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Discovery of a novel lipoxygenase pathway in skinYu, Zheyong 19 October 2005 (has links)
Lipoxygenase (LOX) are non-heme iron dioxygenases that form fatty acid hydroperoxides used in membrane remodeling and cell signaling. Mammalian epidermal LOX type 3 (eLOX3) is distinctive in totally lacking this typical oxygenase activity. Surprisingly, genetic evidence has linked mutations in either eLOX3 or a co-localizing enzyme, 12R-LOX, to an inherited skin disease, non-bullous congenital ichthyosiform erythroderma (NCIE), in which there is a defect in the normal skin permeability barrier (Hum. Mol. Gen. 11, 107-113). Here I identify a logical link of the biochemistry to the genetics. eLOX3 functions as a hydroperoxide isomerase utilizing the product of 12R-LOX, 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE), as the preferred substrate. Using HPLC, GC-MS, NMR and CD spectroscopy, I demonstrated that eLOX3 converts 12R-HPETE to a specific epoxyalcohol, 8R-hydroxy-11R,12R-epoxyeicosa-5Z,9E,14Z-trienoic acid, and 12-ketoeicosatetraenoic acid in a 2:1 ratio. eLOX3 appears to be unique among LOX enzymes in using the ferrous form of the catalytic iron as the active species, initiating reaction by a one electron reduction of the substrate hydroperoxide and completing reaction by rebound hydroxylation to form the epoxyalcohol product. I analyzed the effect of the naturally occurring mutations identified in NCIE on eLOX3 and 12R-LOX catalytic activity; the lipoxygenase activity of 12R-LOX and the hydroperoxide isomerase activity of eLOX3 were totally eliminated. I further demonstrated that the epoxyalcohol formed by human eLOX3 is metabolized by soluble epoxide hydrolase in human keratinocytes to a single trihydroxy isomer, 8R,11S,12R-trihydroxyeicosa-5Z,9E,14Z-trienoic acid. Both the epoxyalcohol and its triol hydrolysis product were then tested for activity in activation of peroxisome proliferator-activated receptors (PPARs). Each selectively caused induction of PPARalpha-dependent transcription with similar activity to 8S-hydroxyeicosatetraenoic acid, a PPARalpha specific agonist. Because human and mouse express a different spectrum of LOX enzymes in skin, I also investigated the substrate selectivity of mouse eLOX3. It uses the product of mouse 8-LOX as its preferred substrate, a coupling consistent with the specific expression of 8-LOX in mouse skin. My results provide strong biochemical evidence for the existence of a novel LOX pathway. Loss of this pathway may contribute to a reduced differentiation in keratinocytes and pathogenesis of NCIE.
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Kinetic analysis of phospholipase D: Allosteric modulation by monomeric GTPases, protein kinase C, and polyphosphoinositidesHenage, Lee Gardner 30 March 2006 (has links)
In mammalian cells, phospholipase D activity is tightly regulated by diverse cellular signals including hormones, neurotransmitters, and growth factors. Multiple signaling pathways converge upon phospholipase D to modulate cellular actions such as cell growth, shape and secretion. The kinetic properties of protein kinase C and G-protein regulation of mammalian phospholipase D1 (PLD1) were examined in order to better understand interactions between PLD1 and its regulators. Activation by Arf-1, RhoA, Rac1, Cdc42, protein kinase Ca, and phosphatidylinositol 4,5-bisphosphate displayed surface dilution kinetics, but these effectors modulated different kinetic parameters. A kinetic description of PLD1 activation by multiple modulators reveals a mechanism for apparent synergy between activators. These findings suggest a role for PLD1 as a signaling node, in which integration of convergent signals occurs within discrete locales of the cellular membrane.
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Autoregulation of ADAR2 function by RNA editing.Feng, Yi 16 December 2005 (has links)
ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-selective conversion of adenosine to inosine. Previous studies from our laboratory have demonstrated that ADAR2 can modify its own pre-mRNA to create a proximal 3=-splice site containing a non-canonical adenosine-inosine (A-I) dinucleotide. Alternative splicing to this proximal acceptor adds 47 nucleotides to the mature ADAR2 transcript, thereby resulting in the loss of functional ADAR2 protein expression due to premature translation termination in an alternate reading frame. To examine whether the editing of ADAR2 transcripts represents a negative autoregulatory strategy to modulate ADAR2 protein expression, we have generated genetically modified mice in which the ability of ADAR2 to edit its own pre-mRNA has been selectively ablated by deletion of a critical sequence (´ECS) required for adenosine-to-inosine (A-to-I) conversion. Here we demonstrate that ADAR2 autoediting and subsequent alternative splicing are abolished in homozygous ´ECS mice and that ADAR2 protein expression is increased in numerous tissues when compared to wild-type animals. The observed increases in ADAR2 protein expression correlate with the extent of ADAR2 autoediting observed in wild-type tissues, and correspond to increases in the editing of ADAR2 substrates, indicating that ADAR2 autoediting is a key regulator of ADAR2 protein expression and activity in vivo.
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REGULATION OF THE TRP CALCIUM CHANNEL BY EYE-PKC IN DROSOPHILAPopescu, Daniela Catalina 27 November 2006 (has links)
The transient receptor potential (TRP) ion channels are implicated in diverse physiological processes, however their regulatory mechanisms remain obscure. In Drosophila, TRP is a calcium channel mediating the light-dependent depolarization of photoreceptor cells. TRP associates with inactivation-no-afterpotential D (INAD), a scaffolding protein that forms a macromolecular complex by also tethering a phospholipase Cbeta, NORPA, and an eye-specific protein kinase C, eye-PKC. It is well established that eye-PKC regulates deactivation of the visual response. Moreover, deactivation is regulated by the interaction between INAD and TRP, as abrogation of this interaction in InaDp215 results in slow deactivation similar to that of inaCp209 lacking eye-PKC. Eye-PKC was shown previously to phosphorylate TRP in vitro. In this thesis we investigated the mechanism by which eye-PKC regulates TRP to achieve fast deactivation of the visual signaling. We identified Ser982 of TRP as an eye-PKC phosphorylation site by an in vitro kinase assay. We show that phosphorylation of TRP by eye-PKC is dependent on both eye-PKC and INAD, suggesting that INAD acts to anchor eye-PKC to TRP. Importantly, we show by mass spectrometry that TRP is indeed phosphorylated by eye-PKC in vivo: phosphorylated peptides spanning Ser982 were observed in TRP isolated from light adapted wild-type but not from inaCp209 flies. To gain insight into the functional significance of phosphorylation at Ser982 of TRP, we generated transgenic flies expressing a modified TRP containing an Ala substitution at Ser982. We demonstrated that transgenic flies exhibited abnormal deactivation, indicating that phosphorylation of TRP at Ser982 by eye-PKC is critical for deactivation of the visual signaling. Interestingly, the slow deactivation defect in the transgenic flies is similar to that of InaDp215 in which TRP fails to associate with INAD, suggesting that the deactivation defect of InaDp215 is due to a loss of TRP phosphorylation by eye-PKC. Taken together, these findings support the notion that the INAD macromolecular complex is important for termination of the visual response, as it positions eye-PKC in close proximity to TRP to promote fast inactivation of the TRP channel.
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Unraveling the Role of G-proteins in Hallucinogenic Drug ActionGarcia, Efrain Eduardo 20 April 2007 (has links)
Extensive evidence suggests that 5-HT2 receptors may play a role in mental disorders including schizophrenia, depression and psychosis. In addition, several studies indicate that Gq-coupled 5-HT2A family of receptors are likely targets for the initiation of events leading to the hallucinogenic behavior elicited by lysergic acid diethylamide (LSD), (+/-)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and related drugs. However, 5-HT2A receptors couple to other G proteins in addition to Gq protein. To evaluate the role of the Gq signaling pathway in DOI-induced behaviors, we utilized two behavioral models of 5-HT2A receptor activation: induction of head-twitches by DOI, a common response to hallucinogenic drugs in rodents, and DOI elicited anxiolytic-like effects in the elevated plus maze. Experimental subjects were genetically modified mice [Gq(-/-)] in which the gene is eliminated by heterologous recombination. Gq(-/-) mice exhibited a decrease in DOI-induced head-twitches, when compared to wild-type littermates. In addition, the DOI-induced increase in anxiolytic-like behavior was abolished in Gq(-/-) mice. These results, combined with our finding that DOI-induced FOS expression in the medial prefrontal cortex was also eliminated in Gq(-/-) mice, suggests a key role for Gq protein in hallucinogenic drug effects.
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STUDYING THE ROLE OF ENDOCANNABINOID SIGNALING IN REPRODUCTIONSun, Xiaofei 16 July 2010 (has links)
Marijuana is the most commonly used illicit drug, and its major active component, Δ9-tetrahydrocannabinol (Δ9-THC) exert its functions by targeting cannabinoid receptors, CNR1 and CNR2. There receptors are also targeted by endocannabinoids, including anandamide.
In this dissertation, we show that genetic loss of Faah, which encodes fatty acid amide hydrolase (FAAH), results in elevated levels of anandamide, an endocannabinoid, in the male reproductive system, leading to compromised fertilizing capacity of sperm. This defect is rescued by superimposing deletion of cannabinoid receptor 1 (Cnr1). Retention of Faah-/- sperm on the egg zona-pellucida provides evidence that sperms capacity to penetrate the zona barrier is dampened by elevated anandamide levels. Collectively, the results show that aberrant endocannabinoid signaling via CNR1 impairs normal sperm function. Besides unveiling a new regulatory mechanism of sperm function, this study has clinical significance in male fertility.
Exposure to marijuana during pregnancy has adverse effects on placentation. Using mice as a model, we found that the endocannabinoid system is also present on the ectoplacental cone and spongiotrophoblast cells in placentas. We also observed that aberrant endocannabinoid signaling confers premature trophoblast stem cell differentiation, and defective trophoblast development and invasion. These defects are reflected in retarded fetal development and elevated spontaneous pregnancy loss. Because the endocannabinoid system is conserved across species, including humans, our study suggests that endocannabinoid signaling is critical to placentation and pregnancy success in humans.
Collectively, my work demonstrated that appropriate endocannabinoid signaling is critical for both male and female reproductive functions.
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Mapping Conformational Changes Along the Activation Pathway of the Heterotrimeric G Protein α Subunit with Site-directed Spin-labelingOldham, William Michael 02 May 2008 (has links)
Heterotrimeric G proteins act as molecular switches in signaling pathways by coupling the activation of heptahelical receptors at the cell surface to intracellular responses. These receptors bind to and activate G proteins by catalyzing GTP for GDP exchange on the Gα subunit, leading to a structural change in Gα(GTP) and Gβγ subunits that allows the activation of a variety of downstream effector proteins. Despite its crucial role in a variety of signal transduction pathways, relatively little is known about the structure of the receptor-G protein complex and how this interaction leads to GDP release from Gα. Thus, the primary goal of this research has been to use the biophysical technique of site-directed spin-labeling to identify and characterize receptor-dependent conformational changes in Gα with electron paramagnetic resonance spectroscopy. With this approach, α5 helix of Gα has been shown to play a key role in coupling receptor-binding to GDP release. In addition, the structure and dynamics of several other important regions of this protein have been explored throughout the G protein activation pathway. These studies enhance the current understanding of G protein structure and function, which has been largely based on high resolution structural data from x-ray crystallography, by providing dynamic information about this protein in solution. This combination of structural approaches should continue to provide important insight into the biomechanics of G protein signaling and will hopefully serve as the starting point for more sophisticated models of the critically important receptor-G protein complex.
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The mechanism and physiological function of epidermal lipoxygenase-3Zheng, Yuxiang 10 December 2010 (has links)
The goal of my thesis research is to elucidate the catalytic mechanism and physiological function of the atypical mammalian lipoxygenase, epidermal lipoxygenase-3. Although named as a lipoxygenase based on sequence homology to other lipoxygenases, epidermal lipoxygenase-3 appears to be devoid of dioxygenase activity with natural polyunsaturated fatty acids and shows instead a prominent hydroperoxide isomerase activity with fatty acid hydroperoxides. Epidermal lipoxygenase-3, together with 12R-lipoxygenase, is implicated through genetics in skin barrier formation, yet the mechanism of action of these two lipoxygenases in the physiological setting is poorly understood. During my research I uncovered dioxygenase activity in epidermal lipoxygenase-3, elucidated its mechanism, and developed a novel model to explain the in vivo action of epidermal lipoxygenase-3 and 12R-lipoxygenase. The mechanistic study of eLOX3 led to a better understanding of the catalysis of epidermal lipoxygenase-3 and lipoxygenases in general, particularly regarding how lipoxygenase is activated by fatty acid hydroperoxides and the role of active site O2 in this activation process. The study on the physiological function of epidermal lipoxygenase-3 led to elucidation of a novel biochemical pathway whereby 12R-lipoxygenase and epidermal lipoxygenase-3 mediate at least part of the long-known and not well-understood effect of essential fatty acids on skin health by oxygenating linoleate-enriched ceramides and allowing subsequent hydrolysis and covalent coupling of the ceramides to proteins, thus providing an explanation for the skin barrier defects exhibited when the gene encoding either epidermal lipoxygenase-3 or 12R-lipoxygenase is deleted, or when essential fatty acids are excluded from the diet.
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Characterization of Mutant Human Brain Sodium Channels Associated with Familial EpilepsyMisra, Sunita N 17 July 2008 (has links)
Investigating genetic forms of epilepsy allows for improved understanding of epilepsy pathophysiology in general. Mutations in voltage-gated sodium channels are a frequent cause of genetic forms of epilepsy. First we constructed a computational model of one sodium channel isoform and an epilepsy-associated mutation that refined our knowledge of how sodium channels inactivate. We next utilized a heterologous expression system to biophysically and biochemically characterize epilepsy-associated mutations. We found that aberrant cell surface expression as well as biophysical abnormalities may underlie some epilepsy syndromes. Finally we performed experiments in a transgenic mouse model system of epilepsy to examine the effect of genetic modifiers on sodium channel function. In summary, this research utilized three different model systems to improve our understanding of genetic forms of epilepsy.
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PROARRHYTHMIC DEFECTS IN TIMOTHY SYNDROME REQUIRE CALMODULIN KINASE IIThiel, William Howard 02 December 2008 (has links)
Ca2+ activated signaling pathways coordinate contraction in the heart, but these pathways cause disease upon excessive activation. Intracellular Ca2+ activates the multifunctional Ca2+ and calmodulin dependent protein kinase II (CaMKII) and activated CaMKII increases Ca2+ entry, by a feed-forward process, through voltage-gated (CaV1.2) Ca2+ channels in cardiac myocytes. Timothy Syndrome an autosomal dominant, monogenic disease that induces cellular Ca2+ overload in cardiomyocytes, due to a loss of voltage dependent inactivation of ICa. Based on the proarrhythmic properties of CaMKII, I hypothesized that excess ICa in Timothy Syndrome would activate CaMKII and that CaMKII would serve as a positive feedback and proarrhythmic signal. A ventricular myocyte model of TS showed significant increases in CaMKII activity and a cellular proarrhythmic phenotype that included action potential prolongation, increased ICa facilitation and afterdepolarizations. A highly specific CaMKII inhibitor, and not an inactive control peptide, reversed the dynamic ICa facilitation increases, normalized the action potential duration and prevented afterdepolarizations in TS ventricular myocytes. The ability of CaMKII to phosphorylate the CaV1.2 C-terminus was also examined.
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