|
1
|
Leiva H, Caro De Silva PL, Painter R, Le VTB, Uychoco P, Figueroa Paniagua D, Endres M, Maltseva N, Joachimiak A, Kuhn ML. A Structurally Diverse Compound Screening Library to Identify Substrates for Diamine, Polyamine, and Related Acetyltransferases. ACS OMEGA 2024; 9:49887-49898. [PMID: 39713696 PMCID: PMC11656603 DOI: 10.1021/acsomega.4c08743] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/23/2024] [Revised: 11/07/2024] [Accepted: 11/12/2024] [Indexed: 12/24/2024]
Abstract
Spermidine/spermine N-acetyltransferases (SSATs) and other types of polyamine acetyltransferases (PAATs) acetylate diamines and/or polyamines. These enzymes are evolutionarily related and belong to the Gcn5-related N-acetyltransferase (GNAT) superfamily, yet we lack a fundamental understanding of their substrate specificity and/or promiscuity toward different compounds. Many of these enzymes are known or are predicted to acetylate polyamines, but in the cell there are other types of compounds that contain moieties derived from polyamines that may be the native substrates for these enzymes. To learn more about the identity of substrates that are acetylated, we selected and screened 17 different GNAT enzymes for activity toward a set of structurally diverse compounds that contained different types of amine moieties (e.g., aminopropyl, aminobutyl, etc.). These compounds included diamines, triamines, and polyamines containing primary amino groups, and they had structural diversity with variation of the chain length and presence or absence of internal amino groups and other functional groups. We found 12 of the 17 enzymes acetylated at least one of the compounds. Some enzymes were selective toward acetylating only one compound while others exhibited substrate promiscuity toward numerous compounds. Our experimental results ultimately allowed us to pinpoint specific substrates that could be further investigated to more fully understand substrate specificity versus promiscuity of GNAT enzymes and the role of acetylated small molecules in cells.
Collapse
Affiliation(s)
- Hazel Leiva
- San
Francisco State University, Department of Chemistry and Biochemistry, San Francisco, California 94132, United States
| | - Pamela L. Caro De Silva
- San
Francisco State University, Department of Chemistry and Biochemistry, San Francisco, California 94132, United States
- Department
of Chemistry, Foothill College, Los Altos, California 94022, United
States
| | - Ron Painter
- San
Francisco State University, Department of Chemistry and Biochemistry, San Francisco, California 94132, United States
| | - Van Thi Bich Le
- San
Francisco State University, Department of Chemistry and Biochemistry, San Francisco, California 94132, United States
| | - Patricia Uychoco
- San
Francisco State University, Department of Chemistry and Biochemistry, San Francisco, California 94132, United States
| | - Daniel Figueroa Paniagua
- San
Francisco State University, Department of Chemistry and Biochemistry, San Francisco, California 94132, United States
| | - Michael Endres
- Center
for Structural Biology of Infectious Diseases, Consortium for Advanced
Science and Engineering, University of Chicago, Chicago, Illinois 60667, United States
- Structural
Biology Center, X-ray Science Division, Argonne National Laboratory, Lemont, Illinois 60439, United States
| | - Natalia Maltseva
- Center
for Structural Biology of Infectious Diseases, Consortium for Advanced
Science and Engineering, University of Chicago, Chicago, Illinois 60667, United States
- Structural
Biology Center, X-ray Science Division, Argonne National Laboratory, Lemont, Illinois 60439, United States
| | - Andrzej Joachimiak
- Center
for Structural Biology of Infectious Diseases, Consortium for Advanced
Science and Engineering, University of Chicago, Chicago, Illinois 60667, United States
- Structural
Biology Center, X-ray Science Division, Argonne National Laboratory, Lemont, Illinois 60439, United States
| | - Misty L. Kuhn
- San
Francisco State University, Department of Chemistry and Biochemistry, San Francisco, California 94132, United States
| |
Collapse
|
|
2
|
Pisani DF, Lettieri-Barbato D, Ivanov S. Polyamine metabolism in macrophage-adipose tissue function and homeostasis. Trends Endocrinol Metab 2024; 35:937-950. [PMID: 38897879 DOI: 10.1016/j.tem.2024.05.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/08/2024] [Revised: 05/23/2024] [Accepted: 05/24/2024] [Indexed: 06/21/2024]
Abstract
Intracellular metabolism is a crucial regulator of macrophage function. Recent evidence revealed that the polyamine pathway and subsequent hypusination of eukaryotic initiation factor 5A (eIF5A) are master regulators of immune cell functions. In brown adipose tissue (BAT), macrophages show an impressive degree of heterogenicity, with specific subsets supporting adaptive thermogenesis during cold exposure. In this review, we discuss the impact of polyamine metabolism on macrophage diversity and function, with a particular focus on their role in adipose tissue homeostasis. Thus, we highlight the exploration of how polyamine metabolism in macrophages contributes to BAT homeostasis as an attractive and exciting new field of research.
Collapse
Affiliation(s)
| | - Daniele Lettieri-Barbato
- Department of Biology, University of Rome Tor Vergata, Rome, Italy; IRCCS-Fondazione Bietti, Rome, Italy.
| | | |
Collapse
|
|
3
|
Mozibullah M, Khatun M, Sikder M, Islam M, Sharmin M. Identifying Oncogenic Missense Single Nucleotide Polymorphisms in Human SAT1 Gene Using Computational Algorithms and Molecular Dynamics Tools. Cancer Rep (Hoboken) 2024; 7:e2130. [PMID: 39041636 PMCID: PMC11264109 DOI: 10.1002/cnr2.2130] [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: 10/29/2023] [Revised: 05/04/2024] [Accepted: 06/30/2024] [Indexed: 07/24/2024] Open
Abstract
BACKGROUND The human SAT1 gene encodes spermidine/spermine N1-acetyltransferase 1 (SSAT1), a regulatory biological catalyst of polyamine catabolism. Numerous essential biological processes, such as cellular proliferation, differentiation, and survival, depend on polyamines like spermidine and spermine. Thus, SSAT1 is involved in key cellular activities such as proliferation and survival of cells and mediates various diseases including cancer. A plethora of studies established the involvement of missense single nucleotide polymorphisms (SNPs) in numerous pathological conditions due to their ability to adversely affect the structure and subsequent function of the protein. AIMS To date, an in silico study to identify the pathogenic missense SNPs of the human SAT1 gene has not been accomplished yet. This study aimed to filter the missense SNPs that were functionally detrimental and pathogenic. METHODS AND RESULTS The rs757435207 (I21N) was ascertained to be the most deleterious and pathogenic by all algorithmic tools. Stability and evolutionary conservation analysis tools also stated that I21N variant decreased the stability and was located in the highly conserved residue. Molecular dynamics simulation revealed that I21N caused substantial alterations in the conformational stability and dynamics of the SSAT1 protein. Consequently, the I21N variant could disrupt the native functional roles of the SSAT1 enzyme. CONCLUSION Therefore, the I21N variant was identified and concluded to be an oncogenic missense variant of the human SAT1 gene. Overall, the findings of this study would be a great directory of future experimental research to develop personalized medicine.
Collapse
Affiliation(s)
- Md. Mozibullah
- Department of Biochemistry and Molecular BiologyMawlana Bhashani Science and Technology UniversityTangailBangladesh
| | - Marina Khatun
- Department of Biochemistry and Molecular BiologyMawlana Bhashani Science and Technology UniversityTangailBangladesh
| | - Md. Asaduzzaman Sikder
- Department of Biochemistry and Molecular BiologyMawlana Bhashani Science and Technology UniversityTangailBangladesh
| | - Mohammod Johirul Islam
- Department of Biochemistry and Molecular BiologyMawlana Bhashani Science and Technology UniversityTangailBangladesh
| | - Mehbuba Sharmin
- Department of Biochemistry and Molecular BiologyMawlana Bhashani Science and Technology UniversityTangailBangladesh
| |
Collapse
|
|
4
|
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.
Collapse
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
| |
Collapse
|
|
5
|
Seoane R, Lama-Díaz T, Romero AM, El Motiam A, Martínez-Férriz A, Vidal S, Bouzaher YH, Blanquer M, Tolosa RM, Castillo Mewa J, Rodríguez MS, García-Sastre A, Xirodimas D, Sutherland JD, Barrio R, Alepuz P, Blanco MG, Farràs R, Rivas C. SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells. Cell Mol Biol Lett 2024; 29:15. [PMID: 38229033 PMCID: PMC10790418 DOI: 10.1186/s11658-024-00533-5] [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: 07/19/2023] [Accepted: 01/04/2024] [Indexed: 01/18/2024] Open
Abstract
BACKGROUND The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown. METHODS SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6-SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1-GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student's t-test, or one-way or two-way analysis of variance (ANOVA). RESULTS We found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells. CONCLUSIONS SUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells.
Collapse
Affiliation(s)
- Rocío Seoane
- Centro de Investigación en Medicina Molecular (CIMUS), IDIS, Universidade de Santiago de Compostela, Avda Barcelona, 15706, Santiago de Compostela, Spain
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Tomás Lama-Díaz
- Centro de Investigación en Medicina Molecular (CIMUS), IDIS, Universidade de Santiago de Compostela, Avda Barcelona, 15706, Santiago de Compostela, Spain
- Departamento de Bioquímica e Bioloxía Molecular, Universidade de Santiago de Compostela, 15706, Santiago de Compostela, Spain
| | - Antonia María Romero
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universitat de València, Burjassot, 46100, Valencia, Spain
- Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), C/ Américo Vespucio 24, Edificio Cabimer, 41092, Seville, Spain
| | - Ahmed El Motiam
- Centro de Investigación en Medicina Molecular (CIMUS), IDIS, Universidade de Santiago de Compostela, Avda Barcelona, 15706, Santiago de Compostela, Spain
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada
| | | | - Santiago Vidal
- Centro de Investigación en Medicina Molecular (CIMUS), IDIS, Universidade de Santiago de Compostela, Avda Barcelona, 15706, Santiago de Compostela, Spain
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Yanis H Bouzaher
- Centro de Investigación en Medicina Molecular (CIMUS), IDIS, Universidade de Santiago de Compostela, Avda Barcelona, 15706, Santiago de Compostela, Spain
| | - María Blanquer
- Centro de Investigación en Medicina Molecular (CIMUS), IDIS, Universidade de Santiago de Compostela, Avda Barcelona, 15706, Santiago de Compostela, Spain
| | - Rocío M Tolosa
- Centro de Investigación en Medicina Molecular (CIMUS), IDIS, Universidade de Santiago de Compostela, Avda Barcelona, 15706, Santiago de Compostela, Spain
| | - Juan Castillo Mewa
- Research Department in Genomics and Proteomics, Instituto Conmemorativo Gorgas de Estudios de la Salud, 0816-02593, Panamá, Republic of Panama
| | - Manuel S Rodríguez
- Laboratoire de Chimie de Coordination LCC-UPR 8241-CNRS, 31400, Toulouse, France
| | - Adolfo García-Sastre
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA
- The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Dimitris Xirodimas
- Montpellier Cell Biology Research Center (CRBM), CNRS-UMR 5237 Université de Montpellier, Montpellier, France
| | - James D Sutherland
- Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160, Derio, Spain
| | - Rosa Barrio
- Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 801A, 48160, Derio, Spain
| | - Paula Alepuz
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universitat de València, Burjassot, 46100, Valencia, Spain
- Instituto Bio TecMed, Universitat de València, Burjassot, 46100, Valencia, Spain
| | - Miguel G Blanco
- Centro de Investigación en Medicina Molecular (CIMUS), IDIS, Universidade de Santiago de Compostela, Avda Barcelona, 15706, Santiago de Compostela, Spain
- Departamento de Bioquímica e Bioloxía Molecular, Universidade de Santiago de Compostela, 15706, Santiago de Compostela, Spain
| | - Rosa Farràs
- Centro de Investigación Príncipe Felipe, 46012, Valencia, Spain
| | - Carmen Rivas
- Centro de Investigación en Medicina Molecular (CIMUS), IDIS, Universidade de Santiago de Compostela, Avda Barcelona, 15706, Santiago de Compostela, Spain.
- Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología (CNB), CSIC, Darwin 3, 28049, Madrid, Spain.
| |
Collapse
|
|
6
|
Tamborlin L, Pereira KD, Guimarães DSPSF, Silveira LR, Luchessi AD. The first evidence of biological activity for free Hypusine, an enigmatic amino acid discovered in the '70s. Amino Acids 2023:10.1007/s00726-023-03283-4. [PMID: 37258638 DOI: 10.1007/s00726-023-03283-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2022] [Accepted: 05/19/2023] [Indexed: 06/02/2023]
Abstract
Hypusine amino acid [Nε-(4-amino-2-hydroxybutyl)-lysine] was first isolated in 1971 from bovine brain extracts. Hypusine originates from a post-translational modification at the eukaryotic translation initiation factor 5A (eIF5A), a protein produced by archaebacteria and eukaryotes. The eIF5A protein is the only one described containing the hypusine residue, which is essential for its activity. Hypusine as a free amino acid is a consequence of proteolytic degradation of eIF5A. Herein, we showed, for the first time, evidence of biological activity for the free hypusine. C6 rat glioma cells were treated with hypusine, and different cellular parameters were evaluated. Hypusine treatment significantly reduced C6 cell proliferation and potently suppressed their clonogenic capacity without leading to apoptosis. Hypusine also decreased the Eif5A transcript content and the global protein synthesis profile that may occur due to negative feedback in response to high hypusine concentration, controlling the content of newly synthesized eIF5A, which can affect the translation process. Besides, hypusine treatment also altered cellular metabolism by changing the pathways for energy production, reducing cellular respiration coupled with oxidative phosphorylation, and increasing the anaerobic metabolism. These observed results and the relationship between eIF5A and tumor processes led us to test the combination of hypusine with the chemotherapeutic drug temozolomide. Combining temozolomide with hypusine reduced the MTT conversion to the same levels as those observed using double temozolomide dosage alone, demonstrating a synergetic action between the compounds. Thus, since 1971, this is the first study showing evidence of biological activity for hypusine not associated with being an essential component of the eiF5A protein. Finding out the molecular targets of hypusine are the following efforts to completely characterize its biological activity.
Collapse
Affiliation(s)
- Leticia Tamborlin
- Laboratory of Biotechnology, School of Applied Sciences, State University of Campinas (UNICAMP), Rua Pedro Zaccaria, 1300, Jardim Santa Luiza, Limeira, São Paulo, 13484-350, Brazil
- Institute of Biosciences, São Paulo State University (UNESP), Rio Claro, São Paulo, Brazil
| | - Karina Danielle Pereira
- Laboratory of Biotechnology, School of Applied Sciences, State University of Campinas (UNICAMP), Rua Pedro Zaccaria, 1300, Jardim Santa Luiza, Limeira, São Paulo, 13484-350, Brazil
| | | | - Leonardo Reis Silveira
- Obesity and Comorbidities Research Center, Department of Structural and Functional Biology, State University of Campinas (UNICAMP), Limeira, São Paulo, Brazil
| | - Augusto Ducati Luchessi
- Laboratory of Biotechnology, School of Applied Sciences, State University of Campinas (UNICAMP), Rua Pedro Zaccaria, 1300, Jardim Santa Luiza, Limeira, São Paulo, 13484-350, Brazil.
- Institute of Biosciences, São Paulo State University (UNESP), Rio Claro, São Paulo, Brazil.
| |
Collapse
|
|
7
|
Wu YY, Wu GQ, Cai NL, Xu YM, Lau ATY. Comparison of Human Eukaryotic Translation Initiation Factors 5A1 and 5AL1: Identification of Amino Acid Residues Important for EIF5A1 Lysine 50 Hypusination and Its Protein Stability. Int J Mol Sci 2023; 24:ijms24076067. [PMID: 37047039 PMCID: PMC10093921 DOI: 10.3390/ijms24076067] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2022] [Revised: 03/07/2023] [Accepted: 03/09/2023] [Indexed: 04/14/2023] Open
Abstract
The human eukaryotic translation initiation factor 5A (EIF5A) family consists of three members, namely EIF5A1, EIF5A2, and EIF5AL1. Recent studies have shown that the expression of EIF5As is related to many human diseases, such as diabetes, viral infection, central nervous system injury, and cancer. Among them, EIF5A1 plays different functions in various cancers, possibly as a tumor-suppressor or oncogene, while EIF5A2 promotes the occurrence and development of cancer. Yet, the biological function of EIF5AL1 is not being studied so far. Interestingly, although there are only three amino acid (at residues 36, 45, and 109) differences between EIF5A1 and EIF5AL1, we demonstrate that only EIF5A1 can be hypusinated while EIF5AL1 cannot, and EIF5AL1 has a tumor-suppressor-like function by inhibiting cell proliferation and migration. We also show that EIF5AL1 protein turnover is mediated through the proteasomal pathway, and EIF5AL1 protein turnover is much faster than that of EIF5A1, which may explain their differential protein expression level in cells. By engineering single and double mutations on these three amino acids, we pinpoint which of these amino acids are critical for hypusination and protein stability. The data of this work should fill in the gaps in EIF5As research and pave the way for future studies on EIF5AL1.
Collapse
Affiliation(s)
- Yu-Yao Wu
- Laboratory of Cancer Biology and Epigenetics, Department of Cell Biology and Genetics, Shantou University Medical College, Shantou 515041, China
| | - Gao-Qi Wu
- Laboratory of Cancer Biology and Epigenetics, Department of Cell Biology and Genetics, Shantou University Medical College, Shantou 515041, China
| | - Na-Li Cai
- Laboratory of Cancer Biology and Epigenetics, Department of Cell Biology and Genetics, Shantou University Medical College, Shantou 515041, China
| | - Yan-Ming Xu
- Laboratory of Cancer Biology and Epigenetics, Department of Cell Biology and Genetics, Shantou University Medical College, Shantou 515041, China
| | - Andy T Y Lau
- Laboratory of Cancer Biology and Epigenetics, Department of Cell Biology and Genetics, Shantou University Medical College, Shantou 515041, China
| |
Collapse
|
|
8
|
Tamaddondoust RN, Wang Y, Jafarnejad SM, Graber TE, Alain T. The highs and lows of ionizing radiation and its effects on protein synthesis. Cell Signal 2021; 89:110169. [PMID: 34662715 DOI: 10.1016/j.cellsig.2021.110169] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Revised: 08/19/2021] [Accepted: 10/06/2021] [Indexed: 11/03/2022]
Abstract
Ionizing radiation (IR) is a constant feature of our environment and one that can dramatically affect organismal health and development. Although the impacts of high-doses of IR on mammalian cells and systems have been broadly explored, there are still challenges in accurately quantifying biological responses to IR, especially in the low-dose range to which most individuals are exposed in their lifetime. The resulting uncertainty has led to the entrenchment of conservative radioprotection policies around the world. Thus, uncovering long-sought molecular mechanisms and tissue responses that are targeted by IR could lead to more informed policymaking and propose new therapeutic avenues for a variety of pathologies. One often overlooked target of IR is mRNA translation, a highly regulated cellular process that consumes more than 40% of the cell's energy. In response to environmental stimuli, regulation of mRNA translation allows for precise and rapid changes to the cellular proteome, and unsurprisingly high-dose of IR was shown to trigger a severe reprogramming of global protein synthesis allowing the cell to conserve energy by preventing the synthesis of unneeded proteins. Nonetheless, under these conditions, certain mRNAs encoding specific proteins are translationally favoured to produce the factors essential to repair the cell or send it down the path of no return through programmed cell death. Understanding the mechanisms controlling protein synthesis in response to varying doses of IR could provide novel insights into how this stress-mediated cellular adaptation is regulated and potentially uncover novel targets for radiosensitization or radioprotection. Here, we review the current literature on the effects of IR at both high- and low-dose on the mRNA translation machinery.
Collapse
Affiliation(s)
- Rosette Niloufar Tamaddondoust
- Molecular Biomedicine Program, Children's Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada; Radiobiology and Health, Canadian Nuclear Laboratories, Chalk River, Ontario, Canada.
| | - Yi Wang
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada; Radiobiology and Health, Canadian Nuclear Laboratories, Chalk River, Ontario, Canada
| | - Seyed Mehdi Jafarnejad
- Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast BT9 7AE, UK
| | - Tyson E Graber
- Molecular Biomedicine Program, Children's Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada
| | - Tommy Alain
- Molecular Biomedicine Program, Children's Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada.
| |
Collapse
|
|
9
|
Kaur J, Goldsmith J, Tankka A, Bustamante Eguiguren S, Gimenez AA, Vick L, Debnath J, Vlahakis A. Atg32-dependent mitophagy sustains spermidine and nitric oxide required for heat-stress tolerance in Saccharomycescerevisiae. J Cell Sci 2021; 134:jcs253781. [PMID: 34096604 PMCID: PMC8214763 DOI: 10.1242/jcs.253781] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2020] [Accepted: 05/06/2021] [Indexed: 11/20/2022] Open
Abstract
In Saccharomyces cerevisiae, the selective autophagic degradation of mitochondria, termed mitophagy, is critically regulated by the adapter protein Atg32. Despite our knowledge about the molecular mechanisms by which Atg32 controls mitophagy, its physiological roles in yeast survival and fitness remains less clear. Here, we demonstrate a requirement for Atg32 in promoting spermidine production during respiratory growth and heat-induced mitochondrial stress. During respiratory growth, mitophagy-deficient yeast exhibit profound heat-stress induced defects in growth and viability due to impaired biosynthesis of spermidine and its biosynthetic precursor S-adenosyl methionine. Moreover, spermidine production is crucial for the induction of cytoprotective nitric oxide (NO) during heat stress. Hence, the re-addition of spermidine to Atg32 mutant yeast is sufficient to both enhance NO production and restore respiratory growth during heat stress. Our findings uncover a previously unrecognized physiological role for yeast mitophagy in spermidine metabolism and illuminate new interconnections between mitophagy, polyamine biosynthesis and NO signaling.
Collapse
|
|
10
|
Smeltzer S, Quadri Z, Miller A, Zamudio F, Hunter J, Stewart NJF, Saji S, Lee DC, Chaput D, Selenica MLB. Hypusination of Eif5a regulates cytoplasmic TDP-43 aggregation and accumulation in a stress-induced cellular model. Biochim Biophys Acta Mol Basis Dis 2021; 1867:165939. [PMID: 32882370 DOI: 10.1016/j.bbadis.2020.165939] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2020] [Revised: 07/27/2020] [Accepted: 08/17/2020] [Indexed: 11/23/2022]
Abstract
TAR DNA-binding protein 43 (TDP-43) is a nuclear RNA/DNA binding protein involved in mRNA metabolism. Aberrant mislocalization to the cytoplasm and formation of phosphorylated/aggregated TDP-43 inclusions remains the hallmark pathology in a spectrum of neurodegenerative diseases, including frontotemporal disorders and Alzheimer's disease. Eukaryotic Translation Initiation Factor 5A undergoes a unique post-translation modification of lysine to hypusine (K50), which determines eIF5A binding partners. We used a sodium arsenite-induced cellular stress model to investigate the role of hypusinated eIF5A (eIF5AHypK50) in governing TDP-43 cytoplasmic mislocalization and accumulation in stress granule. Our proteomics and functional data provide evidence that eIF5A interacts with TDP-43 in a hypusine-dependent manner. Additionally, we showed that following stress TDP-43 interactions with eIF5AHypK50 were induced both in the cytoplasm and stress granules. Pharmacological reduction of hypusination or mutations of lysine residues within the hypusine loop decreased phosphorylated and insoluble TDP-43 levels. The proteomic and biochemical analysis also identified nuclear pore complex importins KPNA1/2, KPNB1, and RanGTP as interacting partners of eIF5AHypK50. These findings are the first to provide a novel pathway and potential therapeutic targets that require further investigation in models of TDP-43 proteinopathies.
Collapse
Affiliation(s)
- Shayna Smeltzer
- Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, FL 33612, USA
| | - Zainuddin Quadri
- Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, FL 33612, USA; Sanders-Brown Center on Aging, Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, KY, USA
| | - Abraian Miller
- Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, FL 33612, USA
| | - Frank Zamudio
- Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, FL 33612, USA
| | - Jordan Hunter
- Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, FL 33612, USA
| | - Nicholas J F Stewart
- Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, FL 33612, USA
| | - Sheba Saji
- Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, FL 33612, USA
| | - Daniel C Lee
- Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, FL 33612, USA; Sanders-Brown Center on Aging, Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, KY, USA
| | - Dale Chaput
- Proteomics and Mass Spectrometry Core Facility, Florida Center of Excellence for Drug Discovery and Innovation (CDDI), University of South Florida, 3720 Spectrum Blvd, Suite 303, Tampa, FL 33612, USA
| | - Maj-Linda B Selenica
- Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, FL 33612, USA; Sanders-Brown Center on Aging, Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, KY, USA.
| |
Collapse
|
|
11
|
Bekebrede AF, Keijer J, Gerrits WJJ, de Boer VCJ. The Molecular and Physiological Effects of Protein-Derived Polyamines in the Intestine. Nutrients 2020; 12:E197. [PMID: 31940783 PMCID: PMC7020012 DOI: 10.3390/nu12010197] [Citation(s) in RCA: 52] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2019] [Revised: 01/08/2020] [Accepted: 01/09/2020] [Indexed: 02/07/2023] Open
Abstract
Consumption of a high-protein diet increases protein entry into the colon. Colonic microbiota can ferment proteins, which results in the production of protein fermentation end-products, like polyamines. This review describes the effects of polyamines on biochemical, cellular and physiological processes, with a focus on the colon. Polyamines (mainly spermine, spermidine, putrescine and cadaverine) are involved in the regulation of protein translation and gene transcription. In this, the spermidine-derived hypusination modification of EIF5A plays an important role. In addition, polyamines regulate metabolic functions. Through hypusination of EIF5A, polyamines also regulate translation of mitochondrial proteins, thereby increasing their expression. They can also induce mitophagy through various pathways, which helps to remove damaged organelles and improves cell survival. In addition, polyamines increase mitochondrial substrate oxidation by increasing mitochondrial Ca2+-levels. Putrescine can even serve as an energy source for enterocytes in the small intestine. By regulating the formation of the mitochondrial permeability transition pore, polyamines help maintain mitochondrial membrane integrity. However, their catabolism may also reduce metabolic functions by depleting intracellular acetyl-CoA levels, or through production of toxic by-products. Lastly, polyamines support gut physiology, by supporting barrier function, inducing gut maturation and increasing longevity. Polyamines thus play many roles, and their impact is strongly tissue- and dose-dependent. However, whether diet-derived increases in colonic luminal polyamine levels also impact intestinal physiology has not been resolved yet.
Collapse
Affiliation(s)
- Anna F. Bekebrede
- Human and Animal Physiology, Wageningen University and Research, 6708 WD Wageningen, The Netherlands; (A.F.B.); (J.K.)
- Animal Nutrition Group, Wageningen University and Research, 6708 WD Wageningen, The Netherlands;
| | - Jaap Keijer
- Human and Animal Physiology, Wageningen University and Research, 6708 WD Wageningen, The Netherlands; (A.F.B.); (J.K.)
| | - Walter J. J. Gerrits
- Animal Nutrition Group, Wageningen University and Research, 6708 WD Wageningen, The Netherlands;
| | - Vincent C. J. de Boer
- Human and Animal Physiology, Wageningen University and Research, 6708 WD Wageningen, The Netherlands; (A.F.B.); (J.K.)
| |
Collapse
|
|
12
|
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: 34] [Impact Index Per Article: 6.8] [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.
Collapse
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
| |
Collapse
|
|
13
|
Spermidine/spermine N1-acetyltransferase 1 is a gene-specific transcriptional regulator that drives brain tumor aggressiveness. Oncogene 2019; 38:6794-6800. [PMID: 31399646 PMCID: PMC6786946 DOI: 10.1038/s41388-019-0917-0] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2018] [Revised: 07/05/2019] [Accepted: 07/07/2019] [Indexed: 11/08/2022]
Abstract
Spermidine/spermine N1-acetyltransferase 1 (SAT1), the rate limiting enzyme in polyamine catabolism, has broad regulatory roles due to near ubiquitous polyamine binding. We describe a novel function of SAT1 as a gene-specific transcriptional regulator through local polyamine acetylation. SAT1 expression is elevated in aggressive brain tumors and promotes resistance to radiotherapy. Expression profiling in glioma cells identified SAT1 target genes that distinguish high and low grade tumors, in support of the prognostic utility of SAT1 expression. We further discovered mechanisms of SAT1-driven tumor aggressiveness through promotion of expression of both DNA damage response pathways as well as cell cycle regulatory genes. Mechanistically, SAT1 associates specifically with the promoter of the MELK gene, which functionally controls other SAT1 targets, and leads biologically to maintenance of neurosphere stemness in conjunction with FOXM1 and EZH2. CRISPR knockin mutants demonstrate the essentiality of the polyamine acetyl transferase activity of SAT1 for its function as a transcriptional regulator. Together, the data demonstrate that gene-specific polyamine removal is a major transcriptional regulatory mechanism active in high grade gliomas that drives poor outcomes.
Collapse
|
|
14
|
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.
Collapse
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
| |
Collapse
|
|
15
|
Myc, Oncogenic Protein Translation, and the Role of Polyamines. Med Sci (Basel) 2018; 6:medsci6020041. [PMID: 29799508 PMCID: PMC6024823 DOI: 10.3390/medsci6020041] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2018] [Revised: 05/19/2018] [Accepted: 05/22/2018] [Indexed: 01/21/2023] Open
Abstract
Deregulated protein synthesis is a common feature of cancer cells, with many oncogenic signaling pathways directly augmenting protein translation to support the biomass needs of proliferating tissues. MYC’s ability to drive oncogenesis is a consequence of its essential role as a governor linking cell cycle entry with the requisite increase in protein synthetic capacity, among other biomass needs. To date, direct pharmacologic inhibition of MYC has proven difficult, but targeting oncogenic signaling modules downstream of MYC, such as the protein synthetic machinery, may provide a viable therapeutic strategy. Polyamines are essential cations found in nearly all living organisms that have both direct and indirect roles in the control of protein synthesis. Polyamine metabolism is coordinately regulated by MYC to increase polyamines in proliferative tissues, and this is further augmented in the many cancer cells harboring hyperactivated MYC. In this review, we discuss MYC-driven regulation of polyamines and protein synthetic capacity as a key function of its oncogenic output, and how this dependency may be perturbed through direct pharmacologic targeting of components of the protein synthetic machinery, such as the polyamines themselves, the eukaryotic translation initiation factor 4F (eIF4F) complex, and the eukaryotic translation initiation factor 5A (eIF5A).
Collapse
|
|
16
|
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: 36] [Impact Index Per Article: 5.1] [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.
Collapse
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
| |
Collapse
|
|
17
|
Pereira KD, Tamborlin L, Meneguello L, de Proença ARG, Almeida ICDPA, Lourenço RF, Luchessi AD. Alternative Start Codon Connects eIF5A to Mitochondria. J Cell Physiol 2016; 231:2682-9. [PMID: 27414022 DOI: 10.1002/jcp.25370] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2015] [Accepted: 03/04/2016] [Indexed: 01/02/2023]
Abstract
Eukaryotic translation initiation factor 5A (eIF5A), a protein containing the amino acid residue hypusine required for its activity, is involved in a number of physiological and pathological cellular processes. In humans, several EIF5A1 transcript variants encode the canonical eIF5A1 isoform B, whereas the hitherto uncharacterized variant A is expected to code for a hypothetical eIF5A1 isoform, referred to as isoform A, which has an additional N-terminal extension. Herein, we validate the existence of eIF5A1 isoform A and its production from transcript variant A. In fact, variant A was shown to encode both eIF5A1 isoforms A and B. Mutagenic assays revealed different efficiencies in the start codons present in variant A, contributing to the production of isoform B at higher levels than isoform A. Immunoblotting and mass spectrometric analyses showed that isoform A can undergo hypusination and acetylation at specific lysine residues, as observed for isoform B. Examination of the N-terminal extension suggested that it might confer mitochondrial targeting. Correspondingly, we found that isoform A, but not isoform B, co-purified with mitochondria when the proteins were overproduced. These findings suggest that eIF5A1 isoform A has a role in mitochondrial function. J. Cell. Physiol. 231: 2682-2689, 2016. © 2016 Wiley Periodicals, Inc.
Collapse
Affiliation(s)
- Karina Danielle Pereira
- Laboratory of Biotechnology, School of Applied Sciences, University of Campinas, Limeira, São Paulo, Brazil
- Institute of Biosciences, São Paulo State University, Rio Claro, São Paulo, Brazil
| | - Letícia Tamborlin
- Laboratory of Biotechnology, School of Applied Sciences, University of Campinas, Limeira, São Paulo, Brazil
| | - Letícia Meneguello
- Laboratory of Biotechnology, School of Applied Sciences, University of Campinas, Limeira, São Paulo, Brazil
- Institute of Biosciences, São Paulo State University, Rio Claro, São Paulo, Brazil
| | | | | | - Rogério Ferreira Lourenço
- Laboratory of Biotechnology, School of Applied Sciences, University of Campinas, Limeira, São Paulo, Brazil
| | - Augusto Ducati Luchessi
- Laboratory of Biotechnology, School of Applied Sciences, University of Campinas, Limeira, São Paulo, Brazil
- Institute of Biosciences, São Paulo State University, Rio Claro, São Paulo, Brazil
| |
Collapse
|
|
18
|
|
|
19
|
Ser/Thr kinases and polyamines in the regulation of non-canonical functions of elongation factor 1A. Amino Acids 2016; 48:2339-52. [DOI: 10.1007/s00726-016-2311-3] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2016] [Accepted: 08/08/2016] [Indexed: 10/21/2022]
|
|
20
|
Abstract
The content of spermidine and spermine in mammalian cells has important roles in protein and nucleic acid synthesis and structure, protection from oxidative damage, activity of ion channels, cell proliferation, differentiation, and apoptosis. Spermidine is essential for viability and acts as the precursor of hypusine, a post-translational addition to eIF5A allowing the translation of mRNAs encoding proteins containing polyproline tracts. Studies with Gy mice and human patients with the very rare X-linked genetic condition Snyder-Robinson syndrome that both lack spermine synthase show clearly that the correct spermine:spermidine ratio is critical for normal growth and development.
Collapse
Affiliation(s)
- Anthony E Pegg
- From the Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
| |
Collapse
|
|
21
|
Barjaktarovic Z, Kempf SJ, Sriharshan A, Merl-Pham J, Atkinson MJ, Tapio S. Ionizing radiation induces immediate protein acetylation changes in human cardiac microvascular endothelial cells. JOURNAL OF RADIATION RESEARCH 2015; 56:623-32. [PMID: 25840449 PMCID: PMC4497387 DOI: 10.1093/jrr/rrv014] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/03/2014] [Revised: 01/21/2015] [Accepted: 02/16/2015] [Indexed: 05/20/2023]
Abstract
Reversible lysine acetylation is a highly regulated post-translational protein modification that is known to regulate several signaling pathways. However, little is known about the radiation-induced changes in the acetylome. In this study, we analyzed the acute post-translational acetylation changes in primary human cardiac microvascular endothelial cells 4 h after a gamma radiation dose of 2 Gy. The acetylated peptides were enriched using anti-acetyl conjugated agarose beads. A total of 54 proteins were found to be altered in their acetylation status, 23 of which were deacetylated and 31 acetylated. Pathway analyses showed three protein categories particularly affected by radiation-induced changes in the acetylation status: the proteins involved in the translation process, the proteins of stress response, and mitochondrial proteins. The activation of the canonical and non-canonical Wnt signaling pathways affecting actin cytoskeleton signaling and cell cycle progression was predicted. The protein expression levels of two nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, sirtuin 1 and sirtuin 3, were significantly but transiently upregulated 4 but not 24 h after irradiation. The status of the p53 protein, a target of sirtuin 1, was found to be rapidly stabilized by acetylation after radiation exposure. These findings indicate that post-translational modification of proteins by acetylation and deacetylation is essentially affecting the radiation response of the endothelium.
Collapse
Affiliation(s)
- Zarko Barjaktarovic
- Helmholtz Zentrum München, Ingolstädter Landstraße 1, Neuherberg 85764, Germany
| | - Stefan J Kempf
- Helmholtz Zentrum München, Ingolstädter Landstraße 1, Neuherberg 85764, Germany
| | | | - Juliane Merl-Pham
- Helmholtz Zentrum München, Ingolstädter Landstraße 1, Neuherberg 85764, Germany
| | - Michael J Atkinson
- Helmholtz Zentrum München, Ingolstädter Landstraße 1, Neuherberg 85764, Germany
| | - Soile Tapio
- Helmholtz Zentrum München, Ingolstädter Landstraße 1, Neuherberg 85764, Germany
| |
Collapse
|
|
22
|
Pällmann N, Braig M, Sievert H, Preukschas M, Hermans-Borgmeyer I, Schweizer M, Nagel CH, Neumann M, Wild P, Haralambieva E, Hagel C, Bokemeyer C, Hauber J, Balabanov S. Biological Relevance and Therapeutic Potential of the Hypusine Modification System. J Biol Chem 2015; 290:18343-60. [PMID: 26037925 DOI: 10.1074/jbc.m115.664490] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2015] [Indexed: 11/06/2022] Open
Abstract
Hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is emerging as a crucial regulator in cancer, infections, and inflammation. Although its contribution in translational regulation of proline repeat-rich proteins has been sufficiently demonstrated, its biological role in higher eukaryotes remains poorly understood. To establish the hypusine modification system as a novel platform for therapeutic strategies, we aimed to investigate its functional relevance in mammals by generating and using a range of new knock-out mouse models for the hypusine-modifying enzymes deoxyhypusine synthase and deoxyhypusine hydroxylase as well as for the cancer-related isoform eIF-5A2. We discovered that homozygous depletion of deoxyhypusine synthase and/or deoxyhypusine hydroxylase causes lethality in adult mice with different penetrance compared with haploinsufficiency. Network-based bioinformatic analysis of proline repeat-rich proteins, which are putative eIF-5A targets, revealed that these proteins are organized in highly connected protein-protein interaction networks. Hypusine-dependent translational control of essential proteins (hubs) and protein complexes inside these networks might explain the lethal phenotype observed after deletion of hypusine-modifying enzymes. Remarkably, our results also demonstrate that the cancer-associated isoform eIF-5A2 is dispensable for normal development and viability. Together, our results provide the first genetic evidence that the hypusine modification in eIF-5A is crucial for homeostasis in mammals. Moreover, these findings highlight functional diversity of the hypusine system compared with lower eukaryotes and indicate eIF-5A2 as a valuable and safe target for therapeutic intervention in cancer.
Collapse
Affiliation(s)
- Nora Pällmann
- From the Department of Oncology, Hematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald Tumor Center, the Heinrich Pette Institute, Leibniz Institute for Experimental Virology, 20251 Hamburg, Germany
| | - Melanie Braig
- From the Department of Oncology, Hematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald Tumor Center, the Division of Hematology and
| | - Henning Sievert
- From the Department of Oncology, Hematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald Tumor Center
| | - Michael Preukschas
- From the Department of Oncology, Hematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald Tumor Center, the Department of Molecular Pathology, Institute for Hematopathology, 22547 Hamburg, Germany
| | | | | | - Claus Henning Nagel
- the Heinrich Pette Institute, Leibniz Institute for Experimental Virology, 20251 Hamburg, Germany
| | - Melanie Neumann
- Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Peter Wild
- Institute of Surgical Pathology, University Hospital Zurich, 8091 Zurich, Switzerland
| | - Eugenia Haralambieva
- Institute of Surgical Pathology, University Hospital Zurich, 8091 Zurich, Switzerland
| | - Christian Hagel
- Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Carsten Bokemeyer
- From the Department of Oncology, Hematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald Tumor Center
| | - Joachim Hauber
- the Heinrich Pette Institute, Leibniz Institute for Experimental Virology, 20251 Hamburg, Germany
| | - Stefan Balabanov
- From the Department of Oncology, Hematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald Tumor Center, the Division of Hematology and
| |
Collapse
|
|
23
|
Depletion of the polyamines spermidine and spermine by overexpression of spermidine/spermine N¹-acetyltransferase 1 (SAT1) leads to mitochondria-mediated apoptosis in mammalian cells. Biochem J 2015; 468:435-47. [PMID: 25849284 DOI: 10.1042/bj20150168] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2015] [Accepted: 04/07/2015] [Indexed: 12/16/2022]
Abstract
The polyamines putrescine, spermidine and spermine are intimately involved in the regulation of cellular growth and viability. Transduction of human embryonic kidney (HEK) 293T cells with an adenovirus encoding a key polyamine catabolic enzyme, spermidine N¹-acetyltransferase 1 (SSAT1)/SAT1 (AdSAT1), leads to a rapid depletion of spermidine and spermine, arrest in cell growth and a decline in cell viability. Annexin V/propidium iodide FACS analyses, terminal uridine nucleotide end-labelling (TUNEL) and caspase 3 assays showed a clear indication of apoptosis in AdSAT1-transduced cells (at 24-72 h), but not in cells transduced with GFP-encoding adenovirus (AdGFP). Apoptosis in the polyamine-depleted cells occurs by the mitochondrial intrinsic pathway, as evidenced by loss of mitochondrial membrane potential, increase in pro-apoptotic Bax, decrease in anti-apoptotic Bcl-xl, Bcl2 and Mcl-1 and release of cytochrome c from mitochondria, upon transduction with AdSAT1. Moreover, TEM images of AdSAT1-transduced cells revealed morphological changes commonly associated with apoptosis, including cell shrinkage, nuclear fragmentation, mitochondrial alteration, vacuolization and membrane blebbing. The apoptosis appears to result largely from depletion of the polyamines spermidine and spermine, as the polyamine analogues α-methylspermidine (α-MeSpd) and N¹,N¹²-dimethylspermine (Me₂Spm) that are not substrates for SAT1 could partially restore growth and prevent apoptosis of AdSAT1-transduced cells. Inhibition of polyamine oxidases did not restore the growth of AdSAT1-transduced cells or block apoptosis, suggesting that the growth arrest and apoptosis were not induced by oxidative stress resulting from accelerated polyamine catabolism. Taken together, these data provide strong evidence that the depletion of the polyamines spermidine and spermine leads to mitochondria-mediated apoptosis.
Collapse
|
|
24
|
Overexpression of spermidine/spermine N 1-acetyltransferase impairs osteoblastogenesis and alters mouse bone phenotype. Transgenic Res 2014; 24:253-65. [DOI: 10.1007/s11248-014-9836-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2014] [Accepted: 09/06/2014] [Indexed: 01/11/2023]
|
|
25
|
Depletion of cellular polyamines, spermidine and spermine, causes a total arrest in translation and growth in mammalian cells. Proc Natl Acad Sci U S A 2013; 110:2169-74. [PMID: 23345430 DOI: 10.1073/pnas.1219002110] [Citation(s) in RCA: 211] [Impact Index Per Article: 17.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The polyamines, putrescine, spermidine, and spermine, are essential polycations, intimately involved in the regulation of cellular proliferation. Although polyamines exert dynamic effects on the conformation of nucleic acids and macromolecular synthesis in vitro, their specific functions in vivo are poorly understood. We investigated the cellular function of polyamines by overexpression of a key catabolic enzyme, spermidine/spermine N(1)-acetyltransferase 1 (SAT1) in mammalian cells. Transient cotransfection of HeLa cells with GFP and SAT1 vectors suppressed GFP protein expression without lowering its mRNA level, an indication that the block in GFP expression was not at transcription, but at translation. Fluorescence single-cell imaging also revealed specific inhibition of endogenous protein synthesis in the SAT1 overexpressing cells, without any inhibition of synthesis of DNA or RNA. Overexpression of SAT1 using a SAT1 adenovirus led to rapid depletion of cellular spermidine and spermine, total inhibition of protein synthesis, and growth arrest within 24 h. The SAT1 effect is most likely due to depletion of spermidine and spermine, because stable polyamine analogs that are not substrates for SAT1 restored GFP and endogenous protein synthesis. Loss of polysomes with increased 80S monosomes in the polyamine-depleted cells suggests a direct role for polyamines in translation initiation. Our data provide strong evidence for a primary function of polyamines, spermidine and spermine, in translation in mammalian cells.
Collapse
|
|
26
|
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.
Collapse
Affiliation(s)
- Nimisha Mittal
- School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
| | | | | | | |
Collapse
|
|
27
|
Soliman ML, Puig KL, Combs CK, Rosenberger TA. Acetate reduces microglia inflammatory signaling in vitro. J Neurochem 2012; 123:555-67. [PMID: 22924711 DOI: 10.1111/j.1471-4159.2012.07955.x] [Citation(s) in RCA: 95] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2012] [Revised: 08/23/2012] [Accepted: 08/24/2012] [Indexed: 12/21/2022]
Abstract
Acetate supplementation increases brain acetyl-CoA and histone acetylation and reduces lipopolysaccharide (LPS)-induced neuroglial activation and interleukin (IL)-1β expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen-activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor-kappa B (NF-κB) in primary and BV-2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS-induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL-6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor-β1 (TGF-β1) and both IL-4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2-4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS-induced elevation of NF-κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non-histone protein acetylation.
Collapse
Affiliation(s)
- Mahmoud L Soliman
- Department of Pharmacology, Physiology and Therapeutics, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND, USA
| | | | | | | |
Collapse
|
|
28
|
Dyshlovoy SA, Naeth I, Venz S, Preukschas M, Sievert H, Jacobsen C, Shubina LK, Gesell Salazar M, Scharf C, Walther R, Krepstakies M, Priyadarshini P, Hauber J, Fedorov SN, Bokemeyer C, Stonik VA, Balabanov S, Honecker F. Proteomic profiling of germ cell cancer cells treated with aaptamine, a marine alkaloid with antiproliferative activity. J Proteome Res 2012; 11:2316-30. [PMID: 22409352 DOI: 10.1021/pr300170p] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Aaptamine is a marine compound isolated from the sponge Aaptos aaptos showing antiproliferative properties via an undefined mode of action. We analyzed the effects of aaptamine treatment on the proliferation and protein expression of the pluripotent human embryonal carcinoma cell line NT2. Effects on proliferation, cell cycle distribution, and induction of apoptosis were analyzed. At lower concentrations, including the IC50 of 50 μM, aaptamine treatment resulted in a G2/M phase cell cycle arrest, whereas at higher concentrations, induction of apoptosis was seen. Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of the most significantly up- and down-regulated proteins. Aaptamine treatment at the IC50 for 48 h resulted in alteration of 10 proteins, of which five each showed up- and down-regulation. Changes in the 2D map were frequently noticed as a result of post-transcriptional modifications, e.g., of the hypusine modification of the eukaryotic initiation factor 5A (eIF5A). Observed alterations such as increased expression of CRABP2 and hypusination of eIF5A have previously been identified during differentiation of pluripotent cells. For the first time, we describe changes in protein expression caused by aaptamine, providing valuable information regarding the mode of action of this compound.
Collapse
Affiliation(s)
- Sergey A Dyshlovoy
- Department of Oncology, Haematology and Bone Marrow Transplantation, Section Pneumology, Hubertus Wald-Tumorzentrum, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
29
|
Proteomics analysis of human umbilical vein endothelial cells treated with resveratrol. Amino Acids 2012; 43:1671-8. [DOI: 10.1007/s00726-012-1248-4] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2011] [Accepted: 02/07/2012] [Indexed: 01/15/2023]
|