| | - Caleb P. Bupp
- Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI 49503, USA; (C.P.B.); (A.F.); (S.M.B.); (D.B.C.); (D.V.); (C.R.S.); (K.L.U.); (S.R.)
- Spectrum Health Medical Genetics, Grand Rapids, MI 49503, USA;
| | - Austin Frisch
- Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI 49503, USA; (C.P.B.); (A.F.); (S.M.B.); (D.B.C.); (D.V.); (C.R.S.); (K.L.U.); (S.R.)
| | - Stephanie M. Bilinovich
- Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI 49503, USA; (C.P.B.); (A.F.); (S.M.B.); (D.B.C.); (D.V.); (C.R.S.); (K.L.U.); (S.R.)
| | - Daniel B. Campbell
- Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI 49503, USA; (C.P.B.); (A.F.); (S.M.B.); (D.B.C.); (D.V.); (C.R.S.); (K.L.U.); (S.R.)
- Center for Research in Autism, Intellectual, and Other Neurodevelopmental Disabilities, Michigan State University, East Lansing, MI 48824, USA
- Neuroscience Program, Michigan State University, East Lansing, MI 48824, USA
| | - Daniel Vogt
- Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI 49503, USA; (C.P.B.); (A.F.); (S.M.B.); (D.B.C.); (D.V.); (C.R.S.); (K.L.U.); (S.R.)
- Center for Research in Autism, Intellectual, and Other Neurodevelopmental Disabilities, Michigan State University, East Lansing, MI 48824, USA
- Neuroscience Program, Michigan State University, East Lansing, MI 48824, USA
| | - Chad R. Schultz
- Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI 49503, USA; (C.P.B.); (A.F.); (S.M.B.); (D.B.C.); (D.V.); (C.R.S.); (K.L.U.); (S.R.)
| | - Katie L. Uhl
- Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI 49503, USA; (C.P.B.); (A.F.); (S.M.B.); (D.B.C.); (D.V.); (C.R.S.); (K.L.U.); (S.R.)
| | | | - Surender Rajasekaran
- Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI 49503, USA; (C.P.B.); (A.F.); (S.M.B.); (D.B.C.); (D.V.); (C.R.S.); (K.L.U.); (S.R.)
- Pediatric Intensive Care Unit, Helen DeVos Children’s Hospital, Grand Rapids, MI 49503, USA
- Office of Research, Spectrum Health, Grand Rapids, MI 49503, USA
| | - André S. Bachmann
- Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI 49503, USA; (C.P.B.); (A.F.); (S.M.B.); (D.B.C.); (D.V.); (C.R.S.); (K.L.U.); (S.R.)
- Correspondence: (J.W.P.); (A.S.B.)
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10
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Tulluri V, Nemmara VV. Role of Antizyme Inhibitor Proteins in Cancers and Beyond. Onco Targets Ther 2021; 14:667-682. [PMID: 33531815 PMCID: PMC7846877 DOI: 10.2147/ott.s281157] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2020] [Accepted: 11/05/2020] [Indexed: 01/30/2023] Open
Abstract
Polyamines are multivalent organic cations essential for many cellular functions, including cell growth, differentiation, and proliferation. However, elevated polyamine levels are associated with a slew of pathological conditions, including multiple cancers. Intracellular polyamine levels are primarily controlled by the autoregulatory circuit comprising two different protein types, Antizymes (OAZ) and Antizyme Inhibitors (AZIN), which regulate the activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC). While OAZ functions to decrease the intracellular polyamine levels by inhibiting ODC activity and exerting a negative control of polyamine uptake, AZIN operates to increase intracellular polyamine levels by binding and sequestering OAZ to relieve ODC inhibition and to increase polyamine uptake. Interestingly, OAZ and AZIN exhibit autoregulatory functions on polyamine independent pathways as well. A growing body of evidence demonstrates the dysregulation of AZIN expression in multiple cancers. Additionally, RNA editing of the Azin1 transcript results in a "gain-of-function" phenotype, which is shown to drive aggressive tumor types. This review will discuss the recent advances in AZIN's role in cancers via aberrant polyamine upregulation and its polyamine-independent protein regulation. This report will also highlight AZIN interaction with proteins outside the polyamine biosynthetic pathway and its potential implication to cancer pathogenesis. Finally, this review will reveal the protein interaction network of AZIN isoforms by analyzing three different interactome databases.
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Affiliation(s)
- Vennela Tulluri
- Department of Chemistry and Biochemistry, Rowan University, Glassboro, NJ08028, USA
| | - Venkatesh V Nemmara
- Department of Chemistry and Biochemistry, Rowan University, Glassboro, NJ08028, USA
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11
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Kapfhamer D, McKenna J, Yoon CJ, Murray-Stewart T, Casero RA, Gambello MJ. Ornithine decarboxylase, the rate-limiting enzyme of polyamine synthesis, modifies brain pathology in a mouse model of tuberous sclerosis complex. Hum Mol Genet 2020; 29:2395-2407. [PMID: 32588887 PMCID: PMC7424721 DOI: 10.1093/hmg/ddaa121] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2020] [Revised: 05/18/2020] [Accepted: 06/11/2020] [Indexed: 12/11/2022] Open
Abstract
Tuberous sclerosis complex (TSC) is a rare autosomal dominant neurodevelopmental disorder characterized by variable expressivity. TSC results from inactivating variants within the TSC1 or TSC2 genes, leading to constitutive activation of mechanistic target of rapamycin complex 1 signaling. Using a mouse model of TSC (Tsc2-RG) in which the Tsc2 gene is deleted in radial glial precursors and their neuronal and glial descendants, we observed increased ornithine decarboxylase (ODC) enzymatic activity and concentration of its product, putrescine. To test if increased ODC activity and dysregulated polyamine metabolism contribute to the neurodevelopmental defects of Tsc2-RG mice, we used pharmacologic and genetic approaches to reduce ODC activity in Tsc2-RG mice, followed by histologic assessment of brain development. We observed that decreasing ODC activity and putrescine levels in Tsc2-RG mice worsened many of the neurodevelopmental phenotypes, including brain growth and neuronal migration defects, astrogliosis and oxidative stress. These data suggest a protective effect of increased ODC activity and elevated putrescine that modify the phenotype in this developmental Tsc2-RG model.
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Affiliation(s)
- David Kapfhamer
- Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia
| | - James McKenna
- Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia
| | - Caroline J Yoon
- Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia
| | - Tracy Murray-Stewart
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Robert A Casero
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Michael J Gambello
- Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia
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12
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Ornithine decarboxylase antizyme inhibitor 2 (AZIN2) is a signature of secretory phenotype and independent predictor of adverse prognosis in colorectal cancer. PLoS One 2019; 14:e0211564. [PMID: 30768610 PMCID: PMC6377119 DOI: 10.1371/journal.pone.0211564] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2018] [Accepted: 01/16/2019] [Indexed: 12/27/2022] Open
Abstract
Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine synthesis. The two ODC antizyme inhibitors (AZIN1) and (AZIN2) are regulators of the catalytic activity of ODC. While AZIN1 is a regulator of cell proliferation, AZIN2 is involved in intracellular vesicle transport and secretion. There are no previous reports on the impact of AZIN2 expression in human cancer. We applied immunohistochemistry with antibodies to human AZIN2 on tissue micro- arrays of colorectal cancers (CRC) from 840 patients with a median follow-up of 5.1 years (range 0-25.8). The 5-year disease-specific survival rate was 58.9% (95% Cl 55.0-62.8%). High AZIN2 expression was associated with mucinous histology (p = 0.002) and location in the right hemicolon (p = 0.021). We found no association with age, gender, stage, or histological tumor grade. High tumor expression of AZIN2 predicted an unfavorable prognosis (p<0.0001, log-rank test), compared to low AZIN2 expression. Cox multivariable analysis identified AZIN2 as an independent factor of an unfavorable prognosis in CRC. The strongest AZIN2 expression was seen in invasive tumor cells having morphological features of epithelial-mesenchymal transition (EMT). Induction of EMT in HT-29 CRC cells lead to upregulated expression of endogenous AZIN2. Given that AZIN2 is a regulator of vesicle transport and secretion, we overexpressed human AZIN2 cDNA in T84 CRC cells, and found strongly enhanced accumulation of CD63-positive exosomes in the culture medium. These findings indicate that AZIN2 expression is a signature of EMT-associated secretory phenotype that is linked to an adverse prognosis in CRC.
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13
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Takeda S, Shigeyasu K, Okugawa Y, Yoshida K, Mori Y, Yano S, Noma K, Umeda Y, Kondo Y, Kishimoto H, Teraishi F, Nagasaka T, Tazawa H, Kagawa S, Fujiwara T, Goel A. Activation of AZIN1 RNA editing is a novel mechanism that promotes invasive potential of cancer-associated fibroblasts in colorectal cancer. Cancer Lett 2018; 444:127-135. [PMID: 30583079 DOI: 10.1016/j.canlet.2018.12.009] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2018] [Revised: 12/03/2018] [Accepted: 12/11/2018] [Indexed: 12/31/2022]
Abstract
Adenosine-to-inosine (A-to-I) RNA editing is a recently described epigenetic modification, which is believed to constitute a key oncogenic mechanism in human cancers. However, its functional role in cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) and its clinical significance remains unclear. Herein, we systematically analyzed a large cohort of 627 colorectal cancer (CRC) specimens, and investigated the expression pattern of ADAR1 and its biological significance on the antizyme inhibitor 1 (AZIN1) RNA editing levels. Both ADAR1 expression and AZIN1 RNA editing levels were significantly elevated in CRC tissues vs. normal mucosa, and these findings correlated with the increased expression of mesenchymal markers, Vimentin (ρ = 0.44) and Fibroblast activation protein (ρ = 0.38). Intriguingly, ADAR1 expression was specifically upregulated in both cancer cells and fibroblasts from cancerous lesions. Conditioned medium from cancer cells led to induction of ADAR1 expression and activation of AZIN1 RNA editing in fibroblasts (p < 0.05). Additionally, edited AZIN1 enhanced the invasive potential of fibroblasts. In conclusion, we provide novel evidence that hyper-editing of AZIN1 enhances the invasive potential of CAFs within the TME in colon and is an important predictor of tumor invasiveness in CRC.
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Affiliation(s)
- Sho Takeda
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Kunitoshi Shigeyasu
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan; Center for Gastrointestinal Research, Center for Translational Genomics and Oncology, Baylor Scott & White Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, TX, USA
| | - Yoshinaga Okugawa
- Center for Gastrointestinal Research, Center for Translational Genomics and Oncology, Baylor Scott & White Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, TX, USA; Department of Gastrointestinal and Pediatric Surgery, Division of Reparative Medicine, Institute of Life Sciences, Mie University Graduate School of Medicine, Mie, Japan
| | - Kazuhiro Yoshida
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan; Center for Gastrointestinal Research, Center for Translational Genomics and Oncology, Baylor Scott & White Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, TX, USA
| | - Yoshiko Mori
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Shuya Yano
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Kazuhiro Noma
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Yuzo Umeda
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Yoshitaka Kondo
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Hiroyuki Kishimoto
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Fuminori Teraishi
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Takeshi Nagasaka
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Hiroshi Tazawa
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Shunsuke Kagawa
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Toshiyoshi Fujiwara
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Ajay Goel
- Center for Gastrointestinal Research, Center for Translational Genomics and Oncology, Baylor Scott & White Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, TX, USA.
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14
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Abstract
The polyamines spermidine, spermine, and their precursor putrescine are organic polycations involved in various cellular processes and are absolutely essential for cellular proliferation. Because of their crucial function in the cell, their intracellular concentration must be maintained at optimal levels. To a large extent, this regulation is achieved through the activity of an autoregulatory loop that involves two proteins, antizyme (Az) and antizyme inhibitor (AzI), that regulate the first enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC), and polyamine uptake activity in response to intracellular polyamine levels. In this Minireview, I will discuss what has been learned about the mechanism of Az expression and its physical interaction with both ODC and AzI in the regulation of polyamines.
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Affiliation(s)
- Chaim Kahana
- From the Department of Molecular Genetics, the Weizmann Institute of Science, Rehovot 76100, Israel
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15
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Ramos-Molina B, Lambertos A, Peñafiel R. Antizyme Inhibitors in Polyamine Metabolism and Beyond: Physiopathological Implications. ACTA ACUST UNITED AC 2018; 6:medsci6040089. [PMID: 30304856 PMCID: PMC6313458 DOI: 10.3390/medsci6040089] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2018] [Revised: 10/02/2018] [Accepted: 10/04/2018] [Indexed: 12/25/2022]
Abstract
The intracellular levels of polyamines, cationic molecules involved in a myriad of cellular functions ranging from cellular growth, differentiation and apoptosis, is precisely regulated by antizymes and antizyme inhibitors via the modulation of the polyamine biosynthetic and transport systems. Antizymes, which are mainly activated upon high polyamine levels, inhibit ornithine decarboxylase (ODC), the key enzyme of the polyamine biosynthetic route, and exert a negative control of polyamine intake. Antizyme inhibitors (AZINs), which are proteins highly homologous to ODC, selectively interact with antizymes, preventing their action on ODC and the polyamine transport system. In this review, we will update the recent advances on the structural, cellular and physiological functions of AZINs, with particular emphasis on the action of these proteins in the regulation of polyamine metabolism. In addition, we will describe emerging evidence that suggests that AZINs may also have polyamine-independent effects on cells. Finally, we will discuss how the dysregulation of AZIN activity has been implicated in certain human pathologies such as cancer, fibrosis or neurodegenerative diseases.
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Affiliation(s)
- Bruno Ramos-Molina
- Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, 30100 Murcia, Spain.
- Laboratory of Cellular and Molecular Endocrinology, Institute of Biomedical Research in Malaga (IBIMA), Virgen de la Victoria University Hospital, 29010 Málaga, Spain.
- CIBER Physiopathology of Obesity and Nutrition (CIBERobn), Institute of Health Carlos III (ISCIII), 28029 Madrid, Spain.
| | - Ana Lambertos
- Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, 30100 Murcia, Spain.
- Biomedical Research Institute of Murcia (IMIB), 30120 Murcia, Spain.
| | - Rafael Peñafiel
- Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, 30100 Murcia, Spain.
- Biomedical Research Institute of Murcia (IMIB), 30120 Murcia, Spain.
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16
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Shigeyasu K, Okugawa Y, Toden S, Miyoshi J, Toiyama Y, Nagasaka T, Takahashi N, Kusunoki M, Takayama T, Yamada Y, Fujiwara T, Chen L, Goel A. AZIN1 RNA editing confers cancer stemness and enhances oncogenic potential in colorectal cancer. JCI Insight 2018; 3:99976. [PMID: 29925690 PMCID: PMC6124399 DOI: 10.1172/jci.insight.99976] [Citation(s) in RCA: 93] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2018] [Accepted: 05/17/2018] [Indexed: 12/30/2022] Open
Abstract
Adenosine-to-inosine (A-to-I) RNA editing, a process mediated by adenosine deaminases that act on the RNA (ADAR) gene family, is a recently discovered epigenetic modification dysregulated in human cancers. However, the clinical significance and the functional role of RNA editing in colorectal cancer (CRC) remain unclear. We have systematically and comprehensively investigated the significance of the expression status of ADAR1 and of the RNA editing levels of antizyme inhibitor 1 (AZIN1), one of the most frequently edited genes in cancers, in 392 colorectal tissues from multiple independent CRC patient cohorts. Both ADAR1 expression and AZIN1 RNA editing levels were significantly elevated in CRC tissues when compared with corresponding normal mucosa. High levels of AZIN1 RNA editing emerged as a prognostic factor for overall survival and disease-free survival and were an independent risk factor for lymph node and distant metastasis. Furthermore, elevated AZIN1 editing identified high-risk stage II CRC patients. Mechanistically, edited AZIN1 enhances stemness and appears to drive the metastatic processes. We have demonstrated that edited AZIN1 functions as an oncogene and a potential therapeutic target in CRC. Moreover, AZIN1 RNA editing status could be used as a clinically relevant prognostic indicator in CRC patients.
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Affiliation(s)
- Kunitoshi Shigeyasu
- Center for Gastrointestinal Research and Center for Translational Genomics and Oncology, Baylor Scott & White Research Institute and Baylor Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, Texas, USA
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Yoshinaga Okugawa
- Center for Gastrointestinal Research and Center for Translational Genomics and Oncology, Baylor Scott & White Research Institute and Baylor Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, Texas, USA
- Department of Gastrointestinal and Pediatric Surgery, Division of Reparative Medicine, Institute of Life Sciences, Mie University Graduate School of Medicine, Mie, Japan
| | - Shusuke Toden
- Center for Gastrointestinal Research and Center for Translational Genomics and Oncology, Baylor Scott & White Research Institute and Baylor Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, Texas, USA
| | - Jinsei Miyoshi
- Department of Gastroenterology and Oncology, University of Tokushima, Tokushima, Japan
| | - Yuji Toiyama
- Department of Gastrointestinal and Pediatric Surgery, Division of Reparative Medicine, Institute of Life Sciences, Mie University Graduate School of Medicine, Mie, Japan
| | - Takeshi Nagasaka
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | | | - Masato Kusunoki
- Department of Gastrointestinal and Pediatric Surgery, Division of Reparative Medicine, Institute of Life Sciences, Mie University Graduate School of Medicine, Mie, Japan
| | - Tetsuji Takayama
- Department of Gastroenterology and Oncology, University of Tokushima, Tokushima, Japan
| | | | - Toshiyoshi Fujiwara
- Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
| | - Leilei Chen
- Cancer Science Institute of Singapore, National University of Singapore, Singapore
- Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Ajay Goel
- Center for Gastrointestinal Research and Center for Translational Genomics and Oncology, Baylor Scott & White Research Institute and Baylor Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, Texas, USA
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17
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Tusup M, Kundig T, Pascolo S. Epitranscriptomics of cancer. World J Clin Oncol 2018; 9:42-55. [PMID: 29900123 PMCID: PMC5997933 DOI: 10.5306/wjco.v9.i3.42] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/07/2018] [Revised: 04/18/2018] [Accepted: 05/23/2018] [Indexed: 02/06/2023] Open
Abstract
The functional impact of modifications of cellular RNAs, including mRNAs, miRNAs and lncRNAs, is a field of intense study. The role of such modifications in cancer has started to be elucidated. Diverse and sometimes opposite effects of RNA modifications have been reported. Some RNA modifications promote, while others decrease the growth and invasiveness of cancer. The present manuscript reviews the current knowledge on the potential impacts of N6-Methyladenosine, Pseudouridine, Inosine, 2’O-methylation or methylcytidine in cancer’s RNA. It also highlights the remaining questions and provides hints on research avenues and potential therapeutic applications, whereby modulating dynamic RNA modifications may be a new method to treat cancer.
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Affiliation(s)
- Marina Tusup
- Department of Dermatology, University Hospital of Zürich, Zurich 8091, Switzerland
- Faculty of Medicine, University of Zurich, Zurich 8091, Switzerland
| | - Thomas Kundig
- Department of Dermatology, University Hospital of Zürich, Zurich 8091, Switzerland
- Faculty of Medicine, University of Zurich, Zurich 8091, Switzerland
| | - Steve Pascolo
- Department of Dermatology, University Hospital of Zürich, Zurich 8091, Switzerland
- Faculty of Medicine, University of Zurich, Zurich 8091, Switzerland
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18
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Characterization of an androgen-responsive, ornithine decarboxylase-related protein in mouse kidney. Biosci Rep 2017; 37:BSR20170163. [PMID: 28607032 PMCID: PMC5518511 DOI: 10.1042/bsr20170163] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2017] [Revised: 06/09/2017] [Accepted: 06/09/2017] [Indexed: 01/26/2023] Open
Abstract
We have investigated and characterized a novel ornithine decarboxylase (ODC) related protein (ODCrp) also annotated as gm853. ODCrp shows 41% amino acid sequence identity with ODC and 38% with ODC antizyme inhibitor 1 (AZIN1). The Odcrp gene is selectively expressed in the epithelium of proximal tubuli of mouse kidney with higher expression in males than in females. Like Odc in mouse kidney, Odcrp is also androgen responsive with androgen receptor (AR)-binding loci within its regulatory region. ODCrp forms homodimers but does not heterodimerize with ODC. Although ODCrp contains 20 amino acid residues known to be necessary for the catalytic activity of ODC, no decarboxylase activity could be found with ornithine, lysine or arginine as substrates. ODCrp does not function as an AZIN, as it neither binds ODC antizyme 1 (OAZ1) nor prevents OAZ-mediated inactivation and degradation of ODC. ODCrp itself is degraded via ubiquination and mutation of Cys363 (corresponding to Cys360 of ODC) appears to destabilize the protein. Evidence for a function of ODCrp was found in ODC assays on lysates from transfected Cos-7 cells where ODCrp repressed the activity of endogenous ODC while Cys363Ala mutated ODCrp increased the enzymatic activity of endogenous ODC.
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19
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Protein degradation, the main hub in the regulation of cellular polyamines. Biochem J 2017; 473:4551-4558. [PMID: 27941031 DOI: 10.1042/bcj20160519c] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2016] [Revised: 09/20/2016] [Accepted: 09/22/2016] [Indexed: 12/15/2022]
Abstract
Ornithine decarboxylase (ODC) is the first and rate-limiting enzyme in the biosynthesis of polyamines, low-molecular-mass aliphatic polycations that are ubiquitously present in all living cells and are essential for fundamental cellular processes. Most cellular polyamines are bound, whereas the free pools, which regulate cellular functions, are subjected to tight regulation. The regulation of the free polyamine pools is manifested by modulation of their synthesis, catabolism, uptake and excretion. A central element that enables this regulation is the rapid degradation of key enzymes and regulators of these processes, particularly that of ODC. ODC degradation is part of an autoregulatory circuit that responds to the intracellular level of the free polyamines. The driving force of this regulatory circuit is a protein termed antizyme (Az). Az stimulates the degradation of ODC and inhibits polyamine uptake. Az acts as a sensor of the free intracellular polyamine pools as it is expressed via a polyamine-stimulated ribosomal frameshifting. Az binds to monomeric ODC subunits to prevent their reassociation into active homodimers and facilitates their ubiquitin-independent degradation by the 26S proteasome. In addition, through a yet unidentified mechanism, Az inhibits polyamine uptake. Interestingly, a protein, termed antizyme inhibitor (AzI) that is highly homologous with ODC, but retains no ornithine decarboxylating activity, seems to regulate cellular polyamines through its ability to negate Az. Overall, the degradation of ODC is a net result of interactions with regulatory proteins and possession of signals that mediate its ubiquitin-independent recognition by the proteasome.
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20
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Mounce BC, Poirier EZ, Passoni G, Simon-Loriere E, Cesaro T, Prot M, Stapleford KA, Moratorio G, Sakuntabhai A, Levraud JP, Vignuzzi M. Interferon-Induced Spermidine-Spermine Acetyltransferase and Polyamine Depletion Restrict Zika and Chikungunya Viruses. Cell Host Microbe 2016; 20:167-77. [PMID: 27427208 DOI: 10.1016/j.chom.2016.06.011] [Citation(s) in RCA: 85] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2016] [Revised: 05/17/2016] [Accepted: 06/13/2016] [Indexed: 01/05/2023]
Abstract
Polyamines are small, positively charged molecules derived from ornithine and synthesized through an intricately regulated enzymatic pathway. Within cells, they are abundant and play several roles in diverse processes. We find that polyamines are required for the life cycle of the RNA viruses chikungunya virus (CHIKV) and Zika virus (ZIKV). Depletion of spermidine and spermine via type I interferon signaling-mediated induction of spermidine/spermine N1-acetyltransferase (SAT1), a key catabolic enzyme in the polyamine pathway, restricts CHIKV and ZIKV replication. Polyamine depletion restricts these viruses in vitro and in vivo, due to impairment of viral translation and RNA replication. The restriction is released by exogenous replenishment of polyamines, further supporting a role for these molecules in virus replication. Thus, SAT1 and, more broadly, polyamine depletion restrict viral replication and suggest promising avenues for antiviral therapies.
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Affiliation(s)
- Bryan C Mounce
- Viral Populations and Pathogenesis Unit, Institut Pasteur, CNRS UMR 3569, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France
| | - Enzo Z Poirier
- Viral Populations and Pathogenesis Unit, Institut Pasteur, CNRS UMR 3569, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France; University of Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, 75013 Paris, France
| | - Gabriella Passoni
- Virologie et Immunologie Moléculaire, INRA, Domaine de Vilvert, 78352 Jouy-en-Josas, France; Macrophages et Développement de l'Immunité, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France; CNRS URA2578, 75724 Paris Cedex 15, France
| | - Etienne Simon-Loriere
- Unité de Génétique Fonctionnelle des Maladies Infectieuses, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France
| | - Teresa Cesaro
- Viral Populations and Pathogenesis Unit, Institut Pasteur, CNRS UMR 3569, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France
| | - Matthieu Prot
- Unité de Génétique Fonctionnelle des Maladies Infectieuses, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France
| | - Kenneth A Stapleford
- Viral Populations and Pathogenesis Unit, Institut Pasteur, CNRS UMR 3569, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France
| | - Gonzalo Moratorio
- Viral Populations and Pathogenesis Unit, Institut Pasteur, CNRS UMR 3569, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France
| | - Anavaj Sakuntabhai
- Unité de Génétique Fonctionnelle des Maladies Infectieuses, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France
| | - Jean-Pierre Levraud
- Macrophages et Développement de l'Immunité, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France; CNRS URA2578, 75724 Paris Cedex 15, France
| | - Marco Vignuzzi
- Viral Populations and Pathogenesis Unit, Institut Pasteur, CNRS UMR 3569, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France.
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21
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Expression of ODC Antizyme Inhibitor 2 (AZIN2) in Human Secretory Cells and Tissues. PLoS One 2016; 11:e0151175. [PMID: 26963840 PMCID: PMC4786150 DOI: 10.1371/journal.pone.0151175] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2016] [Accepted: 02/24/2016] [Indexed: 01/18/2023] Open
Abstract
Ornithine decarboxylase (ODC) antizyme inhibitor 2 (AZIN2), originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and / or secretion, but the ultimate physiological role(s) of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3) to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H,K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated.
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Remaining Mysteries of Molecular Biology: The Role of Polyamines in the Cell. J Mol Biol 2015; 427:3389-406. [DOI: 10.1016/j.jmb.2015.06.020] [Citation(s) in RCA: 401] [Impact Index Per Article: 40.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2015] [Revised: 06/12/2015] [Accepted: 06/29/2015] [Indexed: 11/23/2022]
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Yordanova MM, Wu C, Andreev DE, Sachs MS, Atkins JF. A Nascent Peptide Signal Responsive to Endogenous Levels of Polyamines Acts to Stimulate Regulatory Frameshifting on Antizyme mRNA. J Biol Chem 2015; 290:17863-17878. [PMID: 25998126 PMCID: PMC4505036 DOI: 10.1074/jbc.m115.647065] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2015] [Indexed: 01/06/2023] Open
Abstract
The protein antizyme is a negative regulator of cellular polyamine concentrations from yeast to mammals. Synthesis of functional antizyme requires programmed +1 ribosomal frameshifting at the 3′ end of the first of two partially overlapping ORFs. The frameshift is the sensor and effector in an autoregulatory circuit. Except for Saccharomyces cerevisiae antizyme mRNA, the frameshift site alone only supports low levels of frameshifting. The high levels usually observed depend on the presence of cis-acting stimulatory elements located 5′ and 3′ of the frameshift site. Antizyme genes from different evolutionary branches have evolved different stimulatory elements. Prior and new multiple alignments of fungal antizyme mRNA sequences from the Agaricomycetes class of Basidiomycota show a distinct pattern of conservation 5′ of the frameshift site consistent with a function at the amino acid level. As shown here when tested in Schizosaccharomyces pombe and mammalian HEK293T cells, the 5′ part of this conserved sequence acts at the nascent peptide level to stimulate the frameshifting, without involving stalling detectable by toe-printing. However, the peptide is only part of the signal. The 3′ part of the stimulator functions largely independently and acts at least mostly at the nucleotide level. When polyamine levels were varied, the stimulatory effect was seen to be especially responsive in the endogenous polyamine concentration range, and this effect may be more general. A conserved RNA secondary structure 3′ of the frameshift site has weaker stimulatory and polyamine sensitizing effects on frameshifting.
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Affiliation(s)
- Martina M Yordanova
- School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland
| | - Cheng Wu
- Department of Biology, Texas A&M University, College Station, Texas 77843-3258
| | - Dmitry E Andreev
- Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia
| | - Matthew S Sachs
- Department of Biology, Texas A&M University, College Station, Texas 77843-3258
| | - John F Atkins
- School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland; Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112-5330.
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Silva TM, Cirenajwis H, Wallace HM, Oredsson S, Persson L. A role for antizyme inhibitor in cell proliferation. Amino Acids 2015; 47:1341-52. [PMID: 25813938 PMCID: PMC4458265 DOI: 10.1007/s00726-015-1957-6] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2014] [Accepted: 03/06/2015] [Indexed: 12/20/2022]
Abstract
The polyamines are important for a variety of cellular functions, including cell growth. Their intracellular concentrations are controlled by a complex network of regulatory mechanisms, in which antizyme (Az) has a key role. Az reduces the cellular polyamine content by down-regulating both the enzyme catalysing polyamine biosynthesis, ornithine decarboxylase (ODC), and the uptake of polyamines. The activity of Az is repressed by the binding of a protein, named Az inhibitor (AzI), which is an enzymatically inactive homologue of ODC. Two forms of AzI have been described: AzI1, which is ubiquitous, and AzI2 which is expressed in brain and testis. In the present study, we have investigated the role of AzI1 in polyamine homeostasis and cell proliferation in breast cancer cells. The results obtained showed that the cellular content of AzI increased transiently after induction of cell proliferation by diluting cells in fresh medium. Inhibition of polyamine biosynthesis induced an even larger increase in the cellular AzI content, which remained significantly elevated during the 7-day experimental period. However, this increase was not a consequence of changes in cell cycle progression, as demonstrated by flow cytometry. Instead, the increase appeared to correlate with the cellular depletion of polyamines. Moreover, induced overexpression of AzI resulted in an increased cell proliferation with a concomitant increase in ODC activity and putrescine content. During mitosis, AzI1 was localised in a pattern that resembled that of the two centrosomes, confirming earlier observations. Taken together, the results indicate that AzI fulfils an essential regulatory function in polyamine homeostasis and cell proliferation.
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Affiliation(s)
- Tania M. Silva
- Department of Biology, Lund University, Lund, Sweden
- Present Address: Laboratory of Microbiology and Immunology of Infection, Institute for Molecular and Cell Biology, Porto University, Porto, Portugal
| | - Helena Cirenajwis
- Department of Biology, Lund University, Lund, Sweden
- Present Address: Division of Oncology and Pathology, Department of Clinical Sciences, Lund University, Lund, Sweden
| | - Heather M. Wallace
- Department of Biology, Lund University, Lund, Sweden
- Division of Applied Medicine, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen, UK
| | | | - Lo Persson
- Department of Experimental Medical Science, Lund University, Lund, Sweden
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Park MH, Igarashi K. Polyamines and their metabolites as diagnostic markers of human diseases. Biomol Ther (Seoul) 2014; 21:1-9. [PMID: 24009852 PMCID: PMC3762300 DOI: 10.4062/biomolther.2012.097] [Citation(s) in RCA: 118] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2012] [Accepted: 01/04/2013] [Indexed: 01/31/2023] Open
Abstract
Polyamines, putrescine, spermidine and spermine, are ubiquitous in living cells and are essential for eukaryotic cell growth. These polycations interact with negatively charged molecules such as DNA, RNA, acidic proteins and phospholipids and modulate various cellular functions including macromolecular synthesis. Dysregulation of the polyamine pathway leads to pathological conditions including cancer, inflammation, stroke, renal failure and diabetes. Increase in polyamines and polyamine synthesis enzymes is often associated with tumor growth, and urinary and plasma contents of polyamines and their metabolites have been investigated as diagnostic markers for cancers. Of these, diacetylated derivatives of spermidine and spermine are elevated in the urine of cancer patients and present potential markers for early detection. Enhanced catabolism of cellular polyamines by polyamine oxidases (PAO), spermine oxidase (SMO) or acetylpolyamine oxidase (AcPAO), increases cellular oxidative stress and generates hydrogen peroxide and a reactive toxic metabolite, acrolein, which covalently incorporates into lysine residues of cellular proteins. Levels of protein-conjuagated acrolein (PC-Acro) and polyamine oxidizing enzymes were increased in the locus of brain infarction and in plasma in a mouse model of stroke and also in the plasma of stroke patients. When the combined measurements of PC-Acro, interleukin 6 (IL-6), and C-reactive protein (CRP) were evaluated, even silent brain infarction (SBI) was detected with high sensitivity and specificity. Considering that there are no reliable biochemical markers for early stage of stroke, PC-Acro and PAOs present promising markers. Thus the polyamine metabolites in plasma or urine provide useful tools in early diagnosis of cancer and stroke.
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Affiliation(s)
- Myung Hee Park
- Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, MD, 20892, USA
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Ray RM, Bhattacharya S, Bavaria MN, Viar MJ, Johnson LR. Antizyme (AZ) regulates intestinal cell growth independent of polyamines. Amino Acids 2014; 46:2231-9. [PMID: 24930035 DOI: 10.1007/s00726-014-1777-0] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2014] [Accepted: 06/04/2014] [Indexed: 11/27/2022]
Abstract
Since antizyme (AZ) is known to inhibit cell proliferation and to increase apoptosis, the question arises as to whether these effects occur independently of polyamines. Intestinal epithelial cells (IEC-6) were grown in control medium and medium containing 5 mM difluoromethylornithine (DFMO) to inhibit ODC, DFMO + 5 µM spermidine (SPD), DFMO + 5 µM spermine (SPM), or DFMO + 10 µM putrescine (PUT) for 4 days and various parameters of growth were measured along with AZ levels. Cell counts were significantly decreased and mean doubling times were significantly increased by DFMO. Putrescine restored growth in the presence of DFMO. However, both SPD and SPM when added with DFMO caused a much greater inhibition of growth than did DFMO alone, and both of these polyamines caused a dramatic increase in AZ. The addition of SPD or SPM to media containing DFMO + PUT significantly inhibited growth and caused a significant increase in AZ. IEC-6 cells transfected with AZ-siRNA grew more than twice as rapidly as either control cells or those incubated with DFMO, indicating that removal of AZ increases growth in cells in which polyamine synthesis is inhibited as well as in control cells. In a separate experiment, the addition of SPD increased AZ levels and inhibited growth of cells incubated with DFMO by 50%. The addition of 10 mM asparagine (ASN) prevented the increase in AZ and restored growth to control levels. These results show that cell growth in the presence or absence of ODC activity and in the presence or absence of polyamines depends only on the levels of AZ. Therefore, the effects of AZ on cell growth are independent of polyamines.
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Affiliation(s)
- Ramesh M Ray
- Department of Physiology, University of Tennessee Health Science Center, 894 Union Avenue, Memphis, TN, 38163, USA,
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Qiao JJ, Chan THM, Qin YR, Chen L. ADAR1: a promising new biomarker for esophageal squamous cell carcinoma? Expert Rev Anticancer Ther 2014; 14:865-8. [PMID: 24928581 DOI: 10.1586/14737140.2014.928595] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Esophageal Squamous Cell Carcinoma (ESCC) is a heterogeneous tumor with enormous genetic and epigenetic changes. RNA editing is an epigenetic mechanism that serves as an additional layer of 'RNA mutations' in parallel to DNA mutations. The most frequent type of RNA editing, A-to-I (adenosine-to-inosine) editing catalyzed by Adenosine DeAminase that act on RNA (ADARs), modulates RNA transcripts with profound impact on cellular functions. RNA editing dysregulation has been found to be associated with cancers. Our recent study demonstrated that among all the three RNA editing enzymes, only ADAR1 was overexpressed in primary ESCCs compared with matched non-tumor specimens. In this review, we will discuss current views on the involvement of abnormal A-to-I editing in cancer development, more specifically on the ADAR1-mediated editing in ESCC. Although much is not yet learned about the role of ADAR1 in ESCC, ADAR1 may present an attractive option as a new biomarker for ESCC and as a new molecular therapeutic target.
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Affiliation(s)
- Jun-Jing Qiao
- Department of Clinical Oncology, the First Affiliated Hospital, Zhengzhou University, Zhengzhou, China
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Mutational analysis of the antizyme-binding element reveals critical residues for the function of ornithine decarboxylase. Biochim Biophys Acta Gen Subj 2013; 1830:5157-65. [DOI: 10.1016/j.bbagen.2013.07.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2013] [Revised: 07/04/2013] [Accepted: 07/08/2013] [Indexed: 01/22/2023]
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López-Contreras AJ, de la Morena ME, Ramos-Molina B, Lambertos A, Cremades A, Peñafiel R. The induction of cardiac ornithine decarboxylase by β2 -adrenergic agents is associated with calcium channels and phosphorylation of ERK1/2. J Cell Biochem 2013; 114:1978-86. [PMID: 23519605 DOI: 10.1002/jcb.24540] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2013] [Accepted: 03/05/2013] [Indexed: 12/17/2022]
Abstract
The role that the induction of cardiac ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by beta-adrenergic agents may have in heart hypertrophy is a controversial issue. Besides, the signaling pathways related to cardiac ODC regulation have not been fully elucidated. Here we show that in Balb C mice the stimulation of cardiac ODC activity by adrenergic agents was mainly mediated by β2 -adrenergic receptors, and that this induction was lower in the hypertrophic heart. Interestingly, this stimulation was abolished by the L-calcium channel antagonists verapamil and nifedipine. In addition, whereas the treatment with β2 -adrenergic agents was associated to both the increases in ODC, ODC-antizyme inhibitor 1 (AZIN1), c-fos and c-myc mRNA levels and the phosphorylation of CREB and MAP kinases ERK1 and ERK2 (ERK1/2), the co-treatment with L-calcium channel blockers differentially prevented most of these changes. These results suggest that the stimulation of cardiac ODC by β2 -adrenergic agents is associated with the activation of MAP kinases through the participation of L-calcium channels, and that by itself p-CREB does not appear to be sufficient for the transcriptional activation of ODC. In addition, post-translational mechanisms related with the induction of AZIN1 appear to be related to the increase of cardiac ODC activity.
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Affiliation(s)
- Andrés J López-Contreras
- Faculty of Medicine, Department of Biochemistry and Molecular Biology B and Immunology, University of Murcia, Murcia, Spain
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Multiple forms of mouse antizyme inhibitor 1 mRNA differentially regulated by polyamines. Amino Acids 2013; 46:575-83. [PMID: 24077669 DOI: 10.1007/s00726-013-1598-6] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2013] [Accepted: 09/17/2013] [Indexed: 10/26/2022]
Abstract
Antizyme inhibitor 1 (Azin1), a positive regulator of cellular polyamines, is induced by various proliferative stimuli and repressed by polyamines. It has been reported that the translational repression of Azin1 by polyamines involves an upstream open reading frame on the mRNA, but little has been known about polyamine effect on its transcription or splicing. We found multiple forms of Azin1 transcripts formed by alternative splicing and initiation of transcription from putative alternative start sites. One of the novel splice variants, Azin1-X, has a premature termination codon on 5′ extension of exon 7, encodes a C-terminal truncated form of protein (Azin1ΔC), and is subject to nonsense-mediated mRNA decay. 2-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis, increased both transcription from the canonical transcription start site and the ratio of the full-length mRNA to Azin1-X mRNA, whereas polyamines show the opposite effect. Thus, polyamines regulate two novel steps of Azin1 expression, namely the transcription and a particular splicing pattern, both of which may affect the level of mRNA encoding the full-length active Azin1 protein.
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Structural insight into DFMO resistant ornithine decarboxylase from Entamoeba histolytica: an inkling to adaptive evolution. PLoS One 2013; 8:e53397. [PMID: 23326423 PMCID: PMC3543441 DOI: 10.1371/journal.pone.0053397] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2012] [Accepted: 11/28/2012] [Indexed: 11/19/2022] Open
Abstract
Background Polyamine biosynthetic pathway is a validated therapeutic target for large number of infectious diseases including cancer, giardiasis and African sleeping sickness, etc. α-Difluoromethylornithine (DFMO), a potent drug used for the treatment of African sleeping sickness is an irreversible inhibitor of ornithine decarboxylase (ODC), the first rate limiting enzyme of polyamine biosynthesis. The enzyme ODC of E. histolytica (EhODC) has been reported to exhibit resistance towards DFMO. Methodology/Principal Finding The basis for insensitivity towards DFMO was investigated by structural analysis of EhODC and conformational modifications at the active site. Here, we report cloning, purification and crystal structure determination of C-terminal truncated Entamoeba histolytica ornithine decarboxylase (EhODCΔ15). Structure was determined by molecular replacement method and refined to 2.8 Å resolution. The orthorhombic crystal exhibits P212121 symmetry with unit cell parameters a = 76.66, b = 119.28, c = 179.28 Å. Functional as well as evolutionary relations of EhODC with other ODC homologs were predicted on the basis of sequence analysis, phylogeny and structure. Conclusions/Significance We determined the tetrameric crystal structure of EhODCΔ15, which exists as a dimer in solution. Insensitivity towards DFMO is due to substitution of key substrate binding residues in active site pocket. Additionally, a few more substitutions similar to antizyme inhibitor (AZI), a non-functional homologue of ODCs, were identified in the active site. Here, we establish the fact that EhODC sequence has conserved PLP binding residues; in contrast few substrate binding residues are mutated similar to AZI. Further sequence analysis and structural studies revealed that EhODC may represent as an evolutionary bridge between active decarboxylase and inactive AZI.
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Chen L, Li Y, Lin CH, Chan THM, Chow RKK, Song Y, Liu M, Yuan YF, Fu L, Kong KL, Qi L, Li Y, Zhang N, Tong AHY, Kwong DLW, Man K, Lo CM, Lok S, Tenen DG, Guan XY. Recoding RNA editing of AZIN1 predisposes to hepatocellular carcinoma. Nat Med 2013; 19:209-16. [PMID: 23291631 DOI: 10.1038/nm.3043] [Citation(s) in RCA: 409] [Impact Index Per Article: 34.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2011] [Accepted: 11/21/2012] [Indexed: 01/14/2023]
Abstract
A better understanding of human hepatocellular carcinoma (HCC) pathogenesis at the molecular level will facilitate the discovery of tumor-initiating events. Transcriptome sequencing revealed that adenosine-to-inosine (A→I) RNA editing of AZIN1 (encoding antizyme inhibitor 1) is increased in HCC specimens. A→I editing of AZIN1 transcripts, specifically regulated by ADAR1 (encoding adenosine deaminase acting on RNA-1), results in a serine-to-glycine substitution at residue 367 of AZIN1, located in β-strand 15 (β15) and predicted to cause a conformational change, induced a cytoplasmic-to-nuclear translocation and conferred gain-of-function phenotypes that were manifested by augmented tumor-initiating potential and more aggressive behavior. Compared with wild-type AZIN1 protein, the edited form has a stronger affinity to antizyme, and the resultant higher AZIN1 protein stability promotes cell proliferation through the neutralization of antizyme-mediated degradation of ornithine decarboxylase (ODC) and cyclin D1 (CCND1). Collectively, A→I RNA editing of AZIN1 may be a potential driver in the pathogenesis of human cancers, particularly HCC.
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Affiliation(s)
- Leilei Chen
- Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
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Levillain O, Ramos-Molina B, Forcheron F, Peñafiel R. Expression and distribution of genes encoding for polyamine-metabolizing enzymes in the different zones of male and female mouse kidneys. Amino Acids 2012; 43:2153-63. [PMID: 22562773 DOI: 10.1007/s00726-012-1300-4] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2012] [Accepted: 04/12/2012] [Indexed: 12/16/2022]
Abstract
The role of polyamines in renal physiology is only partially understood. Moreover, most of the data on the enzymes of polyamine metabolism come from studies using whole kidneys. The aim of the present study was to analyze the mRNA abundance of the genes implicated in both the polyamine biosynthetic and catabolic pathways in different renal zones of male and female mice, by means of the quantitative reverse transcription-polymerase chain reaction. Our results indicate that there is an uneven distribution of the different mRNAs studied in the five renal zones: superficial cortex, deep cortex, outer stripe of the outer medulla (OS), inner stripe of the outer medulla (IS), and the inner medulla + papilla (IM). The biosynthetic genes, ornithine decarboxylase (ODC) and spermine synthase, were more expressed in the cortex, whereas the mRNAs of the catabolic genes spermine oxidase (SMO) and diamine oxidase were more abundant in IS and IM. The genes involved in the regulation of polyamine synthesis (AZ1, AZ2 and AZIN1) were expressed in all the renal zones, predominantly in the cortex, while AZIN2 gene was more abundant in the OS. ODC, SMO, spermidine synthase and spermidine/spermine acetyl transferase expression was higher in males than in females. In conclusion, the genes encoding for the polyamine metabolism were specifically and quantitatively distributed along the corticopapillary axis of male and female mouse kidneys, suggesting that their physiological role is essential in defined renal zones and/or nephron segments.
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Affiliation(s)
- Olivier Levillain
- Institut de Biologie et Chimie des Protéines, FRE 3310, Dysfonctionnements de l'homéostasie tissulaire et ingénierie thérapeutique, (DyHTIT), 7 passage du Vercors, 69367, Lyon, France.
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Hsieh JY, Yang JY, Lin CL, Liu GY, Hung HC. Minimal antizyme peptide fully functioning in the binding and inhibition of ornithine decarboxylase and antizyme inhibitor. PLoS One 2011; 6:e24366. [PMID: 21931692 PMCID: PMC3170320 DOI: 10.1371/journal.pone.0024366] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2011] [Accepted: 08/08/2011] [Indexed: 01/10/2023] Open
Abstract
Antizyme (AZ) is a protein with 228 amino acid residues that regulates ornithine decarboxylase (ODC) by binding to ODC and dissociating its homodimer, thus inhibiting its enzyme activity. Antizyme inhibitor (AZI) is homologous to ODC, but has a higher affinity than ODC for AZ. In this study, we quantified the biomolecular interactions between AZ and ODC as well as AZ and AZI to identify functional AZ peptides that could bind to ODC and AZI and inhibit their function as efficiently as the full-length AZ protein. For these AZ peptides, the inhibitory ability of AZ_95-228 was similar to that of AZ_WT. Furthermore, AZ_95-176 displayed an inhibition (IC50: 0.20 µM) similar to that of AZ-95-228 (IC50: 0.16 µM), even though a large segment spanning residues 177–228 was deleted. However, further deletion of AZ_95-176 from either the N-terminus or the C-terminus decreased its ability to inhibit ODC. The AZ_100-176 and AZ_95-169 peptides displayed a noteworthy decrease in ability to inhibit ODC, with IC50 values of 0.43 and 0.37 µM, respectively. The AZ_95-228, AZ_100-228 and AZ_95-176 peptides had IC50 values comparable to that of AZ_WT and formed AZ-ODC complexes with Kd,AZ-ODC values of 1.5, 5.3 and 5.6 µM, respectively. Importantly, our data also indicate that AZI can rescue AZ peptide-inhibited ODC enzyme activity and that it can bind to AZ peptides with a higher affinity than ODC. Together, these data suggest that these truncated AZ proteins retain their AZI-binding ability. Thus, we suggest that AZ_95-176 is the minimal AZ peptide that is fully functioning in the binding of ODC and AZI and inhibition of their function.
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Affiliation(s)
- Ju-Yi Hsieh
- Department of Life Sciences and Institute of Bioinformatics, National Chung Hsing University, Taichung, Taiwan
| | - Jung-Yen Yang
- National Nano Device Laboratories and Department of Electrical Engineering, National Chiao Tung University, Hsinchu, Taiwan
| | - Chih-Li Lin
- Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan
| | - Guang-Yaw Liu
- Institute of Microbiology & Immunology, Chung Shan Medical University, and Division of Allergy, Immunology, and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan
- * E-mail: (H-CH); (G-YL)
| | - Hui-Chih Hung
- Department of Life Sciences and Institute of Bioinformatics, National Chung Hsing University, Taichung, Taiwan
- Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan
- * E-mail: (H-CH); (G-YL)
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Olsen RR, Zetter BR. Evidence of a role for antizyme and antizyme inhibitor as regulators of human cancer. Mol Cancer Res 2011; 9:1285-93. [PMID: 21849468 DOI: 10.1158/1541-7786.mcr-11-0178] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
Antizyme and its endogenous antizyme inhibitor have recently emerged as prominent regulators of cell growth, transformation, centrosome duplication, and tumorigenesis. Antizyme was originally isolated as a negative modulator of the enzyme ornithine decarboxylase (ODC), an essential component of the polyamine biosynthetic pathway. Antizyme binds ODC and facilitates proteasomal ODC degradation. Antizyme also facilitates degradation of a set of cell cycle regulatory proteins, including cyclin D1, Smad1, and Aurora A kinase, as well as Mps1, a protein that regulates centrosome duplication. Antizyme has been reported to function as a tumor suppressor and to negatively regulate tumor cell proliferation and transformation. Antizyme inhibitor binds to antizyme and suppresses its known functions, leading to increased polyamine synthesis, increased cell proliferation, and increased transformation and tumorigenesis. Gene array studies show antizyme inhibitor to be amplified in cancers of the ovary, breast, and prostate. In this review, we summarize the current literature on the role of antizyme and antizyme inhibitor in cancer, discuss how the ratio of antizyme to antizyme inhibitor can influence tumor growth, and suggest strategies to target this axis for tumor prevention and treatment.
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Affiliation(s)
- Rachelle R Olsen
- Vascular Biology Program, Department of Surgery, Children's Hospital Boston, MA, USA
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Fraser AV, Goodwin AC, Hacker-Prietz A, Sugar E, Woster PM, Casero RA. Knockdown of ornithine decarboxylase antizyme 1 causes loss of uptake regulation leading to increased N1, N11-bis(ethyl)norspermine (BENSpm) accumulation and toxicity in NCI H157 lung cancer cells. Amino Acids 2011; 42:529-38. [PMID: 21814790 DOI: 10.1007/s00726-011-1030-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2011] [Accepted: 03/26/2011] [Indexed: 10/17/2022]
Abstract
Ornithine decarboxylase antizyme 1 (AZ1) is a major regulatory protein responsible for the regulation and degradation of ornithine decarboxylase (ODC). To better understand the role of AZ1 in polyamine metabolism and in modulating the response to anticancer polyamine analogues, a small interfering RNA strategy was used to create a series of stable clones in human H157 non-small cell lung cancer cells that expressed less than 5-10% of basal AZ1 levels. Antizyme 1 knockdown clones accumulated greater amounts of the polyamine analogue N (1),N (11)-bis(ethyl)norspermine (BENSpm) and were more sensitive to analogue treatment. The possibility of a loss of polyamine uptake regulation in the knockdown clones was confirmed by polyamine uptake analysis. These results are consistent with the hypothesis that AZ1 knockdown leads to dysregulation of polyamine uptake, resulting in increased analogue accumulation and toxicity. Importantly, there appears to be little difference between AZ1 knockdown cells and cells with normal levels of AZ1 with respect to ODC regulation, suggesting that another regulatory protein, potentially AZ2, compensates for the loss of AZ1. The results of these studies are important for the understanding of both the regulation of polyamine homeostasis and in understanding the factors that regulate tumor cell sensitivity to the anti-tumor polyamine analogues.
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Affiliation(s)
- Alison V Fraser
- Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Bunting-Blaustein Cancer Research Building 1, 1650 Orleans Street, Baltimore, MD 21231, USA
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Ramos-Molina B, López-Contreras AJ, Cremades A, Peñafiel R. Differential expression of ornithine decarboxylase antizyme inhibitors and antizymes in rodent tissues and human cell lines. Amino Acids 2011; 42:539-47. [PMID: 21814789 DOI: 10.1007/s00726-011-1031-y] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2011] [Accepted: 05/30/2011] [Indexed: 11/30/2022]
Abstract
Ornithine decarboxylase antizyme inhibitors, AZIN1 and AZIN2, are regulators and homologous proteins of ornithine decarboxylase (ODC), the rate limiting enzyme in the biosynthesis of polyamines. In this study, we have examined by means of real-time RT-PCR the relative abundance of mRNA of the three ODC paralogs in different rodent tissues, as well as in several cell lines derived from human tumors. With the exception of mouse and rat testes, ODC mRNA was the most expressed gene in all tissues examined (values higher than 60%). AZIN2 was more expressed than AZIN1 in testis, epididymis, brain, adrenal gland and lung, whereas the opposite was found in liver, kidney, heart, intestine and pancreas, as well as in all the cell lines examined. mRNA abundance of the three antizymes (AZ1, AZ2 and AZ3) that interact with ODC and antizyme inhibitors was also analyzed. AZ1 and AZ2 mRNA were ubiquitously expressed, AZ1 mRNA being more abundant than that of AZ2, although the ratio was dependent on the mouse tissue. In carcinoma-derived cells AZ1 was more expressed than AZ2, whereas in neuroblastoma-derived cells AZ2 mRNA was much more abundant than that of AZ1. AZ3 was expressed exclusively in rodent testes, where it was the most abundant of the three antizymes (~80%). This study is the first comparative-quantitative analysis on the expression of antizymes and antizyme inhibitors in different types of mammalian cells.
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Affiliation(s)
- Bruno Ramos-Molina
- Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Campus de Espinardo, 30100, Murcia, Spain
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Murai N, Murakami Y, Matsufuji S. Protocols for studying antizyme expression and function. Methods Mol Biol 2011; 720:237-67. [PMID: 21318878 DOI: 10.1007/978-1-61779-034-8_15] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/12/2023]
Abstract
Antizyme (AZ) is a key molecule in feedback regulation of cellular polyamines. It is induced by polyamines through stimulation of ribosomal frameshifting during its translation. In mammals, AZ is diverged into three paralogs, AZ1-3. Tissue and subcellular distribution are different among the paralogs, as determined by immunochemical methods or expression of fluorescent-tagged proteins. Only AZ2 is known to be phosphorylated. AZ regulates cellular polyamine levels through multiple mechanisms. It binds to ornithine decarboxylase (ODC) to form an inactive complex and to trigger degradation of ODC by 26S proteasomes. The AZ activity to promote ODC degradation can be measured both in vitro and in cells. AZ also inhibits cellular uptake of polyamines. This chapter comprises seven subchapters describing methods for studying expression and function of AZ.
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Affiliation(s)
- Noriyuki Murai
- Department of Molecular Biology, The Jikei University School of Medicine, Minato-ku, Tokyo, Japan
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Chattopadhyay MK, Fernandez C, Sharma D, McPhie P, Masison DC. Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex. Biochem Biophys Res Commun 2011; 406:177-82. [PMID: 21295540 DOI: 10.1016/j.bbrc.2011.01.113] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2011] [Accepted: 01/31/2011] [Indexed: 11/26/2022]
Abstract
Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODC:AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODC:AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% β-sheet, 19% α-helix, and 24% coils. Surface plasmon resonance analyses show that the association constant (K(A)) between yeast AZ and yeast ODC is 6×10(7) (M(-1)). Using purified His-tagged AZ as a binding partner, we have purified the ODC:AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC:AZ complex shows a change in the secondary structure in the complex.
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Affiliation(s)
- Manas K Chattopadhyay
- Laboratory of Biochemistry and Genetics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, 8 Center Drive, Bldg. 8, Bethesda, MD 20892, USA.
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Kahana C. Identification, assay, and functional analysis of the antizyme inhibitor family. Methods Mol Biol 2011; 720:269-78. [PMID: 21318879 DOI: 10.1007/978-1-61779-034-8_16] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Polyamines are small aliphatic polycations present in all living cells. Polyamines are involved in regulating fundamental cellular functions and are absolutely essential for the process of cellular proliferation. Because they fulfill essential cellular functions, their intracellular concentration is tightly regulated via a unique autoregulatory circuit that responds to the intracellular concentration of polyamines. In the heart of this circuit is a small protein called antizyme (Az), whose synthesis is stimulated by polyamines. Az inactivates Ornithine decarboxylase [(ODC), the first key enzyme in the polyamine biosynthetic pathway] and marks it for ubiquitin-independent degradation by the 26S proteasome. In addition, Az inhibits uptake of polyamines via a yet unresolved mechanism. Az itself is subjected to regulation by an ODC-related protein termed antizyme inhibitor (AzI). AzI is highly homologous to ODC, but it lacks ornithine decarboxylating activity. Since its affinity to Az is greater than the affinity Az has for ODC, it rescues ODC from degradation and enables polyamines uptake into the cell.
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Affiliation(s)
- Chaim Kahana
- Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel.
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Yamamoto D, Shima K, Matsuo K, Nishioka T, Chen CY, Hu GF, Sasaki A, Tsuji T. Ornithine decarboxylase antizyme induces hypomethylation of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2) in human oral cancer cell line. PLoS One 2010; 5:e12554. [PMID: 20838441 PMCID: PMC2933235 DOI: 10.1371/journal.pone.0012554] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2010] [Accepted: 07/31/2010] [Indexed: 12/31/2022] Open
Abstract
BACKGROUND Methylation of CpG islands of genome DNA and lysine residues of histone H3 and H4 tails regulates gene transcription. Inhibition of polyamine synthesis by ornithine decarboxylase antizyme-1 (OAZ) in human oral cancer cell line resulted in accumulation of decarboxylated S-adenosylmethionine (dcSAM), which acts as a competitive inhibitor of methylation reactions. We anticipated that accumulation of dcSAM impaired methylation reactions and resulted in hypomethylation of genome DNA and histone tails. METHODOLOGY/PRINCIPAL FINDINGS Global methylation state of genome DNA and lysine residues of histone H3 and H4 tails were assayed by Methylation by Isoschizomers (MIAMI) method and western blotting, respectively, in the presence or absence of OAZ expression. Ectopic expression of OAZ mediated hypomethylation of CpG islands of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2). Protein level of DNA methyltransferase 3B (DNMT3B) and histone H3K9me specific methyltransferase G9a were down-regulated in OAZ transfectant. CONCLUSIONS/SIGNIFICANCE OAZ induced hypomethylation of CpG islands of global genome DNA and H3K9me2 by down-regulating DNMT3B and G9a protein level. Hypomethylation of CpG islands of genome DNA and histone H3K9me2 is a potent mechanism of induction of the genes related to tumor suppression and DNA double strand break repair.
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Affiliation(s)
- Daisuke Yamamoto
- Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America
- Department of Oral and Maxillofacial Surgery, Okayama University Graduate School, Okayama, Japan
| | - Kaori Shima
- Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America
| | - Kou Matsuo
- Division of Oral Pathology, Department of Biosciences, Kyushu Dental College, Kitakyushu, Japan
| | - Takashi Nishioka
- Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America
| | - Chang Yan Chen
- Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America
| | - Guo-fu Hu
- Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America
| | - Akira Sasaki
- Department of Oral and Maxillofacial Surgery, Okayama University Graduate School, Okayama, Japan
| | - Takanori Tsuji
- Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America
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Ivanov IP, Firth AE, Atkins JF. Recurrent Emergence of Catalytically Inactive Ornithine Decarboxylase Homologous Forms That Likely Have Regulatory Function. J Mol Evol 2010; 70:289-302. [DOI: 10.1007/s00239-010-9331-5] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2009] [Accepted: 02/17/2010] [Indexed: 10/19/2022]
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Ivanov IP, Matsufuji S. Autoregulatory Frameshifting in Antizyme Gene Expression Governs Polyamine Levels from Yeast to Mammals. RECODING: EXPANSION OF DECODING RULES ENRICHES GENE EXPRESSION 2010. [DOI: 10.1007/978-0-387-89382-2_13] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
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López-Contreras AJ, Ramos-Molina B, Cremades A, Peñafiel R. Antizyme inhibitor 2: molecular, cellular and physiological aspects. Amino Acids 2009; 38:603-11. [PMID: 19956990 DOI: 10.1007/s00726-009-0419-4] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2009] [Accepted: 09/24/2009] [Indexed: 01/20/2023]
Abstract
Polyamines are small organic polycations essential for cell proliferation and survival. Antizymes (AZs) are small proteins regulated by polyamines that inhibit polyamine biosynthesis and uptake in mammalian cells. In addition, antizyme functions are also regulated by antizyme inhibitors, homologue proteins of ornithine decarboxylase lacking enzymatic activity. There are two antizyme inhibitors (AZIN), known as AZIN1 and AZIN2, that bind to AZs and negate their effects on polyamine metabolism. Here, we review different molecular and cellular properties of the novel AZIN2 with particular emphasis on the role that this protein may have in brain and testis physiology. Whereas AZIN1 is ubiquitously found in mammalian tissues, AZIN2 expression appears to be restricted to brain and testis. In transfected cells, AZIN2 is mainly located in the endoplasmic reticulum-Golgi intermediate compartment and in the cis-Golgi network. AZIN2 is a labile protein that is degraded by the proteasome by a ubiquitin-dependent mechanism. Regarding its physiological role, spatial and temporal analyses of AZIN2 expression in the mouse testis suggest that this protein may have a role in spermiogenesis.
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Affiliation(s)
- Andrés J López-Contreras
- Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Campus de Espinardo, 30100 Murcia, Spain
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45
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Regulation of cellular polyamine levels and cellular proliferation by antizyme and antizyme inhibitor. Essays Biochem 2009; 46:47-61. [DOI: 10.1042/bse0460004] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Polyamines are small aliphatic polycations present in all living cells. Polyamines are essential for cellular viability and are involved in regulating fundamental cellular processes, most notably cellular growth and proliferation. Being such central regulators of fundamental cellular functions, the intracellular polyamine concentration is tightly regulated at the levels of synthesis, uptake, excretion and catabolism. ODC (ornithine decarboxylase) is the first key enzyme in the polyamine biosynthesis pathway. ODC is characterized by an extremely rapid intracellular turnover rate, a trait that is central to the regulation of cellular polyamine homoeostasis. The degradation rate of ODC is regulated by its end-products, the polyamines, via a unique autoregulatory circuit. At the centre of this circuit is a small protein called Az (antizyme), whose synthesis is stimulated by polyamines. Az inactivates ODC and targets it to ubiquitin-independent degradation by the 26S proteasome. In addition, Az inhibits uptake of polyamines. Az itself is regulated by another ODC-related protein termed AzI (antizyme inhibitor). AzI is highly homologous with ODC, but it lacks ornithine-decarboxylating activity. Its ability to serve as a regulator is based on its high affinity to Az, which is greater than the affinity Az has to ODC. As a result, it interferes with the binding of Az to ODC, thus rescuing ODC from degradation and permitting uptake of polyamines.
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López-Contreras AJ, Sánchez-Laorden BL, Ramos-Molina B, de la Morena ME, Cremades A, Peñafiel R. Subcellular localization of antizyme inhibitor 2 in mammalian cells: Influence of intrinsic sequences and interaction with antizymes. J Cell Biochem 2009; 107:732-40. [PMID: 19449338 DOI: 10.1002/jcb.22168] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Ornithine decarboxylase (ODC) and the antizyme inhibitors (AZIN1 and AZIN2), regulatory proteins of polyamine levels, are antizyme-binding proteins. Although it is widely recognized that ODC is mainly a cytosolic enzyme, less is known about the subcellular distribution of AZIN1 and AZIN2. We found that these proteins, which share a high degree of homology in their amino acid sequences, presented differences in their subcellular location in transfected mammalian cells. Whereas ODC was mainly present in the cytosol, and AZIN1 was found predominantly in the nucleus, interestingly, AZIN2 was located in the ER-Golgi intermediate compartment (ERGIC) and in the cis-Golgi network, apparently not related to any known cell-sorting sequence. Our results rather suggest that the N-terminal region may be responsible for this particular location, since its deletion abrogated the incorporation of the mutated AZIN2 to the ERGIC complex and, on the other hand, the substitution of this sequence for the corresponding sequence in ODC, translocated ODC from cytosol to the ERGIC compartment. Furthermore, the coexpression of AZIN2 with any members of the antizyme family induced a shift of AZIN2 from the ERGIC to the cytosol. These findings underline the complexity of the AZs/AZINs regulatory system, supporting early evidence that relates these proteins with additional functions other than regulating polyamine homeostasis.
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Kahana C. Antizyme and antizyme inhibitor, a regulatory tango. Cell Mol Life Sci 2009; 66:2479-88. [PMID: 19399584 PMCID: PMC11115672 DOI: 10.1007/s00018-009-0033-3] [Citation(s) in RCA: 83] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2009] [Revised: 03/29/2009] [Accepted: 04/07/2009] [Indexed: 12/14/2022]
Abstract
The polyamines are small basic molecules essential for cellular proliferation and viability. An autoregulatory circuit that responds to the intracellular level of polyamines regulates their production. In the center of this circuit is a family of small proteins termed antizymes. Antizymes are themselves regulated at the translational level by the level of polyamines. Antizymes bind ornithine decarboxylase (ODC) subunits and target them to ubiquitin-independent degradation by the 26S proteasome. In addition, antizymes inhibit polyamine transport across the plasma membrane via an as yet unresolved mechanism. Antizymes may also interact with and target degradation of other growth-regulating proteins. An inactive ODC-related protein termed antizyme inhibitor regulates polyamine metabolism by negating antizyme functions. The ability of antizymes to degrade ODC, inhibit polyamine uptake and consequently suppress cellular proliferation suggests that they act as tumor suppressors, while the ability of antizyme inhibitors to negate antizyme function indicates their growth-promoting and oncogenic potential.
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Affiliation(s)
- Chaim Kahana
- Department of Molecular Genetics, The Weizmann Institute of Science, 76100 Rehovot, Israel.
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Murakami Y, Suzuki JI, Samejima K, Kikuchi K, Hascilowicz T, Murai N, Matsufuji S, Oka T. The change of antizyme inhibitor expression and its possible role during mammalian cell cycle. Exp Cell Res 2009; 315:2301-11. [PMID: 19426728 DOI: 10.1016/j.yexcr.2009.04.024] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2009] [Revised: 04/24/2009] [Accepted: 04/25/2009] [Indexed: 01/05/2023]
Abstract
Antizyme inhibitor (AIn), a homolog of ODC, binds to antizyme and inactivates it. We report here that AIn increased at the G1 phase of the cell cycle, preceding the peak of ODC activity in HTC cells in culture. During interphase AIn was present mainly in the cytoplasm and turned over rapidly with the half-life of 10 to 20 min, while antizyme was localized in the nucleus. The level of AIn increased again at the G2/M phase along with ODC, and the rate of turn-over of AIn in mitotic cells decreased with the half-life of approximately 40 min. AIn was colocalized with antizyme at centrosomes during the period from prophase through late anaphase and at the midzone/midbody during telophase. Thereafter, AIn and antizyme were separated and present at different regions on the midbody at late telophase. AIn disappeared at late cytokinesis, whereas antizyme remained at the cytokinesis remnant. Reduction of AIn by RNA interference caused the increase in the number of binucleated cells in HTC cells in culture. These findings suggested that AIn contributed to a rapid increase in ODC at the G1 phase and also played a role in facilitating cells to complete mitosis during the cell cycle.
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Affiliation(s)
- Yasuko Murakami
- Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishi-Tokyo, Tokyo 202-8585, Japan
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Tang H, Ariki K, Ohkido M, Murakami Y, Matsufuji S, Li Z, Yamamura KI. Role of ornithine decarboxylase antizyme inhibitor in vivo. Genes Cells 2008; 14:79-87. [PMID: 19077035 DOI: 10.1111/j.1365-2443.2008.01249.x] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Ornithine decarboxylase (ODC) antizyme inhibitor (AZI) has been shown to regulate ODC activity in cell cultures. However, its biological functions in an organism remain unknown. An embryonic stem (ES) cell clone was established, in which the Azin1 gene was disrupted by the gene trap technique. To identify the function of Azin1 gene in vivo, a mutant mouse line was generated using these trapped ES cells. Homozygous mutant mice died at P0 with abnormal liver morphology. Further analysis indicated that the deletion of Azin1 in homozygous mice resulted in the degradation of ODC, and reduced the biosynthesis of putrescine and spermidine. Our results thus show that AZI plays an important role in regulating the levels of ODC, putrescine and spermidine in mice, and is essential for the survival of mice.
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Affiliation(s)
- Hua Tang
- Key Laboratory of Molecular Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing 400016, China
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López-Contreras AJ, Ramos-Molina B, Martínez-de-la-Torre M, Peñafiel-Verdú C, Puelles L, Cremades A, Peñafiel R. Expression of antizyme inhibitor 2 in male haploid germinal cells suggests a role in spermiogenesis. Int J Biochem Cell Biol 2008; 41:1070-8. [PMID: 18973822 DOI: 10.1016/j.biocel.2008.09.029] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2008] [Revised: 09/03/2008] [Accepted: 09/30/2008] [Indexed: 02/07/2023]
Abstract
Recently, we have found that the antizyme inhibitor 2, a novel member of the antizyme binding proteins related to polyamine metabolism, was expressed mainly in the adult testes, although its function in testicular physiology is completely unknown. Therefore, in the present work, the spatial and temporal expression of antizyme inhibitor 2, and other genes related to polyamine metabolism were studied in the mouse testis, in an attempt to understand the role of antizyme inhibitor 2 in testicular functions. For that purpose, the temporal expression of different genes, during the first wave of spermatogenesis in postnatal mice, was studied by real-time RT-PCR, and the spatial distribution of transcripts and protein in the adult testis was examined by both RNA in situ hybridization and immunocytochemistry. The results indicated that antizyme inhibitor 2 was specifically expressed in the haploid germinal cells, similarly to antizyme 3, the testis specific antizyme. Conversely, ornithine decarboxylase mRNA was mainly found in the outer part of the seminiferous tubules where spermatogonia and spermatocytes are located. Functional transfection assays and co-immunoprecipitation experiments corroborated that antizyme inhibitor 2 counteracts the negative action of antizyme 3 on polyamine biosynthesis and uptake. All these results indicate that the expression of antizyme inhibitor 2 is postnatally regulated and strongly suggest that antizyme inhibitor 2 may have a role in spermiogenesis.
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Affiliation(s)
- Andrés J López-Contreras
- Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Campus de Espinardo, 30100 Murcia, Spain.
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