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The Skaggs Institute for Chemical Biology
Scientific Report 1998-1999


Studies in Molecular and Experimental Medicine


E. Beutler, B. Kempf, M. Nishizawa, V. Pasquetto, J. Johnson, S. Vasudevan, M. Suzuki

Barbara Kempf is analyzing a fusion protein, PAX3-FKHR, associated with the soft tissue tumor alveolar rhabdomyosarcoma in children. This protein is generated by a chromosomal rearrangement that results in the fusion of the amino-terminal DNA-binding domains of PAX3 and the carboxy-terminal transcriptional regulatory domain of the winged helix protein FKHR. PAX3-FKHR, expressed by the avian retroviral vector RCAS, induces foci of transformed cells and anchorage-independent growth in cultures of chicken embryo fibroblasts. The fusion protein is a stronger transcriptional activator than is PAX3. The gain-of-function mutation suggests a potential mechanism for oncogenesis.

Dr. Kempf is currently investigating a correlation between DNA binding, transactivation, and transformation in chicken embryo fibroblasts. She constructed a series of deletion mutants of PAX3-FKHR to determine the molecular domains required for transformation of the fibroblasts. The mutants were tested for the ability to bind to DNA and transactivate a reporter gene in vitro. Results suggest that both the DNA-binding domain and the transactivation domain of PAX3-FKHR are required for oncogenic transformation of chicken embryo fibroblasts. Partial truncation of the PAX3 DNA-binding domains was compatible with transformation and resulted in an altered transformed cellular phenotype. This change in cell shape and growth pattern may reflect an alteration in DNA-binding specificity that would result in a different spectrum of target genes. Information gained from these experiments will be important in understanding the method by which the fusion gene alters normal gene expression and causes tumor formation.

Makoto Nishizawa is continuing his study of oncogenes that encode the bZip family of transcription factors, Maf and Jun. He showed that the 2 oncoproteins specifically recognize similar but distinct DNA sequences. He is currently concentrating on the role of protein-protein interaction and DNA binding in oncogenic transformation. For this analysis, he constructed chimeric proteins in which the dimerization and DNA-binding domains of Maf and Jun were replaced with each other or with the corresponding domains of a yeast bZip protein, GCN4. The chimeric proteins differ in their DNA targets and in their ability to interact with other cellular factors.

Recently, Dr. Nishizawa constructed a series of fusion proteins that include the hormone-binding domain of human estrogen receptor and the DNA-binding domain of the bZip factors. These constructs can induce cell transformation only in the presence of estrogen. The information obtained with these constructs will be important in identifying downstream target genes that are upregulated or downregulated by the transcription factors in the development of cancer.

Hepatocellular replication of hepatitis B virus (HBV) is abolished in HBV transgenic mice by inflammatory cytokines induced by HBV-specific T cells and during unrelated viral infections of the liver. Valerie Pasquetto discovered that intrahepatic replication of HBV is also inhibited in mice infected with plasmodium species (Plasmodium yoelii and Plasmodium berghei) that cause murine malaria. When injected into susceptible mice, malaria sporozoites from parasitized mosquitoes infect hepatocytes, develop into merozoites, and are released into the circulation. The merozoites are not infectious for hepatocytes; instead, they infect and multiply in erythrocytes that lyse and release new merozoites to establish a massive erythrocytic infection that is ultimately controlled by the immune response. Lysed erythrocytes, hemoglobin, and malaria pigment are removed from the blood by splenic and hepatic macrophages (Kupffer cells), resulting in macrophage activation, hyperplasia, and the recruitment of inflammatory cells into these organs.

In the current study, transgenic mice that replicate HBV in their hepatocytes were infected with either 106 sporozoites or 107 merozoite-infected erythrocytes, and groups of 3 mice were sacrificed at various intervals between 1 and 20 days after infection. Serum levels of alanine aminotransferase became elevated during the sporozoite infection, in keeping with the ability of this form of the parasite to infect hepatocytes. In contrast, serum levels of the enzyme remained normal during the merozoite infection, consistent with the inability of this form to infect hepatocytes. Nonetheless, in both types of infection, Kupffer cell hyperplasia and a diffuse T-cell infiltrate developed in the liver between 3 and 10 days after inoculation. These events coinci ded with the time course of parasitemia; the induction of 2´,5´-oligoadenylate synthetase, TNF-α, and IFN-γ mRNA; and the disappearance of HBV core antigen and HBV replicative intermediates from the liver. Of note, infection of hepatocytes was not required for this effect. HBV replication was inhibited when the infection was limited to erythrocytes, as it was when the hepatocytes and the erythrocytes were both infected.

These results strongly suggest that during murine malaria, HBV replication is inhibited by inflammatory cytokines secreted primarily by intrahepatic macrophages that are activated by phagocytosis of lysed and merozoite-infected erythrocytes. If this hypothesis is correct, it not only provides insight into the possible impact of malaria on HBV replication in coinfected humans but also suggests that pharmacologic strategies designed to activate intrahepatic macrophages may have therapeutic merit during chronic HBV infection.

Jennifer Johnson found that levels of enzyme activity and phosphorylation of p47phox are much lower in p47phox-deficient B lymphoblasts expressing the p47phox serine(359,370)alanine double mutation than in the same cells expressing wild-type p47phox. In addition, the mutant p47phox does not translocate to the plasma membrane when the cells are stimulated. In contrast, normal phosphorylation and translocation occur in mutants containing aspartate or glutamate at positions 359 and 370, but oxidase activity is still greatly reduced. These results suggest that a negative charge at position 359, position 370, or both positions is sufficient to allow phosphorylation and translocation of p47phox, but that features unique to a phosphorylated hydroxyamino acid are required to support production of superoxide.

Dr. Johnson found a putative new member of the NADPH oxidase complex: p62MCSP. This protein was identified by using the yeast 2-hybrid system and a cDNA that encodes the polypeptide corresponding to residues 245­526 of the cytosolic oxidase component p67phox, a stretch of sequence that contains both SH3 domains of the protein. Dr. Johnson is currently investigating the function of p62MCSP in B lymphocytes transformed by Epstein-Barr virus. For these experiments, cDNAs encoding p62MCSP, and the p62MCSP antisense message, were cloned, and the resulting plasmids were transfected into wild-type B lymphocytes transformed by Epstein-Barr virus. The amounts of recombinant proteins in the various transfectants was determined by immunoblotting. The effect on oxidase activity will be determined by using a chemiluminescence assay to measure production of superoxide induced by phorbol myristate acetate and antibodies to human immunoglobulin. The cytochrome c assay is not sensitive enough for this purpose. Depending on the results of these experiments, further studies may be done on the translocation of p47phox and p67phox and the phosphorylation of p47phox in the transfected cells, as previously were done with transfected p47phox-deficient cells.

Sona Vasudevan is studying the interactions between blood platelets and blood vessels. Adhesion of platelets at the site of vascular lesion initiates formation of thrombus, a process that is crucial for normal hemostasis and thrombosis. The adhesion of platelets under high shear stress conditions is mediated by the binding of the platelet glycoprotein (GP) Ib-IX-V receptor complex to the A1 domain of von Willebrand factor. Although much progress has been made in understanding the structure and function of the A1 domain and its receptor GP Ib, the important aspects of the interaction between the domain and the receptor remain to be explained at the atomic level, and an understanding of the 3-dimensional structure of the complex is required.

Currently, Dr. Vasudevan is trying to crystallize the complex composed of botrocetin (a nonphysiologic modulator) and the A1 domain. Diffractable crystals of botrocetin have been obtained, and attempts are under way to solve the structure of the modulator. In the absence of a 3-dimensional structure of the complex, modeling is a valuable tool for understanding the interactions between the A1 domain and GP Ib. The availability of the structure of the complex of A1 domain with its function-blocking antibody NMC-4 and information from mutagenesis studies enabled us to model a fragment corresponding to residues 271­279 of GP Ibα and to dock the fragment onto the A1 domain of von Willebrand factor.

The results of the docking indicated that residues 559­563 and 596­600 of the A1 domain are the primary binding site of GP Ibα. The results of the modeling and docking procedure agree well with the results of functional studies carried out under flow conditions. A 3-dimensional structure may confirm the results from the modeling.

Makoto Suzuki is searching for receptors on prostate cancer cells, because the detection of specific receptor molecules on the surface of tumor cells provides a potential approach to the cure of prostate cancer. He used the phage display technique to identify ligands that bind specifically to prostate cancer cell lines.

Sequence analysis of the DNA inserts disclosed 7 main peptide sequences. These 7 DNA inserts were mutagenized by error-prone polymerase chain reaction to construct and screen a second-generation library that consisted of variants of the 7 original sequences. The estimated diversity of the second-generation library was 5.6 x 105.

The modified peptides were again selected by cyclic absorption to other normal cells and subsequent binding to prostate cancer cells. Sequence analysis of 22 selected phage clones from the 6th panning of the second-generation library disclosed 17 clones that were identical to 1 of the original 7 peptide clones and 3 mutants of the same clone with a single amino acid change. The affinity of one of the peptides to various normal and cancer cells was measured after biotinylation. The peptide binds not only to prostate cancer cell lines but also other cancer cells, but not to normal prostate epithelial cells. Dr. Suzuki hopes to identify the receptor to which this peptide binds.

Publications

Inanami, O., Johnson, J.L., Babior, B.M. The leukocyte NADPH oxidase subunit p47PHOX: The role of the cysteine residues. Arch. Biochem. Biophys. 350:36, 1998.

Inanami, O., Johnson, J.L., McAdara, J.K., El Benna, J., Faust, L.P., Newburger, P.E., Babior, B.M. Activation of the leukocyte NADPH oxidase by phorbol ester requires the phosphorylation of p47PHOX on serine 303 or 304. J. Biol. Chem. 273:9539, 1998.

Johnson, J.L., Park, J.-W., El Benna, J., Faust, L.P., Inanami, O., Babior, B.M. Activation of p47PHOX, a cytosolic subunit of the leukocyte NADPH oxidase: Phosphorylation of ser-359 or ser-370 precedes phosphorylation at other sites and is required for activity. J. Biol. Chem. 273:35147, 1998.

 

 







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