Dissertations

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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.
Activin induction of the ovine follicle-stimulating hormone beta-subunit is mediated by Smad4 and a forkhead box transcription factor
(2008-10-29) Gore, Arnold Jesse; William L. Miller, Committee Chair; Dennis T. Brown, Committee Member; Jun Ninomiya-Tsuji, Committee Member; Michael B. Goshe, Committee Member
Follicle stimulating hormone (FSH) is a a/b glycoprotein produced in pituitary gonadotropes of all vertebrates and is required for egg maturation, and optimal sperm performance. Activin potently induces FSH by inducing transcription of its rate-limiting b-subunit (FSHB). Four nucleotides of the oFSHB promoter (â€“168 bp to â€“165 bp) are necessary for 99.9 % of oFSHB expression in vivo and 70 % of its progressive induction by activin in LbT2 gonadotropes over a 24 hr period. These 4 nucleotides form part of a putative forkhead box (FOX) binding site juxtaposed upstream to a single-copy (4 bp) Smad binding element (SBE), both of which are associated with activin action. Smad4 from LbT2 cells did not bind the wild type oFSHB SBE in electrophoretic mobility shift assays, but did bind a palindromic SBE derived from the oFSHB SBE. Binding increased 2.6-fold over 20 h without or with activin (25 % additional increase with activin) and was competed 85 % with the native oFSHB sequence indicating Smad4 has high affinity for the native promoter. Additionally, a dominant negative inhibitor of Smad4 reduced activin induction of oFSHB in LBT2 cells by 62 %, indicating that Smad4 is important for activin induction. A second Smad, Smad3, bound transiently to the palindromic SBE (6-fold increase by activin at 2 h). Dominant negative inhibition of Smad3 (3SA) and depletion of Smad2 by siRNA did not alter activin induction of oFSHB suggesting Smads 2 and 3 may not be involved. A p38 inhibitor blocked induction of oFSHB after 8 h, dividing the 24 hr induction by activin into two phases. This suggested that an activin-regulated early gene product is required for the second phase of oFSHB induction. It was found that one forkhead gene, FOXQ1, was increased 4.5-fold 8 h after activin treatment which correlated nicely with the second phase of oFSHB induction uncovered by the p38 inhibitor. FOXQ1 is only one of 43 FOX family members, however, so further studies are required to prove that FOXQ1 is a key driver of FSH production and that it partners with Smad4 to induce oFSHB transcription.
Allosteric Activation of Procaspase-3.
(2012-03-26) Schipper, Joshua Luke; Allan Clark, Chair; William Miller, Member; Robert Rose, Member; Keith Weninger, Member
Allosteric Inactivation of Caspase-3 Without Major Loop Rearrangements: Properties of the Inactive Ensemble.
(2013-10-25) Cade, Christine Elizabeth; Allan Clark, Chair; Robert Rose, Member; Robert Kelly, Member; Dennis Brown, Member
Alphavirus Entry, Assembly, and Egress.
(2016-04-29) Magliocca, Joseph William; Dennis Brown, Chair; Cynthia Hemenway, Member; Robert Rose, Member; Frank Scholle, Member
Analysis of Gonadotrope-specific Expression of Ovine Follicle Stimulating Hormone Beta Subunit Using Adenoviral Expression Constructs in Purified Primary Murine Gonadotropes.
(2010-11-10) Jia, Jingjing; William Miller, Committee Chair; Dennis Brown, Committee Member; Jun Tsuji, Committee Member; Michael Goshe, Committee Member
Application of the Multiple Solvent Crystal Structures Method to Analyze the Protein Binding Surface of H-Ras Protein
(2006-05-02) Buhrman, Gregory Kale; Carla Mattos, Committee Chair
H-Ras is a member of the small, monomeric GTPase protein superfamily. H-Ras functions as a 'molecular switch', using nucleotide dependent conformational changes to relay signals in a number of signal transduction pathways. Mutations in codons 12, 13 and 61 creates an oncogenic version of the protein which does not hydrolyze GTP, resulting in the constitutive activation of downstream effector proteins. Ras proteins participate in multiple protein : protein interactions in the cell, making Ras a good candidate protein to extend the Multiple Solvent Crystal Structures method (MSCS) to the analysis and prediction of protein binding surfaces. MSCS involves solving the crystal structure of the protein after soaking the protein crystal in a variety of organic solvent molecules. Replacing an aqueous solvent with an organic solvent affects the Ras protein structure in several ways. The disordered Switch II region of Ras is ordered in the presence of 2,2,2-trifluoroethanol or 1,6-hexanediol. Polar interactions that stabilize the ordered switch are enhanced in the presence of hydrophobic co-solvents. This suggests that hydrophobic solvents can be used in general to order short biologically relevant segments of disordered regions in protein crystals. We have used MSCS to study two crystal forms of active H-Ras bound to a nonhydrolyzable GTP analog (GMPPNP). We have also solved the structure of an oncogenic mutant of H-Ras (Q61L) in a non-canonical crystal form. This crystal form of H-Ras shows a new conformation for the flexible Switch II region that is not affected by crystal packing forces. This provides a structural explanation for the oncogenic properties of the Q61L mutation, showing that the Q61L mutation stabilizes a non-catalytic conformation of Switch II. MSCS analysis of Ras identifies the known Ras-effector binding domain as a site of protein: protein interaction and predicts a new protein binding site that is located in a large, solvent exposed pocket between Switch II and helix 3. In applying MSCS to the Ras protein, we show that by using polar organic solvent molecules as probes, we can identify binding sites that are highly charged and dynamic.
Arbovirus Entry and Characterization of a new Insect Virus.
(2012-03-23) Vancini, Ricardo; Dennis Brown, Chair; Carla Mattos, Member; Michael Goshe, Member; John Mackenzie, Member
Arbovirus Structure and Interaction with Host Cells.
(2019-06-18) Yang, Sophia; Dennis Brown, Chair; Michael Goshe, Member; Flora Meilleur, Member; Raquel Hernandez, Member; Frank Scholle, Member
Archaeal Box C/D sRNP Assembly, Structure and Function
(2007-11-29) Gagnon, Keith Thomas Jr.; E. Stuart Maxwell, Committee Chair; Paul Agris, Committee Member; Jim Brown, Committee Member; Paul Wollenzien, Committee Member; Clay Clark, Committee Member
Arginyl-tRNA Modifications Modulate Anticodon Domain Structure, Function and Dynamics in Escherichia coli.
(2012-03-20) Cantara, William; Paul Wollenzien, Chair; Earl Maxwell, Member; James Brown, Member; Paul Agris, Member
Assembly and Entry of Alphaviruses.
(2011-03-21) Kononchik, Joseph; Dennis Brown, Chair; Paul Wollenzien, Member; John MacKenzie, Member; John Cavanagh, Member
The Biochemical Characterization of Protein DE and Its Interaction with Rat Epididymal Sperm.
(2001-03-26) Tubbs, Christopher Elliot; Paul L. Wollenzien, Ph.D, Chair; Joseph Hall, Ph.D, Co-Chair; Cynthia Hemenway, Ph.D, Member; Thoyd Melton, Ph.D., Member; William Miller, Ph.D., Member
Using traditional column chromatography, Protein DE has been purified from rat epididymides. Affinity, size exclusion, and ion-exchange chromatography were utilized to purify the protein to homogeneity. Protein DE purity was demonstrated using one and two-dimensional electrophoresis. Using the purified sample, an accurate molecular mass of 27,534 Daltons was determined using electrospray-ionization mass spectrometry. After four chromatographic steps, Protein DE was efficiently separated from all detectable epididymal proteins. This report provides the first rapid and reproducible method for purifying protein DE to homogeneity.Using western blot analysis and immunofluorescence, protein D is initially detected in rat epididymal tissue and associated with sperm from the distal caput region. In contrast, when sperm were recovered from the female reproductive tract seven hours after mating, protein D was not detected by western blot, but did display faint immunofluorescence. Additionally, using photoactivatible cross-linking, a 120 KD sperm membrane protein that specifically interacts with protein D was identified. A population of membrane bound protein D was released from NaCl washed epididymal sperm when incubated in the presence of phosphatidyl-inositol specific phospholipase C. This report is the first demonstrating that both the secretion and sperm-association of protein D occur in the distal caput region of the rat epididymis. It is the only report showing western blot analysis and immunolocalization of sperm-associated protein D on sperm deposited in the female reproductive tract after mating. Additionally, this is the first report that: (a) protein D binds specifically to a 120 KD membrane protein on the surface of epididymal sperm, (b) and that protein D is anchored or associated with a protein that is anchored to the sperm plasma membrane through a glycosylphosphatidyl inositol linkage
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.
Chronic GnRH Inhibits Activin Induction of the ovine FSH Beta Subunit: Involvement of cAMP Response Element Binding Protein (CREB) and Nitric Oxide Synthase typeI (NOSI)
(2008-04-17) Shafiee-Kermani, Farideh; William L. Miller, Committee Chair; Robert R. Anholt, Committee Member; James A. Knopp, Committee Member; Dennis T. Brown, Committee Member
Cis-acting Elements Important for Potato Virus X Minus-strand RNA Synthesis In Vitro and In Vivo
(2007-02-27) Hu, Bin; Paul Wollenzien, Committee Member; Cynthia L. Hemenway, Committee Chair; Barbara Sherry, Committee Member; E. Stuart Maxwell, Committee Member
In order to identify cis-acting elements required for Potato virus X (PVX) minus-strand RNA synthesis, replication in in vitro and in vivo systems was compared. Specifically, RNA dependent RNA polymerase activity using a 850 nt template in PVX infected tobacco plant extract and using infectious full-length transcripts in tobacco protoplasts was analyzed. To facilitate quantitation of results from the in vitro system, the RdRp assay was optimized in several ways. Initial experiments showed that processing of extracts from fresh plant tissue was optimal for the in vitro RdRp assay. Purification of nuclease Bal31 with stable RNase activity was critical for consistency in making and assaying template-dependent plant extracts. Optimal salt concentrations, reaction volume, incubation time, and template concentration conditions were found to ensure template specificity and quantifiable product levels for PVX RdRp. Comparison of data obtained with this optimal extract and the protoplast system showed that the conserved hexanucleotide element and conformation of stem-loop 3 are required for minus-strand RNA synthesis both in vitro and in vivo. More strikingly, we found that long-distance RNA-RNA interactions between conserved internal elements and the hexanucleotide element are required for optimal minus-strand RNA synthesis both in vitro and in vivo. In addition, multiple internal elements can serve as interaction partners. Thus, similar to plus-strand RNA synthesis, PVX minus-strand RNA synthesis requires local elements and long-distance RNA-RNA interactions. A model for RNA synthesis is proposed in which both termini of the genome are paired with internal elements.
Collision-Induced Dissociative Crosslinking Reagents and Methodology for the Structural Analysis of Proteins and Protein-Protein Interactions using Tandem Mass Spectrometry.
(2008-11-13) Soderblom, Erik James; Dr. Steven D. Clouse, Committee Member; Dr. Michael B. Goshe, Committee Chair; Dr. John Cavanagh, Committee Member; Dr. Dennis T. Brown, Committee Member; Dr. Carla Mattos, Committee Member
Chemical crosslinking combined with mass spectrometry is a viable approach to study the low-resolution structure of proteins as well as the topological organization of protein-protein complexes. The identification of the residues involved in the crosslink, however, remains analytically challenging due to the nature of the crosslink itself and its influence on the resulting mass spectrum. To address these challenges, we have developed a novel class of collision-induced dissociative chemical crosslinking reagents which incorporate a gas-phase cleavable aspartyl-prolyl bond into the linker region of the reagent. The specific dissociation of the crosslinked complex allows for a tandem mass spectrometry analysis to be performed on each of the individual peptides involved in the crosslink and therefore simplifies the analysis of the crosslinked products. Following a description of the organic synthesis and purification protocols developed for two collision-induced dissociative crosslinking reagents, a systematic dissociation study of various lysine containing polyalanine peptides was conducted to determine optimized energies for reagent dissociation. Once optimal conditions had been established, methodologies for incorporating the novel crosslinking reagents into automated high-performance liquid chromatography-tandem mass spectrometry analysis were developed on various mass spectrometry platforms and validated with several different model protein systems. To apply our novel crosslinking reagent and methodology to a structurally unknown protein system, a complete crosslinking study of the transition-state regulator AbrB from Bacillus subtilis was conducted. The structural constraints obtained from these crosslinking studies were used to generate a three-dimensional model of full length AbrB which will serve as a platform for additional biochemical studies.
Comparison of the Catalytic Activities of Ricin A-Chain, Maize rproRIP1, MaizeRIP1, and Two Maize rproRIP1 Deletion Mutants Interacting with an RNA 10-mer GAGA Tetraloop.
(2003-07-08) Lieberman, David; Rebecca S. Boston, Committee Member; Paul L. Wollenzien, Committee Member; James A. Knopp, Committee Member; Charles C. Hardin, Committee Chair
Ribosome-inactivating proteins (RIPs) catalytically depurinate a conserved adenine in the ribosomal alpha-sarcin domain, and it is believed that the local structure surrounding the target adenine is (at least transiently) an RNA GAGA tetraloop with the first A in the GAGA sequence being the target adenine. Studies of the catalytic activity of ricin A-chain interacting with ribosomes and with RNA GAGA tetraloops, as well as the catalytic activities of a set of related maize RIPs interacting with ribosomes have been reported in the literature. The purpose of this research project was to extend our understanding of maize RIP1 catalytic activity by measuring and comparing the catalytic activities of ricin A-chain and a set of related maize RIPs interacting with an RNA 10-mer GAGA tetraloop (denoted by A-10) over a range of pH values from pH 3.0 to 6.0. Timecourse experiments of A-10/enzyme reactions were used to measure catalytic activity by quantitative HPLC detection of the adenine released during a succession of elapsed time intervals as the depurination reaction proceeded. The form of the timecourse data did not conform sufficiently to the integrated Michaelis-Menten equation to permit the use of nonlinear curve fitting to characterize the results in terms of the Michaelis-Menten parameters K[subscript M] and k[subscript cat]. Thus, the comparison of the activities of ricin A-chain and the various maize RIP variants both among themselves and as a function of pH, although definitive, is semiquantitative. For one of the enzymes, maizeRIP1, initial velocity experiments at high substrate concentrations yielded an approximate V[subscript max] directly, which was then used to treat V[subscript max] as a constant rather than a variable in fitting the data to the integrated Michaelis-Menten equation, and corresponding values for K[subscript max] were obtained. However, these substrate concentrations were not sufficiently high to yield V[subscript max] values for the other enzymes. One unexpected result, with important ramifications concerning the conformation of the maize proRIP1, was evidence that maize rproRIP1, which is a zymogen (inactive precursor) with respect to ribosomes, may be catalytically active with respect to the small RNA tetraloop A-10.
Detection of Related Solvent Positions and Multiple Solvent Crystal Structures of Trypsin, Chymotrypsin, and Ras-GTPase.
(2012-10-19) Kearney, Bradley Michael; Carla Mattos, Chair; Robert Kelly, Member; Dennis Brown, Member; Allan Clark, Member
Development and Application of Liquid Chromatography-Tandem Mass Spectrometry Methods for Phosphoproteome Analysis of Marek's Disease Virus Infection.
(2011-08-11) Chien, Ko-yi; Michael Goshe, Chair; Dennis Brown, Member; Robert Rose, Member; Hsiao-Ching Liu, Member

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Browsing Dissertations by Discipline "Biochemistry"