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Kuppuswamy S, Watson NJ, Ledford WL, Pavri BA, Zhi W, Gbadebo M, Bonsack F, Xu H, Sukumari-Ramesh S. Brain proteome changes after intracerebral hemorrhage in aged male and female mice. Neurobiol Dis 2025; 212:106936. [PMID: 40320180 DOI: 10.1016/j.nbd.2025.106936] [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: 09/16/2024] [Revised: 03/10/2025] [Accepted: 04/28/2025] [Indexed: 05/19/2025] Open
Abstract
Aging is an independent predictor of adverse outcomes after intracerebral hemorrhage (ICH), a stroke subtype with no effective treatment. Despite the expected increase in the incidence of ICH due to population aging and the widespread use of anticoagulants, preclinical studies with aged animal subjects are lacking, and the pathophysiology of ICH in aged subjects has yet to be defined. Herein, we attempt to characterize the brain proteomic changes after ICH using an unbiased label- free quantitative proteomics approach and bioinformatics. To this end, aged male and female mice (18-24 months old) were subjected to sham/ICH. Mice were euthanized on day 3 post-surgery, and ipsilateral brain tissue was collected and subjected to LC-MS/MS analysis. Considering sex as a biological variable, the data derived from males and females were separately analyzed. The proteomics analysis revealed 133 differentially expressed proteins (DEPs) between the sham and ICH groups in male subjects. Among the DEPs, 98 proteins were downregulated, and 35 proteins were upregulated after ICH, compared to sham. In aged female mice, 315 DEPs were identified, of which 221 proteins were downregulated, and 94 proteins were upregulated after ICH compared to sham. The mass spectrometry data was validated using immunohistochemistry or western blot analysis, and the bioinformatics analysis revealed a comprehensive understanding of the signaling pathways associated with ICH. Some DEPs in both aged male and female mice that could play roles in ICH pathology were 14-3-3 proteins and S100-A9. The study also revealed that mitochondrial dysfunction could be a critical regulator of ICH-induced acute brain damage. Overall, the generated proteomics data could help develop hypothesis-driven functional analysis and delineate the complex pathobiology of ICH.
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Affiliation(s)
- Sivaraman Kuppuswamy
- Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, 1120, 15th Street, CB3515, Augusta, GA 30912, United States of America
| | - Noah J Watson
- Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, 1120, 15th Street, CB3515, Augusta, GA 30912, United States of America
| | - William Luke Ledford
- Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, 1120, 15th Street, CB3515, Augusta, GA 30912, United States of America
| | - Blake A Pavri
- Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, 1120, 15th Street, CB3515, Augusta, GA 30912, United States of America
| | - Wenbo Zhi
- Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, 1120, 15th Street, CB3515, Augusta, GA 30912, United States of America
| | - Mary Gbadebo
- Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, 1120, 15th Street, CB3515, Augusta, GA 30912, United States of America
| | - Frederick Bonsack
- Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, 1120, 15th Street, CB3515, Augusta, GA 30912, United States of America
| | - Hongyan Xu
- Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, 1120, 15th Street, CB3515, Augusta, GA 30912, United States of America
| | - Sangeetha Sukumari-Ramesh
- Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, 1120, 15th Street, CB3515, Augusta, GA 30912, United States of America.
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Rojo-Romero MA, Gutiérrez-Nájera NA, Cruz-Fuentes CS, Romero-Pimentel AL, Mendoza-Morales R, García-Dolores F, Morales-Marín ME, Castro-Martínez X, González-Sáenz E, Torres-Campuzano J, Medina-Sánchez T, Hernández-Fonseca K, Nicolini-Sánchez H, Jiménez-García LF. Proteome analysis of the prefrontal cortex and the application of machine learning models for the identification of potential biomarkers related to suicide. Front Psychiatry 2025; 15:1429953. [PMID: 40051599 PMCID: PMC11882514 DOI: 10.3389/fpsyt.2024.1429953] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/09/2024] [Accepted: 12/24/2024] [Indexed: 03/09/2025] Open
Abstract
Introduction Suicide is a significant public health problem, with increased rates in low- and middle-income countries such as Mexico; therefore, suicide prevention is important. Suicide is a complex and multifactorial phenomenon in which biological and social factors are involved. Several studies on the biological mechanisms of suicide have analyzed the proteome of the dorsolateral prefrontal cortex (DLPFC) in people who have died by suicide. The aim of this work was to analyze the protein expression profile in the DLPFC of individuals who died by suicide in comparison to age-matched controls in order to gain information on the molecular basis in the brain of these individuals and the selection of potential biomarkers for the identification of individuals at risk of suicide. In addition, this information was analyzed using machine learning (ML) algorithms to propose a model for predicting suicide. Methods Brain tissue (Brodmann area 9) was sampled from male cases (n=9) and age-matched controls (n=7). We analyzed the proteomic differences between the groups using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Bioinformatics tools were used to clarify the biological relevance of the differentially expressed proteins. In addition, this information was analyzed using machine learning (ML) algorithms to propose a model for predicting suicide. Results Twelve differentially expressed proteins were also identified (t 14 ≤ 0.5). Using Western blotting, we validated the decrease in expression of peroxiredoxin 2 and alpha-internexin in the suicide cases. ML models were trained using densitometry data from the 2D gel images of each selected protein and the models could differentiate between both groups (control and suicide cases). Discussion Our exploratory pathway analysis highlighted oxidative stress responses and neurodevelopmental pathways as key processes perturbed in the DLPFC of suicides. Regarding ML models, KNeighborsClassifier was the best predicting conditions. Here we show that these proteins of the DLPFC may help to identify brain processes associated with suicide and they could be validated as potential biomarkers of this outcome.
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Affiliation(s)
- Manuel Alejandro Rojo-Romero
- Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico
- Psychiatric and Neurodegenerative Diseases Laboratory, National Institute of Genomic Medicine, Mexico City, Mexico
- National Institute of Psychiatry “Ramón de la Fuente Muñíz”, Mexico City, Mexico
| | - Nora Andrea Gutiérrez-Nájera
- Psychiatric and Neurodegenerative Diseases Laboratory, National Institute of Genomic Medicine, Mexico City, Mexico
| | | | - Ana Luisa Romero-Pimentel
- Psychiatric and Neurodegenerative Diseases Laboratory, National Institute of Genomic Medicine, Mexico City, Mexico
| | - Roberto Mendoza-Morales
- Institute of Expert Services and Forensic Sciences of Mexico City (INCIFO), Mexico City, Mexico
| | - Fernando García-Dolores
- Institute of Expert Services and Forensic Sciences of Mexico City (INCIFO), Mexico City, Mexico
| | - Mirna Edith Morales-Marín
- Psychiatric and Neurodegenerative Diseases Laboratory, National Institute of Genomic Medicine, Mexico City, Mexico
| | - Xóchitl Castro-Martínez
- Psychiatric and Neurodegenerative Diseases Laboratory, National Institute of Genomic Medicine, Mexico City, Mexico
| | | | - Jonatan Torres-Campuzano
- Psychiatric and Neurodegenerative Diseases Laboratory, National Institute of Genomic Medicine, Mexico City, Mexico
| | - Tania Medina-Sánchez
- National Institute of Psychiatry “Ramón de la Fuente Muñíz”, Mexico City, Mexico
| | | | - Humberto Nicolini-Sánchez
- Psychiatric and Neurodegenerative Diseases Laboratory, National Institute of Genomic Medicine, Mexico City, Mexico
| | - Luis Felipe Jiménez-García
- Cell Nanobiology Laboratory, Faculty of Sciences, National Autonomous University of Mexico, Mexico City, Mexico
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Gao X, Gao G, Zheng W, Liu H, Pan W, Xia X, Zhang D, Lin W, Wang Z, Feng B. PARylation of 14-3-3 proteins controls the virulence of Magnaporthe oryzae. Nat Commun 2024; 15:8047. [PMID: 39277621 PMCID: PMC11401899 DOI: 10.1038/s41467-024-51955-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2023] [Accepted: 08/20/2024] [Indexed: 09/17/2024] Open
Abstract
Magnaporthe oryzae is a devastating fungal pathogen that causes the rice blast disease worldwide. The post-translational modification of ADP-ribosylation holds significant importance in various fundamental biological processes. However, the specific function of this modification in M. oryzae remains unknown. This study revealed that Poly(ADP-ribosyl)ation (PARylation) executes a critical function in M. oryzae. M. oryzae Poly(ADP-ribose) polymerase 1 (PARP1) exhibits robust PARylation activity. Disruption of PARylation by PARP1 knock-out or chemical inhibition reveals its involvement in M. oryzae virulence, particularly in appressorium formation. Furthermore, we identified two M. oryzae 14-3-3 proteins, GRF1 and GRF2, as substrates of PARP1. Deletion of GRF1 or GRF2 results in delayed and dysfunctional appressorium, diminished plant penetration, and reduced virulence of the fungus. Biochemical and genetic evidence suggest that PARylation of 14-3-3s is essential for its function in M. oryzae virulence. Moreover, PARylation regulates 14-3-3 dimerization and is required for the activation of the mitogen-activated protein kinases (MAPKs), Pmk1 and Mps1. GRF1 interacts with both Mst7 and Pmk1, and bridges their interaction in a PARylation-dependent manner. This study unveils a distinctive mechanism that PARylation of 14-3-3 proteins controls appressorium formation through MAPK activation, and could facilitate the development of new strategies of rice blast disease control.
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Affiliation(s)
- Xiuqin Gao
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
- College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Gaigai Gao
- College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Weifeng Zheng
- College of Jun Cao Science and Ecology (College of Carbon Neutrality), Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Haibing Liu
- Plant Immunity Center, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
- College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Wenbo Pan
- College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Xi Xia
- College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Dongmei Zhang
- College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, China
| | - Wenwei Lin
- College of Jun Cao Science and Ecology (College of Carbon Neutrality), Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
- Center for Horticultural Biology and Metabolomics, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
| | - Zonghua Wang
- State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
- Fuzhou Institute of Oceanography, Minjiang University, Fuzhou, 350108, China.
| | - Baomin Feng
- Plant Immunity Center, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
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Obsilova V, Obsil T. Look for the Scaffold: Multifaceted Regulation of Enzyme Activity by 14-3-3 Proteins. Physiol Res 2024; 73:S401-S412. [PMID: 38647170 PMCID: PMC11412345 DOI: 10.33549/physiolres.935306] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/04/2024] Open
Abstract
Enzyme activity is regulated by several mechanisms, including phosphorylation. Phosphorylation is a key signal transduction process in all eukaryotic cells and is thus crucial for virtually all cellular processes. In addition to its direct effect on protein structure, phosphorylation also affects protein-protein interactions, such as binding to scaffolding 14-3-3 proteins, which selectively recognize phosphorylated motifs. These interactions then modulate the catalytic activity, cellular localisation and interactions of phosphorylated enzymes through different mechanisms. The aim of this mini-review is to highlight several examples of 14-3-3 protein-dependent mechanisms of enzyme regulation previously studied in our laboratory over the past decade. More specifically, we address here the regulation of the human enzymes ubiquitin ligase Nedd4-2, procaspase-2, calcium-calmodulin dependent kinases CaMKK1/2, and death-associated protein kinase 2 (DAPK2) and yeast neutral trehalase Nth1.
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Affiliation(s)
- V Obsilova
- Institute of Physiology of the Czech Academy of Sciences, Laboratory of Structural Biology of Signaling Proteins, Division BIOCEV, Vestec, Czech Republic. or
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Yan R, Xia Y, Zhou K, Liu J, Sun Y, He C, Ge X, Yang M, Sun C, Yuan L, Li S, Yang B, Meng F, Cao L, Ruan C, Dai K. Essential role of glycoprotein Ibα in platelet activation. Blood Adv 2024; 8:3388-3401. [PMID: 38701351 PMCID: PMC11255362 DOI: 10.1182/bloodadvances.2023012308] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2023] [Revised: 04/03/2024] [Accepted: 04/16/2024] [Indexed: 05/05/2024] Open
Abstract
ABSTRACT Glycoprotein Ibα (GPIbα), the ligand-binding subunit of platelet GPIb-IX complex, interacts with von Willebrand factor (VWF) exposed at the injured vessel wall, initiating platelet adhesion, activation, hemostasis, and thrombus formation. The cytoplasmic tail of GPIbα interacts with 14-3-3ζ, regulating the VWF-GPIbα-elicited signal transduction and VWF binding function of GPIbα. However, we unexpectedly found that the GPIbα-14-3-3ζ association, beyond VWF-dependent function, is essential for general platelet activation. We found that the myristoylated peptide of GPIbα C-terminus MPαC, a potential GPIbα inhibitor, by itself induced platelet aggregation, integrin αIIbβ3 activation, granule secretion, and phosphatidylserine (PS) exposure. Conversely, the deletion of the cytoplasmic tail of GPIbα in mouse platelets (10aa-/-) decreased platelet aggregation, integrin αIIbβ3 activation, granule secretion, and PS exposure induced by various physiological agonists. Phosphoproteome-based kinase activity profiling revealed significantly upregulated protein kinase C (PKC) activity in MPαC-treated platelets. MPαC-induced platelet activation was abolished by the pan-PKC inhibitor and PKCα deletion. Decreased PKC activity was observed in both resting and agonist-stimulated 10aa-/- platelets. GPIbα regulates PKCα activity by sequestering 14-3-3ζ from PKCα. In vivo, the deletion of the GPIbα cytoplasmic tail impaired mouse hemostasis and thrombus formation and protected against platelet-dependent pulmonary thromboembolism. Therefore, our findings demonstrate an essential role for the GPIbα cytoplasmic tail in regulating platelet general activation and thrombus formation beyond the VWF-GPIbα axis.
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Affiliation(s)
- Rong Yan
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Yue Xia
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Kangxi Zhou
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Jun Liu
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Yueyue Sun
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Chunyan He
- Department of Clinical Laboratory, The Second Affiliated Hospital of Soochow University, Suzhou, China
| | - Xinxin Ge
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Mengnan Yang
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Chenglin Sun
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Liuxia Yuan
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Shujun Li
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Biao Yang
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Fanbi Meng
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Lijuan Cao
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Changgeng Ruan
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
| | - Kesheng Dai
- Jiangsu Institute of Hematology, Cyrus Tang Medical Institute, The First Affiliated Hospital and Collaborative Innovation Center of Hematology, Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Health, National Clinical Research Center for Hematological Diseases, Suzhou, China
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Zhou H, Sun X, Dai Y, Wang X, Dai Z, Li X. 14-3-3-η interacts with BCL-2 to protect human endothelial progenitor cells from ox-LDL-triggered damage. Cell Biol Int 2024; 48:290-299. [PMID: 38100125 DOI: 10.1002/cbin.12105] [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: 05/26/2023] [Revised: 10/31/2023] [Accepted: 11/18/2023] [Indexed: 02/15/2024]
Abstract
Oxidized low-density lipoprotein (ox-LDL) causes dysfunction of endothelial progenitor cells (EPCs), and we recently reported that 14-3-3-η can attenuate the damage triggered by ox-LDL in EPCs. However, the molecular mechanisms by which 14-3-3-η protects EPCs from the damage caused by ox-LDL are not fully understood. In this study, we observed that the expression of 14-3-3-η and BCL-2 were downregulated in ox-LDL-treated EPCs. Overexpression of 14-3-3-η in ox-LDL-treated EPC significantly increased BCL-2 level, while knockdown of BCL-2 reduced 14-3-3-η expression and mitigated the protective effect of 14-3-3-η on EPCs. In addition, we discovered that 14-3-3-η colocalizes and interacts with BCL-2 in EPCs. Taken together, these data suggest that 14-3-3-η protects EPCs from ox-LDL-induced damage by its interaction with BCL-2.
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Affiliation(s)
- Hui Zhou
- Department of Cardiovascular Medicine, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
| | - Xiaopei Sun
- Department of Cardiovascular Medicine, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
| | - Yi Dai
- Department of Cardiovascular Medicine, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
| | - Xiaotong Wang
- Department of Cardiovascular Medicine, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
| | - Zhihong Dai
- Department of Cardiovascular Medicine, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
| | - Xiuli Li
- Department of Cardiovascular Medicine, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
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Liu Z, Lyu X, Chen J, Zhang B, Xie S, Yuan Y, Sun L, Yuan S, Yu H, Ding J, Yang M. Arnicolide C Suppresses Tumor Progression by Targeting 14-3-3θ in Breast Cancer. Pharmaceuticals (Basel) 2024; 17:224. [PMID: 38399439 PMCID: PMC10892132 DOI: 10.3390/ph17020224] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2023] [Revised: 02/01/2024] [Accepted: 02/06/2024] [Indexed: 02/25/2024] Open
Abstract
Background: Arnicolide C, which is isolated from Centipeda minima, has excellent antitumor effects. However, the potential impacts and related mechanisms of action of arnicolide C in breast cancer remain unknown. Methods: The viability of breast cancer cells was measured using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and colony formation assays. For analysis of apoptosis and the cell cycle, flow cytometry was used. A molecular docking approach was used to explore the possible targets of arnicolide C. Western blot analysis was used to detect changes in the expression of 14-3-3θ and proteins in related pathways after arnicolide C treatment in breast cancer cells. The anti-breast cancer effect of arnicolide C in vivo was evaluated by establishing cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models. Results: Arnicolide C inhibited proliferation, increased apoptosis, and induced G1 arrest. In particular, molecular docking analysis indicated that arnicolide C binds to 14-3-3θ. Arnicolide C reduced 14-3-3θ expression and inhibited its downstream signaling pathways linked to cell proliferation. Similar results were obtained in the CDX and PDX models. Conclusion: Arnicolide C can have an anti-breast cancer effect both in vitro and in vivo and can induce cell cycle arrest and increase apoptosis in vitro. The molecular mechanism may be related to the effect of arnicolide C on the expression level of 14-3-3θ. However, the specific mechanism through which arnicolide C affects 14-3-3θ protein expression still needs to be determined.
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Affiliation(s)
- Zhengrui Liu
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China
| | - Xiaodan Lyu
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China
| | - Jiaxu Chen
- College of Pharmacy, Lanzhou University, Lanzhou 730000, China
| | - Benteng Zhang
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China
| | - Siman Xie
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China
| | - Yan Yuan
- Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
| | - Li Sun
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China
| | - Shengtao Yuan
- Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing 210009, China
| | - Hong Yu
- Department of Pathology, The Affiliated Taizhou People’s Hospital of Nanjing Medical University, Taizhou 225300, China
| | - Jian Ding
- Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing 210009, China
- State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
| | - Mei Yang
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China
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Abdi G, Jain M, Patil N, Upadhyay B, Vyas N, Dwivedi M, Kaushal RS. 14-3-3 proteins-a moonlight protein complex with therapeutic potential in neurological disorder: in-depth review with Alzheimer's disease. Front Mol Biosci 2024; 11:1286536. [PMID: 38375509 PMCID: PMC10876095 DOI: 10.3389/fmolb.2024.1286536] [Citation(s) in RCA: 6] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2023] [Accepted: 01/05/2024] [Indexed: 02/21/2024] Open
Abstract
Alzheimer's disease (AD) affects millions of people worldwide and is a gradually worsening neurodegenerative condition. The accumulation of abnormal proteins, such as tau and beta-amyloid, in the brain is a hallmark of AD pathology. 14-3-3 proteins have been implicated in AD pathology in several ways. One proposed mechanism is that 14-3-3 proteins interact with tau protein and modulate its phosphorylation, aggregation, and toxicity. Tau is a protein associated with microtubules, playing a role in maintaining the structural integrity of neuronal cytoskeleton. However, in the context of Alzheimer's disease (AD), an abnormal increase in its phosphorylation occurs. This leads to the aggregation of tau into neurofibrillary tangles, which is a distinctive feature of this condition. Studies have shown that 14-3-3 proteins can bind to phosphorylated tau and regulate its function and stability. In addition, 14-3-3 proteins have been shown to interact with beta-amyloid (Aβ), the primary component of amyloid plaques in AD. 14-3-3 proteins can regulate the clearance of Aβ through the lysosomal degradation pathway by interacting with the lysosomal membrane protein LAMP2A. Dysfunction of lysosomal degradation pathway is thought to contribute to the accumulation of Aβ in the brain and the progression of AD. Furthermore, 14-3-3 proteins have been found to be downregulated in the brains of AD patients, suggesting that their dysregulation may contribute to AD pathology. For example, decreased levels of 14-3-3 proteins in cerebrospinal fluid have been suggested as a biomarker for AD. Overall, these findings suggest that 14-3-3 proteins may play an important role in AD pathology and may represent a potential therapeutic target for the disease. However, further research is needed to fully understand the mechanisms underlying the involvement of 14-3-3 proteins in AD and to explore their potential as a therapeutic target.
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Affiliation(s)
- Gholamareza Abdi
- Department of Biotechnology, Persian Gulf Research Institute, Persian Gulf University, Bushehr, Iran
| | - Mukul Jain
- Cell and Developmental Biology Laboratory, Research and Development Cell, Parul University, Vadodara, Gujarat, India
- Department of Life Sciences, Parul Institute of Applied Sciences, Parul University, Vadodara, Gujarat, India
| | - Nil Patil
- Cell and Developmental Biology Laboratory, Research and Development Cell, Parul University, Vadodara, Gujarat, India
- Department of Life Sciences, Parul Institute of Applied Sciences, Parul University, Vadodara, Gujarat, India
| | - Bindiya Upadhyay
- Department of Life Sciences, Parul Institute of Applied Sciences, Parul University, Vadodara, Gujarat, India
| | - Nigam Vyas
- Department of Life Sciences, Parul Institute of Applied Sciences, Parul University, Vadodara, Gujarat, India
- Biophysics and Structural Biology Laboratory, Research and Development Cell, Parul University, Vadodara, Gujarat, India
| | - Manish Dwivedi
- Amity Institute of Biotechnology, Amity University, Lucknow, Uttar Pradesh, India
| | - Radhey Shyam Kaushal
- Department of Life Sciences, Parul Institute of Applied Sciences, Parul University, Vadodara, Gujarat, India
- Biophysics and Structural Biology Laboratory, Research and Development Cell, Parul University, Vadodara, Gujarat, India
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9
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Guo M, He M, Zhang Y, Liu W, Qi M, Liu Z, Yi G, Deng S, Li Y, Sun X, Zhao L, Chen T, Liu Y. Nucleo-cytoplasmic shuttling of 14-3-3 epsilon carrying hnRNP C promotes autophagy. Cancer Biol Ther 2023; 24:2246203. [PMID: 37599448 PMCID: PMC10443976 DOI: 10.1080/15384047.2023.2246203] [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: 10/04/2022] [Revised: 08/03/2023] [Accepted: 08/04/2023] [Indexed: 08/22/2023] Open
Abstract
Translocation of 14-3-3 protein epsilon (14-3-3ε) was found to be involved in Triptolide (Tp)-induced inhibition of colorectal cancer (CRC) cell proliferation. However, the form of cell death induced by 14-3-3ε translocation and mechanisms underlying this effect remain unclear. This study employed label-free LC-MS/MS to identify 14-3-3ε-associated proteins in CRC cells treated with or without Tp. Our results confirmed that heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNP C) were exported out of the nucleus by 14-3-3ε and degraded by ubiquitination. The nucleo-cytoplasmic shuttling of 14-3-3ε carrying hnRNP C mediated Tp-induced proliferation inhibition, cell cycle arrest and autophagic processes. These findings have broad implications for our understanding of 14-3-3ε function, provide an explanation for the mechanism of nucleo-cytoplasmic shuttling of hnRNP C and provide new insights into the complex regulation of autophagy.
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Affiliation(s)
- Manlan Guo
- Department of Neurosurgery & Medical Research Center, Shunde Hospital, Southern Medical University (The First People’s Hospital of Shunde Foshan), Foshan, China
- Transformation Engineering Research Center of Chronic Disease Diagnosis and Treatment, Department of Physiology, School of Basic Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, China
| | - Minyi He
- Center for Clinical Medical Education, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
| | - Yi Zhang
- Transformation Engineering Research Center of Chronic Disease Diagnosis and Treatment, Department of Physiology, School of Basic Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, China
- Department of Oncology, Guizhou Cancer Hospital, Guiyang, Guizhou, China
| | - Weiwen Liu
- Department of Neurosurgery & Medical Research Center, Shunde Hospital, Southern Medical University (The First People’s Hospital of Shunde Foshan), Foshan, China
| | - Min Qi
- Department of Neurosurgery & Medical Research Center, Shunde Hospital, Southern Medical University (The First People’s Hospital of Shunde Foshan), Foshan, China
| | - Zhifeng Liu
- Guangdong Provincial Key Laboratory of Molecular Oncologic Pathology, Southern Medical University, Guangzhou, China
| | - Guozhong Yi
- Department of Neurosurgery & Medical Research Center, Shunde Hospital, Southern Medical University (The First People’s Hospital of Shunde Foshan), Foshan, China
| | - Shengze Deng
- Department of Neurosurgery & Medical Research Center, Shunde Hospital, Southern Medical University (The First People’s Hospital of Shunde Foshan), Foshan, China
| | - Yaomin Li
- Department of Neurosurgery & Medical Research Center, Shunde Hospital, Southern Medical University (The First People’s Hospital of Shunde Foshan), Foshan, China
| | - Xuegang Sun
- School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China
| | - Liang Zhao
- Guangdong Provincial Key Laboratory of Molecular Oncologic Pathology, Southern Medical University, Guangzhou, China
| | - Tengxiang Chen
- Transformation Engineering Research Center of Chronic Disease Diagnosis and Treatment, Department of Physiology, School of Basic Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, China
| | - Yawei Liu
- Department of Neurosurgery & Medical Research Center, Shunde Hospital, Southern Medical University (The First People’s Hospital of Shunde Foshan), Foshan, China
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10
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Bachar-Wikstrom E, Dhillon B, Gill Dhillon N, Abbo L, Lindén SK, Wikstrom JD. Mass Spectrometry Analysis of Shark Skin Proteins. Int J Mol Sci 2023; 24:16954. [PMID: 38069276 PMCID: PMC10707392 DOI: 10.3390/ijms242316954] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2023] [Revised: 11/21/2023] [Accepted: 11/24/2023] [Indexed: 12/18/2023] Open
Abstract
The mucus layer covering the skin of fish has several roles, including protection against pathogens and mechanical damage in which proteins play a key role. While proteins in the skin mucus layer of various common bony fish species have been explored, the proteins of shark skin mucus remain unexplored. In this pilot study, we examine the protein composition of the skin mucus in spiny dogfish sharks and chain catsharks through mass spectrometry (NanoLC-MS/MS). Overall, we identified 206 and 72 proteins in spiny dogfish (Squalus acanthias) and chain catsharks (Scyliorhinus retifer), respectively. Categorization showed that the proteins belonged to diverse biological processes and that most proteins were cellular albeit a significant minority were secreted, indicative of mucosal immune roles. The secreted proteins are reviewed in detail with emphasis on their immune potentials. Moreover, STRING protein-protein association network analysis showed that proteins of closely related shark species were more similar as compared to a more distantly related shark and a bony fish, although there were also significant overlaps. This study contributes to the growing field of molecular shark studies and provides a foundation for further research into the functional roles and potential human biomedical implications of shark skin mucus proteins.
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Affiliation(s)
- Etty Bachar-Wikstrom
- Dermatology and Venereology Division, Department of Medicine (Solna), Karolinska Institutet, 17177 Stockholm, Sweden
- Whitman Center, Marine Biological Laboratory, Woods Hole, MA 02543, USA
| | - Braham Dhillon
- Department of Plant Pathology, Fort Lauderdale Research and Education Center, IFAS, University of Florida, Davie, FL 33314, USA
| | - Navi Gill Dhillon
- Department of Biological Sciences, Nova Southeastern University, Davie, FL 33314, USA
| | - Lisa Abbo
- Whitman Center, Marine Biological Laboratory, Woods Hole, MA 02543, USA
| | - Sara K. Lindén
- Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 40530 Gothenburg, Sweden
| | - Jakob D. Wikstrom
- Dermatology and Venereology Division, Department of Medicine (Solna), Karolinska Institutet, 17177 Stockholm, Sweden
- Whitman Center, Marine Biological Laboratory, Woods Hole, MA 02543, USA
- Dermato-Venereology Clinic, Karolinska University Hospital, 17176 Stockholm, Sweden
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11
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Hui W, Wenhua S, Shuojie Z, Lulin W, Panpan Z, Tongtong Z, Xiaoli X, Juhua D. How does NFAT3 regulate the occurrence of cardiac hypertrophy? IJC HEART & VASCULATURE 2023; 48:101271. [PMID: 37753338 PMCID: PMC10518445 DOI: 10.1016/j.ijcha.2023.101271] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2023] [Revised: 08/24/2023] [Accepted: 09/08/2023] [Indexed: 09/28/2023]
Abstract
Cardiac hypertrophy is initially an adaptive response to physiological and pathological stimuli. Although pathological myocardial hypertrophy is the main cause of morbidity and mortality, our understanding of its mechanism is still weak. NFAT3 (nuclear factor of activated T-cell-3) is a member of the nuclear factor of the activated T cells (NFAT) family. NFAT3 plays a critical role in regulating the expression of cardiac hypertrophy genes by inducing their transcription. Recently, accumulating evidence has indicated that NFAT3 is a potent regulator of the progression of cardiac hypertrophy. This review, for the first time, summarizes the current studies on NFAT3 in cardiac hypertrophy, including the pathophysiological processes and the underlying pathological mechanism, focusing on the nuclear translocation and transcriptional function of NFAT3. This review will provide deep insight into the pathogenesis of cardiac hypertrophy and a theoretical basis for identifying new therapeutic targets in the NFAT3 network.
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Affiliation(s)
- Wang Hui
- Laboratory of Molecular Genetics of Aging & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, China
| | - Su Wenhua
- Laboratory of Molecular Genetics of Aging & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, China
- Department of Cardiology, The First People’s Hospital of Yunnan Province, Kunming, Yunnan, China
| | - Zhang Shuojie
- Laboratory of Molecular Genetics of Aging & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, China
| | - Wang Lulin
- Laboratory of Molecular Genetics of Aging & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, China
| | - Zhao Panpan
- Laboratory of Molecular Genetics of Aging & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, China
| | - Zhang Tongtong
- Laboratory of Molecular Genetics of Aging & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, China
| | - Xie Xiaoli
- Laboratory of Molecular Genetics of Aging & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, China
| | - Dan Juhua
- Laboratory of Molecular Genetics of Aging & Tumor, Medical School, Kunming University of Science and Technology, Kunming, Yunnan, China
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12
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Kongsamut S, Eishingdrelo H. Modulating GPCR and 14-3-3 protein interactions: Prospects for CNS drug discovery. Drug Discov Today 2023; 28:103641. [PMID: 37236523 PMCID: PMC10524340 DOI: 10.1016/j.drudis.2023.103641] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2023] [Revised: 04/29/2023] [Accepted: 05/19/2023] [Indexed: 05/28/2023]
Abstract
The activation of G-protein-coupled receptors (GPCRs) triggers a series of protein-protein interaction events that subsequently induce a chain of reactions, including alteration of receptor structures, phosphorylation, recruitment of associated proteins, protein trafficking and gene expression. Multiple GPCR signaling transduction pathways are evident - two well-studied pathways are the GPCR-mediated G-protein and β-arrestin pathways. Recently, ligand-induced interactions between GPCRs and 14-3-3 proteins have been demonstrated. This linking of GPCRs to 14-3-3 protein signal hubs opens up a whole new realm of signal transduction possibilities. 14-3-3 proteins play a key part in GPCR trafficking and signal transduction. GPCR-mediated 14-3-3 protein signaling can be harnessed for the study of GPCR function and therapeutics.
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Affiliation(s)
- Sathapana Kongsamut
- Research Institute for Scientists Emeriti, Drew University, 36 Madison Avenue, Madison, NJ 07940, USA
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13
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Kumari M, Kapoor R, Devanna BN, Varshney S, Kamboj R, Rai AK, Sharma TR. iTRAQ based proteomic analysis of rice lines having single or stacked blast resistance genes: Pi54/ Pi54rh during incompatible interaction with Magnaporthe oryzae. PHYSIOLOGY AND MOLECULAR BIOLOGY OF PLANTS : AN INTERNATIONAL JOURNAL OF FUNCTIONAL PLANT BIOLOGY 2023; 29:871-887. [PMID: 37520805 PMCID: PMC10382468 DOI: 10.1007/s12298-023-01327-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/19/2022] [Revised: 05/12/2023] [Accepted: 06/08/2023] [Indexed: 08/01/2023]
Abstract
Deployment of single or multiple blast resistance (R) genes in rice plant is considered to be the most promising approach to enhance resistance against blast disease caused by fungus Magnaporthe oryzae. At the proteome level, relatively little information about R gene mediated defence mechanisms for single and stacking resistance characteristics is available. The overall objective of this study is to look at the proteomics of rice plants that have R genes; Pi54, Pi54rh and stacked Pi54 + Pi54rh in response to rice blast infection. In this study 'isobaric tag for relative and absolute quantification' (iTRAQ)-based proteomics analysis was performed in rice plants at 72-h post inoculation with Magnaporthe oryzae and various differentially expressed proteins were identified in these three transgenic lines in comparison to wild type during resistance response to blast pathogen. Through STRING analysis, the observed proteins were further examined to anticipate their linked partners, and it was shown that several defense-related proteins were co-expressed. These proteins can be employed as targets in future rice resistance breeding against Magnaporthe oryzae. The current study is the first to report a proteomics investigation of rice lines that express single blast R gene Pi54, Pi54rh and stacked (Pi54 + Pi54rh) during incompatible interaction with Magnaporthe oryzae. The differentially expressed proteins indicated that secondary metabolites, reactive oxygen species-related proteins, phenylpropanoid, phytohormones and pathogenesis-related proteins have a substantial relationship with the defense response against Magnaporthe oryzae. Supplementary Information The online version contains supplementary material available at 10.1007/s12298-023-01327-3.
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Affiliation(s)
- Mandeep Kumari
- ICAR-National Institute for Plant Biotechnology, New Delhi, India
- Department of Bioscience and Biotechnology, Banasthali Vidyapith, Vanasthali, Rajasthan India
| | - Ritu Kapoor
- National Agri-Food Biotechnology Institute (NABI), Mohali, Punjab India
| | - B. N. Devanna
- ICAR-National Rice Research Institute, Cuttack, Odisha India
| | - Swati Varshney
- CSIR-Institute of Genomics and Integrative Biology, New Delhi, Delhi India
| | - Richa Kamboj
- ICAR-National Institute for Plant Biotechnology, New Delhi, India
- Department of Bioscience and Biotechnology, Banasthali Vidyapith, Vanasthali, Rajasthan India
| | - Amit Kumar Rai
- ICAR-National Institute for Plant Biotechnology, New Delhi, India
| | - T. R. Sharma
- ICAR-National Institute for Plant Biotechnology, New Delhi, India
- Division of Crop Science, Indian Council of Agricultural Research, Krishi Bhavan, New Delhi, India
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14
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Sánchez Viafara JA, de Vasconcelos GL, Maculan R, Alves NG, Ferreira MBD, Sudano MJ, Mingoti GZ, Nunes GB, de Lima RR, Drumond RM, Dos Santos RN, Eberlin MN, Negrão F, Donato MAM, Peixoto CA, Camisão de Souza J. Peroxisome proliferator-activated receptor delta-PPARδ agonist (L-165041) enhances bovine embryo survival and post vitrification viability. Reprod Fertil Dev 2022; 34:658-668. [PMID: 35468312 DOI: 10.1071/rd21245] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2021] [Accepted: 02/09/2022] [Indexed: 11/23/2022] Open
Abstract
The effect of L-165041 (PPARδ-agonist) on decreasing apoptosis and intracellular lipid content was assessed in fresh and vitrified-warmed in vitro -produced bovine embryos. It was hypothesised that the addition of L-165041 to the culture medium enhances development and cryopreservation. Oocytes were allocated to one of two treatments: control-standard culture medium, or L-165041 added to the medium on day1 with no media change. Ultrastructure, cleavage, and blastocyst rates were evaluated in fresh, and in post-vitrification cultured embryos by optical and electronic microscopy. A subset of fresh embryos were fixed for TUNEL assay and for Sudan-Black-B histochemical staining. Vitrified-warmed embryos were assessed using MALDI-MS technique. Cleavage and blastocyst rates (control 49.4±5.2, L-165041 51.8±4.3) were not influenced by L-165041. The proportion of inner cell mass cells (ICM) was higher in fresh embryos, and the rate of total and ICM apoptosis was lower in L-165041. In warmed-embryos, total and ICM apoptosis was lower in L-165041. The overall hatching rate was higher in L-165041 (66.62±2.83% vs 53.19±2.90%). There was less lipid accumulation in fresh L-165041-embryos. In conclusion, the use of L-165041 is recommended to improve the viability of in vitro -derived bovine embryos.
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Affiliation(s)
- Jesús Alfonso Sánchez Viafara
- Departamento de Medicina Veterinária, Universidade Federal de Lavras, Lavras, Minas Gerais, Brasil; and Universidad de Santander, Facultad de Ciencias Agrícolas y Veterinarias, Valledupar, Colombia
| | | | - Renata Maculan
- Instituto Federal do Sul de Minas, Machado, Minas Gerais, Brasil
| | - Nadja Gomes Alves
- Departamento de Zootecnia, Universidade Federal de Lavras, Lavras, Minas Gerais, Brasil
| | | | | | - Gisele Zoccal Mingoti
- Escola de Medicina Veterinária, Laboratório de Fisiologia da Reprodução, Universidade Estadual Paulista, Campus Araçatuba, São Paulo, Brasil
| | - Giovana Barros Nunes
- Escola de Medicina Veterinária, Laboratório de Fisiologia da Reprodução, Universidade Estadual Paulista, Campus Araçatuba, São Paulo, Brasil
| | - Renato Ribeiro de Lima
- Departamento de Estatística, Universidade Federal de Lavras, Lavras, Minas Gerais, Brasil
| | | | | | - Marcos Nogueira Eberlin
- Universidade Estadual de Campinas, Laboratório ThoMSon de Espectrometria de Massas, Campinas, São Paulo, Brasil
| | - Fernanda Negrão
- Universidade Estadual de Campinas, Laboratório ThoMSon de Espectrometria de Massas, Campinas, São Paulo, Brasil
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- NUMPEX-Bio, Universidade Federal do Rio de Janeiro, Campus Duque de Caxias, Rio de Janeiro, Brasil
| | | | | | - José Camisão de Souza
- Departamento de Zootecnia, Universidade Federal de Lavras, Lavras, Minas Gerais, Brasil
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15
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Gao Y, Chen N, Zhang X, Li EY, Luo W, Zhang J, Zhang W, Li S, Wang J, Liu S. Juvenile Hormone Membrane Signaling Enhances its Intracellular Signaling Through Phosphorylation of Met and Hsp83. Front Physiol 2022; 13:872889. [PMID: 35574494 PMCID: PMC9091338 DOI: 10.3389/fphys.2022.872889] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2022] [Accepted: 04/11/2022] [Indexed: 11/30/2022] Open
Abstract
Juvenile hormone (JH) regulates insect development and reproduction through both intracellular and membrane signaling, and the two pathways might crosstalk with each other. Recent studies have reported that JH membrane signaling induces phosphorylation of the JH intracellular receptor Met, thus enhancing its transcriptional activity. To gain more insights into JH-induced Met phosphorylation, we here performed phosphoproteomics to identify potential phosphorylation sites of Met and its paralog Germ-cell expressed (Gce) in Drosophila Kc cells. In vitro experiments demonstrate that JH-induced phosphorylation sites in the basic helix-loop-helix (bHLH) domain, but not in the Per-Arnt-Sim-B (PAS-B) domain, are required for maximization of Met transcriptional activity. Moreover, phosphoproteomics analysis reveale that JH also induces the phosphorylation of Hsp83, a chaperone protein involved in JH-activated Met nuclear import. The JH-induced Hsp83 phosphorylation at S219 facilitates Hsp83-Met binding, thus promoting Met nuclear import and its transcription. By using proteomics, subcellular distribution, and co-immunoprecipitation approaches, we further characterized 14-3-3 proteins as negative regulators of Met nuclear import through physical interaction with Hsp83. These results show that JH membrane signaling induces phosphorylation of the key components in JH intracellular signaling, such as Met and Hsp83, and consequently facilitating JH intracellular signaling.
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Affiliation(s)
- Yue Gao
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, China
| | - Nan Chen
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, China
| | - Xiangle Zhang
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, China
| | - Emma Y. Li
- International Department, The Affiliated High School of South China Normal University, Guangzhou, China
| | - Wei Luo
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, China
| | - Jie Zhang
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, China
| | - Wenqiang Zhang
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, China
| | - Sheng Li
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, China
- Guangmeiyuan R&D Center, Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, South China Normal University, Meizhou, China
| | - Jian Wang
- Department of Entomology, University of Maryland, College Park, MD, United States
| | - Suning Liu
- Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, China
- Guangmeiyuan R&D Center, Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, South China Normal University, Meizhou, China
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16
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Antunes ASLM, Saia-Cereda VM, Crunfli F, Martins-de-Souza D. 14-3-3 proteins at the crossroads of neurodevelopment and schizophrenia. World J Biol Psychiatry 2022; 23:14-32. [PMID: 33952049 DOI: 10.1080/15622975.2021.1925585] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
The 14-3-3 family comprises multifunctional proteins that play a role in neurogenesis, neuronal migration, neuronal differentiation, synaptogenesis and dopamine synthesis. 14-3-3 members function as adaptor proteins and impact a wide variety of cellular and physiological processes involved in the pathophysiology of neurological disorders. Schizophrenia is a psychiatric disorder and knowledge about its pathophysiology is still limited. 14-3-3 have been proven to be linked with the dopaminergic, glutamatergic and neurodevelopmental hypotheses of schizophrenia. Further, research using genetic models has demonstrated the role played by 14-3-3 proteins in neurodevelopment and neuronal circuits, however a more integrative and comprehensive approach is needed for a better understanding of their role in schizophrenia. For instance, we still lack an integrated assessment of the processes affected by 14-3-3 proteins in the dopaminergic and glutamatergic systems. In this context, it is also paramount to understand their involvement in the biology of brain cells other than neurons. Here, we present previous and recent research that has led to our current understanding of the roles 14-3-3 proteins play in brain development and schizophrenia, perform an assessment of their functional protein association network and discuss the use of protein-protein interaction modulators to target 14-3-3 as a potential therapeutic strategy.
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Affiliation(s)
- André S L M Antunes
- Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas, Campinas, Brazil
| | - Verônica M Saia-Cereda
- Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas, Campinas, Brazil
| | - Fernanda Crunfli
- Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas, Campinas, Brazil
| | - Daniel Martins-de-Souza
- Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas, Campinas, Brazil.,Experimental Medicine Research Cluster (EMRC), University of Campinas, Campinas, SP, Brazil.,D'Or Institute for Research and Education (IDOR), São Paulo, Brazil.,Instituto Nacional de Biomarcadores em Neuropsiquiatria (INBION), Conselho Nacional de Desenvolvimento Científico e Tecnológico, São Paulo, Brazil
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17
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Misoprostol treatment prevents hypoxia-induced cardiac dysfunction through a 14-3-3 and PKA regulatory motif on Bnip3. Cell Death Dis 2021; 12:1105. [PMID: 34824192 PMCID: PMC8617186 DOI: 10.1038/s41419-021-04402-3] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2021] [Revised: 11/01/2021] [Accepted: 11/09/2021] [Indexed: 12/31/2022]
Abstract
Systemic hypoxia is a common element in most perinatal emergencies and is a known driver of Bnip3 expression in the neonatal heart. Bnip3 plays a prominent role in the evolution of necrotic cell death, disrupting ER calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests a cardioprotective role for the prostaglandin E1 analog misoprostol during periods of hypoxia, but the mechanisms for this protection are not completely understood. Using a combination of mouse and cell models, we tested if misoprostol is cardioprotective during neonatal hypoxic injury by altering Bnip3 function. Here we report that hypoxia elicits mitochondrial-fragmentation, MPT, reduced ejection fraction, and evidence of necroinflammation, which were abrogated with misoprostol treatment or Bnip3 knockout. Through molecular studies we show that misoprostol leads to PKA-dependent Bnip3 phosphorylation at threonine-181, and subsequent redistribution of Bnip3 from mitochondrial Opa1 and the ER through an interaction with 14-3-3 proteins. Taken together, our results demonstrate a role for Bnip3 phosphorylation in the regulation of cardiomyocyte contractile/metabolic dysfunction, and necroinflammation. Furthermore, we identify a potential pharmacological mechanism to prevent neonatal hypoxic injury.
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18
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Ou M, Xu X, Chen Y, Li L, Zhang L, Liao Y, Sun W, Quach C, Feng J, Tang L. MDM2 induces EMT via the B‑Raf signaling pathway through 14‑3‑3. Oncol Rep 2021; 46:120. [PMID: 33955525 PMCID: PMC8129971 DOI: 10.3892/or.2021.8071] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2020] [Accepted: 03/23/2021] [Indexed: 12/28/2022] Open
Abstract
MDM2 proto‑oncogene, E3 ubiquitin protein ligase (MDM2) is a well‑known oncogene and has been reported to be closely associated with epithelial‑to‑mesenchymal transition (EMT). The present study first demonstrated that the expression levels of MDM2 were markedly increased in TGF‑β‑induced EMT using quantitative PCR and western blotting. In addition, MDM2 was demonstrated to be associated with pathological grade in clinical glioma samples by immunohistochemical staining. Furthermore, overexpression of MDM2 promoted EMT in glioma, lung cancer and breast cancer cell lines using a scratch wound migration assay. Subsequently, the present study explored the mechanism by which MDM2 promoted EMT and revealed that MDM2 induced EMT by upregulating EMT‑related transcription factors via activation of the B‑Raf signaling pathway through tyrosine 3‑monooxygenase activation protein ε using RNA sequencing and western blotting. This mechanism depended on the p53 gene. Furthermore, in vivo experiments and the colony formation experiment demonstrated that MDM2 could promote tumor progression and induce EMT via the B‑Raf signaling pathway. Since EMT contributes to increased drug resistance in tumor cells, the present study also explored the relationship between MDM2 and drug sensitivity using an MTT assay, and identified that MDM2 promoted cell insensitivity to silibinin treatment in an EMT‑dependent manner. This finding is crucial for the development of cancer therapies and can also provide novel research avenues for future biological and clinical studies.
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Affiliation(s)
- Mengting Ou
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, P.R. China
| | - Xichao Xu
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, P.R. China
| | - Ying Chen
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, P.R. China
| | - Li Li
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, P.R. China
| | - Lu Zhang
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, P.R. China
| | - Yi Liao
- Department of Cardiothoracic Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400044, P.R. China
| | - Weichao Sun
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, P.R. China
| | - Christine Quach
- Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA 90033, USA
| | - Jianguo Feng
- Department of Anesthesiology, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P.R. China
| | - Liling Tang
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, P.R. China
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19
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Zhang K, Huang Y, Shi Q. Genome-wide identification and characterization of 14-3-3 genes in fishes. Gene 2021; 791:145721. [PMID: 34010706 DOI: 10.1016/j.gene.2021.145721] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2020] [Revised: 05/11/2021] [Accepted: 05/14/2021] [Indexed: 11/13/2022]
Abstract
The 14-3-3 family genes are highly conserved regulatory factors in eukaryotes with involvement in multiple important cellular processes. However, detailed investigations of this family in fishes are very limited. Here, a comparative genomic and transcriptomic survey were performed to investigate the 14-3-3 family in fishes. We confirmed that the numbers of 14-3-3 genes ranged from 5 to 7 in non-teleost fishes, as well as additional 14-3-3 genes (9 to 11) in teleost fishes. In addition, some special teleost fishes possess 17 to 25 14-3-3s, which undergone the fourth whole-genome duplication (WGD). We also found that six pairs of fish 14-3-3 genes were clustered with mammalian ε, γ, ς, η, τand β isotypes, respectively, while σ was absent with a potential specificity within mammals, on the basis of their phylogenetic and synteny analyses. According to our results, we inferred that the diversity of 14-3-3 genes in fishes seems to be generated from a combination of WGD and gene loss. Comparative transcriptomic analysis revealed that there are differences in tissue distribution, and we speculated that 14-3-3 genes may contribute to terrestrial adaptations in mudskippers. In addition, protein sequence alignments of 14-3-3s supported their differential roles in fishes. In summary, our present comparative genomic and transcriptomic survey will benefit for further functional investigations of these fish genes.
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Affiliation(s)
- Kai Zhang
- College of Animal Science and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China; Department of Ocean Science, Division of Life Science and Hong Kong Branch of the Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), The Hong Kong University of Science and Technology, Hong Kong, China.
| | - Yu Huang
- Shenzhen Key Lab of Marine Genomics, Guangdong Provincial Key Lab of Molecular Breeding in Marine Economic Animals, BGI Academy of Marine Sciences, BGI Marine, BGI, Shenzhen 518083, China.
| | - Qiong Shi
- Shenzhen Key Lab of Marine Genomics, Guangdong Provincial Key Lab of Molecular Breeding in Marine Economic Animals, BGI Academy of Marine Sciences, BGI Marine, BGI, Shenzhen 518083, China; Laboratory of Aquatic Genomics, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China.
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20
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Li S, Zhang N, Liu S, Li J, Liu L, Wang X, Li X, Gong P, Zhang X. Protective Immunity Against Neospora caninum Infection Induced by 14-3-3 Protein in Mice. Front Vet Sci 2021; 8:638173. [PMID: 33748214 PMCID: PMC7965954 DOI: 10.3389/fvets.2021.638173] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2020] [Accepted: 02/03/2021] [Indexed: 11/16/2022] Open
Abstract
Neospora caninum is an apicomplexan parasite that infects many mammals and remains a threatening disease worldwide because of the lack of effective drugs and vaccines. Our previous studies demonstrated that N. caninum 14-3-3 protein (Nc14-3-3), which is included in N. caninum extracellular vesicles (NEVs), can induce effective immune responses and stimulate cytokine expression in mouse peritoneal macrophages. However, whether Nc14-3-3 has a protective effect and its mechanisms are poorly understood. Here, we evaluated the immune responses and protective effects of Nc14-3-3 against exposure to 2 × 107 Nc-1 tachyzoites. Antibody (IgG, IgGl, and IgG2a) levels and Th1-type (IFN-γ and IL-12) and Th2-type (IL-4 and IL-10) cytokines in mouse serum, survival rates, survival times, and parasite burdens were detected. In the present study, the immunostimulatory effect of Nc14-3-3 was confirmed, as it triggered Th1-type cytokine (IFN-γ and IL-12) production in mouse serum 2 weeks after the final immunization. Moreover, the immunization of C57BL/6 mice with Nc14-3-3 induced high IgG antibody levels and significant increases in CD8+ T lymphocytes in the spleens of mice, indicating that the cellular immune response was significantly stimulated. Mouse survival rates and times were significantly prolonged after immunization; the survival rates were 40% for Nc14-3-3 immunization and 60% for NEV immunization, while mice that received GST, PBS, or blank control all died at 13, 9, or 8 days, respectively, after intraperitoneal N. caninum challenge. In addition, qPCR analysis indicated that there was a reduced parasite burden and diminished pathological changes in the mice immunized with Nc14-3-3. Our data demonstrate that vaccination of mice with Nc14-3-3 elicits both cellular and humoral immune responses and provides partial protection against acute neosporosis. Thus, Nc14-3-3 could be an effective antigen candidate for vaccine development for neosporosis.
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Affiliation(s)
- Shan Li
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China.,Department of Social Medicine and Public Health, School of Basic Medicine, Jiujiang University, Jiujiang, China
| | - Nan Zhang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Shaoxiong Liu
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Jianhua Li
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Li Liu
- College of Basic Medicine, Jilin University, Changchun, China
| | - Xiaocen Wang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Xin Li
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Pengtao Gong
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Xichen Zhang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
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21
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Molecular Analysis of 14-3-3 Genes in Citrus sinensis and Their Responses to Different Stresses. Int J Mol Sci 2021; 22:ijms22020568. [PMID: 33430069 PMCID: PMC7826509 DOI: 10.3390/ijms22020568] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Revised: 01/04/2021] [Accepted: 01/05/2021] [Indexed: 12/31/2022] Open
Abstract
14-3-3 proteins (14-3-3s) are among the most important phosphorylated molecules playing crucial roles in regulating plant development and defense responses to environmental constraints. No report thus far has documented the gene family of 14-3-3s in Citrus sinensis and their roles in response to stresses. In this study, nine 14-3-3 genes, designated as CitGF14s (CitGF14a through CitGF14i) were identified from the latest C. sinensis genome. Phylogenetic analysis classified them into ε-like and non-ε groups, which were supported by gene structure analysis. The nine CitGF14s were located on five chromosomes, and none had duplication. Publicly available RNA-Seq raw data and microarray databases were mined for 14-3-3 expression profiles in different organs of citrus and in response to biotic and abiotic stresses. RT-qPCR was used for further examining spatial expression patterns of CitGF14s in citrus and their temporal expressions in one-year-old C. sinensis “Xuegan” plants after being exposed to different biotic and abiotic stresses. The nine CitGF14s were expressed in eight different organs with some isoforms displayed tissue-specific expression patterns. Six of the CitGF14s positively responded to citrus canker infection (Xanthomonas axonopodis pv. citri). The CitGF14s showed expressional divergence after phytohormone application and abiotic stress treatments, suggesting that 14-3-3 proteins are ubiquitous regulators in C. sinensis. Using the yeast two-hybrid assay, CitGF14a, b, c, d, g, and h were found to interact with CitGF14i proteins to form a heterodimer, while CitGF14i interacted with itself to form a homodimer. Further analysis of CitGF14s co-expression and potential interactors established a 14-3-3s protein interaction network. The established network identified 14-3-3 genes and several candidate clients which may play an important role in developmental regulation and stress responses in this important fruit crop. This is the first study of 14-3-3s in citrus, and the established network may help further investigation of the roles of 14-3-3s in response to abiotic and biotic constraints.
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22
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Liu SY, Yuan D, Sun RJ, Zhu JJ, Shan NN. Significant reductions in apoptosis-related proteins (HSPA6, HSPA8, ITGB3, YWHAH, and PRDX6) are involved in immune thrombocytopenia. J Thromb Thrombolysis 2020; 51:905-914. [PMID: 33047245 DOI: 10.1007/s11239-020-02310-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 10/05/2020] [Indexed: 01/24/2023]
Abstract
To investigate differences in the expression of plasma proteins in immune thrombocytopenia (ITP) and normal control groups, bone marrow samples were collected from 20 active ITP patients and 20 healthy controls. Quantitative proteomics analysis based on mass spectrometry was used to measure the protein levels and understand the protein networks. We found differentially expressed proteins in ITP patients and healthy controls. Parallel reaction monitoring (PRM), a targeted proteome quantification technique, was used to quantitatively confirm the identified target proteins and verify the proteomics data. In this study, a total of 829 proteins were identified, and the fold-change cut-off was set at 1.5 (patients vs controls); a total of 26 proteins were upregulated, and 69 proteins were downregulated. The bioinformatics analysis indicated that some differentially expressed proteins were associated with apoptosis. KEGG enrichment analysis showed that the apoptosis-related proteins were closely related to the PI3K-Akt signalling pathway. PRM demonstrated that apoptosis-related proteins were significantly decreased in ITP patients, which further confirmed the important effect of apoptosis on ITP pathogenesis. We hypothesised that apoptosis may be closely related to ITP pathogenesis through the PI3K-Akt signalling pathway.
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Affiliation(s)
- Shu-Yan Liu
- Department of Hematology, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250021, Shandong, China
| | - Dai Yuan
- Department of Hematology, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250021, Shandong, China
- Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, 324 Jing Wu Rd, Jinan, 250021, Shandong, China
| | - Rui-Jie Sun
- Department of Hematology, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250021, Shandong, China
| | - Jing-Jing Zhu
- Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, 324 Jing Wu Rd, Jinan, 250021, Shandong, China
| | - Ning-Ning Shan
- Department of Hematology, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250021, Shandong, China.
- Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, 324 Jing Wu Rd, Jinan, 250021, Shandong, China.
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23
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Hartman AM, Elgaher WAM, Hertrich N, Andrei SA, Ottmann C, Hirsch AKH. Discovery of Small-Molecule Stabilizers of 14-3-3 Protein-Protein Interactions via Dynamic Combinatorial Chemistry. ACS Med Chem Lett 2020; 11:1041-1046. [PMID: 32435423 DOI: 10.1021/acsmedchemlett.9b00541] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2019] [Accepted: 02/28/2020] [Indexed: 12/18/2022] Open
Abstract
Protein-protein interactions (PPIs) play an important role in numerous biological processes such as cell-cycle regulation and multiple diseases. The family of 14-3-3 proteins is an attractive target as they serve as binding partner to various proteins and are therefore capable of regulating their biological activities. Discovering small-molecule modulators, in particular stabilizers, of such complexes via traditional screening approaches is a challenging task. Herein, we pioneered the first application of dynamic combinatorial chemistry (DCC) to a PPI target, to find modulators of 14-3-3 proteins. Evaluation of the amplified hits from the DCC experiments for their binding affinity via surface plasmon resonance (SPR), revealed that the low-micromolar (K D 15-16 μM) acylhydrazones are 14-3-3/synaptopodin PPI stabilizers. Thus, DCC appears to be ideally suited for the discovery of not only modulators but even the more elusive stabilizers of notoriously challenging PPIs.
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Affiliation(s)
- Alwin M. Hartman
- Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands
- Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) − Helmholtz Centre for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany
- Department of Pharmacy, Saarland University, Campus Building E8.1, 66123 Saarbrücken, Germany
| | - Walid A. M. Elgaher
- Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) − Helmholtz Centre for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany
| | - Nathalie Hertrich
- Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) − Helmholtz Centre for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany
| | - Sebastian A. Andrei
- Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands
| | - Christian Ottmann
- Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands
- Department of Chemistry, University of Duisburg-Essen, 47057 Essen, Germany
| | - Anna K. H. Hirsch
- Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands
- Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) − Helmholtz Centre for Infection Research (HZI), Campus Building E8.1, 66123 Saarbrücken, Germany
- Department of Pharmacy, Saarland University, Campus Building E8.1, 66123 Saarbrücken, Germany
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Kwok CSN, Lai KKY, Lam SW, Chan KK, Xu SJL, Lee FWF. Production of high-quality two-dimensional gel electrophoresis profile for marine medaka samples by using Trizol-based protein extraction approaches. Proteome Sci 2020; 18:5. [PMID: 32390769 PMCID: PMC7196234 DOI: 10.1186/s12953-020-00161-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2020] [Accepted: 04/22/2020] [Indexed: 12/25/2022] Open
Abstract
Background Marine medaka is among the most popular models of fish species for ecotoxicology and environmental research and proteomic studies are useful tools for understanding the molecular responses of medaka upon exposure to different environmental stressors. The preparation of high-quality protein samples is the key to producing high-quality two-dimensional gel electrophoresis (2-DE) results for proteomic analysis. In recent years, Trizol-based protein extraction has been gaining popularity because of its promising performance in producing high-quality 2-DE as well as the convenience of the method. Methods Three Trizol-based approaches (Trizol method, Aliquot Trizol method and Trizol method with a commercial clean-up kit) were used to extract proteins from a marine medaka sample and 2-DE profiles were produced. Quality of the 2-DE profiles and effectiveness of the extraction methods were evaluated. For comparison, two common protein extraction methods (lysis buffer method and trichloroacetic acid (TCA)/acetone precipitation extraction) were also applied in parallel to Trizol-based approaches. Results Any of the three Trizol-based approaches produced a high-quality 2-DE profile of marine medaka compared with both lysis buffer method and TCA/acetone precipitation extraction. In addition, Trizol method with a commercial clean-up kit produced the best 2-DE profile in terms of background clarity, number of spots and resolution of proteins. Conclusions Trizol-based approaches offered better choices than traditional protein extraction methods for 2-DE analysis of marine medaka. The modified version of Trizol method with a commercial clean-up kit was shown to produce the best 2-DE profile.
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Affiliation(s)
- Celia Sze-Nga Kwok
- Department of Science, School of Science and Technology, The Open University of Hong Kong, Hong Kong, SAR China
| | - Kaze King-Yip Lai
- Department of Science, School of Science and Technology, The Open University of Hong Kong, Hong Kong, SAR China
| | - Sai-Wo Lam
- Department of Science, School of Science and Technology, The Open University of Hong Kong, Hong Kong, SAR China
| | - Kin-Ka Chan
- Department of Science, School of Science and Technology, The Open University of Hong Kong, Hong Kong, SAR China
| | - Steven Jing-Liang Xu
- Department of Science, School of Science and Technology, The Open University of Hong Kong, Hong Kong, SAR China
| | - Fred Wang-Fat Lee
- Department of Science, School of Science and Technology, The Open University of Hong Kong, Hong Kong, SAR China
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25
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Wang X, Ma G, Zhu H. Regulation of 14-3-3β/α gene expression in response to salinity, thermal, and bacterial stresses in Siberian sturgeon (Acipenser baeri). FISH PHYSIOLOGY AND BIOCHEMISTRY 2020; 46:519-531. [PMID: 31848829 DOI: 10.1007/s10695-019-00702-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/17/2019] [Accepted: 09/04/2019] [Indexed: 06/10/2023]
Abstract
The 14-3-3 proteins are a family of widely expressed acidic proteins, which are involved in the regulation of many biological processes of animals. However, no research regarding 14-3-3 has been described in sturgeon to date, one of the most primitive Actinopterygii species. Here, we identified the first 14-3-3 gene from Siberian sturgeon (Acipenser baeri), named Ab14-3-3β/α (GenBank Accession No. KY094076.1). The cDNA of Ab14-3-3β/α is 1212 bp in length, containing a 5'-untranslated region (UTR) of 82 bp, a 3'UTR of 392 bp, and an open reading frame (ORF) of 738 bp, encoding a polypeptide of 245 amino acids which contains a 14-3-3 homologs domain (PF00244). Phylogenetic analysis showed that the 14-3-3 gene product from Acipenser baeri is a counterpart of vertebrate 14-3-3β/α. The deduced Ab14-3-3β/α protein shares high identities of 46.5-95.5% with the homologs of other species. Ab14-3-3β/α mRNA was constitutively expressed in all examined tissues, with high expression levels in the blood and gill. Furthermore, the expression level of Ab14-3-3β/α mRNA increased significantly in the gill at 1 h under acute salinity shock by transfer of Siberian sturgeons from fresh water (FW) to 15 ppt. In fish subjected to a high temperature (31 °C), Ab14-3-3β/α showed a significant upregulation in the liver at 3 h compared with the control group (24 °C). A 4.85-fold increase of Ab14-3-3β/α expression in the spleen of Siberian sturgeon was observed at 24 h following Aeromonas hydrophila challenge. Collectively, these results indicated that Ab14-3-3β/α might play a certain role in sturgeon in response to some environmental stresses and bacterial challenge.
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Affiliation(s)
- Xiaowen Wang
- Beijing Fisheries Research Institute & Beijing Key Laboratory of Fishery Biotechnology, Beijing, 100068, People's Republic of China
- National Freshwater Fisheries Engineering Technology Research Center, Beijing, 100068, People's Republic of China
| | - Guoqing Ma
- Beijing Fisheries Research Institute & Beijing Key Laboratory of Fishery Biotechnology, Beijing, 100068, People's Republic of China
- National Freshwater Fisheries Engineering Technology Research Center, Beijing, 100068, People's Republic of China
| | - Hua Zhu
- Beijing Fisheries Research Institute & Beijing Key Laboratory of Fishery Biotechnology, Beijing, 100068, People's Republic of China.
- National Freshwater Fisheries Engineering Technology Research Center, Beijing, 100068, People's Republic of China.
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26
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Tissue-Specific Metabolic Regulation of FOXO-Binding Protein: FOXO Does Not Act Alone. Cells 2020; 9:cells9030702. [PMID: 32182991 PMCID: PMC7140670 DOI: 10.3390/cells9030702] [Citation(s) in RCA: 42] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2020] [Revised: 03/09/2020] [Accepted: 03/10/2020] [Indexed: 12/17/2022] Open
Abstract
The transcription factor forkhead box (FOXO) controls important biological responses, including proliferation, apoptosis, differentiation, metabolism, and oxidative stress resistance. The transcriptional activity of FOXO is tightly regulated in a variety of cellular processes. FOXO can convert the external stimuli of insulin, growth factors, nutrients, cytokines, and oxidative stress into cell-specific biological responses by regulating the transcriptional activity of target genes. However, how a single transcription factor regulates a large set of target genes in various tissues in response to a variety of external stimuli remains to be clarified. Evidence indicates that FOXO-binding proteins synergistically function to achieve tightly controlled processes. Here, we review the elaborate mechanism of FOXO-binding proteins, focusing on adipogenesis, glucose homeostasis, and other metabolic regulations in order to deepen our understanding and to identify a novel therapeutic target for the prevention and treatment of metabolic disorders.
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27
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Riedl W, Acharya D, Lee JH, Liu G, Serman T, Chiang C, Chan YK, Diamond MS, Gack MU. Zika Virus NS3 Mimics a Cellular 14-3-3-Binding Motif to Antagonize RIG-I- and MDA5-Mediated Innate Immunity. Cell Host Microbe 2019; 26:493-503.e6. [PMID: 31600501 PMCID: PMC6922055 DOI: 10.1016/j.chom.2019.09.012] [Citation(s) in RCA: 90] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2019] [Revised: 08/29/2019] [Accepted: 09/17/2019] [Indexed: 12/21/2022]
Abstract
14-3-3 protein family members facilitate the translocation of RIG-I-like receptors (RLRs) to organelles that mediate downstream RLR signaling, leading to interferon production. 14-3-3ϵ promotes the cytosolic-to-mitochondrial translocation of RIG-I, while 14-3-3η facilitates MDA5 translocation to mitochondria. We show that the NS3 protein of Zika virus (ZIKV) antagonizes antiviral gene induction by RIG-I and MDA5 by binding to and sequestering the scaffold proteins 14-3-3ϵ and 14-3-3η. 14-3-3-binding is mediated by a negatively charged RLDP motif in NS3 that is conserved in ZIKV strains of African and Asian lineages and is similar to the one found in dengue and West Nile viruses. ZIKV NS3 is sufficient to inhibit the RLR-14-3-3ϵ/η interaction and to suppress antiviral signaling. Mutational perturbation of 14-3-3ϵ/η binding in a recombinant ZIKV leads to enhanced innate immune responses and impaired growth kinetics. Our study provides molecular understanding of immune evasion functions of ZIKV, which may guide vaccine and anti-flaviviral therapy development.
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Affiliation(s)
- William Riedl
- Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA
| | - Dhiraj Acharya
- Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA
| | - Jung-Hyun Lee
- Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA
| | - Guanqun Liu
- Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA
| | - Taryn Serman
- Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA
| | - Cindy Chiang
- Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA
| | - Ying Kai Chan
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
| | - Michael S Diamond
- Department of Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA; Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO 63110, USA; Department of Pathology and Immunology, Washington University School of Medicine, Saint Louis, MO 63110, USA
| | - Michaela U Gack
- Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA.
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Hashemi M, Zali A, Hashemi J, Oraee-Yazdani S, Akbari A. Down-regulation of 14-3-3 zeta sensitizes human glioblastoma cells to apoptosis induction. Apoptosis 2019; 23:616-625. [PMID: 30101359 DOI: 10.1007/s10495-018-1476-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Strong 14-3-3 zeta protein expression plays an important role in tumorigenesis, including in the maintenance of cell growth, resistance increase, and the prevention of apoptosis. In this study, we focus on two targets: (1) the expression of 14-3-3 zeta in the different grades of human astrocytoma (II-IV), (2) suppression of 14-3-3 zeta protein expression in glioblastoma derived astrocytes by 14-3-3 zeta shRNA lentiviral particles. The tissues of human astrocytoma were provided from 30 patients (ten of each grade of astrocytoma). Control tissues were obtained from the peritumoral brain zone of those patients with glioblastoma. The protein and mRNA expression levels of each astrocytoma grade were assessed via western blotting and RT-PCR, respectively. Results indicated that 14-3-3 zeta was significantly expressed in glioblastoma multiforme (grade IV) and 14-3-3 zeta expression levels enhanced according to the increase of astrocytoma malignancy. In the cellular study for knock down of the 14-3-3 zeta protein, surgical biopsy of glioblastoma was used to isolate primary astrocyte. Astrocytes were transduced with 14-3-3 zeta shRNA or non-targeted shRNA lentiviral particles. Furthermore, reduction of the 14-3-3 zeta protein expression in the astrocytes evaluated through qRT-PCR and western blot after transduction of 14-3-3 zeta shRNA lentiviral particles. Moreover, apoptosis properties, including DNA fragmentation and ratio increase of Bax/Bcl-2 were observed in astrocytes following reduction of 14-3-3 zeta protein expression. Further observation indicated that the mitochondrial pathway through release of cytochorome c and caspase-3 activity was involved in the apoptosis induction. Hence, this study demonstrates a key role of the 14-3-3 zeta protein in tumorigenesis but also indicates that 14-3-3 zeta can be considered as a target for the astrocytoma treatment specially glioblastoma.
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Affiliation(s)
- Mansoureh Hashemi
- Functional Neurosurgery Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. .,Shohada Tajrish Hospital, Functional Neurosurgery Research Center, Shahrdari St, Tajrish Square, Tehran, 1989934148, Iran.
| | - Alireza Zali
- Functional Neurosurgery Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Javad Hashemi
- Department of Chemical Engineering, University of Louisville, Louisville, KY40292, USA
| | - Saeed Oraee-Yazdani
- Functional Neurosurgery Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Akhtar Akbari
- Functional Neurosurgery Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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Candelo E, Caicedo G, Mejia L, Pachajoa H. Chromosome 17p13.3 microdeletion syndrome with unaltered PAFAH1B1 gene. NEUROLOGÍA (ENGLISH EDITION) 2019. [DOI: 10.1016/j.nrleng.2018.10.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
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Wei Y, Li X, Zhang D, Liu Y. Comparison of protein differences between high- and low-quality goat and bovine parts based on iTRAQ technology. Food Chem 2019; 289:240-249. [DOI: 10.1016/j.foodchem.2019.03.052] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Revised: 03/01/2019] [Accepted: 03/11/2019] [Indexed: 12/16/2022]
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14-3-3/Tau Interaction and Tau Amyloidogenesis. J Mol Neurosci 2019; 68:620-630. [PMID: 31062171 DOI: 10.1007/s12031-019-01325-9] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2018] [Accepted: 04/22/2019] [Indexed: 01/02/2023]
Abstract
The major function of microtubule-associated protein tau is to promote microtubule assembly in the central nervous system. However, aggregation of abnormally phosphorylated tau is a hallmark of tauopathies. Although the molecular mechanisms of conformational transitions and assembling of tau molecules into amyloid fibril remain largely unknown, several factors have been shown to promote tau aggregation, including mutations, polyanions, phosphorylation, and interactions with other proteins. 14-3-3 proteins are a family of highly conserved, multifunctional proteins that are mainly expressed in the central nervous system. Being a scaffolding protein, 14-3-3 proteins interact with tau and regulate tau phosphorylation by bridging tau with various protein kinases. 14-3-3 proteins also directly regulate tau aggregation via specific and non-specific interactions with tau. In this review, we summarize recent advances in characterization of tau conformation and tau/14-3-3 interaction. We discuss the connection between 14-3-3 binding and tau aggregation with a special emphasis on the regulatory role of 14-3-3 on tau conformation.
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Alblova M, Smidova A, Kalabova D, Lentini Santo D, Obsil T, Obsilova VO. Allosteric activation of yeast enzyme neutral trehalase by calcium and 14-3-3 protein. Physiol Res 2019; 68:147-160. [DOI: 10.33549/physiolres.933950] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Neutral trehalase 1 (Nth1) from Saccharomyces cerevisiae catalyzes disaccharide trehalose hydrolysis and helps yeast to survive adverse conditions, such as heat shock, starvation or oxidative stress. 14-3-3 proteins, master regulators of hundreds of partner proteins, participate in many key cellular processes. Nth1 is activated by phosphorylation followed by 14-3-3 protein (Bmh) binding. The activation mechanism is also potentiated by Ca(2+) binding within the EF-hand-like motif. This review summarizes the current knowledge about trehalases and the molecular and structural basis of Nth1 activation. The crystal structure of fully active Nth1 bound to 14-3-3 protein provided the first high-resolution view of a trehalase from a eukaryotic organism and showed 14-3-3 proteins as structural modulators and allosteric effectors of multi-domain binding partners.
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Affiliation(s)
- M. Alblova
- Department of Structural Biology of Signaling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, Vestec, Czech Republic.
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Surolia R, Li FJ, Wang Z, Li H, Dsouza K, Thomas V, Mirov S, Pérez-Sala D, Athar M, Thannickal VJ, Antony VB. Vimentin intermediate filament assembly regulates fibroblast invasion in fibrogenic lung injury. JCI Insight 2019; 4:123253. [PMID: 30944258 DOI: 10.1172/jci.insight.123253] [Citation(s) in RCA: 65] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Accepted: 02/26/2019] [Indexed: 12/22/2022] Open
Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive disease, with a median survival of 3-5 years following diagnosis. Lung remodeling by invasive fibroblasts is a hallmark of IPF. In this study, we demonstrate that inhibition of vimentin intermediate filaments (VimIFs) decreases the invasiveness of IPF fibroblasts and confers protection against fibrosis in a murine model of experimental lung injury. Increased expression and organization of VimIFs contribute to the invasive property of IPF fibroblasts in connection with deficient cellular autophagy. Blocking VimIF assembly by pharmacologic and genetic means also increases autophagic clearance of collagen type I. Furthermore, inhibition of expression of collagen type I by siRNA decreased invasiveness of fibroblasts. In a bleomycin injury model, enhancing autophagy in fibroblasts by an inhibitor of VimIF assembly, withaferin A (WFA), protected from fibrotic lung injury. Additionally, in 3D lung organoids, or pulmospheres, from patients with IPF, WFA reduced the invasiveness of lung fibroblasts in the majority of subjects tested. These studies provide insights into the functional role of vimentin, which regulates autophagy and restricts the invasiveness of lung fibroblasts.
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Affiliation(s)
- Ranu Surolia
- Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine
| | - Fu Jun Li
- Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine
| | - Zheng Wang
- Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine
| | - Huashi Li
- Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine
| | - Kevin Dsouza
- Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine
| | - Vinoy Thomas
- Department of Materials Science and Engineering, and
| | - Sergey Mirov
- Department of Physics, University of Alabama at Birmingham (UAB), Birmingham, Alabama, USA
| | - Dolores Pérez-Sala
- Department of Structural and Chemical and Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
| | - Mohammad Athar
- Department of Dermatology, UAB, Birmingham, Alabama, USA
| | - Victor J Thannickal
- Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine
| | - Veena B Antony
- Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine
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Lu YC, Wang P, Wu QG, Zhang RK, Kong A, Li YF, Lee SC. Hsp74/14-3-3σ Complex Mediates Centrosome Amplification by High Glucose, Insulin, and Palmitic Acid. Proteomics 2019; 19:e1800197. [PMID: 30688006 DOI: 10.1002/pmic.201800197] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2018] [Revised: 12/26/2018] [Indexed: 01/08/2023]
Abstract
It has been reported recently that type 2 diabetes promotes centrosome amplification via 14-3-3σ/ROCK1 complex. In the present study, 14-3-3σ interacting proteins are characterized and their roles in the centrosome amplification by high glucose, insulin, and palmitic acid are investigated. Co-immunoprecipitation in combination with MS analysis identified 134 proteins that interact with 14-3-3σ, which include heat shock 70 kDa protein 4 (Hsp74). Gene ontology analyses reveal that many of them are enriched in binding activity. Kyoto Encyclopedia of Genes and Genomes analysis shows that the top three enriched pathways are ribosome, carbon metabolism, and biosynthesis of amino acids. Molecular and functional investigations show that the high glucose, insulin, and palmitic acid increase the expression and binding of 14-3-3σ and Hsp74 as well as centrosome amplification, all of which are inhibited by knockdown of 14-3-3σ or Hsp74. Moreover, molecular docking analysis shows that the interaction between the 14-3-3σ and the Hsp74 is mainly through hydrophobic contacts and a lesser degree ionic interactions and hydrogen bond by different amino acids residues. In conclusion, the results suggest that the experimental treatment triggers centrosome amplification via upregulations of expression and binding of 14-3-3σ and Hsp74.
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Affiliation(s)
- Yu Cheng Lu
- School of Life Sciences, Shanxi University, Taiyuan, Shanxi, 030006, P. R. China.,Central Laboratory, Linyi People's Hospital, Linyi, Shandong, 276000, P. R. China
| | - Pu Wang
- School of Life Sciences, Shanxi University, Taiyuan, Shanxi, 030006, P. R. China
| | - Qi Gui Wu
- School of Life Sciences, Shanxi University, Taiyuan, Shanxi, 030006, P. R. China
| | - Rui Kai Zhang
- School of Life Sciences, Shanxi University, Taiyuan, Shanxi, 030006, P. R. China
| | - Alice Kong
- Department of Medicine and Therapeutics, the Chinese University of Hong Kong, Shatin, Hong Kong SAR, 999077, P. R. China
| | - Yuan Fei Li
- Department of Oncology, First Clinical Hospital of Shanxi Medical University, Taiyuan, Shanxi, 030001, P. R. China
| | - Shao Chin Lee
- School of Life Sciences, Shanxi University, Taiyuan, Shanxi, 030006, P. R. China.,School of Life Sciences, Jiangsu Normal University, Xuzhou, Jiangsu, 221010, P. R. China
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Papanikolopoulou K, Grammenoudi S, Samiotaki M, Skoulakis EMC. Differential effects of 14-3-3 dimers on Tau phosphorylation, stability and toxicity in vivo. Hum Mol Genet 2019; 27:2244-2261. [PMID: 29659825 DOI: 10.1093/hmg/ddy129] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2018] [Accepted: 04/06/2018] [Indexed: 01/09/2023] Open
Abstract
Neurodegenerative dementias collectively known as Tauopathies involve aberrant phosphorylation and aggregation of the neuronal protein Tau. The largely neuronal 14-3-3 proteins are also elevated in the central nervous system (CNS) and cerebrospinal fluid of Tauopathy patients, suggesting functional linkage. We use the simplicity and genetic facility of the Drosophila system to investigate in vivo whether 14-3-3s are causal or synergistic with Tau accumulation in precipitating pathogenesis. Proteomic, biochemical and genetic evidence demonstrate that both Drosophila 14-3-3 proteins interact with human wild-type and mutant Tau on multiple sites irrespective of their phosphorylation state. 14-3-3 dimers regulate steady-state phosphorylation of both wild-type and the R406W mutant Tau, but they are not essential for toxicity of either variant. Moreover, 14-3-3 elevation itself is not pathogenic, but recruitment of dimers on accumulating wild-type Tau increases its steady-state levels ostensibly by occluding access to proteases in a phosphorylation-dependent manner. In contrast, the R406W mutant, which lacks a putative 14-3-3 binding site, responds differentially to elevation of each 14-3-3 isoform. Although excess 14-3-3ζ stabilizes the mutant protein, elevated D14-3-3ɛ has a destabilizing effect probably because of altered 14-3-3 dimer composition. Our collective data demonstrate the complexity of 14-3-3/Tau interactions in vivo and suggest that 14-3-3 attenuation is not appropriate ameliorative treatment of Tauopathies. Finally, we suggest that 'bystander' 14-3-3s are recruited by accumulating Tau with the consequences depending on the composition of available dimers within particular neurons and the Tau variant.
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Affiliation(s)
- Katerina Papanikolopoulou
- Division of Neuroscience, Biomedical Sciences Research Centre 'Alexander Fleming', Vari 16672, Greece
| | - Sofia Grammenoudi
- Division of Neuroscience, Biomedical Sciences Research Centre 'Alexander Fleming', Vari 16672, Greece
| | - Martina Samiotaki
- Proteomics Facility, Biomedical Sciences Research Centre 'Alexander Fleming', Vari 16672, Greece
| | - Efthimios M C Skoulakis
- Division of Neuroscience, Biomedical Sciences Research Centre 'Alexander Fleming', Vari 16672, Greece
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Li J, Xu H, Wang Q, Wang S, Xiong N. 14-3-3ζ promotes gliomas cells invasion by regulating Snail through the PI3K/AKT signaling. Cancer Med 2019; 8:783-794. [PMID: 30656845 PMCID: PMC6382716 DOI: 10.1002/cam4.1950] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2018] [Revised: 11/12/2018] [Accepted: 12/10/2018] [Indexed: 12/16/2022] Open
Abstract
14-3-3ζ has been reported to function as critical regulators of diverse cellular responses. However, the role of 14-3-3ζ in gliomas progression remains largely unknown. The expression level of 14-3-3ζ and Snail was detected by Western blot analysis and quantitative polymerase chain reaction in different grades of human gliomas. The effect of 14-3-3ζ on gliomas progression was measured using cell migration and invasion assay, the colony formation experiment, and CCK-8 assay. The effect of 14-3-3ζ on PI3K/AKT/Snail signaling protein expression levels was tested by Western blotting. Firstly, 14-3-3ζ was often up-regulated in high-grade gliomas relative to low-grade gliomas, and this overexpression was significantly related to tumor size, Karnofsky Performance Scale score and weaker disease-free survival. Secondly, the overexpression of 14-3-3ζ promoted gliomas cells proliferation, migration, and invasion. Conversely, the knockdown of 14-3-3ζ suppressed gliomas cells proliferation, migration, and invasion. Furthermore, subsequent mechanistic studies showed that 14-3-3ζ could activate PI3K/AKT/Snail signaling pathway to facilitate gliomas cells proliferation, migration, and invasion. This study shows that the overexpression of 14-3-3ζ can promote remarkably gliomas cells proliferation, migration, and invasion by regulating the Snail protein expression through activating PI3K/AKT signaling, and it may serve as a potential prognostic marker and therapeutic target for gliomas.
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Affiliation(s)
- Junjun Li
- Department of NeurosurgeryUnion HospitalTongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Hao Xu
- Department of NeurosurgeryUnion HospitalTongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Qiangping Wang
- Department of NeurosurgeryUnion HospitalTongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Sihua Wang
- Department of Thoracic surgeryUnion HospitalTongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Nanxiang Xiong
- Department of NeurosurgeryUnion HospitalTongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
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Xu Y, Ren J, He X, Chen H, Wei T, Feng W. YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization. Autophagy 2019; 15:1017-1030. [PMID: 30653408 DOI: 10.1080/15548627.2019.1569928] [Citation(s) in RCA: 58] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
As a master regulator of the macroautophagy/autophagy-lysosomal pathway, TFEB (transcription factor EB) plays a prominent role in regulating neurodegenerative diseases and cancer. The transcription activity of TFEB is tightly controlled by phosphorylation and dephosphorylation. Phosphorylated S211 (p-S211) of TFEB can be recognized by YWHA/14-3-3 proteins for TFEB cytoplasmic localization. Here, we characterized the interactions between phosphorylated TFEB and YWHA/14-3-3 proteins and determined the structures of YWHA/14-3-3 proteins in complex with a TFEB p-S211-peptide. Although the critical arginine for YWHA/14-3-3 recognition is missing in the N terminus of the TFEB p-S211-peptide, the C-terminal additional hydrophobic residues of the peptide unexpectedly occupy nearly half of the target-binding groove of YWHA/14-3-3 proteins, which compensates for the N-terminal defect and is distinct from the canonical YWHA/14-3-3-binding mode. Mutations of essential residues in the interaction interface between TFEB and YWHA/14-3-3 proteins disrupted their interactions and severely impaired the cytoplasmic localization of TFEB, which altered the expression of TFEB target genes and affected autophagy. Thus, YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization and its activity. Abbreviation: ACTB: actin beta; ALP: autophagy-lysosomal pathway; ATP6V1H: ATPase H+ transporting V1 subunit H; bHLH: basic helix-loop-helix; CLEAR: coordinated lysosomal expression and regulation; Co-IP: co-immunoprecipitation; CTSB: cathepsin B; CTSD: cathepsin D; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MITF: melanocyte inducing transcription factor; NLS: nuclear localization signal; TFEB: transcription factor EB; YWHA/14-3-3: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein.
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Affiliation(s)
- Yang Xu
- a National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics , Chinese Academy of Sciences , Beijing , China.,b College of Life Sciences , University of Chinese Academy of Sciences , Beijing , China
| | - Jinqi Ren
- a National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics , Chinese Academy of Sciences , Beijing , China
| | - Xiaolong He
- a National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics , Chinese Academy of Sciences , Beijing , China.,b College of Life Sciences , University of Chinese Academy of Sciences , Beijing , China
| | - Han Chen
- a National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics , Chinese Academy of Sciences , Beijing , China.,b College of Life Sciences , University of Chinese Academy of Sciences , Beijing , China
| | - Taotao Wei
- a National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics , Chinese Academy of Sciences , Beijing , China.,b College of Life Sciences , University of Chinese Academy of Sciences , Beijing , China
| | - Wei Feng
- a National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics , Chinese Academy of Sciences , Beijing , China.,b College of Life Sciences , University of Chinese Academy of Sciences , Beijing , China
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Li S, Gong P, Zhang N, Li X, Tai L, Wang X, Yang Z, Yang J, Zhu X, Zhang X, Li J. 14-3-3 Protein of Neospora caninum Modulates Host Cell Innate Immunity Through the Activation of MAPK and NF-κB Pathways. Front Microbiol 2019; 10:37. [PMID: 30740096 PMCID: PMC6355710 DOI: 10.3389/fmicb.2019.00037] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Accepted: 01/10/2019] [Indexed: 12/30/2022] Open
Abstract
Neospora caninum is an obligate intracellular apicomplexan parasite, the etiologic agent of neosporosis, and a major cause of reproductive loss in cattle. There is still a lack of effective prevention and treatment measures. The 14-3-3 protein is a widely expressed acidic protein that spontaneously forms dimers within apicomplexan parasites. This protein has been isolated and sequenced in many parasites; however, there are few reports about the N. caninum 14-3-3 protein. Here, we successfully expressed and purified a recombinant fusion protein of Nc14-3-3 (rNc14-3-3) and prepared a polyclonal antibody. Immunofluorescence and immunogold electron microscopy studies of tachyzoites or N. caninum-infected cells suggested that 14-3-3 was localized in the cytosol and the membrane. Western blotting analysis indicated that rNc14-3-3 could be recognized by N. caninum-infected mouse sera, suggesting that 14-3-3 may be an infection-associated antigen that is involved in the host immune response. We demonstrated that rNc14-3-3 induced cytokine expression by activating the MAPK and AKT signaling pathways, and inhibitors of p38, ERK, JNK, and AKT could significantly decrease the production of IL-6, IL-12p40, and TNF-α. In addition, phosphorylated nuclear factor-κB (NF-κB/p65) was observed in wild-type peritoneal macrophages (PMs) treated with rNc14-3-3, and the protein level of NF-κB/p65 was reduced in the cytoplasm but increased correspondingly in the nucleus after 2 h of treatment. These results were also observed in deficient in TLR2-/- PMs. Taken together, our results indicated that the N. caninum 14-3-3 protein can induce effective immune responses and stimulate cytokine expression by activating the MAPK, AKT, and NF-κB signaling pathways but did not dependent TLR2, suggesting that Nc14-3-3 is a novel vaccine candidate against neosporosis.
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Affiliation(s)
- Shan Li
- Key Laboratory of Zoonosis Research by Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Pengtao Gong
- Key Laboratory of Zoonosis Research by Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Nan Zhang
- Key Laboratory of Zoonosis Research by Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Xin Li
- Key Laboratory of Zoonosis Research by Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Lixin Tai
- Key Laboratory of Zoonosis Research by Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Xu Wang
- Key Laboratory of Zoonosis Research by Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Zhengtao Yang
- Key Laboratory of Zoonosis Research by Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Ju Yang
- Key Laboratory of Zoonosis Research by Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Xingquan Zhu
- State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Xichen Zhang
- Key Laboratory of Zoonosis Research by Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Jianhua Li
- Key Laboratory of Zoonosis Research by Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China
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Yun D, Wang H, Wang Y, Chen Y, Zhao Z, Ma J, Ji Y, Huang Q, Chen J, Chen H, Lu D. Shuttling SLC2A4RG is regulated by 14-3-3θ to modulate cell survival via caspase-3 and caspase-6 in human glioma. EBioMedicine 2019; 40:163-175. [PMID: 30686753 PMCID: PMC6413354 DOI: 10.1016/j.ebiom.2019.01.030] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2018] [Revised: 01/10/2019] [Accepted: 01/11/2019] [Indexed: 12/27/2022] Open
Abstract
Background Glioma is the most common and aggressive primary brain tumor with polygenic susceptibility. The cytoplasmic/nuclear shuttling protein, SLC2A4RG (SLC2A4 regulator), has been identified in the 20q13.33 region influencing glioma susceptibility by genome-wide association studies (GWAS) and fine mapping analyses. Methods To discover the expression of SLC2A4RG and its relationship with patient prognosis, tissue microarray containing glioma samples and normal brains was constructed followed by immunohistochemical staining. The role of SLC2A4RG on cell proliferation, cell cycle, and apoptosis was evaluated by gain- and loss-of-function assays in vivo, and subcutaneous and intracranial xenografts were performed to assess its functional effects. The mechanism underlying SLC2A4RG was further investigated via luciferase reporter analyses, ChIP, mass spectrometry, Co-IP, immunofluorescence, etc. Findings The potential tumor suppressor role of SLC2A4RG was further validated by in vitro and in vivo experiments that SLC2A4RG could attenuate cell proliferation via G2/M phase arrest and induce glioma cell apoptosis by direct transactivation of caspase-3 and caspase-6. Moreover, its function displaying showed to depend on the nuclear transportation of SLC2A4RG, however, bound with 14-3-3θ, it would be sequestered in the cytoplasm followed by reversal effect. Interpretation We identify a new pro-oncogenic mechanism whereby 14-3-3θ negatively regulates the nuclear function of the tumor suppressor SLC2A4RG, with significant therapeutic implications for the intervention of human glioma. Fund This work was supported by the National Natural Science Foundation of China (81372706, 81572501, and 81372235).
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Affiliation(s)
- Dapeng Yun
- State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, 2005 Songhu Road, Shanghai 200438, China
| | - Hongxiang Wang
- Department of Neurosurgery, Shanghai Institute of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China
| | - Yuqi Wang
- State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, 2005 Songhu Road, Shanghai 200438, China
| | - Yuanyuan Chen
- Department of Critical Care Medicine, Wuxi No'2 People's Hospital, Wuxi, Jiangsu Province, China
| | - Zhipeng Zhao
- State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, 2005 Songhu Road, Shanghai 200438, China
| | - Jiawei Ma
- Division of Molecular Thoracic Oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Yuanyuan Ji
- State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, 2005 Songhu Road, Shanghai 200438, China
| | - Qilin Huang
- Department of Neurosurgery, Shanghai Institute of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China
| | - Juxiang Chen
- Department of Neurosurgery, Shanghai Institute of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
| | - Hongyan Chen
- State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, 2005 Songhu Road, Shanghai 200438, China.
| | - Daru Lu
- State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, 2005 Songhu Road, Shanghai 200438, China.
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Zhao S, Li B, Li C, Gao H, Miao Y, He Y, Wang H, Gong L, Li D, Zhang Y, Feng J. The Apoptosis Regulator 14-3-3η and Its Potential as a Therapeutic Target in Pituitary Oncocytoma. Front Endocrinol (Lausanne) 2019; 10:797. [PMID: 31849836 PMCID: PMC6893364 DOI: 10.3389/fendo.2019.00797] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/25/2019] [Accepted: 11/01/2019] [Indexed: 11/24/2022] Open
Abstract
The 14-3-3 protein family has attracted much attention in research into the pathogenesis of human tumors because of its involvement in tumorigenesis. In previous studies, we found that 14-3-3η was highly expressed in pituitary oncocytoma. However, the mechanism by which 14-3-3η regulates tumorigenesis in pituitary oncocytoma is unclear. 14-3-3η-binding proteins were investigated in pituitary oncocytoma by immunoprecipitation and proteomic analysis. A total of 443 proteins were identified as 14-3-3η binding proteins. The interactions of 14-3-3η and its binding partners were identified by a network analysis using the STRING database. The network included 433 nodes and 564 edges. PRAS40 (AKT1S1) was a binding protein of 14-3-3η and showed experimental interactions with 14-3-3η in the STRING database. The combined score was 0.407, which suggested a functional link. The 443 binding proteins of 14-3-3η showed enriched molecular signatures in GSEA and GO analysis. PRAS40 (AKT1S1) was enriched in the mTOR signaling pathway. Western blot analysis showed that the relative expression of p-PRAS40 (T246)/PRAS40 was significantly higher in pituitary oncocytoma than in normal pituitary tissues (p < 0.05). R18, a 14-3-3 protein inhibitor, inhibited MMQ cell proliferation after treatment with 8 μM R18 for 48 h compared to the control group (p < 0.01). These results suggest that 14-3-3η may be involved in promoting tumorigenesis in pituitary oncocytoma by interacting with PRAS40 (T246) via the mTOR signaling pathway.
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Affiliation(s)
- Sida Zhao
- Cell Biology, Beijing Neurosurgical Institute, Capital Medical University, Beijing, China
- *Correspondence: Sida Zhao
| | - Bin Li
- Beijing Tiantan Hospital, Capital Medical University, Beijing, China
| | - Chuzhong Li
- Cell Biology, Beijing Neurosurgical Institute, Capital Medical University, Beijing, China
| | - Hua Gao
- Cell Biology, Beijing Neurosurgical Institute, Capital Medical University, Beijing, China
| | - Yazhou Miao
- Cell Biology, Beijing Neurosurgical Institute, Capital Medical University, Beijing, China
| | - Yue He
- Cell Biology, Beijing Neurosurgical Institute, Capital Medical University, Beijing, China
| | - Hongyun Wang
- Cell Biology, Beijing Neurosurgical Institute, Capital Medical University, Beijing, China
| | - Lei Gong
- Cell Biology, Beijing Neurosurgical Institute, Capital Medical University, Beijing, China
| | - Dan Li
- Cell Biology, Beijing Neurosurgical Institute, Capital Medical University, Beijing, China
| | - Yazhuo Zhang
- Cell Biology, Beijing Neurosurgical Institute, Capital Medical University, Beijing, China
- Beijing Tiantan Hospital, Capital Medical University, Beijing, China
- Beijing Institute for Brain Disorders Brain Tumor Center, Capital Medical University, Beijing, China
- China National Clinical Research Center for Neurological Diseases, Beijing, China
- Chinese Medical Association, Beijing, China
| | - Jie Feng
- Cell Biology, Beijing Neurosurgical Institute, Capital Medical University, Beijing, China
- Jie Feng
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Long S, Guo W, Hu S, Su F, Zeng Y, Zeng J, Tan EK, Ross CA, Pei Z. G2019S LRRK2 Increases Stress Susceptibility Through Inhibition of DAF-16 Nuclear Translocation in a 14-3-3 Associated-Manner in Caenorhabditis elegans. Front Neurosci 2018; 12:782. [PMID: 30464741 PMCID: PMC6234837 DOI: 10.3389/fnins.2018.00782] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2018] [Accepted: 10/09/2018] [Indexed: 01/17/2023] Open
Abstract
Mutations in leucine-rich repeat kinase 2 (LRRK2) are common causes of familial Parkinson’s disease (PD). Oxidative stress plays a key role in the pathogenesis of PD. Mutations in LRRK2 have been shown to increase susceptibility to oxidative stress. To explore mechanisms underlying susceptibility to oxidative stress in LRRK2 mutants, we generated stable Caenorhabditis elegans (C. elegans) strains in which human LRRK2 proteins including wild type LRRK2 (WT), G2019S LRRK2 (G2019S), and G2019S-D1994A kinase-dead LRRK2 (KD) were expressed in all neurons. Human 14-3-3 β was injected into LRRK2 transgenic worms to allow co-expression of 14-3-3 β and LRRK2 proteins. We found that G2019S transgenic worms had increased sensitivity to stress (heat and juglone treatment) and impaired stress-induced nuclear translocation of DAF-16. In addition, G2019S inhibited ftt2 (a 14-3-3 gene homolog in C. elegans) knockdown-associated nuclear translocation of DAF-16. Comparably, overexpression of human 14-3-3 β could attenuate G2019S-associated toxicity in response to stress and rescued G2019S-mediated inhibition of sod-3 and dod-3 expression. Taken together, our study provides evidence suggesting that 14-3-3-associated inhibition of DAF-16 nuclear translocation could be a mechanism for G2019S LRRK2-induced oxidative stress and cellular toxicity. Our findings may give a hint that the potential of 14-3-3 proteins as neuroprotective targets in PD patients carrying LRRK2 mutations.
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Affiliation(s)
- Simei Long
- Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Wenyuan Guo
- Department of Neurology, The First Affiliated Hospital, Guangzhou Medical University, Guangzhou, China
| | - Sophie Hu
- Cumming School of Medicine, University of Calgary, Calgary, AB, Canada
| | - Fengjuan Su
- Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Yixuan Zeng
- Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, China
| | - Jinsheng Zeng
- Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Eng-King Tan
- Department of Neurology, Singapore General Hospital, Singapore, Singapore.,National Neuroscience Institute, Singapore, Singapore.,Duke-NUS Graduate Medical School, Singapore, Singapore
| | - Christopher A Ross
- Division of Neurobiology, Department of Psychiatry-Departments of Neuroscience, Neurology, and Pharmacology, Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Zhong Pei
- Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
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Structural snapshots of RAF kinase interactions. Biochem Soc Trans 2018; 46:1393-1406. [PMID: 30381334 DOI: 10.1042/bst20170528] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2018] [Revised: 07/25/2018] [Accepted: 07/30/2018] [Indexed: 02/07/2023]
Abstract
RAF (rapidly accelerated fibrosarcoma) Ser/Thr kinases (ARAF, BRAF, and CRAF) link the RAS (rat sarcoma) protein family with the MAPK (mitogen-activated protein kinase) pathway and control cell growth, differentiation, development, aging, and tumorigenesis. Their activity is specifically modulated by protein-protein interactions, post-translational modifications, and conformational changes in specific spatiotemporal patterns via various upstream regulators, including the kinases, phosphatase, GTPases, and scaffold and modulator proteins. Dephosphorylation of Ser-259 (CRAF numbering) and dissociation of 14-3-3 release the RAF regulatory domains RAS-binding domain and cysteine-rich domain for interaction with RAS-GTP and membrane lipids. This, in turn, results in RAF phosphorylation at Ser-621 and 14-3-3 reassociation, followed by its dimerization and ultimately substrate binding and phosphorylation. This review focuses on structural understanding of how distinct binding partners trigger a cascade of molecular events that induces RAF kinase activation.
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The Eukaryotic Host Factor 14-3-3 Inactivates Adenylate Cyclase Toxins of Bordetella bronchiseptica and B. parapertussis, but Not B. pertussis. mBio 2018; 9:mBio.00628-18. [PMID: 30154257 PMCID: PMC6113625 DOI: 10.1128/mbio.00628-18] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Bordetella pertussis, Bordetella bronchiseptica, and Bordetella parapertussis share highly homologous virulence factors and commonly cause respiratory infections in mammals; however, their host specificities and disease severities differ, and the reasons for this remain largely unknown. Adenylate cyclase toxin (CyaA) is a homologous virulence factor that is thought to play crucial roles in Bordetella infections. We herein demonstrate that CyaAs function as virulence factors differently between B. bronchiseptica/B. parapertussis and B. pertussis. B. bronchiseptica CyaA bound to target cells, and its enzyme domain was translocated into the cytosol similarly to B. pertussis CyaA. The hemolytic activity of B. bronchiseptica CyaA on sheep erythrocytes was also preserved. However, in nucleated target cells, B. bronchiseptica CyaA was phosphorylated at Ser375, which constitutes a motif (RSXpSXP [pS is phosphoserine]) recognized by the host factor 14-3-3, resulting in the abrogation of adenylate cyclase activity. Consequently, the cytotoxic effects of B. bronchiseptica CyaA based on its enzyme activity were markedly attenuated. B. parapertussis CyaA carries the 14-3-3 motif, indicating that its intracellular enzyme activity is abrogated similarly to B. bronchiseptica CyaA; however, B. pertussis CyaA has Phe375 instead of Ser, and thus, was not affected by 14-3-3. In addition, B. pertussis CyaA impaired the barrier function of epithelial cells, whereas B. bronchiseptica CyaA did not. Rat infection experiments suggested that functional differences in CyaA are related to differences in pathogenicity between B. bronchiseptica/B. parapertussis and B. pertussis. Bordetella pertussis, B. bronchiseptica, and B. parapertussis are bacterial respiratory pathogens that are genetically close to each other and produce many homologous virulence factors; however, their host specificities and disease severities differ, and the reasons for this remain unknown. Previous studies attempted to explain these differences by the distinct virulence factors produced by each Bordetella species. In contrast, we indicated functional differences in adenylate cyclase toxin, a homologous virulence factor of Bordetella. The toxins of B. bronchiseptica and presumably B. parapertussis were inactivated by the host factor 14-3-3 after phosphorylation in target cells, whereas the B. pertussis toxin was not inactivated because of the lack of the phosphorylation site. This is the first study to show that 14-3-3 inactivates the virulence factors of pathogens. The present results suggest that pathogenic differences in Bordetella are attributed to the different activities of adenylate cyclase toxins.
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Ulloa-Aguirre A, Reiter E, Crépieux P. FSH Receptor Signaling: Complexity of Interactions and Signal Diversity. Endocrinology 2018; 159:3020-3035. [PMID: 29982321 DOI: 10.1210/en.2018-00452] [Citation(s) in RCA: 70] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/08/2018] [Accepted: 06/27/2018] [Indexed: 12/20/2022]
Abstract
FSH is synthesized in the pituitary by gonadotrope cells. By binding to and interacting with its cognate receptor [FSH receptor (FSHR)] in the gonads, this gonadotropin plays a key role in the control of gonadal function and reproduction. Upon activation, the FSHR undergoes conformational changes leading to transduction of intracellular signals, including dissociation of G protein complexes into components and activation of several associated interacting partners, which concertedly regulate downstream effectors. The canonical Gs/cAMP/protein kinase A pathway, considered for a long time as the sole effector of FSHR-mediated signaling, is now viewed as one of several mechanisms employed by this receptor to transduce intracellular signals in response to the FSH stimulus. This complex network of signaling pathways allows for a fine-tuning regulation of the gonadotropic stimulus, where activation/inhibition of its multiple components vary depending on the cell context, cell developmental stage, and concentration of associated receptors and corresponding ligands. Activation of these multiple signaling modules eventually converge to the hormone-integrated biological response, including survival, proliferation and differentiation of target cells, synthesis and secretion of paracrine/autocrine regulators, and, at the molecular level, functional selectivity and differential gene expression. In this mini-review, we discuss the complexity of FSHR-mediated intracellular signals activated in response to ligand stimulation. A better understanding of the signaling pathways involved in FSH action might potentially influence the development of new therapeutic strategies for reproductive disorders.
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Affiliation(s)
- Alfredo Ulloa-Aguirre
- Red de Apoyo a la Investigación, Universidad Nacional Autónoma de México-Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico
| | - Eric Reiter
- Biology and Bioinformatics of Signaling Systems Group, Unité Mixtes de Recherche 85, Unité Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, Nouzilly, France
- Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7247, Nouzilly, France
- Université François Rabelais, Nouzilly, France
| | - Pascale Crépieux
- Biology and Bioinformatics of Signaling Systems Group, Unité Mixtes de Recherche 85, Unité Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, Nouzilly, France
- Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7247, Nouzilly, France
- Université François Rabelais, Nouzilly, France
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Sharma S, Čermáková K, De Rijck J, Demeulemeester J, Fábry M, El Ashkar S, Van Belle S, Lepšík M, Tesina P, Duchoslav V, Novák P, Hubálek M, Srb P, Christ F, Řezáčová P, Hodges HC, Debyser Z, Veverka V. Affinity switching of the LEDGF/p75 IBD interactome is governed by kinase-dependent phosphorylation. Proc Natl Acad Sci U S A 2018; 115:E7053-E7062. [PMID: 29997176 PMCID: PMC6065015 DOI: 10.1073/pnas.1803909115] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.
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Affiliation(s)
| | - Kateřina Čermáková
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 166 10 Prague 6, Czech Republic
- Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX 77030
- Center for Precision Environmental Health, Baylor College of Medicine, Houston, TX 77030
- Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030
| | - Jan De Rijck
- Molecular Virology and Gene Therapy, KU Leuven, 3000 Leuven, Belgium;
| | | | - Milan Fábry
- Institute of Molecular Genetics of the Czech Academy of Sciences, 142 20 Prague 4, Czech Republic
| | - Sara El Ashkar
- Molecular Virology and Gene Therapy, KU Leuven, 3000 Leuven, Belgium
| | - Siska Van Belle
- Molecular Virology and Gene Therapy, KU Leuven, 3000 Leuven, Belgium
| | - Martin Lepšík
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 166 10 Prague 6, Czech Republic
| | - Petr Tesina
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 166 10 Prague 6, Czech Republic
- Institute of Molecular Genetics of the Czech Academy of Sciences, 142 20 Prague 4, Czech Republic
| | - Vojtěch Duchoslav
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 166 10 Prague 6, Czech Republic
| | - Petr Novák
- Institute of Microbiology of the Czech Academy of Sciences, 142 20 Prague 4, Czech Republic
| | - Martin Hubálek
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 166 10 Prague 6, Czech Republic
| | - Pavel Srb
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 166 10 Prague 6, Czech Republic
| | - Frauke Christ
- Molecular Virology and Gene Therapy, KU Leuven, 3000 Leuven, Belgium
| | - Pavlína Řezáčová
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 166 10 Prague 6, Czech Republic
- Institute of Molecular Genetics of the Czech Academy of Sciences, 142 20 Prague 4, Czech Republic
| | - H Courtney Hodges
- Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX 77030
- Center for Precision Environmental Health, Baylor College of Medicine, Houston, TX 77030
- Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030
- Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030
| | - Zeger Debyser
- Molecular Virology and Gene Therapy, KU Leuven, 3000 Leuven, Belgium;
| | - Václav Veverka
- Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 166 10 Prague 6, Czech Republic;
- Department of Cell Biology, Faculty of Science, Charles University, 116 36 Prague 1, Czech Republic
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46
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Cai K, Shao W, Chen X, Campbell YL, Nair MN, Suman SP, Beach CM, Guyton MC, Schilling MW. Meat quality traits and proteome profile of woody broiler breast (pectoralis major) meat. Poult Sci 2018; 97:337-346. [PMID: 29053841 DOI: 10.3382/ps/pex284] [Citation(s) in RCA: 84] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2017] [Accepted: 09/07/2017] [Indexed: 01/03/2023] Open
Abstract
Woody breast meat has recently become prevalent in the broiler industry in both the United States and European Union. Recent publications have described the meat quality characteristics of woody breast meat as having hardened areas and pale ridge-like bulges at both the caudal and cranial regions of the breast. The present study investigated the meat quality (pH, color, cooking loss, and shear force) and protein quality characteristics (protein and salt-soluble protein content) in woody breast meat as compared to normal breast meat. In addition, the differences in the muscle proteome profiles of woody and normal breast meat were characterized. Results indicated that woody breast meat had a greater average pH (P < 0.0001) and cooking loss (P = 0.001) than normal breast meat, but woody breast meat did not differ in shear force (P > 0.05) in comparison to normal breast meat samples. The L*, a*, and b* values of woody breast fillets were greater than normal breast fillets (P < 0.0001 to L*; P = 0.002 to a*; P = 0.016 to b*). The woody breast meat had more fat (P < 0.0001) and moisture (P < 0.021) and less protein (P < 0.0001) and salt-soluble protein (P < 0.0001) when compared with normal breast fillets. Whole muscle proteome analysis indicated 8 proteins that were differentially expressed (P < 0.05) between normal and woody breast meat samples. The differences in muscle proteome between normal and woody breast meat indicated an increased oxidative stress in woody breast meat when compared to normal meat. In addition, the abundance of some glycolytic enzymes, which are critical to the regeneration of adenosine triphosphate (ATP) in postmortem muscles, was lower in woody breast meat than in normal breast meat. Proteomic differences provide additional information on the biochemical pathways and genetic variations that lead to woody breast meat. Further research should be conducted to elucidate the genetic and nutritional contributions to the proliferation of woody breast meat in the United States.
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Affiliation(s)
- K Cai
- School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009 PRA
| | - W Shao
- Department of Food Science, Nutrition, and Health Promotion, Mississippi State University, Mississippi State 39762
| | - X Chen
- Department of Poultry Science, Mississippi State University, Mississippi State 39762
| | - Y L Campbell
- Department of Food Science, Nutrition, and Health Promotion, Mississippi State University, Mississippi State 39762
| | - M N Nair
- Department of Animal and Food Sciences, University of Kentucky, Lexington 40546
| | - S P Suman
- Department of Animal and Food Sciences, University of Kentucky, Lexington 40546
| | - C M Beach
- Proteomics Core Facility, University of Kentucky, Lexington 40506
| | - M C Guyton
- Department of Food Science, Nutrition, and Health Promotion, Mississippi State University, Mississippi State 39762
| | - M W Schilling
- Department of Food Science, Nutrition, and Health Promotion, Mississippi State University, Mississippi State 39762
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Kim JA, Kim JC, Min JS, Kang I, Oh J, Ahn JK. HSV-1 ICP27 induces apoptosis by promoting Bax translocation to mitochondria through interacting with 14-3-3θ. BMB Rep 2018; 50:257-262. [PMID: 28256197 PMCID: PMC5458675 DOI: 10.5483/bmbrep.2017.50.5.023] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2017] [Indexed: 12/16/2022] Open
Abstract
The subcellular localization of Bax plays a crucial role during apoptosis. In response to apoptotic stimuli, Bax translocates from the cytoplasm to the mitochondria, where it promotes the release of cytochrome c to the cytoplasm. In cells infected with HSV-1, apoptosis is triggered or blocked by diverse mechanisms. In this study, we demonstrate how HSV-1 ICP27 induces apoptosis and modulates mitochondrial membrane potential in HEK 293T cells. We found that ICP27 interacts with 14-3-3θ which sequesters Bax to the cytoplasm. In addition, ICP27 promotes the translocation of Bax to the mitochondria by inhibiting the interaction between 14-3-3θ and Bax. Our findings may provide a novel apoptotic regulatory pathway induced by ICP27 during HSV-1 infection.
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Affiliation(s)
- Ji Ae Kim
- Department of Microbiology & Molecular Biology, College of Biological Science and Biotechnology, Chungnam National University, Daejeon 34134, Korea
| | - Jin Chul Kim
- Department of Microbiology & Molecular Biology, College of Biological Science and Biotechnology, Chungnam National University, Daejeon 34134, Korea
| | - Jung Sun Min
- Department of Microbiology & Molecular Biology, College of Biological Science and Biotechnology, Chungnam National University, Daejeon 34134, Korea
| | - Inho Kang
- Department of Microbiology & Molecular Biology, College of Biological Science and Biotechnology, Chungnam National University, Daejeon 34134, Korea
| | - Jeongho Oh
- Department of Microbiology & Molecular Biology, College of Biological Science and Biotechnology, Chungnam National University, Daejeon 34134, Korea
| | - Jeong Keun Ahn
- Department of Microbiology & Molecular Biology, College of Biological Science and Biotechnology, Chungnam National University, Daejeon 34134, Korea
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Subcellular localization of FOXO3a as a potential biomarker of response to combined treatment with inhibitors of PI3K and autophagy in PIK3CA-mutant cancer cells. Oncotarget 2018; 8:6608-6622. [PMID: 28036259 PMCID: PMC5351656 DOI: 10.18632/oncotarget.14245] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2016] [Accepted: 12/01/2016] [Indexed: 01/01/2023] Open
Abstract
Autophagy is the process of lysosome-mediated degradation and recycling that functions as an adaptive survival mechanism during anti-cancer therapy. Aberrant activation of the phosphoinositide-3-kinase (PI3K) pathway frequently occurs in solid tumors, including cervical cancer. However, single-agent PI3K inhibitors show modest anti-tumor efficacy in clinics. To see whether autophagy inhibition improves the efficacy of PI3K inhibitor in PIK3CA-mutant cancer cells, cells were treated with BKM120, a pan-PI3K inhibitor, and the autophagy inhibitor hydroxychloroquine (HCQ). Autophagy inhibition augmented the efficacy of BKM120 depending on PIK3CA-mutant cancer cell type. BKM120 treatment led to the nuclear accumulation of forkhead box O3 (FOXO3a) in Caski and T47D cells, which showed a synergistic effect of BKM120 and HCQ and the strong induction of autophagy. However, most FOXO3a remained in cytoplasm in C33A and ME180 cells, which did not exhibit synergy. These data suggest that BKM120-induced nuclear translocation of FOXO3a might elicit autophagy and be a critical factor determining the synergistic activity of BKM120 and HCQ in PIK3CA-mutant cancer cells. The release of FOXO3a from 14-3-3 by BV02 or 14-3-3 knockdown induced autophagy by BKM120 in C33A cells and sensitized the cells to the combined BKM120 and HCQ treatment, suggesting that cytoplasmic retention of FOXO3a by 14-3-3 even in the presence of BKM120 inhibit autophagy induction and synergistic effect of BKM120 and HCQ combination. Taken together, our study shows that subcellular localization of FOXO3a might be a potential biomarker for predicting response to the combination treatment with PI3K and autophagy inhibitors in PIK3CA-mutant cervical cancer patients.
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YWHAE silencing induces cell proliferation, invasion and migration through the up-regulation of CDC25B and MYC in gastric cancer cells: new insights about YWHAE role in the tumor development and metastasis process. Oncotarget 2018; 7:85393-85410. [PMID: 27863420 PMCID: PMC5356744 DOI: 10.18632/oncotarget.13381] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2016] [Accepted: 10/27/2016] [Indexed: 12/16/2022] Open
Abstract
We previously observed reduced YWHAE (14-3-3ε) protein expression in a small set of gastric cancer samples. YWHAE may act as a negative regulator of the cyclin CDC25B, which is a transcriptional target of MYC oncogene. The understanding of YWHAE role and its targets is important for the better knowledge of gastric carcinogenesis. Thus, we aimed to evaluate the relationship among YWHAE, CDC25B, and MYC in vitro and in vivo. For this, we analyzed the YWHAE, CDC25B, and MYC expression in YWHA-silenced, CDC25B-silenced, and MYC-silenced gastric cancer cell lines, as well as in gastric cancer and non-neoplastic gastric samples. In gastric cancer cell lines, YWHAE was able to inhibit the cell proliferation, invasion and migration through the reduction of MYC and CDC25B expression. Conversely, MYC induced the cell proliferation, invasion and migration through the induction of CDC25B and the reduction of YWHAE. Most of the tumors presented reduced YWHAE and increased CDC25B expression, which seems to be important for tumor development. Increased MYC expression was a common finding in gastric cancer and has a role in poor prognosis. In the tumor initiation, the opposite role of YWHAE and CDC25B in gastric carcinogenesis seems to be independent of MYC expression. However, the inversely correlation between YWHAE and MYC expression seems to be important for gastric cancer cells invasion and migration. The interaction between YWHAE and MYC and the activation of the pathways related to this interaction play a role in the metastasis process.
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50
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Group-I PAKs-mediated phosphorylation of HACE1 at serine 385 regulates its oligomerization state and Rac1 ubiquitination. Sci Rep 2018; 8:1410. [PMID: 29362425 PMCID: PMC5780496 DOI: 10.1038/s41598-018-19471-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2017] [Accepted: 12/28/2017] [Indexed: 12/21/2022] Open
Abstract
The regulation of Rac1 by HACE1-mediated ubiquitination and proteasomal degradation is emerging as an essential element in the maintenance of cell homeostasis. However, how the E3 ubiquitin ligase activity of HACE1 is regulated remains undetermined. Using a proteomic approach, we identified serine 385 as a target of group-I PAK kinases downstream Rac1 activation by CNF1 toxin from pathogenic E. coli. Moreover, cell treatment with VEGF also promotes Ser-385 phosphorylation of HACE1. We have established in vitro that HACE1 is a direct target of PAK1 kinase activity. Mechanistically, we found that the phospho-mimetic mutant HACE1(S385E), as opposed to HACE1(S385A), displays a lower capacity to ubiquitinate Rac1 in cells. Concomitantly, phosphorylation of Ser-385 plays a pivotal role in controlling the oligomerization state of HACE1. Finally, Ser-385 phosphorylated form of HACE1 localizes in the cytosol away from its target Rac1. Together, our data point to a feedback inhibition of HACE1 ubiquitination activity on Rac1 by group-I PAK kinases.
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