Evidence supporting a role for N-epsilon-(3-formyl-3,4-dehydropiperidino)lysine accumulation in Muller glia dysfunction and death in diabetic retinopathy

Purpose: Recent evidence suggests that neuroglial dysfunction and degeneration contributes to the etiology and progression of diabetic retinopathy. Advanced lipoxidation end products (ALEs) have been implicated in the pathology of various diseases, including diabetes and several neurodegenerative disorders. The purpose of the present study was to investigate the possible link between the accumulation of ALEs and neuroretinal changes in diabetic retinopathy.

Methods: Retinal sections obtained from diabetic rats and age-matched controls were processed for immunohistochemistry using antibodies against several well defined ALEs. In vitro experiments were also performed using a human Muller (Moorfields/Institute of Ophthalmology-Muller 1 [ MIO-M1]) glia cell line. Western blot analysis was used to measure the accumulation of the acrolein-derived ALE adduct N epsilon-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) in Muller cells preincubated with FDP-lysine-modified human serum albumin (FDP-lysine-HSA). Responses of Muller cells to FDP-lysine accumulation were investigated by analyzing changes in the protein expression of heme oxygenase-1 (HO-1), glial fibrillary acidic protein (GFAP), and the inwardly rectifying potassium channel Kir4.1. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha) were determined by reverse transcriptase PCR (RT-PCR). Apoptotic cell death was evaluated by fluorescence-activated cell sorting (FACS) analysis after staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide.

Results: No significant differences in the levels of malondialdehyde-, 4-hydroxy-2-nonenal-, and 4-hydroxyhexenal-derived ALEs were evident between control and diabetic retinas after 4 months of diabetes. By contrast, FDP-lysine immunoreactivity was markedly increased in the Muller glia of diabetic rats. Time-course studies revealed that FDP-lysine initially accumulated within Muller glial end feet after only a few months of diabetes and thereafter spread distally throughout their inner radial processes. Exposure of human Muller glia to FDP-lysine-HSA led to a concentration-dependent accumulation of FDP-lysine-modified proteins across a broad molecular mass range. FDP-lysine accumulation was associated with the induction of HO-1, no change in GFAP, a decrease in protein levels of the potassium channel subunit Kir4.1, and upregulation of transcripts for VEGF, IL-6, and TNF-alpha. Incubation of Muller glia with FDP-lysine-HSA also caused apoptosis at high concentrations.

Conclusions: Collectively, these data strongly suggest that FDP-lysine accumulation could be a major factor contributing to the Muller glial abnormalities occurring in the early stages of diabetic retinopathy.

Contribution of the C-terminal tri-lysine regions of human immunodeficiency virus type 1 integrase for efficient reverse transcription and viral DNA nuclear import

Affiliation: Zhujun Ao, Éric Cohen & Xiaojian Yao : Département de microbiologie et immunologie, Faculté de Médecine, Université de Montréal

Étude de la polyubiquitination en lysine 63 dans les effets proinflammatoires de l'angiotensine II in vitro

Les évidences scientifiques révèlent l’implication des actions proinflammatoires de l’angiotensine II (Ang II) dans le développement de l’athérosclérose. Cependant, la caractérisation des bases moléculaires de l’Ang II sur le tissu vasculaire n’est pas totalement élucidée. La majorité des actions de l’Ang II implique l’activation d’une variété de cascades de signalisation dont les voies mitogen-activated protein kinases (MAPKs) ; c-Jun N-terminal kinases (JNKs), p38 kinases et extracellular signal-regulated kinases (ERK) et l’activation du facteur de transcription NF-κB via le complexe IKK. Récemment, une nouvelle modification post-traductionnelle dans les actions de l’Ang II, soit la polyubiquitination de la sous-unité NF-κB essential modulator (NEMO) du complexe IKK, a été révélée. L’objectif de mon projet de recherche est de vérifier l’importance de la polyubiquitination en K63 tout en caractérisant les protéines impliquées dans la modification de NEMO dans des cellules musculaires lisses vasculaires (CMLV) exposées à l’Ang II. Notre étude suggère, selon une approche siARN combinant Ubc7 et Ubc13, la diminution de la phosphorylation du complexe IKK, de Akt et des MAPKs. De plus, nos résultats illustrent l’implication de TRAF6 dans la signalisation cellulaire de l’Ang II. Finalement, notre étude révèle la présence de la polyubiquitination en K63 dans la signalisation cellulaire de l’Ang II par chromatographie d’affinité. Cette étude met en évidence l’implication de la polyubiquitination en K63 dans la signalisation de l’Ang II dans des CMLV et implique Ubc13 et Ubc7 dans le remodelage vasculaire et l’inflammation dépendante de l’Ang II dans des CMLV.

Study of histone H3 lysine 56 deacetylation in saccharomyces cerevisiae

Chez la levure Saccharomyces cerevisiae, l'acétylation de l'histone H3 sur la lysine 56 (H3K56ac) est présente sur les histones néo-synthétisées déposées derrière les fourches de réplication et est essentielle pour préserver la viabilité cellulaire en réponse au dommage à l'ADN. La désacétylation d'H3K56 sur l'ensemble du génome catalysée par Hst3 et Hst4 et a lieu en phase G2 ou M. H3K56ac est une lame à double tranchant. L'absence d'H3K56ac rend les cellules sensibles aux dommages à l'ADN. En revanche, un excès d'acétylation d'H3K56 dans un mutant hst3Δ hst4Δ a des conséquences encore plus sévères tels que la thermo-sensibilité, l'hypersensibilité aux agents génotoxiques, l'instabilité génomique ainsi qu'une courte durée de vie réplicative. Les désacétylases Hst3 et Hst4 sont étroitement régulées au cours du cycle cellulaire afin de permettre à l'H3K56ac d'exercer son rôle en réponse aux dommages à l'ADN tout en évitant les conséquences néfastes de l'hyperacétylation d'H3K56. Dans cette thèse, nous avons identifié la machinerie moléculaire responsable de la dégradation de Hst3. De plus, nous avons exploré les raisons pour lesquelles l'absence de désacétylation donne lieu aux phénotypes du mutant hst3Δ hst4Δ. Au chapitre 2, nous démontrons que la dégradation d'Hst3 peut être complétée avant l'anaphase. Ceci suggère que la désacétylation de H3K56 a lieu durant une courte fenêtre du cycle cellulaire se situant entre la complétion de la phase S et la métaphase. De plus, nous avons identifié deux sites de phosphorylation d'Hst3 par la kinase cycline-dépendante 1 (Cdk1) et démontré que ces évènements de phosphorylation conduisent à la dégradation d'Hst3 in vivo. Nous avons aussi démontré que l'ubiquityltransférase Cdc34 et l'ubiquitine ligase SCFCdc4 sont requises pour la dégradation d'Hst3. Finalement, nous avons montré que la phosphorylation d'Hst3 par la kinase mitotique Clb2-Cdk1 peut directement entraîner l'ubiquitylation d'Hst3 par SCFCdc4 in vitro. Au chapitre 3, nous avons étudié les mécanismes moléculaires sous-jacents à la sensibilité extrême du mutant hst3Δ hst4Δ aux agents qui endommagent l'ADN. Nous avons établi qu'en raison de la présence anormale d'H3K56ac devant les fourches de réplication, le mutant hst3Δ hst4Δ exhibe une forte perte de viabilité lorsqu'exposé au méthyl méthanesulfonate (MMS) durant un seul passage à travers la phase S. Nous avons aussi découvert que, malgré le fait que le point de contrôle de réponse aux dommages à l'ADN est activé normalement dans le mutant hst3Δ hst4Δ, ce mutant est incapable de compléter la réplication de l'ADN et d'inactiver le point de contrôle pour une longue période de temps après exposition transitoire au MMS. L'ensemble de nos résultats suggère que les lésions à l'ADN induites par le MMS dans le mutant hst3Δ hst4Δ causent une forte perte de viabilité parce que ce mutant est incapable de compléter la réplication de l'ADN après une exposition transitoire au MMS. Dans la deuxième section du chapitre 3, nous avons employé une approche génétique afin d'identifier de nouveaux mécanismes de suppression de deux phénotypes prononcés du mutant hst3Δ hst4Δ. Nous avons découvert que la délétion de plusieurs gènes impliqués dans la formation de frontières entre l'hétérochromatine et de l'euchromatine atténue les phénotypes du mutant hst3Δ hst4Δ sans réduire l'hyperacétylation d'H3K56. Nos résultats indiquent aussi que l'abondante acétylation de l'histone H4 sur la lysine 16 (H4K16ac) est néfaste au mutant hst3Δ hst4Δ. Ce résultat suggère un lien génétique intriguant entre l'acétylation d'H3K56 et celle d'H4K16. L'existence de ce lien était jusqu'à présent inconnu. Nous avons identifié un groupe de suppresseurs spontanés où H3K56ac est indétectable, mais la majorité de nos suppresseurs ne montrent aucune réduction flagrante d'H3K56ac ou d'H4 K16ac par rapport aux niveaux observés dans le mutant hst3Δ hst4Δ. Une étude plus approfondie de ce groupe de suppresseurs est susceptible de mener à la découverte de nouveaux mécanismes génétiques ou épigénétiques permettant d'éviter les conséquences catastrophiques de l'hyperacétylation d'H3K56 chez le mutant hst3Δ hst4Δ. En résumé, cette thèse identifie la machinerie moléculaire responsable de la dégradation d'Hst3 (une désacétylase d'H3K56) durant une fenêtre de temps situées entre la fin de la phase S et la métaphase. Nos résultats permettent aussi d'expliquer pourquoi la dégradation d'Hst3 précède le début de la phase S durant laquelle l'acétylation d'H3K56 s'accumule derrière les fourches de réplication afin d'exercer son rôle de mécanisme de défense contre le dommage à l'ADN. De plus, nous avons identifié plusieurs suppresseurs qui permettent de contourner le rôle important d'Hst3 et Hst4 en réponse au dommage à l'ADN. Plusieurs suppresseurs révèlent un lien génétique inattendu entre deux formes abondantes d'acétylation des histones chez Saccharomyces cerevisiae, soit H3K56ac et H4K16ac.

S6K1 is acetylated at lysine 516 in response to growth factor stimulation

The 70kDa ribosomal protein S6 kinase 1 (S6K1) plays important roles in the regulation of protein synthesis, cell growth and metabolism. S6K1 is activated by the phosphorylation of multiple serine and threonine residues in response to stimulation by a variety of growth factors and cytokines. In addition to phosphorylation, we have recently shown that S6K1 is also targeted by lysine acetylation. Here, using tandem mass spectrometry we have mapped acetylation of S6K1 to lysine 516, a site close to the C-terminus of the kinase that is highly conserved amongst vertebrate S6K1 orthologues. Using acetyl-specific K516 antibodies, we show that acetylation of endogenous S6K1 at this site is potently induced upon growth factor stimulation. Although S6K1 acetylation and phosphorylation are both induced by growth factor stimulation, these events appear to be functionally independent. Indeed, experiments using inhibitors of S6K1 activation and exposure of cells to various stresses indicate that S6K1 acetylation can occur in the absence of phosphorylation and vice versa. We propose that K516 acetylation may serve to modulate important kinase-independent functions of S6K1 in response to growth factor signalling.

Effects of cottonseed cake-based diets supplemented with blood meal, alone or with lysine, on the growth of pigs

An experiment was carried out to establish the effect on the growth of pigs of including blood meal or lysine in diets containing gossypol from cottenseed cake. Forty Landrace x Large White pigs (20 of each sex) were randomly allocated to 5 treatments of 8 pigs each in a 2x2 factorial design with two levels of lysine or two levels of blood meal in the diets plus a control diet. The pigs were fed different diets and slaughtered at 75.0+/-2.0 kg live weight for carcase analysis. Supplementing the diets with blood meal resulted in higher live weight gains (p<0.001) and improved feed conversion ratios (p<0.001) than supplementing with lysine. Pigs fed the higher level of cottonseed cake showed a significant (p<0.001) depression in live weight gain and feed conversion ratio compared to those fed a low level of the cake. There was no significant difference (p>0.05) in intake in the pigs fed diets with cottonseed cake including blood meal or synthetic lysine. The kidney and liver weights of the pigs fed the diets with a higher level of cottonseed cake were significantly greater (p<0.001) than in those fed the lower level, but when the diets containing cottonseed cake were supplemented with blood meal or lysine at the same level there was no significant difference (p>0.05) in the weights of these organs. Lysine or other factors derived from blood meal appear to be more efficient than synthetic lysine in reducing the adverse effects of gossypol.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Spontaneous condensation in DNA-polystyrene-b-poly(l-lysine) polyelectrolyte block copolymer mixtures

We investigated the condensation of calf thymus DNA by amphiphilic polystyrene(m)-b-poly(l-lysine)(n) block copolymers (PSm-b- PLys(n), m, n = degree of polymerization), using small-angle X-ray scattering, polarized optical microscopy and laser scanning confocal microscopy. Microscopy studies showed that the DNA condenses in the form of fibrillar precipitates, with an irregular structure, due to electrostatic interactions between PLys and DNA. This is not modified by the presence of hydrophobic PS block. Scattering experiments show that the structure of the polyplexes corresponds to a local order of DNA rods which becomes more compact upon increasing n. It can be concluded that for DNA/ PSm-b- PLys(n) polyplexes, the balance between the PLys block length and the excess charge in the system plays an essential role in the formation of a liquid crystalline phase.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes, Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Nonspherical assemblies generated from polystyrene-b-poly(L-lysine) polyelectrolyte block copolymers

This report describes the aqueous solution self-assembly of a series of polystyrene(m)-b-poly(L-lysine)n block copolymers (m = 8-10; n = 10-70). The polymers are prepared by ring-opening polymerization of epsilon-benzyloxycarbonyl-L-lysine N-carboxyanhydride using amine terminated polystyrene macroinitiators, followed by removal of the benzyloxycarbonyl side chain protecting groups. The critical micelle concentration of the block copolymers determined using the pyrene probe technique shows a parabolic dependence on peptide block length exhibiting a maximum at n = approximately 20 (m = 8) or n = approximately 60 (m = 10). The shape and size of the aggregates has been studied by dynamic and static light scattering, small-angle neutron scattering (SANS), and analytical ultracentrifugation (AUC). Surprisingly, Holtzer and Kratky analysis of the static light scattering results indicates the presence of nonspherical, presumably cylindrical objects independent of the poly(L-lysine)n block length. This is supported by SANS data, which can be fitted well by assuming cylindrical scattering objects. AUC analysis allows the molecular weight of the aggregates to be estimated as several million g/mol, corresponding to aggregation numbers of several 10s to 100s. These aggregation numbers agree with those that can be estimated from the length and diameter of the cylinders obtained from the scattering results.

The adsorption geometry and chemical state of lysine on Cu{110}

Chemisorbed layers of lysine adsorbed on Cu{110} have been studied using X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. XPS indicates that the majority (70%) of the molecules in the saturated layer at room temperature (coverage 0.27 ML) are in their zwitterionic state with no preferential molecular orientation. After annealing to 420 K a less densely packed layer is formed (0.14 ML), which shows a strong angular dependence in the characteristic π-resonance of oxygen K edge NEXAFS and no indication of zwitterions in XPS. These experimental results are best compatible with molecules bound to the substrate through the oxygen atoms of the (deprotonated) carboxylate group and the two amino groups involving Cu atoms in three different close packed rows. This μ4 bonding arrangement with an additional bond through the !-amino group is different from geometries previously suggested for lysine on Cu{110}.

Electrospinning of Hyperbranched Poly-L-Lysine/Polyaniline Nanofibers for Application in Cardiac Tissue Engineering

Electrospun polyaniline nanofibers are one of the most promising materials for cardiac tissue engineering due to their tunable electroactive properties. Moreover, the biocompatibility of polyaniline nanofibes can be improved by grafting of adhesive peptides during the synthesis. In this paper, we describe the biocompatible properties and cardiomyocytes proliferation on polyaniline electrospun nanofibers modified by hyperbranched poly-L-lysine dendrimers (HPLys). The microstructure characterization of the HPLys/polyaniline nanofibers was carried out by scanning electron microscopy (SEM). It was observed that the application of electrical current stimulates the differentiation of cardiac cells cultured on the nanofiber scaffolds. Both electroactivity and biocompatibility of the HPLys based nanofibers suggest the use this material for culture of cardiac cells and opens the possibility of using this material as a biocompatible electroactive 3-D matrix in cardiac tissue engineering.