Biomedical subjects

S R Grossman

Publications and source records attributed to S R Grossman.

21 records · Page 2Linked to original sources

PubMed1989

Intracellular localization and DNA-binding properties of human papillomavirus type 18 E6 protein expressed with a baculovirus vector.

The E6 protein of human papillomavirus type 18 (HPV-18) is a putative zinc-finger protein that is expressed in HPV-18-induced genital neoplasias. We have studied the biochemical properties of E6 protein synthesized in large amounts with a baculovirus expression vector. When E6 protein was synthesized in insect cells infected with an E6-expressing baculovirus, the protein was localized to both nuclear and membrane fractions, with half-lives of 4 and 2 h, respectively. Changing the first five amino acids of E6 did not alter the pattern of cellular localization of the protein but dramatically increased the half-life of the nuclear component to longer than 30 h and increased the half-life of the membrane component to 8 h. Although the baculovirus-expressed E6 protein bound to double-stranded DNA with high affinity, no sequence specificity for HPV-18 DNA was detected.

Animals↗

PubMed1989

Identification of human papillomavirus type 18 transforming genes in immortalized and primary cells.

The selective retention and expression of the E6-E7 region of human papillomavirus (HPV) types 16 and 18 in cervical carcinomas suggests that these viral sequences play a role in the development of genital neoplasia. Each of three possible gene products, E6, E6*, and E7, from this region of HPV-18 were examined for transforming properties in several types of rodent cells. We have found that in immortalized fibroblasts, both E6 and E7 (but not E6*) are capable of inducing anchorage-independent growth. In rat embryo cells, the HPV-18 E7 open reading frame was an effective immortalizing agent and complemented an activated ras oncogene for transformation. In both immortalized and primary cells, transformation was observed when the HPV-18 sequences were expressed from either the HPV-18 promoter or a heterologous promoter. The E6-E7 region is not, however, the sole transforming domain of HPV-18, since another portion of the early region, possibly E5, also exhibited transforming capability in immortalized fibroblasts. The development of human cervical carcinomas may therefore involve a series of steps involving multiple viral and cellular gene products.

Animals↗

PubMed1989

E6 protein of human papillomavirus type 18 binds zinc.

The E6 open reading frames of human and animal papillomaviruses encode a transforming protein containing a conserved pattern of repeating cysteine doublets (cys-x-x-cys) similar to that found in steroid receptor zinc finger proteins. The spacing between the cysteine doublets, however, is twice as long as in any other zinc finger protein. To demonstrate that an E6 protein could indeed bind zinc, we synthesized the human papillomavirus type 18 E6 protein in insect cells with a baculovirus vector and analysed the protein for zinc-binding activity by a zinc-blot assay. Probing of E6 protein blotted to nitrocellulose from SDS-polyacrylamide gels with radioactive [65Zn]Cl2 demonstrated that it possessed zinc-binding activity. Reduction of cysteines with DTT prior to the addition of zinc greatly increased the zinc-binding activity of blotted E6 protein. Competition experiments showed that Cd2+ and Co2+ were more effective competitors for zinc-binding than Mg2+ or Ca2+, indicating that zinc atoms may be tetrahedrally coordinated in E6-zinc complexes. We mapped zinc-binding protein domains by proteolytic cleavage and demonstrated that a small 4kDa fragment of the protein retained zinc-binding activity, consistent with a model of individual 29 amino acid zinc-binding 'fingers'.

Binding, Competitive↗