Browsing by Subject "regulation"

Now showing 1 - 9 of 9
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    A Structural and Kinetic Study into the Role of the Quaternary Shift in Bacillus stearothermophilus Phosphofructokinase
    (2011-10-21) Mosser, Rockann Elizabeth
    Bacillus stearothermophilus phosphofructokinase (BsPFK) is a homotetramer that is allosterically inhibited by phosphoenolpyruvate (PEP), which binds along one dimer-dimer interface. The substrate, fructose-6-phosphate (F6P), binds along the other dimer-dimer interface. The different functional forms BsPFK can take when in the presence of F6P and PEP can be described by the following diproportionation equilibrium: XE + EA <--> XEA + E where XE is the enzyme bound to PEP, EA is the enzyme bound to F6P, E represents the apo enzyme, and XEA is the ternary complex formed when both substrate and inhibitor are bound. Currently in the Protein Data Bank (PDB) there are two relevant forms of wild-type BsPFK, the EA form and the X'E form, which represents the enzyme bound to the PEP analog, phosphoglycolate (PGA). When comparing the EA and the X'E structures, a 7? rotation about the substrate-binding interface is observed and is termed the quaternary shift. The current study uses methyl TROSY NMR to examine the different liganded states of BsPFK, and for the first time structural data for the XEA species is shown. In addition, crystallography was used to obtain the first apo structure of BsPFK. To distinguish between changes associated with the quaternary shift and those associated with the intra-subunit tertiary changes, the variant D12A BsPFK was studied using kinetics, crystallography, and NMR. Crystal structures of apo and PEP bound forms of D12A BsPFK both indicate a shifted structure similar to the X'E form of wild-type. Kinetic studies of D12A BsPFK, when compared to wild-type, show a 50-fold diminished F6P binding affinity, 100-fold enhanced binding affinity, and a similar coupling constant. A conserved hydrogen bond between D12 and T156 takes place across the substrate binding interface in the EA form of BsPFK. The variant T156A BsPFK shows similar binding, coupling, and structural characteristics to D12A BsPFK. PEP still inhibits these variants of BsPFK despite the fact that the enzymes are in the quaternary shifted position prior to PEP binding. Therefore the quaternary shift of BsPFK primarily perturbs ligand binding but does not directly contribute to heterotropic allosteric inhibition.
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    Center of the periphery
    (2009-05) Thrond, Matthew Dale; Levack, Brian P.; Kamil, Neil, 1954-
    Print culture was a fundamental site in which new ideas about England’s role in world affairs were debated in the latter half of the sixteenth century. Print changed the ways in which new discoveries, proposals, grievances, and questions were assessed, and not always to the desired effect. In the face of the sphinx-like power of the press, a wide array of strategies emerged to control it. But people at many levels of the publishing process could use the rhetoric of the text, and of the printed book, to rearrange the relationships between authors and readers, to upset the thrust of a particular line of argument, to alter the aesthetic, moral, or pragmatic judgment a reader might exercise, or in a more subtle way to change the terms of the issue at hand. In view of the diversity of these possibilities, this report follows figures known to the London print world, some authors, some printers, and examines how they acted, reacted, and worked through, issues that arose from being on the cusp of England’s relationship with a wider world.
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    Characterization of the Tri10 gene from Fusarium sporotrichioides
    (Texas A&M University, 2004-09-30) Tag, Andrew George
    The trichothecene mycotoxins are secondary metabolites produced by a variety of fungal genera including Fusarium, Myrothecium, Trichothecium, and Stachybotyris, that are toxic to humans and animals that ingest them by consumption of contaminated grain. This work details the characterization of a novel regulatory gene from Fusarium sporotrichioides, Tri10, which is located in the trichothecene gene cluster. Northern analysis of Tri10 deletion strains, Tri10 overexpressing strains, and a Tri6 deletion strain indicated that Tri10 is required for wild-type trichothecene gene expression and for wild-type expression of a primary metabolic gene, Fpps. Analysis of these mutants also provided evidence for a regulatory feedback loop where Tri10 is required for the expression of Tri6 and Tri6 negatively regulates Tri10. Furthermore, under certain growth conditions the sensitivity of ?Tri10 and ?Tri6 strains to T-2 toxin was increased. Analysis of mutants altered in the expression and genomic position of Tri10 revealed that placing Tri10 under the control of an exogenous promoter resulted in the overexpression of Tri10 and the other Tri genes whether this construct was located inside or outside of the Tri gene cluster. Work outside of this study has shown that in addition to Fpps, three other primary metabolic genes from the isoprenoid pathway feeding into trichothecene biosynthesis (Acat, Mk, Hmgs) are also influenced by the expression of Tri10 and Tri6. In the present study, targeted cDNA microarrays were used in conjunction with multiple mutants to reveal a large group of genes, containing both trichothecene and primary metabolic genes, which were positively influenced by Tri10 expression. At the same time, a small group of genes negatively influenced by Tri10 expression was observed. These results were in agreement with observations made outside of this study and validated the use of targeted cDNA microarrays for further studies. Additional analysis of the regulatory network linking trichothecene secondary metabolism and isoprenoid primary metabolism revealed that in a mutant blocked in the first step of the pathway, and therefore in the absence of trichothecene production, this regulatory link is mediated by Tri10 and Tri6.
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    Epigenetic modifications and conserved, non-coding DNA play a role in regulation of type IV collagen gene expression
    (2009-05-15) Moody, Jessica Ashley
    Type IV collagens are components of basement membranes throughout the body and are involved in maintenance of the structural integrity of tissues as well as cellular differentiation, growth, and adhesion. Members of this collagen family are uniquely arranged in pairs in a head-to-head orientation and share a proximal promoter region. The COL4A5-COL4A6 gene pair is involved in numerous human diseases and cancer metastasis. For these reasons, defining the mechanisms that regulate collagen gene expression is of specific interest. To study type IV collagens, an in vitro model system was characterized. Comparative genomics was utilized to identify conserved, non-coding DNA in COL4A5 and COL4A6. These sequences were transfected into cell lines differing in type IV collagen expression and tested for the ability to regulate transcription of a reporter gene. Each cell line was also treated with the epigenetic modifying agents, 5-Aza and TSA. The effects on type IV collagen expression were determined. The COL4A5-COL4A6 promoter region was extensively characterized using ChIP analysis; antibodies against RNAPII, acetylated histone H3, and H3K9me2 were used. Additionally, bisulfite sequencing was carried out on each cell line to determine the methylation status of CpG dinucleotides in the promoter. Cell lines differing in expression of COL4A5 and COL4A6 were identified: 1) SCC-25 keratinocytes and HEK-293 cells transcribed both COL4A5 and COL4A6, 2) HT-1080 cells selectively activated COL4A5, and 3) SK-N-SH neuroblastoma cells did not express either gene. In SK-N-SH cells, histone modifications were shown to facilitate formation of condensed chromatin to prevent transcription initiation; repression was independent of DNA methylation. Activation of COL4A5 and COL4A6 in SCC-25 and HEK-293 cells involved acetylation of histones, although differences between the two cell types were seen. In addition, conserved, non-coding sequences were shown to affect transcription of a reporter gene; these sequences may be interacting with the transcription machinery to modulate collagen expression. Finally, repression of COL4A6 in HT-1080 cells appeared to be mediated through DNA methylation of the promoter; selective activation of COL4A5 may involve conserved, non-coding DNA. In summary, epigenetic modifications as well as conserved sequences are intimately involved in regulation of type IV collagen gene expression.
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    Identification of novel regulatory mechanisms controlling heterocyst development in Anabaena Sp. strain PCC 7120
    (2009-05-15) Aldea, Maria Ramona
    The regulatory mechanisms that govern heterocyst development in Anabaena sp. strain PCC 7120 have been continuously refined over the last two decades. In this work, we show that three of the sigma factor genes present in the Anabaena sp. strain PCC 7120 genome are developmentally regulated. Time-lapse microscopy of gfp reporter strains indicated that expression of sigC, sigG, and sigE is upregulated specifically in differentiating cells at 4 h, 9 h and 16 h, respectively, after induction of heterocyst development. We proposed that the sigma factors encoded by these genes are involved in regulation of heterocyst-specific genes whose expression is relatively coincident with that of sigC, sigG, or sigE. Indeed, inactivation of the sigC gene caused delayed and reduced expression of genes required for the early stages of heterocyst development, and caused delayed development. Inactivation of the sigE gene caused a considerable drop in expression of nifH, a late gene required for nitrogen fixation. We also provide evidence that c-di-GMP, a novel bacterial second messenger, is involved in regulating heterocyst development. The all2874 gene encodes a bona fide diguanylate cyclase, which synthesizes c-di-GMP, and the gene's inactivation resulted in a decreased tendency to form heterocysts; this phenotype was exacerbated by high light intensity. We hypothesize that the putative operon all2875-all2874 senses and relays information about light conditions and this information is integrated into the decision to form heterocysts. Finally, we identified the all0187 gene, which is expressed at 9 h, a time when cells that have initiated differentiation commit to complete the process. In nitrogen-free medium, all0187 mutant filaments formed abnormally long heterocysts and were unable to grow diazotrophically. Septum formation between heterocysts and their flanking vegetative cells was incomplete, leaving one or both poles of the heterocysts more opened and potentially more permeable to oxygen. Despite having nitrogenase activity, the all0187 mutant was unable to grow diazotrophically. We hypothesize that the diazotrophic growth defect is caused by the inability of the heterocysts to transport fixed nitrogen to the neighboring vegetative cells.
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    Identification of Structural Changes Associated with Regulation of Tyrosine Hydroxylase
    (2011-10-21) Wang, Shanzhi
    Tyrosine hydroxylase (TyrH) is the first and rate-limiting enzyme of catecholamine synthetic pathway, and its regulation is critical for controlling catecholamine synthesis. The well recognized regulatory mechanisms are inhibition by catecholamine binding and re-activation upon Ser40 phosphorylation. Catecholamines bind to TyrH tightly, while phosphorylation of TyrH at Ser40 decreases the binding affinity by several hundred-fold. Regulation of TyrH is accompanied by conformational changes of the protein. This study focuses on the identification of the conformational changes of TyrH upon dopamine binding and Ser40 phosphorylation, using hydrogen deuterium exchange mass spectrometry (HDMS) and fluorescence spectroscopy. HDMS identifies three peptides undergoing conformational changes upon dopamine binding, peptide 35-41, 42-71 and 295-299. Peptides 35-41 and 42-71 are on the regulatory domain, while peptide 295-299 is at the active site entrance. Upon dopamine binding, all three peptides are protected from exchange; phosphorylation of TyrH at Ser40 has opposite effects on the exchange kinetics of peptide 295-299, but peptides 35-41 and 42-71 could not be detected by MS after phosphorylation. This suggests that the structural effects of dopamine binding and Ser40 phosphorylation are opposite. The fluorescence spectroscopy of mutant enzymes containing a single tryptophan at position 14, 34 or 74 was performed before and after phosphorylation. F34W/F3W TyrH has a significant decrease in steady-state fluorescence anisotropy, an increase in the bimolecular quenching rate constant kq and dynamic anisotropy upon phosphorylation at Ser40, while F14W/F3W TyrH and F74W/F3W TyrH exhibit much smaller differences. This suggests that phosphorylation of TyrH at Ser40 increases the flexibility of the regulatory domain. The results are consistent with TyrH existing in two conformations, a closed conformation stabilized by dopamine in which the N-terminal regulator domain of TyrH covers the active site entrance and an open conformation stabilized by phosphorylation in which the regulatory domain has moved away from the active site entrance.
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    Online regulations of low order systems under bounded control
    (Texas A&M University, 2004-09-30) Arora, Sumit
    Time-optimal solutions provide us with the fastest means to regulate a system in presence of input constraints. This advantage of time-optimal control solutions is offset by the fact that their real-time implementation involves computationally intensive iterative techniques. Moreover, time-optimal controls depend on the initial state and have to be recalculated for even the slightest perturbation. Clearly time-optimal controls are not good candidates for online regulation. Consequently, the search for alternatives to time-optimal solutions is a very active area of research. The work described here is inspired by the simplicity of optimal-aim concept. The "optimal-aim strategies" provide online regulation in presence of bounded inputs with minimal computational effort. These are based purely on state-space geometry of the plant and are inherently adaptive in nature. Optimal-aim techniques involve aiming of trajectory derivative (or the state velocity vector) so as to approach the equilibrium state in the best possible manner. This thesis documents the efforts to develop an online regulation algorithm for systems with input constraints. Through a number of hypotheses focussed on trying to reproduce the exact time-optimal solution, the diffculty associated with this task is demonstrated. A modification of optimal-aim concept is employed to develop a novel regulation algorithm. In this algorithm, aim directions are chosen in a special manner to generate the time-optimal control approximately. The control scheme thus developed is shown to be globally stabilizing for systems having eigenvalues in the CLHP (closed left half-plane). It is expected that this method or its modifications can be extended to higher dimensional systems as a part of future research. An alternative control algorithm involving a simple state-space aiming concept is also developed and discussed.
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    Role of new virulence mechanisms/factors (type 3 secretion system and TOX-R regulated lipoprotein [TAGA]) in the pathogenesis of the emerging human pathogen Aeromonas hydrophila
    (2006-08-10) Lakshmi Pillai; Dr. Ashok K. Chopra, Ph.D. C.Sc.; Dr. Vladimir Motin, Ph.D.; Dr. Golda A.K. Leonard, Ph.D.; Dr. Eric M. Smith, Ph.D.; Dr. Chandrasekha Yallampalli, Ph.D.; Dr. Amy Horneman, Ph.D., SM(ASCP)
    Aeromonas hydrophila, a gram-negative bacterium that causes gastroenteritis, wound infections, septicemia, and other diseases in humans, produces many different virulence factors. A clinical isolate SSU of A. hydrophila possesses a cytotoxic enterotoxin Act, a potent virulence factor that is secreted into the environment through the bacterium’s type 2 secretion system (T2SS) and possesses several biological activities, including cytotoxicity, enterotoxicity, and lethality in a mouse model. The purpose of this study was to identify new virulence factors that contribute to the pathogenesis of this bacterium. We identified and characterized a type 3 secretion system (T3SS) in A. hydrophila SSU. By marker-exchange mutagenesis of the aopB gene, a crucial gene involved in the formation of the translocon apparatus, the functionality of the T3SS was elucidated, both in in vitro and in vivo models. Further, the characterization of the regulatory gene DNA adenine methyltransferase (Dam) from SSU and its role in modulating the function of both the T3SS and Act was investigated. The role of the T3SS in influencing the phenomenon of quorum sensing (QS) in A. hydrophila SSU was also conducted. This study highlights a unique link between the T3SS and Act of A. hydrophila and the production of QS molecules or lactones. While searching for potential effector proteins secreted through the T3SS of A. hydrophila SSU, the identification of a new virulence factor, ToxR regulated lipoprotein (TagA), was revealed. TagA is a zinc metalloprotease which has only been identified in the gram-negative pathogens, Escherichia coli O157:H7 and Vibrio cholerae. In A. hydrophila, TagA has been shown to play a role in the inhibition of complement by binding to and cleaving the serpin C1-INH. By recruiting C1-INH to the surface of the bacteria and cleaving it, TagA is able to significantly prevent the activation of complement at the cell surface, ultimately increasing the serum resistance of the pathogen. TagA can also target C1-INH to erythrocyte surfaces and decrease the lysis that occurs in the presence of serum. Confocal fluorescence microscopy revealed that the serpin C1-INH binds to TagA on the surface of the bacteria.
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    Studies of the relationship of protein structure to regulation and catalysis in tyrosine hydroxylase
    (Texas A&M University, 2007-09-17) Sura, Giri Raju
    Tyrosine hydroxylase (TyrH) catalyzes the rate-limiting step in the synthesis of the catecholamine neurotransmitters dopamine, epinephrine, and norepinephrine. Phosphorylation of Ser40 of rat TyrH activates the enzyme by decreasing the affinity for catecholamines. In humans, there are four different TyrH isoforms with varying lengths for the regulatory domain. DOPA and dopamine binding studies were performed on the phosphorylated and unphosphorylated human isoforms. The Kd for DOPA was increased two times upon phosphorylation of hTyrH1, but no change was seen for hTyrH4; the Kd value decreased with the increase in the size of regulatory domain. The small effect on the Kd value for DOPA upon phosphorylation of hTyrH suggests that DOPA does not regulate the activity of hTyrH. Dopamine binds very tightly and upon phosphorylation the affinity for dopamine is decreased. This Kd value decreases with the increase in the length of the regulatory domain. The crystal structures of substrate complexes of the homologous enzyme phenylalanine hydroxylase (PheH) show a large movement of a surface loop (residues 131-155) upon amino acid binding. The corresponding loop residues (175-200) in TyrH play an important role in DOPA formation. This conformational change in TyrH loop was studied with fluorescence anisotropy. Three tryptophan residues in the TyrH, at positions 166, 233, and 372, were mutated to phenylalanine, and Phe184 was mutated to tryptophan. An increase in anisotropy was observed in the presence of phenylalanine and 6-methyl-5-deazatetrahydropterin (6M5DPH4), but the magnitude of the change of anisotropy with 6M5DPH4 was greater than that with phenylalanine. Further characterization of the sole tryptophan in the loop showed a decrease in the amplitude of the local motion only in the presence of 6M5DPH4 alone. The conformational change in wild type TyrH was examined by H/D exchange LC/MS spectroscopy in the presence of the natural ligands. Time-course dependent deuterium incorporation into the loop in the presence of ligands indicated that the pterin alone can induce the conformational change in the loop irrespective of whether iron is reduced or oxidized. From these results, one can conclude that the loop undergoes a conformational change upon pterin binding, making the active site better for amino acid binding.