News and Publications
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,
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
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
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
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
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.
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.