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乙肝病毒X与Tab1蛋白相互作用的体内外验证 *
({{custom_author.role_cn}}), {{javascript:window.custom_author_cn_index++;}}Identification of Protein-protein Interaction of Hepatitis B Virus X Protein and Tab1 in Vivo and in Vitro
({{custom_author.role_en}}), {{javascript:window.custom_author_en_index++;}}目的:借助实验室前期质谱分析技术和数据分析研究基础,采用免疫共沉淀(Co-IP)和蛋白质沉降实验(GST pull-down)验证HBV X蛋白与Tab1蛋白的相互作用,为进一步研究HBx在HBV慢性感染致癌机制中的作用提供一定的实验依据。方法:成功构建pGEX-2TK-GST-HBx质粒,对GST-HBx融合蛋白进行诱导表达,与GST-beads结合孵育,构建pcDNA3.1/myc-His(-)B-Tab1,转染293T细胞使其表达,然后GST pull-down体外试验验证二者的相互作用;构建pcDNA3.1/myc-His(-)B-Tab1和pcDNA3.1-3×flag-HBx真核表达质粒,共转染293T和HepG2细胞使其表达,通过Co-IP实验验证抗Myc抗体可以将HBx从细胞裂解液中沉淀下来,证实了它们在两种细胞系中存在相互作用。结果:显示了HBx和Tab1在体内外条件下能够发生相互作用,为进一步明确HBV X蛋白功能及作用机制奠定基础。
With the help of analysis of mass spectrometry and data analysis according to the preliminary laboratory basic research, Co-IP and GST pull-down methods was used to prove the interaction of HBV X protein with Tab1. In order to study the carcinogenic mechanism of HBx protein, Some experimental evidence for further study of the role of HBx in the carcinogenic mechanism of HBV chronic infection were provided. pGEX-2TK-GST-HBx plasmid was successfully constructed, GST-HBx fusion protein was induced and incubated with GST-beads. pcDNA3.1/myc-His (-) B-Tab1 plasmid was constructed,then was transfected 293T cells to express Myc-Tab1. Finally the interaction of GST-HBx and Myc-Tab1 was verified in GST pull-down test. Moreover, the eukaryotic expression plasmids including pcDNA3.1/myc-His (-) B-Tab1 and pcDNA3.1-3×flag-HBx were successfully constructed, then were co-transfected into human embryonic kideny 293T cells and HepG2 cells to express fusion protein, further Co-IP test was demonstrated that the anti-Myc antibody could precipitate HBx from the cell lysate, and confirmed their intracellular interaction in two human cell lines. In conclusion, all results show that HBx and Tab1 could interact in vitro and in vivo, also established foundation to reveal the function and mechanism of HBV X protein.
HBV X蛋白 / Tab1 / 免疫共沉淀实验 / GST沉降实验 / 相互作用 {{custom_keyword}} /
HBV X protein / Tab1 / Co-IP / GST pull-down / Interaction {{custom_keyword}} /
图1 Tab1的PCR产物Fig.1 PCR products of human Tab1 M1:Trans 15 000 DNA marker,Tab1 of PCR;M2: Trans 2 000 DNA marker |
图2 pcDNA3.1/myc-His(-)B-Tab1的酶切鉴定和菌落PCR鉴定Fig.2 Restriction enzyme digestion and colony PCR identification of pcDNA3.1/myc-His(-)B-Tab1 (a)Double enzyme digestion identification M1: Trans 15 000 DNA marker; 1:Empty vector;2: Double digestion products;M2:Trans 2 000 DNA marker (b)Colony PCR identification M1:Trans 15 000 DNA marker; Clone 1-5: Selecting five monoclonal colonies;M2:Trans 2 000 DNA marker |
图4 HBx的PCR产物Fig.4 PCR products of HBx M1:Trans 2 000 DNA marker; HBx: Products of PCR |
图5 pGEX-2TK-GST-HBx的酶切鉴定和菌落PCR鉴定Fig.5 Restriction enzyme digestion and colony PCR identification of pGEX-2TK-GST-HBx (a)Double enzyme digestion identification M1: Trans 15 000 DNA marker; 1:Empty vector;2: Double digestion products;M2:Trans 2 000 DNA marker (b)Colony PCR identification M1: Trans 15 000 DNA marker; Clone 1-6: Selecting five monoclonal colonies;M2:Trans 2 000 DNA marker |
图6 GST-HBx融合蛋白的诱导表达Fig.6 IPTG Induction of GST-HBx fusion protein expression M:Protein marker; 1-3:IPTG induction of GST-HBx; 4-6: IPTG induction of GST |
图8 Tab1与HBx在293T细胞内的相互作用Fig.8 Interaction between Tab1 and HBx in 293T cells by Co-IP |
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The hepatitis B virus-encoded protein, HBx, may contribute to carcinogenesis by perturbing cell growth and differentiation. There is some evidence indicating that HBx represses the nuclear import of the tumor repressor p53 and p53-dependent trans -activation and that HBx activates members of the basic region-leucine zipper (bZIP) family.
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Hepatitis B virus (HBV) contributes to hepatocellular carcinoma (HCC) development through direct and indirect mechanisms. HBV DNA integration into the host genome occurs at early steps of clonal tumor expansion and induces both genomic instability and direct insertional mutagenesis of diverse cancer-related genes. Prolonged expression of the viral regulatory protein HBx and/or altered versions of the preS/S envelope proteins dysregulates cell transcription and proliferation control and sensitizes liver cells to carcinogenic factors. Accumulation of preS1 large envelope proteins and/or preS2/S mutant proteins activates the unfold proteins response, that can contribute to hepatocyte transformation. Epigenetic changes targeting the expression of tumor suppressor genes occur early in the development of HCC. A major role is played by the HBV protein, HBx, which is recruited on cellular chromatin and modulates chromatin dynamics at specific gene loci. Compared with tumors associated with other risk factors, HBV-related tumors have a higher rate of chromosomal alterations, p53 inactivation by mutations and overexpression of fetal liver/hepatic progenitor cells genes. The WNT/ -catenin pathway is also often activated but HBV-related tumors display a low rate of activating -catenin mutations. HBV-related HCCs may arise on non-cirrhotic livers, further supporting the notion that HBV plays a direct role in liver transformation by triggering both common and etiology specific oncogenic pathways in addition to stimulating the host immune response and driving liver chronic necro-inflammation.
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Abstract BACKGROUND/AIMS: Hepatitis B virus (HBV), a major causative agent of hepatocellular carcinoma (HCC), encodes an oncogenic X protein (HBx) that transcriptionally activates multiple viral and cellular promoters. How this regulation influences HCC is unclear. METHODS: Global gene expression in HBx-expressing and non-expressing hepatoma cells was analyzed using cDNA microarrays. RESULTS: Genes that were markedly up- or down-regulated in the presence of HBx included those involved in signal transduction, metabolism, apoptosis, cytokine production, cell cycle, cell adhesion and oncogenesis. Other genes of ill-defined function were also affected. Trans-activation proficient HBx up-regulated the transcription, translation and secretion of matrix metalloproteinase-3 (MMP-3), manifest as a cell migratory phenotype. This HBx effect was abrogated in the presence of a MMP-3 specific peptide inhibitor. CONCLUSIONS: HBx exerts selective transcriptional control in hepatoma cells and induces cellular migration through the activation of MMP-3.
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Abstract Hepatocellular carcinoma (HCC) affects males in a significantly higher proportion than females and is one of the human cancers etiologically related to viral factors. Many studies provide strong evidence of the direct role that hepatitis B virus (HBV) plays in hepatic carcinogenesis, but the functions of HBV surface antigen ( HBsAg ) and X protein ( HBx ) in hepatocarcinogenesis through direct or indirect mechanisms are still being debated. We generated two HBV gene knock-in transgenic mouse lines by homologous recombination. HBsAg and HBx genes were integrated into the mouse p21 locus. Both male and female p21-HBx transgenic mice developed HCC after the age of 18 months; however, male p21-HBsAg transgenic mice began to develop HCC 3 months earlier. The expression of a number of genes related to metabolism and genomic instability largely resembled the molecular changes during the development of HCC in humans. ER- (estrogen receptor- ) was extremely up-regulated only in tumor tissues of male p21-HBsAg mice, providing genetic evidence that HBsAg might be the major risk factor affecting the gender difference in the causes of HCC. In conclusion, these mice might serve as good models for studying the different roles of HBsAg and HBx in early events of HBV-related hepatocarcinogenesis. (H EPATOLOGY 2004;39:318 324.)
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Abstract With an estimate of 350 million people chronically infected with the hepatitis B virus (HBV) worldwide, it is critically important to understand how persistent HBV infection is maintained and linked to chronic hepatitis, cirrhosis, and de- velopment of liver cancer (hepatocellular carcinoma (HCC)) (39). This review will focus on the HBV nonstructural X pro- tein (HBx), a key regulatory protein of the virus that is at the intersection of HBV infection, replication, pathogenesis, and possibly carcinogenesis. The exact role of HBx in viral repli- cation has yet to be established, and its link to the progression of HCC remains controversial. Moreover, it is still unclear whether development of HCC associated with chronic infec- tion by HBV involves a viral protein, is solely the consequence of a continual inflammatory response to infection, or requires both. Understanding the role of HBx in HBV replication and its effect on hepatocyte biology may help resolve this issue. This review describes key studies and activities of HBx, often interpreted within the context of the viral life cycle. The reader is referred to several comprehensive reviews on the reported biological properties of HBx (3, 78, 137). HBV
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Abstract Transforming growth factor-beta (TGF-beta) regulates many aspects of cellular function. A member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, TAK1, was previously identified as a mediator in the signaling pathway of TGF-beta superfamily members. The yeast two-hybrid system has now revealed two human proteins, termed TAB1 and TAB2 (for TAK1 binding protein), that interact with TAK1. TAB1 and TAK1 were co-immunoprecipitated from mammalian cells. Overproduction of TAB1 enhanced activity of the plasminogen activator inhibitor 1 gene promoter, which is regulated by TGF-beta, and increased the kinase activity of TAK1. TAB1 may function as an activator of the TAK1 MAPKKK in TGF-beta signal transduction.
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Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family that is activated by growth factors and cytokines such as TGF-β, IL-1β, and TNF-α, and mediates a wide range of biological processes through activation of the nuclear factor-κB (NF-κB) and the mitogen-activated protein (MAP) kinase signaling pathways. It is well established that activation status of TAK1 is tightly regulated by forming a complex with its binding partners, TAK1-binding proteins (TAB1, TAB2, and TAB3). Interestingly, recent evidence indicates the importance of post-translational modifications (PTMs) of TAK1 and TABs in the regulation of TAK1 activation. To date, a number of PTMs of TAK1 and TABs have been revealed, and these PTMs appear to fine-tune and coordinate TAK1 activities depending on the cellular context. This review therefore focuses on recent advances in the understanding of the PTMs of the TAK1-TAB complex.
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In addition to coding for virion structural proteins and the reverse tanscriptase, all mammalian hepadnaviruses also contain an open-reading frame called X, because its function could not be fathomed from the deduced amino acid sequence. While it is now known that the woodchuck virus (and presumably the other hepadnaviruses, as well) cannot replicate in the animal host if the X gene is mutated, the exact function of the X protein in the viral life cycle is still unknown. In transient transfection studies, X protein has been shown to trans-activate a wide variety of promoters. The mechanism of this activation is also unclear, although there is accumulating evidence in favor of both a cytoplasmic effect involving signal transduction pathways, and a nuclear effect directly on the transcriptional machinery. It appears that the X protein is at least as complex as many of the other, better studied viral trans-activators, and may well yield new insights into mammalian cell functions in the future.
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Abstract With an estimate of 350 million people chronically infected with the hepatitis B virus (HBV) worldwide, it is critically important to understand how persistent HBV infection is maintained and linked to chronic hepatitis, cirrhosis, and de- velopment of liver cancer (hepatocellular carcinoma (HCC)) (39). This review will focus on the HBV nonstructural X pro- tein (HBx), a key regulatory protein of the virus that is at the intersection of HBV infection, replication, pathogenesis, and possibly carcinogenesis. The exact role of HBx in viral repli- cation has yet to be established, and its link to the progression of HCC remains controversial. Moreover, it is still unclear whether development of HCC associated with chronic infec- tion by HBV involves a viral protein, is solely the consequence of a continual inflammatory response to infection, or requires both. Understanding the role of HBx in HBV replication and its effect on hepatocyte biology may help resolve this issue. This review describes key studies and activities of HBx, often interpreted within the context of the viral life cycle. The reader is referred to several comprehensive reviews on the reported biological properties of HBx (3, 78, 137). HBV
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Abstract Transforming growth factor-beta (TGF-beta) regulates many aspects of cellular function. A member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, TAK1, was previously identified as a mediator in the signaling pathway of TGF-beta superfamily members. The yeast two-hybrid system has now revealed two human proteins, termed TAB1 and TAB2 (for TAK1 binding protein), that interact with TAK1. TAB1 and TAK1 were co-immunoprecipitated from mammalian cells. Overproduction of TAB1 enhanced activity of the plasminogen activator inhibitor 1 gene promoter, which is regulated by TGF-beta, and increased the kinase activity of TAK1. TAB1 may function as an activator of the TAK1 MAPKKK in TGF-beta signal transduction.
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The authors have declared that no competing interests exist.
作者已声明无竞争性利益关系。
PDF(621 KB)
图1 Tab1的PCR产物图2 pcDNA3.1/myc-His(-)B-Tab1的酶切鉴定和菌落PCR鉴定图3 pcDNA3.1/myc-His(-)B-Tab1的表达鉴定图4 HBx的PCR产物图5 pGEX-2TK-GST-HBx的酶切鉴定和菌落PCR鉴定图6 GST-HBx融合蛋白的诱导表达图7 琼脂糖Beads纯化GST-HBx融合蛋白的蛋白质电泳检测图8 Tab1与HBx在293T细胞内的相互作用图9 Tab1与HBx在HepG2细胞内的相互作用图10 Tab1与HBx在体外的相互作用/
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