G -rich oligodeoxyribonucleotides: Triplexes and transcription

Formation of a triple helix resulting from oligonucleotide binding to the DNA double helix offers new possibilities to control gene expression at the transcriptional level. Purine-motif triplexes can be formed under physiological pH. Nevertheless, this formation was inhibited by certain monovalent cations during the association but not during dissociation. Since triplexes are very stable, it was possible to assemble them in the absence of KCl and have them survive throughout the course of an in vitro transcription reaction. As for the design of a better triplex-forming oligonucleotide, 12 nucleotides in length afforded the highest binding affinity. G/T-rich oligonucleotides can be very polymorphic in solution. The conditions for forming purine-motif triplexes, duplexes or G-quartets were determined. Understanding these parameters will be important for the practical use of G-rich oligonucleotides in the development of DNA aptamers where the structure of the oligonucleotide is paramount in dictating its function. Finally, purine-motif triplexes were demonstrated to significantly inhibit gene transcription in vitro. The optimal effect on this process was dependent on the location of triplexes within the promoter, i.e., whether upstream or proximally downstream of the transcription start site. The mechanism for the inhibition of transcription appeared to be interference with initiation through preventing engagement by RNA polymerase. This finding is revolutionary when compared to the conventional model where triplexes inhibit transcription only by occluding binding by trans-acting proteins. Our findings broaden the utility of triplexes and support a strategy for antigene therapy by triplexes. ^

New insights into the role of the duodenal vitamin D receptor in calcium homeostasis

It is generally believed that 1,25(OH)2D3, bound to its receptor (VDR) contributes to calcium homeostasis by regulating active calcium absorption in the proximal small intestine. However, studying patients with hereditary vitamin D-resistant rickets (HVDRR) provided investigators with a better understanding of VDR's role in calcium homeostasis. HVDRR patients have inactivating mutations in the VDR, and as a consequence they develop hypocalcemia, hyperparathyroidism and severe rickets. However, these phenotypes can be corrected if the patients are given IV infusions of calcium or dietary calcium. This raises the question of what is the physiological significance of VDR-regulated active calcium absorption if calcium homeostasis can be restored independently of the VDR. ^ In order to distinguish the contribution of VDR in the proximal small intestine to overall calcium homeostasis, I generated transgenic mice expressing the human VDR (hVDR) exclusively in the proximal small intestine of mVDR-/- mice by using an hVDR-expressing transgene driven by the duodenal-specific adenosine deaminase enhancer (hVDR+/mVDR-/-). hVDR+/mVDR-/- mice expressed transcriptionally active hVDR only in the proximal small intestine and responded to 1,25(OH)2D3 by up-regulating expression of TRPV6 and calbindin D9K, genes involved in calcium absorption. Furthermore, ligated duodenal loop assays determined that calcium absorption in hVDR+/mVDR-/- mice was as responsive to 1,25(OH)2D3 as in WT mice. Despite having a functional hVDR in the proximal small intestine, hVDR+/mVDR-/- mice were hypocalcemic, had hyperparathyroidism, and were rachitic when fed a normal rodent diet at weaning, as were the mVDR-/- mice. However, when fed a high calcium, phosphorus, and lactose diet (rescue diet), the hVDR+/mVDR-/- mice responded more effectively than the mVDR-/- mice by down-regulation of parathyroid hormone production and by a greater increase in bone mineralization. Furthermore, when three-month-old rachitic mice were fed a rescue diet for 3 weeks, serum calcium and bone mineral content were normalized in hVDR+/mVDR-/- mice, but not in mVDR-/- mice. ^ In conclusion, hVDR expression enabled young mice to better use the rescue diet than mVDR-/- mice. Expression of transgenic hVDR also protected the ability of older mice to respond to the rescue diet despite the absence of the VDR elsewhere in the intestinal tract. I propose that because hVDR+/mVDR-/- mice responded better than mVDR-/- mice to the rescue diet, it is likely that VDR expression in the proximal small intestine is necessary in nutritional (insufficient dietary calcium) and physiological (age) conditions when passive calcium absorption is inadequate. ^

Organization and function of platelet glycoprotein Ib-IX complex

Glycoprotein (GP) Ib-IX complex, the second most abundant receptor expressed on the platelet surface, plays critical roles in haemostasis and thrombosis by binding to its ligand, von Willebrand factor (vWF). Defect or malfunction of the complex leads to severe bleeding disorders, heart attack or stroke. Comprised of three type I transmembrane subunits—GPIbα, GPIbβ and GPIX, efficient expression of the GPIb-IX complex requires all three subunits, as evident from genetic mutations identified in the patients and reproduced in transfected Chinese hamster ovary (CHO) cells. However, how the subunits are assembled together and how the complex function is regulated is not fully clear. By probing the interactions among the three subunits in transfected cells, we have demonstrated that the transmembrane domains of the three subunits interact with one another, facilitating formation of the two membrane-proximal disulfide bonds between GPIbα and GPIbβ. We have also identified the interface between extracellular domains of GPIbβ and GPIX, and provided evidence suggesting a direct interaction between extracellular domains of GPIbα and GPIX. All of these interactions are not only critical for correct assembly and consequently efficient expression of the GPIb-IX complex on the cell surface, but also for its function, such as the proper ligand binding, since removing the two inter-subunit disulfide bonds significantly hampers vWF binding to the complex under both static and physiological flow conditions. The two inter-subunit disulfide bonds are also critical for regulating the ectodomain shedding of GPIbα by the GPIbβ cytoplasmic domain. Mutations in the juxtamembrane region of the GPIbβ cytoplasmic domain deregulate GPIbα shedding, and such deregulation is further enhanced when the two inter-subunit disulfide bonds are removed. In summary, we have established the overall organization of the GPIb-IX complex, and the importance of proper organization on its function. ^

Homology Cloning, Heterologous Expression and Characterization of a New Channelrhodopsin

Channelrhodopsins are phototaxis receptors in the plasma membranes of motile unicellular algae. They function as light-gated cation channels and this channel activity has been exploited to trigger action potentials in neurons with light to control neural circuits (“optogenetics"). Four channelrhodopsins were identified in two algal species, Chlamydomonas reinhardtii and Volvox carteri, with known genome sequences; each species contains 2 channelrhodopsins, one absorbing at longer wavelengths and one at shorter wavelengths, named CrChR1 and CrChR2, respectively. Our goals are to expand knowledge of channelrhodopsin mechanisms and also to identify new channelrhodopsins from various algal species with improved properties for optogenetic use. For these aims we are targeting algae from extreme environments to establish the natural diversity of their properties. We cloned a new channelrhodopsin from the psychrophilic (cold-loving) alga, Chlamydomonas augustae, with degenerate primers based on the 4 known homologs. The new protein is 48% and 52% identical to CrChR1 and CrChR2, respectively. We expressed the channelrhodopsin in HEK293 cells and measured light-induced currents to assess their kinetics and action spectrum. Based on the primary structure, kinetics of light-induced photocurrents in HEK293 cells, and action spectrum maximum of 520 nm near that of the two previously found CrChR1, we named the new channelrhodopsin CaChR1. The properties of robust channel activity at physiological pH, fast on-and-off kinetics, and greatly red-shifted action spectrum maximum from that of CrChR2, make CaChR1 advantageous as an optogenetic tool. To know this new channelrhodopsin better, we expressed His-tagged CaChR1 in Pichia pastoris and the yield is about 6 mg/L. The purified His-tagged CaChR1 exhibited an absorption spectrum identical to the action spectrum of CaChR1-generated photocurrents. The future work will be measurement of the photocycles of CaChR1 by flash photolysis, crystallization of CaChR1 for the structure and mutagenesis of CaChR1 to find the critical amino acids accounting for red-shifted spectra, slow inactivation and rapid on-and-off kinetics. Seven new channelrhodopsins including CaChR1 from different algal species have been cloned in our lab at this time, bringing the total known to 13. The work of cloning of these new channelrhodopsins along with the expression of CaChR1 was published in Photochemistry and Photobiology in January 2012

THE EQUILIBRIUM CONSTANT FOR THE GLYCINE SYNTHASE REACTION (ONE-CARBON, METABOLISM, FOLATE)

The equilibrium constant (K(,c)) under physiological conditions (38(DEGREES)C, 0.25 M ionic strength (I), pH 7.0) for the glycine synthase (GS) reaction (E C 2.1.2.1.0) (Equation 1) has been determined. (UNFORMATTED TABLE FOLLOWS)^ 5,10-CH(,2)-H(,4)Folate NADH NH (,4)+ CO(,2) ^ K(,c) = Eq. 1^ H(,4)Folate NAD('+) GLY ^(TABLE ENDS)^ The enzymatic instability of the GS enzyme complex itself has made it necessary to determine the overall K(,c) from the product of constants for the partial reactions of GS determined separately under the same conditions. The partial reactions are the H(,4)Folate-formaldehyde (CH(,2)(OH)(,2)) condensation reaction (Reaction 1) the K(,c) for which has been reported by this laboratory (3.0 x 10('4)), the lipoate (LipS(,2)) dehydrogenase reaction (LipDH) (Reaction 2) and the Gly-Lip^ decarboxylase reaction (Reaction 3) forming reduced lipoate (Lip(SH)(,2)), NH(,4)('+), CO(,2) and CH(,2)(OH)(,2.) (UNFORMATTED TABLE FOLLOWS)(,)^ H(,4)Fote + CH(,2)(OH)(,2) 5,10-CH(,2)-H(,4)Folate (1)^ Lip(SH)(,2) + NAD('+) LipS(,2) + NADH + H('+) (2)^ H('+) + Gly + LipS(,2) Lip(SH)(,2) + NH(,4)('+) CO(,2) + CH(,2)(OH)(,2) (3)^(TABLE ENDS)^ In this work the K(,c) for Reactions 2 and 3 are reported.^ The K(,c)' for the LipDH reaction described by other authors was reported with unexplainable conclusions regarding the pH depend- ence for the reaction. These conclusions would imply otherwise unexpected acid dissociation constants for reduced and oxidized lipoate. The pK(,a)',s for these compounds have been determined to resolve discrepancy. The conclusions are as follows: (1) The K(,c) for the LipDH reaction is 2.08 x 10('-8); (2) The pK(,a)',s for Lip(SH)(,2) are 4.77(-COOH), 9.91(-SH), 11.59(-SH); for LipS(,2) the carboxyl pK(,a)' is 4.77; (3) Contrary to previous literature, the log K(,c)' for the LipDH reaction is a linear function of the pH, a conclusion supported by the values for the dissociation constants.^ The K(,c) for Reaction 3 is the product of constants for Reactions 4-7. (UNFORMATTED TABLE FOLLOWS)^ LipSHSCH(,2)OH + H(,2)O Lip(SH)(,2) + CH(,2)(OH)(,2) (4)^ H(,2)O + LipSHSCH(,2)NH(,3)('+) LipSHSCH(,2)OH + NH(,4)('+) (5)^ LipSHSCH(,2)NH(,2) + H('+) LipSHSCH(,2)NH(,3)('+) (6)^ Gly + LipS(,2) LipSHSCH(,2)NH(,2) + CO(,2) (7)^(TABLE ENDS)^ Reactions 4-6 are non-enzymatic reactions whose constants were determined spectrophotometrically. Reaction 7 was catalyzed by the partially purified P-protein of GS with equilibrium approached from both directions. The value for K(,c) for this reaction is 8.15 x 10('-3). The combined K(,c) for Reactions 4-7 or Reaction 3 is 2.4 M.^ The overall K(,c) for the GS reaction determined by combination of values for Reactions 1-3 is 1.56 x 10('-3). ^

A physiological role of cytochromes P450 4Fs: Mouse model

Cytochromes P450 4Fs (CYP4F) are a subfamily of enzymes involved in arachidonic acid metabolism with highest catalytic activity towards leukotriene B 4 (LTB4), a potent chemoattractant involved in prompting inflammation. CYP4F-mediated metabolism of LTB4 leads to inactive ω-hydroxy products incapable of initiating chemotaxis and the inflammatory stimuli that result in the influx of inflammatory cells. Our hypothesis is based on the catalytic ability of CYP4Fs to inactivate pro-inflammatory LTB4 which assures these enzymes a pivotal role in the process of inflammation resolution. ^ To test this hypothesis and evaluate the changes in CYP4F expression under complex inflammatory conditions, we designed two mouse models, one challenged with lipopolysaccharide (LPS) as a sterile model of sepsis and the other challenged with a systemic live bacterial infection of Citrobacter rodentium, an equivalent of the human enterobacterium E. coli pathogen invasion. Based on the evidence that Peroxisome Proliferator Activated Receptors (PPARs) play an active role in inflammation regulation, we also examined PPARs as a regulation mechanism in CYP4F expression during inflammation using PPARα knockout mice under LPS challenge. Using the Citrobacter rodentium model of inflammation, we studied CYP4F levels to compare them to those in LPS challenged animals. LPS-triggered inflammation signal is mediated by Toll-like 4 (TLR4) receptors which specifically respond to LPS in association with several other proteins. Using TLR4 knockout mice challenged with Citrobacter rodentium we addressed possible mediation of CYP4F expression regulation via these receptors. ^ Our results show isoform- and tissue-specific CYP4F expression in all the tissues examined. The Citrobacter rodentium inflammation model revealed significant reduction in liver expression of CYP4F14 and CYP4F15 and an up-regulation of gene expression of CYP4F16 and CYP4F18. TLR4 knockout studies showed that the decrease in hepatic CYP4F15 expression is TLR4-dependent. CYP4F expression in kidney shows down-regulation of CYP4F14 and CYP4F15 and up-regulation of CYP4F18 expression. In the LPS inflammation model, we showed similar patterns of CYP4F changes as in Citrobacter rodentium -infected mice. The renal profile of CYP4Fs in PPARα knockout mice with LPS challenge showed CYP4F15 down-regulation to be PPARα dependent. Our study confirmed tissue- and isoform-specific regulation of CYP4F isoforms in the course of inflammation. ^

Acetate metabolism and the control of environmental pH in Candida albicans and Saccharomyces cerevisiae

Candida albicans is the most common opportunistic fungal pathogen of humans. The balance between commensal and pathogenic C. albicans is maintained largely by phagocytes of the innate immune system. Analysis of transcriptional changes after macrophage phagocytosis indicates the C. albicans response is broadly similar to starvation, including up-regulation of alternate carbon metabolism. Systems known and suspected to be part of acetate/acetyl-CoA metabolism were also up-regulated, importantly the ACH and ACS genes, which manage acetate/acetyl-CoA interconversion, and the nine-member ATO gene family, thought to participate in transmembrane acetate transport and also linked to the process of environmental alkalinization. ^ Studies into the roles of Ach, Acs1 and Acs2 function in alternate carbon metabolism revealed a substantial role for Acs2 and lesser, but distinct roles, for Ach and Acs1. Deletion mutants were made in C. albicans and were phenotypically evaluated both in vitro and in vivo. Loss of Ach function resulted in mild growth defects on ethanol and acetate and no significant attenuation in virulence in a disseminated mouse model of infection. While loss of Acs1 did not produce any significant phenotypes, loss of Acs2 greatly impaired growth on multiple carbon sources, including glucose, ethanol and acetate. We also concluded that ACS1 and ACS2 likely comprise an essential gene pair. Expression analyses indicated that ACS2 is the predominant form under most growth conditions. ^ ATO gene function had been linked to the process of environmental alkalinization, an ammonium-mediated phenomenon described here first in C. albicans. During growth in glucose-poor, amino acid-rich conditions C. albicans can rapidly change its extracellular pH. This process was glucose-repressible and was accompanied by hyphal formation and changes in colony morphology. We showed that introduction of the ATO1G53D point mutant to C. albicans blocked alkalinization, as did over-expression of C. albicans ATO2, the only C. albicans ATO gene to lack the conserved N-terminal domain. A screen for alkalinization-deficient mutants revealed that ACH1 is essential for alkalinization. However, addition of acetate to the media restored alkalinization to the ach1 mutant. We proposed a model of ATO function in which Atos regulated the cellular co-export of ammonium and acetate. ^

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.

Development and Characterization of an in vitro Four-species Anaerobic Dental Biofilm Model

Dental caries is the most common chronic disease worldwide. It is characterized by the demineralization of tooth enamel caused by acid produced by cariogenic dental bacteria growing on tooth surfaces, termed bacterial biofilms. Cariogenesis is a complex biological process that is influence by multiple factors and is not attributed to a sole causative agent. Instead, caries is associated with multispecies microbial biofilm communities composed of some bacterial species that directly influence the development of a caries lesion and other species that are seemingly benign but must contribute to the community in an uncharacterized way. Clinical analysis of dental caries and its microbial populations is challenging due to many factors including low sensitivity of clinical measurement tools, variability in saliva chemistry, and variation in the microbiota. Our laboratory has developed an in vitro anaerobic biofilm model for dental carries to facilitate both clinical and basic research-based analyses of the multispecies dynamics and individual factors that contribute to cariogenicity. The rational for development of this system was to improve upon the current models that lack key elements. This model places an emphasis on physiological relevance and ease of maintenance and reproducibility. The uniqueness of the model is based on integrating four critical elements: 1) a biofilm community composed of four distinct and representative species typically associated with dental caries, 2) a semi-defined synthetic growth medium designed to mimic saliva, 3) physiologically relevant biofilm growth substrates, and 4) a novel biofilm reactor device designed to facilitate the maintenance and analysis. Specifically, human tooth sections or hydroxyapatite discs embedded into poly(methyl methacrylate) (PMMA) discs are incubated for an initial 24 hr in a static inverted removable substrate (SIRS) biofilm reactor at 37°C under anaerobic conditions in artificial saliva (CAMM) without sucrose in the presence of 1 X 106 cells/ml of each Actinomyces odontolyticus, Fusobacterium nucleatum, Streptococcus mutans, and Veillonella dispar. During days 2 and 3 the samples are maintained continually in CAMM with various exposures to 0.2% sucrose; all of the discs are transferred into fresh medium every 24 hr. To validate that this model is an appropriate in vitro representation of a caries-associated multispecies biofilm, research aims were designed to test the following overarching hypothesis: an in vitro anaerobic biofilm composed of four species (S. mutans, V. dispar, A. odontolyticus, and F. nucleatum) will form a stable biofilm with a community profile that changes in response to environmental conditions and exhibits a cariogenic potential. For these experiments the biofilms as described above were exposed on days 2 and 3 to either CAMM lacking sucrose (no sucrose), CAMM with 0.2% sucrose (constant sucrose), or were transferred twice a day for 1 hr each time into 0.2% sucrose (intermittent sucrose). Four types of analysis were performed: 1) fluorescence microscopy of biofilms stained with Syto 9 and hexidium idodine to determine the biofilm architecture, 2) quantitative PCR (qPCR) to determine the cell number of each species per cm2, 3) vertical scanning interferometry (VSI) to determine the cariogenic potential of the biofilms, and 4) tomographic pH imaging using radiometric fluorescence microscopy after exposure to pH sensitive nanoparticles to measure the micro-environmental pH. The qualitative and quantitative results reveal the expected dynamics of the community profile when exposed to different sucrose conditions and the cariogenic potential of this in vitro four-species anaerobic biofilm model, thus confirming its usefulness for future analysis of primary and secondary dental caries.

Inactivation and self -association of CaM kinase: Effects of ATP, substrate binding domains, and isozymic forms

Calcium/calmodulin-dependent protein kinase II (CaM kinase) is a multifunctional Ser/Thr protein kinase, that is highly enriched in brain and is involved in regulating many aspects of neuronal function. We observed that forebrain CaM kinase from crude homogenates, cytosolic fractions and purified preparations inactivates and translocates into the particulate fraction following autophosphorylation. Using purified forebrain CaM kinase as well as recombinant $\alpha$ isozyme, we determined that the formation of particulate enzyme was due to enzyme self-association. The conditions of autophosphorylation determine whether enzyme self-association and/or inactivation will occur. Self-association of CaM kinase is sensitive to pH, ATP concentration, and enzyme autophosphorylation. This process is prevented by saturating concentrations of ATP. However, in limiting ATP, pH is the dominant factor, and enzyme self-association occurs at pH values $\rm{<}7.0.$ Site-specific mutants were produced by substituting Ala for Thr286, Thr253, or Thr305,306 to determine whether these sites of autophosphorylation affect enzyme inactivation and self-association. The only mutation that influenced these processes was Ala286, which removed the protective effect afforded by autophosphorylation in saturating ATP. Enzyme inactivation occurs in the presence and absence of self-association and appears predominantly sensitive to nucleotide concentration, because saturating concentrations of $\rm Mg\sp{2+}/ADP$ or $\rm Mg\sp{2+}/ATP$ prevent this process. These data implicate the ATP binding pocket in both inactivation and self-association. We also observed that select peptide substrates and peptide inhibitors modeled after the autoregulatory domain of CaM kinase prevented these processes. The $\alpha$ and $\beta$ isozymes of CaM kinase were characterized independently, and were observed to exhibit differences in both enzyme inactivation and self-association. The $\beta$ isozyme was less sensitive to inactivation, and was never observed to self-associate. Biophysical characterization, and transmission electron microscopy coupled with image analysis indicated both isozymes were multimeric, however, the $\alpha$ and $\beta$ isozymes appeared structurally different. We hypothesize that the $\alpha$ subunit of CaM kinase plays both a structural and enzymatic role, and the $\beta$ subunit plays an enzymatic role. The ramifications for the functional differences observed for inactivation and self-association are discussed based on potential structural differences and autoregulation of the $\alpha$ and $\beta$ isozymes in both calcium-induced physiological and pathological processes. ^