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T Dobner

Publications and source records attributed to T Dobner.

At least 19 recordsLinked to original sources
PubMed2004

Cell transformation by human adenoviruses.

The last 40 years of molecular biological investigations into human adenoviruses have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of their productive infection cycle in permissive host cells. Also, initial observations concerning the carcinogenic potential of human adenoviruses subsequently revealed decisive insights into the molecular mechanisms of the origins of cancer, and established adenoviruses as a model system for explaining virus-mediated transformation processes. Today it is well established that cell transformation by human adenoviruses is a multistep process involving several gene products encoded in early transcription units 1A (E1A) and 1B (E1B). Moreover, a large body of evidence now indicates that alternative or additional mechanisms are engaged in adenovirus-mediated oncogenic transformation involving gene products encoded in early region 4 (E4) as well as epigenetic changes resulting from viral DNA integration. In particular, detailed studies on the tumorigenic potential of subgroup D adenovirus type 9 (Ad9) E4 have now revealed a new pathway that points to a novel, general mechanism of virus-mediated oncogenesis. In this chapter, we summarize the current state of knowledge about the oncogenes and oncogene products of human adenoviruses, focusing particularly on recent findings concerning the transforming and oncogenic properties of viral proteins encoded in the E1B and E4 transcription units.

Adenovirus E1 Proteins↗
PubMed2004

Transcription factor AP-2alpha triggers apoptosis in cardiac myocytes.

Idiopathic-dilated cardiomyopathy (IDC) is a common primary myocardial disease of unknown etiology associated with apoptosis, cardiac dilatation, progressive heart failure and increased mortality. An elevation of the transcription factor activator protein 2alpha (AP-2alpha) is involved in vertebrate embryonic development and oncogenesis. Here, we show that AP-2alpha protein is expressed in the human heart and increased in human failing myocardium with IDC. Adenovirus-mediated overexpression of human AP-2alpha triggered apoptosis and increased mRNA levels of Bcl-2 family members Bax and Bcl-x in rat cardiomyocytes. Immunohistological analysis of human myocardium revealed an increased percentage of AP-2alpha-positive nuclei in IDC and, interestingly, a colocalization of AP-2alpha-positive but not -negative cells with a caspase-cleaved fragment of poly(ADP-ribose)polymerase. We suggest AP-2alpha as a novel cardiac regulator implicated in the activation of apoptosis in IDC.

Adenoviridae↗
PubMed2002

[A new human cellular protein AUP1. I. In vitro interaction of AUP1 with adenoviral proteins E4ORF3 and E1A].

The 11-kDa product of adenovirus early region 4 (E4) open reading frame (ORF) 3 participates in many processes occurring in infected cell, including post-transcriptional steps in late viral gene expression and viral DNA synthesis. In addition, E4ORF3 from adenovirus type 5 (Ad5) displays the features of a viral oncoprotein. It initiates focal transformation of primary rat cells in cooperation with Ad5 El genes and confers multiple additional transformed properties on E1-expressing cells. Biochemical details of E4ORF3 activities in these processes are not well understood. A large body of evidence indicates that its lytic and transforming functions are mediated by physical interactions with viral and cellular components involved in DNA transcription and repair, as well as by host cell factors that regulate the integrity of nuclear multiprotein complexes known as PML oncogenic domains (PODs). In this study we have employed the two-hybrid screen in yeast to isolate human cDNAs encoding for E4ORF3-interacting proteins. Among 15 positive clones five cDNAs encode for a cellular protein called AUP1. In vitro-binding assays demonstrated that AUP1 fused to glutathione S-transferase (GST) specifically binds to E4ORF3 from Ad5, Ad9 and Ad40 generated in a coupled transcription-translation system, whereas no interactions was observed with ORF3 from Ad12. Interestingly, GST-AUP1 interacted also specifically with in vitro translated Ad5 E1A proteins. Regions involved in the Ad5 E4ORF3/AUP1 interaction in vitro map to the central part of E4 protein and the carboxy-terminal region of AUP1, while E1A binds to an amino-terminal segment of the cell protein. Taken together, these studies indicate that AUP1 may represent a cellular target of both adenovirus E4ORF3 and E1A proteins. Additional studies are currently under way to confirm the significance of these interactions in living cells in vivo.

Adenovirus E1A Proteins↗
PubMed2002

[A new human cellular protein AUP1. II. cDNA cloning, genomic organization of Aup1 gene ans preliminary characterization of human AUP1 protein].

We report here the cloning of human cDNA of Aup1 gene (ancient uniquitous protein 1), its genomic organization and preliminary characterization of human AUP1 protein. Genomic length of the human Aup1 gene is composed of 12 exons and 11 introns spanning 3047 bp. A single open reading frame of 1230 bp starts with the first ATG from nucleotide 5630 of the genomic sequence, in accordance with the Kazak rule, and stops with TGA on nucleotide 8500 of the genomic sequence AC005041.2. The 3 noncoding region has a 152 bp length followed by poly(A)tail. All exon-intron junctions conform to 5' donor and 3' acceptor consensus. Exon and intron sizes range from 69 to 187 bp, and from 180 to 262 bp, resp. We have cloned a full-length human Aup1 cDNA. For preliminary characterization of human AUP1 protein, we cloned the full-length human Aup1 cDNA in pcDNA3 vector. In vitro transcription/translation of the cloned cDNA yielded a 45 kDa protein. Human Aup1 coding region demonstrates 90% homology to its rat and mouse homologous at the nucleotide level, and 82% at the amine acid level.

Amino Acid Sequence↗
PubMed2002

[A new human cellular protein AUP1. III. The intracellular localization of AUP1 protein in different human and rat cell lines].

Previously, we cloned a full-length cDNA of human Aup1 and showed that AUP1 may represent a new cellular target for the two adenovirus oncoproteins, E1A Ad5 and E4ORF3. In this study, we generated a polyclonal anti-AUP1 antibody and examined the subcellular localization of AUP1 in MCF7 cells, HeLa cells, H1299 cells, 293 cells, BRK1 cells and transfectants expressing adenoviruse E1 genes. Double staining of AUP1 and various markers for cytoplasmic structures showed that the pattern of AUP1 distribution in the cytoplasm was puctuate and diffuse and without any colocalization with Golgi apparatus or endoplasmic reticulum. Additional studies with ectopically expressed AUP1, fused with red fluorescent protein (RFP) in H1299 and McG7 human cell lines and BRK1 rat cell line, showed cytoplasmic localization of RFP-AUP1. Western blot analysis revealed that AUP1 was expressed at similar levels in all tested cell lines and had the same molecular weight as the rat protein (45 kDa). Taken together, these results suggest that AUP1 is a cytoplasmic protein that is expressed in all cell lines we examined.

Adenovirus E1 Proteins↗
PubMed2001

Adenovirus early E4 genes in viral oncogenesis.

Previous investigations into potential transforming activities of adenovirus (Ad) early genes were largely overshadowed by the more obvious roles of E1A and E1B products. One exception was an Ad9 E4 protein (ORF1) shown to enhance transformation of cultured cells and promote mammary tumors in female rats. Recently, significant advances in understanding Ad E4 gene products at the molecular level have revealed that these proteins possess an unexpectedly diverse collection of functions, which not only orchestrate many viral processes, but overlap with oncogenic transformation of primary mammalian cells. Operating through a complex network of protein interactions with key viral and cellular regulatory components, Ad E4 products are apparently involved in transcription, apoptosis, cell cycle control, DNA repair, cell signaling, posttranslational modifications and the integrity of nuclear multiprotein complexes known as PML oncogenic domains (PODs). Some of these functions directly relate to known transforming and oncogenic processes, or implicate mechanisms such as modulating the function and subcellular localization of cellular PDZ domain-containing proteins, POD reorganization, targeted proteolytic degradation, inhibition of DNA double-strand break repair and 'hit-and-run' mutagenesis. Here, we summarize the recent data and discuss how E4 gene product interactions may contribute to viral oncogenesis.

Adenoviridae↗
PubMed2001

Molecular regulation and biological function of adenovirus early genes: the E4 ORFs.

Over the past few years there have been a number of interesting advances in our understanding of the functions encoded by the adenovirus early transcription unit 4 (Ad E4). A large body of recent data demonstrates that E4 proteins encompass an unexpectedly diverse collection of functions required for efficient viral replication. E4 gene products operate through a complex network of protein interactions with key viral and cellular regulatory components involved in transcription, apoptosis, cell cycle control and DNA repair, as well as host cell factors that regulate cell signaling, posttranslational modifications and the integrity of nuclear multiprotein complexes known as nuclear bodies (NBs) or PML oncogenic domains (PODs). As understood at present, some of the lytic functions overlap with roles in oncogenic transformation of primary mammalian cells. These observations, together with findings that E4 proteins substantially affect cell toxicity and the immune response of the host have profound implications for the development of Ad vectors for gene therapy. In this article we will summarize recent findings regarding the diverse functions of E4 gene products in the context of earlier work. We will emphasize the interaction of E4 proteins with cellular and viral interaction partners, the role of these interactions for lytic virus growth and how these interactions may contribute to viral oncogenesis. Finally, we will discuss their role in Ad vector and adeno-associated virus infections.

Adenovirus E4 Proteins↗
PubMed2001

SUMO-1 modification required for transformation by adenovirus type 5 early region 1B 55-kDa oncoprotein.

SUMO-1 is a small ubiquitin-related modifier protein that is covalently linked to many cellular and viral protein targets. Modification by SUMO-1 is proposed to play a role in protein targeting and/or stability. We show here that adenovirus type 5 early region 1B 55-kDa (E1B-55kDa) oncoprotein can be covalently modified by SUMO-1 in vivo through a major attachment site comprising a single lysine residue at amino acid position 104. The sequence surrounding this lysine matches the proposed PsiKxE consensus motif required for SUMO-1 conjugation. A single mutation (K104R) that abolishes SUMOylation of E1B-55kDa dramatically reduces the ability of the adenovirus type 5 protein to transform primary baby rat kidney cells in cooperation with E1A and to inhibit p53-mediated transactivation. Overexpression of SUMO-1 in adenovirus type 5 E1A/E1B-55kDa-transformed baby rat kidney cells causes the relocalization of E1B-55kDa from the cytoplasm to the nucleus, where it accumulates with SUMO-1 in dot- or track-like structures. Significantly, when SUMO-1 is ectopically expressed in transformed rat cells no effect on the cytoplasmic localization of the E1B-K104R mutant protein is observed. Our results demonstrate that SUMO-1 modification is required for transformation by adenovirus type 5 E1B-55kDa and provide further evidence for the idea that this posttranslational modification plays a role in protein targeting to specific subcellular sites.

Adenoviridae↗
PubMed2001

Heterogeneous nuclear ribonucleoprotein E1B-AP5 is methylated in its Arg-Gly-Gly (RGG) box and interacts with human arginine methyltransferase HRMT1L1.

The heterogeneous nuclear ribonucleoprotein (hnRNP) family includes predominantly nuclear proteins acting at different stages of mRNA metabolism. A characteristic feature of hnRNPs is to undergo post-translational asymmetric arginine methylation catalysed by different type 1 protein arginine methyltransferases (PRMTs). A novel mammalian hnRNP, E1B-AP5, recently identified by its interaction with adenovirus early protein E1B-55 kDa, has been proposed to have a regulatory role in adenoviral and host-cell mRNA processing/nuclear export [Gabler, Schutt, Groitl, Wolf, Shenk and Dobner (1998) J. Virol. 72, 7960-7971]. Here we report that E1B-AP5 is methylated in vivo in its Arg-Gly-Gly (RGG)-box domain, known to mediate protein-RNA interactions. The activity responsible for E1B-AP5 methylation forms a complex with E1B-AP5 in vivo. The predominant mammalian arginine methyltransferase HRMT1L2 (hPRMT1) did not detectably methylate endogenous E1B-AP5 despite efficiently methylating a recombinant RGG-box domain of E1B-AP5. Using yeast two-hybrid screening we identified HRMT1L1 (PRMT2) as one of the proteins interacting with E1B-AP5. By in situ immunofluorescence we demonstrated that E1B-AP5 co-localizes with the nuclear fraction of HRMT1L1. The Src homology 3 (SH3) domain of HRMT1L1 was essential for its interaction with E1B-AP5 in vivo. We suggest that HRMT1L1 is responsible for specific E1B-AP5 methylation in vivo.

Amino Acid Motifs↗
PubMed2001

Induction of AP-2alpha expression by adenoviral infection involves inactivation of the AP-2rep transcriptional corepressor CtBP1.

AP-2 transcription factors execute important functions during embryonic development and malignant transformation. Recently, we have isolated a transcriptional repressor of AP-2alpha expression, the novel Krüppel-related zinc finger protein AP-2rep (Klf12). Here, we show that repression of AP-2alpha transcription by AP-2rep is dependent on an N-terminal PVDLS motif that interacts specifically with the corepressor CtBP1 both in vivo and in vitro. This interaction motif was previously identified in the C-terminal region of the adenoviral oncoprotein E1A. Infection of both HeLa and PA-1 cells with adenovirus type 5 strongly induced AP-2alpha mRNA. Consistently, E1A was necessary and sufficient to mediate up-regulation of AP-2alpha. Transiently transfected wild-type E1A protein activated an AP-2rep sensitive cis-regulatory element of the AP-2alpha promoter, but E1A protein harboring a mutation in the PVDLS motif failed to activate. In summary, we conclude that the adenoviral oncoprotein E1A activates transcription from the endogenous AP-2alpha gene, an effect that involves transcriptional derepression of the AP-2alpha promoter by interaction of E1A with the AP-2rep corepressor CtBP1.

Adenoviridae↗
PubMed2001

The adenovirus type 5 E1B-55K oncoprotein actively shuttles in virus-infected cells, whereas transport of E4orf6 is mediated by a CRM1-independent mechanism.

The E1B-55K and E4orf6 proteins of adenovirus type 5 are involved in viral mRNA export. Here we demonstrate that adenovirus infection does not inhibit the function of the E1B-55K nuclear export signal and that E1B-55K also shuttles in infected cells. Even during virus infection, E1B-55K was exported by the leptomycin B-sensitive CRM1 pathway, whereas E4orf6 transport appeared to be mediated by an alternative mechanism. Our results strengthen the potential role of E1B-55K as the "driving force" for adenoviral late mRNA export.

Adenovirus E1B Proteins↗
PubMed2001

"Hit-and-run" transformation by adenovirus oncogenes.

According to classical concepts of viral oncogenesis, the persistence of virus-specific oncogenes is required to maintain the transformed cellular phenotype. In contrast, the "hit-and-run" hypothesis claims that viruses can mediate cellular transformation through an initial "hit," while maintenance of the transformed state is compatible with the loss ("run") of viral molecules. It is well established that the adenovirus E1A and E1B gene products can cooperatively transform primary human and rodent cells to a tumorigenic phenotype and that these cells permanently express the viral oncogenes. Additionally, recent studies have shown that the adenovirus E4 region encodes two novel oncoproteins, the products of E4orf6 and E4orf3, which cooperate with the viral E1A proteins to transform primary rat cells in an E1B-like fashion. Unexpectedly, however, cells transformed by E1A and either E4orf6 or E4orf3 fail to express the viral E4 gene products, and only a subset contain E1A proteins. In fact, the majority of these cells lack E4- and E1A-specific DNA sequences, indicating that transformation occurred through a hit-and-run mechanism. We provide evidence that the unusual transforming activities of the adenoviral oncoproteins may be due to their mutagenic potential. Our results strongly support the possibility that even tumors that lack any detectable virus-specific molecules can be of viral origin, which could have a significant impact on the use of adenoviral vectors for gene therapy.

Adenoviridae↗
PubMed2001

p300 acts as a transcriptional coactivator for mammalian Notch-1.

Notch-1 belongs to a family of transmembrane receptor proteins that direct the decisions as to various cell fates. After ligand binding, a proteolytic cleavage step occurs and the intracellular part of Notch-1, Notch-1-IC, translocates into the nucleus, where it targets the DNA binding protein RBP-J kappa/CBF1. RBP-J kappa mediates repression through recruitment of a histone deacetylase-containing complex. The Notch-1-IC/RBP-J kappa complex overcomes repression and activates the transcription of Notch target genes. We have identified a novel domain in Notch-1-IC, the EP domain, which is indispensable for full transcriptional activation. This transactivation domain is localized adjacent to the ankyrin repeats of Notch-1-IC. In cotransfection experiments, Notch-1-IC-mediated transcriptional activation was inhibited by E1A12S and p53, two proteins, which interfere with the function of the common coactivator p300. Protein-protein interaction assays demonstrated the association of Notch-1-IC and the CH3 region of p300. In addition, the interaction of mammalian Notch-1-IC with p300 was destabilized after deletion of the EP domain of Notch-1-IC. Based on physical interaction with Notch-1-IC and coactivator functions of p300, we propose a model for Notch-1-mediated gene regulation via p300.

Adenovirus E1A Proteins↗
PubMed2001

Specific nucleoprotein complexes within adenovirus capsids.

Adenoviral DNA was examined within capsids by dimethyl sulfate footprinting. Protein-DNA interactions were visualized through ligation-mediated PCR (LM-PCR). Signals for protein binding were found adjacent to both inverted terminal repeats (ITR). There were no indications of close protein binding at several other loci of the viral genome. Therefore, adenovirus type 5 seems to contain sequence- or locus-specific DNA binding proteins within the virion.

Adenoviridae↗
PubMed2000

An ovine adenovirus vector lacks transforming ability in cells that are transformed by AD5 E1A/B sequences.

Adenoviruses of the Mastadenovirus and Aviadenovirus genera are able to transform certain cell types and induce tumor formation in susceptible animals. For the mastadenoviruses the E1A/B sequences are largely responsible for these properties but E4 sequences may also be involved. The transforming sequences of the aviadenoviruses, which lack E1A/B and E4 homologues, have not yet been fully identified. The recent proposal for a third genus of adenoviruses, which apparently lack an E1A homologue and have weak E1B homology, prompted an examination of the transforming properties of ovine adenovirus OAV287 (OAV), the prototype member of the new group. When OAV and human adenovirus type 5 (Ad5) were used to infect primary rat embryo cells, transformed foci developed in Ad5- but not in OAV-infected cultures. Similarly, after plasmid transfection, baby rat kidney cells were transformed by Ad5 E1A/B but not by OAV sequences. When CSL503 cells, an ovine cell line that is permissive for OAV, were transfected with Ad5 E1A/B sequences, transformed foci again appeared. However, plasmids or fragments containing complete or partial OAV genome sequences did not detectably transform CSL503 cells under the same conditions. When Ad5 E1A/B sequences were incorporated into the complete OAV genome and transfected, transformed clones were again obtained, showing that the gene dosage and transfection conditions were not limiting for transformation. The provision of Ad5 E1A and OAV sequences in combination marginally increased the number of morphologically altered foci in baby rat kidney cells but failed to induce multilayered focus formation. The data suggest that OAV lacks transforming functions in the cell types examined. Additional information suggesting that OAV may have a fundamentally distinct strategy for replication compared with other Ads is discussed.

Adenoviridae↗
PubMed2000

The adenovirus type 5 E1B-55K oncoprotein is a highly active shuttle protein and shuttling is independent of E4orf6, p53 and Mdm2.

The E1B-55K and E4orf6 oncoproteins of adenovirus type 5 are involved in the export of viral mRNAs. Previously, it was suggested that a complex composed of E1B-55K and E4orf6 serves as a nucleocytoplasmic transporter for viral mRNAs in which the E4orf6 protein directs both nuclear import and export. We now demonstrate that the E1B-55K protein itself shuttles efficiently in the absence of E4orf6. In addition, E1B-55K trafficking was independent of the defined shuttle proteins Mdm2 or p53, which interacts with E1B-55K. The identified N-terminal E1B-55K leucine-rich nuclear-export signal (NES) conferred rapid nuclear export even in a heterologous system in contrast to the postulated E4orf6NES. Interestingly, although shuttling was blocked by inhibitors of the CRM1 mediated export pathway, E1B-55K inhibited neither the activity nor the trafficking of the retroviral shuttle proteins HIV-1 Rev and HTLV-1 Rex. In contrast, Rev or Rex blocked the nuclear export of E1B-55K, most likely by competing for essential export factors. Our results provide new insights into the regulation of the adenovirus mRNA export system and the processes of adenovirus mediated transformation. Oncogene (2000) 19, 850 - 857.

3T3 Cells↗
PubMed2000

Two distinct activities contribute to the oncogenic potential of the adenovirus type 5 E4orf6 protein.

Previous studies have shown that the adenovirus type 5 (Ad5) E4orf6 gene product displays features of a viral oncoprotein. It initiates focal transformation of primary rat cells in cooperation with Ad5 E1 genes and confers multiple additional transformed properties on E1-expressing cells, including profound morphological alterations and dramatically accelerated tumor growth in nude mice. It has been reported that E4orf6 binds to p53 and, in the presence of the Ad5 E1B-55kDa protein, antagonizes p53 stability by targeting the tumor suppressor protein for active degradation. In the present study, we performed a comprehensive mutant analysis to assign transforming functions of E4orf6 to distinct regions within the viral polypeptide and to analyze a possible correlation between E4orf6-dependent p53 degradation and oncogenesis. Our results show that p53 destabilization maps to multiple regions within both amino- and carboxy-terminal parts of the viral protein and widely cosegregates with E4orf6-dependent acceleration of tumor growth, indicating that both effects are related. In contrast, promotion of focus formation and morphological transformation require only a carboxy-terminal segment of the E4 protein. Thus, these effects are completely independent of p53 stability, but may involve other interactions with the tumor suppressor. Our results demonstrate that at least two distinct activities contribute to the oncogenic potential of Ad5 E4orf6. Although genetically separable, both activities are largely mediated through a novel highly conserved, cysteine-rich motif and a recently described arginine-faced amphipathic alpha helix, which resides within a carboxy-terminal "oncodomain" of the viral protein.

Adenovirus E1A Proteins↗