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Role of mammalian ubiquitin ligases UBR1 and UBR2 in cytosolic protein quality control
MST. RASHEDA SULTANA
Year of Dissertation:
UBR1 and UBR2 ubiquitin ligases function in the N-end rule degradation pathway in lower and higher eukaryotic cells. In yeast, the Ubr1 homologue also functions by N-end rule independent means to promote degradation of misfolded proteins generated via stress or with Hsp90 inhibitor GA. Based on these studies I examined the role of mammalian UBR1 in the degradation of protein kinase clients upon Hsp90 inhibition. I provide evidence that mammalian UBR1 promotes protein kinase quality control and sensitizes the cells to Hsp90 inhibition. The UBR1 deleted MEF cells showed reduced degradation of several protein kinases in the presence of GA. My findings also showed that Akt, p-Akt and Cdk4 the Hsp90 client protein kinases are still degraded in mouse UBR1 -/- cells treated with GA, but their levels recovered within 12-18 hours, in contrast to the wild type cells. The same findings were observed for human BT474 breast cancer cells with knocked down UBR1 by shRNA. These findings correlate with increased induction of Hsp90 expression in the Ubr1-/- cells compared with wild type cells. In addition, deletion of UBR1 and UBR2 showed resistance in terms of cell viability compared to wild type cells in the presence of GA and PU-H71. I also observed a reduction of UBR1 protein levels in GA-treated MEF and BT474 cells, suggesting that UBR1 is an Hsp90 client. I propose the existance of a novel feedback loop, where UBR1 negatively controls Hsp90 expression, while Hsp90 controls UBR1 stability. Further studies with CHIP reveal that CHIP and UBR1 have some functional overlap with respect to their E3 activities while UBR1 also affects the function of the Hsp90 chaperone machinery. My studies with other Hsp90 clients showed that UBR1 promotes degradation of steroid hormone receptors GR and AR but not the ER- £\. Co-expression of rUBR1 with hGR led to reduce the levels of hGR in the presence and absence of GA. There is a direct correlation between increasing UBR1 concentration and decreasing GR levels. Further studies addressed the specificity of that function with analysis of hAR and hER-fÑ. In this case, there was a significant reduction of the hAR levels when UBR1 was overexpressed, even in the absence of GA. By contrast, similar experiments with transfected hER-£\ suggest that UBR1 does not play a similar role in the degradation of this receptor. My combined findings suggest that UBR1 acts specifically in the clearance of GR and AR but not in ER-fÑ. All of these findings suggest that UBR1 is involved in the cytosolic protein quality control in mammalian system and it also plays a role in determining the sensitivity of the cells to the Hsp90 inhibitors.
BIOPHYSICAL CHARACTERIZATION OF HCV Q65H NS5B AND EIF4B : PROTEIN - NUCLEIC ACID INTERACTIONS
Year of Dissertation:
Hepatitis C Virus (HCV) infection is one of the most common chronic blood borne infections. The mechanism underlying persistence of HCV infections are not well understood. Mutation of Gln65His (Q65H), which develops in the NS5B dependent RNA polymerase (RdRp) during chronic infection, has 1.8 fold enhanced invitro RdRp activity, may have major implications in the efficiency of HCV RNA replication and viral persistence. The reason for this increase in activity is not well understood (Virology 2003 Dec 5; 317(1):65-72). We used endogenous tryptophan fluorescence to characterize the interactions of Q65H protein with GTP, GDP, magnesium and a 19mer RNA template. The Q65H protein exhibits 3 fold lower Kd (11.6 ± 1.2 µM) than wildtype (36.8 ± 1.0 µM) for GTP, whereas the Kd values of the mutant for magnesium and 19 mer RNA template were similar to wildtype protein. Thermodynamic studies on the binding reaction of the Q65H variant were also performed to provide details of the energetic and entropic characteristics. The second part of this study involved interaction of eIF4B with zinc. eIF4B promotes RNA dependent ATP hydrolysis and ATP dependent RNA helicase activity of eIF4A and eIF4F. It has recently been reported that eIF4B also organizes assembly of the RNA, eIFiso4G, eIF4A, and poly-(A) binding protein and that zinc enhances the RNA binding and interaction with PABP. We previously reported that zinc binds tightly with eIF4B with a Kd of 19.7 ± 1.6 nM (J Biol Chem. 2008 Dec 26; 283(52):36140-53). The Kd of binding of eIF4B with 20mer poly (A) RNA is 77 ± 7 nM and in presence of zinc is reduced down to 45 ± 3 nM. Circular dichroism showed that addition of zinc resulted in a more than 50% decrease in alpha content of the eIF4B protein. To better understand the role of eIF4B in the selection of RNA, we also studied the interaction of eIF4B with structurally different RNAs and the effect eIF4A on this interaction using fluorescence anisotropy. This overall study on eIF4B helps towards the better understanding of basic translation initiation machinery and some of its regulators.
Green Drugs: Anticancer Properties of Clerodendrum viscosum and Curcumin Conjugates
Year of Dissertation:
This thesis aims to find potential anti-cancer drug from natural sources like plants. (i) The objectives are to find and characterize the anticancer components from the plant Clerodendrum viscosum. (ii) the synthesis of curcumin derivatives with increased solubility and amplified bioactivity. Clerodendrum viscosum (Cv) is a traditional medicine plant in India for a long period of time employed to promote wound healing, and recently it has been found have some positive effects on cervical cancer. However, there were no direct v molecular and biochemical evidences for that. In this thesis, we made aqueous extracts (Cv-ap) from the Cv by ammonium sulfate precipitation. It was found that this extract had bioactivity against Hela cells - a human cervical cancer cell line. Caspase-3 immuno-staining proved that the Cv-ap can induce Hela cells apoptosis. It is also interesting that the Cv-ap can inhibit the movement of the cells at lower concentration without inducing cells apoptosis, which means it may stop the invasion of the cancer cells. MTT assays showed that at lower concentration the Cv-ap may not only inhibit the movement of the cells but also inhibit proliferation of the Hela cells. For the characterization of the Cv-ap, it was found that it had glycoproteins confirmed by SDS-PAGE staining with the Pro-Q emerald 300, and by binding to the concavalin A column. For further purification, the bio-assay guided method was used to purify the bioactivity components. After Cv-ap was passed through the column, the fraction with the most activity fraction was selected and followed by heating, and finally by precipitation with 66.7% ethanol. In the end we got the vi bioactivity component EPHP3 fraction which has a 30 fold increasing activity when compared to the Cv-ap. My second project aimed to overcome the difficulties associated with the low water/plasma solubility of the potent anti-oxidant, anti-inflammatory, anti-carcinogenic, anti- Alzheimer's active curcumin. We have successfully synthesized a curcumin derivate BSA-curcumin via a one-step successfully synthesized. BSA-curcumin had the high solubility in water and good cytotoxicity to the Hela cells. In conclusion, my research has characterized and produced novel plant derived components that have anti-cancer activity and may eventually be used in clinical therapies.
Identification and Mechanistic Study of Immunostimulatory Lipids in Juzen-Taiho-To
Year of Dissertation:
Juzen–taiho–to (JTT) is an herbal medicine known to exhibit safe and effective immunostimulatory activity. It is clinically used to improve the immune functions of cancer patients undergoing chemotherapy and radiation therapy in Japan. The chemical constituents responsible for its therapeutic effects are poorly characterized due to the chemical complexity of this formulation and possible synergism. Identification of the active constituents would be the first step to systemically characterize the safety and efficacy of JTT and to understand the mechanism. The main objective of this thesis was the identification of macrophage (MΦ)–stimulating compound(s) in JTT by biomarker–guided screening, and the characterization of individual components to identify possible new biological effects of those compounds. In this thesis, chapter 1 describes the establishment of the technique we used, biomarker–guided screening, to identify therapeutically relevant compounds from herbal medicines. The technique was established in our lab and refined using two different herbal medicines, namely Keishi–bukuryo–gan and Toki–shakuyaku–san. Chapter 2 describes the identification of common chemical components in MΦ–stimulating fractions from two different batches of JTT. This is an important achievement because batch–to–batch variability is a major concern in the natural product research. Chapter 3 describes the characterization of individual compounds identified in MΦ–stimulating fractions, which led to the discovery of one system that exhibit significant synergistic MΦ–stimulating activity, i.e. β–Glucocerebroside (GlcCer) and C16:0–ceramide (Cer). This finding is important because it is a rare example of a structurally defined, small molecule mixture that exhibited synergistic MΦ–stimulating activity. In addition, the activity assay showed that MΦ–stimulating activity of GlcCer/Cer mixture was time–sensitive and diminished within 3 hr of sample reconstitution in DMSO. Further mechanistic study of GlcCer/Cer mixture using transmission electron microscope (TEM) and dynamic light scattering (DLS) revealed that GlcCer/Cer mixture forms nanoparticles in both DMSO and an aqueous environment, and the size of the nanoparticles correlated with the MΦ–stimulating activity. Our study provided a new basis of novel combinational therapy, and served as an important step to systemically characterize the safe and effective immunostimulatory herbal medicine, JTT.
BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF DI-ASPARTYL INTRAMEMBRANE PROTEASES
Celia Mirtha Torres Arancivia
Year of Dissertation:
A new era in the study of proteases was initiated with the discovery of intramembrane-cleaving proteases (I-CLiPs). In an unusual mechanism, I-CLiPs are able to catalyze peptide bond cleavage of substrates within the membrane bilayer, an environment poorly permeable to water. Here we will focus on gamma-secretase and presenilin(PS)-like proteases, membrane proteases that use two aspartic acids as catalytic residues and constitute the most relevant family of I-CLiPs. We first describe the purification of gamma-secretase from a mammalian expression system. We used this preparation for negative-stain single-particle electron microscopy and determined the structure of a native-like catalytically active gamma-secretase complex at a resolution of 25 Å. Antibody labeling of the extracellular domain of Nicastrin was employed to ascertain the topology of the reconstruction. In addition, active site labeling with a gold-coupled inhibitor demonstrated that gamma-secretase contains a single active site facing a large conical internal cavity. Secondly, we used the yeast P. pastoris as an expression system to isolate milligram quantities of active and fully mature gamma-secretase. The higher yield of gamma-secretase obtained from this system allowed the addition of a size exclusion chromatography step to the purification procedure to reduce the structural heterogeneity of the preparation and thereby improve the structural analyses. Finally, we provide novel proof for the existence of a presenilin-like protease in the euryarchaeota M. marisnigri (MCMJR1). We tested in vitro protease activity on chimeric protein substrates containing transmembrane domain (TMD) regions and found that MCMJR1 was indeed able to generate a proteolytic product. Mutagenesis confirmed that two aspartic acid residues were essential for the activity of the protease. Also, the activity of MCMJR1 was blocked when an inhibitor of gamma-secretase was incubated with the enzyme. Mass spectrometry analysis revealed that MCMJR1 cleaves the chimeric substrates close to the middle of the TMD with promiscuous peptide bond selectivity. In conclusion, our study has enhanced the understanding of PS/gamma-secretase and provided new routes for functional and structural studies on di-aspartyl intramembrane proteases, both of which are pivotal in unraveling the mechanistic details of intramembrane proteolysis in biology and disease.
CHARACTERIZING MIGRATORY SIGNALING PATHWAYS OF TRANSPLANTABLE RETINAL PROGENITOR CELLS AND PHOTORECEPTOR PRECURSOR CELLS TOWARD RESTORATION OF DEGENERATIVE RETINA – A SYSTEMS BIOLOGY APPROACH
Year of Dissertation:
A common feature of several heterogeneous diseases that result in retinal degeneration (RD) is photoreceptor loss, leading to an irreversible decline in visual function [15-17]. There are no cell replacement treatments available for RD diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Although many RD cases are of a genetic origin, a promising strategy to treat diseased phenotypes is by replacing lost photoreceptor cells, for synaptic integration and restoration of visual function. To advance photoreceptor-replacement strategies as a practical therapy, in light of highly restricted integration rates reported across studies, this body of research focused on defining the molecular mechanisms facilitating migration of transplantable photoreceptor precursors in the retinal microenvironment. To accomplish this work we utilized bioinformatics, bioengineering and molecular biologic techniques for a systems level approach. Guided by classic neuronal migration models, we hypothesized that transplanted photoreceptor precursors navigate to specific retinal lamina in part due to cell surface receptor expression and in response to spatially gradated directional ligand cues provided by the host retinal microenvironment. Given the neural origin of the mammalian retinal system, we also predicted that these chemotactic receptor-ligand pairs trigger intracellular signaling events in migrating photoreceptors analogous to canonical migration pathways exhibited by neuronal precursors. For a comprehensive account of these motility-deterministic biochemical interactions, we first performed in silico bioinformatics modeling of PPC transplantation into light-damaged retina by matching microarray datasets between PPC receptors and ligands in the light-damaged retinal microenvironment. We then refined the gene expression network data to focus on motility deterministic interactions at the interface of the PPC cell-surface receptors and extracellular ligands of the damaged retina. Our in silico network modeling generated a library of ligand-receptor pairs associated with cellular movement specific for this retinal transplantation paradigm and the intracellular signaling pathways induced by candidate chemotactic ligands. Working from predicted interactions of in silico paired PPC receptors and retinal ligands, we then performed cell migration analysis to evaluate whether exposure to stromal derived factor-1α (SDF-1α) would guide the motility of PPCs and RPCs in vitro. We also assessed the chemotactic effects of epidermal growth factor (EGF) on RPCs. Cell surface expression of C-X-C chemokine receptor type 4 (CXCR4) receptors on PPCs and RPCs, and EGF receptor expression on RPCs were verified via immunocytochemical staining and validated by Western blot analysis. Boyden chamber analysis was used as an initial high-throughput screen to verify the motogenic effects of the ligands on PPCs and RPCs. We determined that RPC motility was optimally stimulated in these chambers by EGF concentrations in the range of 20-400ng/ml, with decreased stimulation at higher concentrations, suggesting concentration-dependence of EGF-induced motility. Both RPCs and PPCs also demonstrated a concentration-dependent chemotactic response to an optimal SDF-1α concentration of 100ng/ml. Using bioinformatics downstream signaling pathway analysis of the EGF and SDF-1α ligands in a retina-specific gene network, we predicted a chemotactic function for EGF involving the MAPK and JAK-STAT intracellular signaling pathways. Based on targeted inhibition studies, we show that ligand binding, phosphorylation of EGFR and activation of the intracellular STAT3 and PI3Kinase signaling pathways are necessary to drive RPC motility. The JAK-STAT pathway was also implicated in transducing similar motogenic effects on PPCs with SDF-1α induction. To test our hypothesis of the gradated nature of ECM ligand effects on both ontogenetic retinal cell types, we employed engineered microfluidic devices to generate quantifiable steady-state gradients of EGF and SDF-1α coupled with live-cell tracking, and analyzed the dynamics of individual RPC and PPC motility. Microfluidic analysis, including center of mass and maximum accumulated distance, revealed that EGF induced motility is chemokinetic in EGFR expressing RPCs with optimal activity observed in response to low concentration gradients. On the other hand, PPCs and RPCs exhibited significant chemotaxis towards the source of SDF-1α with longer accumulated Euclidean distances and Center of Mass (COM) compared to controls. We also ascertained that receptor mediated signaling was requisite for ligand-induced motility by using the CXCR4 inhibitor, AMD 3100, to antagonize the SDF-1α receptor. CXCR4 receptor inhibition resulted in decreases of PPC and RPC movement in uniform and steady state gradients for a number of migration indices measured. To advance translational application of the characterized chemotactic signaling potential of transplantable photoreceptor precursors, we performed computational drug analysis of our newly identified motility-deterministic networks, to develop a library of FDA approved drugs and small molecules predicted to potentially influence the expression of target motility signaling mechanisms in photoreceptor progenitor cells. Using the Expression2Kinases software and LINCS drug computational algorithm, we were able to identify pharmacological drug targets that modulate the biochemical activity of transcriptional regulatory genes which govern the expression of candidate receptor protein targets, and provide preliminary results validating the up-regulatory effect of candidate drug aminophenazone on SDF-1α receptor CXCR4 expression. Results from this study demonstrate the applicability of our systems level in silico modeling of matched transplantable cell surface-receptors and transplantation site ligands to predict molecular signaling guiding migration. Verification of in silico predictions, using molecular and microfluidic analysis provide important data for defining cell response properties to specific ligands present during transplantation into the retinal microenvironment. The drug computational analysis provides a translational perspective to our in silico modeling paradigms extending its applicability. Future studies will validate the functionality of resolved ligand-receptor pairs from our in silico library and characterize down-stream signaling guiding motility and homing. This systems level paradigm can effectively be applied to defining the molecular basis of transplantable cell migration in vivo toward improved efficiency for repair of retina and other neural tissue types.
A coarse-grained view of protein-protein recognition
Rodney Versace Babilonia
Year of Dissertation:
One of the most important characteristics of proteins is their ability to specifically interact with other proteins and with other types of molecules to build supramolecular assemblies in order to perform different kinds of functions. Protein-protein interaction has been a subject of study in several sciences including biochemistry, structural biology, and computational biophysics/biology. One limitation that delays our understanding of molecular recognition is the lack of high-resolution three-dimensional structures of the protein-protein complexes. Because of this, the methods for computational prediction have gained popularity and importance but in many cases, the predicted complex is not accurate. Predicting the native three-dimensional conformation of protein-protein complexes still remains a big challenge. Most biological processes in the cell involve a huge number of atoms and happen at time-scales that are frequently beyond the current limits of classical atomistic simulations. The ELNEDIN approach is a new and powerful coarse-grained representation with the ability to overcome size and time limits without deforming the overall shape of a protein. Previous studies have shown that the quality of the ELNEDIN scaffold influences the ability of the modeled proteins to experience structural transitions and to associate and form a stable complex. The main objective of this thesis is to test if the ELNEDIN approach is able to discriminate native interfaces from non-native. The Barnase/Barstar complex, the RNase/Barnase complex and the Ubiquitin/Ubiquitin ligase complex were chosen to test this hypothesis. Each individual protein model was simulated using the ELNEDIN approach and the potential of mean force for the dissociation of the complex was calculated. Our results show: 1) It is possible to obtain accurate energy-profiles using the ELNEDIN approach. 2) The shape of the free energy landscape around a protein receptor has a funnel-like shape where the bottom of the funnel is the global minimum and it starts to increase smoothly. 3) The solvent plays an important role in the shape of the free energy profile. 4) The ELNEDIN approach is able to recognize the native conformation for hydrophilic interfaces. To further support the last result, an additional complex was chosen: the Nuclease A/Nuclease inhibitor A complex.
Venomic Characterization of the Terebridae and Novel Terebrid Neuropeptides
Year of Dissertation:
Unravelling the complex mixture of neuropeptides produced by the terebrid venom duct holds the promise of discovering tomorrow's therapeutics. Cone snails have already demonstrated the potential found in the venom of these unusual marine organisms, through the commercial approval of drugs for pain and other indications. Terebrids, as the sister family to the cone snails, have been much less investigated, but have a species richness that makes them very attractive in the search for novel neuropeptides. The venomics research described in this work encompasses the first comprehensive characterization of the terebrid venom duct transcriptomes of two species, Cinguloterebra anilis and Terebra subulata. De novo assembly and analysis were performed using next generation sequencing technology and state of the art bioinformatics tools to identify the cocktail of peptides, teretoxins, produced by the venom duct. These disulfide rich peptides often show a remarkable specificity for subtypes of ion channels and ligand-gated receptors, giving them therapeutic potential, but they are only available in vanishingly small amounts from the natural source. As a result, identification of teretoxins using next generation sequencing is a prelude to synthesizing them in sufficient quantities to test for bioactivity. Here recombinant expression and solid phase peptide synthesis have both been utilized for the synthesis of four different teretoxins, with a special focus on recombinant synthesis to design a reproducible strategy for synthesizing disulfide rich peptides greater than 40 amino acids in length. Preliminary characterization of bioactivity was performed by injecting synthesized toxin into the polychaete N. virens. A teretoxin identified from Terebra guttata, Tg77, has produced promising results in this assay, with repeated trials showing the effects of uncoordinated movement and rigid paralysis. Further testing on Tg77 and other teretoxins will be carried forward, with an evident need for high throughput assays to provide an efficient means for the testing of novel compounds with a variety of potential molecular targets.
Identification and characterization of protein phopshatases regulating the Sma/Mab pathway in C. elegans
Year of Dissertation:
Identification and characterization of protein phosphatases regulating the Sma/Mab signaling pathway in C. elegans by Sheng Xiong Advisor: Professor Cathy Savage-Dunn TGF-beta signaling is a conserved signaling pathway among eukaryotes, which controls various normal cellular responses from cell proliferation to cell death. The mutations in its components are found in developmental disorders and cancer. Therefore, this signaling pathway is extensively investigated so that new therapeutic targets could be discovered and novel drugs could be developed. Previous studies suggested the involvement of phosphatases in regulation of TGF-beta signaling, but these studies were performed in cell culture rather than intact organisms. C. elegans is a tractable organism in which to study signaling in vivo. In C. elegans, growth is controled by a conserved TGFβ pathway, the Sma/Mab pathway. We used a C. elegans RNA interference library of phosphatases to identify genes that cause a body size phenotype. Library-wide screening was carried out in an RNAi-hypersensitive mutant background, rrf-3. To further narrow the candidate pool, we analyzed the body size phenotypes of these candidates using different genetic backgrounds. These analyses allow us to narrow the candidate pool down to 80-candidates. A Sma/Mab pathway reporter, RAD-SMAD reporter (a kind gift from Dr. Jun Liu, Cornell University), was used to assess whether the candidates regulate body size phenotype in a Sma/Mab pathway-dependent manner or not. The reporter assay revealed 22 likely candidates regulate Sma/Mab signaling directly or indirectly. Among them, 11 candidates were verified as protein phosphatases by sequencing. These include homologues of mammalian PPM1A/B/G and PP1. Our study revealed that ppm-1 (metal-ion dependent protein phosphatase-1), a human PPM1A/B/G homolog, might act as a potential SMA-3-specific linker phosphatase to regulate the Sma/Mab pathway. This is the first time to show that a homolog of human PPM1A/B/G might act as a potential linker phosphatase of R-Smads to promote TGFβ signaling. Our studies also showed that a homolog of the inhibitory regulatory subunit of human PP1 might synergize with the catalytic subunit in the regulation of TGFβ signaling for the first time. We also discovered the regulatory role of several protein tyrosine phosphatases in this signaling cascade. In summary, this study sheds a light on elucidating the regulatory mechanism of TGFβ signaling pathway, therefore providing insight in various TGFβ signaling-involved human developmental disorders and cancer, and contributing to the development of potential diagnostic markers and therapeutic targets in human diseases.
Investigate the Effects of Osteogenesis Imperfecta Mutations on the Conformation of Collagen Triple Helix
Year of Dissertation:
The clinical severity of Osteogenesis Imperfecta (OI), also known as the brittle bone disease, relates to the extent of conformational changes in the collagen triple helix induced by Gly substitution mutations. The lingering question is why Gly substitutions at different locations of collagen cause different disruptions of the triple helix. Here, we describe markedly different conformational changes of the triple helix induced by two Gly substitution mutations placed only 12 residues apart. The effects of the Gly substitutions were characterized using a recombinant collagen fragment modeling the 63-residue segment of the _1 chain of type I collagen containing no Hyp (residues 877-939) obtained from Escherichia coli. Two Gly3Ser substitutions at Gly-901 and Gly-913 associated with, respectively, mild and severe OI variants were introduced by site-directed mutagenesis. Biophysical characterization and limited protease digestion experiments revealed that while the substitution at Gly-901 causes relatively minor destabilization of the triple helix, the substitution at Gly-913 induces large scale unfolding of an unstable region C-terminal to the mutation site. This extensive unfolding is caused by the intrinsic low stability of the C-terminal region of the helix and the mutation induced disruption of a set of salt bridges, which functions to lock this unstable region into the triple helical conformation. The extensive conformational changes associated with the loss of the salt bridges highlight the long range impact of the local interactions of triple helix and suggest a new mechanism by which OI mutations cause severe conformational damages in collagen. In addition to the biomedical studies, the recombinant collagen fragments have also proven to be a good system to produce nano-templates. The rigidity of the helix backbone, the linear conformation, and the largely exposed side chains of residues at the X and Y positions for chemical modification make the triple helix an ideal template for nanowires