Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53

1993 ◽  
Vol 13 (2) ◽  
pp. 775-784 ◽  
Author(s):  
J M Huibregtse ◽  
M Scheffner ◽  
P M Howley
Keyword(s):  
High Risk ◽  
P53 Protein ◽  
Growth Control ◽  
Human Papillomaviruses ◽  
Cloning And Expression ◽  
Hpv16 E6 ◽  
Reading Frame ◽  
E6 Oncoprotein ◽  
Protein Complex Formation

The E6 oncoproteins of the cancer-associated or high-risk human papillomaviruses (HPVs) target the cellular p53 protein. The association of E6 with p53 leads to the specific ubiquitination and degradation of p53 in vitro, suggesting a model by which E6 deregulates cell growth control by the elimination of the p53 tumor suppressor protein. Complex formation between E6 and p53 requires an additional cellular factor, designated E6-AP (E6-associated protein), which has a native and subunit molecular mass of approximately 100 kDa. Here we report the purification of E6-AP and the cloning of its corresponding cDNA, which contains a novel open reading frame encoding 865 amino acids. E6-AP, translated in vitro, has the following properties: (i) it associates with wild-type p53 in the presence of the HPV16 E6 protein and simultaneously stimulates the association of E6 with p53, (ii) it associates with the high-risk HPV16 and HPV18 E6 proteins in the absence of p53, and (iii) it induces the E6- and ubiquitin-dependent degradation of p53 in vitro.

scholarly journals Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53.

1993 ◽  
Vol 13 (2) ◽  
pp. 775-784 ◽  
Author(s):  
J M Huibregtse ◽  
M Scheffner ◽  
P M Howley
Keyword(s):  
High Risk ◽  
P53 Protein ◽  
Growth Control ◽  
Human Papillomaviruses ◽  
Cloning And Expression ◽  
Hpv16 E6 ◽  
Reading Frame ◽  
E6 Oncoprotein ◽  
Protein Complex Formation

The E6 oncoproteins of the cancer-associated or high-risk human papillomaviruses (HPVs) target the cellular p53 protein. The association of E6 with p53 leads to the specific ubiquitination and degradation of p53 in vitro, suggesting a model by which E6 deregulates cell growth control by the elimination of the p53 tumor suppressor protein. Complex formation between E6 and p53 requires an additional cellular factor, designated E6-AP (E6-associated protein), which has a native and subunit molecular mass of approximately 100 kDa. Here we report the purification of E6-AP and the cloning of its corresponding cDNA, which contains a novel open reading frame encoding 865 amino acids. E6-AP, translated in vitro, has the following properties: (i) it associates with wild-type p53 in the presence of the HPV16 E6 protein and simultaneously stimulates the association of E6 with p53, (ii) it associates with the high-risk HPV16 and HPV18 E6 proteins in the absence of p53, and (iii) it induces the E6- and ubiquitin-dependent degradation of p53 in vitro.

scholarly journals The E6 Oncoproteins of High-Risk Papillomaviruses Bind to a Novel Putative GAP Protein, E6TP1, and Target It for Degradation

1999 ◽  
Vol 19 (1) ◽  
pp. 733-744 ◽  
Author(s):  
Qingshen Gao ◽  
Seetha Srinivasan ◽  
Sarah N. Boyer ◽  
David E. Wazer ◽  
Vimla Band
Keyword(s):  
High Risk ◽  
P53 Protein ◽  
Single Gene ◽  
Human Papillomaviruses ◽  
Dominant Negative ◽  
Hpv16 E6 ◽  
Hpv E6 ◽  
E6 Oncoprotein ◽  
E6 Protein

ABSTRACT The high-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors. Previous studies have identified two viral oncoproteins, E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high-risk HPV E6 protein to immortalize human mammary epithelial cells (MECs) has provided a single-gene model to study the mechanisms of E6-induced oncogenic transformation. In this system, the E6 protein targets the p53 tumor suppressor protein for degradation, and mutational analyses have shown that E6-induced degradation of p53 protein is required for MEC immortalization. However, the inability of most dominant-negative p53 mutants to induce efficient immortalization of MECs suggests the existence of additional targets of the HPV E6 oncoprotein. Using the yeast two-hybrid system, we have isolated a novel E6-binding protein. This polypeptide, designated E6TP1 (E6-targeted protein 1), exhibits high homology to GTPase-activating proteins for Rap, including SPA-1, tuberin, and Rap1GAP. The mRNA for E6TP1 is widely expressed in tissues and in vitro-cultured cell lines. The gene for E6TP1 localizes to chromosome 14q23.2-14q24.3 within a locus that has been shown to undergo loss of heterozygosity in malignant meningiomas. Importantly, E6TP1 is targeted for degradation by the high-risk but not the low-risk HPV E6 proteins both in vitro and in vivo. Furthermore, the immortalization-competent but not the immortalization-incompetent HPV16 E6 mutants target the E6TP1 protein for degradation. Our results identify a novel target for the E6 oncoprotein and provide a potential link between HPV E6 oncogenesis and alteration of a small G protein signaling pathway.

scholarly journals Human Papillomavirus Type 16 E6 induces cell competition

2021 ◽  
Author(s):  
Nicole Brimer ◽  
Scott Vande Pol
Keyword(s):  
Human Papillomavirus ◽  
High Risk ◽  
Epithelial Cells ◽  
Human Papillomaviruses ◽  
Cell Competition ◽  
Vitro Assay ◽  
Epithelial Tumors ◽  
E6 Oncoprotein ◽  
E6 And E7

High risk human papillomavirus (HPV) infections induce squamous epithelial tumors in which the virus replicates.  Initially, the virus-infected epithelial cells are untransformed, but expand in both number and area at the expense of normal squamous epithelial cells.  How this occurs is unknown, but is presumed to be due to viral oncogene expression.  We have developed an  in vitro  assay in which colonies of post-confluent HPV16 expressing cells outcompete confluent surrounding normal keratinocytes for surface area.   The enhanced cell competition induced by the complete HPV16 genome is conferred by E6 expression alone, and not by individual expression of E5 or E7.   In traditional oncogene assays, E7 is a more potent oncogene than E6, but such assays do not include interaction with normal surrounding cells.  These new results separate classic oncogenicity that is primarily conferred by E7, from cell competition that we show is primarily conferred by E6, and provides a new biological role for E6 oncoproteins from high risk human papillomaviruses.   Importance High risk papillomavirus infections induce epithelial tumors, some of which evolve into malignancies.   The development and maintenance of cancer is due to the virally encoded E6 and E7 oncoproteins.  How a virally infected keratinocyte out-competes normal uninfected keratinocytes has been unknown.  The present work shows that the enhanced competition of HPV16-infected cells is primarily due to the expression of the E6 oncoprotein and not the E7 or E5 oncoproteins.   This work shows the importance of measuring oncoprotein traits in the context of cell competition with uninfected cells, and shows the potential of papillomavirus oncoproteins to be novel genetic probes for the analysis of cell competition

scholarly journals High Risk α-HPV E6 Impairs Translesion Synthesis by Blocking POLη Induction

Cancers ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 28
Author(s):  
Sebastian O. Wendel ◽  
Jazmine A. Snow ◽  
Tyler Bastian ◽  
Laura Brown ◽  
Candy Hernandez ◽  
...  
Keyword(s):  
Dna Damage ◽  
High Risk ◽  
Replication Fork ◽  
Ex Vivo ◽  
Translesion Synthesis ◽  
Replication Stress ◽  
Human Papillomaviruses ◽  
Hpv16 E6 ◽  
Hpv E6

High risk genus α human papillomaviruses (α-HPVs) express two versatile oncogenes (α-HPV E6 and E7) that cause cervical cancer (CaCx) by degrading tumor suppressor proteins (p53 and RB). α-HPV E7 also promotes replication stress and alters DNA damage responses (DDR). The translesion synthesis pathway (TLS) mitigates DNA damage by preventing replication stress from causing replication fork collapse. Computational analysis of gene expression in CaCx transcriptomic datasets identified a frequent increased expression of TLS genes. However, the essential TLS polymerases did not follow this pattern. These data were confirmed with in vitro and ex vivo systems. Further interrogation of TLS, using POLη as a representative TLS polymerase, demonstrated that α-HPV16 E6 blocks TLS polymerase induction by degrading p53. This doomed the pathway, leading to increased replication fork collapse and sensitivity to treatments that cause replication stress (e.g., UV and Cisplatin). This sensitivity could be overcome by the addition of exogenous POLη.

scholarly journals Cloning and expression of human liver dehydroepiandrosterone sulphotransferase

1993 ◽  
Vol 289 (1) ◽  
pp. 233-240 ◽  
Author(s):  
K A Comer ◽  
J L Falany ◽  
C N Falany
Keyword(s):  
Molecular Mass ◽  
Bile Acids ◽  
Blot Analysis ◽  
Human Liver ◽  
Molecular Size ◽  
Cloning And Expression ◽  
Reading Frame ◽  
Acid Protein ◽  
Liver Cdna Library

Dehydroepiandrosterone sulphotransferase (DHEA-ST) catalyses the 3′-phosphoadenosine 5′-phosphosulphate-dependent sulphation of a wide variety of steroids in human liver and adrenal tissue and is responsible for most, if not all, of the sulphation of bile acids in human liver. This report describes the isolation, characterization and expression of a cDNA which encodes human liver DHEA-ST. The DHEA-ST cDNA, designated DHEA-ST8, was isolated from a Uni-Zap XR human liver cDNA library and is composed of 1060 bp and contains an open reading frame encoding a 285-amino-acid protein with a molecular mass of approx. 33765 Da. Translation of DHEA-ST8 in vitro generated a protein identical in molecular size with that of DHEA-ST. Expression of DHEA-ST8 in COS-7 cells produces an active DHEA-ST protein which is capable of sulphating DHEA, has the same molecular mass as human liver DHEA-ST and is recognized by rabbit anti-(human liver DHEA-ST) antibodies. Northern-blot analysis of human liver RNA detects the presence of three different size transcripts; however, Southern-blot analysis of human DNA suggests that only one gene may be present in the genome. These results describe the cloning of a human ST which has an important role in the sulphation of steroids and bile acids in human liver and adrenals.

scholarly journals Examination of the pRb-Dependent and pRb-Independent Functions of E7 In Vivo

2005 ◽  
Vol 79 (17) ◽  
pp. 11392-11402 ◽  
Author(s):  
Scott Balsitis ◽  
Fred Dick ◽  
Denis Lee ◽  
Linda Farrell ◽  
R. Katherine Hyde ◽  
...  
Keyword(s):  
Cell Cycle ◽  
High Risk ◽  
Genetically Engineered ◽  
Human Papillomaviruses ◽  
Epidermal Differentiation ◽  
Mutant Form ◽  
Human Papillomavirus 16 ◽  
Independent Functions

ABSTRACT High-risk human papillomaviruses encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. Although E7 protein is best known for its ability to inactivate the retinoblastoma tumor suppressor protein, pRb, many other activities for E7 have been proposed in in vitro studies. Herein, we describe studies that allowed us to define unambiguously the pRb-dependent and -independent activities of E7 for the first time in vivo. In these studies, we crossed mice transgenic for human papillomavirus 16 E7 to knock-in mice genetically engineered to express a mutant form of pRb (pRbΔLXCXE) that is selectively defective for binding E7. pRb inactivation was necessary for E7 to induce DNA synthesis and to overcome differentiation-dependent cell cycle withdrawal and DNA damage-induced cell cycle arrest. While most of E7's effects on epidermal differentiation were found to require pRb inactivation, a modest delay in terminal differentiation with resulting hyperplasia was observed in E7 mice on the Rb ΔLXCXE mutant background. E7-induced p21 upregulation was also pRb dependent, and genetic Rb inactivation was sufficient to reproduce this effect. While E7-mediated p21 induction was partially p53 dependent, neither p53 nor p21 induction by E7 required p19ARF. These data show that E7 upregulates the expression of p53 and p21 via pRb-dependent mechanisms distinct from the proposed p19-Mdm2 pathway. These results extend our appreciation of the importance of pRb as a relevant target for high-risk E7 oncoproteins.

scholarly journals Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidylethanolamine

1999 ◽  
Vol 339 (2) ◽  
pp. 291-298 ◽  
Author(s):  
Annette L. HENNEBERRY ◽  
Christopher R. McMASTER
Keyword(s):  
De Novo ◽  
Active Form ◽  
Fatty Acyl ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Kennedy Pathway ◽  
Membrane Spanning

Cholinephosphotransferase catalyses the final step in the synthesis of phosphatidylcholine (PtdCho) via the Kennedy pathway by the transfer of phosphocholine from CDP-choline to diacylglycerol. Ethanolaminephosphotransferase catalyses an analogous reaction with CDP-ethanolamine as the phosphobase donor for the synthesis of phosphatidylethanolamine (PtdEtn). Together these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PtdCho and PtdEtn synthesized de novo. A human choline/ethanolaminephosphotransferase cDNA (hCEPT1) was cloned, expressed and characterized. Northern blot analysis revealed one hCEPT1 2.3 kb transcript that was ubiquitous and not enriched, with respect to actin, in any particular cell type. The open reading frame predicts a protein (hCEPT1p) of 416 amino acid residues with a molecular mass of 46550 Da containing seven membrane-spanning domains. A predicted amphipathic helix resides within the active site of the enzyme with the final two aspartic residues of the CDP-alcohol phosphotransferase motif, DG(X)2AR(X)8G(X)3D(X)3D, positioned within this helix. hCEPT1p was successfully expressed in a full-length, active form in Saccharomyces cerevisiae cells devoid of endogenous cholinephosphotransferase or ethanolaminephosphotransferase activities (HJ091, cpt1::LEU2 ept1-). In vitro, hCEPT1p displayed broad substrate specificity, utilizing both CDP-choline and CDP-ethanolamine as phosphobase donors to a broad range of diacylglycerols, resulting in the synthesis of both PtdCho and PtdEtn. In vivo, S. cerevisiae cells (HJ091, cpt1::LEU2 ept1-) expressing hCEPT1 efficiently incorporated both radiolabelled choline and ethanolamine into phospholipids, demonstrating that hCEPT1p has the ability to synthesize both choline- and ethanolamine- containing phospholipids in vitro and in vivo.

scholarly journals Isolation and characterization of the first American bottlenose dolphin papillomavirus: Tursiops truncatus papillomavirus type 2

2006 ◽  
Vol 87 (12) ◽  
pp. 3559-3565 ◽  
Author(s):  
Manuela Rehtanz ◽  
Shin-je Ghim ◽  
Annabel Rector ◽  
Marc Van Ranst ◽  
Patricia A. Fair ◽  
...  
Keyword(s):  
High Risk ◽  
Bottlenose Dolphin ◽  
Tursiops Truncatus ◽  
Phylogenetic Analyses ◽  
Human Papillomaviruses ◽  
Binding Motif ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
Free Ranging

A novel papillomavirus (PV) was isolated from a genital condyloma of a free-ranging bottlenose dolphin inhabiting the coastal waters of Charleston Harbor, SC, USA: Tursiops truncatus papillomavirus type 2 (TtPV2). This novel virus represents the first isolated North American cetacean PV and the first American bottlenose dolphin PV. After the viral genome was cloned, sequenced and characterized genetically, phylogenetic analyses revealed that TtPV2 is most similar to the only published cetacean PV isolated and characterized thus far, Phocoena spinipinnis PV type 1 (PsPV1). A striking feature of the genome of TtPV2, as well as that of PsPV1, is the lack of an E7 open reading frame, which typically encodes one of the oncogenic proteins believed to be responsible for malignant transformation in the high-risk mucosotropic human papillomaviruses (HPVs). TtPV2 E6 contains a PDZ-binding motif that has been shown to be involved in transformation in the case of high-risk genital HPVs.

scholarly journals Human Papillomavirus Oncoprotein E6 Inactivates the Transcriptional Coactivator Human ADA3

2002 ◽  
Vol 22 (16) ◽  
pp. 5801-5812 ◽  
Author(s):  
Ajay Kumar ◽  
Yongtong Zhao ◽  
Gaoyuan Meng ◽  
Musheng Zeng ◽  
Seetha Srinivasan ◽  
...  
Keyword(s):  
High Risk ◽  
P53 Protein ◽  
Human Papillomaviruses ◽  
Transcriptional Coactivator ◽  
Interacting Protein ◽  
Hpv E6 ◽  
Cycle Arrest ◽  
Evolutionarily Conserved ◽  
Novel Strategy ◽  
High Risk Hpv

ABSTRACT High-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors. The HPV oncoprotein E6 is essential for oncogenic transformation. We identify here hADA3, human homologue of the yeast transcriptional coactivator yADA3, as a novel E6-interacting protein and a target of E6-induced degradation. hADA3 binds selectively to the high-risk HPV E6 proteins and only to immortalization-competent E6 mutants. hADA3 functions as a coactivator for p53-mediated transactivation by stabilizing p53 protein. Notably, three immortalizing E6 mutants that do not induce direct p53 degradation but do interact with hADA3 induced the abrogation of p53-mediated transactivation and G1 cell cycle arrest after DNA damage, comparable to wild-type E6. These findings reveal a novel strategy of HPV E6-induced loss of p53 function that is independent of direct p53 degradation. Given the likely role of the evolutionarily conserved hADA3 in multiple coactivator complexes, inactivation of its function may allow E6 to perturb numerous cellular pathways during HPV oncogenesis.

Cloning and expression ofSpodoptera lituranucleopolyhedrovirusgp41gene inEscherichia coliand preparation of antibody

2005 ◽  
Vol 2 (2) ◽  
pp. 113-117
Author(s):  
Pan Li-Jing ◽  
Li Zhao-Fei ◽  
Yin Chong ◽  
Lv Lei ◽  
Pang Yi
Keyword(s):  
Fusion Protein ◽  
Prokaryotic Expression ◽  
Recombinant Plasmid ◽  
Nitrilotriacetic Acid ◽  
Cloning And Expression ◽  
Reading Frame ◽  
Pcr Product ◽  
Prokaryotic Expression Vector ◽  
Polymerase Chain

AbstractGP41, a major glycoprotein, identified in the occlusion-derived virions (ODV) of baculoviruses, is required for the egress of nucleocapsids from the nucleus in the pathway of budded virion (BV) synthesis. Using the polymerase chain reaction (PCR), the open reading frame (ORF) ofSpodoptera lituranucleopolyhedrovirus (SpltMNPV)gp41gene was obtained from SpltMNPV genomic DNA. The PCR product was cloned into pMD18-T vector to get the recombinant plasmid (pT-gp41). Thegp41gene was recombinedin vitrowith prokaryotic expression vector pQE30 and transformed intoEscherichia coliM15 [pREP4]. The M15 [pREP4] strain, containinggp41recombinant plasmid, expressed a 37.9 kDa 6×His-tag fusion protein after induction with 1 mmol/l isopropylthio-β-d-galactoside (IPTG). The fusion protein was purified with a nickel-nitrilotriacetic acid (Ni–NTA) resin column and used as the immunogen to raise GP41-specific antibody. Western blotting analysis indicated that the antibody was suitable to be used for further analysis of GP41 protein.

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