Browsing by Author "Carla Mattos, Committee Member"

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Activin Induction of Follicle Stimulating Hormone is Mediated by Transforming Growth Factor Beta Activated Kinase-1 (TAK-1) in Pituitary Gonadotropes.
(2006-05-04) Safwat, Nedal Wafik; Carla Mattos, Committee Member; Dennis Brown, Committee Member; David Schomberg, Committee Member; William L. Miller, Committee Member; Carla Mattos, Committee Member; Dennis Brown, Committee Member; David Schomberg, Committee Member; William L. Miller, Committee Member
Follicle stimulating hormone (FSH) is an essential hormone for female folliculogenesis and plays an important role in male spermatogenesis. The hormone is secreted by pituitary gonadotropes in the anterior pituitary lobe, and its overall production is regulated by expression of the FSHβ subunit. The regulation of FSHβ subunit is achieved by combined actions of neurocrine, endocrine, and pituitary paracrine/autocrine factors. Activin shown to be produced locally within the pituitary is a potent stimulator of the FSHβ subunit. This study used 4.7 kb of the ovine FSHβ promoter linked to luciferase (oFSHβLuc) plus a well characterized activins responsive construct, p3TPLuc, to investigate the hypothesis that Smad3, TAK1 (TGFβ activated kinase1), or both cause activin-mediated induction of FSH. Over-expression of either Smad3 or TAK1 induced oFSHβLuc in gonadotrope-derived LβT2 cells as much as activin itself. Induction of p3TPLuc by activin is known to require Smad3 activation in many cell types and this was true in LβT2 cells where 10-fold induction by activin (2-8 h after activin treatment) was blocked > 90% by two dominant negative (DN) inhibitors of Smad3 [DN-Smad3 (3SA) and DN-Smad3 (D407E)]. By contrast, 6.5-fold induction of oFSHβLuc by activin (10-24 h after activin treatment) was not blocked by either DN-Smad inhibitor, suggesting that activation of Smad3 did not trigger induction of oFSHβLuc. By contrast, inhibition of TAK1 by a DN-TAK1 construct led to a 50% decrease in activin-mediated induction of oFSHβLuc, and a specific inhibitor of TAK1 (5Z-7-Oxozeanol) blocked induction by 100% indicating that TAK1 is necessary for activin induction of oFSHβLuc. Finally, inhibiting p38-mitogen activated protein kinase (p38-MAPK; often activated by TAK1) blocked induction of oFSHβLuc by 60%. In conclusion, the data presented here indicate that activation of TAK1 (and probably p38-MAPK), but not Smad3, is necessary for triggering induction of oFSHβ by activin. In addition, a method was developed in our laboratory for purifying primary gonadotropes to study the solitary role of factors regulating FSHβ gene. We were able to isolate primary gonadotropes from pituitary with purities higher than 95%. The data show that gonadotropes are able to produce activin and/or activin-like molecule(s), however paracrine factors from pituitary non-gonadotropes play a major role in controlling FSHβ at the pituitary level. Overall, the study presented provides an understanding to the TAK1 signaling pathway mediating activin induction of FSHβ, and shows that primary gonadotropes rely on paracrine factors to produce activin.
Characterization of Furin Protease Sensitive Site Processing and Its Effects on Sindbis Virus Assembly and Budding
(2006-02-27) Nelson, Steevenson; Carla Mattos, Committee Member; Fred Fuller, Committee Member; Dennis Brown, Committee Chair; John Cavanagh, Committee Member
Sindbis virus particles are composed of three structural proteins (C/E2/E1). The E1 glycoprotein is organized into a highly constrained, energy-rich conformation. Its hypothesized that this energy is utilized to drive events that deliver the viral genome to the cytoplasm of a host cell. The extraction of the E1 glycoprotein from virus membranes results in disulfide-bridge rearrangement and the collapse of the protein to a low-energy, non-native configuration. In a new approach to the production of membrane glycoproteins, furin protease recognition motifs were installed at various positions in the E1 glycoprotein ectodomain. Proteins containing the furin sensitive sites undergo normal folding and assembly in the endoplasmic reticulum and only experience the consequence of the mutation after transport to the cell surface. Processing by furin in the Golgi results in the release of the protein from the membrane from which they are assembled. This processing also impacts the envelopment of the nucleocapsid in the modified plasma membrane. E2 has been shown to be responsible for host receptor recognition and thus plays a critical role in the virus lifecycle. To expand on our previously characterize E1 furin mutant study, we installed furin protease recognition motifs at various positions in the ectodomain of E2. Mutants were analyzed for production of truncated proteins and the effect of the mutations on virus assembly and budding was also characterized. Processing of the E2 mutants by the enzyme furin results in the release of the truncated proteins from the membrane in a fashion that is similar to the processing observed in the E1 furin sensitive mutants. This processing was also observed to impact envelopment of the nucleocapsid with virus protein modified plasma membrane at a step consistent with an early event in the envelopment process. Overall, this technique provides a unique method for studying the mechanism of virus assembly and protein structure without altering crucial early events in protein assembly, folding and maturation.
Computational Biology of Ras Proteins
(2008-04-07) Dellinger, Andrew Everette; William R. Atchley, Committee Chair; Carla Mattos, Committee Member; Jeffrey Thorne, Committee Member; Jon Doyle, Committee Member
In this research, computational biology is used to elucidate how evolutionary history has changed roles of structure and function among Ras proteins, with a focus on the Ras family. This dissertation begins with phylogenetic analyses of the Ras superfamily and Ras family. Phylogenetic trees of the Ras family were estimated using Neighbor-Joining, Weighted Neighbor-joining, Parsimony, Quartet Puzzling, Maximum Likelihood and Bayesian methods. In nearly all cases, each clade represented a subfamily. Clade members and clade divisions were consistent among all the trees, increasing the probability of a correct estimation of the evolutionary history. Further investigation into the evolution of sequence involved decomposing sequence covariation into its respective components. The roles of the functional and structural components of covariation were the focus of several multivariate analyses. Decision tree analysis, a data mining method, found that sequence divergence in critical sites of the hydrophobic core, dimerization regions and ligand binding regions were sufficient to divide Ras subfamilies. Alignments of GDP-bound and GTP-bound crystal structures revealed that only Ral and M-Ras proteins have structural variation in the effector binding switch I regions, while all Ras structures vary in the protein binding switch II region. Di-Ras2-GDP was shown to have a unique C-terminal loop which binds to the interswitch region. Last, a common factor analysis was computed. The factors contain the set of sites that both discriminate among the subfamilies and have a unique functional or structural role, such as Ral tree-determinant sites. Finally, sequence signatures were developed for each of the families of the Ras superfamily using Boltzmann-Shannon entropy. This method was compared to the PROSITE signature, profile hidden Markov model and MEME position-specific scoring matrix methods. The Entropy method identified approximately 8% fewer proteins than the best of the other methods, MEME. Comparative analyses of these sequence signatures determined which sites and amino acids played important roles in the changes in protein function and structure among Ras families.
Computer Simulations of Protein Folding and Aggregation
(2004-05-13) Nguyen, Hung Duc; Carla Mattos, Committee Member; Steven W. Peretti, Committee Member; Jason M. Haugh, Committee Member; Carol K. Hall, Committee Chair
Computer simulation is used to study the competition between protein folding and aggregation, especially the formation of ordered structures that are also known as amyloid fibrils. Employing simplified protein models, we simulate multi-protein systems at a greater level of detail than has previously been possible, probe the fundamental physics that govern protein folding and aggregation, and explore the energetic and structural characteristics of amorphous and fibrillar protein aggregates. We first tackle the aggregation problem by using a low-resolution model called the lattice HP model developed by Lau and Dill. Dynamic Monte Carlo simulations are conducted on a system of simple, two-dimensional lattice protein molecules. We investigate how changing the rate of chemical or thermal renaturation affects the folding and aggregation behavior of the model protein molecule by simulating three renaturation methods: infinitely slow cooling, slow but finite cooling, and quenching. We find that the infinitely slow cooling method provides the highest refolding yields. We then study how the variation of protein concentration affects the refolding yield by simulating the pulse renaturation method, in which denatured proteins are slowly added to the refolding simulation box in a stepwise manner. We observe that the pulse renaturation method provides refolding yields that are substantially higher than those observed in the other three methods even at high packing fractions. We then investigate the folding of a polyalanine peptide with the sequence Ac-KA14K-NH2 using a novel off-lattice, intermediate-resolution protein model originally developed by Smith and Hall. The thermodynamics of a system containing a single Ac-KA14K-NH2 molecule is explored by employing the replica exchange simulation method to map out the conformational transitions as a function of temperature. We also explore the influence of solvent type on the folding process by varying the relative strength of the sidechain's hydrophobic interactions and backbone hydrogen bonding interactions. The peptide in our simulations tends to mimic a real polyalanine in that it can exist in three distinct structural states: alpha-helix, beta-structure, and random coil, depending upon the solvent conditions. We next examine the formation of fibrillar protein aggregates, which have been implicated in the pathology of several neurodegenerative diseases including Alzheimer's and Parkinson's, using the Smith/Hall intermediate-resolution protein model. Simulations were conducted on systems containing 12 to 96 Ac-KA14K-NH2 peptides at a wide variety of concentrations and temperatures. We are able to observe the formation of fibrils from random coils within just a few days on a single processor of an AMD Athlon MP 2200+ workstation. To our knowledge, these are the first simulations to span the whole process of fibril formation from the random coil state to the fibril state on such a large system. We find that fibril formation strongly depends upon the peptide concentration, the temperature, and the hydrophobic interaction strength of non-polar sidechains. The fibrils observed in our simulations mimic the structural characteristics observed in experiments in that most peptides in our fibrils were arranged in an in-register parallel orientation with intra-sheet and inter-sheet distances that are similar to those observed in experiments, and are disproportionately long along the fibril axis with about six beta-sheets, each of which contains many peptides. We also investigate the kinetics of fibril formation by performing constant-temperature simulations on systems containing 48 Ac-KA_14K-NH2 peptides with the Smith/Hall intermediate-resolution protein model. We find that fibril formation is nucleation dependent with an ordered nucleus of two beta-sheets, each with two to three peptides. The lag time before fibril formation commences decreases with increasing concentration and increases with increasing temperature. In addition, fibril formation appears to be a nucleated conformational conversion process in which small amorphous aggregates --> beta-sheets --> ordered nucleus --> subsequent rapid growth of a stable fibril. Fibril growth in our simulations involves both beta-sheet elongation, in which the fibril grows by adding individual peptides to the end of each beta-sheet and lateral addition, in which the fibril grows by adding already-formed beta-sheets to its side. Moreover, the rate of fibril formation increases with increasing concentration and decreases with increasing temperature. Finally, we examine the thermodynamics of systems containing 96 Ac-KA14K-NH2 peptides by performing replica exchange simulations over a wide range of temperatures and peptide concentrations. We map out a phase diagram in the temperature-concentration plane delineating the regions where random coils, alpha-helices, beta-sheets, fibrils, and amorphous aggregates are stable.
Dependence Among Sites in Protein and RNA Evolution
(2005-11-13) Yu, Jiaye; William R. Atchley, Committee Member; Carla Mattos, Committee Member; Jeffrey L. Thorne, Committee Chair; Bruce S. Weir, Committee Member
Widely used models of molecular evolution assume independent change among sequence sites. This assumption facilitates computation but it is biologically unrealistic. RNA secondary structure and protein tertiary structure both change more slowly over time than do the encoding DNA sequences. The constraints upon sequence evolution that serve to maintain structure induce dependent change among molecular sequence positions. The object of this thesis is to characterize the impact of structure on sequence evolution. The dependence among sites in protein evolution is first studied. Two simple and not very parametric hypothesis tests are introduced to study the spatial clustering of amino acid replacements within protein tertiary structure. Results of applying these tests to 273 protein families support the expectation that spatial clustering of amino acid replacements within tertiary structure is a ubiquitous phenomenon. More importantly, patterns of amino acid replacements do not seem to be solely attributable to spatial clustering of sequence positions that are independently evolving and have high rates of change. Instead, application of the newly introduced simple hypothesis tests yields evidence for dependent change among spatially clustered protein positions. This portion of the thesis work thereby casts doubt upon widely used methods for phylogeny inference. The second focus of this thesis is the impact of RNA secondary structure on RNA evolution. A model of RNA evolution incorporating RNA secondary structure is developed. The model introduces dependence among sites in RNA evolution via the effects of sequence changes on the approximate free energy of the resulting RNA secondary structure. This approximate free energy information can be thought as surrogate of fitness that serves as a link between genotype and phenotype in the model. Analysis of eukaryotic 5S ribosomal RNA sequences with this model shows the importance of RNA secondary structure on evolution. This analysis also confirms the value of the new model for studying adaptive evolution and for inferring ancestral sequences.
Equilibrium and Kinetic folding properties of alpha-helical Greek key protein domains
(2003-11-13) Chen, Yun-Ru; Carla Mattos, Committee Member; A. Clay Clark, Committee Chair; Harold Swaisgood, Committee Member; Dennis T. Brown, Committee Member
I have characterized the equilibrium and kinetic folding properties of members of alpha-helical Greek key protein domain, caspase recruitment domains of RICK (RICK-CARD) and procaspase-1 (Pro-1-CARD). At equilibrium, folding of both RICK-CARD and Pro-1-CARD is well described by a two-state mechanism representing the native and unfolded ensembles. The proteins are marginally stable,with the Gibbs free energy of 3.0 and 1.1 kcal/mol and m-values of 1.27 and 0.68 kcal/mol/M for RICK-CARD and Pro-1-CARD, respectively (30 mM Tris-HCl, pH 8, 1 mM DTT, 25 degree Celsius). The folding pathways of RICK-CARD are complex and contain at least two or three non-native conformations. The major folding event of RICK-CARD has a folding rate constant of 30 s⁻¹. Studies on seven mutants of RICK-CARD suggest that the three prolyl residues contribute to its structural stability and folding complexity. The folding of Pro-1-CARD is different than that of RICK-CARD. The folding and unfolding kinetics of Pro-1-CARD are fast but with kinetically trapped intermediates present, which appear to be similar to that of RICK-CARD. The work suggests that different alpha-helical Greek key protein domains fold differently but may share some similar folding pathways.
Fc-binding Hexamer Peptide Ligands for Immunoglobulin Purification
(2008-08-18) Yang, Haiou; Carla Mattos, Committee Member; Patrick V. Gurgel, Committee Member; Jason M. Haugh, Committee Member; Ruben G. Carbonell, Committee Chair; Peter K. Kilpatrick, Committee Member
Antibodies and their fragments have found a wide array of applications as pharmaceutical compounds, in addition to their common usage in the purification and localization of proteins. Antibody-based therapeutics accounts for roughly 20% of the therapeutic products in development in the USA, with the majority being of the immunoglobulin G (IgG) isotype. For IgG purification, affinity chromatography has been greatly used where Staphylococcus aureus Protein A and Streptococcus Protein G are the most common affinity ligands for IgG. However, the drawbacks associated with these two proteins have given rise to the searching for alternative affinity ligands for antibody purification. We therefore set our research goal as to find a small peptide that can bind hIgG through its Fc portion and can be used in purification of antibodies and Fc-fusion proteins. Small peptides are interesting for their advantages of being more stable, less immunogenic, less expensive and milder in elution than protein ligands. Peptides were searched by a radiolabeled-screening technique in a combinatorial linear hexamer peptide library built on solid phase Toyopearl AF-Amino resins. The screening identified a family of ligands with homogenous composition of His + aromatic group + positively charge group having the ability to match Protein A in binding human IgG (hIgG) through its Fc portion. The selectivity to the Fc portion is comparable to Protein A. The HWRGWV ligand of the Fc-binding peptide family has been investigated in many aspects and exhibits some interesting tributes. It has broad binding spectrum. It can bind all subclasses of hIgG, human IgD, IgE, IgM, and to a less extent human secretory IgA. It also displays the ability to retain chicken and several mammalian IgGs. Deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry data that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids SNGQPEN in the loop Ser383 — Asn389 by specific interactions. The selectivity of the HWRGWV resin to Fc over Fab is affected by its peptide density which also influences the equilibrium constant for hIgG. Increasing density increases both the association constant which is in the order of 105 M-1 and the binding of the Fab fragment. A ligand density of around 0.1 meq—g was determined to have both the specificity and appropriate affinity to Fc. HWRGWV demonstrates the ability to purify IgG. It can isolate hIgG from mammalian cell culture media containing 10% fetal calf serum (cMEM) with more than 95% of both purity and yield. The ligand was also used to isolate hIgG from Cohn II+III paste and an yield of 82% and purity of 73% were obtained in one step. The bound IgG can be recovered using phosphate buffer at pH 4 and its binding capacity for hIgG is 130 mg⁄g-dry-resin. Acetylation of the N-terminal amine does not pose any influence on either the static binding or the dynamic isolation of hIgG. Temperature has no significant influence on hIgG isolation from cMEM. Increasing peptide density improve the yield but with a compensation of the purity to a similar degree. Feed hIgG concentration in the range of 0.5 — 10 mg⁄mL affects the recovery yield where the yield is favored with higher IgG concentration. The separation of hIgG from cMEM by HWRGWV is comparable to Protein A at an initial hIgG concentration of 10 mg⁄mL and to A2P agarose gel at both 10 and 0.5 mg⁄mL, but using a milder pH 4 elution condition. HWRGWV was immobilized on Sepharose CL-4B. HWRGWV-modified Sepharose CL-4B can isolate hIgG from cMEM with similar purity to and a lower yield than on Toyopearl AF-Amino under the conditions optimized on the latter matrix. Our experiments also show that 2% AcOH is not strong enough to completely remove the bound proteins on HWRGWV. A better column regeneration and sanitation procedure is needed for longer column lifetime. This work demonstrates the possibility to use a peptide as short as six amino acids to mimic Protein A in IgG isolation by binding through the Fc portion.
Insect response to Alphavirus infection
(2007-11-03) Mudiganti, Usharani; Dennis T. Brown, Committee Chair; Carla Mattos, Committee Member; Linda K. Hanley-Bowdoin, Committee Member; Gregory C. Gibson, Committee Member
Invertebrate cells survive Alphavirus infections to establish viral persistence, in contrast to cell death seen soon after infection in mammalian cells. Invertebrate response to prototype alphavirus, Sindbis, has been studied to a certain extent, using mosquitoes and cell lines derived from mosquitoes. Some of the observations made in studies using mosquito systems include formation of intracellular vesicles soon after infection with Sindbis, identification of antiviral activity in the media used to grow the mosquito cell lines and in Sindbis-infected mosquito cell lysates, controlled levels of virus production as persistence is established and superinfection exclusion by Sindbis-infected cells. The study presented here is designed to utilize array of genomic and genetic information available in Drosophila model to identify the candidate genes ⁄ gene products playing a role in establishment of alphavirus persistence. Observations described in Chapter I establish Drosophila S2 cells as a suitable invertebrate system to study alphavirus-insect interactions. Gene expression analysis identified increased expression of 18 transcripts coding for membrane trafficking and cytoskeletal components and 10 transcripts coding for Notch pathway components, at 5 days post-infection. Identification of upregulation of Notch pathway suggests similarities between mechanism of establishment of persistence of Alphaviruses and Herpesviruses. Transcript coding for TEP II, a wide-spectrum protease inhibitor is increased in expression at 5 days post-infection and upon superinfection at 5 days post-infection. We probed for inhibition of viral protease activity during early persistence and upon superinfection of Sindbis-infected cells with Sindbis. Inhibition of Sindbis viral protease nsP2 is identified to be involved in establishment of viral persistence and superinfection exclusion in cells derived from Mosquito and Drosophila
Interactions in the Active Site Loops are Important for Maintaining the Active Site Environment of (Pro)caspase-3
(2004-04-16) Feeney, Brett; E. Stuart Maxwell, Committee Member; Dr. A. Clay Clark, Committee Chair; Carla Mattos, Committee Member
Apoptosis is obligatory to development and in maintaining the vital balance between cell growth and death. Commitment to apoptosis involves a proteolytic cascade by a family of cysteine proteases named caspases. Caspases are split it into two general classes, those involved in the proinflammatory response and those involved in apoptosis. Of those involved in apoptosis, there are two further subdivisions, the apical caspases and the executioner caspases. The irreversible commitment to apoptosis involves activation of executioner caspases, namely caspase-3. Procaspase-3 exists in the cell as a dimeric zymogen, where upon limited proteolytic cleavage at specific aspartate residues, it is activated and apoptosis results. We have previously shown the (pro)caspase-3 undergoes pH dependent conformational changes monitored by fluorescence emission (Bose, et al 2003). In this study, we unambiguously assign dimer dissociation to one of the pH dependent transitions observed in this assay by using size exclusion chromatography. We have also examined the effects of breaking specific salt bridges and hydrogen bonds by mutating residues in context of caspase-3, an inactive procaspase-3(C163S) and an uncleavable procaspase-3(D3A). We show that there are a number of stabilizing contacts that are required in order to ensure proper processing during maturation, ensure enzyme fidelity and maintain overall structure. At present, the function of the prodomain of executioner caspases has elucidated researchers. We have also hypothesized that the effector caspase prodomain may have some role as an intramolecular chaperone during maturation. We show that the caspase-3 prodomain does play a role in pH dependent folding of the (pro)caspase-3 dimer. Salt has also been well described as having effects on caspase-3 activity and stability. There is a lack of knowledge as to the direct effects that different ions have on both procaspase-3 zymogen and mature caspase-3. In this study, we describe direct effects of different cations on (pro)caspase-3 activity and active site environment. We also study stabilizing effects of salt on (pro)caspase-
Intracellular Signaling Networks in the Immune Response: Pathways Activated by Interleukin-2 and Interleukin-4 Receptors and their Roles in T Cell Proliferation
(2006-09-20) Comfort, Kristen Krupa; Robert Kelly, Committee Member; David Ollis, Committee Member; Jason M. Haugh, Committee Chair; Carla Mattos, Committee Member
Cells sense and respond to chemical and physical stimuli through signal transduction pathways, which mediate cell proliferation, differentiation, migration, and survival. The cytokines interleukin-2 (IL-2) and interleukin-4 (IL-4) are key regulators of the adaptive immune system, particularly influencing the clonal expansion and differentiation of T cells. At least in culture, both synergistic and antagonistic effects of IL-2 and -4 co-stimulation have been reported; the antagonism, when observed, is thought to arise from the utilization of a common subunit shared by IL-2 and IL-4 receptors. We have sought to characterize IL-2 and IL-4 signaling at the level of intracellular pathways activated by these receptors. IL-2 receptors are known to activate the Ras/extracellular signal-regulated kinase (Erk) and phosphoinositide (PI) 3-kinase pathways as well as the STAT5 transcription factor. IL-4 is unique among cytokines in that it does not activate Ras⁄Erk; it does activate PI 3-kinase⁄Akt as well as a distinct STAT, STAT6. The HT-2 mouse T cell line responds to both IL-2 and -4. We found that IL-4 initially antagonizes, and later synergizes with, IL-2-stimulated HT-2 cell proliferation in a dose-dependent manner. IL-4 also stimulates cell adhesion in static cultures. At the level of intracellular signaling, IL-4 antagonizes IL-2-stimulated activation of Akt, possibly through competition for limiting amounts of common receptor subunit and/or PI 3-kinase. Because IL-4 alone does not promote any Erk activation, we were surprised to find that IL-4 enhances IL-2-stimulated activation of Erk. IL-2⁄IL-4 co-stimulation provokes transient activation of STAT5 and prolonged activation of STAT6. This extended STAT6 activation may be critical in the IL-2⁄IL-4 induced synergy in T cell growth. Currently, we are investigating the crosstalk between these pathways and their functional roles in IL-2 and IL-4-stimulated T cell responses.
Kinetic Analysis of PDGF Receptor Signaling Pathways in Fibroblasts
(2005-01-24) Park, Chang Shin; Steven W. Peretti, Committee Member; Robert M. Kelly, Committee Member; Jason M. Haugh, Committee Chair; Carla Mattos, Committee Member
Living cells perceive information about their surroundings and decode it in order to decide how to respond. All of this information flow is processed through intracellular signaling pathways. However, research on these intracellular processes is difficult because signaling pathways are connected to each other in numerous ways. Due to the complicated nature of the interactions among signaling molecules, the overall signal transduction network has hardly been investigated quantitatively or systematically. In order to reveal the underlying control of the signaling network, we designed mechanistic experiments to quantitatively measure the activity of signaling molecules and formulated a mathematical model consistent with these data. In this manner, we approached the study of the platelet-derived growth factor (PDGF) receptor mediated phosphoinositide (PI) 3-kinase/Akt signaling pathway and crosstalk between Ras and PI 3-kinase. The linear signaling pathway, PDGF receptor/PI 3-kinase/Akt, was systematically investigated by using biochemical assays and mathematical modeling. We find that PDGF receptor phosphorylation shows positive cooperativity with respect to PDGF concentration, and the receptor dimerization process is consistent with association of two 1:1 ligand-receptor intermediate complexes. The kinetics of receptor phosphorylation are transient at high PDGF concentrations. Activation of PI 3-kinase, and thus Akt, are saturated with respect to the number of activated receptors. At higher PDGF concentrations, the kinetics of 3¬°¬Ø PI production are governed by the first-order turnover rate constant and those of Akt are further controlled by its deactivation rate constant. To investigate the crosstalk between Ras and PI 3-kinase, we systematically designed experiments to assess the effect of Ras on PI 3-kinase activation and developed a simple mathematical model to describe the molecular assembly of complexes involving PDGF receptors, PI 3-kinase, and Ras. From the experimental results, we find that the Ras effectively enhances the affinity of PI 3-kinase towards the receptor. In our model, this can only be explained if a ternary receptor/PI 3-kinase/Ras complex forms in two steps, where the second step is greatly enhanced through induced proximity and possibly allosteric effects.
Monogenic Diabetes and Regulation of the Transcription Factor, HNF1.
(2008-01-23) Wan, Cheng; William L. Miller, Committee Member; Robert C. Smart, Committee Member; Carla Mattos, Committee Member; Robert B. Rose, Committee Chair
Nonparametric, Parametric and Semiparametric Models for Screening and Decoding Pools of Chemical Compounds
(2002-12-18) Yi, Bingming; Jacqueline M. Hughes-Oliver, Committee Chair; Sidney Stanley Young, Committee Co-Chair; Anastasios Tsiatis, Committee Member; Carla Mattos, Committee Member
During High Throughput Screening (HTS), large collections of chemical compounds are tested for potency with respect to one or more assays. In reality, only a very small fraction of the compounds in a collection will be potent enough to act as lead molecules in later drug discovery phases. Testing all compounds is neither cost-effective nor desirable. Based on the belief that chemical structure is highly related to potency of compounds, structure activity relationships (SARs) can be very helpful for selecting a handful of chemical compounds for testing. This work investigates SARs using four different statistical methods. The first uses a latent class cell-based method. The second benefits from a fractional factorial design for optimizing the cell-based method to significantly increase hit rates. The third improves HTS efficiency by considering pooling experiments for chemical compounds in the presence of interaction and dilution. Rather than testing one compound at a time, chemical compounds are mixed together and tested by groups. Likelihood models are built and hit rates are shown to be higher than for traditional methods. The fourth solves the estimation problem in a pooling experiment by treating the pooling data as missing at random. Semiparametric models are implemented and estimators are shown to be more efficient than likelihood methods based on the same data.
Stochastic Modeling of the Behavior of Dynein
(2005-04-11) Goedecke, David Michael; John F. Monahan, Committee Member; Kevin Gross, Committee Member; Carla Mattos, Committee Member; Sharon R. Lubkin, Committee Co-Chair; Timothy C. Elston, Committee Co-Chair
Molecular motors are proteins that convert stored energy into physical work inside cells, and thus are the engines that drive many cellular functions. An individual motor can be studied using a laser trap to measure its response to working against an external force. Axonemal dynein is the molecular motor responsible for the rhythmic beating of eukaryotic cilia and flagella. An individual axonemal dynein molecule is capable of both unidirectional, processive motion and bidirectional motion when placed under a load (Shingyoji et al., 1998). This capability may be an important underlying factor in the mechanism for flagellar and ciliary motion. A detailed stochastic model is proposed which links the physical motion of a two-headed dynein molecule to the biochemical steps of its ATP hydrolysis cycle. Forward motion is driven by ATP hydrolysis, while backward motion is due to a passive process of biased diffusion. The model exhibits both processive and bidirectional behaviors. A simplified model which can be more easily analyzed is derived, as is an alternate version which steps backward actively, rather than sliding passively. The simplified models are then used to predict motor characteristics such as the load-velocity profile, the stall force, and the effective diffusion coefficient, which can be determined experimentally and used to distinguish among competing mechanisms.
Systematic Analysis of Crosstalk in the PDGF Receptor Signal Transduction Network
(2009-08-19) Wang, Chun-Chao; Carla Mattos, Committee Member; Jason M. Haugh, Committee Chair; Robert M. Kelly, Committee Member; Balaji Rao, Committee Member
Intracellular signal transduction is traditionally characterized in terms of pathways, comprised of serial activation processes. Although it is appreciated that canonical signaling pathways are simply dominant routes of regulation embedded in larger interaction networks, relatively little has been done to quantify pathway crosstalk in such networks. Through quantitative measurements that systematically canvas an array of stimulation and molecular perturbation conditions, together with computational modeling and analysis, we have elucidated crosstalk mechanisms in the platelet-derived growth factor (PDGF) receptor signaling network, in which phosphoinositide 3-kinase (PI3K) and Ras/extracellular signal-regulated kinase (Erk) pathways are prominently activated. We show that, while PI3K signaling is insulated from crosstalk, PI3K enhances Erk activation in multiple ways. Whereas simultaneously blocking Ras and PI3K abolishes PDGF-stimulated Erk phosphorylation, each pathway makes an independent contribution to Erk activation, and PI3K affects Ras activation as well. The magnitudes of these effects depend strongly on the stimulation conditions, subject to saturation effects in the respective pathways and negative feedback loops. Motivated by those dynamics, a kinetic model of the network was formulated and used to precisely quantify the relative contributions of PI3K-dependent and -independent modes of Ras/Erk activation.
Towards Predictive Molecular Dynamics Simulations of DNA: Role of Electrostatics and of the Cell Environment.
(2007-01-11) Baucom, Jason Butler; Celeste Sagui, Committee Chair; Christopher Roland, Committee Member; Carla Mattos, Committee Member; Steffen Heber, Committee Member
Molecular dynamics simulations of the DNA duplex d(CCAACGTTGG)2 were used to study the relationship between DNA sequence and structure in a crystal environment. Three different force fields were used: a traditional description based on atomic point charges, a polarizable force field, and an "extra-point" force field (with additional charges on extranuclear sites). It is found that all the force fields reproduce fairly well the sequence-dependent features of the experimental structure. The polarizable force field, however, provides the most accurate representation of the crystal structure and the sequence-dependent effects observed in the experiment. These results point out to the need of the inclusion of polarization for accurate descriptions of DNA. This work has also investigated to what extent molecular dynamics (MD) simulations can reproduce DNA sequence-specific features, given different electrostatic descriptions and different cell environments. For this purpose, we have carried out multiple unrestrained MD simulations of the DNA duplex d(CCAACGTTGG)2. With respect to the electrostatic descriptions, two different force fields are studied: a traditional description based on atomic point charges and a polarizable force field. With respect to the cell environment, the difference between crystal and solution environments is emphasized, as well as the structural importance of divalent ions. By imposing the correct experimental unit cell environment, an initial configuration with two ideal B-DNA duplexes in the unit cell, is shown to converge to the crystallographic structure. This convergence is measured by the appearance of sequence-dependent features that very closely resemble the crystallographic ones, as well as by the decay of the all-atom root-mean-squared coordinates deviations (RMSD) with respect to the crystallographic structure. Given the appropriate crystallographic constraints, this is first example of multiple nanosecond molecular dynamics trajectory that shows an ideal B-DNA model converging to an experimental structure, with a significant decay of RMSD. At later times, the polarizable force field is able to maintain this lower RMSD while the nonpolarizable force field starts to drift away.
Transcriptional Profiling of Geminivirus Infection in Arabidopsis thaliana Col-0
(2007-04-03) Ascencio-Ibanez, Jose Trinidad; Carla Mattos, Committee Member; Dennis Brown, Committee Member; Linda Hanley-Bowdoin, Committee Chair; Dominique Robertson, Committee Member
Geminiviruses are small DNA viruses that replicate in the nucleus and use plant replication machinery to amplify their single-stranded genomes. Geminiviruses replicate through a combination of rolling-circle and recombination-dependent replication. Earlier studies showed that the geminivirus, Cabbage leaf curl virus (CaLCuV), induces transcription of a host gene encoding a replication factor. To scrutinize the global impact of geminivirus infection on host gene expression, Arabidopsis transcriptome in response to CaLCuV infection at 12 days post inoculation was examined. These experiments uncovered 5241 Arabidopsis genes with changes in transcript levels (q < 0.004, p < 0.002) in response to infection. Data mining of the differentially expressed genes revealed that CaLCuV triggers a pathogen response via the salicylic acid pathway and inhibits the jasmonate pathway. CaLCuV also induced genes associated with programmed cell death and genotoxic stress, including components of the host DNA repair apparatus. CaLCuV also impacted expression of cell cycle-associated genes, preferentially activating genes with peak expression in S and G2 and inhibiting genes that peak in G1 and M. A limited set of core cell cycle genes expressed during cell cycle re-entry, late G1, S and early G2 had increased RNA levels, while core cell cycle genes expressed in early G1 and late G2 had reduced transcript levels. FACS analysis of nuclei from infected leaves showed an increase in 8C, 16C and 32C DNA content relative to nuclei from control leaves. Together, these results underscore the complexity of geminivirus/host interactions and establish that geminiviruses alter cell cycle controls to induce plant cells to move into an endocycle and support viral replication. We also standardized a previously reported method for inoculating geminiviruses using plasmid DNA rubbed onto leaves in the presence of an abrasive (DNA abrasion). We can obtain 100% inoculation efficiency with this method in solanaceous plants. Silencing of an endogenous gene was successful when a silencing vector was inoculated by DNA abrasion.