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The Skaggs Institute for Chemical Biology
Scientific Report 1999-2000

Studies in Molecular and Experimental Medicine

E. Beutler, J. Johnson, K. Kato, M. Nishizawa, V. Pasquetto, G. Subbarao, M. Suzuki

Bcl-2 protein family members play a key role in determining the apoptotic response of a cell. The antiapoptosis member Bcl-2 is essential for normal development and homeostasis, while mutations in the gene are involved in many disease states, including cancer. Over the past year Jennifer Johnson found direct evidence that the bcl-2 gene is transcriptionally regulated by nuclear factor-κB (NF-κB). The transcriptional activity of the bcl-2 p2 promoter was increased 6-fold by overexpression of NF-κB. Furthermore, this transactivation localized to an NF-κB site approximately 200 bp upstream of the translation start site. In view of the increasing evidence of an antiapoptosis function for NF-κB, this finding appears to be an important one and is the first direct evidence linking the NF-κB signaling pathway to Bcl-2 expression, and thus to a prosurvival response.

During the previous year, Kazunobu Kato initiated studies to delete the mouse gene for the ß-subunit of the platelet glycoprotein (GP) Ib-IX-V receptor. Progress includes the successful generation of a gene-targeting vector, homologous recombination in pluripotent embryonic stem cells, and the generation of mice carrying a single defective ß-subunit gene. Currently, heterozygous mice are being bred to generate the null ß-subunit phenotype. These mice will be characterized to determine if they elicit the mouse Bernard-Soulier syndrome and then used as a model for the in vivo expression of a variant platelet ß-subunit lacking a cytoplasmic tail. These mice will determine the physiologic relevance, if any, of signaling through the ß-subunit of the GP Ib-IX-V complex in the platelet response to vascular injury.

Dr. Kato also initiated a project to generate mice lacking the recently characterized human platelet GP VI collagen receptor. His progress includes the generation of a mouse bone marrow cDNA library and the isolation of full-length cDNA clones for the mouse GP VI mRNA. The complete mouse GP VI gene is being characterized to facilitate the generation of the GP VI targeting vector. Experiments for the next year will include targeted homologous recombination at the mouse GP VI locus and the generation of mice lacking the GP VI collagen receptor. The physiologic relevance of the mouse GP VI receptor in this model of platelet dysfunction will be investigated.

Makoto Nishizawa continues 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 identification of target genes of these 2 oncoproteins. For this purpose, he constructed a series of fusion proteins that include the hormone-binding domain of human estrogen receptor and the DNA-binding domain of the oncoproteins. These constructs can induce cell transformation only in the presence of estrogen. Dr. Nishizawa also constructed several fusions of the transrepression domain of MxiI and the DNA-binding domain of the oncoproteins. These molecules suppressed growth of both normal and transformed cells. These constructs should be useful to identify target genes that are upregulated or downregulated by the transcription factors in the process of oncogenic transformation.

Valerie Pasquetto and coworkers previously showed that hepatitis B virus (HBV) replication is abolished in the liver of HBV transgenic mice by inflammatory cytokines induced by HBV-specific cytotoxic T cells and during unrelated viral infections of the liver. To define the cytokine-inducible mechanisms that inhibit HBV replication in the liver of HBV transgenic mice, she and her coworkers produced a system that reproduces the effects in vitro. HBV transgenic mice were crossed with transgenic mice that constitutively express a truncated version of the human MET protooncogene in their hepatocytes, rendering them constitutively resistant to apoptosis and reproducibly susceptible to immortalization. From these HBV-MET double transgenic mice, Dr. Pasquetto and her colleagues derived a panel of highly differentiated immortalized hepatocyte cell lines that stably express all of the HBV viral RNAs, secrete HBsAg and HBeAg, display cytoplasmic HBcAg, produce all of the viral replication, and express the liver-specific genes for transthyretin and albumin. HBV gene expression and replication are inhibited by a cytokine cocktail containing recombinant IFN-α/ß, IFN-γ, and TNF-α, coinciding with the induction of interferon-inducible genes (e.g., the gene for 2´,5´-oligoadenylate synthetase) and with the downregulation of negative acute-phase genes (genes for transthyretin and albumin). These results suggest that the cytokines trigger the same antiviral events in these cells in vitro as they do in vivo, thereby making it possible to study the mechanism(s) responsible for the antiviral effects of the cytokines in ways that are not possible in vivo.

Gowtham Subbarao's investigations are aimed toward understanding the structural basis of the interaction between the A1 domain of von Willebrand factor and the human platelet membrane GP Ib-IX complex, as the first step in thrombus formation, consequently enabling dissection of the underlying mechanism at the molecular level. His efforts have been directed toward crystallization of the dimeric form of the A1 domain with a fragment of the D3 domain of von Willebrand factor, comprising residues 445-733. A preliminary x-ray diffraction data set of these crystals was collected to a resolution of 2.8 Å; subsequent efforts resulted in higher quality crystals that diffract to a resolution of 2.2 Å. Efforts are under way to improve crystal quality and eventually to collect data to higher resolutions at the Stanford Synchrotron Radiation Laboratory. Botrocetin has also been purified by a method adapted from that of Andrews et al. (Biochemistry 28:8317, 1989) and subsequently complexed to the 445-733 dimeric fragment of von Willebrand factor. It is envisioned that crystallization trials will subsequently lead to a structural characterization of the complex.

Makoto Suzuki is focusing on a molecular screening of galactosemia, an inherited deficiency of galactose-1-phosphate uridyltransferase. He has screened the DNA of a panethnic population of more than 4100 subjects by using allele-specific oligonucleotide hybridization designed to detect 5 galactose-1-phosphate uridyltransferase mutations. On the basis of the gene frequency of the Q188R mutation, the most common mutation in the white population, the birth incidence of classic galactosemia is estimated at 1:71,000 in the Caucasian population.

In addition, Dr. Suzuki is continuing his study of prostate cancer ligands. He showed that 2 peptides from a 12mer peptide phage library bound to DU145 cells preabsorbed with normal fibroblasts and specific cell lines. Selection was from a library with a diversity of about 2 x 109. He has confirmed that phage libraries presenting random peptides can be used to select cell-binding peptides in the absence of any prior knowledge of cellular receptor. Dr. Suzuki is continuing to work on isolating the membrane protein to which the ligand binds to show its affinity and specificity by flow cytometry and enzyme-linked immunosorbent assay. If receptors of sufficient specificity are found, tumoricidal drugs attached to the ligand could be targeted to receptors found on tumor cells. This targeting of drugs could eventually lead to more effective treatment of prostate cancer with fewer side effects.



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