Disruption of the histidine triad nucleotide-binding hint2 gene in mice affects glycemic control and mitochondrial function

The histidine triad nucleotide-binding (Hint2) protein is a mitochondrial adenosine phosphoramidase expressed in liver and pancreas. Its physiological function is unknown. To elucidate the role of Hint2 in liver physiology, the Hint2 gene was deleted. Hint2(-/-) and Hint2(+/+) mice were generated in a mixed C57Bl6/J x 129Sv background. At 20 weeks, the phenotypic changes in Hint2(-/-) relative to Hint2(+/+) mice were an accumulation of hepatic triglycerides, decreased tolerance to glucose, a defective counter-regulatory response to insulin-provoked hypoglycaemia, an increase in plasma interprandial insulin but a decrease in glucose stimulated insulin secretion and defective thermoregulation upon fasting. Leptin mRNA in adipose tissue and plasma leptin were elevated. In mitochondria from Hint2(-/-) hepatocytes, state 3 respiration was decreased, a finding confirmed in HepG2 cells where HINT2 mRNA was silenced. The linked complex II to III electron transfer was decreased in Hint2(-/-) mitochondria, which was accompanied by a lower content of coenzyme Q. HIF-2α expression and the generation of reactive oxygen species were increased. Electron microscopy of mitochondria in Hint2(-/-) mice aged 12 months revealed clustered, fused organelles. The hepatic activities of 3-hydroxyacyl-CoA dehydrogenase short chain and glutamate dehydrogenase (GDH) were decreased by 68% and 60%, respectively, without a change in protein expression. GDH activity was similarly decreased in HINT2-silenced HepG2 cells. When measured in the presence of purified sirtuin 3, latent GDH activity was recovered (126% in Hint2(-/-) vs. 83% in Hint2(+/+) ). This suggests a greater extent of acetylation in Hint2(-/-) than in Hint2(+/+) . Conlusions: Hint2 positively regulates mitochondrial lipid metabolism and respiration, and glucose homeostasis. The absence of Hint2 provokes mitochondrial deformities and a change in the pattern of acetylation of selected proteins. (HEPATOLOGY 2012.).

Absence of the calcium-binding protein calretinin, not of calbindin D-28k, causes a permanent impairment of murine adult hippocampal neurogenesis

Calretinin (CR) and calbindin D-28k (CB) are cytosolic EF-hand Ca(2+)-binding proteins and function as Ca(2+) buffers affecting the spatiotemporal aspects of Ca(2+) transients and possibly also as Ca(2+) sensors modulating signaling cascades. In the adult hippocampal circuitry, CR and CB are expressed in specific principal neurons and subsets of interneurons. In addition, CR is transiently expressed within the neurogenic dentate gyrus (DG) niche. CR and CB expression during adult neurogenesis mark critical transition stages, onset of differentiation for CR, and the switch to adult-like connectivity for CB. Absence of either protein during these stages in null-mutant mice may have functional consequences and contribute to some aspects of the identified phenotypes. We report the impact of CR- and CB-deficiency on the proliferation and differentiation of progenitor cells within the subgranular zone (SGZ) neurogenic niche of the DG. Effects were evaluated (1) two and four weeks postnatally, during the transition period of the proliferative matrix to the adult state, and (2) in adult animals (3 months) to trace possible permanent changes in adult neurogenesis. The absence of CB from differentiated DG granule cells has no retrograde effect on the proliferative activity of progenitor cells, nor affects survival or migration/differentiation of newborn neurons in the adult DG including the SGZ. On the contrary, lack of CR from immature early postmitotic granule cells causes an early loss in proliferative capacity of the SGZ that is maintained into adult age, when it has a further impact on the migration/survival of newborn granule cells. The transient CR expression at the onset of adult neurogenesis differentiation may thus have two functions: (1) to serve as a self-maintenance signal for the pool of cells at the same stage of neurogenesis contributing to their survival/differentiation, and (2) it may contribute to retrograde signaling required for maintenance of the progenitor pool.

Lactococcus lactis HemW (HemN) is a haem-binding protein with a putative role in haem trafficking

Lactococcus lactis cannot synthesize haem, but when supplied with haem, expresses a cytochrome bd oxidase. Apart from the cydAB structural genes for this oxidase, L. lactis features two additional genes, hemH and hemW (hemN), with conjectured functions in haem metabolism. While it appears clear that hemH encodes a ferrochelatase, no function is known for hemW. HemW-like proteins occur in bacteria, plants and animals, and are usually annotated as CPDHs (coproporphyrinogen III dehydrogenases). However, such a function has never been demonstrated for a HemW-like protein. We here studied HemW of L. lactis and showed that it is devoid of CPDH activity in vivo and in vitro. Recombinantly produced, purified HemW contained an Fe-S (iron-sulfur) cluster and was dimeric; upon loss of the iron, the protein became monomeric. Both forms of the protein covalently bound haem b in vitro, with a stoichiometry of one haem per monomer and a KD of 8 μM. In vivo, HemW occurred as a haem-free cytosolic form, as well as a haem-containing membrane-associated form. Addition of L. lactis membranes to haem-containing HemW triggered the release of haem from HemW in vitro. On the basis of these findings, we propose a role of HemW in haem trafficking. HemW-like proteins form a distinct phylogenetic clade that has not previously been recognized.

Alteration in 5HT1A Receptor Activity from a Prenatal Exposure to Dexamethasone in a Stressed and Non-Stressed Adult Male Rat

Synthetic glucocorticoids (GC) are used as a clinical therapeutic to stimulate lung development in fetuses that present the risk of preterm delivery. Previous studies have shown that a prenatal exposure to Dexamethasone (DEX) causes a disturbance in normal GC mediation of neuritic outgrowth, cell signaling, and serotonergic systems. Our hypothesis is that a prenatal exposure to DEX during the third trimester of pregnancy alters 5HT1A receptor function. Pregnant dams were injected daily with 150μg/ml/kg of DEX from gestation day 14 through 19. Control dams were treated with and equal volume of saline. Swim stress followed by elevated plus maze testing was conducted on male rats an hour and a half prior to being sacrificed to induce postnatal acute stress. The non-stressed group was also tested and allowed to return to baseline before sacrifice. Hippocampi were analyzed using a radioligand-receptor binding assay and GTPγS35 incorporation (3H-MPPF antagonist and 8-OH-DPAT agonist, respectively). A significant increase in Kd was found in non-stressed DEX-exposed animals compared to non-stressed controls (p

Probing The Active And Allosteric Binding Sites Of Soybean Lipoxygenase-1

Soybean lipoxygenase-1 is a model for lipoxygenase activity. While the mechanism of oxygenation is understood, the substrate binding mechanism has not yet been elucidated. Two putative binding mechanisms are the ¿head-first¿ and ¿tail-first¿ models, in which the carboxy-terminus or the methyl terminus of the fatty acid substrate is inserted into the active site while the remainder of the molecule protrudes from the surface, respectively. Previous work has demonstrated that derivatization of fatty acid substrates with D-tryptophan increases active site affinity. It has also been shown that while polyunsaturated fatty acids are the natural substrates of lipoxygenases, monounsaturated fatty acids can be oxygenated at a much slower rate. Starting with a monounsaturated fatty acid, oleic acid, as a platform, the molecule N-oleoyl-D-tryptophan (ODT) was synthesized with the anticipation of it being a potent competitive substrate-analogue inhibitor that could be used to discern the substrate binding mechanism. Inhibition kinetics demonstrated that this molecule functions as a partially competitive inhibitor, through an unknown mechanism. The implication behind partially competitive inhibition is that substrate and inhibitor molecules can bind simultaneously to the enzyme, which alludes to the presence of an allosteric binding domain. To investigate the possibility of an inhibitor binding site on the non-catalytic subunit, limited proteolysis was used to cleave the subunits apart which should have eliminated inhibition. Interestingly, it was observed that at high substrate concentrations the inhibitor was completely ineffective, but at low substrate concentrations the inhibitor maintained its standard efficacy. A satisfactory explanation for these results has not yet been determined.

Crosslinking Studies To Probe Substrate Binding By Soybean Lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids into conjugated diene hydroperoxides. The three dimensional structure of SBLO-1 is known, but it is not certain how substrates bind. One hypothesis involves the transient separation of helix-2 and helix-11 located on the exterior of the molecule in front of the active site iron. A second hypothesis involves a conformational change in the side chains of residues leucine 541 and threonine 259. To test these hypotheses, site directed mutagenesis was used to create a cysteine mutation on each helix, which could allow for the formation of a disulfide linkage. Disulfide formation between the two cysteines in the T259C,S545C mutant was found to be unfavorable, but later shown to be present at higher pH values using SDS-PAGE. Treatment of the T259C,S545C with the crosslinker 2,3-dibromomaleimide (DBM) resulted in a 50% reduction in catalytic activity. No loss of activity was observed when the single mutant, S545C, or the wild type was treated with DBM. Single mutants T259C and L541C both showed approximately 20% reduction in the rate after addition of DBM. Double mutants T259C,L541C and S263C,S545C showed approximately 30% reduction in the rate after addition of DBM. Single mutants T259C and L541C showed an increase in activity after incubation with NEM. Double mutants T259C,S545C and T259C,L541C showed an increase in activity after incubation with NEM. The S263C,S545C double mutant showed a slight decrease in activity in the presence of NEM. It is unclear how the NEM and DBM are interacting with the molecule, but this can easily be determined through mass spectrometry experiments.

Enzymatic Activity of Soybean Lipoxygenase-1 on Linoleoyl-Derivatized Agarose

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of linoleic acid to form 13(S) and 9(R) hydroperoxides. The manner in which substrates bind to the lipoxygenase family of enzymes is not known. It is believed fatty acid substrates may bind either with the aliphatic end first or with the carboxylate group facing the interior of the protein. This thesis tested a potential methyl-end first substrate binding mechanism by studying the activity of SBLO-1 to oxygenate immobilized linoleoyl residues attached to an insoluble polymer. Linoleic acid was attached to aminohexyl agarose in the presence of N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS). The concentration of the covalently attached residues was facilitated by enriching linoleic acid with a small amount of the radioactive 14C-isotope. Functionalization yields of 3% available primary amines on the resin were obtained. Enzymatic oxygenation of the linoleoyl-residues was verified using the ferrous oxidation in xylenol orange (FOX) assay. Approximately 30% of the attached linoleoyl moieties were converted to hydroperoxides in the presence of SBLO-1. A disulfide-containing cleavable linker, cystamine, was used as part of an improved method to isolate the product in a facile manner. Cystamine was attached to NHS-activated agarose with approximately 5% overall functionalization yield of available functional groups. 14C-linoleic acid was successfully covalently linked to the cystamine moieties in the presence of EDC and NHS. The FOX assay verified the enzymatic oxygenation of the linoleoyl residues attached to cystamine-derivatized agarose. The isolation of the peroxide product was attempted in a series of extractions in organic solvents. The product was analyzed using GC/MS which did not show a new peak indicative of product. Further work is needed to successfully analyze the stereoand regiochemistry of the oxygenated product. The presence of the peroxides in this study indicated the linoleoyl residues behave as substrates of SBLO-1. It is unknown how bulky substrates bind to the active site; however, it is difficult to rationalize a carboxylate group-first binding mode. Discovery of the 13(S)-hydroperoxide product on the linoleoyl-agarose would support the claim of a potential methyl-end first binding mechanism.

Increased titers of anti-Saccharomyces cerevisiae antibodies in Crohn's disease patients with reduced H-ficolin levels but normal MASP-2 activity

Mannan-binding lectin (MBL) and ficolins are microbial pattern recognition molecules that activate the lectin pathway of complement. We previously reported the association of MBL deficiency with anti-Saccharomyces cerevisiae antibodies (ASCA) in patients with Crohn's disease (CD). However, ASCA are also frequently found in MBL-proficient CD patients. Here we addressed expression/function of ficolins and MBL-associated serine protease-2 (MASP-2) regarding potential association with ASCA.

Regulatory mechanism of the light-activable allosteric switch LOV-TAP for the control of DNA binding: a computer simulation study

The spatio-temporal control of gene expression is fundamental to elucidate cell proliferation and deregulation phenomena in living systems. Novel approaches based on light-sensitive multiprotein complexes have recently been devised, showing promising perspectives for the noninvasive and reversible modulation of the DNA-transcriptional activity in vivo. This has lately been demonstrated in a striking way through the generation of the artificial protein construct light-oxygen-voltage (LOV)-tryptophan-activated protein (TAP), in which the LOV-2-Jα photoswitch of phototropin1 from Avena sativa (AsLOV2-Jα) has been ligated to the tryptophan-repressor (TrpR) protein from Escherichia coli. Although tremendous progress has been achieved on the generation of such protein constructs, a detailed understanding of their functioning as opto-genetical tools is still in its infancy. Here, we elucidate the early stages of the light-induced regulatory mechanism of LOV-TAP at the molecular level, using the noninvasive molecular dynamics simulation technique. More specifically, we find that Cys450-FMN-adduct formation in the AsLOV2-Jα-binding pocket after photoexcitation induces the cleavage of the peripheral Jα-helix from the LOV core, causing a change of its polarity and electrostatic attraction of the photoswitch onto the DNA surface. This goes along with the flexibilization through unfolding of a hairpin-like helix-loop-helix region interlinking the AsLOV2-Jα- and TrpR-domains, ultimately enabling the condensation of LOV-TAP onto the DNA surface. By contrast, in the dark state the AsLOV2-Jα photoswitch remains inactive and exerts a repulsive electrostatic force on the DNA surface. This leads to a distortion of the hairpin region, which finally relieves its tension by causing the disruption of LOV-TAP from the DNA.

Regulation of ecto-5'-nucleotidase activity via Ca2+-dependent, annexin 2-mediated membrane rearrangement?

The spatial segregation of the plasma membrane plays a prominent role in distinguishing and sorting a large number of signals a cell receives simultaneously. The plasma membrane comprises regions known as lipid rafts, which serve as signal-transduction hubs and platforms for sorting membrane-associated proteins. Ca(2+)-binding proteins of the annexin family have been ascribed a role in the regulation of raft dynamics. Glycosylphosphatidylinositol-anchored 5'-nucleotidase is an extracellular, raft-associated enzyme responsible for conversion of extracellular ATP into adenosine. Our results point to a regulation of ecto-5'-nucleotidase activity by Ca(2+)-dependent, annexin-mediated stabilization of membrane rafts.

Differences in catalytic activity between rat testicular and ovarian carbonyl reductases are due to two amino acids

The sequences of rat testis carbonyl reductase (rCR1) and rat ovary carbonyl reductase (rCR2) are 98% identical, differing only at amino acids 140, 141, 143, 235 and 238. Despite such strong sequence identity, we find that rCR1 and rCR2 have different catalytic constants for metabolism of menadione and 4-benzoyl-pyridine. Compared to rCR1, rCR2 has a 20-fold lower K(m) and 5-fold lower k(cat) towards menadione and a 7-fold lower K(m) and 7-fold lower k(cat) towards 4-benzoyl-pyridine. We constructed hybrids of rCR1 and rCR2 that were changed at either residues 140, 141 and 143 or residues 235 and 238. rCR1 with residues 140, 141 and 143 of rCR2 has similar catalytic efficiency for menadione and 4-benzoyl-pyridine as rCR1. rCR1 with Thr-235 and Glu-238 of rCR2 has the catalytic constants of rCR2, indicating that it is this part of rCR2 that contributes to its lower K(m) for menadione and 4-benzoyl-pyridine. Comparisons of three-dimensional models of rCR1 and rCR2 show how Thr-235 and Glu-238 stabilize rCR2 binding of NADPH and menadione.

A novel mutation in the steroidogenic acute regulatory protein gene promoter leading to reduced promoter activity

We have identified a novel cytosine/thymidine polymorphism of the human steroidogenic acute regulatory (StAR) gene promoter located 3 bp downstream of the steroidogenic factor-1 (SF-1)-binding site and 9 bp upstream of the TATA box (ATTTAAG). Carriers of this mutation have a high prevalence of primary aldosteronism. In transfection experiments, basal StAR promoter activity was unaltered by the mutation in murine Y-1 cells and human H295R cells. In Y-1 cells, forskolin (25 microM, 6 h) significantly increased wild-type promoter activity to 230+/-33% (P<0.05, n=4). In contrast, forskolin increased mutated promoter activity only to 150+/-27%, with a significant 35% reduction compared to wild type (P<0.05, n=3). In H295R cells, angiotensin II (AngII; 10 nM) increased wild-type StAR promoter activity to 265+/-22% (P<0.01, n=3), while mutated StAR promoter activity in response to AngII only reached 180+/-29% of controls (P< 0.01, n=3). Gel mobility shift assays show the formation of two additional complexes with the mutated promoter: one with the transcription repressor DAX-1 and another with a yet unidentified factor, which strongly binds the SF-1 response element. Thus, this novel mutation in the human StAR promoter is critically involved in the regulation of StAR gene expression and is associated with reduced promoter activity, a finding relevant for adrenal steroid response to physiological stimulators.

Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity

- Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

Induction of EROD activity by 1-phenylimidazole and beta-naphthoflavone in rainbow trout cultured hepatocytes: a comparative study

The classical pathway for induction of cytochrome P4501A (CYP1A) by xenobiotics is ligand binding to the aryl hydrocarbon receptor (AhR). However, several studies with mammalian cell systems point out a range of xenobiotics including imidazole derivatives, which are able to activate CYP1A through non-classical mechanisms. The objective of the present work is to compare induction of CYP1A (determined at the catalytic level as 7-ethoxyresorufin-O-deethylase, EROD) in rainbow trout (Oncorhynchus mykiss) hepatocytes by the prototypic AhR ligand, beta-naphthoflavone (betaNF), and by the imidazole derivative, 1-phenylimidazole (PIM). PIM was able to induce EROD activity although its potency was clearly lower than that of betaNF. In order to assess the relative importance of classical AhR ligand binding and alternative signaling pathways in CYP1A induction by PIM, co-exposure experiments with the partial AhR antagonist alpha-naphthoflavone (alphaNF) or with inhibitors of protein kinase C (staurosporine) and tyrosine kinases (genistein, herbimicine) were performed. alphaNF and herbimicin provoked a decrease of EROD induction both by betaNF and PIM, whereas staurosporine and genistein remained without effect. The overall similarities in the response of betaNF and PIM to the various inhibitors suggest that both compounds, in apparent contrast to the behaviour of some other imidazole derivatives, induce CYP1A following similar mechanisms.

Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish

Many natural and synthetic compounds present in the environment exert a number of adverse effects on the exposed organisms, leading to endocrine disruption, for which they were termed endocrine disrupting chemicals (EDCs). A decrease in reproduction success is one of the most well-documented signs of endocrine disruption in fish. Estrogens are steroid hormones involved in the control of important reproduction-related processes, including sexual differentiation, maturation and a variety of others. Careful spatial and temporal balance of estrogens in the body is crucial for proper functioning. At the final step of estrogen biosynthesis, cytochrome P450 aromatase, encoded by the cyp19 gene, converts androgens into estrogens. Modulation of aromatase CYP19 expression and function can dramatically alter the rate of estrogen production, disturbing the local and systemic levels of estrogens. In the present review, the current progress in CYP19 characterization in teleost fish is summarized and the potential of several classes of EDCs to interfere with CYP19 expression and activity is discussed. Two cyp19 genes are present in most teleosts, cyp19a and cyp19b, primarily expressed in the ovary and brain, respectively. Both aromatase CYP19 isoforms are involved in the sexual differentiation and regulation of the reproductive cycle and male reproductive behavior in diverse teleost species. Alteration of aromatase CYP19 expression and/or activity, be it upregulation or downregulation, may lead to diverse disturbances of the above mentioned processes. Prediction of multiple transcriptional regulatory elements in the promoters of teleost cyp19 genes suggests the possibility for several EDC classes to affect cyp19 expression on the transcriptional level. These sites include cAMP responsive elements, a steroidogenic factor 1/adrenal 4 binding protein site, an estrogen-responsive element (ERE), half-EREs, dioxin-responsive elements, and elements related to diverse other nuclear receptors (peroxisome proliferator activated receptor, retinoid X receptor, retinoic acid receptor). Certain compounds including phytoestrogens, xenoestrogens, fungicides and organotins may modulate aromatase CYP19 activity on the post-transcriptional level. As is shown in this review, diverse EDCs may affect the expression and/or activity of aromatase cyp19 genes through a variety of mechanisms, many of which need further characterization in order to improve the prediction of risks posed by a contaminated environment to teleost fish population.