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E J Androphy

Publications and source records attributed to E J Androphy.

At least 37 records · Page 2Linked to original sources
PubMed1998

Identification of an alpha helical motif sufficient for association with papillomavirus E6.

We recently identified a cellular protein named E6BP or ERC-55 that binds cancer-related papillomavirus E6 proteins (Chen, J. J., Reid, C. E., Band, V., and Androphy, E. J. (1995) Science 269, 529-531). By construction of a series of deletion mutants, the region of E6BP that is necessary and sufficient for complex formation with human papillomavirus type 16 E6 has been mapped to a 25-amino acid domain. The corresponding peptide was synthesized and found by nuclear magnetic resonance spectroscopy to bind calcium and fold into a classical helix-loop-helix EF-hand conformation. Additional deletion mutagenesis showed that 13 amino acids that form the second alpha helix mediated E6 association. Alanine replacement mutagenesis indicated that amino acids of this helix were most important for E6 binding. Alignment of this alpha helical E6 binding peptide with the 18-amino acid E6 binding region of E6AP (Huibregtse, J. M., Scheffner, M., and Howley, P. M. (1993) Mol. Cell. Biol. 13, 4918-4927) and the first LD repeat of another E6-binding protein, paxillin (Tong, X., and Howley, P. M. (1997) J. Biol. Chem. 272, 33373-33376), revealed substantial similarities among these E6 binding domains. The extent of homology and the mutational data define the peptide as an E6 binding motif.

Amino Acid Sequence↗
PubMed1998

Polyomavirus large T can support DNA replication in human cells.

Human cells are generally thought to be nonpermissive for polyomavirus (Py) DNA replication. Using transient transfection, we show that Py large T-antigen (LT) was able to support replication of a Py origin-containing plasmid in two human cell lines. Replication supported by LT in human cells was specific for the Py origin and required its enhancer sequences, as well as the previously reported critical phosphorylation sites within LT. Py replication efficiency was comparable to that of papillomavirus E1 and E2 activated DNA replication in transient assays performed in human 293 and C-33A cells. Previous analysis of DNA replication in vitro has pointed to polymerase alpha-primase as a specificity determinant for polyomavirus. The data presented here imply that in certain cellular environments, Py LT must functionally interact with human polymerase alpha-primase to permit DNA replication.

3T3 Cells↗
PubMed1998

SMN oligomerization defect correlates with spinal muscular atrophy severity.

Spinal muscular atrophy (SMA) is a motor-neuron disorder resulting from anterior-horn-cell death. The autosomal recessive form has a carrier frequency of 1 in 50 and is the most common genetic cause of infant death. SMA is categorized as types I-III, ranging from severe to mild, based upon age of onset and clinical course. Two closely flanking copies of the survival motor neuron (SMN) gene are on chromosome 5q13 (ref. 1). The telomeric SMN (SMN1) copy is homozygously deleted or converted in >95% of SMA patients, while a small number of SMA disease alleles contain missense mutations within the carboxy terminus. We have identified a modular oligomerization domain within exon 6 of SMN1. All previously identified missense mutations map within or immediately adjacent to this domain. Comparison of wild-type to mutant SMN proteins of type I, II and III SMA patients showed a direct correlation between oligomerization and clinical type. Moreover, the most abundant centromeric SMN product, which encodes exons 1-6 but not 7, demonstrated reduced self-association. These findings identify decreased SMN self-association as a biochemical defect in SMA, and imply that disease severity is proportional to the intracellular concentration of oligomerization-competent SMN proteins.

Cyclic AMP Response Element-Binding Protein↗
PubMed1998

The domain encoded by exon 2 of the survival motor neuron protein mediates nucleic acid binding.

Spinal muscular atrophy (SMA) is a motor neuron disorder resulting from anterior horn cell death. Survival motor neuron ( SMN ) is the SMA-determining gene and is deleted or gene converted in >95% of SMA patients. The SMN protein has a role in spliceosomal snRNP biogenesis and has therefore been implicated indirectly in general cellular RNA processing due to its unique sub-nuclear localization within structures termed 'gems', which co-localize with spliceosomal factors within coiled bodies. In this report, direct SMN RNA-binding activity, in addition to ssDNA and dsDNA binding is demonstrated. The region of SMN encoded by exon 2 is necessary and sufficient to mediate its nucleic acid-binding activities. This domain is homologous to several nucleic acid-binding factors, including several high mobility group (HMG) proteins. Additionally, previously reported SMN missense mutations isolated from SMA patients demonstrated reduced RNA-binding activity, suggesting that nucleic acid binding is functionally significant.

Amino Acid Sequence↗
PubMed1998

Functional interaction of the bovine papillomavirus E2 transactivation domain with TFIIB.

Induction of gene expression by the papillomavirus E2 protein requires its approximately 220-amino-acid amino-terminal transactivation domain (TAD) to interact with cellular factors that lead to formation of an activated RNA polymerase complex. These interaction partners have yet to be identified and characterized. The E2 protein localizes the transcription complex to the target promoter through its carboxy-terminal sequence-specific DNA binding domain. This domain has been reported to bind the basal transcription factors TATA-binding protein and TFIIB. We present evidence establishing a direct interaction between amino acids 74 to 134 of the E2 TAD and TFIIB. Within this region, the E2 point mutant N127Y was partially defective and W99C was completely defective for TFIIB binding in vitro, and these mutants displayed reduced or no transcriptional activity, respectively, upon transfection into C33A cells. Overexpression of TFIIB specifically restored transactivation by N127Y to close to wild-type levels, while W99C remained inactive. To further demonstrate the functional interaction of TFIIB with the wild-type E2 TAD, this region was fused to a bacterial DNA binding domain (LexA:E2:1-216). Upon transfection with increasing amounts of LexA:E2:1-216, there was reduction of its transcriptional activity, a phenomenon thought to result from titration of limiting factors, or squelching. Squelching of LexA:E2:1-216, or the wild-type E2 activator, was partially relieved by overexpression of TFIIB. We conclude that a specific region of the E2 TAD functionally interacts with TFIIB.

Adenovirus E2 Proteins↗
PubMed1998

Transactivation-competent bovine papillomavirus E2 protein is specifically required for efficient repression of human papillomavirus oncogene expression and for acute growth inhibition of cervical carcinoma cell lines.

The papillomavirus E2 proteins can function as sequence-specific transactivators or transrepressors of transcription and as cofactors in viral DNA replication. We previously demonstrated that acute expression of the bovine papillomavirus type 1 (BPV1) E2 protein in HeLa and HT-3 cervical carcinoma cell lines greatly reduced cellular proliferation by imposing a specific G1/S phase growth arrest. In this report, we analyzed the effects of a panel of point mutations in the BPV1 E2 protein to identify the functional requirements for acute growth inhibition. Disruption of E2-specific transactivation by mutations within either the transactivation domain or the DNA binding domain severely impaired E2-mediated growth inhibition in HeLa and HT-3 cells, even though these mutants retain various other E2 activities. This result indicates that functional transactivation activity is required for acute E2-mediated growth inhibition. HeLa cells, which contain a wild-type p53 gene, and HT-3 cells, which contain a transactivation-defective p53 gene, exhibited similar responses to the E2 mutants, suggesting that identical functions of the E2 protein were required for growth arrest regardless of p53 status. Replacement of the E2 transactivation domain with that of the herpes simplex virus VP16 generated a chimeric transactivator that efficiently stimulated expression of an E2-responsive reporter plasmid yet was completely defective for growth inhibition, suggesting that an E2-specific transactivation function is required for growth arrest. Surprisingly, the transactivation-defective E2 mutants were also markedly defective in their ability to repress transcription of the native human papillomavirus type 18 (HPV18) E6/E7 oncogenes in HeLa cells and of the HPV18 promoter present in a transfected reporter plasmid. These mutants were also defective in their ability to increase p53 levels. Therefore, efficient repression of the HPV18 promoter in HeLa cells is not merely a consequence of the binding of an E2 protein to appropriately situated binding sites in the promoter.

Animals↗
PubMed1997

Mutational analysis of transcriptional activation by the bovine papillomavirus type 1 E6.

While the bovine papillomavirus type 1 (BPV-1) E6 induces tumorigenic transformation of murine C127 cells, it does not bind or promote the degradation of p53. We recently showed the cellular protein ERC-55/E6BP binds BPV-1 E6 as well as the cancer-related human papillomavirus (HPV) E6 proteins. BPV-1 E6 also binds E6-AP, a ubiquitin ligase necessary for HPV E6-induced p53 degradation. We previously reported that the transforming activity of a set of BPV-1 E6 mutants correlated with their E6BP-binding ability. Another function of BPV-1 E6 is stimulation of transcription when targeted to a promoter, although cellular promoters responsive to BPV-1 E6 have not been identified. To examine whether its transcriptional function is required for oncogenic activity, or is related to its interactions with E6-AP or E6BP, a series of BPV-1 E6 mutants were analyzed as fusions to a sequence-specific DNA binding domain for activity in yeast and in mammalian cells. We show that some transformation defective mutants retained substantial levels of transcriptional activation activity. These mutants also distinguish transcriptional activation from E6-AP and E6BP binding. These results suggest the transcriptional activation function of BPV-1 E6 is not sufficient for cell transformation.

3T3 Cells↗
PubMed1997

The survival motor neuron protein in spinal muscular atrophy.

The 38 kDa survival motor neuron (SMN) protein is encoded by two ubiquitously expressed genes: telomeric SMN (SMN(T)) and centromeric SMN (SMN(C)). Mutations in SMN(T), but not SMN(C), cause proximal spinal muscular atrophy (SMA), an autosomal recessive disorder that results in loss of motor neurons. SMN is found in the cytoplasm and nucleus. The nuclear form is located in structures termed gems. Using a panel of anti-SMN antibodies, we demonstrate that the SMN protein is expressed from both the SMN(T) and SMN(C) genes. Western blot analysis of fibroblasts from SMA patients with various clinical severities of SMA showed a moderate reduction in the amount of SMN protein, particularly in type I (most severe) patients. Immunocytochemical analysis of SMA patient fibroblasts indicates a significant reduction in the number of gems in type I SMA patients and a correlation of the number of gems with clinical severity. This correlation to phenotype using primary fibroblasts may serve as a useful diagnostic tool in an easily accessible tissue. SMN is expressed at high levels in brain, kidney and liver, moderate levels in skeletal and cardiac muscle, and low levels in fibroblasts and lymphocytes. In SMA patients, the SMN level was moderately reduced in muscle and lymphoblasts. In contrast, SMN was expressed at high levels in spinal cord from normals and non-SMA disease controls, but was reduced 100-fold in spinal cord from type I patients. The marked reduction of SMN in type I SMA spinal cords is consistent with the features of this motor neuron disease. We suggest that disruption of SMN(T) in type I patients results in loss of SMN from motor neurons, resulting in the degeneration of these neurons.

Animals↗
PubMed1997

Sequences flanking the core DNA-binding domain of bovine papillomavirus type 1 E2 contribute to DNA-binding function.

We have compared a series of molecular constructs that contain the minimal DNA-binding and dimerization domain of bovine papillomavirus type 1 (BPV-1) E2 alone or this binding domain plus the adjacent 16 or 40 amino acids to test the role of the flanking sequences in E2 function. The presence of these sequences resulted in an up to eightfold increase in the affinity of E2 for its target DNA and stabilized the protein against denaturation both in the absence of DNA and in the form of DNA-protein complexes. In addition, an aspartic acid-to-tyrosine mutation within the flanking region blocked DNA binding and function. These data demonstrate that sequences flanking the core domain contribute to E2 function and are, in fact, an integral part of the DNA-binding domain of BPV-1 E2.

Animals↗
PubMed1997

Functional interaction of a novel cellular protein with the papillomavirus E2 transactivation domain.

The transactivation domain (AD) of bovine papillomavirus type 1 E2 stimulates gene expression and DNA replication. To identify cellular proteins that interact with this 215-amino-acid domain, we used a transactivation-defective mutant as bait in the yeast two-hybrid screen. In vitro and in vivo results demonstrate that the cDNA of one plasmid isolated in this screen encodes a 37-kDa nuclear protein that specifically binds to an 82-amino-acid segment within the E2 AD. Mutants with point mutations within this E2 domain were isolated based on their inability to interact with AMF-1 and were found to be unable to stimulate transcription. These mutants also exhibited defects in viral DNA replication yet retained binding to the viral E1 replication initiator protein. Overexpression of AMF-1 stimulated transactivation by both wild-type E2 and a LexA fusion to the E2 AD, indicating that AMF-1 is a positive effector of the AD of E2. We conclude that interaction with AMF-1 is necessary for the transcriptional activation function of the E2 AD in mammalian cells.

Animals↗
PubMed1996

Genetic analysis of the bovine papillomavirus E2 transcriptional activation domain.

The bovine papillomavirus type 1 E2 transactivator has a large amino-terminal 215-residue transcriptional activation domain (TAD) that is active in Saccharomyces cerevisiae and higher eukaryotic cells. Comparison to other transcriptional activators suggests that its functions may be mediated in part through two acidic regions, A1 and A2, in this domain. We have characterized the functional elements within the E2 TAD using LexA-E2 fusions and by screening randomly generated libraries of E2 mutations for transcriptional activation in yeast. The A1 region was highly sensitive to substitutions that reduce negative charge, although there was not a perfect correlation between overall charge and transcriptional activity. Mutations were isolated within a hydrophobic amino acid motif that overlaps the A2 region and resembles elements described in other viral and cellular transactivation domains. When fused to the LexA DNA binding domain, this hydrophobic motif within the acidic A2 region was unable to activate transcription in S. cerevisiae. Multiple highly defective mutations primarily altering hydrophobic amino acids were identified in the distal third of the E2 TAD. The transcription phenotype of many of these E2 TAD mutations was similar in yeast and COS cells.

Amino Acid Sequence↗
PubMed1996

The BPV-1 E2 DNA-contact helix cysteine is required for transcriptional activation but not replication in mammalian cells.

The papillomavirus E2 protein contains an amino-terminal region thought necessary and sufficient to support transcriptional activation and a carboxy-terminal region shown to direct sequence-specific DNA binding and dimerization. A cysteine residue in the center of the E2 DNA recognition helix is highly conserved among papillomavirus E2 proteins. Mutations of this cysteine in bovine papillomavirus type 1 E2 to serine and glycine resulted in proteins which failed to activate E2-dependent promoters in mammalian cells. These E2 mutants were DNA-binding competent, dimeric, and nuclear. When fused to the VP16 transactivation domain, C-terminal regions of E2 containing the mutations at 340 supported transcriptional activation, indicating that the heterologous trans-activation domain did not require cysteine in the DNA-binding helix as did the full-length E2 transactivating protein. Although cysteine-340 was required for transcriptional activation it was not required for DNA replication in vivo. Together, these results suggest that the E2 DNA-binding domain may directly contribute to functions of transcriptional activation previously thought limited to the N-terminal domain.

3T3 Cells↗
PubMed1996

Transcriptional activation function is not required for stimulation of DNA replication by bovine papillomavirus type 1 E2.

Bovine papillomavirus type 1 replication was previously shown to require both the E1 initiator protein and the E2 transactivator protein. We show here that E1, in the absence of E2, is sufficient for low-level bovine papillomavirus type 1 DNA replication in C-33A cells. In addition, studies of genetically isolated E2 point mutants demonstrate that enhancement of replication by E2 does not require its transcriptional activation function. The uncoupling of the E2 functions suggests that stimulation of transcription and replication by enhancer proteins occurs via divergent mechanisms.

Animals↗
PubMed1996

Mutational analysis of human papillomavirus type 16 E6 demonstrates that p53 degradation is necessary for immortalization of mammary epithelial cells.

We have previously demonstrated that normal human mammary epithelial cells (MECs) are efficiently immortalized by human papillomavirus type 16 (HPV16) E6. HPV16 E6 binds to and induces p53 degradation in vitro and induces a marked reduction of p53 protein in MECs. Low-risk HPV6 E6 is defective for p53 binding and degradation in vitro but immortalized MECs at low efficiency. The HPV6 E6-immortalized MECs had markedly reduced levels of p53. To directly investigate whether the ability of HPV16 E6 to stimulate p53 degradation is required for E6-induced immortalization, a series of HPV16 E6 mutants were analyzed for the ability to bind and degrade p53 in vitro, induce a reduction in p53 levels in vivo, and immortalize MECs. We observed that one set of mutants efficiently immortalized MECs, caused a reduction in p53 levels in vivo, and degraded p53 in vitro. Other mutants immortalized MECs with low efficiency and either induced p53 degradation at low levels or were unable to induce p53 degradation in vitro; however, all of the immortal clones displayed low levels of p53. A third class of mutants did not immortalize MECs and failed to induce a reduction in p53 levels in vivo or degrade p53 in vitro. These results demonstrate that a reduction in p53 protein levels due to enhanced degradation is essential for MEC immortalization by HPV16 E6.

Base Sequence↗
PubMed1995

Interaction of papillomavirus E6 oncoproteins with a putative calcium-binding protein.

Human papillomaviruses (HPVs) are associated with the majority of cervical cancers and encode a transforming protein, E6, that interacts with the tumor suppressor protein p53. Because E6 has p53-independent transforming activity, the yeast two-hybrid system was used to search for other E6-binding proteins. One such protein, E6BP, interacted with cancer-associated HPV E6 and with bovine papillomavirus type 1 (BPV-1) E6. The transforming activity of BPV-1 E6 mutants correlated with their E6BP-binding ability. E6BP is identical to a putative calcium-binding protein, ERC-55, that appears to be localized in the endoplasmic reticulum.

Bovine papillomavirus 1↗
PubMed1995

The domain of p53 required for binding HPV 16 E6 is separable from the degradation domain.

The E6 proteins of specific cancer-associated human papillomaviruses (HPVs) complex with and mediate degradation of the cellular anti-oncogene p53 in vitro. A critical property of p53 is its ability to stimulate transcription from promoters containing its recognition sequence. HPV E6, mutant p53 proteins, and several DNA tumor virus oncogenes inhibit the transcriptional activity of wild-type p53. In this report, the structural requirements for the interaction between HPV 16 E6 and p53 were examined both in vivo and in vitro. p53-stimulated transcription was efficiently inhibited by wild-type HPV 16 E6 and E6 mutants competent for p53 binding and degradation. A series of p53 deletions and hybrid proteins with heterologous DNA binding, dimerization and transactivation domains were analysed for transcriptional interaction with HPV 16 E6 to determine the domains of p53 required for transcriptional inhibition. These chimeric proteins were also analysed for E6 binding and E6-mediated degradation in vitro. In both assays, complex formation with E6 was mediated through the amino-terminal 345 amino acids of p53 without a specific requirement for its C-terminus. Hybrid proteins containing residues 161-345 of p53 also bound E6, but this segment of p53 was not susceptible to E6 induced proteolysis. A second region of p53, within its N-terminal 160 aa, is required for E6 induced degradation of complexed p53. Taken together, these results suggest that the complex formation between E6 and p53 is not mediated through the C-terminus of p53 and that binding and degradation are separable.

Animals↗