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Arteaga-Resendiz NK, Rodea GE, Ribas-Aparicio RM, Olivares-Cervantes AL, Castelán-Vega JA, Olivares-Trejo JDJ, Mendoza-Elizalde S, López-Villegas EO, Colín C, Aguilar-Rodea P, Reyes-López A, Salazar García M, Velázquez-Guadarrama N. HP0953 - hypothetical virulence factor overexpresion and localization during Helicobacter pylori infection of gastric epithelium. World J Gastroenterol 2022; 28:3886-3902. [PMID: 36157534 PMCID: PMC9367236 DOI: 10.3748/wjg.v28.i29.3886] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/29/2022] [Revised: 04/26/2022] [Accepted: 07/11/2022] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND The high prevalence and persistence of Helicobacter pylori (H. pylori) infection, as well as the diversity of pathologies related to it, suggest that the virulence factors used by this microorganism are varied. Moreover, as its proteome contains 340 hypothetical proteins, it is important to investigate them to completely understand the mechanisms of its virulence and survival. We have previously reported that the hypothetical protein HP0953 is overexpressed during the first hours of adhesion to inert surfaces, under stress conditions, suggesting its role in the environmental survival of this bacterium and perhaps as a virulence factor.
AIM To investigate the expression and localization of HP0953 during adhesion to an inert surface and against gastric (AGS) cells.
METHODS Expression analysis was performed for HP0953 during H. pylori adhesion. HP0953 expression at 0, 3, 12, 24, and 48 h was evaluated and compared using the Kruskal-Wallis equality-of-populations rank test. Recombinant protein was produced and used to obtain polyclonal antibodies for immunolocalization. Immunogold technique was performed on bacterial sections during adherence to inert surfaces and AGS cells, which was analyzed by transmission electron microscopy. HP0953 protein sequence was analyzed to predict the presence of a signal peptide and transmembrane helices, both provided by the ExPASy platform, and using the GLYCOPP platform for glycosylation sites. Different programs, via, I-TASSER, RaptorX, and HHalign-Kbest, were used to perform three-dimensional modeling.
RESULTS HP0953 exhibited its maximum expression at 12 h of infection in gastric epithelium cells. Immunogold technique revealed HP0953 localization in the cytoplasm and accumulation in some peripheral areas of the bacterial body, with greater expression when it is close to AGS cells. Bioinformatics analysis revealed the presence of a signal peptide that interacts with the transmembrane region and then allows the release of the protein to the external environment. The programs also showed a similarity with the Tip-alpha protein of H. pylori. Tip-alpha is an exotoxin that penetrates cells and induces tumor necrosis factor alpha production, and HP0953 could have a similar function as posttranslational modification sites were found; modifications in turn require enzymes located in eukaryotic cells. Thus, to be functional, HP0953 may necessarily need to be translocated inside the cell where it can trigger different mechanisms producing cellular damage.
CONCLUSION The location of HP0953 around infected cells, the probable posttranslational modifications, and its similarity to an exotoxin suggest that this protein is a virulence factor.
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Affiliation(s)
- Nancy K Arteaga-Resendiz
- Laboratorio de Investigación en Enfermedades Infecciosas, Hospital Infantil de México Federico Gómez, Mexico City 06720, Mexico
- Posgrado en Biomedicina y Biotecnología Molecular, Laboratorio de Producción y Control de Biológicos, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City 11340, Mexico
| | - Gerardo E Rodea
- Laboratorio de Investigación en Enfermedades Infecciosas, Hospital Infantil de México Federico Gómez, Mexico City 06720, Mexico
| | - Rosa María Ribas-Aparicio
- Posgrado en Biomedicina y Biotecnología Molecular, Laboratorio de Producción y Control de Biológicos, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City 11340, Mexico
| | - Alma L Olivares-Cervantes
- Laboratorio de Investigación en Enfermedades Infecciosas, Hospital Infantil de México Federico Gómez, Mexico City 06720, Mexico
| | - Juan Arturo Castelán-Vega
- Posgrado en Biomedicina y Biotecnología Molecular, Laboratorio de Producción y Control de Biológicos, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City 11340, Mexico
| | - José de Jesús Olivares-Trejo
- Laboratorio de Adquisición de Hierro, Universidad Autónoma de la Ciudad México, Posgrado Ciencias Genómica, Mexico City 03100, Mexico
| | - Sandra Mendoza-Elizalde
- Laboratorio de Investigación en Enfermedades Infecciosas, Hospital Infantil de México Federico Gómez, Mexico City 06720, Mexico
| | - Edgar O López-Villegas
- Laboratorio Central de Microscopía, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City 11340, Mexico
| | - Christian Colín
- Laboratorio de Investigación en Enfermedades Infecciosas, Hospital Infantil de México Federico Gómez, Mexico City 06720, Mexico
| | - Pamela Aguilar-Rodea
- Laboratorio de Investigación en Enfermedades Infecciosas, Hospital Infantil de México Federico Gómez, Mexico City 06720, Mexico
| | - Alfonso Reyes-López
- Centro de estudios económicos y sociales en salud, Hospital Infantil de México Federico Gómez, Mexico City 06720, Mexico
| | - Marcela Salazar García
- Laboratorio de Investigación en Biología del Desarrollo y Teratogénesis Experimental, Hospital Infantil de México Federico Gómez, Mexico City 06720, Mexico
| | - Norma Velázquez-Guadarrama
- Laboratorio de Investigación en Enfermedades Infecciosas, Hospital Infantil de México Federico Gómez, Mexico City 06720, Mexico
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Suganuma M, Watanabe T, Sueoka E, Lim IK, Fujiki H. Role of TNF-α-Inducing Protein Secreted by Helicobacter pylori as a Tumor Promoter in Gastric Cancer and Emerging Preventive Strategies. Toxins (Basel) 2021; 13:181. [PMID: 33804551 PMCID: PMC7999756 DOI: 10.3390/toxins13030181] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2021] [Revised: 02/20/2021] [Accepted: 02/21/2021] [Indexed: 12/24/2022] Open
Abstract
The tumor necrosis factor-α (TNF-α)-inducing protein (tipα) gene family, comprising Helicobacter pylori membrane protein 1 (hp-mp1) and tipα, has been identified as a tumor promoter, contributing to H. pylori carcinogenicity. Tipα is a unique H. pylori protein with no similarity to other pathogenicity factors, CagA, VacA, and urease. American H. pylori strains cause human gastric cancer, whereas African strains cause gastritis. The presence of Tipα in American and Euro-Asian strains suggests its involvement in human gastric cancer development. Tipα secreted from H. pylori stimulates gastric cancer development by inducing TNF-α, an endogenous tumor promoter, through its interaction with nucleolin, a Tipα receptor. This review covers the following topics: tumor-promoting activity of the Tipα family members HP-MP1 and Tipα, the mechanism underlying this activity of Tipα via binding to the cell-surface receptor, nucleolin, the crystal structure of rdel-Tipα and N-terminal truncated rTipα, inhibition of Tipα-associated gastric carcinogenesis by tumor suppressor B-cell translocation gene 2 (BTG2/TIS21), and new strategies to prevent and treat gastric cancer. Thus, Tipα contributes to the carcinogenicity of H. pylori by a mechanism that differs from those of CagA and VacA.
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Affiliation(s)
- Masami Suganuma
- Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan
| | - Tatsuro Watanabe
- Department of Drug Discovery and Biomedical Sciences, Faculty of Medicine, Saga University, Nabeshima, Saga 849-8501, Japan;
| | - Eisaburo Sueoka
- Department of Clinical Laboratory Medicine, Faculty of Medicine, Saga University, Nabeshima, Saga 849-8501, Japan; (E.S.); (H.F.)
| | - In Kyoung Lim
- Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon 16499, Gyeonggi-do, Korea;
| | - Hirota Fujiki
- Department of Clinical Laboratory Medicine, Faculty of Medicine, Saga University, Nabeshima, Saga 849-8501, Japan; (E.S.); (H.F.)
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3
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Berlamont H, De Witte C, Bauwens E, Min Jou H, Ducatelle R, De Meester E, Gansemans Y, Deforce D, Van Nieuwerburgh F, Haesebrouck F, Smet A. Distinct transcriptome signatures of Helicobacter suis and Helicobacter heilmannii strains upon adherence to human gastric epithelial cells. Vet Res 2020; 51:62. [PMID: 32381076 PMCID: PMC7206758 DOI: 10.1186/s13567-020-00786-w] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2020] [Accepted: 04/21/2020] [Indexed: 12/18/2022] Open
Abstract
The porcine Helicobacter suis and canine-feline H. heilmannii are gastric Helicobacter species with zoonotic potential. However, little is known about the pathogenesis of human infections with these Helicobacter species. To gain more insight into the interactions of both zoonotic Helicobacter species with human gastric epithelial cells, we investigated bacterial genes that are differentially expressed in a H. suis and H. heilmannii strain after adhesion to the human gastric epithelial cell line MKN7. In vitro Helicobacter-MKN7 binding assays were performed to obtain bacterial RNA for sequencing analysis. H. suis and H. heilmannii bacteria attached to the gastric epithelial cells (i.e. cases) as well as unbound bacteria (i.e. controls) were isolated, after which prokaryotic RNA was purified and sequenced. Differentially expressed genes were identified using the DESeq2 package and SARTools pipeline in R. A list of 134 (83 up-regulated and 51 down-regulated) and 143 (60 up-regulated and 83 down-regulated) differentially expressed genes (padj ≤ 0.01; fold change ≥ 2) were identified for the adherent H. suis and H. heilmannii strains, respectively. According to BLASTp analyses, only 2 genes were commonly up-regulated and 4 genes commonly down-regulated in both pathogens. Differentially expressed genes of the H. suis and H. heilmannii strains belonged to multiple functional classes, indicating that adhesion of both strains to human gastric epithelial cells evokes pleiotropic adaptive responses. Our results suggest that distinct pathways are involved in human gastric colonization of H. suis and H. heilmannii. Further research is needed to elucidate the clinical significance of these findings.
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Affiliation(s)
- Helena Berlamont
- Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
| | - Chloë De Witte
- Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
| | - Eva Bauwens
- Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
| | - Hannah Min Jou
- Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
| | - Richard Ducatelle
- Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
| | - Ellen De Meester
- Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium
| | - Yannick Gansemans
- Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium
| | - Dieter Deforce
- Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium
| | - Filip Van Nieuwerburgh
- Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium
| | - Freddy Haesebrouck
- Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
| | - Annemieke Smet
- Translational Research in Immunology and Inflammation, Laboratory of Experimental Medicine and Pediatrics, Faculty of Medicine and Health Sciences, Antwerp University, 2610, Antwerp, Belgium.
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Multiplex serology of Helicobacter pylori antigens in detection of current infection and atrophic gastritis - A simple and cost-efficient method. Microb Pathog 2018; 119:137-144. [PMID: 29665437 DOI: 10.1016/j.micpath.2018.04.018] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2018] [Revised: 04/10/2018] [Accepted: 04/10/2018] [Indexed: 02/07/2023]
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Helicobacter pylori antigenic Lpp20 is a structural homologue of Tipα and promotes epithelial-mesenchymal transition. Biochim Biophys Acta Gen Subj 2017; 1861:3263-3271. [PMID: 28947343 DOI: 10.1016/j.bbagen.2017.09.017] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2017] [Revised: 09/07/2017] [Accepted: 09/21/2017] [Indexed: 02/07/2023]
Abstract
BACKGROUND Helicobacter pylori is a bacterium that affects about 50% of the world population and, despite being often asymptomatic, it is responsible of several gastric diseases, from gastritis to gastric cancer. The protein Lpp20 (HP1456) plays an important role in bacterium survival and host colonization, but the possibility that it might be involved in the etiology of H. pylori-related disorders is an unexplored issue. Lpp20 is a lipoprotein bound to the external membrane of the bacterium, but it is also secreted inside vesicles along with other two proteins of the same operon, i.e. HP1454 and HP1457. RESULTS In this study we determined the crystal structure of Lpp20 and we found that it has a fold similar to a carcinogenic factor released by H. pylori, namely Tipα. We demonstrate that Lpp20 promotes cell migration and E-cadherin down-regulation in gastric cancer cells, two events recalling the epithelial-mesenchymal transition (EMT) process. Differently from Tipα, Lpp20 also stimulates cell proliferation. CONCLUSIONS This identifies Lpp20 as a new pathogenic factor produced by H. pylori that promotes EMT and thereby the progression of cancer to the metastatic state.
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Kaplan-Türköz B. A putative Toll/interleukin-1 receptor domain protein fromHelicobacter pyloriis dimeric in solution and interacts with human Toll-like receptor adaptor myeloid differentiation primary response 88. Microbiol Immunol 2017; 61:85-91. [DOI: 10.1111/1348-0421.12469] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2016] [Revised: 02/03/2017] [Accepted: 02/10/2017] [Indexed: 12/26/2022]
Affiliation(s)
- Burcu Kaplan-Türköz
- Department of Food Engineering; Faculty of Engineering; Ege University; 35100, Bornova İzmir Turkey
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Rosadi F, Fiorentini C, Fabbri A. Bacterial protein toxins in human cancers. Pathog Dis 2015; 74:ftv105. [DOI: 10.1093/femspd/ftv105] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/29/2015] [Indexed: 12/16/2022] Open
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Quarantini S, Cendron L, Zanotti G. Crystal structure of the secreted protein HP1454 from the human pathogen Helicobacter pylori. Proteins 2014; 82:2868-73. [DOI: 10.1002/prot.24608] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2014] [Revised: 04/30/2014] [Accepted: 05/09/2014] [Indexed: 12/23/2022]
Affiliation(s)
- Sandra Quarantini
- Department of Biomedical Sciences; University of Padua; Viale G. Colombo 3 Padua 35131 Italy
| | - Laura Cendron
- Department of Biomedical Sciences; University of Padua; Viale G. Colombo 3 Padua 35131 Italy
| | - Giuseppe Zanotti
- Department of Biomedical Sciences; University of Padua; Viale G. Colombo 3 Padua 35131 Italy
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9
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Zanotti G, Cendron L. Structural and functional aspects of the Helicobacter pylori secretome. World J Gastroenterol 2014; 20:1402-1423. [PMID: 24587618 PMCID: PMC3925851 DOI: 10.3748/wjg.v20.i6.1402] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/28/2013] [Accepted: 01/06/2014] [Indexed: 02/06/2023] Open
Abstract
Proteins secreted by Helicobacter pylori (H. pylori), an important human pathogen responsible for severe gastric diseases, are reviewed from the point of view of their biochemical characterization, both functional and structural. Despite the vast amount of experimental data available on the proteins secreted by this bacterium, the precise size of the secretome remains unknown. In this review, we consider as secreted both proteins that contain a secretion signal for the periplasm and proteins that have been detected in the external medium in in vitro experiments. In this way, H. pylori’s secretome appears to be composed of slightly more than 160 proteins, but this number must be considered very cautiously, not only because the definition of secretome itself is ambiguous but also because the included proteins were observed as secreted in in vitro experiments that were not representative of the environmental situation in vivo. The proteins that appear to be secreted can be grouped into different classes: enzymes (48 proteins), outer membrane proteins (43), components of flagella (11), members of the cytotoxic-associated genes pathogenicity island or other toxins (8 and 5, respectively), binding and transport proteins (9), and others (11). A final group, which includes 28 members, is represented by hypothetical uncharacterized proteins. Despite the large amount of data accumulated on the H. pylori secretome, a considerable amount of work remains to reach a full comprehension of the system at the molecular level.
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He C, Chen M, Liu J, Yuan Y. Host genetic factors respond to pathogenic step-specific virulence factors of Helicobacter pylori in gastric carcinogenesis. MUTATION RESEARCH. REVIEWS IN MUTATION RESEARCH 2014; 759:14-26. [PMID: 24076409 DOI: 10.1016/j.mrrev.2013.09.002] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/28/2013] [Revised: 09/13/2013] [Accepted: 09/13/2013] [Indexed: 12/18/2022]
Abstract
The interindividual differences in risk of Helicobacter pylori (H. pylori)-associated gastric cancer involve significant heterogeneities of both host genetics and H. pylori strains. Several recent studies proposed a distinct sequence for H. pylori exerting its virulence in the host stomach: (i) adhering to and colonizing the surface of gastric epithelial cells, (ii) evading and attenuating the host defense, and (iii) invading and damaging the gastric mucosa. This review focuses on several key issues that still need to be clarified, such as which virulence factors of H. pylori are involved in the three pathogenic steps, which host genes respond to the step-specific virulence factors, and whether and/or how the corresponding host genetic variations influence the risk of gastric carcinogenesis. Urease, BabA and SabA in the adhesion-step, PGN and LPS in the immune evasion-step, and CagA, VacA and Tipα in the mucosal damage-step were documented to play an important role in step-specific pathogenicity of H. pylori infection. There is evidence further supporting a role of potentially functional polymorphisms of host genes directly responding to these pathogenic step-specific virulence factors in the susceptibility of gastric carcinogenesis, especially for urease-interacting HLA class II genes, BabA-interacting MUC1, PGN-interacting NOD1, LPS-interacting TLR4, and CagA-interacting PTPN11 and CDH1. With the continuous improvement of understanding the genetic profile of H. pylori-associated gastric carcinogenesis, a person at increased risk for gastric cancer may benefit from several aspects of efforts: (i) prevent H. pylori infection with a vaccine targeting certain step-specific virulence factor; (ii) eradicate H. pylori infection by blocking step-specific psychopathological characteristics of virulence factors; and (iii) adjust host physiological function to resist the carcinogenic role of step-specific virulence factors or interrupt the cellular signal transduction of the interplay between H. pylori and host in each pathogenic step, especially for the subjects with precancerous lesions in the stomach.
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Affiliation(s)
- Caiyun He
- Tumor Etiology and Screening Department of Cancer Institute and General Surgery, The First Affiliated Hospital of China Medical University; Key Laboratory of Cancer Etiology and Prevention (China Medical University), Liaoning Provincial Education Department, Shenyang 110001, China
| | - Moye Chen
- Tumor Etiology and Screening Department of Cancer Institute and General Surgery, The First Affiliated Hospital of China Medical University; Key Laboratory of Cancer Etiology and Prevention (China Medical University), Liaoning Provincial Education Department, Shenyang 110001, China
| | - Jingwei Liu
- Tumor Etiology and Screening Department of Cancer Institute and General Surgery, The First Affiliated Hospital of China Medical University; Key Laboratory of Cancer Etiology and Prevention (China Medical University), Liaoning Provincial Education Department, Shenyang 110001, China
| | - Yuan Yuan
- Tumor Etiology and Screening Department of Cancer Institute and General Surgery, The First Affiliated Hospital of China Medical University; Key Laboratory of Cancer Etiology and Prevention (China Medical University), Liaoning Provincial Education Department, Shenyang 110001, China.
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Tang CL, Hao B, Zhang GX, Shi RH, Cheng WF. Helicobacter pylori tumor necrosis factor-α inducing protein promotes cytokine expression via nuclear factor-κB. World J Gastroenterol 2013; 19:399-403. [PMID: 23372364 PMCID: PMC3554826 DOI: 10.3748/wjg.v19.i3.399] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/12/2012] [Revised: 12/10/2012] [Accepted: 12/20/2012] [Indexed: 02/06/2023] Open
Abstract
AIM To study the effects of Helicobacter pylori (H. pylori) tumor necrosis factor-α (TNF) inducing protein (Tip-α) on cytokine expression and its mechanism. METHODS We cloned Tip-α from the H. pylori strain 26695, transformed Escherichia coli with an expression plasmid, and then confirmed the expression product by Western blotting. Using different concentrations of Tip-α that affected SGC7901 and GES-1 cells at different times, we assessed cytokine levels using enzyme-linked immunosorbent assay. We blocked SGC7901 cells with pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of nuclear factor κB (NF-κB). We then detected interleukin (IL)-1β and TNF-α levels in SGC7901 cells. RESULTS Western blot analysis using an anti-Tip-α antibody revealed a 23-kDa protein, which indicated that recombinant Tip-α protein was recombined successfully. The levels of IL-1β, IL-8 and TNF-α were significantly higher following Tip-α interference, whether GES-1 cells or SGC-7901 cells were used (P < 0.05). However, the levels of cytokines (including IL-1β, IL-8 and TNF-α) secreted by SGC-7901 cells were greater than those secreted by GES-1 cells following treatment with Tip-α at the same concentration and for the same duration (P < 0.05). After blocking NF-κB with PDTC, the cells (GES-1 cells and SGC-7901 cells) underwent interference with Tip-α. We found that IL-1β and TNF-α levels were significantly decreased compared to cells that only underwent Tip-α interference (P < 0.05). CONCLUSION Tip-α plays an important role in cytokine expression through NF-κB.
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12
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Suganuma M, Watanabe T, Yamaguchi K, Takahashi A, Fujiki H. Human gastric cancer development with TNF-α-inducing protein secreted from Helicobacter pylori. Cancer Lett 2012; 322:133-8. [PMID: 22459353 DOI: 10.1016/j.canlet.2012.03.027] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2012] [Revised: 03/13/2012] [Accepted: 03/21/2012] [Indexed: 12/12/2022]
Abstract
TNF-α-inducing protein (Tipα) is a unique carcinogenic factor of Helicobacter pylori, which is secreted into culture broth. The biological activities of Tipα and deletion mutant were studied. Tipα protein specifically binds to cell-surface nucleolin and then enters the gastric cancer cells, where TNF-α and chemokine gene expressions are induced by NF-κB activation. Nucleolin localizes on the surface of gastric cancer cells, and interaction between Tipα and cell-surface nucleolin causes a cancer-oriented microenvironment that increases the risk of gastric cancer. This paper discusses a new mechanism of gastric cancer development with H. pylori and provides a new preventive strategy.
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Affiliation(s)
- Masami Suganuma
- Research Institute for Clinical Oncology, Saitama Cancer Center, Japan.
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13
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Gao M, Li D, Hu Y, Zhang Y, Zou Q, Wang DC. Crystal structure of TNF-α-inducing protein from Helicobacter pylori in active form reveals the intrinsic molecular flexibility for unique DNA-binding. PLoS One 2012; 7:e41871. [PMID: 22860022 PMCID: PMC3409205 DOI: 10.1371/journal.pone.0041871] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2012] [Accepted: 06/29/2012] [Indexed: 01/26/2023] Open
Abstract
Tipα (TNF-α-inducing protein) from Helicobacter pylori is a carcinogenic effector. Studies on this protein revealed that a homodimer linked by a pair of intermolecular disulfide bridges (Cys25-Cys25 and Cys27-Cys27) was absolutely necessary for its biological functions. The activities of Tipα would be abolished when both disulfide bridges were disrupted. The crystal structures of Tipα reported to date, however, were based on inactive, monomeric mutants with their N-terminal, including residues Cys25 and Cys27, truncated. Here we report the crystal structure of H. pylori Tipα protein, TipαN25, at 2.2Å resolution, in which Cys25 and Cys27 form a pair of inter-chain disulfide bridges linking an active dimer. The disulfide bridges exhibit structural flexibility in the present structure. A series of structure-based mutagenesis, biochemical assays and molecular dynamic simulations on DNA-Tipα interactions reveal that Tipα utilizes the dimeric interface as the DNA-binding site and that residues His60, Arg77 and Arg81 located at the interface are crucial for DNA binding. Tipα could bind to one ssDNA, two ssDNA or one dsDNA in experiments, respectively, in the native or mutant states. The unique DNA-binding activities of Tipα indicate that the intrinsic flexible nature of disulfide bridges could endow certain elasticity to the Tipα dimer for its unique bioactivities. The results shed light on the possible structural mechanism for the functional performances of Tipα.
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Affiliation(s)
- Mingming Gao
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People’s Republic of China
- Graduate University of Chinese Academy of Sciences, Beijing, People’s Republic of China
| | - Defeng Li
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People’s Republic of China
| | - Yonglin Hu
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People’s Republic of China
| | - Ying Zhang
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People’s Republic of China
| | - Quanming Zou
- Department of Clinical Microbiology and Immunology, Third Military Medical University, Chongqing, People’s Republic of China
| | - Da-Cheng Wang
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People’s Republic of China
- * E-mail:
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Hernandez JA, Phillips AH, Erbil WK, Zhao D, Demuez M, Zeymer C, Pelton JG, Wemmer DE, Rubio LM. A sterile alpha-motif domain in NafY targets apo-NifDK for iron-molybdenum cofactor delivery via a tethered domain. J Biol Chem 2011; 286:6321-8. [PMID: 21156797 PMCID: PMC3057858 DOI: 10.1074/jbc.m110.168732] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2010] [Revised: 10/18/2010] [Indexed: 12/20/2022] Open
Abstract
NafY participates in the final steps of nitrogenase maturation, having a dual role as iron-molybdenum cofactor (FeMo-co) carrier and as chaperone to the FeMo-co-deficient apo-NifDK (apo-dinitrogenase). NafY contains an N-terminal domain of unknown function (n-NafY) and a C-terminal domain (core-NafY) necessary for FeMo-co binding. We show here that n-NafY and core-NafY have very weak interactions in intact NafY. The NMR structure of n-NafY reveals that it belongs to the sterile α-motif (SAM) family of domains, which are frequently involved in protein-protein interactions. The presence of a SAM domain in NafY was unexpected and could not be inferred from its amino acid sequence. Although SAM domains are very commonly found in eukaryotic proteins, they have rarely been identified in prokaryotes. The n-NafY SAM domain binds apo-NifDK. As opposed to full-length NafY, n-NafY impaired FeMo-co insertion when present in molar excess relative to FeMo-co and apo-NifDK. The implications of these observations are discussed to offer a plausible mechanism of FeMo-co insertion. NafY domain structure, molecular tumbling, and interdomain motion, as well as NafY interaction with apo-NifDK are consistent with the function of NafY in FeMo-co delivery to apo-NifDK.
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Affiliation(s)
- Jose A. Hernandez
- From the Department of Biochemistry, Arizona College of Osteopathic Medicine, Midwestern University Arizona, Glendale, Arizona 85308
| | - Aaron H. Phillips
- the Department of Chemistry
- QB3 Institute, and
- Division of Physical Biosciences of Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720
| | - W. Kaya Erbil
- the Department of Chemistry
- QB3 Institute, and
- Division of Physical Biosciences of Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720
| | - Dehua Zhao
- the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, and
| | - Marie Demuez
- the Instituto Madrileño de Estudios Avanzados (IMDEA) Energía, 28223 Madrid, Spain
| | - Cathleen Zeymer
- the Department of Chemistry
- QB3 Institute, and
- Division of Physical Biosciences of Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720
| | - Jeffery G. Pelton
- QB3 Institute, and
- Division of Physical Biosciences of Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720
| | - David E. Wemmer
- the Department of Chemistry
- QB3 Institute, and
- Division of Physical Biosciences of Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720
| | - Luis M. Rubio
- the Instituto Madrileño de Estudios Avanzados (IMDEA) Energía, 28223 Madrid, Spain
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15
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Watanabe T, Tsuge H, Imagawa T, Kise D, Hirano K, Beppu M, Takahashi A, Yamaguchi K, Fujiki H, Suganuma M. Nucleolin as cell surface receptor for tumor necrosis factor-alpha inducing protein: a carcinogenic factor of Helicobacter pylori. J Cancer Res Clin Oncol 2010; 136:911-21. [PMID: 20049476 DOI: 10.1007/s00432-009-0733-y] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2009] [Accepted: 11/13/2009] [Indexed: 12/16/2022]
Abstract
PURPOSE Tumor necrosis factor-alpha inducing protein (Tipalpha) is a unique carcinogenic factor released from Helicobacter pylori (H. pylori). Tipalpha specifically binds to cells and is incorporated into cytosol and nucleus, where it strongly induces expression of TNF-alpha and chemokine genes mediated through NF-kappaB activation, resulting in tumor development. To elucidate mechanism of action of Tipalpha, we studied a binding protein of Tipalpha in gastric epithelial cells. METHODS Tipalpha binding protein was found in cell lysates of mouse gastric cancer cell line MGT-40 by FLAG-pull down assay and identified to be cell surface nucleolin by flow cytometry using anti-nucleolin antibody. Incorporation of Tipalpha into the cells was determined by Western blotting and expression of TNF-alpha gene was quantified by RT-PCR. RESULTS Nucleolin was co-precipitated with Tipalpha-FLAG, but not with del-Tipalpha-FLAG (an inactive mutant). After treatment with Tipalpha-FLAG, incorporated Tipalpha was co-immunoprecipitated with endogenous nucleolin using anti-nucleolin antibody. The direct binding of Tipalpha to recombinant His-tagged nucleolin fragment (284-710) was also confirmed. Although nucleolin is an abundant non-ribosomal protein of the nucleolus, we found that nucleolin is present on the cell surface of MGT-40 cells. Pretreatment with anti-nucleolin antibody enhanced Tipalpha-incorporation into the cells through nucleolin internalization. In addition, pretreatment with tunicamycin, an inhibitor of N-glycosylation, decreased the amounts of cell surface nucleolin and inhibited both internalization of Tipalpha and expression of TNF-alpha gene. CONCLUSIONS All the results indicate that nucleolin acts as a receptor for Tipalpha and shuttles Tipalpha from cell surface to cytosol and nuclei. These findings provide a new mechanistic insight into gastric cancer development with Tipalpha.
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Affiliation(s)
- Tatsuro Watanabe
- Saitama Cancer Center, Research Institute for Clinical Oncology, Kitaadachi-gun, Saitama 362-0806, Japan
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16
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Structural basis for the Helicobacter pylori-carcinogenic TNF-alpha-inducing protein. Biochem Biophys Res Commun 2009; 388:193-8. [PMID: 19643085 DOI: 10.1016/j.bbrc.2009.07.121] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2009] [Accepted: 07/22/2009] [Indexed: 12/16/2022]
Abstract
Stomach cancer is strongly associated with infection by Helicobacter pylori. In 2005, we identified a new H. pylori gene encoding a TNF-alpha inducing protein (Tipalpha) that acts as a carcinogenic factor. Tipalpha is secreted from H. pylori as a homodimer whose subunits are linked by disulfide bonds. We also characterized a Tipalpha deletion mutant (del-Tipalpha) that lacks the N-terminal six amino acid residues (LQACTC), including two cysteines (C5 and C7) that form disulfide bonds, but nonetheless shows a weak ability to induce TNF-alpha expression. Here we report that del-Tipalpha has a novel elongated structure containing a 40-A-long alpha helix, and forms a heart-shaped homodimer via non-covalent bonds. Moreover, their circular dichroism spectra strongly suggest that the structures of the del-Tipalpha and Tipalpha homodimers are very similar. del-Tipalpha's unique mode of dimer formation provides important insight into protein-protein interactions and into the mechanism underlying the carcinogenicity of H. pylori infection.
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17
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Jang JY, Yoon HJ, Yoon JY, Kim HS, Lee SJ, Kim KH, Kim DJ, Jang S, Han BG, Lee BI, Suh SW. Crystal structure of the TNF-alpha-Inducing protein (Tipalpha) from Helicobacter pylori: Insights into Its DNA-binding activity. J Mol Biol 2009; 392:191-7. [PMID: 19596016 DOI: 10.1016/j.jmb.2009.07.010] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2009] [Revised: 07/01/2009] [Accepted: 07/06/2009] [Indexed: 12/16/2022]
Abstract
Helicobacter pylori infection is one of the highest risk factors for gastroduodenal diseases including gastric cancer. Tumor necrosis factor-alpha (TNF-alpha) is one of the essential cytokines for tumor promotion, and thus, an H. pylori protein that induces TNF-alpha is believed to play a significant role in gastric cancer development in humans. The HP0596 gene product of H. pylori strain 26695 was identified as the TNF-alpha-inducing protein (Tipalpha). Tipalpha is secreted from H. pylori as dimers and enters the gastric cells. It was shown to have a DNA-binding activity. Here, we have determined the crystal structure of Tipalpha from H. pylori. Its monomer consists of two structural domains ("mixed domain" and "helical domain"). Tipalpha exists as a dimer in the crystal, and the dimeric structure represents a novel scaffold for DNA binding. A positively charged surface patch formed across the two monomers of the Tipalpha dimer by the loop between helices alpha1 and alpha2 may be important in DNA binding.
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Affiliation(s)
- Jun Young Jang
- Department of Chemistry, Seoul National University, Korea
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