Interactions between cyclosporin A, low-density lipoprotein and the low-density lipoprotein receptor

Cyclosporine A (CSA) is a cyclic eleven amino acid, lipophilic molecule used therapeutically as an immunosuppressive agent. Cyclosporine can specifically inhibit the transcription of a number of different genes. It is known that CSA is bound almost exclusively to lipoproteins in plasma, however, the relationship between the low density lipoprotein (LDL), the LDL receptor, and CSA has not been fully elucidated. The exact mechanism of cellular uptake of CSA is unknown, but it is believed to be by simple passive diffusion across the cell membrane. In addition, it has been recently shown that the frequent finding of hypercholesterolemia seen in patients treated with CSA can be explained by a CSA-induced effect. The mechanism by which CSA induces hypercholesterolemia is not known. We have used an LDL receptor-deficient animal model, the Watanabe Heritable Hyperlipidemic (WHHL) rabbit to investigate the role of LDL and the LDL receptor in the cellular uptake of CSA. Using this animal model, we have shown that CSA uptake by lymphocytes is predominantly LDL receptor-mediated. Chemical modification of apoB-100 on LDL particles abolishes their ability to bind to the LDL receptor. When CSA is incubated with modified LDL much less is taken-up than when native LDL is incubated with CSA. Treatment of two human cell lines with CSA results in a dose-dependent decrease in LDL receptor mRNA levels. Using a novel transfection system involving the 5$\sp\prime$-flanking region of the LDL receptor gene, we have found that CSA decreases the number of transcripts, but is dependent on whether or not cholesterol is present and the stage of growth of the cells. ^

Defective pituitary function and gamete interactions in $\beta$1,4-galactosyltransferase null mice

Despite much attention, the function of oligosaccharide chains of glycoproteins remains largely unknown. Our understanding of oligosaccharide function in vivo has been limited to the use of reagents and targeted mutations that eliminate entire oligosaccharide chains. However, most, if not all biological functions for oligosaccharides have been attributed to specific terminal sequences on these oligosaccharides, yet there have been few studies to examine the consequences of modifying terminal oligosaccharide structures in vivo. To address this issue, mice were created bearing a targeted mutation in $\beta$1,4-galactosyltransferase, an enzyme responsible for elaboration of many of the proposed biologically-active carbohydrate epitopes. Most galactosyltransferase-null mice died within the first few weeks after birth and were characterized by stunted growth, thin skin, sparse hair, and dehydration. In addition, the adrenal cortices were poorly stratified and spermatogenesis was delayed. The few surviving adults had puffy skin (myxedema), difficulty delivering pups at birth (dystocia), and failed to lactate (agalactosis). All of these defects are consistant with endocrine insufficiency, which was confirmed by markedly decreased levels of serum thyroxine. The anterior pituitary gland appeared functionally delayed in newborn mutant mice, since the constituent cells were quiescent and nonsecretory, unlike that of control littermates. However, the anterior pituitary acquired a normal secretory phenotype during neonatal development, although it remained abnormally small and its glycoprotein hormones were devoid of $\beta$1,4-galactosyl residues. These results support in vitro studies suggesting that incomplete glycosylation of pituitary hormones leads to the creation of hormone antagonists that down regulate subsequent endocrine function producing polyglandular endocrine insufficiency. More surprisingly, the fact that some mice survive this neonatal period indicates the presence of a previously unrecognized compensatory pathway for glycoprotein hormone glycosylation and/or action.^ In addition to its well-studied biosynthetic function in the Golgi complex, a GalTase isoform is also expressed on the sperm surface where it functions as a gamete receptor during fertilization by binding to its oligosaccharide ligand on the egg coat glycoprotein, ZP3. Aggregation of GalTase by multivalent ZP3 oligosaccharides activates a G-protein cascade leading to the acrosome reaction. Although GalTase-null males are fertile, the mutant sperm bind less ZP3 than wild-type sperm, and are unable to undergo the acrosome reaction in response to either zona pellucida glycoproteins or to anti-GalTase anti-serum, as do wild-type sperm. However, mutant and wild-type sperm undergo the acrosome reaction normally in response to calcium ionophore which bypasses the requirement for ZP3 binding. Interestingly, the phenotype of the GalTase-null sperm is reciprocal to that of sperm that overexpress surface GalTAse and which bind more ZP3 leading to precocious acrosome reactions. These results confirm that GalTase functions as at least one of the sperm receptors for ZP3, and that GalTase participates in the ZP3-induced signal transduction pathway during zona pellucida-induced acrosome reactions. ^

Functions and intramolecular interactions of ribosomal RNA in decoding of termination codons

Two regions in the 3$\prime$ domain of 16S rRNA (the RNA of the small ribosomal subunit) have been implicated in decoding of termination codons. Using segment-directed PCR random mutagenesis, I isolated 33 translational suppressor mutations in the 3$\prime$ domain of 16S rRNA. Characterization of the mutations by both genetic and biochemical methods indicated that some of the mutations are defective in UGA-specific peptide chain termination and that others may be defective in peptide chain termination at all termination codons. The studies of the mutations at an internal loop in the non-conserved region of helix 44 also indicated that this structure, in a non-conserved region of 16S rRNA, is involved in both peptide chain termination and assembly of 16S rRNA.^ With a suppressible trpA UAG nonsense mutation, a spontaneously arising translational suppressor mutation was isolated in the rrnB operon cloned into a pBR322-derived plasmid. The mutation caused suppression of UAG at two codon positions in trpA but did not suppress UAA or UGA mutations at the same trpA positions. The specificity of the rRNA suppressor mutation suggests that it may cause a defect in UAG-specific peptide chain termination. The mutation is a single nucleotide deletion (G2484$\Delta$) in helix 89 of 23S rRNA (the large RNA of the large ribosomal subunit). The result indicates a functional interaction between two regions of 23S rRNA. Furthermore, it provides suggestive in vivo evidence for the involvement of the peptidyl-transferase center of 23S rRNA in peptide chain termination. The $\Delta$2484 and A1093/$\Delta$2484 (double) mutations were also observed to alter the decoding specificity of the suppressor tRNA lysT(U70), which has a mutation in its acceptor stem. That result suggests that there is an interaction between the stem-loop region of helix 89 of 23S rRNA and the acceptor stem of tRNA during decoding and that the interaction is important for the decoding specificity of tRNA.^ Using gene manipulation procedures, I have constructed a new expression vector to express and purify the cellular protein factors required for a recently developed, realistic in vitro termination assay. The gene for each protein was cloned into the newly constructed vector in such a way that expression yielded a protein with an N-terminal affinity tag, for specific, rapid purification. The amino terminus was engineered so that, after purification, the unwanted N-terminal tag can be completely removed from the protein by thrombin cleavage, yielding a natural amino acid sequence for each protein. I have cloned the genes for EF-G and all three release factors into this new expression vector and the genes for all the other protein factors into a pCAL-n expression vector. These constructs will allow our laboratory group to quickly and inexpensively purify all the protein factors needed for the new in vitro termination assay. (Abstract shortened by UMI.) ^

Molecular mechanism of PUVA-induced apoptosis in mouse epidermal cells

A combination of psoralens and ultraviolet-A radiation referred to as PUVA, is widely used in the treatment of psoriasis. PUVA therapy is highly effective in killing hyperproliferative cells, but its mechanism of action has not been fully elucidated. Psoralen binds to DNA, and upon photoactivation by UVA, it forms monofunctional adducts and interstrand cross-links. PUVA treatment has been shown to be mutagenic and to produce tumors in animals. In addition, epidemiological studies have reported a 10 to 15 percent increased risk of developing squamous cell carcinoma in individuals treated chronically with PUVA. However, it remains a treatment for skin disorders such as psoriasis because its benefits outweigh its risks. The widespread use of PUVA therapy and its associated cancer risk requires us to understand the molecular mechanisms by which PUVA induces cell death. Immortalized JB6 mouse epidermal cells, p53−/− mice, and Fas Ligand−/− (gld) mice were used to investigate the molecular mechanism by which PUVA kills cells. Treatment of JB6 cells with 10 μg/ml 8-methoxypsoralen followed by irradiation with 20 kJ/m2 UVA resulted in cell death. The cells exhibited morphological and biochemical characteristics of apoptosis such as chromatin condensation, DNA ladder formation, and TUNEL-positivity. PUVA treatment stabilized and phosphorylated p53 leading to its activation, as measured by nuclear localization and induction of p21Waf/Cip1, a transcriptional target of p53. Subsequent in vivo studies revealed that there was statistically significantly less apoptosis in p53 −/− mice than in p53+/+ mice at 72 hours after PUVA. In addition, immunohistochemical analysis revealed more Fas and FasL expression in p53+/+ mice than in p53−/− mice, suggesting that p53 is required to transcriptionally activate Fas, which in turn causes the cells to undergo apoptosis. Studies with gld mice confirmed a role for Fas/FasL interactions in PUVA-induced apoptosis. There was statistically significantly less apoptosis in gld mice compared with wild-type mice 24, 48, and 72 hours after PUVA. These results demonstrate that PUVA-induced apoptosis in mouse epidermal cells requires p53 and Fas/FasL interactions. These findings may be important for designing effective treatments for diseases such as psoriasis without increasing the patient's risk for skin cancer. ^

4HPR-X radiation mechanisms of interactions in non-small cell lung cancer (NSCLC) cells in vitro

Lung cancer is the leading cause of cancer death. However, poor survival using conventional therapies fuel the search for more rational interventions. The objective of this study was to design and implement a 4HPR-radiation interaction model in NSCLC, employing a traditional clinical modality (radiation), a relatively new, therapeutically unexplored agent (4HPR) and rationally combining them based on molecular mechanistic findings pertaining to their interactions. To test the hypothesis that 4HPR sensitizes cells to radiation-induced cell death via G2+M accumulation, we designed a working model consisting of H522 adenocarcinoma cells (p53, K-ras mutated) derived from an NSCLC patient; 4HPR at concentrations up to 10 μM; and X radiation up to 6 Gy generated by a patient-dedicated Phillips RT-250 X ray unit at 250 KV, 15 mA, 1.85 Gy/min. We found that 4HPR produced time- and dose-dependent morphological changes, growth inhibition, and DNA damage-inducing enhancement of reactive oxygen species. A transient G2+M accumulation of cells maximal at 24 h of continuous 4HPR exposure was used for irradiation time scheduling. Our data demonstrated enhanced cell death (both apoptotic and necrotic) in irradiated cells pre-treated with 4HPR versus those with either stressor alone. 4HPR's effect of increased NSCLC cells' radioresponse was confirmed by clonogenic assay. To explore these practical findings from a molecular mechanistic perspective, we further investigated and showed that levels of cyclin B1 and p34cdc2 kinase—both components of the mitosis promoting factor (MPF) regulating the G2/M transition—did not change following 4HPR treatment. Likewise, cdc25C phosphatase was not altered. However, enhanced p34cdc2 phosphorylation on its Thr14Tyr15 residues—indicative of its inactivation and increased expression of MPF negative regulators chk1 and wee1 kinases—were supportive of explaining 4HPR-treated cells' accumulation. Hence, p34cdc2 phosphorylation, chk1, and wee1 warrant further evaluation as potential molecular targets for 4HPR-X radiation combination. In summary, we (1) demonstrated that 4HPR not only induces cell death by itself, but also increases NSCLC cells' subsequent radioresponse, indicative of potential clinical applicability, and (2) for the first time, shed light on deciphering 4HPR-X radiation molecular mechanisms of interaction, including the finding of 4HPR's role as a p34cdc2 inactivator via Thr14Tyr15 phosphorylation. ^

Delineating the molecular basis of human genetic diseases: Epigenetic and functional studies

Identifying and characterizing the genes responsible for inherited human diseases will ultimately lead to a more holistic understanding of disease pathogenesis, catalyze new diagnostic and treatment modalities, and provide insights into basic biological processes. This dissertation presents research aimed at delineating the genetic and molecular basis of human diseases through epigenetic and functional studies and can be divided into two independent areas of research. The first area of research describes the development of two high-throughput melting curve based methods to assay DNA methylation, referred to as McMSP and McCOBRA. The goal of this project was to develop DNA methylation methods that can be used to rapidly determine the DNA methylation status at a specific locus in a large number of samples. McMSP and McCOBRA provide several advantages over existing methods, as they are simple, accurate, robust, and high-throughput making them applicable to large-scale DNA methylation studies. McMSP and McCOBRA were then used in an epigenetic study of the complex disease Ankylosing spondylitis (AS). Specifically, I tested the hypothesis that aberrant patterns of DNA methylation in five AS candidate genes contribute to disease susceptibility. While no statistically significant methylation differences were observed between cases and controls, this is the first study to investigate the hypothesis that epigenetic variation contributes to AS susceptibility and therefore provides the conceptual framework for future studies. ^ In the second area of research, I performed experiments to better delimit the function of aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), which when mutated causes various forms of inherited blindness such as Leber congenital amaurosis. A yeast two-hybrid screen was performed to identify putative AIPL1-interacting proteins. After screening 2 × 106 bovine retinal cDNA library clones, 6 unique putative AIPL1-interacting proteins were identified. While these 6 AIPL1 protein-protein interactions must be confirmed, their identification is an important step in understanding the functional role of AIPL1 within the retina and will provide insight into the molecular mechanisms underlying inherited blindness. ^

Molecular and genetic analyses of Fgf signaling during cardiac outflow tract development

Epithelial-mesenchymal tissue interactions regulate the development of derivatives of the caudal pharyngeal arches (PAs) to govern the ultimate morphogenesis of the aortic arch and outflow tract (OFT) of the heart. Disruption of these signaling pathways is thought to contribute to the pathology of a significant proportion of congenital cardiovascular defects in humans. In this study, I tested whether Fibroblast Growth Factor 15 (Fgf15), a secreted signaling molecule expressed within the PAs, is an extracellular mediator of tissue interactions during PA and OFT development. Analyses of Fgf15−/− mouse embryonic hearts revealed abnormalities primarily localized to the OFT, correlating with aberrant cardiac neural crest cell behavior. The T-box-containing transcription factor Tbx1 has been implicated in the cardiovascular defects associated with the human 22q11 Deletion Syndromes, and regulates the expression of other Fgf family members within the mouse PAs. However, expression and genetic interaction studies incorporating mice deficient for Tbx1, its upstream regulator, Sonic Hedgehog (Shh), or its putative downstream effector, Fgf8, indicated that Fgf15 functions during OFT development in a manner independent of these factors. Rather, analyses of compound mutant mice indicated that Fgf15 and Fgf9, an additional Fgf family member expressed within the PAs, genetically interact, providing insight into the factors acting in conjunction with Fgf15 during OFT development. Finally, in an effort to further characterize this Fgf15-mediated developmental pathway, promoter deletion analyses were employed to isolate a 415bp sequence 7.1Kb 5′ to the Fgf15 transcription start site both necessary and sufficient to drive reporter gene expression within the epithelium of the PAs. Sequence comparisons among multiple mammalian species facilitated the identification of evolutionarily conserved potential trans-acting factor binding sites within this fragment. Subsequent studies will investigate the molecular pathway(s) through which Fgf15 functions via identification of factors that bind to this element to govern Fgf15 gene expression. Furthermore, targeted deletion of this element will establish the developmental requirement for pharyngeal epithelium-derived Fgf15 signaling function. Taken as a whole, these data demonstrate that Fgf15 is a component of a novel, Tbx1-independent molecular pathway, functioning within the PAs in a manner cooperative with Fgf9, required for proper development of the cardiac OFT. ^

Molecular genetic analyses of extracellular signaling pathways during murine retinal development

Extracellular signaling pathways initiated by secreted proteins are important in the co-ordination of tissue interactions in multi-cellular organisms, particularly during embryonic development. These signaling cascades direct diverse cellular events, including proliferation, differentiation and migration, in both autocrine and paracrine modes. In adult animals, abnormal function of these proteins often results in degenerative and tumourigenic syndromes. In this study, I have focused on elucidating the role of Bone Morphogenetic Protein (Bmp) signal transduction during neuronal specification and differentiation in the vertebrate embryo, using the mouse retina as a model. Using tissue-specific conditional knock-out approaches, the consequences of genetic loss-of-function of this signaling pathway on retinal physiology were examined. Mutant mice lacking Bmp type I receptor function displayed a range of retinal phenotypes, each of which appeared to be regulated at a different threshold of Bmp receptor activity. Novel essential functions for Bmp signaling were uncovered for retinal neurogenesis, cell survival, and axonal pathfinding at the optic disc. Further, BmprIa and BmprIa exhibited genetic interactions suggestive of functional redundancy. To further characterize the underlying molecular bases for the pleiotropic effects of Bmp receptors, retina-specific loss-of-function mutants of the obligate Bmp-activated transcriptional mediator Smad4 were generated. A comparison of the retina-specific Smad4 mutant phenotypes with those of the Bmp receptor mutant retina revealed that only a subset of retinal phenotypes, namely optic disc axon pathfinding and axial patterning were common for both classes of mutant animals. Thus, these results suggest that, contrary to the classic scheme of Bmp signal transduction, Smad4-independent pathways may be operative downstream of the type I receptors. Indeed, such alternative intracellular signaling cascades may constitute a molecular basis for the multiple cellular responses elicited by Bmp signaling. Finally, I tested whether the potential Bmp pathway targets, the extracellular ligands Fgf9 and Fgf15, mediate essential cellular processes in the retina. The analyses of Fgf9 −/−; Fgf15−/− mutant mice posit a novel shared role for these genes in intra-retinal axon pathfinding. Collectively, these studies have elucidated part of the molecular machinery directing mammalian neuro-retinal development, and provided useful in vivo models to study visual function. ^

Novel molecular insights into human tooth agenesis

The development of dentition is a fascinating process that involves a complex series of epithelial-mesenchymel signaling interactions. That such a precise process frequently goes awry is not surprising. Indeed, tooth agenesis is one of the most commonly inherited disorders in humans that affects up to twenty percent of the population and imposes significant functional, emotional and financial burdens on patients. Mutations in the paired box domain containing transcription factor PAX9 result in autosomal dominant tooth agenesis that primarily involves posterior dentition. Despite these advances, little is known about how PAX9 mediates key signaling actions in tooth development and how aberrations in PAX9 functions lead to tooth agenesis. As an initial step towards providing evidence for the pathogenic role of mutant PAX9 proteins, I performed a series of molecular genetic analyses aimed at resolving the structural and functional defects produced by a number of PAX9 mutations causing non-syndromic posterior tooth agenesis. It is likely that the pathogenic mechanism underlying tooth agenesis for the first two mutations studied (219InsG and IIe87Phe) is haploinsufficiency. For the six paired domain missense mutations studied, the lack of functional defects observed for three of the mutant proteins suggests that these mutations altered PAX9 function through alternate mechanisms. Next, I explored further the nature of the partnership between Pax9 and the Msx1 homeoprotein and their role in the expression of a downstream effector molecule, Bmp4. When viewed in the context of events occurring in dental mesenchyme, the results of these studies indicate that the Pax9-Msx1 protein interaction involves the localized up-regulation of Bmp4 activity that is mediated by synergistic interactions between the two transcription factors. Importantly, these assays corroborate in vivo data from mouse genetic studies and support reports of Pax9-dependent expression of Bmp4 in dental mesenchyme. Taken together, these results suggest that PAX9 mutations cause an early developmental defect due to an inability to maintain the inductive potential of dental mesenchyme through involvement in a pathway involving Msx1 and Bmp4. ^

Biochemical and genetic characterization of the Saccharomyces cerevisiae Hsp110 molecular chaperone Sse1

The Ssel/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a carboxyl-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an amino-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Δ phenotypes. Surprisingly, all mutants predicted to abolish ATP hydrolysis complemented the temperature sensitivity of sse1Δ, whereas mutations in predicted ATP binding residues were non-functional. Remarkably, the two domains of Ssel when expressed in trans functionally complement the sse1Δ growth phenotype and interact by coimmunoprecipitation analysis, indicative of a novel type of interdomain communication. ^ Relatively little is known regarding the interactions and cellular functions of Ssel. Through co-immunoprecipitation analysis, we found that Ssel forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. The ATPase domains of Ssel and the Hsp70s were found to be critical for interaction as inactivating point mutations severely reduced interaction efficiency. Ssel stimulated Ssal ATPase activity synergistically with the co-chaperone Ydj1 via a novel nucleotide exchange activity. Furthermore, FES1, another Ssa nucleotide exchange factor, can functionally substitute for SSE1/2 when overexpressed, suggesting that Hsp70 nucleotide exchange is the fundamental role of the Sse proteins in yeast, and by extension, the Hsp110 homologs in mammals. ^ Cells lacking SSE1 were found to accumulate prepro-α-factor, but not the cotranslationally imported protein Kar2, similar to mutants in the Ssa chaperones. This indicates that the interaction between Ssel and Ssa is functionally significant in vivo. In addition, sse10 cells are compromised for cell wall strength, likely a result of decreased Hsp90 chaperone activity with the cell integrity MAP kinase SIC. Taken together, this work established that the Hsp110 family must be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.^

An integrated molecular biology and computational approach to CYP1A1 expression in human brain

The cytochromes P450 comprise a superfamily of heme-containing mono-oxygenases. These enzymes metabolize numerous xenobiotics, but also play a role in metabolism of endogenous compounds. The P450 1A1 enzyme generally metabolizes polycyclic aromatic hydrocarbons, and its expression can be induced by aryl hydrocarbon receptor (AhR) activation. CYP1A1 is an exception to the generality that the majority of CYPs demonstrate highest expression in liver; CYP1Al is present in numerous extrahepatic tissues, including brain. This P450 has been observed in two forms, wildtype (WT) and brain variant (BV), arising from alternatively spliced mRNA transcripts. The CYP1A1 BV mRNA presented an exon deletion and was detected in human brain but not liver tissue of the same individuals. ^ Quantitative PCR analyses were performed to determine CYP1A1 WT and BV transcript expression levels in normal, bipolar disorder or schizophrenic groups. In our samples, we show that CYP1A1 BV mRNA, when present, is found alongside the full-length form. Furthermore, we demonstrate a significant decrease in expression of CYP1A1 in patients with bipolar disorder or schizophrenia. The expression level was not influenced by post-mortem interval, tissue pH, age, tobacco use, or lifetime antipsychotic medication load. ^ There is no indication of increased brain CYP1A1 expression in normal smokers versus non-smokers in these samples. We observed slightly increased CYP1A1 expression only in bipolar and schizophrenic smokers versus non-smokers. This may be indicative of complex interactions between neuronal chemical environments and AhR-mediated CYP1A1 induction in brain. ^ Structural homology modeling demonstrated that P450 1A1 BV has several alterations to positions/orientations of substrate recognition site residues compared to the WT isoform. Automated substrate docking was employed to investigate the potential binding of neurological signaling molecules and neurotropic drugs, as well as to differentiate specificities of the two P450 1A1 isoforms. We consistently observed that the BV isoform produced energetically favorable substrate dockings in orientations not observed for the same substrate in the WT isoform. These results demonstrated that structural differences, namely an expanded substrate access channel and active site, confer greater capacity for unique compound docking positions suggesting a metabolic profile distinct from the wildtype form for these test compounds. ^

Characterization of the interactions between fibronectin and the Borrelia burgdorferi lipoprotein, BBK32

The findings presented in this dissertation detail the complex interaction between BBK32 and fibronectin and describe novel consequences of the interaction. BBK32 is a fibronectin-binding protein on Borrelia burgdorferi, the causative agent of Lyme disease. We found that BBK32 contains multiple fibronectin-binding motifs, recognizing the fibronectin N-terminal domain (NTD) and the gelatin binding domain (GBD) in an anti-parallel order, where corresponding sites in BBK32 and fibronectin are aligned so that there is a one-to-one interaction between the proteins. While characterizing this interaction, we discovered that binding of BBK32 to the GBD inhibits the migration stimulating factor's (MSF) motogenic activity. In the presence of BBK32, endothelial cells do not migrate in response to increasing concentrations of MSF or the GBD. MSF is found under wound healing conditions, and inhibition of its activity may allow the tick-transmitted spirochetes to delay wound healing and to establish an infection. ^ Biophysical structural studies, designed to identify a mechanism of interaction, revealed that BBK32 binding to the NTD leads to the unfolding of plasma fibronectin, which exposes α5β1 integrin recognition motifs. Binding assays demonstrate that the BBK32-NTD interaction enhances the plasma fibronectin-α5β1 integrin interaction, which may allow B. burgdorferi to invade host cells, and thereby evade the host immune system. ^ We also determined that BBK32 binds fibronectin F3 modules, which leads to plasma fibronectin aggregation and induction of superfibronectin. The resulting superfibronectin is conformationally distinct from plasma and cellular fibronectin, and can inhibit endothelial cell proliferation. BBK32's active superfibronectin-forming motif has been located to a region between residues 160 and 175, which contains two sequence motifs that are also found in anastellin, the only other known superfibronectin-inducing protein. ^ A potential consequence of BBK32-induced superfibronectin formation was identified. BBK32-induced superfibronectin formation results in the exposure of α4β1 integrin recognition sequences in fibronectin. The α4β1 integrin is required for leukocyte transendothelial cell migration. BBK32-induced superfibronectin inhibits this activity. The inhibition of leukocyte recruitment to the infection site may slow the activity of the host immune system, and permit the spirochetes to establish an infection. ^

Molecular mechanisms by which p53/LSD1 and ERalpha/Trim24 complexes mediate gene regulation

In this dissertation, I identify two molecular mechanisms by which transcription factors cooperate with their co-regulators to mediate gene regulation. In the first part, I demonstrate that p53 directly recruits LSD1, a histone demethylase, to AFP chromatin to demethylate methylated H3K4 and actively mediate transcription repression. Loss of p53 and LSD1 interaction at chromatin leads to derepression of AFP in hepatic cells. In the second part, I reveal that Trim24 functions as an important co-activator in ERα-mediated gene activation in response to estrogen stimulation. Trim24 is recruited by ligand-bound ERα to chromatin and stabilizes ERα-chromatin interactions by binding to histone H3 via its PHD finger, which preferentially recognizes unmethylated H3K4. ^

GENETIC INTERACTIONS OF FACTORS THAT REGULATE ALTERNATIVE RNA SPLICING IN THE MALE GERM LINE OF DROSOPHILA

Alternative RNA splicing is a critical process that contributes variety to protein functions, and further controls cell differentiation and normal development. Although it is known that most eukaryotic genes produce multiple transcripts in which splice site selection is regulated, how RNA binding proteins cooperate to activate and repress specific splice sites is still poorly understood. In addition how the regulation of alternative splicing affects germ cell development is also not well known. In this study, Drosophila Transformer 2 (Tra2) was used as a model to explore both the mechanism of its repressive function on its own pre-mRNA splicing, and the effect of the splicing regulation on spermatogenesis in testis. Half-pint (Hfp), a protein known as splicing activator, was identified in an S2 cell-based RNAi screen as a co-repressor that functions in combination with Tra2 in the splicing repression of the M1 intron. Its repressive splicing function is found to be sequence specific and is dependent on both the weak 3’ splice site and an intronic splicing silencer within the M1 intron. In addition we found that in vivo, two forms of Hfp are expressed in a cell type specific manner. These alternative forms differ at their amino terminus affecting the presence of a region with four RS dipeptides. Using assays in Drosophila S2 cells, we determined that the alternative N terminal domain is necessary in repression. This difference is probably due to differential localization of the two isoforms in the nucleus and cytoplasm. Our in vivo studies show that both Hfp and Tra2 are required for normal spermatogenesis and cooperate in repression of M1 splicing in spermatocytes. But interestingly, Tra2 and Hfp antagonize each other’s function in regulating germline specific alternative splicing of Taf1 (TBP associated factor 1). Genetic and cytological studies showed that mutants of Hfp and Taf1 both cause similar defects in meiosis and spermatogenesis. These results suggest Hfp regulates normal spermatogenesis partially through the regulation of taf1 splicing. These observations indicate that Hfp regulates tra2 and taf1 activity and play an important role in germ cell differentiation of male flies.

Characterization of Jak, STAT, and Src interactions in Head and Neck Squamous Cell Carcinoma

Recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is common; thus, it is essential to improve the effectiveness and reduce toxicity of current treatments. Proteins in the Src/Jak/STAT pathway represent potential therapeutic targets, as this pathway is hyperactive in HNSCC and it has roles in cell migration, metastasis, proliferation, survival, and angiogenesis. During short-term Src inhibition, Janus kinase (Jak) 2, and signal transducer and activator of transcription (STAT) 3 and STAT5 are dephosphorylated and inactivated. Following sustained Src inhibition, STAT5 remains inactive, but Jak2 and STAT3 are reactivated following their early inhibition. To further characterize the mechanism of this novel feedback pathway we performed several experiments to look at the interactions between Src, Jak2, STAT5 and STAT3. We attempted to develop a non-radioactive kinase assay using purified recombinant Jak2 and Src proteins, but found that phospho-tyrosine antibodies were non-specifically binding to purified recombinant proteins. We then performed in vitro kinase assays (IVKAs) using purified recombinant Jak2, Src, STAT3, and STAT5 proteins with and without Src and Jak2 pharmacologic inhibitors. We also examined the interactions of these proteins in intact HNSCC cells. We found that recombinant Jak2, STAT3, and STAT5 are direct substrates of Src and that recombinant Src, STAT3, and STAT5 are direct substrates of Jak2 in the IVKA. To our knowledge, the finding that Src is a Jak substrate is novel and has not been shown before. In intact HNSCC cells we find that STAT3 can be reactivated despite continuous Src inhibition and that STAT5 continues to be inhibited despite Jak2 reactivation. Also, Jak2 inhibition did not affect Src or STAT5 activity but it did cause STAT3 inhibition. We hypothesized that the differences between the intact cells and the IVKA assays were due to a potential need for binding partners in intact HNSCC cells. One potential binding partner that we examined is the epidermal growth factor receptor (EGFR). We found that EGFR activation caused increased activation of Src and STAT5 but not Jak2. Our results demonstrate that although STAT3 and STAT5 are capable of being Src and Jak2 substrates, in intact HNSCC cells Src predominantly regulates STAT5 and Jak2 regulates STAT3. Regulation of STAT5 by Src may involve interactions between Src and EGFR. This knowledge along with future studies will better define the mechanisms of STAT regulation in HNSCC cells and ultimately result in an ideal combination of therapeutic agents for HNSCC.