Real-time Measurement of Glial Progenitor Chemotactic Migration
Year of Dissertation:
2012
Gliomas are the most commonly diagnosed form of central nervous system tumors, occurring primarily in adults. Like many malignant cancers, gliomas pathologically exhibit very aggressive spreading and lead to an average diagnosed survival expectancy of twelve months. This prognosis is due in large part to the uncontrolled division and migration of malignant tumor cells within healthy brain, which makes complete surgical resection impossible. Gliomas are known to contain numerous genotypic and phenotypic alterations that affect cell proliferation and survival. Previous research has indicated that both gliomas and their precursor cells exhibit distinct migration patterns in brain tissue, which may be induced by specific cytokines and their concentration gradients. Here, we investigated the migration of four brain tumor cell (BTCs) lines (U-87 MG; U-251 MG; Daoy; and XFMPDGF) and three RCAS-infected glial progenitor cell (GPCs) populations (GPCLacZ, GPCPDGF, and GPCkRas) toward various growth factors, including but not limited to: EGF, HGF/SF, PDGF-BB, and TGF-α.
COMPUTATIONAL STUDIES ON INTERACTION BETWEEN SOME RECEPTORS AND LIGANDS OF TRANSFORMING GROWTH FACTOR-BETA SUPERFAMILY: DESIGN OF INHIBITOR(S) TO PROMOTE OSTEOGENESIS
Year of Dissertation:
2010
Transforming growth factor beta (TGF-β) superfamily members execute distinct and intricate roles in numerous biological events such as cell growth, differentiation, embryogenesis, immune responses, and morphogenesis etc. Diverse cellular responses are instigated by binding of these superfamily members to specific transmembrane serine/threonine kinase receptors on the cell surface and thereby activating specific pathways. As a result receptor-regulated Smad proteins are phosphorylated, followed by their complex formation with Co-Smad that together translocates into nucleus and leads to the initiation of transcription of target genes. Bone morphogenic proteins (BMPs), activins, Inhibins are the most important members of TGF-β family. When they exert their biological activity, they are sternly regulated by extracellular antagonists such as noggin, follistatin, CV2, and so forth that are expressed in close temporal and spatial proximity. Blocking these antagonists' interaction with their target receptor proteins helps to promote their respective biological responses when needed. This work is an attempt to use the current understanding of some of the receptors and ligands of TGF- superfamily members, namely BMPs, activins and inhibins and their antagonists such as noggin, follistatin, and crossveinless 2 (CV2) interactions through the analysis of their available complex structures to design small molecular inhibitors with an ultimate goal of promoting their respective biological responses.
Dissecting the role of human PPR motif proteins in mitochondrial gene expression
Author:
Catherine Bangeranye
Year of Dissertation:
2010
Advisor:
Serafin Piñol-Roma
Pentatricopeptide repeat (PPR) motif proteins constitute a growing superfamily of proteins that are broadly defined by the presence of one or more copies of a conserved 35 amino acid sequence, the PPR motif. They are particularly abundant in plants, and those whose function has been characterized have been implicated in several aspects of RNA metabolism in mitochondria and chloroplasts. In humans, PPR motif proteins are fewer in number. They include LRPPRC (Leucine-Rich PPR-motif -Containing protein), an RNA-binding protein that is a component of nuclear ribonucleoprotein (RNP) complexes that contain spliced mRNAs. Most of the LRPPRC, however, localizes predominantly to mitochondria, where it binds polyadenylated RNAs. Mutations in the lrpprc gene cause cytochrome c oxidase deficiency in Leigh Syndrome (LSFC), which is accompanied by a decrease in COXI and COXIII mitochondrial mRNAs. Our hypothesis is that LRPPRC is an essential trans-acting factor in mitochondrial mRNA metabolism. In order to address the function of LRPPRC in mitochondria, we isolated LRPPRC-associated mitochondrial RNP complexes (mtRNPs). Analysis of isolated mtRNPs shows that the mitochondrially-encoded mRNAs associate with LRPPRC. A reduction in LRPPRC levels using RNAi causes a parallel reduction in steady-state levels of mitochondrially-encoded mRNAs, but not of nuclear-encoded mRNAs. Thus, LRPPRC is an important factor for mitochondrial gene expression and is necessary for the accumulation of the mitochondrial mRNAs to which it binds. Using LRPPRC as a paradigm, we sought and analyzed other members of the PPR motif family in humans. Four other human PPR-motif proteins, PTCD1, PTCD2, PTCD3 and PTCD4, also localize in mitochondria. Moreover, some of these proteins also bind RNA and exist in the same complexes as LRPPRC. This indicates that the human PTCD proteins, as is the case with LRPPRC, are also involved in mitochondrial RNA metabolism, pointing to PPR motif proteins in humans as a novel family of trans-acting factors in mitochondrial gene expression. These findings open the way for an expanded and more detailed understanding of human mitochondrial gene expression, and for an exploration of the potential involvement of human PPR motif proteins in mitochondrial diseases, as has already been determined for LRPPRC.
The role of Mdm2 in estrogen-mediated breast cancer cell proliferation
Year of Dissertation:
2011
Estrogen signaling is important in breast cancer development and progression. Mdm2, a negative regulator of the p53 tumor suppressor, is often over-expressed in estrogen receptor positive breast cancers. To study the role of Mdm2 in the estrogen-mediated breast cancer cell proliferation, we examined the effect of estrogen on the p53-Mdm2 pathway in estrogen receptor positive and p53 wild-type MCF-7 breast cancer cells. Estrogen-mediated increase in cell proliferation correlated with increased Mdm2, but no concomitant decrease in the p53 protein level. Blocking Mdm2 expression with inducible shRNA inhibited estrogen-mediated cell proliferation and colony formation in soft agar. Mdm2 knockdown in the presence of estrogen increased p21 and the percent of cells in the G1 phase. Interestingly, knockdown of p53 had no effect on the estrogen-mediated cell proliferation. Estrogen also up-regulated the Mdm2 protein levels in cells exposed to the DNA damaging agent, etoposide, and the Mdm2 inhibitor, Nutlin-3. In turn, estrogen inhibited etoposide- and Nutlin-3-induced transcription of puma, a pro-apoptotic p53 target gene, without changing the p53 protein levels or p53 recruitment to the chromatin. The decrease in puma gene transcription correlated with a decrease in Puma protein and an increase in Bcl-2 protein, an anti-apoptotic estrogen receptor target. Overall, our findings suggest that estrogen signals to an Mdm2-mediated pathway to provoke cell proliferation and that this pathway is associated with inhibition of the G1 checkpoint.
Expression, Purification, Circular Dichroism, and NMR Analysis of Triple Transmembrane Domain Containing Fragments of a GPCR
Year of Dissertation:
2013
G protein-coupled receptors (GPCRs) are the largest class of signaling molecules in eukaryotes and are important pharmacological targets. Structural characterization of GPCRs is of paramount importance to the discovery of more efficient drugs; however, these studies are hindered by the inherent hydrophobicity, flexibility, and large size of these signaling proteins. Since their flexibility makes crystallization difficult, stabilizing mutations or substitutions are required to facilitate crystal-packing contacts. The size of the receptor/membrane mimetic complex required for solution-state nuclear magnetic resonance (NMR) analysis is too large to enable efficient isotropic tumbling. For these reasons, high-resolution structural information is available for only thirteen of the ~1000 GPCRs identified to date.
Computational Studies of the Functional States Associated with Epidermal Growth Factor Receptor Activation
Year of Dissertation:
2011
Epidermal growth factor receptors (EGFR) belong to the ErbB family of receptor tyrosine
Polyadenylation/Deadenylation/Tumor Suppressor Factors Regulate 3' End Processing Under Different Cellular Conditions
Year of Dissertation:
2009
Under DNA damaging conditions the steady-state levels of cellular mRNAs change as a result of regulation of either or both their biosynthesis and turnover. mRNA 3' end cleavage, involved in the regulation of mRNA stability, is strongly but transiently inhibited upon UV treatment. This inhibition is mediated by both the formation of the BRCA1/BARD1/CstF complex and the proteasomal-mediated degradation of RNA polymerase II (RNAP II). As CstF-50 interacts with the tumor suppressor BARD1 to inhibit 3' processing and with RNAP II to activate 3' cleavage, it has been proposed that this cleavage factor plays a coordinating role in the DNA damage response.
STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF RHOMBOID INTRAMEMBRANE PROTEASES
Year of Dissertation:
2011
Intramembrane proteases are involved in multiple biological processes including cell growth and development, and apoptosis. There is no conservancy between hydrosoluble and membrane proteases. However, the catalytic residues and surrounding amino acids are absolutely conserved, suggesting that they both protein families share catalytic mechanisms but with two remarkable differences. (1) The ability of intramembrane proteases to cleave their substrates in the hydrophobic interior of the lipid bilayer, and (2) to do so in regions where the substrate displays a-helical conformation. D. melanogaster rhomboid-1 cleaves within the transmembrane domain region of epidermic growth factor receptor (EGFR) ligands Gurken, Keren and Spitz, resulting in their extracellular export. We designed substrate chimeras in which the transmembrane and cytoplasmic regions of Gurken, Keren and Spitz were preserved, while their EGFR ligand ectodomain was replaced by maltose binding protein. In vitro activity assays in detergent using purified components showed that rhomboid-1, H. sapiens RHBDL2, P. aeruginosa PA3086 and E. coli GlpG display comparable activity against these substrate chimeras. Mass spectrometry analysis of the N-terminal reaction product identified a single cleavage site after Ala138 for the Spitz chimeras, after Ala122 for the Keren chimeras, and after Ala245 for the Gurken chimeras that was identical for all rhomboids tested, suggesting a conservation of proteolytic profiles among prokaryotic and eukaryotic rhomboids. The identified cleavage site was located towards the N-terminal end of the transmembrane domain of each substrate. Positions that were sensitive to alanine scanning were further studied by introducing additional mutations to show that aside of ala in position P1, amino acids with low-helical propensities are necessary in the positions P2 and P1'. Finally, a bulky hydrophobic residue with a high helical propensity is important in P2' position to control the location of cleavage. We also carried out structural work and solved the N-terminal domain of Rhomboid and showed it displays high-affinity for membranes. Our work is put in a more general context by comparison with other intramembrane proteases and future work to unravel the mechanism of substrate binding and unwinding is also discussed.
Protein Kinase C Substrates That Drive Motility of Cancer Cells
Year of Dissertation:
2010
As the intracellular receptor of tumor promoting phorbol esters, protein kinase C (PKC) is functionally linked to carcinogenesis and metastasis. Therefore, it is crucial to identify substrates of PKC in order to understand the mechanisms by which these substrate proteins participate in cancer-related phenotypes such as motile behavior. The work to be described consists of two projects: 1) new PKC substrates that contribute to the motility phenotype of human breast cells, and 2) the role of a known PKC substrate, MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate) in the motility pathway of mouse melanoma cells.
Structure Determination of a Double Transmembrane Fragment of the G protein-coupled Receptor Ste2p in Membrane Mimetic Environments
Year of Dissertation:
2010
G-protein coupled receptors (GPCRs) are relevant in cellular signal transduction pathways and are targets for disease therapeutics. Since the sequencing of the human genome, there have been close to 1000 GPCRs predicted and many have been characterized by biological and biochemical analysis. Though these integral membrane proteins (IMPs) have little sequence similarity, they show strong putative structural similarities. All GPCRs contain an N-terminal extracellular domain (NT), 7 transmembrane helical regions (TM) connected by intra- and extracellular loops (IL and EL, respectively), and a C-terminal intracellular tail (CT). The extracellular domains are thought to play a role in ligand-receptor interactions and together with the TM domains form the ligand binding site for many GPCRs.