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Gao Y, Xu S, Qiao J, Wang C, Wang K, Sun J, Liu L, Li L, Liang M, Hu K. AZIN2 is associated with apoptosis of germ cells in undescended testis. Cells Dev 2024; 179:203925. [PMID: 38797332 DOI: 10.1016/j.cdev.2024.203925] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2023] [Revised: 04/24/2024] [Accepted: 05/03/2024] [Indexed: 05/29/2024]
Abstract
Undescended testis (UDT), known as cryptorchidism (CRY), is a common congenital disorder in which one or both testicles do not descend normally into the scrotum. A unilateral UDT model was established by inducing UDT in mice through surgery. The results showed that the testis in the UDT model group was abnormal; the lumen of the seminiferous tubule was atrophic; apoptosis, necrosis and shedding were observed in many of the germ cells; the level of sex hormones was abnormal; and mature sperm was reduced. Subsequently, transcriptome sequencing was conducted on the testicular tissue of UDT model mice. Through analysis and verification of differential genes, AZIN2 was identified as playing a key role in the decline in male fertility caused by cryptorchidism. AZIN2 expression and spermine content was down-regulated in the testis of the UDT group. We then used a combination of hypoxanthine and xanthine to create a GC-1 cell damage model. In this model, AZIN2 expression and spermine content was down-regulated. When si-Azin2 transfected GC-1 cells, cell viability and proliferation were decreased. However, in the GC-1 cell damage model transfected with Azin2 over-expressed plasmid, AZIN2 expression and spermine content was up-regulated, reversing the cell damage caused by hypoxanthine and xanthine, and restoring the proliferation ability of GC-1 cells. These results indicate that in UDT, down-regulated AZIN2 expression is a factor in testicular damage. This discussion of the connection between AZIN2 and germ cells has important clinical significance as it provides an important reference for the diagnosis and treatment of cryptorchidism.
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Affiliation(s)
- Yuanyuan Gao
- School of Life Science, Bengbu Medical University, Bengbu, Anhui, People's Republic of China
| | - Shumin Xu
- School of Life Science, Bengbu Medical University, Bengbu, Anhui, People's Republic of China
| | - Jiajun Qiao
- School of Life Science, Bengbu Medical University, Bengbu, Anhui, People's Republic of China
| | - Chen Wang
- School of Life Science, Bengbu Medical University, Bengbu, Anhui, People's Republic of China
| | - Kaixian Wang
- School of Life Science, Bengbu Medical University, Bengbu, Anhui, People's Republic of China
| | - Junpei Sun
- First Affiliated Hospital, Bengbu Medical University, Bengbu, Anhui, People's Republic of China
| | - Lei Liu
- First Affiliated Hospital, Bengbu Medical University, Bengbu, Anhui, People's Republic of China
| | - Leina Li
- School of Life Science, Bengbu Medical University, Bengbu, Anhui, People's Republic of China
| | - Meng Liang
- School of Life Science, Bengbu Medical University, Bengbu, Anhui, People's Republic of China.
| | - Ke Hu
- School of Life Science, Bengbu Medical University, Bengbu, Anhui, People's Republic of China.
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Baicalein, 7,8-Dihydroxyflavone and Myricetin as Potent Inhibitors of Human Ornithine Decarboxylase. Nutrients 2020; 12:nu12123867. [PMID: 33348871 PMCID: PMC7765794 DOI: 10.3390/nu12123867] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2020] [Revised: 12/14/2020] [Accepted: 12/15/2020] [Indexed: 11/23/2022] Open
Abstract
Background: Human ornithine decarboxylase (ODC) is a well-known oncogene, and the discovery of ODC enzyme inhibitors is a beneficial strategy for cancer therapy and prevention. Methods: We examined the inhibitory effects of a variety of flavone and flavonol derivatives on ODC enzymatic activity, and performed in silico molecular docking of baicalein, 7,8-dihydroxyflavone and myricetin to the whole dimer of human ODC to investigate the possible binding site of these compounds on ODC. We also examined the cytotoxic effects of these compounds with cell-based studies. Results: Baicalein, 7,8-dihydroxyflavone and myricetin exhibited significant ODC suppression activity with IC50 values of 0.88 µM, 2.54 µM, and 7.3 µM, respectively, which were much lower than that of the active-site irreversible inhibitor α-DL-difluoromethylornithine (IC50, the half maximal inhibitory concentration, of approximately 100 µM). Kinetic studies and molecular docking simulations suggested that baicalein, and 7,8-dihydroxyflavone act as noncompetitive inhibitors that are hydrogen-bonded to the region near the active site pocket in the dimer interface of the enzyme. Baicalein and myricetin suppress cell growth and induce cellular apoptosis, and both of these compounds suppress the ODC-evoked anti-apoptosis of cells. Conclusions: Therefore, we suggest that the flavone or flavonol derivatives baicalein, 7,8-dihydroxyflavone, and myricetin are potent chemopreventive and chemotherapeutic agents that target ODC.
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Lambertos A, Peñafiel R. Polyamine biosynthesis in Xenopus laevis: the xlAZIN2/xlODC2 gene encodes a lysine/ornithine decarboxylase. PLoS One 2019; 14:e0218500. [PMID: 31509528 PMCID: PMC6738921 DOI: 10.1371/journal.pone.0218500] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2019] [Accepted: 08/26/2019] [Indexed: 11/29/2022] Open
Abstract
Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines, organic cations that are implicated in many cellular processes. The enzyme is regulated at the post-translational level by an unusual system that includes antizymes (AZs) and antizyme inhibitors (AZINs). Most studies on this complex regulatory mechanism have been focused on human and rodent cells, showing that AZINs (AZIN1 and AZIN2) are homologues of ODC but devoid of enzymatic activity. Little is known about Xenopus ODC and its paralogues, in spite of the relevance of Xenopus as a model organism for biomedical research. We have used the information existing in different genomic databases to compare the functional properties of the amphibian ODC1, AZIN1 and AZIN2/ODC2, by means of transient transfection experiments of HEK293T cells. Whereas the properties of xlODC1 and xlAZIN1 were similar to those reported for their mammalian orthologues, the former catalyzing the decarboxylation of L-ornithine preferentially to that of L-lysine, xlAZIN2/xlODC2 showed important differences with respect to human and mouse AZIN2. xlAZIN2 did not behave as an antizyme inhibitor, but it rather acts as an authentic decarboxylase forming cadaverine, due to its higher affinity to L-lysine than to L-ornithine as substrate; so, in accordance with this, it should be named as lysine decarboxylase (LDC) or lysine/ornithine decarboxylase (LODC). In addition, AZ1 stimulated the degradation of xlAZIN2 by the proteasome, but the removal of the 21 amino acid C-terminal tail, with a sequence quite different to that of mouse or human ODC, made the protein resistant to degradation. Collectively, our results indicate that in Xenopus there is only one antizyme inhibitor (xlAZIN1) and two decarboxylases, xlODC1 and xlLDC, with clear preferences for L-ornithine and L-lysine, respectively.
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Affiliation(s)
- Ana Lambertos
- Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Murcia, Spain
- Biomedical Research Institute of Murcia (IMIB), Murcia, Spain
| | - Rafael Peñafiel
- Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Murcia, Spain
- Biomedical Research Institute of Murcia (IMIB), Murcia, Spain
- * E-mail:
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New insights of polyamine metabolism in testicular physiology: A role of ornithine decarboxylase antizyme inhibitor 2 (AZIN2) in the modulation of testosterone levels and sperm motility. PLoS One 2018; 13:e0209202. [PMID: 30566531 PMCID: PMC6300296 DOI: 10.1371/journal.pone.0209202] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2018] [Accepted: 12/01/2018] [Indexed: 12/19/2022] Open
Abstract
The specific role of polyamines in the testis physiology is not fully understood. Antizymes (OAZs) and antizyme inhibitors (AZINs) are modulators of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis and polyamine uptake. Although the three known OAZs are expressed in the testis, only OAZ3 is testis specific and has been proven to have an essential role in male fertility. Regarding the two existing AZINs, AZIN2 is the most abundantly expressed member in this gonad. Whereas previous studies suggested that AZIN2 might participate in mouse spermatogenesis, immunohistological analysis of human testicular sections revealed that AZIN2 is also detected in the steroidogenic Leydig cells but not in the germinal epithelium. In the present study, we found a close ontogenic similarity in the mRNA levels of OAZs and AZINs between mice and rats, but an opposite expression pattern of ODC activity. Further analysis of AZIN2 and OAZ3 in the testis of mice with different alterations in spermatogenesis and fertility, induced either genetically or pharmacologically, corroborated that both AZIN2 and OAZ3 are mainly expressed in the haploid germinal cells. Finally, by using transgenic mice with a truncated Azin2 gene fused to the bacterial lacZ gene, we studied the expression of Azin2 in testes, epididymides and spermatozoa. AZIN2 was detected in spermatids and spermatozoa, as well as in Leydig cells, and in epithelial epidydimal cells. Azin2 knock-out male mice were fertile; however, they showed marked decreases in testicular putrescine and plasma and testicular testosterone levels, and a dramatic reduction in the sperm motility. These results suggest an important role for AZIN2 in testicular cells by modulating polyamine concentrations, testosterone synthesis and sperm function. Overall, our data corroborate the relevance of polyamine regulation in testis functions, where both AZIN2 and OAZ3 play fundamental roles.
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Abstract
This paper is in recognition of the 100th birthday of Dr. Herbert Tabor, a true pioneer in the polyamine field for over 70 years, who served as the editor-in-chief of the Journal of Biological Chemistry from 1971 to 2010. We review current knowledge of MYC proteins (c-MYC, MYCN, and MYCL) and focus on ornithine decarboxylase 1 (ODC1), an important bona fide gene target of MYC, which encodes the sentinel, rate-limiting enzyme in polyamine biosynthesis. Although notable advances have been made in designing inhibitors against the "undruggable" MYCs, their downstream targets and pathways are currently the main avenue for therapeutic anticancer interventions. To this end, the MYC-ODC axis presents an attractive target for managing cancers such as neuroblastoma, a pediatric malignancy in which MYCN gene amplification correlates with poor prognosis and high-risk disease. ODC and polyamine levels are often up-regulated and contribute to tumor hyperproliferation, especially of MYC-driven cancers. We therefore had proposed to repurpose α-difluoromethylornithine (DFMO), an FDA-approved, orally available ODC inhibitor, for management of neuroblastoma, and this intervention is now being pursued in several clinical trials. We discuss the regulation of ODC and polyamines, which besides their well-known interactions with DNA and tRNA/rRNA, are involved in regulating RNA transcription and translation, ribosome function, proteasomal degradation, the circadian clock, and immunity, events that are also controlled by MYC proteins.
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Affiliation(s)
- André S Bachmann
- From the Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, Michigan 49503 and
| | - Dirk Geerts
- the Department of Medical Biology, Amsterdam University Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
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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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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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8
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Lambertos A, Ramos-Molina B, Cerezo D, López-Contreras AJ, Peñafiel R. The mouse Gm853 gene encodes a novel enzyme: Leucine decarboxylase. Biochim Biophys Acta Gen Subj 2017; 1862:365-376. [PMID: 29108956 DOI: 10.1016/j.bbagen.2017.11.007] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2017] [Revised: 10/31/2017] [Accepted: 11/02/2017] [Indexed: 12/18/2022]
Abstract
Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines. ODC-antizyme inhibitors (AZINs) are homologous proteins of ODC, devoid of enzymatic activity but acting as regulators of polyamine levels. The last paralogue gene recently incorporated into the ODC/AZINs family is the murine Gm853, which is located in the same chromosome as AZIN2, and whose biochemical function is still unknown. By means of transfection assays of HEK293T cells with a plasmid containing the coding region of Gm853, we show here that unlike ODC, GM853 was a stable protein that was not able to decarboxylate l-ornithine or l-lysine and that did not act as an antizyme inhibitor. However, GM853 showed leucine decarboxylase activity, an enzymatic activity never described in animal cells, and by acting on l-leucine (Km=7.03×10-3M) it produced isopentylamine, an aliphatic monoamine with unknown function. The other physiological branched-chain amino acids, l-valine and l-isoleucine were poor substrates of the enzyme. Gm853 expression was mainly detected in the kidney, and as Odc, it was stimulated by testosterone. The conservation of Gm853 orthologues in different mammalian species, including primates, underlines the possible biological significance of this new enzyme. In this study, we describe for the first time a mammalian enzyme with leucine decarboxylase activity, therefore proposing that the gene Gm853 and its protein product should be named as leucine decarboxylase (Ldc, LDC).
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Affiliation(s)
- Ana Lambertos
- Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Spain; Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain
| | - Bruno Ramos-Molina
- Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Spain
| | - David Cerezo
- Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Spain
| | - Andrés J López-Contreras
- Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Spain
| | - Rafael Peñafiel
- Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Spain; Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain.
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Hussain T, Tan B, Ren W, Rahu N, Dad R, Kalhoro DH, Yin Y. Polyamines: therapeutic perspectives in oxidative stress and inflammatory diseases. Amino Acids 2017; 49:1457-1468. [PMID: 28733904 DOI: 10.1007/s00726-017-2447-9] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2017] [Accepted: 06/01/2017] [Indexed: 11/29/2022]
Abstract
Polyamines are naturally occurring aliphatic compounds, particularly essential elements for biological functions. These compounds play a central role in regulating molecular pathways which are responsible for cellular proliferation, growth, and differentiation. Importantly, excessive polyamine catabolism can lead to a prominent source of oxidative stress which increases inflammatory response and thought to be involved in several diseases including stroke, renal failure, neurological disease, liver disease, and even cancer. Moreover, polyamine supplementation increases life span in model organisms and may encounter oxidative stress via exerting its potential anti-oxidant and anti-inflammatory properties. The revealed literature indicates that an emerging role of polyamine biosynthetic pathway could be a novel target for drug development against inflammatory diseases. In this review, we expand the knowledge on the metabolism of polyamines, and its anti-oxidant and anti-inflammatory activities which might have future implications against inflammatory diseases in humans and animals.
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Affiliation(s)
- Tarique Hussain
- National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, Hunan, People's Republic of China.,University of the Chinese Academy of Sciences, Beijing, 10008, People's Republic of China
| | - Bie Tan
- National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, Hunan, People's Republic of China.
| | - Wenkai Ren
- National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, Hunan, People's Republic of China.,University of the Chinese Academy of Sciences, Beijing, 10008, People's Republic of China
| | - Najma Rahu
- Department of Veterinary Microbiology, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam, 70050, Sindh, Pakistan
| | - Rahim Dad
- Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People's Republic of China
| | - Dildar Hussain Kalhoro
- Department of Veterinary Microbiology, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam, 70050, Sindh, Pakistan
| | - Yulong Yin
- National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, Hunan, People's Republic of China.
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11
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Liu YC, Lee CY, Lin CL, Chen HY, Liu GY, Hung HC. Multifaceted interactions and regulation between antizyme and its interacting proteins cyclin D1, ornithine decarboxylase and antizyme inhibitor. Oncotarget 2016; 6:23917-29. [PMID: 26172301 PMCID: PMC4695161 DOI: 10.18632/oncotarget.4469] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2015] [Accepted: 06/16/2015] [Indexed: 11/25/2022] Open
Abstract
Ornithine decarboxylase (ODC), cyclin D1 (CCND1) and antizyme inhibitor (AZI) promote cell growth. ODC and CCND1 can be degraded through antizyme (AZ)-mediated 26S proteasomal degradation. This paper describes a mechanistic study of the molecular interactions between AZ and its interacting proteins. The dissociation constant (Kd) of the binary AZ-CCND1 complex and the respective binding sites of AZ and CCND1 were determined. Our data indicate that CCND1 has a 4-fold lower binding affinity for AZ than does ODC and an approximately 40-fold lower binding affinity for AZ than does AZI. The Kd values of AZ-CCND1, AZ-ODC and AZ-AZI were 0.81, 0.21 and 0.02 μM, respectively. Furthermore, the Kd values for CCND1 binding to the AZ N-terminal peptide (AZ34–124) and AZ C-terminal peptide (AZ100–228) were 0.92 and 8.97 μM, respectively, indicating that the binding site of CCND1 may reside at the N-terminus of AZ, rather than the C-terminus. Our data also show that the ODC-AZ-CCND1 ternary complex may exist in equilibrium. The Kd values of the [AZ-CCND1]-ODC and [AZ-ODC]-CCND1 complexes were 1.26 and 4.93 μM, respectively. This is the first paper to report the reciprocal regulation of CCND1 and ODC through AZ-dependent 26S proteasomal degradation.
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Affiliation(s)
- Yen-Chin Liu
- Department of Life Sciences, National Chung Hsing University (NCHU), Taichung, Taiwan
| | - Chien-Yun Lee
- Department of Life Sciences, National Chung Hsing University (NCHU), Taichung, Taiwan.,Graduate Institute of Biotechnology, National Chung-Hsing University (NCHU), Taichung, Taiwan.,Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan
| | - Chi-Li Lin
- Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan
| | - Hui-Yi Chen
- Biotechnology Center, National Chung-Hsing University (NCHU), Taichung, Taiwan.,Agricultural Biotechnology Center (ABC), National Chung-Hsing University (NCHU), Taichung, Taiwan
| | - Guang-Yaw Liu
- Institute of Microbiology & Immunology, Chung Shan Medical University, Taichung, Taiwan.,Division of Allergy, Immunology, and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan
| | - Hui-Chih Hung
- Department of Life Sciences, National Chung Hsing University (NCHU), Taichung, Taiwan.,Agricultural Biotechnology Center (ABC), National Chung-Hsing University (NCHU), Taichung, Taiwan.,Institute of Genomics and Bioinformatics, National Chung Hsing University (NCHU), Taichung, Taiwan
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12
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Biosynthesis of polyamines and polyamine-containing molecules. Biochem J 2016; 473:2315-29. [DOI: 10.1042/bcj20160185] [Citation(s) in RCA: 98] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2016] [Accepted: 04/22/2016] [Indexed: 12/16/2022]
Abstract
Polyamines are evolutionarily ancient polycations derived from amino acids and are pervasive in all domains of life. They are essential for cell growth and proliferation in eukaryotes and are essential, important or dispensable for growth in bacteria. Polyamines present a useful scaffold to attach other moieties to, and are often incorporated into specialized metabolism. Life has evolved multiple pathways to synthesize polyamines, and structural variants of polyamines have evolved in bacteria, archaea and eukaryotes. Among the complex biosynthetic diversity, patterns of evolutionary reiteration can be distinguished, revealing evolutionary recycling of particular protein folds and enzyme chassis. The same enzyme activities have evolved from multiple protein folds, suggesting an inevitability of evolution of polyamine biosynthesis. This review discusses the different biosynthetic strategies used in life to produce diamines, triamines, tetra-amines and branched and long-chain polyamines. It also discusses the enzymes that incorporate polyamines into specialized metabolites and attempts to place polyamine biosynthesis in an evolutionary context.
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Greenwood MP, Greenwood M, Paton JFR, Murphy D. Control of Polyamine Biosynthesis by Antizyme Inhibitor 1 Is Important for Transcriptional Regulation of Arginine Vasopressin in the Male Rat Hypothalamus. Endocrinology 2015; 156:2905-17. [PMID: 25961839 PMCID: PMC4511134 DOI: 10.1210/en.2015-1074] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
The polyamines spermidine and spermine are small cations present in all living cells. In the brain, these cations are particularly abundant in the neurons of the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus, which synthesize the neuropeptide hormones arginine vasopressin (AVP) and oxytocin. We recently reported increased mRNA expression of antizyme inhibitor 1 (Azin1), an important regulator of polyamine synthesis, in rat SON and PVN as a consequence of 3 days of dehydration. Here we show that AZIN1 protein is highly expressed in both AVP- and oxytocin-positive magnocellular neurons of the SON and PVN together with antizyme 1 (AZ1), ornithine decarboxylase, and polyamines. Azin1 mRNA expression increased in the SON and PVN as a consequence of dehydration, salt loading, and acute hypertonic stress. In organotypic hypothalamic cultures, addition of the irreversible ornithine decarboxylase inhibitor DL-2-(difluoromethyl)-ornithine hydrochloride significantly increased the abundance of heteronuclear AVP but not heteronuclear oxytocin. To identify the function of Azin1 in vivo, lentiviral vectors that either overexpress or knock down Azin1 were stereotaxically delivered into the SON and/or PVN. Azin1 short hairpin RNA delivery resulted in decreased plasma osmolality and had a significant effect on food intake. The expression of AVP mRNA was also significantly increased in the SON by Azin1 short hairpin RNA. In contrast, Azin1 overexpression in the SON decreased AVP mRNA expression. We have therefore identified AZIN1, and hence by inference, polyamines as novel regulators of the expression of the AVP gene.
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Affiliation(s)
- Michael P Greenwood
- School of Clinical Sciences (M.P.G., M.G., D.M.), University of Bristol, Bristol BS1 3NY, United Kingdom; School of Physiology and Pharmacology (J.F.R.P.), University of Bristol, Bristol BS8 1TD, United Kingdom; and Department of Physiology (D.M.), University of Malaya, Kuala Lumpur, Malaysia 50603
| | - Mingkwan Greenwood
- School of Clinical Sciences (M.P.G., M.G., D.M.), University of Bristol, Bristol BS1 3NY, United Kingdom; School of Physiology and Pharmacology (J.F.R.P.), University of Bristol, Bristol BS8 1TD, United Kingdom; and Department of Physiology (D.M.), University of Malaya, Kuala Lumpur, Malaysia 50603
| | - Julian F R Paton
- School of Clinical Sciences (M.P.G., M.G., D.M.), University of Bristol, Bristol BS1 3NY, United Kingdom; School of Physiology and Pharmacology (J.F.R.P.), University of Bristol, Bristol BS8 1TD, United Kingdom; and Department of Physiology (D.M.), University of Malaya, Kuala Lumpur, Malaysia 50603
| | - David Murphy
- School of Clinical Sciences (M.P.G., M.G., D.M.), University of Bristol, Bristol BS1 3NY, United Kingdom; School of Physiology and Pharmacology (J.F.R.P.), University of Bristol, Bristol BS8 1TD, United Kingdom; and Department of Physiology (D.M.), University of Malaya, Kuala Lumpur, Malaysia 50603
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14
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Stegehake D, Kurosinski MA, Schürmann S, Daniel J, Lüersen K, Liebau E. Polyamine-independent Expression of Caenorhabditis elegans Antizyme. J Biol Chem 2015; 290:18090-18101. [PMID: 26032421 DOI: 10.1074/jbc.m115.644385] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2015] [Indexed: 11/06/2022] Open
Abstract
Degradation of ornithine decarboxylase, the rate-limiting enzyme of polyamine biosynthesis, is promoted by the protein antizyme. Expression of antizyme is positively regulated by rising polyamine concentrations that induce a +1 translational frameshift required for production of the full-length protein. Antizyme itself is negatively regulated by the antizyme inhibitor. In our study, the regulation of Caenorhabditis elegans antizyme was investigated, and the antizyme inhibitor was identified. By applying a novel GFP-based method to monitor antizyme frameshifting in vivo, we show that the induction of translational frameshifting also occurs under stressful conditions. Interestingly, during starvation, the initiation of frameshifting was independent of polyamine concentrations. Because frameshifting was also prevalent in a polyamine auxotroph double mutant, a polyamine-independent regulation of antizyme frameshifting is suggested. Polyamine-independent induction of antizyme expression was found to be negatively regulated by the peptide transporter PEPT-1, as well as the target of rapamycin, but not by the daf-2 insulin signaling pathway. Stress-dependent expression of C. elegans antizyme occurred morely slowly than expression in response to increased polyamine levels, pointing to a more general reaction to unfavorable conditions and a diversion away from proliferation and reproduction toward conservation of energy. Interestingly, antizyme expression was found to drastically increase in aging individuals in a postreproductive manner. Although knockdown of antizyme did not affect the lifespan of C. elegans, knockdown of the antizyme inhibitor led to a significant reduction in lifespan. This is most likely caused by an increase in antizyme-mediated degradation of ornithine decarboxylase-1 and a resulting reduction in cellular polyamine levels.
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Affiliation(s)
- Dirk Stegehake
- Department of Molecular Physiology, Institute for Animal Physiology, University of Muenster, 48143 Muenster, Germany
| | - Marc-André Kurosinski
- Department of Molecular Physiology, Institute for Animal Physiology, University of Muenster, 48143 Muenster, Germany
| | - Sabine Schürmann
- Department of Molecular Physiology, Institute for Animal Physiology, University of Muenster, 48143 Muenster, Germany
| | - Jens Daniel
- Department of Molecular Physiology, Institute for Animal Physiology, University of Muenster, 48143 Muenster, Germany
| | - Kai Lüersen
- Department of Molecular Physiology, Institute for Animal Physiology, University of Muenster, 48143 Muenster, Germany
| | - Eva Liebau
- Department of Molecular Physiology, Institute for Animal Physiology, University of Muenster, 48143 Muenster, Germany.
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15
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Ma R, Jiang D, Kang B, Bai L, He H, Chen Z, Yi Z. Molecular cloning and mRNA expression analysis of antizyme inhibitor 1 in the ovarian follicles of the Sichuan white goose. Gene 2015; 568:55-60. [PMID: 25959024 DOI: 10.1016/j.gene.2015.05.014] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2014] [Revised: 04/03/2015] [Accepted: 05/06/2015] [Indexed: 11/18/2022]
Abstract
Antizyme inhibitor 1 (Azin1) plays critical roles in various cellular pathways, including ornithine decarboxylase regulation, polyamine anabolism and uptake and cell proliferation. However, the molecular characteristics of the AZIN1 gene and its expression profile in goose tissues and ovarian follicles have not been reported. In this study, the AZIN1 cDNA of the Sichuan white goose (Anser cygnoides) was cloned, and analyzed for its phylogenetic and physiochemical properties. The expression profile of AZIN1 mRNA in geese tissues and ovarian follicles were examined using quantitative real-time PCR. The results showed that the open reading frame of the AZIN1 cDNA is 1,353 bp in length, encoding a 450 amino acid protein with a molecular weight of 50 kDa. Out of all tissues examined, AZIN1 expression was highest in the adrenal gland and lowest in breast muscle. There was also a high expression of AZIN1 in the cerebellum and isthmus of oviduct. With follicular development, AZIN1 gene expression gradually increased, and its expression in F1 was significantly higher than in F5 (P<0.05). AZIN1 expression was also significantly higher in the POF1 than in the other follicles (P<0.05), and there was a low mRNA expression of AZIN1 in atretic follicles. The results of AZIN1 expression profiling in ovarian follicles suggest that AZIN1 may play an important role in the progression of follicular development, potentially through regulating polyamine levels.
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Affiliation(s)
- Rong Ma
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China; Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China
| | - Dongmei Jiang
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China; Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China
| | - Bo Kang
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China; Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China.
| | - Lin Bai
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China; Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China
| | - Hui He
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China; Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China
| | - Ziyu Chen
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China; Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China
| | - Zhixin Yi
- College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China; Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China
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16
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He H, Kang B, Jiang D, Ma R, Bai L. Molecular cloning and mRNA expression analysis of ornithine decarboxylase antizyme 2 in ovarian follicles of the Sichuan white goose (Anser cygnoides). Gene 2014; 545:247-52. [DOI: 10.1016/j.gene.2014.05.022] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2014] [Accepted: 05/10/2014] [Indexed: 11/26/2022]
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17
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Agostinelli E. Polyamines and transglutaminases: biological, clinical, and biotechnological perspectives. Amino Acids 2014; 46:475-85. [PMID: 24553826 DOI: 10.1007/s00726-014-1688-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2014] [Accepted: 01/27/2014] [Indexed: 01/08/2023]
Affiliation(s)
- Enzo Agostinelli
- Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, SAPIENZA University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy,
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18
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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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19
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Eight new mtDNA sequences of glass sponges reveal an extensive usage of +1 frameshifting in mitochondrial translation. Gene 2013; 535:336-44. [PMID: 24177232 DOI: 10.1016/j.gene.2013.10.041] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2013] [Revised: 10/17/2013] [Accepted: 10/21/2013] [Indexed: 11/22/2022]
Abstract
Three previously studied mitochondrial genomes of glass sponges (phylum Porifera, class Hexactinellida) contained single nucleotide insertions in protein coding genes inferred as sites of +1 translational frameshifting. To investigate the distribution and evolution of these sites and to help elucidate the mechanism of frameshifting, we determined eight new complete or nearly complete mtDNA sequences from glass sponges and examined individual mitochondrial genes from three others. We found nine new instances of single nucleotide insertions in these sequences and analyzed them both comparatively and phylogenetically. The base insertions appear to have been gained and lost repeatedly in hexactinellid mt protein genes, suggesting no functional significance for the frameshifting sites. A high degree of sequence conservation, the presence of unusual tRNAs, and a distinct pattern of codon usage suggest the "out-of-frame pairing" model of translational frameshifting. Additionally, we provide evidence that relaxed selection pressure on glass sponge mtDNA - possibly a result of their low growth rates and deep-water lifestyle - has allowed frameshift insertions to be tolerated for hundreds of millions of years. Our study provides the first example of a phylogenetically diverse and extensive usage of translational frameshifting in animal mitochondrial coding sequences.
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20
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Lange I, Geerts D, Feith DJ, Mocz G, Koster J, Bachmann AS. Novel interaction of ornithine decarboxylase with sepiapterin reductase regulates neuroblastoma cell proliferation. J Mol Biol 2013; 426:332-46. [PMID: 24096079 DOI: 10.1016/j.jmb.2013.09.037] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2013] [Revised: 09/21/2013] [Accepted: 09/25/2013] [Indexed: 01/24/2023]
Abstract
Ornithine decarboxylase (ODC) is the sentinel enzyme in polyamine biosynthesis. Both ODC and polyamines regulate cell division, proliferation, and apoptosis. Sepiapterin reductase (SPR) catalyzes the last step in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase, and has been implicated in neurological diseases but not yet in cancer. In this study, we present compelling evidence that native ODC and SPR physically interact, and we defined the individual amino acid residues involved in both enzymes using in silico protein-protein docking simulations. The resulting heterocomplex is a surprisingly compact structure, featuring two energetically and structurally equivalent binding modes both in monomer and in dimer conformations. The novel interaction between ODC and SPR proteins was confirmed under physiological conditions by co-immunoprecipitation and co-localization in neuroblastoma (NB) cells. Importantly, we showed that siRNA (small interfering RNA)-mediated knockdown of SPR expression significantly reduced endogenous ODC enzyme activity in NB cells, thus demonstrating the biological relevance of the ODC-SPR interaction. Finally, in a cohort of 88 human NB tumors, we found that high SPR mRNA expression correlated significantly with poor survival prognosis using a Kaplan-Meier analysis (log-rank test, P=5 × 10(-4)), suggesting an oncogenic role for SPR in NB tumorigenesis. In conclusion, we showed that ODC binds SPR and thus propose a new concept in which two well-characterized biochemical pathways converge via the interaction of two enzymes. We identified SPR as a novel regulator of ODC enzyme activity and, based on clinical evidence, present a model in which SPR drives ODC-mediated malignant progression in NB.
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Affiliation(s)
- Ingo Lange
- Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720, USA
| | - Dirk Geerts
- Department of Pediatric Oncology/Hematology, Sophia Children's Hospital, Erasmus University Medical Center, 3015 GE Rotterdam, The Netherlands
| | - David J Feith
- Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
| | - Gabor Mocz
- Pacific Biosciences Research Center, University of Hawaii at Manoa, Honolulu, HI 96822, USA
| | - Jan Koster
- Department of Oncogenomics, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
| | - André S Bachmann
- Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720, USA; Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA.
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21
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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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22
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López-Garcia C, Ramos-Molina B, Lambertos A, López-Contreras AJ, Cremades A, Peñafiel R. Antizyme inhibitor 2 hypomorphic mice. New patterns of expression in pancreas and adrenal glands suggest a role in secretory processes. PLoS One 2013; 8:e69188. [PMID: 23874910 PMCID: PMC3709932 DOI: 10.1371/journal.pone.0069188] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2012] [Accepted: 06/07/2013] [Indexed: 01/21/2023] Open
Abstract
The intracellular levels of polyamines, polycations implicated in proliferation, differentiation and cell survival, are regulated by controlling their biosynthesis, catabolism and transport. Antizymes and antizyme inhibitors are key regulatory proteins of polyamine levels by affecting ornithine decarboxylase, the rate-limiting biosynthetic enzyme, and polyamine uptake. We recently described the molecular function of a novel antizyme inhibitor (AZIN2). However, the physiological function of AZIN2 in mammals is mostly unknown. To gain insight on the tissue expression profile of AZIN2 and to find its possible physiological role, we have generated, transgenic mice with severe Azin2 hypomorphism. This mouse model expresses transgenic bacterial β-D-galactosidase as a reporter gene, under the control of the Azin2 endogenous promoter, what allows a very sensitive and specific detection of the expression of the gene in the different tissues of transgenic mice. The biochemical and histochemical analyses of β-D-galactosidase together with the quantification of Azin2 mRNA levels, corroborated that AZIN2 is mainly expressed in testis and brain, and showed for the first time that AZIN2 is also expressed in the adrenal glands and pancreas. In these tissues, AZIN2 was not expressed in all type of cells, but rather in specific type of cells. Thus, AZIN2 was mainly found in the haploid germinal cells of the testis and in different brain regions such as hippocampus and cerebellum, particularly in specific type of neurons. In the adrenal glands and pancreas, the expression was restricted to the adrenal medulla and to the Langerhans islets, respectively. Interestingly, plasma insulin levels were significantly reduced in the transgenic mice. These results support the idea that AZIN2 may have a role in the modulation of reproductory and secretory functions and that this mouse model might be an interesting tool for the progress of our understanding on the role of AZIN2 and polyamines in specific mammalian cells.
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Affiliation(s)
- Carlos López-Garcia
- Department of Biochemistry and Molecular Biology B and Immunology, School of Medicine, University of Murcia, Murcia, Spain
| | - Bruno Ramos-Molina
- Department of Biochemistry and Molecular Biology B and Immunology, School of Medicine, University of Murcia, Murcia, Spain
| | - Ana Lambertos
- Department of Biochemistry and Molecular Biology B and Immunology, School of Medicine, University of Murcia, Murcia, Spain
| | | | - Asunción Cremades
- Department of Pharmacology, School of Medicine, University of Murcia, Murcia, Spain
- Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain
| | - Rafael Peñafiel
- Department of Biochemistry and Molecular Biology B and Immunology, School of Medicine, University of Murcia, Murcia, Spain
- Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain
- * E-mail:
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23
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Peng L, Guo J, Zhang Z, Liu L, Cao Y, Shi H, Wang J, Wang J, Friedman SL, Sninsky JJ. A candidate gene study for the association of host single nucleotide polymorphisms with liver cirrhosis risk in chinese hepatitis B patients. Genet Test Mol Biomarkers 2013; 17:681-6. [PMID: 23844940 DOI: 10.1089/gtmb.2013.0058] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
BACKGROUND AND AIMS Recently, genetic association studies have linked a number of single nucleotide polymorphisms (SNPs) with liver fibrosis risk of hepatitis C. The present study was designed to validate the association of emerging SNPs with development of liver cirrhosis and chronicity in a Chinese population infected with hepatitis B virus (HBV). METHODS 714 Chinese subjects with persistent HBV infection (429 with evident liver cirrhosis and 285 without cirrhosis clinically or pathologically) and 280 subjects with spontaneous HBV clearance were studied. Six SNPs in five candidate genes were detected with the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method. The distribution of each polymorphism was compared between the age-matched cirrhotic and noncirrhotic subjects, and between subjects with persistent infection and spontaneous HBV clearance. RESULTS The rs2679757 polymorphism of antizyme inhibitor 1 (AZIN1) gene was associated with the risk of cirrhosis (odds ratio [OR] for GG+AG versus AA=1.47, 95% confidence interval [CI]=1.08-2.01, p=0.01). So was rs886277 in the transient receptor potential cation channel subfamily M, member 5 (TRPM5) gene (OR for CC versus CT+TT=1.63, 95% CI=1.20-2.22, p=0.002). The frequencies of these two SNPs were also associated with the severity of decompensated cirrhosis based on the Child-Pugh classification. Genotype frequencies of other SNPs were not different between the cirrhotic and noncirrhotic groups. No SNPs were associated with the outcome of spontaneous HBV clearance. CONCLUSIONS AZIN1 rs2679757 and TRPM5 rs886277 are associated with the risk of HBV-related liver cirrhosis in Chinese. The emerging SNPs warrant further clinical validation in other cohorts or ethnic groups, and could lead to mechanistic studies to reveal their contributions to fibrosis progression.
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Affiliation(s)
- Lijun Peng
- Division of Digestive Diseases, Department of Internal Medicine, Zhong Shan Hospital, Shanghai Medical College, Fu Dan University, Shanghai, China
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24
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Kim DH, Barrett MP. Metabolite-dependent regulation of gene expression in Trypanosoma brucei. Mol Microbiol 2013; 88:841-5. [PMID: 23668674 DOI: 10.1111/mmi.12243] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/16/2013] [Indexed: 12/21/2022]
Abstract
Mechanisms regulating gene expression in trypanosomatid protozoa differ significantly from those in other eukaryotes. Transcription of the genome appears to be more or less constitutive with the polyadenylation and trans-splicing of large polycistronic RNAs producing monocistronic RNAs whose translation may then depend upon information within their 3' untranslated regions (3'UTRs). Various 3'UTR sequences involved in life-cycle stage-dependent differential gene expression have been described. Moreover, several RNA-binding proteins have been implicated in regulating expression of these transcripts through altering either their stability or their ability to interact with ribosomes. In this issue of Molecular Microbiology Xiao et al. report on a regulatory element within the 3'UTR of the transcript that encodes the polyamine pathway regulatory protein called prozyme. It appears that the RNA element controls translation of the prozyme RNA causing expression to be upregulated when levels of decarboxylated S-adenosylmethionine (dcAdoMet) are depleted. Since prozyme activates the enzyme S-adenosylmethionine decarboxylase (AdoMetDC), which is responsible for the production of dcAdoMet, losing this metabolite leads to upregulation of prozyme, activation of AdoMetDC and restoration of optimal levels of dcAdomet. The system thus represents a novel metabolite-sensing regulatory circuit that maintains polyamine homeostasis in these cells.
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25
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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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26
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Gamble LD, Hogarty MD, Liu X, Ziegler DS, Marshall G, Norris MD, Haber M. Polyamine pathway inhibition as a novel therapeutic approach to treating neuroblastoma. Front Oncol 2012. [PMID: 23181218 PMCID: PMC3499881 DOI: 10.3389/fonc.2012.00162] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Polyamines are highly regulated essential cations that are elevated in rapidly proliferating tissues, including diverse cancers. Expression analyses in neuroblastomas suggest that up-regulation of polyamine pro-synthetic enzymes and down-regulation of catabolic enzymes is associated with poor prognosis. Polyamine sufficiency may be required for MYCN oncogenicity in MYCN amplified neuroblastoma, and targeting polyamine homeostasis may therefore provide an attractive therapeutic approach. ODC1, an oncogenic MYCN target, is rate-limiting for polyamine synthesis, and is overexpressed in many cancers including neuroblastoma. Inhibition of ODC1 by difluoromethylornithine (DFMO) decreased tumor penetrance in TH-MYCN mice treated pre-emptively, and extended survival and synergized with chemotherapy in treating established tumors in both TH-MYCN and xenograft models. Efforts to augment DFMO activity, or otherwise maximally reduce polyamine levels, are focused on antagonizing polyamine uptake or augmenting polyamine export or catabolism. Since polyamine inhibition appears to be clinically well tolerated, these approaches, particularly when combined with chemotherapy, have great potential for improving neuroblastoma outcome in both MYCN amplified and non-MYCN amplified neuroblastomas.
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Affiliation(s)
- Laura D Gamble
- Children's Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre Sydney, NSW, Australia
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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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Neuroblastoma: Ornithine Decarboxylase and Polyamines are Novel Targets for Therapeutic Intervention. PEDIATRIC CANCER 2012. [DOI: 10.1007/978-94-007-2418-1_9] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
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Role of polyamines, their analogs and transglutaminases in biological and clinical perspectives. Amino Acids 2011; 42:397-409. [DOI: 10.1007/s00726-011-1129-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2011] [Accepted: 09/26/2011] [Indexed: 01/07/2023]
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Liu YC, Hsu DH, Huang CL, Liu YL, Liu GY, Hung HC. Determinants of the differential antizyme-binding affinity of ornithine decarboxylase. PLoS One 2011; 6:e26835. [PMID: 22073206 PMCID: PMC3207831 DOI: 10.1371/journal.pone.0026835] [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: 03/16/2011] [Accepted: 10/05/2011] [Indexed: 01/26/2023] Open
Abstract
Ornithine decarboxylase (ODC) is a ubiquitous enzyme that is conserved in all species from bacteria to humans. Mammalian ODC is degraded by the proteasome in a ubiquitin-independent manner by direct binding to the antizyme (AZ). In contrast, Trypanosoma brucei ODC has a low binding affinity toward AZ. In this study, we identified key amino acid residues that govern the differential AZ binding affinity of human and Trypanosoma brucei ODC. Multiple sequence alignments of the ODC putative AZ-binding site highlights several key amino acid residues that are different between the human and Trypanosoma brucei ODC protein sequences, including residue 119, 124,125, 129, 136, 137 and 140 (the numbers is for human ODC). We generated a septuple human ODC mutant protein where these seven bases were mutated to match the Trypanosoma brucei ODC protein sequence. The septuple mutant protein was much less sensitive to AZ inhibition compared to the WT protein, suggesting that these amino acid residues play a role in human ODC-AZ binding. Additional experiments with sextuple mutants suggest that residue 137 plays a direct role in AZ binding, and residues 119 and 140 play secondary roles in AZ binding. The dissociation constants were also calculated to quantify the affinity of the ODC-AZ binding interaction. The Kd value for the wild type ODC protein-AZ heterodimer ([ODC_WT]-AZ) is approximately 0.22 μM, while the Kd value for the septuple mutant-AZ heterodimer ([ODC_7M]-AZ) is approximately 12.4 μM. The greater than 50-fold increase in [ODC_7M]-AZ binding affinity shows that the ODC-7M enzyme has a much lower binding affinity toward AZ. For the mutant proteins ODC_7M(-Q119H) and ODC_7M(-V137D), the Kd was 1.4 and 1.2 μM, respectively. These affinities are 6-fold higher than the WT_ODC Kd, which suggests that residues 119 and 137 play a role in AZ binding.
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Affiliation(s)
- Yen-Chin Liu
- Department of Life Sciences and Institute of Genomics and Bioinformatics, National Chung-Hsing University, Taichung, Taiwan
| | - Den-Hua Hsu
- Department of Life Sciences and Institute of Genomics and Bioinformatics, National Chung-Hsing University, Taichung, Taiwan
| | - Chi-Liang Huang
- Department of Life Sciences and Institute of Genomics and Bioinformatics, National Chung-Hsing University, Taichung, Taiwan
| | - Yi-Liang Liu
- Institute of Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan
- Division of Allergy, Immunology, and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan
| | - Guang-Yaw Liu
- Institute of Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan
- Division of Allergy, Immunology, and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan
- * E-mail: (HCH); (GYL)
| | - Hui-Chih Hung
- Department of Life Sciences and Institute of Genomics and Bioinformatics, National Chung-Hsing University, Taichung, Taiwan
- Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan
- * E-mail: (HCH); (GYL)
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31
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Olsen RR, Chung I, Zetter BR. Knockdown of antizyme inhibitor decreases prostate tumor growth in vivo. Amino Acids 2011; 42:549-58. [PMID: 21909979 DOI: 10.1007/s00726-011-1032-x] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2011] [Accepted: 06/07/2011] [Indexed: 10/17/2022]
Abstract
The endogenous protein antizyme inhibitor (AZI) is a potential oncogene which promotes cell growth by both inhibiting antizyme (AZ) activity and releasing ornithine decarboxylase (ODC) from AZ-mediated degradation. High levels of ODC and polyamines are associated with numerous types of neoplastic transformation, and the genomic region including AZI is frequently amplified in tumors of the ovary and prostate. To determine whether AZI functionally promotes prostate tumor growth, we made PC3M-LN4 (human) and AT6.1 (rat) cancer cell lines stably expressing shRNA to knockdown antizyme inhibitor 1 (AZI). AZI knockdown was confirmed by western blot, quantitative real-time PCR, and immunofluorescence. To examine the ability of these cells to form tumors in vivo, 1 × 10(6) cells were injected subcutaneously into nude mice either with (PC3M-LN4) or without (AT6.1) Matrigel. Tumor growth was measured two times per week by caliper. We found that cells in which AZI levels had been knocked down by shRNA formed significantly smaller tumors in vivo in both human and rat prostate cancer cell lines. These results suggest that not only does AZI promote tumor growth, but also that AZI may be a valid therapeutic target for cancer treatment.
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Affiliation(s)
- Rachelle R Olsen
- Vascular Biology Program, Department of Surgery, Children's Hospital Boston, Boston, MA 02115, USA
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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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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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Liu YC, Liu YL, Su JY, Liu GY, Hung HC. Critical factors governing the difference in antizyme-binding affinities between human ornithine decarboxylase and antizyme inhibitor. PLoS One 2011; 6:e19253. [PMID: 21552531 PMCID: PMC3084279 DOI: 10.1371/journal.pone.0019253] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2010] [Accepted: 03/24/2011] [Indexed: 12/18/2022] Open
Abstract
Both ornithine decarboxylase (ODC) and its regulatory protein, antizyme inhibitor (AZI), can bind with antizyme (AZ), but the latter has a higher AZ-binding affinity. The results of this study clearly identify the critical amino acid residues governing the difference in AZ-binding affinities between human ODC and AZI. Inhibition experiments using a series of ODC mutants suggested that residues 125 and 140 may be the key residues responsible for the differential AZ-binding affinities. The ODC_N125K/M140K double mutant demonstrated a significant inhibition by AZ, and the IC50 value of this mutant was 0.08 µM, three-fold smaller than that of ODC_WT. Furthermore, the activity of the AZ-inhibited ODC_N125K/M140K enzyme was hardly rescued by AZI. The dissociation constant (Kd) of the [ODC_N125K/M140K]-AZ heterodimer was approximately 0.02 µM, which is smaller than that of WT_ODC by approximately 10-fold and is very close to the Kd value of AZI_WT, suggesting that ODC_N125K/M140K has an AZ-binding affinity higher than that of ODC_WT and similar to that of AZI. The efficiency of the AZI_K125N/K140M double mutant in the rescue of AZ-inhibited ODC enzyme activity was less than that of AZI_WT. The Kd value of [AZI_K125N/K140M]-AZ was 0.18 µM, nine-fold larger than that of AZI_WT and close to the Kd value of ODC_WT, suggesting that AZI_K125N/K140M has an AZ-binding affinity lower than that of AZI_WT and similar to that of ODC. These data support the hypothesis that the differences in residues 125 and 140 in ODC and AZI are responsible for the differential AZ-binding affinities.
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Affiliation(s)
- Yen-Chin Liu
- Department of Life Sciences and Institute of Genomics and Bioinformatics, National Chung-Hsing University, Taichung, Taiwan
| | - Yi-Liang Liu
- Department of Life Sciences and Institute of Genomics and Bioinformatics, National Chung-Hsing University, Taichung, Taiwan
- Division of Allergy, Immunology and Rheumatology and Institute of Immunology, Chung-Shan Medical University and Hospital, Taichung, Taiwan
| | - Jia-Yang Su
- Department of Life Sciences and Institute of Genomics and Bioinformatics, National Chung-Hsing University, Taichung, Taiwan
| | - Guang-Yaw Liu
- Division of Allergy, Immunology and Rheumatology and Institute of Immunology, Chung-Shan Medical University and Hospital, Taichung, Taiwan
- * E-mail: (HCH); (GYL)
| | - Hui-Chih Hung
- Department of Life Sciences and Institute of Genomics and Bioinformatics, National Chung-Hsing University, Taichung, Taiwan
- * E-mail: (HCH); (GYL)
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Tassoni A, Bagni N, Ferri M, Franceschetti M, Khomutov A, Marques MP, Fiuza SM, Simonian AR, Serafini-Fracassini D. Helianthus tuberosus and polyamine research: past and recent applications of a classical growth model. PLANT PHYSIOLOGY AND BIOCHEMISTRY : PPB 2010; 48:496-505. [PMID: 20172735 DOI: 10.1016/j.plaphy.2010.01.019] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/01/2009] [Revised: 01/21/2010] [Accepted: 01/27/2010] [Indexed: 05/27/2023]
Abstract
The earliest studies concerning polyamines (PAs) in plants were performed by using in vitro cultured explants of Helianthus tuberosus dormant tuber. This parenchyma tissue was particularly useful due to its susceptibility to several growth substances, including PAs. During tuber dormancy, PA levels are too low to sustain cell division; thus Helianthus represents a natural PA-deficient model. When cultivated in vitro in the presence of auxins, Helianthus tuber dormant parenchyma cells at the G(0) stage start to divide synchronously acquiring meristematic characteristics. The requirement for auxins to induce cell division can be substituted by aliphatic PAs such as putrescine, spermidine or spermine. Cylinders or slices of explanted homogeneous tuber parenchyma were cultured in liquid medium for short-term studies on the cell cycle, or on solid agar medium for long-term experiments. Morphological and physiological modifications of synchronously dividing cells were studied during the different phases of the cell cycle in relation to PAs biosynthesis and oxidation. Long-term experiments led to the identification of the PAs as plant growth regulators, as the sole nitrogen source, as tuber storage substances and as essential factors for morphogenetic processes and cell homeostasis. More recently this system was used to study the effects on plant cell proliferation of platinum- or palladium-derived drugs (cisplatin and platinum or palladium bi-substituted spermine) that are used in human cancer cell lines as antiproliferative and cytotoxic agents. Cisplatin was the most active both in cell proliferation inhibition and on PA metabolism. Similar experiments were performed using three agmatine analogous. Different effects of these compounds were observed on cell proliferation, free PA levels and enzyme activities, leading to a hypothesis of a correlation between their chemical structure and the agmatine metabolism in plants.
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Affiliation(s)
- Annalisa Tassoni
- Department of Experimental Evolutionary Biology, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy.
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Geerts D, Koster J, Albert D, Koomoa DLT, Feith DJ, Pegg AE, Volckmann R, Caron H, Versteeg R, Bachmann AS. The polyamine metabolism genes ornithine decarboxylase and antizyme 2 predict aggressive behavior in neuroblastomas with and without MYCN amplification. Int J Cancer 2010; 126:2012-24. [PMID: 19960435 DOI: 10.1002/ijc.25074] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
High polyamine (PA) levels and ornithine decarboxylase (ODC) overexpression are well-known phenomena in many aggressive cancer types. We analyzed the expression of ODC and ODC-activity regulating genes antizymes 1-3 (OAZ1-3) and antizyme inhibitors 1-2 (AZ-IN1-2) in human neuroblastoma (NB) tumors and correlated these with genetic and clinical features of NB. Since ODC is a known target gene of MYCN, the correlation between ODC and MYCN was of special interest. Data were obtained from Affymetrix micro-array analysis of 88 NB tumor samples. In addition, mRNA expression levels of ODC, OAZ2 and MYCN in a MYCN-inducible NB cell line were determined by quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). ODC mRNA expression in NB tumors was significantly predictive of decreased overall survival probability and correlated with several unfavorable clinical NB characteristics (all p < 0.005). Interestingly, high ODC mRNA expression also showed significant correlation with poor survival prognosis in Kaplan-Meier analyses stratified for patients without MYCN amplification, suggesting an additional role for ODC independent of MYCN. Conversely, high OAZ2 mRNA expression correlated with increased survival and with several favorable clinical NB characteristics (all p < 0.003). In addition, we provide first evidence of a role for MYCN-associated transcription factors MAD2 and MAD7 in ODC regulation. In NB cell cultures, ectopic overexpression of MYCN altered ODC but not OAZ2 mRNA levels. In conclusion, these data suggest that elevated ODC and low OAZ2 mRNA expression levels correlate with several unfavorable genetic and clinical features in NB, offering new insights into PA pathways and PA metabolism-targeting therapy in NB.
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Affiliation(s)
- Dirk Geerts
- Department of Human Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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Dulloo I, Gopalan G, Melino G, Sabapathy K. The antiapoptotic DeltaNp73 is degraded in a c-Jun-dependent manner upon genotoxic stress through the antizyme-mediated pathway. Proc Natl Acad Sci U S A 2010; 107:4902-7. [PMID: 20185758 PMCID: PMC2841924 DOI: 10.1073/pnas.0906782107] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
p73, the structural and functional homologue of p53, exists as two major forms: the transactivation-proficient, proapoptotic TAp73 or the transactivation-deficient, antiapoptotic DNp73. Expectedly, expression of both these major forms has to be coordinated precisely to achieve the desired cellular outcome. Genotoxic insults resulting in cell death lead to the stabilization of TAp73, mainly through posttranslational modifications, and the concomitant degradation of DNp73, through poorly understood mechanisms. We have therefore investigated the possible mechanisms of stress-induced DNp73 degradation and show here that c-Jun, the AP-1 family member activated by stress signals and involved in stabilizing TAp73, promotes DNp73 degradation. Genotoxic stress-mediated DNp73 degradation was found to occur in a c-Jun-dependent manner through a ubiquitin-independent but proteasome-dependent mechanism. Absence or down-regulation of c-Jun expression abrogated the reduction of DNp73 levels upon stress insults, whereas overexpression of c-Jun led to its degradation. c-Jun controlled DNp73 degradation through the nonclassical, polyamine-induced antizyme (Az) pathway by regulating the latter's processing during stress response. Consistently, expression of c-Jun or Az, or addition of polyamines, promoted DNp73 degradation, whereas silencing Az expression or inhibiting Az activity in cells exposed to stress reduced c-Jun-dependent DNp73 degradation. Moreover, Az was able to bind to DNp73. These data together demonstrate the existence of a c-Jun-dependent mechanism regulating the abundance of the antiapoptotic DNp73 in response to genotoxic stress.
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Affiliation(s)
| | - Ganesan Gopalan
- Laboratory of Gene Structure and Expression, Division of Cellular and Molecular Research, Humphrey Oei Institute of Cancer Research, National Cancer Centre Singapore, 11, Hospital Drive, Singapore 169610, Singapore
| | - Gerry Melino
- Laboratory of Apoptosis and Cancer, Medical Research Council Toxicology Unit, University of Leicester, Hodgkin Building, PO Box 138, Lancaster Road, LE1 9HN, Leicester, United Kingdom
- Department of Biology, Instituto Dermopatico dell’Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico, University of Rome Tor Vergata, 00133 Rome, Italy
| | - Kanaga Sabapathy
- Laboratory of Molecular Carcinogenesis and
- Department of Biochemistry, National University of Singapore, 8, Medical Drive, Singapore 117597, Singapore
- Cancer and Stem Cell Biology Program, Duke–National University of Singapore Graduate Medical School, 8, College Road, Singapore 169857, Singapore
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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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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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40
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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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41
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Yonghong L, Chang M, Abar O, Garcia V, Rowland C, Catanese J, Ross D, Broder S, Shiffman M, Cheung R, Wright T, Friedman SL, Sninsky J. Multiple variants in toll-like receptor 4 gene modulate risk of liver fibrosis in Caucasians with chronic hepatitis C infection. J Hepatol 2009; 51:750-7. [PMID: 19586676 PMCID: PMC2883297 DOI: 10.1016/j.jhep.2009.04.027] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/16/2009] [Revised: 04/03/2009] [Accepted: 04/16/2009] [Indexed: 12/24/2022]
Abstract
BACKGROUND/AIMS Seven genomic loci, implicated by single nucleotide polymorphisms (SNPs), have recently been associated with progression to advanced fibrosis (fibrosis risk) in patients with chronic hepatitis C virus. Other variants in these loci have not been examined but may be associated with fibrosis risk independently of or due to linkage disequilibrium with the original polymorphisms. METHODS We carried out dense genotyping and association testing of additional SNPs in each of the 7 regions in Caucasian case control samples. RESULTS We identified several SNPs in the toll-like receptor 4 (TLR4) and syntaxin binding protein 5-like (STXBP5L) loci that were associated with fibrosis risk independently of the original significant SNPs. Haplotypes consisting of these SNPs in TLR4 and STXBP5L were strongly associated with fibrosis risk (global P=3.04 x 10(-5) and 4.49 x 10(-6), respectively). CONCLUSIONS Multiple variants in TLR4 and STXBP5L genes modulate risk of liver fibrosis. These findings are of relevance for understanding the pathogenesis of HCV-induced liver disease in Caucasians and may be extended to other ethnicities as well.
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Affiliation(s)
- Li Yonghong
- Celera Corporation, 1401 Harbor Bay Parkway, Alameda, CA 94502, USA,Corresponding author. Tel.: +1 510 7496283; fax: +1 510 7496200.
| | - Monica Chang
- Celera Corporation, 1401 Harbor Bay Parkway, Alameda, CA 94502, USA
| | - Olivia Abar
- Celera Corporation, 1401 Harbor Bay Parkway, Alameda, CA 94502, USA
| | - Veronica Garcia
- Celera Corporation, 1401 Harbor Bay Parkway, Alameda, CA 94502, USA
| | - Charles Rowland
- Celera Corporation, 1401 Harbor Bay Parkway, Alameda, CA 94502, USA
| | - Joseph Catanese
- Celera Corporation, 1401 Harbor Bay Parkway, Alameda, CA 94502, USA
| | - David Ross
- Celera Corporation, 1401 Harbor Bay Parkway, Alameda, CA 94502, USA
| | - Samuel Broder
- Celera Corporation, 1401 Harbor Bay Parkway, Alameda, CA 94502, USA
| | | | | | | | - Scott L. Friedman
- Mount Sinai School of Medicine, Division of Liver Diseases, New York, NY, USA
| | - John Sninsky
- Celera Corporation, 1401 Harbor Bay Parkway, Alameda, CA 94502, USA
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42
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Su KL, Liao YF, Hung HC, Liu GY. Critical factors determining dimerization of human antizyme inhibitor. J Biol Chem 2009; 284:26768-77. [PMID: 19635796 DOI: 10.1074/jbc.m109.007807] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer K(d), suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (K(d) value = 1.3 microm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a K(d) value of approximately 0.1 microm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained.
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Affiliation(s)
- Kuo-Liang Su
- Department of Life Sciences and Institute of Genomics and Bioinformatics, National Chung-Hsing University, Taichung
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RNA interference-mediated silencing of ornithine decarboxylase and spermidine synthase genes in Trypanosoma brucei provides insight into regulation of polyamine biosynthesis. EUKARYOTIC CELL 2009; 8:747-55. [PMID: 19304951 DOI: 10.1128/ec.00047-09] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Polyamine biosynthesis is a drug target for the treatment of African sleeping sickness; however, mechanisms regulating the pathway in Trypanosoma brucei are not well understood. Recently, we showed that RNA interference (RNAi)-mediated gene silencing or the inhibition of S-adenosylmethionine decarboxylase (AdoMetDC) led to the upregulation of the AdoMetDC activator, prozyme, and ornithine decarboxylase (ODC) proteins. To determine if this regulatory response is specific to AdoMetDC, we studied the effects of the RNAi-induced silencing of the spermidine synthase (SpdSyn) and ODC genes in bloodstream form T. brucei. The knockdown of either gene product led to the depletion of the polyamine and trypanothione pools and to cell death. Decarboxylated AdoMet levels were elevated, while AdoMet was not affected. There was no significant effect on the protein levels of other polyamine pathway enzymes. The treatment of parasites with the ODC inhibitor alpha-difluoromethylornithine gave similar results to those observed for ODC knockdown. Thus, the cellular response to the loss of AdoMetDC activity is distinctive, suggesting that AdoMetDC activity controls the expression levels of the other spermidine biosynthetic enzymes. RNAi-mediated cell death occurred more rapidly for ODC than for SpdSyn. Further, the ODC RNAi cells were rescued by putrescine, but not spermidine, suggesting that the depletion of both putrescine and spermidine is more detrimental than the depletion of spermidine alone. This finding may contribute to the effectiveness of ODC as a target for the treatment of African sleeping sickness, thus providing important insight into the mechanism of action of a key antitrypanosomal agent.
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Lopez-Garcia C, Lopez-Contreras AJ, Cremades A, Castells MT, Peñafiel R. Transcriptomic analysis of polyamine-related genes and polyamine levels in placenta, yolk sac and fetus during the second half of mouse pregnancy. Placenta 2009; 30:241-9. [PMID: 19131104 DOI: 10.1016/j.placenta.2008.12.004] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2008] [Revised: 12/02/2008] [Accepted: 12/05/2008] [Indexed: 11/25/2022]
Abstract
In mammals, polyamines are essential for the maintenance of cell growth. Although early studies reported the highest values of mammalian ornithine decarboxylase (ODC) activity, a key enzyme in polyamine biosynthesis, in rodent placenta, the role of this enzyme in the second half of rodent pregnancy is still controversial. In order to get new insights on polyamine metabolism during this period of pregnancy, we studied polyamine levels, ODC expression and activity and transcript profile of different polyamine-related genes in mouse placenta, fetus and yolk sac. Results indicated that ODC activity and protein levels were higher in placenta than in fetus and yolk sac, especially in the labyrinth, although no correlation between ODC activity and polyamine levels were observed. The half-life of placental ODC ( approximately 190 min) was also higher than the fetal one ( approximately 24 min). Messenger RNAs of all biosynthetic and retroconversion enzymes of polyamine metabolism were present in the three gestational compartments analyzed, as well as those of antizymes 1 and 2 and antizyme inhibitor 1. However, no expression of antizyme 3 and antizyme inhibitor 2 was detected. The catabolic enzyme diamine oxidase was expressed only in the maternal part of placenta but not in the fetal part or in the fetus. The expansion of polyamine pools in the fetus was markedly higher than in placenta, in spite of its lower biosynthetic activity. Our results suggest that the elevated polyamine biosynthetic activity of mouse placenta is required to satisfy the high demand of polyamines required by the growing fetus, during the later period of pregnancy.
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Affiliation(s)
- C Lopez-Garcia
- 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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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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López-Contreras AJ, Ramos-Molina B, Cremades A, Peñafiel R. Antizyme inhibitor 2 (AZIN2/ODCp) stimulates polyamine uptake in mammalian cells. J Biol Chem 2008; 283:20761-9. [PMID: 18508777 PMCID: PMC3258956 DOI: 10.1074/jbc.m801024200] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2008] [Revised: 05/01/2008] [Indexed: 01/08/2023] Open
Abstract
One of the processes that regulate intracellular levels of polyamines in mammalian cells is polyamine uptake. We have measured polyamine uptake in COS7 cells for putrescine, spermidine, and spermine, obtaining K(m) values of 4.5, 1.0, and 0.8 mum, respectively. Treatment of nonconfluent cells with cycloheximide stimulated polyamine uptake and prevented the inhibitory effect found in cells preloaded with polyamines, suggesting the existence of a feedback repression mechanism mediated by antizymes. Transient transfected cells with mutated antizyme forms of AZ1, AZ2, and AZ3, which do not require frameshifting, showed a total blockade of polyamine uptake. Transfection of COS7 cells with mouse or human AZIN2, a novel member of the antizyme inhibitor family, recently characterized by our group, markedly stimulated polyamine uptake and counteracted the action of any of the three antizymes in co-transfected cells. The stimulatory effect of AZIN2 on polyamine uptake was abrogated when the putative antizyme binding sequence, formed by residues 117-140 in AZIN2, was deleted. Real time reverse transcription-PCR analysis of antizyme inhibitor transcripts revealed that in brain and testes AZIN2 is more expressed than AZIN1, especially in the testes where the relative expression was about 25-fold higher. Collectively, our results clearly indicate that AZIN2 affects polyamine homeostasis not only by increasing ornithine decarboxylase activity but also by stimulating polyamine uptake, through negating the inhibitory effect of the antizymes. This finding may have physiological relevance, mostly in testes where AZ3 and AZIN2 are mainly expressed.
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Affiliation(s)
- Andrés J. López-Contreras
- Department of Biochemistry and Molecular
Biology B and Immunology and Department of
Pharmacology, Faculty of Medicine, University of Murcia, 30100 Murcia,
Spain
| | - Bruno Ramos-Molina
- Department of Biochemistry and Molecular
Biology B and Immunology and Department of
Pharmacology, Faculty of Medicine, University of Murcia, 30100 Murcia,
Spain
| | - Asunción Cremades
- Department of Biochemistry and Molecular
Biology B and Immunology and Department of
Pharmacology, Faculty of Medicine, University of Murcia, 30100 Murcia,
Spain
| | - Rafael Peñafiel
- Department of Biochemistry and Molecular
Biology B and Immunology and Department of
Pharmacology, Faculty of Medicine, University of Murcia, 30100 Murcia,
Spain
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Giannakis M, Chen SL, Karam SM, Engstrand L, Gordon JI. Helicobacter pylori evolution during progression from chronic atrophic gastritis to gastric cancer and its impact on gastric stem cells. Proc Natl Acad Sci U S A 2008; 105:4358-4363. [PMID: 18332421 PMCID: PMC2393758 DOI: 10.1073/pnas.0800668105] [Citation(s) in RCA: 92] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2007] [Indexed: 02/07/2023] Open
Abstract
We have characterized the adaptations of Helicobacter pylori to a rarely captured event in the evolution of its impact on host biology-the transition from chronic atrophic gastritis (ChAG) to gastric adenocarcinoma-and defined the impact of these adaptations on an intriguing but poorly characterized interaction between this bacterium and gastric epithelial stem cells. Bacterial isolates were obtained from a single human host colonized with a single dominant strain before and after his progression from ChAG to gastric adenocarcinoma during a 4-year interval. Draft genome assemblies were generated from two isolates, one ChAG-associated, the other cancer-associated. The cancer-associated strain was less fit in a gnotobiotic transgenic mouse model of human ChAG and better able to establish itself within a mouse gastric epithelial progenitor-derived cell line (mGEP) that supports bacterial attachment. GeneChip-based comparisons of the transcriptomes of mGEPs and a control mouse gastric epithelial cell line revealed that, upon infection, the cancer-associated strain regulates expression of GEP-associated signaling and metabolic pathways, and tumor suppressor genes associated with development of gastric cancer in humans, in a manner distinct from the ChAG-associated isolate. The effects on GEP metabolic pathways, some of which were confirmed in gnotobiotic mice, together with observed changes in the bacterial transcriptome are predicted to support aspects of an endosymbiosis between this microbe and gastric stem cells. These results provide insights about how H. pylori may adapt to and influence stem cell biology and how its intracellular residency could contribute to gastric tumorigenesis.
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Affiliation(s)
- Marios Giannakis
- *Center for Genome Sciences, Washington University School of Medicine, St. Louis, MO 63108
| | - Swaine L. Chen
- *Center for Genome Sciences, Washington University School of Medicine, St. Louis, MO 63108
| | - Sherif M. Karam
- Department of Anatomy, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain 17666, United Arab Emirates
| | - Lars Engstrand
- Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, 171 77 Stockholm, Sweden; and
- Swedish Institute for Infectious Disease Control, 171 82 Solna, Sweden
| | - Jeffrey I. Gordon
- *Center for Genome Sciences, Washington University School of Medicine, St. Louis, MO 63108
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Abstract
Studies over many years have suggested that increased polyamine synthesis may be necessary for neoplastic growth. This review summarizes recent work on the regulation of putrescine production both de novo and via the degradation of higher polyamines and provides a summary of studies using transgenic mice in which the levels of proteins that regulate these processes (L-ornithine decarboxylase, antizyme and spermidine/spermine-N(1)-acetyltransferase) are altered.
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Affiliation(s)
- A E Pegg
- Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
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50
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Willert EK, Fitzpatrick R, Phillips MA. Allosteric regulation of an essential trypanosome polyamine biosynthetic enzyme by a catalytically dead homolog. Proc Natl Acad Sci U S A 2007; 104:8275-80. [PMID: 17485680 PMCID: PMC1895940 DOI: 10.1073/pnas.0701111104] [Citation(s) in RCA: 73] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
African sleeping sickness is a fatal disease that is caused by the protozoan parasite Trypanosoma brucei. Polyamine biosynthesis is an essential pathway in the parasite and is a validated drug target for treatment of the disease. S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes a key step in polyamine biosynthesis. Here, we show that trypanosomatids uniquely contain both a functional AdoMetDC and a paralog designated prozyme that has lost catalytic activity. The T. brucei prozyme forms a high-affinity heterodimer with AdoMetDC that stimulates its activity by 1,200-fold. Both genes are expressed in T. brucei, and analysis of AdoMetDC activity in T. brucei extracts supports the finding that the heterodimer is the functional enzyme in vivo. Thus, prozyme has evolved to be a catalytically dead but allosterically active subunit of AdoMetDC, providing an example of how regulators of multimeric enzymes can evolve through gene duplication and mutational drift. These data identify a distinct mechanism for regulating AdoMetDC in the parasite that suggests new strategies for the development of parasite-specific inhibitors of the polyamine biosynthetic pathway.
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Affiliation(s)
- Erin K. Willert
- *Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390-9041; and
| | - Richard Fitzpatrick
- Chemistry Research Department, Genzyme Drug and Biomaterial R & D, 153 Second Avenue, Waltham, MA 02134
| | - Margaret A. Phillips
- *Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390-9041; and
- To whom correspondence should be addressed. E-mail:
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