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Xiong X, Du Y, Liu P, Li X, Lai X, Miao H, Ning B. Unveiling EIF5A2: A Multifaceted Player in Cellular Regulation, Tumorigenesis and Drug Resistance. Eur J Pharmacol 2025:177596. [PMID: 40194645 DOI: 10.1016/j.ejphar.2025.177596] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2025] [Revised: 03/28/2025] [Accepted: 04/02/2025] [Indexed: 04/09/2025]
Abstract
The eukaryotic initiation factor 5A2 gene (EIF5A2) is a highly conserved and multifunctional gene that significantly influences various cellular processes, including translation elongation, RNA binding, ribosome binding, protein binding and post-translational modifications. Overexpression of EIF5A2 is frequently observed in multiple cancers, where it functions as an oncoprotein. Additionally, EIF5A2 is implicated in drug resistance through the regulation of various molecular pathways. In the review, we describe the structure and functions of EIF5A2 in normal cells and its role in tumorigenesis. We also elucidate the molecular mechanisms associated with EIF5A2 in the context of tumorigenesis and drug resistance. We propose that the biological roles of EIF5A2 in regulating diverse cellular processes and tumorigenesis are clinically significant and warrant further investigation.
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Affiliation(s)
- Xifeng Xiong
- Guangzhou Institute of Traumatic Surgery, Guangzhou Red Cross Hospital of Jinan University, Guangzhou 510220, Guangdong, China; Guangzhou Institute of Burn Clinical Medicine, Guangzhou Red Cross Hospital of Jinan University, Guangzhou 510220, Guangdong, China
| | - Yanli Du
- Guangdong Medical University, Zhanjiang 524023, Guangdong, China; Department of Orthopedic, Guangzhou Red Cross Hospital of Jinan University, Guangzhou 510220, Guangdong, China
| | - Peng Liu
- Departments of Burn and Plastic, Guangzhou Red Cross Hospital of Jinan University, Guangzhou 510220, Guangdong, China
| | - Xinye Li
- Guangdong Medical University, Zhanjiang 524023, Guangdong, China; Department of Orthopedic, Guangzhou Red Cross Hospital of Jinan University, Guangzhou 510220, Guangdong, China
| | - Xudong Lai
- Department of infectious disease, Guangzhou Red Cross Hospital of Jinan University, Guangzhou 510220, Guangdong, China
| | - Haixiong Miao
- Department of Orthopedic, Guangzhou Red Cross Hospital of Jinan University, Guangzhou 510220, Guangdong, China.
| | - Bo Ning
- Department of Neurosurgery, Guangzhou Red Cross Hospital of Jinan University, Guangzhou 510220, Guangdong, China.
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2
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Wątor-Wilk E, Wilk P, Grudnik P. The structural biology of deoxyhypusination complexes. Structure 2025; 33:221-227. [PMID: 39809274 DOI: 10.1016/j.str.2024.12.011] [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: 07/11/2024] [Revised: 10/22/2024] [Accepted: 12/13/2024] [Indexed: 01/16/2025]
Abstract
Deoxyhypusination is the first rate-limiting step of the unique post-translational modification-hypusination-that is catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). This modification is essential for the activation of translation factor 5A in eukaryotes (eIF5A) and Archaea (aIF5A). This perspective focuses on the structural biology of deoxyhypusination complexes in eukaryotic and archaeal organisms. Based on recently published crystal and cryogenic electron microscopy (cryo-EM) structures of deoxyhypusination complexes from three different organisms, we compare the structural features and stoichiometries of DHS-IF5A complexes across different species. We discuss conserved elements in the active site architecture and binding interfaces as well as significant differences in their stoichiometry and regulation mechanisms. The structural insights provide a comprehensive understanding of the deoxyhypusination process and highlight evolutionary adaptations across the domains of life. Future research should focus on the regulatory mechanisms governing DHS activity and the functional implications of stoichiometric variations in different organisms.
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Affiliation(s)
- Elżbieta Wątor-Wilk
- Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland; Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Kraków, Poland
| | - Piotr Wilk
- Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland
| | - Przemysław Grudnik
- Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland.
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3
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Wątor E, Wilk P, Kochanowski P, Grudnik P. Structural characterization of the (deoxy)hypusination in Trichomonas vaginalis questions the bifunctionality of deoxyhypusine synthase. FEBS J 2024; 291:3856-3869. [PMID: 38923395 DOI: 10.1111/febs.17207] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2024] [Revised: 04/14/2024] [Accepted: 06/12/2024] [Indexed: 06/28/2024]
Abstract
Trichomonas vaginalis, the causative agent of trichomoniasis, is a prevalent anaerobic protozoan parasite responsible for the most common nonviral sexually transmitted infection globally. While metronidazole and its derivatives are approved drugs for this infection, rising resistance necessitates the exploration of new antiparasitic therapies. Protein posttranslational modifications (PTMs) play crucial roles in cellular processes, and among them, hypusination, involving eukaryotic translation factor 5A (eIF5A), has profound implications. Despite extensive studies in various organisms, the role of hypusination in T. vaginalis and its potential impact on parasite biology and pathogenicity remain poorly understood. This study aims to unravel the structural basis of the hypusination pathway in T. vaginalis using X-ray crystallography and cryo-electron microscopy. The results reveal high structural homology between T. vaginalis and human orthologs, providing insights into the molecular architecture of eIF5A and deoxyhypusine synthase (DHS) and their interaction. Contrary to previous suggestions of bifunctionality, our analyses indicate that the putative hydroxylation site in tvDHS is nonfunctional, and biochemical assays demonstrate exclusive deoxyhypusination capability. These findings challenge the notion of tvDHS functioning as both deoxyhypusine synthase and hydroxylase. The study enhances understanding of the hypusination pathway in T. vaginalis, shedding light on its functional relevance and potential as a drug target, and contributing to the development of novel therapeutic strategies against trichomoniasis.
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Affiliation(s)
- Elżbieta Wątor
- Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland
| | - Piotr Wilk
- Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland
| | - Paweł Kochanowski
- Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland
- Doctoral School of Exact and Natural Sciences, Jagiellonian University, Kraków, Poland
| | - Przemysław Grudnik
- Małopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland
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D'Agostino M, Simonetti A, Motta S, Wolff P, Romagnoli A, Piccinini A, Spinozzi F, Di Marino D, La Teana A, Ennifar E. Crystal structure of archaeal IF5A-DHS complex reveals insights into the hypusination mechanism. Structure 2024; 32:878-888.e4. [PMID: 38582076 DOI: 10.1016/j.str.2024.03.008] [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: 12/18/2023] [Revised: 02/12/2024] [Accepted: 03/12/2024] [Indexed: 04/08/2024]
Abstract
The translation factor IF5A is highly conserved in Eukarya and Archaea and undergoes a unique post-translational hypusine modification by the deoxyhypusine synthase (DHS) enzyme. DHS transfers the butylamine moiety from spermidine to IF5A using NAD as a cofactor, forming a deoxyhypusine intermediate. IF5A is a key player in protein synthesis, preventing ribosome stalling in proline-rich sequences during translation elongation and facilitating translation elongation and termination. Additionally, human eIF5A participates in various essential cellular processes and contributes to cancer metastasis, with inhibiting hypusination showing anti-proliferative effects. The hypusination pathway of IF5A is therefore an attractive new therapeutic target. We elucidated the 2.0 Å X-ray crystal structure of the archaeal DHS-IF5A complex, revealing hetero-octameric architecture and providing a detailed view of the complex active site including the hypusination loop. This structure, along with biophysical data and molecular dynamics simulations, provides new insights into the catalytic mechanism of the hypusination reaction.
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Affiliation(s)
- Mattia D'Agostino
- Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy; Architecture et Réactivité de l'ARN, CNRS UPR 9002, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France
| | - Angelita Simonetti
- Architecture et Réactivité de l'ARN, CNRS UPR 9002, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France
| | - Stefano Motta
- Department of Earth and Environmental Sciences, University of Milano Bicocca, Piazza della Scienza 1, 20126 Milan, Italy
| | - Philippe Wolff
- Architecture et Réactivité de l'ARN, CNRS UPR 9002, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France
| | - Alice Romagnoli
- Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy; New York-Marche Structural Biology Center (Ny-Masbic), Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy
| | - Astra Piccinini
- Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy
| | - Francesco Spinozzi
- Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy
| | - Daniele Di Marino
- Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy; New York-Marche Structural Biology Center (Ny-Masbic), Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy; Neuronal Death and Neuroprotection Unit, Department of Neuroscience, Mario Negri Institute for Pharmacological Research-IRCCS, Via Mario Negri 2, 20156 Milano, Italy.
| | - Anna La Teana
- Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy; New York-Marche Structural Biology Center (Ny-Masbic), Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy.
| | - Eric Ennifar
- Architecture et Réactivité de l'ARN, CNRS UPR 9002, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France.
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Wu B, Liu S. Structural Insights into the Mechanisms Underlying Polyaminopathies. Int J Mol Sci 2024; 25:6340. [PMID: 38928047 PMCID: PMC11203672 DOI: 10.3390/ijms25126340] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2024] [Revised: 06/01/2024] [Accepted: 06/05/2024] [Indexed: 06/28/2024] Open
Abstract
Polyamines are ubiquitous in almost all biological entities and involved in various crucial physiological processes. They are also closely associated with the onset and progression of many diseases. Polyaminopathies are a group of rare genetic disorders caused by alterations in the function of proteins within the polyamine metabolism network. Although the identified polyaminopathies are all rare diseases at present, they are genetically heritable, rendering high risks not only to the carriers but also to their descendants. Meanwhile, more polyaminopathic patients might be discovered with the increasing accessibility of gene sequencing. This review aims to provide a comprehensive overview of the structural variations of mutated proteins in current polyaminopathies, in addition to their causative genes, types of mutations, clinical symptoms, and therapeutic approaches. We focus on analyzing how alterations in protein structure lead to protein dysfunction, thereby facilitating the onset of diseases. We hope this review will offer valuable insights and references for the future clinical diagnosis and precision treatment of polyaminopathies.
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Affiliation(s)
- Bing Wu
- Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Key Laboratory of Fermentation Engineering (Ministry of Education), Wuhan 430068, China
- Hubei Key Laboratory of Industrial Microbiology, National “111” Center for Cellular Regulation and Molecular Pharmaceutics, Hubei University of Technology, Wuhan 430068, China
| | - Sen Liu
- Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Key Laboratory of Fermentation Engineering (Ministry of Education), Wuhan 430068, China
- Hubei Key Laboratory of Industrial Microbiology, National “111” Center for Cellular Regulation and Molecular Pharmaceutics, Hubei University of Technology, Wuhan 430068, China
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6
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Liu Y, Peng L, Chen J, Chen L, Wu Y, Cheng M, Chen M, Ye X, Jin Y. EIF5A2 specifically regulates the transcription of aging-related genes in human neuroblastoma cells. BMC Geriatr 2023; 23:83. [PMID: 36750933 PMCID: PMC9906866 DOI: 10.1186/s12877-023-03793-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2022] [Accepted: 02/02/2023] [Indexed: 02/09/2023] Open
Abstract
BACKGROUND Post-transcriptional regulation plays a critical role in controlling biological processes such as aging. Previous studies have shown that eukaryotic initiation factor 5A (EIF5A) might play a crucial role in aging. It is unknown whether EIF5A2, a second isoform of EIF5A, could impact aging through post-transcriptional regulation. METHODS In the present study, EIF5A2 overexpression (EIF5A2-OE) was induced in SH-SY5Y cells. RNA-seq, bioinformatics analysis and RT-qPCR validation experiments were then performed to explore the molecular mechanism of EIF5A2-mediated transcriptional regulation. Cell viability, proportion of senescent cells and the cell cycle were respectively determined by Cell Counting Kit-8, SA-β‑galactosidase and flow cytometry to evaluate the cell senescence. RESULTS A total of 190 downregulated and 126 upregulated genes related to EIF5A2-OE were identified. Genes closely related to cellular aging processes such as unfolded protein response (UPR), cell adhesion and calcium signaling pathway were under global transcriptional regulation. Moreover, EIF5A2-OE promoted the viability of SH-SY5Y cells and reduced cell senescence in vitro. Among 30 genes with the most significant expression differences in EIF5A2-OE cells, we identified eight genes, including ASNS, ATF3, ATF4, CEBPB, DDIT3, HERPUD1, HSPA5 and XBP1, enriched in the UPR. Through EIF5A2-tanscription factors (TFs)-targets regulation network in EIF5A2-OE cells, we found three TFs, BHLHE40, RHOXF1 and TBX20, that targeted at these eight UPR-related genes. Verification test via the published database of human glial cell tissue showed only BHLHE40 and RHOXF1 were significantly associated with EIF5A2. CONCLUSIONS Our findings suggest that EIF5A2 may alleviate cell senescence in vitro and mediate UPR-related genes via specific TFs. Thus, EIF5A2 could function as a regulator of aging via the regulation of transcription, which greatly expands the current understanding of the mechanisms of EIF5A2-mediated gene regulation.
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Affiliation(s)
- Yuwei Liu
- grid.49470.3e0000 0001 2331 6153Department of Internal Medicine and Geriatrics, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, Hubei China ,grid.49470.3e0000 0001 2331 6153Department of General Practice, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, Hubei China
| | - Li Peng
- grid.49470.3e0000 0001 2331 6153Department of Internal Medicine and Geriatrics, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, Hubei China ,grid.49470.3e0000 0001 2331 6153Department of Cardiology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, Hubei China
| | - Jing Chen
- grid.49470.3e0000 0001 2331 6153Department of Internal Medicine and Geriatrics, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, Hubei China
| | - Ling Chen
- grid.49470.3e0000 0001 2331 6153Department of Internal Medicine and Geriatrics, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, Hubei China
| | - Ying Wu
- grid.49470.3e0000 0001 2331 6153Department of Internal Medicine and Geriatrics, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, Hubei China
| | - Mengxin Cheng
- grid.49470.3e0000 0001 2331 6153Department of Internal Medicine and Geriatrics, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, Hubei China
| | - Min Chen
- grid.49470.3e0000 0001 2331 6153Department of Internal Medicine and Geriatrics, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, Hubei China
| | - Xujun Ye
- Department of Internal Medicine and Geriatrics, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, Hubei, China.
| | - Yalei Jin
- Department of General Practice, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, Hubei, China.
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Mudryi V, Peske F, Rodnina M. Translation Factor Accelerating Peptide Bond Formation on the Ribosome: EF-P and eIF5A as Entropic Catalysts and a Potential Drug Targets. BBA ADVANCES 2023; 3:100074. [PMID: 37082265 PMCID: PMC10074943 DOI: 10.1016/j.bbadva.2023.100074] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2022] [Revised: 01/06/2023] [Accepted: 01/07/2023] [Indexed: 01/11/2023] Open
Abstract
Elongation factor P (EF-P) and its eukaryotic homolog eIF5A are auxiliary translation factors that facilitate peptide bond formation when several sequential proline (Pro) residues are incorporated into the nascent chain. EF-P and eIF5A bind to the exit (E) site of the ribosome and contribute to favorable entropy of the reaction by stabilizing tRNA binding in the peptidyl transferase center of the ribosome. In most organisms, EF-P and eIF5A carry a posttranslational modification that is crucial for catalysis. The chemical nature of the modification varies between different groups of bacteria and between pro- and eukaryotes, making the EF-P-modification enzymes promising targets for antibiotic development. In this review, we summarize our knowledge of the structure and function of EF-P and eIF5A, describe their modification enzymes, and present an approach for potential drug screening aimed at EarP, an enzyme that is essential for EF-P modification in several pathogenic bacteria.
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8
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Farache D, Liu L, Lee ASY. Eukaryotic Initiation Factor 5A2 Regulates Expression of Antiviral Genes. J Mol Biol 2022; 434:167564. [PMID: 35358571 PMCID: PMC11906106 DOI: 10.1016/j.jmb.2022.167564] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2021] [Revised: 03/22/2022] [Accepted: 03/22/2022] [Indexed: 12/14/2022]
Abstract
Translation factors are essential for regulation of protein synthesis. The eukaryotic translation initiation factor 5A (eIF5A) family is made up of two paralogues - eIF5A1 and eIF5A2 - which display high sequence homology but distinct tissue tropism. While eIF5A1 directly binds to the ribosome and regulates translation initiation, elongation, and termination, the molecular function of eIF5A2 remains poorly understood. Here, we engineer an eIF5A2 knockout allele in the SW480 colon cancer cell line. Using ribosome profiling and RNA-Sequencing, we reveal that eIF5A2 is functionally distinct from eIF5A1 and does not regulate transcript-specific or global protein synthesis. Instead, eIF5A2 knockout leads to decreased intrinsic antiviral gene expression, including members of the IFITM and APOBEC3 family. Furthermore, cells lacking eIF5A2 display increased permissiveness to virus infection. Our results uncover eIF5A2 as a factor involved regulating the antiviral transcriptome, and reveal an example of how gene duplications of translation factors can result in proteins with distinct functions.
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Affiliation(s)
- Dorian Farache
- Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA; Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
| | - Luochen Liu
- Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA; Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
| | - Amy S Y Lee
- Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA; Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
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9
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Barba-Aliaga M, Alepuz P. Role of eIF5A in Mitochondrial Function. Int J Mol Sci 2022; 23:1284. [PMID: 35163207 PMCID: PMC8835957 DOI: 10.3390/ijms23031284] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2021] [Revised: 01/19/2022] [Accepted: 01/21/2022] [Indexed: 12/17/2022] Open
Abstract
The eukaryotic translation initiation factor 5A (eIF5A) is an evolutionarily conserved protein that binds ribosomes to facilitate the translation of peptide motifs with consecutive prolines or combinations of prolines with glycine and charged amino acids. It has also been linked to other molecular functions and cellular processes, such as nuclear mRNA export and mRNA decay, proliferation, differentiation, autophagy, and apoptosis. The growing interest in eIF5A relates to its association with the pathogenesis of several diseases, including cancer, viral infection, and diabetes. It has also been proposed as an anti-aging factor: its levels decay in aged cells, whereas increasing levels of active eIF5A result in the rejuvenation of the immune and vascular systems and improved brain cognition. Recent data have linked the role of eIF5A in some pathologies with its function in maintaining healthy mitochondria. The eukaryotic translation initiation factor 5A is upregulated under respiratory metabolism and its deficiency reduces oxygen consumption, ATP production, and the levels of several mitochondrial metabolic enzymes, as well as altering mitochondria dynamics. However, although all the accumulated data strongly link eIF5A to mitochondrial function, the precise molecular role and mechanisms involved are still unknown. In this review, we discuss the findings linking eIF5A and mitochondria, speculate about its role in regulating mitochondrial homeostasis, and highlight its potential as a target in diseases related to energy metabolism.
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Affiliation(s)
- Marina Barba-Aliaga
- Instituto de Biotecnología y Biomedicina (Biotecmed), Universitat de València, 46100 València, Spain
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universitat de València, 46100 València, Spain
| | - Paula Alepuz
- Instituto de Biotecnología y Biomedicina (Biotecmed), Universitat de València, 46100 València, Spain
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universitat de València, 46100 València, Spain
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10
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Tauc M, Cougnon M, Carcy R, Melis N, Hauet T, Pellerin L, Blondeau N, Pisani DF. The eukaryotic initiation factor 5A (eIF5A1), the molecule, mechanisms and recent insights into the pathophysiological roles. Cell Biosci 2021; 11:219. [PMID: 34952646 PMCID: PMC8705083 DOI: 10.1186/s13578-021-00733-y] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2021] [Accepted: 12/14/2021] [Indexed: 11/29/2022] Open
Abstract
Since the demonstration of its involvement in cell proliferation, the eukaryotic initiation factor 5A (eIF5A) has been studied principally in relation to the development and progression of cancers in which the isoform A2 is mainly expressed. However, an increasing number of studies report that the isoform A1, which is ubiquitously expressed in normal cells, exhibits novel molecular features that reveal its new relationships between cellular functions and organ homeostasis. At a first glance, eIF5A can be regarded, among other things, as a factor implicated in the initiation of translation. Nevertheless, at least three specificities: (1) its extreme conservation between species, including plants, throughout evolution, (2) its very special and unique post-translational modification through the activating-hypusination process, and finally (3) its close relationship with the polyamine pathway, suggest that the role of eIF5A in living beings remains to be uncovered. In fact, and beyond its involvement in facilitating the translation of proteins containing polyproline residues, eIF5A is implicated in various physiological processes including ischemic tolerance, metabolic adaptation, aging, development, and immune cell differentiation. These newly discovered physiological properties open up huge opportunities in the clinic for pathologies such as, for example, the ones in which the oxygen supply is disrupted. In this latter case, organ transplantation, myocardial infarction or stroke are concerned, and the current literature defines eIF5A as a new drug target with a high level of potential benefit for patients with these diseases or injuries. Moreover, the recent use of genomic and transcriptomic association along with metadata studies also revealed the implication of eIF5A in genetic diseases. Thus, this review provides an overview of eIF5A from its molecular mechanism of action to its physiological roles and the clinical possibilities that have been recently reported in the literature.
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Affiliation(s)
- Michel Tauc
- LP2M, CNRS, Université Côte d'Azur, Nice, France. .,Laboratories of Excellence Ion Channel Science and Therapeutics, Nice, France. .,Laboratoire de Physiomédecine Moléculaire, UMR7370, Faculté de Médecine, CNRS, Université Côte d'Azur, 28 Avenue de Valombrose, 06107, Nice Cedex, France.
| | - Marc Cougnon
- LP2M, CNRS, Université Côte d'Azur, Nice, France.,Laboratories of Excellence Ion Channel Science and Therapeutics, Nice, France
| | - Romain Carcy
- Service de Réanimation Polyvalente et Service de Réanimation des Urgences Vitales, CHU Nice, Hôpital Pasteur 2, Nice, France
| | - Nicolas Melis
- Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, 20892, USA
| | - Thierry Hauet
- INSERM, IRTOMIT, CHU de Poitiers, Université de Poitiers, La Milétrie, Poitiers, France
| | - Luc Pellerin
- INSERM, IRTOMIT, CHU de Poitiers, Université de Poitiers, La Milétrie, Poitiers, France
| | - Nicolas Blondeau
- Laboratories of Excellence Ion Channel Science and Therapeutics, Nice, France.,IPMC, CNRS, Université Côte d'Azur, Valbonne, France
| | - Didier F Pisani
- LP2M, CNRS, Université Côte d'Azur, Nice, France.,Laboratories of Excellence Ion Channel Science and Therapeutics, Nice, France
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11
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Silva SF, Klippel AH, Ramos PZ, Santiago ADS, Valentini SR, Bengtson MH, Massirer KB, Bilsland E, Couñago RM, Zanelli CF. Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major. PLoS Negl Trop Dis 2020; 14:e0008762. [PMID: 33044977 PMCID: PMC7581365 DOI: 10.1371/journal.pntd.0008762] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2020] [Revised: 10/22/2020] [Accepted: 08/31/2020] [Indexed: 01/03/2023] Open
Abstract
Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections. Target-based drug discovery strategies hold the promise to discover safer and more effective treatments for Neglected Tropical Diseases (NTDs). Genetic manipulation techniques have been used to successfully identify essential genes in eukaryotic parasites. Unfortunately, the fact that a gene is essential under controlled laboratory conditions does not automatically make the corresponding gene-product vulnerable to pharmacological intervention in a clinical setting within the human host. To allow the discovery and development of small molecule tool compounds that can be used to validate pharmacologically vulnerable targets, one must first establish compound screening assays and obtain structural information for the candidate target. Eukaryotic cells lacking deoxyhypusine synthase (DHS) function are not viable. DHS catalyzes the first step in a post-translational modification that is critical for the function of eIF5A. Presence of mature eIF5A is also essential for eukaryotic cell viability. Here we reported compound screening assays (yeast-based for Brugia malayi and Leishmania major; in vitro for B. malayi only) and provided further regulatory and structural insights we hope will aid in the identification and development of inhibitors for the DHS enzymes from two NTD-causing organisms—B. malayi, the causative agent of lymphatic filariasis and L. major, the causative agent of cutaneous leishmaniasis.
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Affiliation(s)
| | | | - Priscila Zonzini Ramos
- Molecular Biology and Genetic Engineering Center (CBMEG), Medicinal Chemistry Center (CQMED), Structural Genomics Consortium (SGC-UNICAMP), University of Campinas-UNICAMP, Campinas, SP, Brazil
- Department of Structural and Functional Biology, Institute of Biology, University of Campinas—UNICAMP, Campinas, SP, Brazil
| | - André da Silva Santiago
- Molecular Biology and Genetic Engineering Center (CBMEG), Medicinal Chemistry Center (CQMED), Structural Genomics Consortium (SGC-UNICAMP), University of Campinas-UNICAMP, Campinas, SP, Brazil
- Department of Structural and Functional Biology, Institute of Biology, University of Campinas—UNICAMP, Campinas, SP, Brazil
| | | | - Mario Henrique Bengtson
- Molecular Biology and Genetic Engineering Center (CBMEG), Medicinal Chemistry Center (CQMED), Structural Genomics Consortium (SGC-UNICAMP), University of Campinas-UNICAMP, Campinas, SP, Brazil
- Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas—UNICAMP, Campinas, SP, Brazil
| | - Katlin Brauer Massirer
- Molecular Biology and Genetic Engineering Center (CBMEG), Medicinal Chemistry Center (CQMED), Structural Genomics Consortium (SGC-UNICAMP), University of Campinas-UNICAMP, Campinas, SP, Brazil
- Department of Structural and Functional Biology, Institute of Biology, University of Campinas—UNICAMP, Campinas, SP, Brazil
| | - Elizabeth Bilsland
- Department of Structural and Functional Biology, Institute of Biology, University of Campinas—UNICAMP, Campinas, SP, Brazil
| | - Rafael Miguez Couñago
- Molecular Biology and Genetic Engineering Center (CBMEG), Medicinal Chemistry Center (CQMED), Structural Genomics Consortium (SGC-UNICAMP), University of Campinas-UNICAMP, Campinas, SP, Brazil
- * E-mail: (RMC); (CFZ)
| | - Cleslei Fernando Zanelli
- School of Pharmaceutical Sciences, São Paulo State University—UNESP, Araraquara, SP, Brazil
- * E-mail: (RMC); (CFZ)
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12
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Tanaka Y, Kurasawa O, Yokota A, Klein MG, Saito B, Matsumoto S, Okaniwa M, Ambrus-Aikelin G, Uchiyama N, Morishita D, Kimura H, Imamura S. New Series of Potent Allosteric Inhibitors of Deoxyhypusine Synthase. ACS Med Chem Lett 2020; 11:1645-1652. [PMID: 34345355 PMCID: PMC8323115 DOI: 10.1021/acsmedchemlett.0c00331] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2020] [Accepted: 07/30/2020] [Indexed: 02/06/2023] Open
Abstract
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Deoxyhypusine synthase (DHPS) is
the primary enzyme responsible
for the hypusine modification and, thereby, activation of the eukaryotic
translation initiation factor 5A (eIF5A), which is key in regulating
the protein translation processes associated with tumor proliferation.
Although DHPS inhibitors could be a promising therapeutic option for
treating cancer, only a few studies reported druglike compounds with
this inhibition property. Thus, in this work, we designed and synthesized
a new chemical series possessing fused ring scaffolds designed from
high-throughput screening hit compounds, discovering a 5,6-dihydrothieno[2,3-c]pyridine derivative (26d) with potent inhibitory
activity; furthermore, the X-ray crystallographic analysis of the
DHPS complex with 26d demonstrated a distinct allosteric
binding mode compared to a previously reported inhibitor. These findings
could be significantly useful in the functional analysis of conformational
changes in DHPS as well as the structure-based design of allosteric
inhibitors.
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Affiliation(s)
- Yuta Tanaka
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Osamu Kurasawa
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Akihiro Yokota
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Michael G. Klein
- Department of Structural Biology, Takeda California, 10410 Science Center Drive, San Diego, California 92121, United States
| | - Bunnai Saito
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Shigemitsu Matsumoto
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Masanori Okaniwa
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Geza Ambrus-Aikelin
- Department of Structural Biology, Takeda California, 10410 Science Center Drive, San Diego, California 92121, United States
| | - Noriko Uchiyama
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Daisuke Morishita
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Hiromichi Kimura
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Shinichi Imamura
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
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Kaiser A, Heiss K, Mueller AK, Fimmers R, Matthes J, Njuguna JT. Inhibition of EIF-5A prevents apoptosis in human cardiomyocytes after malaria infection. Amino Acids 2020; 52:693-710. [PMID: 32367435 DOI: 10.1007/s00726-020-02843-2] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2019] [Accepted: 04/11/2020] [Indexed: 10/24/2022]
Abstract
In this study, a determination of Troponin I and creatine kinase activity in whole-blood samples in a cohort of 100 small infants in the age of 2-5 years from Uganda with complicated Plasmodium falciparum malaria suggests the prevalence of cardiac symptoms in comparison to non-infected, healthy patients. Troponin I and creatine kinase activity increased during infection. Different reports showed that complicated malaria coincides with hypoxia in children. The obtained clinical data prompted us to further elucidate the underlying regulatory mechanisms of cardiac involvement in human cardiac ventricular myocytes. Complicated malaria is the most common clinical presentation and might induce cardiac impairment by hypoxia. Eukaryotic initiation factor 5A (eIF-5A) is involved in hypoxia induced factor (HIF-1α) expression. EIF-5A is a protein posttranslationally modified by hypusination involving catalysis of the two enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase. Treatment of human cardiomyocytes with GC7, an inhibitor of DHS, catalyzing the first step in hypusine biosynthesis led to a decrease in proinflammatory and proapoptotic myocardial caspase-1 activity in comparison to untreated cardiomyocytes. This effect was even more pronounced after co-administration of GC7 and GPI from P. falciparum simulating the pathology of severe malaria. Moreover, in comparison to untreated and GC7-treated cardiomyocytes, co-administration of GC7 and GPI significantly decreased the release of cytochrome C and lactate from damaged mitochondria. In sum, coadministration of GC7 prevented cardiac damage driven by hypoxia in vitro. Our approach demonstrates the potential of the pharmacological inhibitor GC7 to ameliorate apoptosis in cardiomyocytes in an in vitro model simulating severe malaria. This regulatory mechanism is based on blocking EIF-5A hypusination.
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Affiliation(s)
- Annette Kaiser
- Medical Research Centre, University Duisburg-Essen, Hufelandstrasse 55, 45147, Essen, Germany.
| | - Kirsten Heiss
- Centre for Infectious Diseases, Parasitology Unit, University Hospital Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany
- German Center for Infectious Diseases (DZIF), Heidelberg, Germany
| | - Ann-Kristin Mueller
- Centre for Infectious Diseases, Parasitology Unit, University Hospital Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany
- German Center for Infectious Diseases (DZIF), Heidelberg, Germany
| | - Rolf Fimmers
- Institut für Medizinische Biometrie, Informatik Und Epedimologie, Sigmund-Freud-Strasse 25, 53107, Bonn, Germany
| | - Jan Matthes
- Centre of Pharmcology, University of Cologne, Gleueler Strasse 24, 50931, Köln, Germany
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14
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Half Way to Hypusine-Structural Basis for Substrate Recognition by Human Deoxyhypusine Synthase. Biomolecules 2020; 10:biom10040522. [PMID: 32235505 PMCID: PMC7226451 DOI: 10.3390/biom10040522] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2020] [Revised: 03/23/2020] [Accepted: 03/26/2020] [Indexed: 02/08/2023] Open
Abstract
Deoxyhypusine synthase (DHS) is a transferase enabling the formation of deoxyhypusine, which is the first, rate-limiting step of a unique post-translational modification: hypusination. DHS catalyses the transfer of a 4-aminobutyl moiety of polyamine spermidine to a specific lysine of eukaryotic translation factor 5A (eIF5A) precursor in a nicotinamide adenine dinucleotide (NAD)-dependent manner. This modification occurs exclusively on one protein, eIF5A, and it is essential for cell proliferation. Malfunctions of the hypusination pathway, including those caused by mutations within the DHS encoding gene, are associated with conditions such as cancer or neurodegeneration. Here, we present a series of high-resolution crystal structures of human DHS. Structures were determined as the apoprotein, as well as ligand-bound states at high-resolutions ranging from 1.41 to 1.69 Å. By solving DHS in complex with its natural substrate spermidine (SPD), we identified the mode of substrate recognition. We also observed that other polyamines, namely spermine (SPM) and putrescine, bind DHS in a similar manner as SPD. Moreover, we performed activity assays showing that SPM could to some extent serve as an alternative DHS substrate. In contrast to previous studies, we demonstrate that no conformational changes occur in the DHS structure upon spermidine-binding. By combining mutagenesis and a light-scattering approach, we show that a conserved “ball-and-chain” motif is indispensable to assembling a functional DHS tetramer. Our study substantially advances our knowledge of the substrate recognition mechanism by DHS and may aid the design of pharmacological compounds for potential applications in cancer therapy.
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15
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Tanaka Y, Kurasawa O, Yokota A, Klein MG, Ono K, Saito B, Matsumoto S, Okaniwa M, Ambrus-Aikelin G, Morishita D, Kitazawa S, Uchiyama N, Ogawa K, Kimura H, Imamura S. Discovery of Novel Allosteric Inhibitors of Deoxyhypusine Synthase. J Med Chem 2020; 63:3215-3226. [PMID: 32142284 DOI: 10.1021/acs.jmedchem.9b01979] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Deoxyhypusine synthase (DHPS) utilizes spermidine and NAD as cofactors to incorporate a hypusine modification into the eukaryotic translation initiation factor 5A (eIF5A). Hypusine is essential for eIF5A activation, which, in turn, plays a key role in regulating protein translation of selected mRNA that are associated with the synthesis of oncoproteins, thereby enhancing tumor cell proliferation. Therefore, inhibition of DHPS is a promising therapeutic option for the treatment of cancer. To discover novel lead compounds that target DHPS, we conducted synthetic studies with a hit obtained via high-throughput screening. Optimization of the ring structures of the amide compound (2) led to bromobenzothiophene (11g) with potent inhibitory activity against DHPS. X-ray crystallographic analysis of 11g complexed with DHPS revealed a dramatic conformational change in DHPS, which suggests the presence of a novel allosteric site. These findings provide the basis for the development of novel therapy distinct from spermidine mimetic inhibitors.
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Affiliation(s)
- Yuta Tanaka
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Osamu Kurasawa
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Akihiro Yokota
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Michael G Klein
- Department of Structural Biology, Takeda California, 10410 Science Center Drive, San Diego, California 92121, United States
| | - Koji Ono
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Bunnai Saito
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Shigemitsu Matsumoto
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Masanori Okaniwa
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Geza Ambrus-Aikelin
- Department of Structural Biology, Takeda California, 10410 Science Center Drive, San Diego, California 92121, United States
| | - Daisuke Morishita
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Satoshi Kitazawa
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Noriko Uchiyama
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Kazumasa Ogawa
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Hiromichi Kimura
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
| | - Shinichi Imamura
- Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
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16
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Wu GQ, Xu YM, Lau ATY. Recent insights into eukaryotic translation initiation factors 5A1 and 5A2 and their roles in human health and disease. Cancer Cell Int 2020; 20:142. [PMID: 32368188 PMCID: PMC7191727 DOI: 10.1186/s12935-020-01226-7] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2020] [Accepted: 04/20/2020] [Indexed: 02/05/2023] Open
Abstract
The eukaryotic translation initiation factor 5A1 (eIF5A1) and its homolog eIF5A2 are the only two human proteins containing the unique post-translational modification-hypusination, which is essential for the function of these two proteins. eIF5A1 was initially identified as a translation initiation factor by promoting the first peptide bond formation of protein during translation; however, recent results suggest that eIF5A1 also functions as a translation elongation factor. It has been shown that eIF5A1 is implicated in certain human diseases, including diabetes, several human cancer types, viral infections and diseases of neural system. Meanwhile, eIF5A2 is overexpressed in many cancers, and plays an important role in the development and progression of cancers. As multiple roles of these two factors were observed among these studies, therefore, it remains unclear whether they act as oncogene or tumor suppressor. In this review, the recent literature of eIF5As and their roles in human diseases, especially in human cancers, will be discussed.
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Affiliation(s)
- Gao-Qi Wu
- Laboratory of Cancer Biology and Epigenetics, Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong 515041 People’s Republic of China
| | - Yan-Ming Xu
- Laboratory of Cancer Biology and Epigenetics, Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong 515041 People’s Republic of China
| | - Andy T. Y. Lau
- Laboratory of Cancer Biology and Epigenetics, Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong 515041 People’s Republic of China
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17
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Zhong X, Xiu H, Bi Y, Zhang H, Chang L, Diao H. Targeting eIF5A2 inhibits prostate carcinogenesis, migration, invasion and metastasis in vitro and in vivo. Bioengineered 2020; 11:619-627. [PMID: 32522053 PMCID: PMC8291822 DOI: 10.1080/21655979.2020.1774993] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2020] [Revised: 05/24/2020] [Accepted: 05/25/2020] [Indexed: 02/07/2023] Open
Abstract
Overexpression of eukaryotic initiation factor- 5A2 (eIF5A2) has been implicated in promoting tumor cell migration and invasion in many cancers. However, whether eIF5A2 could be as the target for prostate cancer (PCa) treatment is still unknown. In this study, small interfering RNA specific for eIF5A2 (eIF5A2 siRNA) and lentivector for eIF5A2 shRNA (Lv-eIF5A2 shRNA) was performed to down-regulate eIF5A2 expression in PCa PC-3 M IE8 cells and in animal tumor model, respectively. The biological function of eIF5A2 siRNA or Lv-eIF5A2 shRNA on PC-3 M IE8 cell growth, apoptosis, migration, invasion and lung metastasis were explored. The results showed that targeting eIF5A2 inhibited PC-3 M IE8 cell invasion, migration, proliferation and increased cell apoptosis in vitro, and inhibited tumor growth and lung metastasis in vivo. Analysis of eIF5A2 signaling pathways in the clonal derivatives showed a decrease in MMP-2 and MMP-9 activation and increase in bcl-2 expression. We therefore concluded that therapies targeting the eIF5A2 signaling pathway may be more effective to prevent organ metastasis and primary tumor formation.
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Affiliation(s)
- Xiulong Zhong
- Department of Urology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China
| | - Hong Xiu
- Department of Nursing, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China
| | - Yanan Bi
- Department of Nursing, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China
| | - Hongmei Zhang
- Department of Nursing, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China
| | - Laizhen Chang
- Department of Medicine, Huaou Group Hospital, Qingdao, Shandong, China
| | - Huifeng Diao
- Department of Urology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China
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18
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Scarlata S. The role of phospholipase Cβ on the plasma membrane and in the cytosol: How modular domains enable novel functions. Adv Biol Regul 2019; 73:100636. [PMID: 31409535 DOI: 10.1016/j.jbior.2019.100636] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2019] [Revised: 07/14/2019] [Accepted: 07/25/2019] [Indexed: 01/14/2023]
Abstract
Phospholipase Cβ (PLCβ) is a signaling enzyme activated by G proteins to generate calcium signals. The catalytic core of PLCβ is surrounded by modular domains that mediate the interaction of the enzyme with known protein partners on the plasma membrane. The C-terminal region PLCβ contains a novel coiled-coil domain that is required for Gαq binding and activation. Recent work has shown that this domain also binds a number of cytosolic proteins that regulate protein translation, and that these proteins compete with Gαq for PLCβ binding. The ability of PLCβ to shuttle between the cytosol to impact protein translation and the plasma membrane to mediate calcium signals puts PLCβ in a central role in cell function.
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Affiliation(s)
- Suzanne Scarlata
- Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, 100 Institute Rd., Worcester, MA, 01609, United States.
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19
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Meng QB, Peng JJ, Qu ZW, Zhu XM, Wen Z, Kang WM. Eukaryotic initiation factor 5A2 and human digestive system neoplasms. World J Gastrointest Oncol 2019; 11:449-458. [PMID: 31236196 PMCID: PMC6580320 DOI: 10.4251/wjgo.v11.i6.449] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/31/2018] [Revised: 04/17/2019] [Accepted: 05/04/2019] [Indexed: 02/05/2023] Open
Abstract
Eukaryotic initiation factor 5A2 (eIF5A2), as one of the two isoforms in the family, is reported to be a novel oncogenic protein that is involved in multiple aspects of many types of human cancer. Overexpression or gene amplification of EIF5A2 has been demonstrated in many cancers. Accumulated evidence shows that eIF5A2 initiates tumor formation, enhances cancer cell growth, increases cancer cell metastasis, and promotes treatment resistance through multiple means, including inducing epithelial–mesenchymal transition, cytoskeletal rearrangement, angiogenesis, and metabolic reprogramming. Expression of eIF5A2 in cancer correlates with poor survival, advanced disease stage, as well as metastasis, suggesting that eIF5A2 function is crucial for tumor development and maintenance but not for normal tissue homeostasis. All these studies suggest that eIF5A2 is a useful biomarker in the prediction of cancer prognosis and serves as an anticancer molecular target. This review focuses on the expression, subcellular localization, post-translational modifications, and regulatory networks of eIF5A2, as well as its biochemical functions and evolving clinical applications in cancer, especially in human digestive system neoplasms.
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Affiliation(s)
- Qing-Bin Meng
- Department of Gastrointestinal Surgery, the First Hospital of Wuhan City, Wuhan 430022, Hubei Province, China
| | - Jing-Jing Peng
- Department of Gastroenterology, General Hospital of the Yangtze River Shipping, Wuhan 430015, Hubei Province, China
| | - Zi-Wei Qu
- Department of Gastrointestinal Surgery, the First Hospital of Wuhan City, Wuhan 430022, Hubei Province, China
| | | | - Zhang Wen
- Department of Hepato-Biliary-Pancreatic Surgery and Liver Transplantation, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
| | - Wei-Ming Kang
- Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
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Aksu M, Trakhanov S, Vera Rodriguez A, Görlich D. Structural basis for the nuclear import and export functions of the biportin Pdr6/Kap122. J Cell Biol 2019; 218:1839-1852. [PMID: 31023722 PMCID: PMC6548137 DOI: 10.1083/jcb.201812093] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2018] [Revised: 03/26/2019] [Accepted: 03/28/2019] [Indexed: 12/26/2022] Open
Abstract
Importins ferry proteins into nuclei while exportins carry cargoes to the cytoplasm. In the accompanying paper in this issue (Vera Rodriguez et al. 2019. J. Cell Biol. https://doi.org/10.1083/jcb.201812091), we discovered that Pdr6 is a biportin that imports, e.g., the SUMO E2 ligase Ubc9 while depleting the translation factor eIF5A from the nuclear compartment. In this paper, we report the structures of key transport intermediates, namely, of the Ubc9•Pdr6 import complex, of the RanGTP•Pdr6 heterodimer, and of the trimeric RanGTP•Pdr6•eIF5A export complex. These revealed nonlinear transport signals, chaperone-like interactions, and how the RanGTPase system drives Pdr6 to transport Ubc9 and eIF5A in opposite directions. The structures also provide unexpected insights into the evolution of transport selectivity. Specifically, they show that recognition of Ubc9 by Pdr6 differs fundamentally from that of the human Ubc9-importer Importin 13. Likewise, Pdr6 recognizes eIF5A in a nonhomologous manner compared with the mammalian eIF5A-exporter Exportin 4. This suggests that the import of Ubc9 and active nuclear exclusion of eIF5A evolved in different eukaryotic lineages more than once and independently from each other.
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Affiliation(s)
- Metin Aksu
- Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
| | - Sergei Trakhanov
- Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
| | - Arturo Vera Rodriguez
- Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
| | - Dirk Görlich
- Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
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Abstract
Advances in our understanding of the metabolism and molecular functions of polyamines and their alterations in cancer have led to resurgence in the interest of targeting polyamine metabolism as an anticancer strategy. Increasing knowledge of the interplay between polyamine metabolism and other cancer-driving pathways, including the PTEN-PI3K-mTOR complex 1 (mTORC1), WNT signalling and RAS pathways, suggests potential combination therapies that will have considerable clinical promise. Additionally, an expanding number of promising clinical trials with agents targeting polyamines for both therapy and prevention are ongoing. New insights into molecular mechanisms linking dysregulated polyamine catabolism and carcinogenesis suggest additional strategies that can be used for cancer prevention in at-risk individuals. In addition, polyamine blocking therapy, a strategy that combines the inhibition of polyamine biosynthesis with the simultaneous blockade of polyamine transport, can be more effective than therapies based on polyamine depletion alone and may involve an antitumour immune response. These findings open up new avenues of research into exploiting aberrant polyamine metabolism for anticancer therapy.
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Affiliation(s)
- Robert A Casero
- Department of Oncology, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA.
| | - Tracy Murray Stewart
- Department of Oncology, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA
| | - Anthony E Pegg
- Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA, USA
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22
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Park MH, Wolff EC. Hypusine, a polyamine-derived amino acid critical for eukaryotic translation. J Biol Chem 2018; 293:18710-18718. [PMID: 30257869 DOI: 10.1074/jbc.tm118.003341] [Citation(s) in RCA: 136] [Impact Index Per Article: 19.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
The natural amino acid hypusine (N ϵ-4-amino-2-hydroxybutyl(lysine)) is derived from the polyamine spermidine, and occurs only in a single family of cellular proteins, eukaryotic translation factor 5A (eIF5A) isoforms. Hypusine is formed by conjugation of the aminobutyl moiety of spermidine to a specific lysine residue of this protein. The posttranslational synthesis of hypusine involves two enzymatic steps, catalyzed by deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Hypusine is essential for eIF5A activity. Inactivation of either the eIF5A or the DHPS gene is lethal in yeast and mouse, underscoring the vital role of eIF5A hypusination in eukaryotic cell growth and animal development. The long and basic side chain of the hypusine residue promotes eIF5A-mediated translation elongation by facilitating peptide bond formation at polyproline stretches and at many other ribosome-pausing sites. It also enhances translation termination by stimulating peptide release. By promoting translation, the hypusine modification of eIF5A provides a key link between polyamines and cell growth regulation. eIF5A has been implicated in several human pathological conditions. Recent genetic data suggest that eIF5A haploinsufficiency or impaired deoxyhypusine synthase activity is associated with neurodevelopmental disorders in humans.
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Affiliation(s)
- Myung Hee Park
- From the NIDCR, National Institutes of Health, Bethesda, Maryland 20892
| | - Edith C Wolff
- From the NIDCR, National Institutes of Health, Bethesda, Maryland 20892
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23
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Chen C, Zhang B, Wu S, Song Y, Li J. Knockdown of EIF5A2 inhibits the malignant potential of non-small cell lung cancer cells. Oncol Lett 2018. [PMID: 29541224 DOI: 10.3892/ol.2018.7832] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Eukaryotic translation initiation factor 5A2 (EIF5A2) has been demonstrated to be upregulated in numerous types of human cancer and is associated with cancer progression. However, the expression and role of EIF5A2 in non-small cell lung cancer (NSCLC) remains unclear. In the present study, the role of EIF5A2 in NSCLC was investigated, in addition to the underlying molecular mechanisms by which EIF5A2 acts. Relative EIF5A2 expression levels were determined in NSCLC cells and compared with levels in non-cancerous lung tissues. Short interfering (si)RNA targeted against EIF5A2 was used to knock down EIF5A2 levels in NSCLC cells. Cell proliferation, apoptosis rate, migration ability and invasion ability were determined in untreated and siRNA-treated NSCLC cells, in addition to the relative protein expression levels of various tumorigenic proteins and E-cadherin. EIF5A2 expression was significantly higher in NSCLC tissues compared with adjacent normal tissues. Knockdown of EIF5A2 in the NSCLC cells significantly inhibited cell proliferation and induced apoptosis. Furthermore, EIF5A2 silencing suppressed cell migratory and invasive capacities in vitro. Silencing of EIF5A2 in the NSCLC cells resulted in the downregulation of the tumorigenic proteins, apoptosis regulator Bcl-2 and myc proto-oncogene protein, and upregulation of E-cadherin, suggesting that EIF5A2 promotes proliferation and metastasis through these proteins. EIF5A2 may therefore serve as a novel therapeutic target for the treatment of NSCLC.
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Affiliation(s)
- Cheng Chen
- Department of Thoracic Surgery, Affiliated Hospital of Zunyi Medical College, Zunyi, Guizhou 563000, P.R. China
| | - Bojia Zhang
- Department of Infectious Disease, Shanghai 10th People's Hospital, Tongji University School of Medicine, Shanghai 200072, P.R. China
| | - Shanshan Wu
- Department of Nursing, Affiliated Hospital of Zunyi Medical College, Zunyi, Guizhou 563000, P.R. China
| | - Yongxiang Song
- Department of Thoracic Surgery, Affiliated Hospital of Zunyi Medical College, Zunyi, Guizhou 563000, P.R. China
| | - Jian Li
- Department of Thoracic Surgery, Affiliated Hospital of Zunyi Medical College, Zunyi, Guizhou 563000, P.R. China
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24
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Moretti NS, Cestari I, Anupama A, Stuart K, Schenkman S. Comparative Proteomic Analysis of Lysine Acetylation in Trypanosomes. J Proteome Res 2018; 17:374-385. [PMID: 29168382 DOI: 10.1021/acs.jproteome.7b00603] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
Protein acetylation is a post-translational modification regulating diverse cellular processes. By using proteomic approaches, we identified N-terminal and ε-lysine acetylated proteins in Trypanosoma cruzi and Trypanosoma brucei, which are protozoan parasites that cause significant human and animal diseases. We detected 288 lysine acetylation sites in 210 proteins of procyclic form, an insect stage of T. brucei, and 380 acetylation sites in 285 proteins in the form of the parasite that replicates in mammalian bloodstream. In T. cruzi insect proliferative form we found 389 ε-lysine-acetylated sites in 235 proteins. Notably, we found distinct acetylation profiles according to the developmental stage and species, with only 44 common proteins between T. brucei stages and 18 in common between the two species. While K-ac proteins from T. cruzi are enriched in enzymes involved in oxidation/reduction balance, required for the parasite survival in the host, in T. brucei, most K-ac proteins are enriched in metabolic processes, essential for its adaptation in its hosts. We also identified in both parasites a quite variable N-terminal acetylation sites. Our results suggest that protein acetylation is involved in differential regulation of multiple cellular processes in Trypanosomes, contributing to our understanding of the essential mechanisms for parasite infection and survival.
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Affiliation(s)
- Nilmar Silvio Moretti
- Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo , R. Pedro de Toledo 669 L6A, 04039-032 São Paulo, SP, Brazil.,Center for Infectious Disease Research , 307 Westlake Avenue North, Suite 500, Seattle, Washington 98109, United States
| | - Igor Cestari
- Center for Infectious Disease Research , 307 Westlake Avenue North, Suite 500, Seattle, Washington 98109, United States
| | - Atashi Anupama
- Center for Infectious Disease Research , 307 Westlake Avenue North, Suite 500, Seattle, Washington 98109, United States
| | - Ken Stuart
- Center for Infectious Disease Research , 307 Westlake Avenue North, Suite 500, Seattle, Washington 98109, United States
| | - Sergio Schenkman
- Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo , R. Pedro de Toledo 669 L6A, 04039-032 São Paulo, SP, Brazil
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25
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Barbosa NM, Boldrin PEG, Rossi D, Yamamoto PA, Watanabe TF, Serrão VH, Hershey JWB, Fraser CS, Valentini SR, Zanelli CF. Mapping surface residues of eIF5A that are important for binding to the ribosome using alanine scanning mutagenesis. Amino Acids 2016; 48:2363-74. [PMID: 27388480 PMCID: PMC5897047 DOI: 10.1007/s00726-016-2279-z] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2016] [Accepted: 06/11/2016] [Indexed: 01/15/2023]
Abstract
The translation elongation factor eIF5A is conserved through evolution and is necessary to rescue the ribosome during translation elongation of polyproline-containing proteins. Although the site of eIF5A binding to the ribosome is known, no systematic analysis has been performed so far to determine the important residues on the surface of eIF5A required for ribosome binding. In this study, we used clustered charged-to-alanine mutagenesis and structural modeling to address this question. We generated four new mutants of yeast eIF5A: tif51A-4, tif51A-6, tif51A-7 and tif51A-11, and complementation analysis revealed that tif51A-4 and tif51A-7 could not sustain cell growth in a strain lacking wild-type eIF5A. Moreover, the allele tif51A-4 also displayed negative dominance over wild-type eIF5A. Both in vivo GST-pulldowns and in vitro fluorescence anisotropy demonstrated that eIF5A from mutant tif51A-7 exhibited an importantly reduced affinity for the ribosome, implicating the charged residues in cluster 7 as determinant features on the eIF5A surface for contacting the ribosome. Notably, modified eIF5A from mutant tif51A-4, despite exhibiting the most severe growth phenotype, did not abolish ribosome interactions as with mutant tif51A-7. Taking into account the modeling eIF5A + 80S + P-tRNA complex, our data suggest that interactions of eIF5A with ribosomal protein L1 are more important to stabilize the interaction with the ribosome as a whole than the contacts with P-tRNA. Finally, the ability of eIF5A from tif51A-4 to bind to the ribosome while potentially blocking physical interaction with P-tRNA could explain its dominant negative phenotype.
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Affiliation(s)
- Natália M Barbosa
- Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University-UNESP, Rod Araraquara-Jaú Km01, Araraquara, SP, 14800-903, Brazil
| | - Paulo E G Boldrin
- Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University-UNESP, Rod Araraquara-Jaú Km01, Araraquara, SP, 14800-903, Brazil
| | - Danuza Rossi
- Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University-UNESP, Rod Araraquara-Jaú Km01, Araraquara, SP, 14800-903, Brazil
| | - Priscila A Yamamoto
- Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University-UNESP, Rod Araraquara-Jaú Km01, Araraquara, SP, 14800-903, Brazil
| | - Tatiana F Watanabe
- Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University-UNESP, Rod Araraquara-Jaú Km01, Araraquara, SP, 14800-903, Brazil
| | - Vitor H Serrão
- Physics and Interdisciplinary Science Department, Physics Institute of Sao Carlos, University of Sao Paulo-USP, Sao Carlos, SP, 13563-120, Brazil
| | - John W B Hershey
- Molecular and Cellular Biology Department, University of California, Davis, CA, 95616, USA
| | - Christopher S Fraser
- Molecular and Cellular Biology Department, University of California, Davis, CA, 95616, USA
| | - Sandro R Valentini
- Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University-UNESP, Rod Araraquara-Jaú Km01, Araraquara, SP, 14800-903, Brazil
| | - Cleslei F Zanelli
- Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University-UNESP, Rod Araraquara-Jaú Km01, Araraquara, SP, 14800-903, Brazil.
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26
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The molecular choreography of protein synthesis: translational control, regulation, and pathways. Q Rev Biophys 2016; 49:e11. [PMID: 27658712 DOI: 10.1017/s0033583516000056] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Translation of proteins by the ribosome regulates gene expression, with recent results underscoring the importance of translational control. Misregulation of translation underlies many diseases, including cancer and many genetic diseases. Decades of biochemical and structural studies have delineated many of the mechanistic details in prokaryotic translation, and sketched the outlines of eukaryotic translation. However, translation may not proceed linearly through a single mechanistic pathway, but likely involves multiple pathways and branchpoints. The stochastic nature of biological processes would allow different pathways to occur during translation that are biased by the interaction of the ribosome with other translation factors, with many of the steps kinetically controlled. These multiple pathways and branchpoints are potential regulatory nexus, allowing gene expression to be tuned at the translational level. As research focus shifts toward eukaryotic translation, certain themes will be echoed from studies on prokaryotic translation. This review provides a general overview of the dynamic data related to prokaryotic and eukaryotic translation, in particular recent findings with single-molecule methods, complemented by biochemical, kinetic, and structural findings. We will underscore the importance of viewing the process through the viewpoints of regulation, translational control, and heterogeneous pathways.
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27
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Aksu M, Trakhanov S, Görlich D. Structure of the exportin Xpo4 in complex with RanGTP and the hypusine-containing translation factor eIF5A. Nat Commun 2016; 7:11952. [PMID: 27306458 PMCID: PMC4912631 DOI: 10.1038/ncomms11952] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2016] [Accepted: 05/16/2016] [Indexed: 12/28/2022] Open
Abstract
Xpo4 is a bidirectional nuclear transport receptor that mediates nuclear export of eIF5A and Smad3 as well as import of Sox2 and SRY. How Xpo4 recognizes such a variety of cargoes is as yet unknown. Here we present the crystal structure of the RanGTP·Xpo4·eIF5A export complex at 3.2 Å resolution. Xpo4 has a similar structure as CRM1, but the NES-binding site is occluded, and a new interaction site evolved that recognizes both globular domains of eIF5A. eIF5A contains hypusine, a unique amino acid with two positive charges, which is essential for cell viability and eIF5A function in translation. The hypusine docks into a deep, acidic pocket of Xpo4 and is thus a critical element of eIF5A's complex export signature. This further suggests that Xpo4 recognizes other cargoes differently, and illustrates how Xpo4 suppresses - in a chaperone-like manner - undesired interactions of eIF5A inside nuclei.
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Affiliation(s)
- Metin Aksu
- Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
| | - Sergei Trakhanov
- Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
| | - Dirk Görlich
- Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
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28
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Ermert AL, Mailliet K, Hughes J. Holophytochrome-Interacting Proteins in Physcomitrella: Putative Actors in Phytochrome Cytoplasmic Signaling. FRONTIERS IN PLANT SCIENCE 2016; 7:613. [PMID: 27242820 PMCID: PMC4867686 DOI: 10.3389/fpls.2016.00613] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/01/2016] [Accepted: 04/21/2016] [Indexed: 05/26/2023]
Abstract
Phytochromes are the principle photoreceptors in light-regulated plant development, primarily acting via translocation of the light-activated photoreceptor into the nucleus and subsequent gene regulation. However, several independent lines of evidence indicate unambiguously that an additional cytoplasmic signaling mechanism must exist. Directional responses in filament tip cells of the moss Physcomitrella patens are steered by phy4 which has been shown to interact physically with the blue light receptor phototropin at the plasma membrane. This complex might perceive and transduce vectorial information leading to cytoskeleton reorganization and finally a directional growth response. We developed yeast two-hybrid procedures using photochemically functional, full-length phy4 as bait in Physcomitrella cDNA library screens and growth assays under different light conditions, revealing Pfr-dependent interactions possibly associated with phytochrome cytoplasmic signaling. Candidate proteins were then expressed in planta with fluorescent protein tags to determine their intracellular localization in darkness and red light. Of 14 candidates, 12 were confirmed to interact with phy4 in planta using bimolecular fluorescence complementation. We also used database information to study their expression patterns relative to those of phy4. We discuss the likely functional characteristics of these holophytochrome-interacting proteins (HIP's) and their possible roles in signaling.
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29
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Schmidt C, Becker T, Heuer A, Braunger K, Shanmuganathan V, Pech M, Berninghausen O, Wilson DN, Beckmann R. Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome. Nucleic Acids Res 2016; 44:1944-51. [PMID: 26715760 PMCID: PMC4770232 DOI: 10.1093/nar/gkv1517] [Citation(s) in RCA: 95] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2015] [Revised: 12/16/2015] [Accepted: 12/17/2015] [Indexed: 12/31/2022] Open
Abstract
During protein synthesis, ribosomes become stalled on polyproline-containing sequences, unless they are rescued in archaea and eukaryotes by the initiation factor 5A (a/eIF-5A) and in bacteria by the homologous protein EF-P. While a structure of EF-P bound to the 70S ribosome exists, structural insight into eIF-5A on the 80S ribosome has been lacking. Here we present a cryo-electron microscopy reconstruction of eIF-5A bound to the yeast 80S ribosome at 3.9 Å resolution. The structure reveals that the unique and functionally essential post-translational hypusine modification reaches toward the peptidyltransferase center of the ribosome, where the hypusine moiety contacts A76 of the CCA-end of the P-site tRNA. These findings would support a model whereby eIF-5A stimulates peptide bond formation on polyproline-stalled ribosomes by stabilizing and orienting the CCA-end of the P-tRNA, rather than by directly contributing to the catalysis.
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Affiliation(s)
- Christian Schmidt
- Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany
| | - Thomas Becker
- Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany
| | - André Heuer
- Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany
| | - Katharina Braunger
- Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany
| | - Vivekanandan Shanmuganathan
- Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany
| | - Markus Pech
- Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany
| | - Otto Berninghausen
- Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany
| | - Daniel N Wilson
- Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany
| | - Roland Beckmann
- Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany
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30
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Tariq M, Ito A, Ishfaq M, Bradshaw E, Yoshida M. Eukaryotic translation initiation factor 5A (eIF5A) is essential for HIF-1α activation in hypoxia. Biochem Biophys Res Commun 2016; 470:417-424. [PMID: 26773503 DOI: 10.1016/j.bbrc.2016.01.024] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2015] [Accepted: 01/05/2016] [Indexed: 12/27/2022]
Abstract
The eukaryotic initiation factor 5A (eIF5A) is an essential protein involved in translation elongation and cell proliferation. eIF5A undergoes several post-translational modifications including hypusination and acetylation. Hypusination is indispensable for the function of eIF5A. On the other hand, the precise function of acetylation remains unknown, but it may render the protein inactive since hypusination blocks acetylation. Here, we report that acetylation of eIF5A increases under hypoxia. During extended hypoxic periods an increase in the level of eIF5A acetylation correlated with a decrease in HIF-1α, suggesting involvement of eIF5A activity in HIF-1α expression under hypoxia. Indeed, suppression of eIF5A by siRNA oligo-mediated knockdown or treatment with GC7, a deoxyhypusine synthase inhibitor, led to significant reduction of HIF-1α activity. Furthermore, knockdown of eIF5A or GC7 treatment reduced tumor spheroid formation with a concomitant decrease in HIF-1α expression. Our results suggest that functional, hypusinated eIF5A is necessary for HIF-1α expression during hypoxia and that eIF5A is an attractive target for cancer therapy.
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Affiliation(s)
- Mohammad Tariq
- Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Graduate School of Science and Engineering, Saitama University, 645 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan
| | - Akihiro Ito
- Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Japan Agency for Medical Research and Development, AMED-CREST, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004, Japan.
| | - Muhammad Ishfaq
- Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Graduate School of Science and Engineering, Saitama University, 645 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan
| | - Elliot Bradshaw
- Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Graduate School of Science and Engineering, Saitama University, 645 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan
| | - Minoru Yoshida
- Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Graduate School of Science and Engineering, Saitama University, 645 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan; Japan Agency for Medical Research and Development, AMED-CREST, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004, Japan
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31
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Liu W, Shang FF, Xu Y, Belegu V, Xia L, Zhao W, Liu R, Wang W, Liu J, Li CY, Wang TH. eIF5A1/RhoGDIα pathway: a novel therapeutic target for treatment of spinal cord injury identified by a proteomics approach. Sci Rep 2015; 5:16911. [PMID: 26593060 PMCID: PMC4655360 DOI: 10.1038/srep16911] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2015] [Accepted: 10/22/2015] [Indexed: 02/05/2023] Open
Abstract
Spinal cord injury (SCI) is frequently accompanied by a degree of spontaneous functional recovery. The underlying mechanisms through which such recovery is generated remain elusive. In this study, we observed a significant spontaneous motor function recovery 14 to 28 days after spinal cord transection (SCT) in rats. Using a comparative proteomics approach, caudal to the injury, we detected difference in 20 proteins. Two of these proteins, are eukaryotic translation initiation factor 5A1 (eIF5A1) that is involved in cell survival and proliferation, and Rho GDP dissociation inhibitor alpha (RhoGDIα), a member of Rho GDI family that is involved in cytoskeletal reorganization. After confirming the changes in expression levels of these two proteins following SCT, we showed that in vivo eIF5A1 up-regulation and down-regulation significantly increased and decreased, respectively, motor function recovery. In vitro, eIF5A1 overexpression in primary neurons increased cell survival and elongated neurite length while eIF5A1 knockdown reversed these results. We found that RhoGDIα up-regulation and down-regulation rescues the effect of eIF5A1 down-regulation and up-regulation both in vivo and in vitro. Therefore, we have identified eIF5A1/RhoGDIα pathway as a new therapeutic target for treatment of spinal cord injured patients.
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Affiliation(s)
- Wei Liu
- Institute of Neurological Disease, The state key laboratory of Biotherapy, Department of Anesthesiology and Translational Neuroscience Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, 61041, P.R. China
| | - Fei-Fei Shang
- Institute of Neurological Disease, The state key laboratory of Biotherapy, Department of Anesthesiology and Translational Neuroscience Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, 61041, P.R. China
| | - Yang Xu
- Institute of Neurological Disease, The state key laboratory of Biotherapy, Department of Anesthesiology and Translational Neuroscience Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, 61041, P.R. China
| | - Visar Belegu
- Department of Neurology, Johns Hopkins School of Medicine, Baltimore, MD, USA
| | - Lei Xia
- Institute of Neurological Disease, The state key laboratory of Biotherapy, Department of Anesthesiology and Translational Neuroscience Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, 61041, P.R. China
| | - Wei Zhao
- Institute of Neurological Disease, The state key laboratory of Biotherapy, Department of Anesthesiology and Translational Neuroscience Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, 61041, P.R. China
| | - Ran Liu
- Institute of Neurological Disease, The state key laboratory of Biotherapy, Department of Anesthesiology and Translational Neuroscience Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, 61041, P.R. China
| | - Wei Wang
- Institute of Neurological Disease, The state key laboratory of Biotherapy, Department of Anesthesiology and Translational Neuroscience Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, 61041, P.R. China
| | - Jin Liu
- Institute of Neurological Disease, The state key laboratory of Biotherapy, Department of Anesthesiology and Translational Neuroscience Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, 61041, P.R. China
| | - Chen-Yun Li
- Key Laboratory of Agro-Biodiversity and Pest Management of Education Ministry of China, Yunnan Agricultural University, Kunming, 650000, P.R. China
| | - Ting-Hua Wang
- Institute of Neurological Disease, The state key laboratory of Biotherapy, Department of Anesthesiology and Translational Neuroscience Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, 61041, P.R. China
- Institute of Neuroscience, Kunming medical University, Kunming 650031, P.R. China
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32
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Lassak J, Wilson DN, Jung K. Stall no more at polyproline stretches with the translation elongation factors EF-P and IF-5A. Mol Microbiol 2015; 99:219-35. [PMID: 26416626 DOI: 10.1111/mmi.13233] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/25/2015] [Indexed: 01/18/2023]
Abstract
Synthesis of polyproline proteins leads to translation arrest. To overcome this ribosome stalling effect, bacteria depend on a specialized translation elongation factor P (EF-P), being orthologous and functionally identical to eukaryotic/archaeal elongation factor e/aIF-5A (recently renamed 'EF5'). EF-P binds to the stalled ribosome between the peptidyl-tRNA binding and tRNA-exiting sites, and stimulates peptidyl-transferase activity, thus allowing translation to resume. In their active form, both EF-P and e/aIF-5A are post-translationally modified at a positively charged residue, which protrudes toward the peptidyl-transferase center when bound to the ribosome. While archaeal and eukaryotic IF-5A strictly depend on (deoxy-) hypusination (hypusinylation) of a conserved lysine, bacteria have evolved diverse analogous modification strategies to activate EF-P. In Escherichia coli and Salmonella enterica a lysine is extended by β-lysinylation and subsequently hydroxylated, whereas in Pseudomonas aeruginosa and Shewanella oneidensis an arginine in the equivalent position is rhamnosylated. Inactivation of EF-P, or the corresponding modification systems, reduces not only bacterial fitness, but also impairs virulence. Here, we review the function of EF-P and IF-5A and their unusual posttranslational protein modifications.
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Affiliation(s)
- Jürgen Lassak
- Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, D-81377, Munich, Germany.,Department of Biology I, Microbiology, Ludwig-Maximilians-Universität München, D-82152, Martinsried, Germany
| | - Daniel N Wilson
- Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, D-81377, Munich, Germany.,Gene Center, Department for Biochemistry, Ludwig-Maximilians-Universität München, 81377, Munich, Germany
| | - Kirsten Jung
- Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, D-81377, Munich, Germany.,Department of Biology I, Microbiology, Ludwig-Maximilians-Universität München, D-82152, Martinsried, Germany
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Mathews MB, Hershey JWB. The translation factor eIF5A and human cancer. BIOCHIMICA ET BIOPHYSICA ACTA 2015; 1849:836-44. [PMID: 25979826 PMCID: PMC4732523 DOI: 10.1016/j.bbagrm.2015.05.002] [Citation(s) in RCA: 132] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/06/2015] [Accepted: 05/06/2015] [Indexed: 12/14/2022]
Abstract
The eukaryotic initiation factor eIF5A is a translation factor that, unusually, has been assigned functions in both initiation and elongation. Additionally, it is implicated in transcription, mRNA turnover and nucleocytoplasmic transport. Two eIF5A isoforms are generated from distinct but related genes. The major isoform, eIF5A1, is considered constitutive, is abundantly expressed in most cells, and is essential for cell proliferation. The second isoform, eIF5A2, is expressed in few normal tissues but is highly expressed in many cancers and has been designated a candidate oncogene. Elevated expression of either isoform carries unfavorable prognostic implications for several cancers, and both have been advanced as cancer biomarkers. The amino acid hypusine, a presumptively unique eIF5A post-translational modification, is required for most known eIF5A functions and it renders eIF5A susceptible to inhibitors of the modification pathway as therapeutic targets. eIF5A has been shown to regulate a number of gene products specifically, termed the eIF5A regulon, and its role in translating proline-rich sequences has recently been identified. A model is advanced that accommodates eIF5A in both the initiation and elongation phases of translation. We review here the biochemical functions of eIF5A, the relationship of its isoforms with human cancer, and evolving clinical applications. This article is part of a Special Issue entitled: Translation and Cancer.
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Affiliation(s)
- Michael B Mathews
- Department of Medicine, Rutgers New Jersey Medical School, Newark, NJ 07103, USA.
| | - John W B Hershey
- Department of Biochemistry and Molecular Medicine, University of California Davis School of Medicine, Davis, CA 95616, USA.
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Kobayashi K, Katz A, Rajkovic A, Ishii R, Branson OE, Freitas MA, Ishitani R, Ibba M, Nureki O. The non-canonical hydroxylase structure of YfcM reveals a metal ion-coordination motif required for EF-P hydroxylation. Nucleic Acids Res 2014; 42:12295-305. [PMID: 25274739 PMCID: PMC4231759 DOI: 10.1093/nar/gku898] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
EF-P is a bacterial tRNA-mimic protein, which accelerates the ribosome-catalyzed polymerization of poly-prolines. In Escherichia coli, EF-P is post-translationally modified on a conserved lysine residue. The post-translational modification is performed in a two-step reaction involving the addition of a β-lysine moiety and the subsequent hydroxylation, catalyzed by PoxA and YfcM, respectively. The β-lysine moiety was previously shown to enhance the rate of poly-proline synthesis, but the role of the hydroxylation is poorly understood. We solved the crystal structure of YfcM and performed functional analyses to determine the hydroxylation mechanism. In addition, YfcM appears to be structurally distinct from any other hydroxylase structures reported so far. The structure of YfcM is similar to that of the ribonuclease YbeY, even though they do not share sequence homology. Furthermore, YfcM has a metal ion-coordinating motif, similar to YbeY. The metal ion-coordinating motif of YfcM resembles a 2-His-1-carboxylate motif, which coordinates an Fe(II) ion and forms the catalytic site of non-heme iron enzymes. Our findings showed that the metal ion-coordinating motif of YfcM plays an essential role in the hydroxylation of the β-lysylated lysine residue of EF-P. Taken together, our results suggested the potential catalytic mechanism of hydroxylation by YfcM.
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Affiliation(s)
- Kan Kobayashi
- Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan Global Research Cluster, RIKEN, 2-1, Hirosawa, Wako, Saitama, 351-0198, Japan
| | - Assaf Katz
- Department of Microbiology, Ohio State University, Columbus, OH 43210, USA
| | - Andrei Rajkovic
- Molecular, Cell, and Developmental Biology Program, Ohio State University, Columbus, OH 43210, USA
| | - Ryohei Ishii
- Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan Global Research Cluster, RIKEN, 2-1, Hirosawa, Wako, Saitama, 351-0198, Japan
| | - Owen E Branson
- Department of Biochemistry, Ohio State University, Columbus, OH 43210, USA
| | - Michael A Freitas
- Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, OH 43210, USA
| | - Ryuichiro Ishitani
- Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan Global Research Cluster, RIKEN, 2-1, Hirosawa, Wako, Saitama, 351-0198, Japan
| | - Michael Ibba
- Department of Microbiology, Ohio State University, Columbus, OH 43210, USA Ohio State Biochemistry Program, Center for RNA Biology, Ohio State University, Columbus, OH 43210, USA
| | - Osamu Nureki
- Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan Global Research Cluster, RIKEN, 2-1, Hirosawa, Wako, Saitama, 351-0198, Japan
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Abstract
In addition to the small and large ribosomal subunits, aminoacyl-tRNAs, and an mRNA, cellular protein synthesis is dependent on translation factors. The eukaryotic translation initiation factor 5A (eIF5A) and its bacterial ortholog elongation factor P (EF-P) were initially characterized based on their ability to stimulate methionyl-puromycin (Met-Pmn) synthesis, a model assay for protein synthesis; however, the function of these factors in cellular protein synthesis has been difficult to resolve. Interestingly, a conserved lysine residue in eIF5A is post-translationally modified to hypusine and the corresponding lysine residue in EF-P from at least some bacteria is modified by the addition of a β-lysine moiety. In this review, we provide a summary of recent data that have identified a novel role for the translation factor eIF5A and its hypusine modification in the elongation phase of protein synthesis and more specifically in stimulating the production of proteins containing runs of consecutive proline residues.
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Affiliation(s)
- Thomas E. Dever
- Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
| | - Erik Gutierrez
- Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
- Department of Biology, Johns Hopkins University, Baltimore, MD, USA
| | - Byung-Sik Shin
- Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
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Rossi D, Kuroshu R, Zanelli CF, Valentini SR. eIF5A and EF-P: two unique translation factors are now traveling the same road. WILEY INTERDISCIPLINARY REVIEWS. RNA 2014; 5:209-22. [PMID: 24402910 DOI: 10.1002/wrna.1211] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/01/2013] [Revised: 11/01/2013] [Accepted: 11/06/2013] [Indexed: 11/09/2022]
Abstract
Translational control is extremely important in all organisms, and some of its aspects are highly conserved among all primary kingdoms, such as those related to the translation elongation step. The previously classified translation initiation factor 5A (eIF5A) and its bacterial homologue elongation factor P (EF-P) were discovered in the late 70's and have recently been the object of many studies. eIF5A and EF-P are the only cellular proteins that undergo hypusination and lysinylation, respectively, both of which are unique posttranslational modifications. Herein, we review all the important discoveries related to the biochemical and functional characterization of these factors, highlighting the implication of eIF5A in translation elongation instead of initiation. The findings that eIF5A and EF-P are important for specific cellular processes and play a role in the relief of ribosome stalling caused by specific amino acid sequences, such as those containing prolines reinforce the hypothesis that these factors are involved in specialized translation. Although there are some divergences between these unique factors, recent studies have clarified that they act similarly during protein synthesis. Further studies may reveal their precise mechanism of ribosome activity modulation as well as the mRNA targets that require eIF5A and EF-P for their proper translation.
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Affiliation(s)
- Danuza Rossi
- Department of Biological Sciences, School of Pharmaceutical Sciences, Univ Estadual Paulista (UNESP), Araraquara, SP, Brazil
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Wang FW, Guan XY, Xie D. Roles of eukaryotic initiation factor 5A2 in human cancer. Int J Biol Sci 2013; 9:1013-20. [PMID: 24250246 PMCID: PMC3831114 DOI: 10.7150/ijbs.7191] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2013] [Accepted: 09/26/2013] [Indexed: 12/15/2022] Open
Abstract
Eukaryotic initiation factor 5A (eIF5A), the only known cellular protein containing the amino acid hypusine, is an essential component of translation elongation. eIF5A2, one of the two isoforms in the eIF5A family, is reported to be a novel oncogenic protein in many types of human cancer. Both in vitro and in vivo studies showed that eIF5A2 could initiate tumor formation, enhance cancer cell growth, and increase cancer cell motility and metastasis by inducing epithelial-mesenchymal transition. Accumulatied evidence suggests that eIF5A2 is a useful biomarker in the prediction of cancer prognoses and serves as an anticancer molecular target. In this review, we will focus on updating current knowledge of the EIF5A2 gene in human cancers. The molecular mechanisms of EIF5A2 related to tumorigenesis will also be discussed.
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Affiliation(s)
- Feng-wei Wang
- 1. Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China. Collaborative Innovation Center of Cancer Medicine
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38
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Galvão FC, Rossi D, Silveira WDS, Valentini SR, Zanelli CF. The deoxyhypusine synthase mutant dys1-1 reveals the association of eIF5A and Asc1 with cell wall integrity. PLoS One 2013; 8:e60140. [PMID: 23573236 PMCID: PMC3613415 DOI: 10.1371/journal.pone.0060140] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2012] [Accepted: 02/21/2013] [Indexed: 11/19/2022] Open
Abstract
The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1) catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1) and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1Δ and overexpression of TIF51A (eIF5A) or DYS1 is toxic for an asc1Δ strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1Δ mutants in comparison with the pkc1Δ mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of mRNAs associated with cell integrity.
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Affiliation(s)
- Fabio Carrilho Galvão
- Department of Biological Sciences, Univ Estadual Paulista – UNESP, Araraquara-Saõ Paulo, Brazil
| | - Danuza Rossi
- Department of Biological Sciences, Univ Estadual Paulista – UNESP, Araraquara-Saõ Paulo, Brazil
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Dias CAO, Garcia W, Zanelli CF, Valentini SR. eIF5A dimerizes not only in vitro but also in vivo and its molecular envelope is similar to the EF-P monomer. Amino Acids 2013; 44:631-44. [PMID: 22945904 DOI: 10.1007/s00726-012-1387-7] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2012] [Accepted: 08/01/2012] [Indexed: 11/28/2022]
Abstract
The protein eukaryotic initiation factor 5A (eIF5A) is highly conserved among archaea and eukaryotes, but not in bacteria. Bacteria have the elongation factor P (EF-P), which is structurally and functionally related to eIF5A. eIF5A is essential for cell viability and the only protein known to contain the amino acid residue hypusine, formed by post-translational modification of a specific lysine residue. Although eIF5A was initially identified as a translation initiation factor, recent studies strongly support a function for eIF5A in the elongation step of translation. However, the mode of action of eIF5A is still unknown. Here, we analyzed the oligomeric state of yeast eIF5A. First, by using size-exclusion chromatography, we showed that this protein exists as a dimer in vitro, independent of the hypusine residue or electrostatic interactions. Protein-protein interaction assays demonstrated that eIF5A can form oligomers in vitro and in vivo, in an RNA-dependent manner, but independent of the hypusine residue or the ribosome. Finally, small-angle X-ray scattering (SAXS) experiments confirmed that eIF5A behaves as a stable dimer in solution. Moreover, the molecular envelope determined from the SAXS data shows that the eIF5A dimer is L-shaped and superimposable on the tRNA(Phe) tertiary structure, analogously to the EF-P monomer.
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Affiliation(s)
- Camila Arnaldo Olhê Dias
- Department of Biological Sciences, School of Pharmaceutical Sciences, UNESP-Univ Estadual Paulista, Rodovia Araraquara-Jaú, km 01, Araraquara, SP 14801-902, Brazil
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40
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Mittal N, Subramanian G, Bütikofer P, Madhubala R. Unique posttranslational modifications in eukaryotic translation factors and their roles in protozoan parasite viability and pathogenesis. Mol Biochem Parasitol 2013; 187:21-31. [PMID: 23201129 DOI: 10.1016/j.molbiopara.2012.11.001] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2012] [Revised: 11/04/2012] [Accepted: 11/05/2012] [Indexed: 11/21/2022]
Abstract
Protozoan parasites are one of the major causes of diseases worldwide. The vector transmitted parasites exhibit complex life cycles involving interactions between humans, protozoa, and arthropods. In order to adapt themselves to the changing microenvironments, they have to undergo complex morphological and metabolic changes. These changes can be brought about by expressing a new pool of proteins in the cell or by modifying the existing repertoire of proteins via posttranslational modifications (PTMs). PTMs involve covalent modification and processing of proteins thereby modulating their functions. Some of these changes may involve PTMs of parasite proteins to help the parasite survive within the host and the vector. Out of many PTMs known, three are unique since they occur only on single proteins: ethanolamine phosphoglycerol (EPG) glutamate, hypusine and diphthamide. These modifications occur on eukaryotic elongation factor 1A (eEF1A), eukaryotic initiation factor 5A (eIF5A) and eukaryotic elongation factor 2 (eEF2), respectively. Interestingly, the proteins carrying these unique modifications are all involved in the elongation steps of translation. Here we review these unique PTMs, which are well conserved in protozoan parasites, and discuss their roles in viability and pathogenesis of parasites. Characterization of these modifications and studying their roles in physiology as well as pathogenesis will provide new insights in parasite biology, which may also help in developing new therapeutic interventions.
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Affiliation(s)
- Nimisha Mittal
- School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
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41
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Ishfaq M, Maeta K, Maeda S, Natsume T, Ito A, Yoshida M. Acetylation regulates subcellular localization of eukaryotic translation initiation factor 5A (eIF5A). FEBS Lett 2012; 586:3236-41. [PMID: 22771473 DOI: 10.1016/j.febslet.2012.06.042] [Citation(s) in RCA: 57] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2012] [Revised: 06/22/2012] [Accepted: 06/22/2012] [Indexed: 10/28/2022]
Abstract
Eukaryotic translation initiation factor 5A (eIF5A) is a protein subject to hypusination, which is essential for its function. eIF5A is also acetylated, but the role of that modification is unknown. Here, we report that acetylation regulates the subcellular localization of eIF5A. We identified PCAF as the major cellular acetyltransferase of eIF5A, and HDAC6 and SIRT2 as its major deacetylases. Inhibition of the deacetylases or impaired hypusination increased acetylation of eIF5A, leading to nuclear accumulation. As eIF5A is constitutively hypusinated under physiological conditions, we suggest that reversible acetylation plays a major role in controlling the subcellular localization of eIF5A.
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Affiliation(s)
- Muhammad Ishfaq
- Chemical Genetic Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, Japan
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42
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Schwentke A, Krepstakies M, Mueller AK, Hammerschmidt-Kamper C, Motaal BA, Bernhard T, Hauber J, Kaiser A. In vitro and in vivo silencing of plasmodial dhs and eIf-5a genes in a putative, non-canonical RNAi-related pathway. BMC Microbiol 2012; 12:107. [PMID: 22694849 PMCID: PMC3438091 DOI: 10.1186/1471-2180-12-107] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2012] [Accepted: 05/31/2012] [Indexed: 01/21/2023] Open
Abstract
BACKGROUND Deoxyhypusine synthase (DHS) catalyzes the first step in hypusine biosynthesis of eukaryotic initiation factor 5A (eIF-5A) in Plasmodium falciparum. Target evaluation of parasitic DHS has recently been performed with CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease. CNI-1493 prevented infected mice from experimental cerebral malaria by decreasing the levels in hypusinated eIF-5A and serum TNF, implicating a link between cytokine signaling and the hypusine pathway.Therefore we addressed the question whether either DHS itself or eIF-5A is required for the outcome of severe malaria. In a first set of experiments we performed an in vitro knockdown of the plasmodial eIF-5A and DHS proteins by RNA interference (RNAi) in 293 T cells. Secondly, transfection of siRNA constructs into murine Plasmodium schizonts was performed which, in turn, were used for infection. RESULTS 293 T cells treated with plasmodial DHS- and eIF-5A specific siRNAs or control siRNAs were analyzed by RT-PCR to determine endogenous dhs -and eIF-5A mRNA levels. The expressed DHS-shRNA and EIF-5A-shRNA clearly downregulated the corresponding transcript in these cells. Interestingly, mice infected with transgenic schizonts expressing either the eIF-5A or dhs shRNA showed an elevated parasitemia within the first two days post infection which then decreased intermittently. These results were obtained without drug selection. Blood samples, which were taken from the infected mice at day 5 post infection with either the expressed EIF-5A-shRNA or the DHS-shRNA were analyzed by RT-PCR and Western blot techniques, demonstrating the absence of either the hypusinated form of eIF-5A or DHS. CONCLUSIONS Infection of NMRI mice with schizonts from the lethal P. berghei ANKA wildtype strain transgenic for plasmodial eIF-5A-specific shRNA or DHS-specific shRNA resulted in low parasitemia 2-9 days post infection before animals succumbed to hyperparasitemia similar to infections with the related but non-lethal phenotype P. berghei strain NK65. RT-PCR and Western blot experiments performed with blood from the transfected erythrocytic stages showed that both genes are important for the proliferation of the parasite. Moreover, these experiments clearly demonstrate that the hypusine pathway in Plasmodium is linked to human iNos induction.
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Affiliation(s)
- Andreas Schwentke
- University Duisburg-Essen, Medical Research Centre, Institute of Pharmacogenetics, Hufelandstrasse 55, 45147, Essen, Germany
| | - Marcel Krepstakies
- Heinrich Pette Institute - Leibniz Institute for Experimental Virology, Martinistrasse 52, 20251, Hamburg, Germany
| | - Ann-Kristin Mueller
- Department of Infectious Diseases, Parasitology Unit, University Hospital Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany
| | - Christiane Hammerschmidt-Kamper
- Department of Infectious Diseases, Parasitology Unit, University Hospital Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany
| | - Basma A Motaal
- Heinrich Pette Institute - Leibniz Institute for Experimental Virology, Martinistrasse 52, 20251, Hamburg, Germany
| | - Tina Bernhard
- University Duisburg-Essen, Medical Research Centre, Institute of Pharmacogenetics, Hufelandstrasse 55, 45147, Essen, Germany
| | - Joachim Hauber
- Heinrich Pette Institute - Leibniz Institute for Experimental Virology, Martinistrasse 52, 20251, Hamburg, Germany
| | - Annette Kaiser
- University Duisburg-Essen, Medical Research Centre, Institute of Pharmacogenetics, Hufelandstrasse 55, 45147, Essen, Germany
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Nishimura K, Lee SB, Park JH, Park MH. Essential role of eIF5A-1 and deoxyhypusine synthase in mouse embryonic development. Amino Acids 2012; 42:703-10. [PMID: 21850436 PMCID: PMC3220921 DOI: 10.1007/s00726-011-0986-z] [Citation(s) in RCA: 75] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2011] [Accepted: 05/10/2011] [Indexed: 10/17/2022]
Abstract
The eukaryotic initiation factor 5A (eIF5A) contains a polyamine-derived amino acid, hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed post-translationally by the addition of the 4-aminobutyl moiety from the polyamine spermidine to a specific lysine residue, catalyzed by deoxyhypusine synthase (DHPS), and subsequent hydroxylation by deoxyhypusine hydroxylase (DOHH). The eIF5A precursor protein and both of its modifying enzymes are highly conserved, suggesting a vital cellular function for eIF5A and its hypusine modification. To address the functions of eIF5A and the first modification enzyme, DHPS, in mammalian development, we knocked out the Eif5a or the Dhps gene in mice. Eif5a heterozygous knockout mice and Dhps heterozygous knockout mice were viable and fertile. However, homozygous Eif5a1 (gt/gt) embryos and Dhps (gt/gt) embryos died early in embryonic development, between E3.5 and E7.5. Upon transfer to in vitro culture, homozygous Eif5a (gt/gt) or Dhps (gt/gt) blastocysts at E3.5 showed growth defects when compared to heterozygous or wild type blastocysts. Thus, the knockout of either the eIF5A-1 gene (Eif5a) or of the deoxyhypusine synthase gene (Dhps) caused early embryonic lethality in mice, indicating the essential nature of both eIF5A-1 and deoxyhypusine synthase in mammalian development.
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Affiliation(s)
| | - Seung Bum Lee
- Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Bldg 30, Room 211, MD 20892-4340, USA
| | - Jong Hwan Park
- Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Bldg 30, Room 211, MD 20892-4340, USA
| | - Myung Hee Park
- Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Bldg 30, Room 211, MD 20892-4340, USA
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44
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Park JH, Johansson HE, Aoki H, Huang BX, Kim HY, Ganoza MC, Park MH. Post-translational modification by β-lysylation is required for activity of Escherichia coli elongation factor P (EF-P). J Biol Chem 2011; 287:2579-90. [PMID: 22128152 DOI: 10.1074/jbc.m111.309633] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Bacterial elongation factor P (EF-P) is the ortholog of archaeal and eukaryotic initiation factor 5A (eIF5A). EF-P shares sequence homology and crystal structure with eIF5A, but unlike eIF5A, EF-P does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical β-lysyl-lysine. Here we present biochemical evidence for β-lysyl-lysine modification in Escherichia coli EF-P and for its role in EF-P activity by characterizing native and recombinant EF-P proteins for their modification status and activity in vitro. Mass spectrometric analyses confirmed the lysyl modification at lysine 34 in native and recombinant EF-P proteins. The β-lysyl-lysine isopeptide was identified in the exhaustive Pronase digests of native EF-P and recombinant EF-P isolated from E. coli coexpressing EF-P, YjeA, and YjeK but not in the digests of proteins derived from the vectors encoding EF-P alone or EF-P together with YjeA, indicating that both enzymes, YjeA and YjeK, are required for β-lysylation of EF-P. Endogenous EF-P as well as the recombinant EF-P preparation containing β-lysyl-EF-P stimulated N-formyl-methionyl-puromycin synthesis ∼4-fold over the preparations containing unmodified EF-P and/or α-lysyl-EF-P. The mutant lacking the modification site lysine (K34A) was inactive. This is the first report of biochemical evidence for the β-lysylation of EF-P in vivo and the requirement for this modification for the activity of EF-P.
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Affiliation(s)
- Jong-Hwan Park
- Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA
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Park JH, Dias CAO, Lee SB, Valentini SR, Sokabe M, Fraser CS, Park MH. Production of active recombinant eIF5A: reconstitution in E.coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes. Protein Eng Des Sel 2011; 24:301-9. [PMID: 21131325 PMCID: PMC3038461 DOI: 10.1093/protein/gzq110] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2010] [Revised: 11/03/2010] [Accepted: 11/03/2010] [Indexed: 11/12/2022] Open
Abstract
Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the polyamine-modified lysine, hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Hypusine occurs only in eukaryotes and certain archaea, but not in eubacteria. It is formed post-translationally by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. eIF5A binds to the ribosome and stimulates translation in a hypusine-dependent manner, but its mode of action in translation is not well understood. Since quantities of highly pure hypusine-modified eIF5A is desired for structural studies as well as for determination of its binding sites on the ribosome, we have used a polycistronic vector, pST39, to express eIF5A alone, or to co-express human eIF5A-1 with DHS or with both DHS and DOHH in Escherichia coli cells, to engineer recombinant proteins, unmodified eIF5A, deoxyhypusine- or hypusine-modified eIF5A. We have accomplished production of three different forms of recombinant eIF5A in high quantity and purity. The recombinant hypusine-modified eIF5A was as active in methionyl-puromycin synthesis as the native, eIF5A (hypusine form) purified from mammalian tissue. The recombinant eIF5A proteins will be useful tools in future structure/function and the mechanism studies in translation.
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Affiliation(s)
- Jong Hwan Park
- Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4340, USA
| | - Camila A. O. Dias
- Department of Biological Sciences, School of Pharmaceutical Sciences, University of Estadual Paulista – UNESP, Bldg 30 Rm 211, Araraquara, SP, Brazil
| | - Seung Bum Lee
- Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4340, USA
| | - Sandro R. Valentini
- Department of Biological Sciences, School of Pharmaceutical Sciences, University of Estadual Paulista – UNESP, Bldg 30 Rm 211, Araraquara, SP, Brazil
| | | | - Christopher S. Fraser
- Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA
| | - Myung Hee Park
- Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4340, USA
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Ma Y, Miura E, Ham BK, Cheng HW, Lee YJ, Lucas WJ. Pumpkin eIF5A isoforms interact with components of the translational machinery in the cucurbit sieve tube system. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2010; 64:536-50. [PMID: 20807213 DOI: 10.1111/j.1365-313x.2010.04347.x] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/13/2023]
Abstract
In yeast, eIF5A, in combination with eEF2, functions at the translation step, during the protein elongation cycle. This result is of significance with respect to functioning of the enucleate sieve tube system, as eIF5A was recently detected in Cucurbita maxima (pumpkin) phloem sap. In the present study, we further characterized four CmeIF5A isoforms, encoding three proteins, all of which were present in the phloem sap. Although hypusination of CmeIF5A was not necessary for entry into the sieve elements, this unique post-translational modification was necessary for RNA binding. The two enzymes required for hypusination were detected in pumpkin phloem sap, where presumably this modification takes place. A combination of gel-filtration chromatography and protein overlay assays demonstrated that, as in yeast, CmeIF5A interacts with phloem proteins, like eEF2, known to be involved in protein synthesis. These findings are discussed in terms of a potential role for eIF5A in regulating protein synthesis within the enucleate sieve tube system of the angiosperms.
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Affiliation(s)
- Yi Ma
- Department of Plant Biology, College of Biological Sciences, University of California, Davis, CA 95616, USA
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Park MH, Nishimura K, Zanelli CF, Valentini SR. Functional significance of eIF5A and its hypusine modification in eukaryotes. Amino Acids 2010; 38:491-500. [PMID: 19997760 PMCID: PMC2829442 DOI: 10.1007/s00726-009-0408-7] [Citation(s) in RCA: 240] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2009] [Accepted: 09/09/2009] [Indexed: 10/20/2022]
Abstract
The unusual basic amino acid, hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)-lysine], is a modified lysine with the addition of the 4-aminobutyl moiety from the polyamine spermidine. This naturally occurring amino acid is a product of a unique posttranslational modification that occurs in only one cellular protein, eukaryotic translation initiation factor 5A (eIF5A, eIF-5A). Hypusine is synthesized exclusively in this protein by two sequential enzymatic steps involving deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The deoxyhypusine/hypusine synthetic pathway has evolved in archaea and eukaryotes, and eIF5A, DHS and DOHH are highly conserved suggesting a vital cellular function of eIF5A. Gene disruption and mutation studies in yeast and higher eukaryotes have provided valuable information on the essential nature of eIF5A and the deoxyhypusine/hypusine modification in cell growth and in protein synthesis. In view of the extraordinary specificity and functional significance of hypusine-containing eIF5A in mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes are novel potential targets for intervention in aberrant cell proliferation.
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Affiliation(s)
- M H Park
- Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bldg 30, Room 211, Bethesda, MD 20892-4340, USA.
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