Structure Determination of a Double Transmembrane Fragment of the G protein-coupled Receptor Ste2p in Membrane Mimetic Environments
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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. The number of structures in the protein structural database is increasing exponentially, but there are only a few high-resolution GPCR structures, those of rhodopsin, the β-adrenergic receptors and the adenosine A2A receptor. GPCRs are difficult to crystallize and the protein:detergent complex size can be restrictive for solution NMR. As is true of GPCRs in general, limited structural information is available for Ste2p, a yeast GPCR which recognizes the α-factor tridecapeptide mating pheromone. Many groups, including the Naider lab, have been working with smaller fragments of GPCRs in an attempt to elucidate the structure of individual regions by either crystallization or NMR and to combine these to achieve a better picture of GPCR structure. The use of fragments of GPCRs to study the structure of these large membrane receptors remains controversial. I have used Ste2p as a paradigm to answer the following questions: 1) Can GPCR fragments fold into a tertiary structure without the context of the full protein? and 2) Do organic:aqueous solvents such as trifluoroethanol(TFE):water result in GPCR fragment structures similar to those found in detergents and lipids? I have cloned and expressed two 2TM regions of Ste2p, G31-T110 (TM1-TM2) and R231-S339 (TM6-TM7-CT40). The constructs were chosen based on the biological relevance of the domain and a buried surface area analysis that was performed using a rhodopsin-templated model of Ste2p and the NACCESS software package. Expression in Escherichia coli in minimal media and CNBr cleavage conditions for these 2TM fusion proteins were optimized and the fusion protein was labeled with [15N], [15N,13C], [15N,13C,2H] and with selectively [15N]-labeled amino acids to aid in NMR assignments. Purification of isotopically labeled peptides was performed using RP-HPLC with a Zorbax C3 column and an acetonitrile:water gradient containing isopropanol and 0.1% TFA. Yields of Ste2p(G31-T110) were 6-20 mg per L culture. Far UV CD analysis indicated that Ste2p(G31-T110) formed a highly helical peptide in TFE:water and various micellar environments, whereas the helicity was reduced for Ste2p(R231-S339). Initial [15N,1H]-HSQC analysis showed that CD can be used as an NMR screening technique to determine conditions for high resolution NMR analysis. Ste2p(G31-T110) was used to determine a high-resolution structure of a 2TM GPCR fragment in TFE:water(+0.1% TFA) (1:1; v/v) by multidimensional NMR spectroscopy. The 80-residue polypeptide was folded into a helical hairpin, but the overall convergence of the structure bundle was poor with an RMSD of ~ 6.7 Å. Comparison to an NMR study of TM1-TM2 in LPPG micelles concluded that very different structures occurred in the two membrane mimetic media and it was observed that relative conformation of TM2 to TM1 in TFE:water was twisted in comparison to that in the LPPG micelle so that the interactions between helices were on opposite sides. Although Ste2p(G31-T110) folds into a tertiary structure in TFE:water (1:1; v/v) the structure is quite flexible compared to that in LPPG micelles.
Mechanism of Phospho-alpha-Tubulin-driven Motility in Human Breast Epithelial Cells
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Protein kinase C (PKC), an enzyme important in signaling pathways that give rise to various cell phenotypes, has been closely associated with metastatic phenotypes of breast cancer. Tissues from patients of varying degrees of tumorgenicity have shown an elevated expression of the PKCα isoform as well as other diacylglycerol (DAG)-sensitive isoforms. Moreover, independent studies with highly invasive breast cell lines, such as MDA-MB-231 cells, have also shown an elevated level of PKCα. Therefore, PKCα has long been a therapeutic target for breast cancer. Among its known substrates (recently identified by our laboratory), α6-tubulin will, upon phosphorylation by PKC, enhance migration in breast epithelial cells. The mechanistic pathway as to how a phosphorylated state of α-tubulin can confer motility in breast epithelial cells was addressed in the present work. Different approaches were undertaken to dissect the role(s) played by phospho-α6-tubulin in enhancing cell migration: i) extensive live cell imaging to quantitate microtubule dynamics, ii) immunofluorescence of microtubules in fixed cells, iii) partitioning of phospho-α6-tubulin between particulate and soluble phases, and iv) pull-down assays to determine effects on the small GTPases. For these studies, two mutants of α6-tubulin namely, S165D (pseudo-phosphorylated) and S165N (phosphorylation-resistant), were used extensively. In addition, cells expressing wildtype (WT)-α6-tubulin were treated with either the PKC-activating DAG-lactone or the pan-PKC inhibitor bis-indoleylmaleimide (BIM). Experiments were conducted in non-motile MCF-10A cells that have a low expression level of PKC and therefore offer an ideal platform for studying PKC-mediated changes. Some experiments were also conducted in MDA-MB-231 metastatic breast epithelial cells to re-affirm the role of phospho-α-tubulin in motility and related phenotypes. Functionality of phospho-α-tubulin was also explored by examining its interaction with the microtubule cargo protein MARCKS (a PKC-substrate), and membrane proteins like IQGAP1 and Rac1 having known involvement in cell migration. Each α6-tubulin mutant was studied for its effects on the distinct morphological glandular structures that it produced in a 3D growth environment, in order to simulate an in vivo condition. This study is the first of its kind to establish a detailed mechanism of cell movement driven by the PKC substrate α-tubulin in human breast cells. The novel role of phospho-α-tubulin in promoting motility will establish it as an important predictive marker for metastatic breast cancer.
The microtubule associated protein tau renders breast cancer cells TNF-alpha resistant by inhibiting TNF-receptor signaling.
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The pro-inflammatory cytokine Tumor Necrosis Factor &alpha is often found in elevated concentration within the microenvironment of breast tumors. A number of findings have now established that TNF&alpha can exert opposing effects on tumor cells - acting either as an anti-cancer agent or as a promoter of tumor progression. To date, mechanisms underlying these divergent outcomes have not been elucidated. Here, we demonstrate that tau, classically considered as a microtubule-associated protein, plays a key role to determine whether cancer cells respond negatively (apoptosis) or positively (proliferation) to TNF&alpha exposure. Using RNAi knockdown experiments we show that up-regulation of tau protein in breast cancer cells is necessary for the acquisition of resistance to TNF&alpha mediated cytotoxicity. In contrast, an analysis of generated stable cell lines overexpressing full-length tau indicates that tau can inhibit TNF&alpha induced caspase activation and NFκB nuclear translocation. Site-directed mutagenesis has revealed that the N-terminal portion of tau, which does not bind to tubulin, is sufficient for this inhibition of TNF&alpha signaling. Finally, mechanistic studies have uncovered that tau inhibits TNF-receptor trimerization and receptor clustering thereby blocking subsequent signaling. Taken together, we conclude that acquisition of TNF&alpha resistance requires a previously undescribed mechanism involving up-regulation of tau, which in turn inhibits receptor trimerization and thus attenuates TNF&alpha downstream signaling in tumor cells.
Turnip Mosaic Virus Genome-Linked Protein (VPg) Inhibits Pokeweed Antiviral Protein (PAP)-Mediated Depurination of RNA
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Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and is an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is a cap-binding protein, and it was suggested that it inhibits translation of RNA by binding to the 5' m7G cap structure of eukaryotic mRNA, and depurinating the mRNA at sites downstream of the cap structure. PAP is a potent antiviral agent against many plant, animal, and human viruses. Depurination of capped viral RNA may be the primary mechanism for PAP's antiviral activity. However, the above mechanism does not clarify the inhibitory effect of PAP on the replication of uncapped viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and Turnip mosaic virus (TuMV) genome linked protein (VPg) were investigated. VPg is important in the initiation of protein synthesis, functioning as a cap analog. VPg stimulates the in vitro translation of uncapped IRES-containing RNA and inhibits capped RNA translation in wheat germ extracts. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP-VPg interactions. PAP interacts strongly with VPg, thus the effect of VPg on the PAP catalyzed depurination of several different RNA molecules was determined to investigate whether VPg binding to PAP influences selectivity of depurination. PAP binds to and depurinates both m7GpppG-capped and uncapped S/R oligo nucleotide and TEV RNAs, supporting previous conclusions that the cap structure is not the only determinant for PAP depurination of RNA. VPg decreases depurination of the above capped and uncapped RNAs and competes with TEV RNA for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the defense mechanisms of the plant. Depurination inhibition of PAP by VPg also suggests the possible use of this protein against cytotoxic activity of RIPs and inhibition of their biological potency.
p53 and Beclin 1 in Caenorhabditis elegans are involved in cell death and DNA damage repair
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p53 is a key regulator of growth arrest, DNA repair, apoptosis, and autophagy in response to cellular stress in animals as diverse as Caenorhabditis elegans and humans. In mammals, p53 functions as a key tumor suppressor protein that promotes removal of potentially tumorigenic cells. p53 is mutated or deleted in over 50% of all mammalian tumors and these tumors are highly resistant to available therapies. Therefore, identifying therapeutic agents and cell death pathways that promote p53-independent cell death is vital to the future of cancer therapy. We are interested in identifying how the alkylating agent, 10-decarbomyl mitomycin C (DMC) promotes p53-indpendent cell death. To answer this question, we used a C. elegans as a model system to identify known and novel cell death genes involved in DMC-induced death. C. elegans have an orthologue of p53, cep-1, that functions in germline cell death in a similar way to p53 in apoptosis. The germline in C. elegans has both mitotic and meiotic cells and displays CEP-1/p53-dependent cell death in response to UV. We examined both germline cell death and transcript levels of CEP-1 target genes in C. elegans. We found that DMC increased CED-1::GFP positive cells and DNA lesions in the absence of CEP-1 while UV required CEP-1 for germline cell death but not nuclear lesion formation. More lesions were seen over time in cep-1(gk138) mutant worms leading to the idea that CEP-1 is involved in DNA repair. Additionally, we examined the role of autophagy in cell death and DNA damage and saw that initially the knockdown of bec-1 required CEP-1 for increases in germ cell death. However in the F1 generation of bec-1 RNAi knockdown animals, the observed increase in cell death was due to a lack of clearance. Furthermore, bec-1 knockdown resulted in the increase of DNA lesions in worms with UV damage.
PROTEASOME FUNCTION DURING AGING IN DROSOPHILA
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Aging is associated with dysfunction of protein homeostasis and increased protein damage caused by oxidative stress, but the detailed molecular mechanisms are unknown. The ubiquitin-proteasome pathway (UPP) is critical to the protein quality control system as it degrades a majority of intracellular proteins in eukaryotic cells. Age-related decline in proteasome function has been shown in a variety of mammalian tissues. However the role of UPP during the aging process still remains a puzzle. In this study, we compared the UPP function between two Drosophila melanogaster strains, the longevity mutant methuselah (mth) and the wild type w1118 at different ages, which would help answer this question. The first part of the thesis evaluated the UPP function in mth and w1118 during the natual aging process and under oxidative stress condition. Mth encodes a GTP binding protein-coupled receptor (GPCR). The mth fly with a single Mth gene mutant has been shown to increase ~35% lifespan, but the cellular role of Mth remained elusive. We are the first to report that mth displays lower proteasome activity, lower proteasome levels and lower ATP steady state levels at young ages, but relatively higher levels at old ages during normal aging, when compared with control strain w1118. Under oxidative stress conditions, proteasome activity remained nearly unchanged in w1118 after 5 days of treatment with 1% H2O2, but it was elevated in mth. Moreover, while both strains exhibited a gradual increase in ubiquitinated protein conjugates and aggregates during normal aging process, mth produced fewer conjugates and aggregates at the comparative ages than w1118. Under oxidative stress conditions, the levels of ubiquitinated conjugates and aggregates were elevated in both strains, but less were observed in mth. Together, these data suggest that mth exhibits higher proteasome plasticity and maintains a more efficient protein homeostasis, contributing to longer lifespan. We propose that maintaining a steady state protein turnover rate by the ubiquitin/proteasome pathway will delay the aging process. In the second part of the thesis, we modulated different lifespans using non-genetic approach- Dietary Restriction (DR), which is the only intervention known so far to reliably increase lifespan in a variety of organisms. Currently, it is unclear that what effects aging and DR have on proteasome-mediated protein degradation. We compared the UPP function in three Drosophila strains, wild type Oregon R (OR), w1118 and longevity mutant mth under two diets - DR and ad libitum (AL). All three fly strains responded to DR with extended lifespan. In OR strain, DR significantly reduced proteasome activity in male and female flies at young ages (old ages not tested) and ATP level at all age groups in male flies. These data indicate higher proteasome activity and ATP level do not always lead to longer lifespan. In addition, DR significantly reduced age-related decline in proteasome activity, and ameliorated age-related increases in ubiquitinated protein in the wild type w1118. However, in mth, DR reduced proteasome activity and displayed a slightly higher level of ubiquitinated protein conjugates under both young and old ages. Our data indicate that DR has many beneficial effects towards the function of the UPP in wild type w1118, and that a preservation of the UPP may be a potential mechanism by which DR extend lifespan in w1118. But in longevity mutant mth, DR can not further ameliorate proteasome function, which suggests that other mechanisms might be responsible for the longer lifespan caused by DR. Cumulatively, these data have implications for understanding the effects of aging and DR on protein turnover and the mechanism of lifespan extension in longevity mutant mth.
Dictyostelium discoideum RnoA Interprets cAMP Mediated Signals to Influence Actin Organization
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Tight control of actin cytoskeletal dynamics is essential for proper cell function. ARNO, a guanine nucleotide exchange factor for Arf, is associated with actin cytoskeletal regulation but its exact role is unknown. To explore ARNO's role in this regulation and in actin mediated processes, the Dictyostelium discoideum homolog RnoA was examined. RnoA is involved in development, as the reduction of RnoA by antisense prolongs aggregation, delaying development. Also, RnoA overproduction arrests development at the mound stage. This arrest is rescued by the addition of wild type cells. In these chimeric mixtures, rnoA overexpressing cells (RnoA OE) preferentially sort to the stalk, suggesting RnoA plays a role in cell sorting. RnoA antisense and RnoA OE cells fail to stream during aggregation. Chemotaxis assays reveal these mutants do not chemotax toward cAMP, indicating RnoA is part of the cellular response to cAMP. This defect is specific to cAMP-directed chemotaxis, as both RnoA mutants effectively chemotax to folate and exhibit normal cell motility. The chemotactic defects of the RnoA mutants may be due to an impaired cAMP response as evidenced by altered cell polarity and F-actin polymerization after cAMP stimulation. RnoA OE cells have increased filopodia compared to wild type cells, implying altered F-actin localization. Thus, RnoA likely organizes F-actin during development. Phospholipase D (PLD), the enzyme responsible for phosphatidic acid production, and paxillin, a cytoskeletal adaptor protein, are also involved in actin cytoskeletal organization. Given their communal association with the actin cytoskeleton, we explored the interactions between RnoA and the D. discoideum PLD and paxillin homologs, PldB and PaxB, respectively. PaxB and PldB regulate calcium dependent and calcium independent cell-cell cohesion, respectively. Cells lacking PaxB are known to have reduced cell-substrate adhesion. We find that overexpression of PldB rescues the adhesion defect of paxB null cells, implying a genetic interaction between paxB and pldB. Co-immunoprecipitation studies suggest RnoA, PldB, and PaxB physically interact. This interaction does not depend on PaxB, PldB, or F-actin organization. Taken together, the results suggest that RnoA, in complex with PldB and PaxB, coordinates F-actin organization during actin mediated processes such as adhesion.
New Families of Beta-Lactamase Inhibitors
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A traditional approach to improving the efficacy of â-lactam antibiotics has been to modify natural antibiotics by modifying the nucleus and adding various side chains. It is generally believed that â-lactam based antibiotics have a limited future given increased resistance demonstrated by many strains of bacteria. Therefore the lack of performance among commonly used antibiotics against the rise of resistant bacterial strains has propelled researchers to look for novel types of antimicrobial agents and enzyme inhibitors as tools to combat this serious and growing threat. The focus of this research is to investigate novel small-molecules from different families with antibacterial properties such as peptides, aromatic ketones, biphenyl and stilbene compounds and boronic acids that demonstrate an inhibitory effect on class A â-lactamase. Some of these inhibitors were found by screening natural products other were found by screening libraries of compounds and yet others were found by computational approaches involving molecular docking. Of all the compounds tested as long as the molecule was within hydrogen bonding distance of Ser84, Lys87 Ser142, Asn186, Gly252, Glu182 or Asp142, they demonstrated inhibitory effect which demonstrates that docking for this class of compounds is highly correlated with successful experimental inhibition.
VEGF signaling mediates neuroprotection against oxidative stress in hippocampal neurons in vivo and in vitro
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Vascular endothelia growth factor (VEGF) signaling through its cognate receptor VEGFR-2 has been shown to be neuroprotective against stressful stimuli including oxidative stress. Using a rat pilocarpine model of status epilepticus (SE), pilocarpine-induced SE was associated with the induction of the oxidative stress marker Heme oxygenase-1 (HO-1) and increased expression of the pro-inflammatory enzyme Cyclooxygenase-2 (COX-2). Treatments with exogenous VEGF attenuated the induction in HO-1 and the increase in COX-2 levels. Previous studies showed that VEGF protected hippocampal cell death in this model and these results suggest that oxidative stress and inflammation contribute to this neuronal loss. Similarly, in cultured hippocampal neurons, pilocarpine induced neuronal cell death accompanied with increased HO-1 expression, reactive oxygen species (ROS) production, caspase-3 cleavage and a loss of mitochondrial membrane potential (ÄØm) that were all suppressed by VEGF. To delineate the protective mechanisms associated with VEGF-mediated neuroprotection, VEGFR-2 inhibition was investigated with respect to oxidative stress in primary cultures of hippocampal neurons. Treatments with the pharmacological inhibitor SU1498 elicited a cytotoxicity that was prevented by the antioxidant N-acetyl-cysteine (NAC) and accompanied by induction of HO-1, cleavage of caspase-3, production of ROS together with a loss of ÄØm. Knockdown of VEGFR-2 by siRNA generated a similar pattern of ROS and ÄØm loss. Treatments with VEGF or VEGF-B prevented neurons from the cell death and mitochondrial dysfunction induced by SU1498 or siRNA, suggesting that a molecular switch occurs where both growth factors may signal through VEGFR-1 when VEGFR-2 activity is blocked. Consistent with a general role for pro-apoptotic BAD and anti-apoptotic Bcl-xL in neuronal cell death and oxidative stress, the phosphorylation of MEK/ERK1/2 and BAD (inactivation) and the protein levels of Bcl-xL that were attenuated by SU1498 were somewhat increased by VEGF. Our findings support a role for VEGF in signaling BAD inactivation and increased Bcl-xL as a specific mechanism that protects hippocampal neurons against oxidative stress. These findings also suggest that VEGF activates VEGFR-2 to protect neurons against oxidative stress but can signal through alternate receptors to serve the same function.
Isolation and Characterization of Immunomodulatory Compounds from Juzen-Taiho-To: Novel Understanding of Phytosteryl Glucosides Nano-Aggregates and Synergism
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This thesis describes the realization of the immunomodulatory properties of four phytosteryl-glucosides betta;-sitosterol-D-Glucoside, campesteryl betta-D-glucoside, 24-epicampestryl betta-D-glucoside, Stigmasteryl betta-D-glucoside) purified from a Japanese Kampo herbal formulation termed Juzen-Taiho-To (JTT). JTT has been used for almost a thousand years and currently is prescribed to people with an impaired immune system. Our screening methodology termed Genomic-Screening allowed us to investigate the immunomodulatory properties of JTT in a new and exciting way. Genomic Screening employs the use of GeneChip® oligonucleotide array, and quantitative reverse-transcriptase polymerase chain reaction qRT-PCR. THP-1 human cancerous monocytic cell line was used in our study with JTT due to their accurate representation of healthy monocytes as well as a large variety of receptors. THP-1 treatment with JTT revealed many genes to be differentially regulated most of which were down stream elements of NFkappa B pathway. We chose (ICAM-1) for our screening since it was the most reproducible assay and it also had a high fold change. By monitoring ICAM-1 response to JTT treatment we were able to uncover known phytosteryl-glucosides that previously were not known for their immunostimulatory properties. In addition our study discovered that a specific ratio of our phytosteryl glucosides that synergistically induced ICAM-1 expression. We have also discovered that the purification approach of the four phytosteryl glucosides greatly affects the immunostimulatory properties. Using Atomic Force Microscopy (AFM) and Dynamic Light Scattering (DLS) we were able to show that nano-aggregated of betta-sitosterol-D-Glucoside formed in a water containing solution diminished ICAM-1 expression in THP-1 cells. These findings underline the importance of natural product chemistry as well as the approach to purifying small organic molecule from natural resources. The underlining mechanism of how our phytosteryl glucosides induce an immune response is still unknown. Nevertheless our finding has opened the door to new research avenues with a family of compound that contain a novel class of pathogen associated molecular patterns.