Serum-free transient protein production system based on adenoviral vector and PER.C6 technology: high yield and preserved bioactivity.

Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an DeltaE1/DeltaE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed DeltaE1/DeltaE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The DeltaE1/DeltaE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of DeltaE1/DeltaE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that DeltaE1/DeltaE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.

CDC25A phosphatase is a target of E2F and is required for efficient E2F-induced S phase.

Functional inactivation of the pRB pathway is a very frequent event in human cancer, resulting in deregulated activity of the E2F transcription factors. To understand the functional role of the E2Fs in cell proliferation, we have developed cell lines expressing E2F-1, E2F-2, and E2F-3 fused to the estrogen receptor ligand binding domain (ER). In this study, we demonstrated that activation of all three E2Fs could relieve the mitogen requirement for entry into S phase in Rat1 fibroblasts and that E2F activity leads to a shortening of the G(0)-G(1) phase of the cell cycle by 6 to 7 h. In contrast to the current assumption that E2F-1 is the only E2F capable of inducing apoptosis, we showed that deregulated E2F-2 and E2F-3 activities also result in apoptosis. Using the ERE2F-expressing cell lines, we demonstrated that several genes containing E2F DNA binding sites are efficiently induced by the E2Fs in the absence of protein synthesis. Furthermore, CDC25A is defined as a novel E2F target whose expression can be directly regulated by E2F-1. Data showing that CDC25A is an essential target for E2F-1, since its activity is required for efficient induction of S phase by E2F-1, are provided. Finally, our results show that expression of two E2F target genes, namely CDC25A and cyclin E, is sufficient to induce entry into S phase in quiescent fibroblasts. Taken together, our results provide an important step in defining how E2F activity leads to deregulated proliferation.

Cell cycle-regulated expression of mammalian CDC6 is dependent on E2F.

The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis. The CDC6 gene is a target for MBF and SBF-regulated transcription. S. cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication. Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S. pombe homolog of MBF. By cloning the promoter for the human homolog of Cdc6p and Cdc18p, we demonstrate here that the cell cycle-regulated transcription of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase. Our data also demonstrate that the human CDC6 protein (hCDC6) is essential and limiting for DNA synthesis, since microinjection of an anti-CDC6 rabbit antiserum blocks DNA synthesis and CDC6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments. Furthermore, E2F is sufficient to induce expression of the endogenous CDC6 gene even in the absence of de novo protein synthesis. In conclusion, our results provide a direct link between regulated progression through G1 controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication.

Cloning, expression and chromosomal localization of a new putative receptor-like protein tyrosine phosphatase.

We have isolated a mouse cDNA of 5.7 kb, encoding a new member of the family of receptor-like protein tyrosine phosphatases, termed mRPTP mu. The cDNA predicts a protein of 1432 amino acids (not including signal peptide) with a calculated Mr of 161,636. In addition, we have cloned the human homologue, hRPTP mu, which shows 98.7% amino acid identity to mRPTP mu. The predicted mRPTP mu protein consists of a 722 amino acid extracellular region, containing 13 potential N-glycosylation sites, a single transmembrane domain and a 688 amino acid intracellular part containing 2 tandem repeats homologous to the catalytic domains of other tyrosine phosphatases. The N-terminal extracellular part contains a region of about 170 amino acids with no sequence similarities to known proteins, followed by one Ig-like domain and 4 fibronectin type III-like domains. The intracellular part is unique in that the region between the transmembrane domain and the first catalytic domain is about twice as large as in other receptor-like protein tyrosine phosphatases. RNA blot analysis reveals a single transcript, that is most abundant in lung and present in much lower amounts in brain and heart. Transfection of the mRPTP mu cDNA into COS cells results in the synthesis of a protein with an apparent Mr of 195,000, as detected in immunoblots using an antipeptide antibody. The human RPTP mu gene is localized on chromosome 18pter-q11, a region with frequent abnormalities implicated in human cancer.

mUBC9, a novel adenovirus E1A-interacting protein that complements a yeast cell cycle defect.

Adenovirus E1A encodes two nuclear phosphoproteins that can transform primary rodent fibroblasts in culture. Transformation by E1A is mediated at least in part through binding to several cellular proteins, including the three members of the retinoblastoma family of growth inhibitory proteins. We report here the cloning of a novel murine cDNA whose encoded protein interacts with both adenovirus type 5 and type 12 E1A proteins. The novel E1A-interacting protein shares significant sequence homology with ubiquitin-conjugating enzymes, a family of related proteins that is involved in the proteasome-mediated proteolysis of short-lived proteins. Highest homology was seen with a Saccharomyces cerevisiae protein named UBC9. Importantly, the murine E1A-interacting protein complements a cell cycle defect of a S. cerevisiae mutant which harbors a temperature-sensitive mutation in UBC9. We therefore named this novel E1A-interacting protein mUBC9. We mapped the region of E1A that is required for mUBC9 binding and found that the transformation-relevant conserved region 2 of E1A is required for interaction.

Degradation of E2F by the ubiquitin-proteasome pathway: regulation by retinoblastoma family proteins and adenovirus transforming proteins.

E2F transcription factors are key regulators of transcription during the cell cycle. E2F activity is regulated at the level of transcription and DNA binding and by complex formation with the retinoblastoma pocket protein family. We show here that free E2F-1 and E2F-4 transcription factors are unstable and that their degradation is mediated by the ubiquitin-proteasome pathway. Both E2F-1 and E2F-4 are rendered unstable by an epitope in the carboxyl terminus of the proteins, in close proximity to their pocket protein interaction surface. We show that binding of E2F-1 to pRb or E2F-4 to p107 or p130 protects E2Fs from degradation, causing the complexes to be stable. The increased stability of E2F-4 pocket protein complexes may contribute to the maintenance of active transcriptional repression in quiescent cells. Surprisingly, adenovirus transforming proteins, which release pocket protein-E2F complexes, also inhibit breakdown of free E2F. These data reveal an additional level of regulation of E2F transcription factors by targeted proteolysis, which is inhibited by pocket protein binding and adenovirus early region 1 transforming proteins.

Crystal structure of murine/human Ubc9 provides insight into the variability of the ubiquitin-conjugating system.

Murine/human ubiquitin-conjugating enzyme Ubc9 is a functional homolog of Saccharomyces cerevisiae Ubc9 that is essential for the viability of yeast cells with a specific role in the G2-M transition of the cell cycle. The structure of recombinant mammalian Ubc9 has been determined from two crystal forms at 2.0 A resolution. Like Arabidopsis thaliana Ubc1 and S. cerevisiae Ubc4, murine/human Ubc9 was crystallized as a monomer, suggesting that previously reported hetero- and homo-interactions among Ubcs may be relatively weak or indirect. Compared with the known crystal structures of Ubc1 and Ubc4, which regulate different cellular processes, Ubc9 has a 5-residue insertion that forms a very exposed tight beta-hairpin and a 2-residue insertion that forms a bulge in a loop close to the active site. Mammalian Ubc9 also possesses a distinct electrostatic potential distribution that may provide possible clues to its remarkable ability to interact with other proteins. The 2-residue insertion and other sequence and structural heterogeneity observed at the catalytic site suggest that different Ubcs may utilize catalytic mechanisms of varying efficiency and substrate specificity.

BS69, a novel adenovirus E1A-associated protein that inhibits E1A transactivation.

The adenovirus E1A gene products are nuclear phosphoproteins that can transactivate the other adenovirus early genes as well as several cellular genes, and can transform primary rodent cells in culture. Transformation and transactivation by E1A proteins is most likely to be mediated through binding to several cellular proteins, including the retinoblastoma gene product pRb, the pRb-related p107 and p130, and the TATA box binding protein TBP. We report here the cloning of BS69, a novel protein that specifically interacts with adenovirus 5 E1A. BS69 has no significant homology to known proteins and requires the region that is unique to the large (289R) E1A protein for high affinity binding. BS69 and E1A proteins coimmunoprecipitate in adenovirus-transformed 293 cells, indicating that these proteins also interact in vivo. BS69 specifically inhibits transactivation by the 289R E1A protein, but not by the 243R E1A protein. BS69 also suppressed the E1A-stimulated transcription of the retinoic acid receptor in COS cells, but did not affect the cellular E1A-like activity that is present in embryonic carcinoma cells. Our data indicate that BS69 is a novel and specific suppressor of E1A-activated transcription.

TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein.

Using a protein binding assay, we show that the amino-terminal 204 amino acids of the c-Myc protein interact directly with a key component of the basal transcription factor TFIID, the TATA box-binding protein (TBP). Essentially the same region of the c-Myc protein also binds the product of the retinoblastoma gene, the RB protein. c-Myc protein coimmunoprecipitates with TBP in lysates of mammalian cells, demonstrating that the proteins are also complexed in vivo. A short peptide that spans the RB binding site of the E7 protein of human papilloma virus type 16 interferes with the binding of c-Myc to TBP. The same peptide also blocks binding of adenovirus E1A protein to TBP, suggesting that c-Myc and E1A bind to RB and TBP through overlapping epitopes. Furthermore, we show that binding of RB to E1A prevents association of E1A with TBP. Our data suggest that one of the functions of RB and RB-like proteins is to prevent interaction of viral and cellular oncoproteins, such as c-Myc and E1A, with TBP.

MDMX: a novel p53-binding protein with some functional properties of MDM2.

Here we report the isolation of a cDNA encoding a new p53-associating protein. This new protein has been called MDMX on the basis of its structural similarity to MDM2, which is especially notable in the p53-binding domain. In addition, the putative metal binding domains in the C-terminal part of MDM2 are completely conserved in MDMX. The middle part of the MDMX and MDM2 proteins shows a low degree of conservation. We can show by co-immunoprecipitation that the MDMX protein interacts specifically with p53 in vivo. This interaction probably occurs with the N-terminal part of p53, because the activity of the transcription activation domain of p53 was inhibited by co-transfection of MDMX. Northern blotting showed that MDMX, like MDM2, is expressed in all tissues tested, and that several mRNAs for MDMX can be detected. Interestingly, the level of MDMX mRNA is unchanged after UV irradiation, in contrast to MDM2 transcription. This observation suggests that MDMX may be a differently regulated modifier of p53 activity in comparison with MDM2. Our study indicates that at least one additional member of the MDM protein family exists which can modulate p53 function.