Browsing by Author "William L. Miller, 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.
Estrogen Response Element and the Promoter Context of the Human and Mouse Lactoferrin Genes Influence Estrogen Receptor alpha-Mediated Transactivation Activity in Mammary Gland Cells
(2004-12-01) Stokes, Kenya; William L. Miller, Committee Member; Mark Conkling, Committee Member; Jonathan Allen, Committee Member; Christina T. Teng, Committee Co-Chair; Brenda Alston-Mills, Committee Co-Chair
The purpose of this research has been to determine whether an extended estrogen response element half-site (ERRE) contributes to the differential estrogen responses of the human and mouse lactoferrin estrogen response element (ERE) in the context of their natural promoters. This research utilized molecular biology techniques to evaluate gene activation. Transfections of MCF-7 cells showed that liganded ER-alpha activates transcription of the human lactoferrin ERE 4-fold higher than the mouse lactoferrin ERE in the context of their natural promoters. Since the ERRE of the human lactoferrin gene naturally occurs 18 bp upstream from the ERE and is absent in the mouse lactoferrin gene promoter, we created a chimeric mouse lactoferrin CAT reporter, which now encodes the ERRE in the identical location as in the human lactoferrin gene. The addition of the ERRE in the mouse lactoferrin gene rendered this reporter extremely responsive to estrogen stimulation. We also demonstrated that the conformation of the estrogen receptor bound to the ERE alone or in the presence of ERRE differed and that the ERRE influenced the selectivity of coactivators in liganded ER-alpha-mediated transcriptional activity. Like the lactoferrin gene ERE, most known natural estrogen response elements are imperfect palindromes that differ from the consensus by at least a 1 base pair change and confer different levels of ER transcriptional activation compared to the consensus ERE. In contrast to our transient transfection data showing a lower estrogen response of the mouse lactoferrin ERE compared to the human lactoferrin ERE in the context of their natural promoters, in vivo data showed that the gene is robustly transcribed in response of estrogen in both species. Therefore, this research model can be applied to studies of genes that have different hormone responses in vivo versus in vitro in an attempt to identify non-typical estrogen response elements that influence ER-alpha-mediated transactivation.
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
The Role of Estrogen Receptor-a and Estrogen Receptor-b in the Hyperluteinized Mouse Ovary
(2004-09-13) Couse, John Floyd; Robert C. Smart, Committee Chair; Kenneth S. Korach, Committee Co-Chair; Gerald A. LeBlanc, Committee Member; William L. Miller, Committee Member
The hypothalamic-pituitary-gonadal (HPG) axis was characterized in female mice lacking one or both forms of estrogen receptor (ERa, ERb) with the aim of elucidating the contribution of each receptor form to gonadotropin homeostasis and ovarian function. These studies consisted of a thorough evaluation of gene expression for the gonadotropin subunits in the pituitary and the components necessary for steroidogenesis in the ovary. These data were corroborated with evaluations of the plasma levels for each of the relevant pituitary and gonadal hormones. Females lacking ERb (bERKO) exhibit minimal disruption in HPG axis function but do exhibit deficits in gonadotropin responsiveness in the ovary. Females lacking ERa (aERKO) exhibit dramatic ovarian phenotypes of hemorrhagic and cystic follicles and exaggerated steroid synthesis in the ovaries. The phenotypes in the aERKO ovary are attributable to chronically elevated LH due to the loss of ERa function in the hypothalamus. Pharmacologic reduction of plasma LH levels in aERKO females abates the ovarian phenotypes. These studies indicate that the hypothalamic functions of ERa are most critical to ovarian function. To better understand the contribution of ERb to the manifestations of LH-hyperstimulation in the ovary, females lacking functional ERb but possessing elevated LH via a transgene (bERKOLHCTP) were generated. Characterization of bERKOLHCTP animals indicates the intraovarian functions of ERb are necessary for the induction of LH-associated cystic follicles but not amplified steroidogenesis. An additional novel finding in aERKO ovaries was ectopic expression of the Leydig cell specific enzyme, 17b-HSD III and correlating male-like testosterone synthesis. This phenotype is dependent on LH-hyperstimulation of ovaries lacking ERa function to manifest. In summary, the predominant contribution of ERa to ovarian function occurs in the hypothalamus, whereas ERb is more important within the ovary itself. Presence of Leydig cell specific gene expression in aERKO ovaries indicates a potential role for estradiol and ERa in gonadal differentiation.
Structural Characterization of Two-Component Signal Transduction Proteins and Calbindin D28k
(2006-11-17) Kojetin, Douglas John; Dennis T. Brown, Committee Member; William L. Miller, Committee Member; Ronald A. Venters, Committee Member; Charles H. Opperman, Committee Member; John Cavanagh, Committee Chair
The studies described involve the structural analysis of proteins involved in signal transduction pathways. In the first study, the metal binding properties of the initiation of sporulation response regulator Spo0F was studied using a variety of biophysical and biochemical techniques. The experiments show that most of the divalent metals studied, including and Ca²⁺, Mg²⁺ and Mn²⁺, which display primarily 1:1 binding, allow for favorable conditions for phosphotransfer between Spo0F and its cognate kinase KinA. In contrast, Spo0F binds up to three Cu²⁺ ions and the presence of this metal does not allow for the phosphotransfer reaction to occur. In the second study, a comparative modeling study of the OmpR sub-family of response regulators from B. subtilis and E. coli was performed and used to suggest the possibility of sub-classes within this related domain family based on regions of the response regulator regulatory domain that is known to interact with cognate four-helix bundle HisKA/Hpt domains. In the third study, the structural refinement of the four-helix bundle LuxU phosphotransferase from V. harveyi is described using a combination of dipolar couplings and water-based explicit refinement. In the fourth and last study, the development of a solution structure of Ca²⁺-loaded calbindin D[subscript 28k], an EF-hand calcium binding protein, and the interaction between peptides derived from ran-binding protein M and myo-inositol monophosphatase are described.
Study of Protein Binding Sites on the GTPase RalA and the Sugar-binding Protein Hen Egg White Lysozyme
(2006-08-10) Nicely, Nathan; Robert Kelly, Committee Member; Carla Mattos, Committee Chair; Robert Rose, Committee Member; Dennis Brown, Committee Member; William L. Miller, Committee Member
Hen egg white lysozyme and simian RalA are two very different proteins by function and category. Lysozyme is an extracellular enzyme that catalyzes the hydrolysis of the β-linkage between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) subunits in the peptidoglycan polymers that compose some Gram-positive bacterial cell walls. RalA is a Ras-related GTPase involved in multiple distinct signaling pathways. The structure and sequence of its core domain is similar to Ras and Rap (another Ras-related GTPase), but they have mutually exclusive sets of upstream activators and downstream effectors. Furthermore, RalA is activated by calcium-loaded Calmodulin through its carboxy-terminal domain, and it binds phospholipase D constitutively through its amino-terminal domain; both traits are unique within the Ras subfamily. Lysozyme has a deep active site cleft between two subdomains which is responsible for binding the saccharide substrate. This binding site is small in its surface area compared to the total accessible surface area of lysozyme. It is relatively well-ordered and pre-formed, with good shape complementarity to the substrate. We employ the Multiple Solvent Crystal Structures method which uses small organic solvent molecules as probes to map the functional surface of the protein. Of ten solvent-soaked crystal structures, 11 solvent molecules were identified as bound to lysozyme in a total of six sites. Nine of these 11 solvent molecules bound in the active site cleft in well defined clusters corresponding to the established subsites in which the NAM/NAG subunits of the natural substrates bind. Five of these nine bind in subsite C, which has the most favorable binding energy of the six subsites. Two bind in subsite D and one each in subsites E and F. The positions and orientations of the bound solvent molecules mimic the acetamido functional groups on the NAM/NAG subunits, especially in subsite C. Of the two organic solvent molecules which bound outside the active site cleft, one bound at a two-fold crystal contact and the other on the edge of the epitope for an anti-lysozyme antibody. RalA has two large segments, termed the switch regions (I & II), that experience disorder-to-order transitions upon complexation with binding partners. These regions are responsible for significant structural changes across a large patch of the protein's accessible surface. We have solved the crystal structures of RalA in both its GDP- ("off;" inactivated) and GTP analog-bound ("on;" activated) forms. Disorder-to-order transitions occur in both switch regions upon protein-protein interaction in the form of crystallographic and noncrystallographic symmetry contacts; however, in the absence of such protein-protein contacts, both switches are disordered. This indicates a departure from the behavior of Ras in which the presence of GTP analog alone is sufficient to order switch I. Also, we identify two possible sites for protein-protein interaction on the surface of RalA by comparing structural features of the protein with the available data regarding amino acid residues important for its biochemical functions and including the experimental functionality map for Ras generated by the Multiple Solvent Crystal Structures method. A thorough analysis of the binding sites on RalA and lysozyme reveal some trends which agree with recent hypotheses on the nature of protein-ligand interfaces. First, all the binding sites on both proteins tend to have centers which are relatively invariant in terms of structural plasticity. These cores are surrounded by residues which exhibit conformational flexibility. Second, the binding sites are sparsely hydrated; any bound water molecules at our binding sites can be displaced by solvent molecules. Conversely, the switch regions of RalA are well hydrated at protein-protein contacts, reflecting the ability of water molecules to contribute to the close packing of atoms and charge complementarity in protein-ligand interfaces.