Gerard W.
Dougherty
Department of Pathology
Doctor of
Philosophy
2000
Major Advisor: Mary Lou Cutler, Ph.D., Department of Pathology
Thesis Title: Identification of an in vitro binding protein for and the in vivo phosphorylation sites of the Ras Suppressor Rsu-l
ABSTRACT
The Ras Suppressor Rsu-l is a 33 kDa protein originally discovered as a cDNA that suppressed v-Ras transformation of NIH-3T3 fibroblasts. Rsu-l overexpression in several cell lines (NIH-3T3, U251, PC12, MCF7) increased ERK activity and levels of p21CIP, and inhibited growth rate and decreased JNK activity. Stabilization of p53 and inhibition of CDK2 and cyclin D-associated kinase activity has also been observed in MCF7 cells stably transfected with Rsu-l. In this study we attempted to identify the binding proteins to Rsu-l by the Far Western Cloning method. Preliminary experiments with a fusion protein containing the glutathione-S-transferase (GST) tag and the carboxy-terminus of Rsu-l showed binding to a protein of approximately 30 kDa both in solution and immobilized on nitrocellulose. A K562 human leukemic cell library was screened with 32P-labeled GST-C-terminal Rsu-l. Results indicate that one subclone from approximately one million plaques screened bound to the Rsu-l protein in vitro. This subclone, designated PI-4, expressed a protein of approximately 30 kDa. Sequence analysis of this subclone suggests homology to an as yet uncharacterized gene sequence on the long arm of chromosome 21 and also the WAS protein. Additional studies were done to ascertain the role of Rsu-l phosphorylation on its biological activity. The Rsu-l sequence contains several consensus phosphorylation sites for protein kinase C (PKC), protein kinase A (PKA) and casein kinase II. Previous experiments showed that Rsu-l is phosphorylated in vivo in response to growth factor and TPA, a known activator of PKC. Phosphoamino acid analysis of Rsu-l suggests that in vivo phosphorylation occurs on serine residues. In this study site-directed mutagenesis of individual Rsu-l consensus PKC sites revealed that two serine residues (serine-4 and serine-163) are phosphorylated in vivo in response to TPA. This is supported by evidence that Rsu-l phosphorylation can be specifically blocked by the PKC inhibitor bisindolylmaleimide I (HIM), but not by inhibitors of tyrosine kinase, MEK and PI3K. Stable transfectants of non- phosphorylatable Rsu-l mutants in MCF7 were isolated and tested for growth rate and effect on apoptosis. Previous studies with Rsu-l have demonstrated an enhancement of apoptosis in response to TNF-" and staurosporine in MCF7 cells, accompanied by stabilization of p53 protein levels. Results indicate that non-phosphorylatable mutants of Rsu-l still exhibit growth inhibition but do not enhance apoptosis in MCF7 cells. Together these results support the hypothesis that PKC or a PKC-dependent serine kinase mediates. the proapoptotic effect but not growth inhibitory effect of Rsu-l.
Thomas W.
Geisbert
Department of Pathology
Doctor of
Philosophy
2003
Major Advisor: Elliott Kagan, M.D., F.R.C.Path., Department of Pathology
Thesis Title:
"Pathogenesis of Ebola Hemorrhagic Fever in Primate Models
In Vivo and In Vitro
ABSTRACT
Ebola virus (EBOV) causes severe hemorrhagic fever (HF) with high mortality in humans and nonhuman primates. Despite progress made during the last decade to identify key modulators of EBOV pathogenesis, cultural mores, and a range of logistical problems, have hindered the systematic pathogenetic analysis of human EBOV infections. Nonhuman primate models of EBOV HF were developed, but with few exceptions, previous investigations examined animals naturally infected or killed when moribund, and shed little light on the pathogenesis of infection during times before death. In this study, we investigated the process(es) triggering the coagulation abnormalities characteristic of primate EBOV infections and attempted to identify the sequence of key morphologic, virologic, and inflammatory events. This study examined tissues of 21 nonhuman primates over time and also temporally evaluated EBOV infection of primary human monocytes/macrophages (PHM) and endothelial cells in vitro. Results showed that tissue factor plays an important role in triggering the hemorrhagic complications that characterize EBOV infections, and dysregulation of protein C exacerbates disease. Increased levels of TF were associated with lymphoid macrophages, while analysis of peripheral blood mononuclear cell RNA showed increased tissue factor transcripts by day 3. Analysis of PHM RNA at 1, 24, and 48 hours showed increased tissue factor transcripts while levels of tissue factor protein were dramatically increased by day 2. A rapid drop in plasma protein C levels was evident in all monkeys by day 2. Moreover, replication of EBOV in endothelial cells was not consistently observed until the latter stages of disease, well after the onset of disseminated intravascular coagulation, suggesting that the characteristic coagulation abnormalities are not the direct result of EBOV-induced cytolysis of endothelial cells. Dendritic cells in lymphoid tissues were identified as early and sustained targets of EBOV implicating their role in the immunosuppression characteristic of EBOV infections. Bystander apoptosis and loss of NK cells was a prominent finding suggesting the importance of innate immunity in determining the fate of the host. Accordingly, primate models have been invaluable in identifying several new targets for chemotherapeutic interventions that may ameliorate the effects of EBOV HF.
Roseann Marie
Waterhouse
Department of Pathology
Doctor of
Philosophy
2001
Major Advisor: Gabriela Dveksler, Associate Professor, Department of Pathology
Thesis Title: Identification of the First Receptor for a Pregnancy Specific Glycoprotein. Tetraspanins find their Ligand
ABSTRACT
Pregnancy
specific glycoproteins (PSGs) are a family of secreted proteins produced by
the placenta, which have been shown to be essential for pregnancy success.
The ability of human PSGs to induce anti-inflammatory cytokines has led to
the hypothesis that these proteins may function to protect the fetus from
attack by the maternal immune system. With the purpose of developing an animal
model to study the function of PSGs, we have studied the effects of murine
PSG17 on macrophages and we have cloned its receptor. RAW 264.7 cells and
peritoneal macrophages were treated with recombinant PSG17N, which consists
of the N-domain of PSG17. PSG17N induced production of IL-l 0 and IL-6 at
the protein and RNA levels in these cells. Secretion of TGF$1 and PGE2 was
also induced upon treatment with PSG17N. We then examined the PSG17-RAW cell
surface binding interaction. Scatchard analysis revealed that there are approximately
1770 binding sites per cell with a Kd of 2.2 X 10-11 M. For the purpose of
cloning the PSG17 receptor, we screened a RAW cell cDNA library by panning.
The receptor was identified as CD9, a member of the tetraspanin superfamily.
The specificity of the CD9-PSG17 interaction was confirmed by ELISA and flow
cytometry in CD9-transfected cells. Furthermore, binding of PSG17 to CD9-expressing
cells was blocked with anti-CD9 antibodies. We then tested whether murine
PSG18 and 19, which share with PSG17 the ability to induce cytokines in macrophages,
use CD9 as their receptor. These proteins do not bind to CD9. In conclusion,
we have identified the first receptor for a PSG as well as the first natural
ligand for a member of the tetraspanin superfamily.