The redoxomics of PTEN: walking a fine line between damage and signaling:mass spectrometry-based approaches to study the effect of oxidation on PTEN function, structure, and protein-protein interactions

The research described in this PhD thesis focuses on proteomics approaches to study the effect of oxidation on the modification status and protein-protein interactions of PTEN, a redox-sensitive phosphatase involved in a number of cellular processes including metabolism, apoptosis, cell proliferation, and survival. While direct evidence of a redox regulation of PTEN and its downstream signaling has been reported, the effect of cellular oxidative stress or direct PTEN oxidation on PTEN structure and interactome is still poorly defined. In a first study, GST-tagged PTEN was directly oxidized over a range of hypochlorous acid (HOCl) concentration, assayed for phosphatase activity, and oxidative post-translational modifications (oxPTMs) were quantified using LC-MS/MS-based label-free methods. In a second study, GSTtagged PTEN was prepared in a reduced and reversibly H2O2-oxidized form, immobilized on a resin support and incubated with HCT116 cell lysate to capture PTEN interacting proteins, which were analyzed by LC-MS/MS and comparatively quantified using label-free methods. In parallel experiments, HCT116 cells transfected with a GFP-tagged PTEN were treated with H2O2 and PTENinteracting proteins immunoprecipitated using standard methods. Several high abundance HOCl-induced oxPTMs were mapped, including those taking place at amino acids known to be important for PTEN phosphatase activity and protein-protein interactions, such as Met35, Tyr155, Tyr240 and Tyr315. A PTEN redox interactome was also characterized, which identified a number of PTEN-interacting proteins that vary with the reversible inactivation of PTEN caused by H2O2 oxidation. These included new PTEN interactors as well as the redox proteins peroxiredoxin-1 (Prdx1) and thioredoxin (Trx), which are known to be involved in the recycling of PTEN active site following H2O2-induced reversible inactivation. The results suggest that the oxidative modification of PTEN causes functional alterations in PTEN structure and interactome, with fundamental implications for the PTEN signaling role in many cellular processes, such as those involved in the pathophysiology of disease and ageing.

Functional genomic and molecular analyses of transcripts related to glycerol synthesis and trafficking in rainbow smelt (Osmerus mordax) using cold temperature-induced models of glycerol production

The rainbow smelt (Osmerus mordax) is an anadromous teleost that produces type II antifreeze protein (AFP) and accumulates modest urea and high glycerol levels in plasma and tissues as adaptive cryoprotectant mechanisms in sub-zero temperatures. It is known that glyceroneogenesis occurs in liver via a branch in glycolysis and gluconeogenesis and is activated by low temperature; however, the precise mechanisms of glycerol synthesis and trafficking in smelt remain to be elucidated. The objective of this thesis was to provide further insight using functional genomic techniques [e.g. suppression subtractive hybridization (SSH) cDNA library construction, microarray analyses] and molecular analyses [e.g. cloning, quantitative reverse transcription - polymerase chain reaction (QPCR)]. Novel molecular mechanisms related to glyceroneogenesis were deciphered by comparing the transcript expression profiles of glycerol (cold temperature) and non-glycerol (warm temperature) accumulating hepatocytes (Chapter 2) and livers from intact smelt (Chapter 3). Briefly, glycerol synthesis can be initiated from both amino acids and carbohydrate; however carbohydrate appears to be the preferred source when it is readily available. In glycerol accumulating hepatocytes, levels of the hepatic glucose transporter (GLUT2) plummeted and transcript levels of a suite of genes (PEPCK, MDH2, AAT2, GDH and AQP9) associated with the mobilization of amino acids to fuel glycerol synthesis were all transiently higher. In contrast, in glycerol accumulating livers from intact smelt, glycerol synthesis was primarily fuelled by glycogen degradation with higher PGM and PFK (glycolysis) transcript levels. Whether initiated from amino acids or carbohydrate, there were common metabolic underpinnings. Increased PDK2 (an inhibitor of PDH) transcript levels would direct pyruvate derived from amino acids and / or DHAP derived from G6P to glycerol as opposed to oxidation via the citric acid cycle. Robust LIPL (triglyceride catabolism) transcript levels would provide free fatty acids that could be oxidized to fuel ATP synthesis. Increased cGPDH (glyceroneogenesis) transcript levels were not required for increased glycerol production, suggesting that regulation is more likely by post-translational modification. Finally, levels of a transcript potentially encoding glycerol-3-phosphatase, an enzyme not yet characterized in any vertebrate species, were transiently higher. These comparisons also led to the novel discoveries that increased G6Pase (glucose synthesis) and increased GS (glutamine synthesis) transcript levels were part of the low temperature response in smelt. Glucose may provide increased colligative protection against freezing; whereas glutamine could serve to store nitrogen released from amino acid catabolism in a non-toxic form and / or be used to synthesize urea via purine synthesis-uricolysis. Novel key aspects of cryoprotectant osmolyte (glycerol and urea) trafficking were elucidated by cloning and characterizing three aquaglyceroporin (GLP)-encoding genes from smelt at the gene and cDNA levels in Chapter 4. GLPs are integral membrane proteins that facilitate passive movement of water, glycerol and urea across cellular membranes. The highlight was the discovery that AQP10ba transcript levels always increase in posterior kidney only at low temperature. This AQP10b gene paralogue may have evolved to aid in the reabsorption of urea from the proximal tubule. This research has contributed significantly to a general understanding of the cold adaptation response in smelt, and more specifically to the development of a working scenario for the mechanisms involved in glycerol synthesis and trafficking in this species.

Caractérisation de l’activité Nur77 et de ses complexes en essais BRET par complémentation

Dans le système nerveux central, la dopamine joue un rôle crucial dans de nombreuses fonctions physiologiques telles que : l’apprentissage, le mouvement volontaire, la motivation, la cognition et la production hormonale. Il a été aussi démontré que le système de signalisation dopaminergique est altéré dans plusieurs maladies neurologiques et psychiatriques comme la maladie de Parkinson et la schizophrénie. Des études, effectuées dans le laboratoire du Dr.Daniel Lévesque (laboratoire d’accueil), ont montré que les récepteurs nucléaires Nur77 (NR4A1, NGFI-B) et RXRγ (retinoid X receptors γ) sont impliqués dans la régulation des effets de la dopamine dans le système nerveux central. De plus, ces données suggèrent que le complexe Nur77 et RXR joueraient un rôle crucial dans l’effet des médicaments antipsychotiques et antiparkinsoniens. Toutefois, très peu de médicaments ciblant Nur77 ont été identifiés à ce jour et les médicaments agissant sur RXRγ restent mal caractérisés. En outre, les analyses actuellement disponibles ne peuvent pas résumer la complexité des activités des NRs et génèrent des mesures indirectes des activités des drogues. Afin de mieux comprendre comment est régulée l’interaction Nur77/RXRγ dans ces processus, mon projet a été de mettre au point un essai BRET (Bioluminescence Resonance Energy Transfer) et PCA-BRET (Protein Complementation Assay-BRET) basé sur le recrutement d'un motif mimant un co-activateur fusionné avec la YFP. Nos différents essais ont été validés par courbes dose-réponse en utilisant différents composés RXR . Les EC50 (concentration efficace médiane, qui permet de mesurer l'efficacité d'un composé) obtenues étaient très semblables aux valeurs précédemment rapportées dans la littérature. Nous avons aussi pu identifier un composé le SR11237 (BMS649) qui semble posséder une sélectivité pour le complexe Nur77/RXRγ par rapport aux complexes Nurr1/RXRγ et RXRγ /RXRγ. Nos résultats indiquent que ces essais de BRET peuvent être utilisés pour évaluer la sélectivité de nouveaux composés pour les complexes Nur77/RXRγ, Nurr1/RXRγ et RXRγ /RXRγ. Un autre aspect de mon projet de doctorat a été de mettre en évidence par BRET l’importance de la SUMOylation dans la régulation de l'activité de Nur77 dans sa forme monomèrique, homodimèrique et hétérodimèrique. Nous avons ainsi identifié que Nur77 recrute principalement SUMO2 sur sa lysine 577. Il est intéressant de noté que le recrutement de la SUMO2 à Nur77 est potentialisé en présence de la SUMO E3 Ligase PIASγ. Aussi, la perte de la SUMOylation sur la lysine 577 entraîne l'incapacité de Nur77 de recruter divers motifs de co-activation mais pas pour ses formes homo- et hétérodimèrique. Cependant, la présence de PIASγ ne potentialise pas le recrutement du co-activateur, suggérant que cette SUMO E3 Ligase est seulement impliqué dans le processus de recrutement de la SUMO mais pas dans celui du co-activateur. Nous avons ainsi déterminé une nouvelle modification post-traductionnelle sur Nur77 régulant spécifiquement son activité monomérique Ces projets pourraient donc apporter de nouvelles données cruciales pour l’amélioration du traitement de la maladie de Parkinson ou de la schizophrénie, ainsi que d'obtenir une meilleure compréhension sur les mécanismes permettant la régulation de la fonction de Nur77

Dual roles of DNA repair enzymes in RNA biology/post-transcriptional control

Despite consistent research into the molecular principles of the DNA damage repair pathway for almost two decades, it has only recently been found that RNA metabolism is very tightly related to this pathway, and the two ancient biochemical mechanisms act in alliance to maintain cellular genomic integrity. The close links between these pathways are well exemplified by examining the base excision repair pathway, which is now well known for dual roles of many of its members in DNA repair and RNA surveillance, including APE1, SMUG1, and PARP1. With additional links between these pathways steadily emerging, this review aims to provide a summary of the emerging roles for DNA repair proteins in the post-transcriptional regulation of RNAs. 

Étude fonctionnelle d’un nouveau complexe multi-enzymatique régulant l’épigénome

L’ubiquitination, une modification post-traductionnelle importante pour le contrôle de nombreux processus cellulaires, est une réaction réversible. La réaction inverse, nommée déubiquitination est catalysée par les déubiquitinases (DUB). Nous nous sommes intéressés dans nos travaux à étudier l’ubiquitination de l’histone H2A (H2Aub), au niveau des résidus lysines 118 et 119 (K118/K119), une marque épigénétique impliquée dans la régulation de la prolifération cellulaire et la réparation de l’ADN. Le régulateur transcriptionnel BAP1, une déubiquitinase nucléaire, a été initialement identifié pour sa capacité à promouvoir la fonction suppressive de tumeurs de BRCA1. BAP1 forme un complexe multi-protéique avec plusieurs facteurs transcriptionnels et sa fonction principale est la déubiquitination de H2Aub. Plusieurs études ont démontré que BAP1 est un gène suppresseur de tumeurs majeur et qu’il est largement muté et inactivé dans une multitude de cancers. En effet, BAP1 émerge comme étant la DUB la plus mutée au niveau des cancers. Cependant, le ou les mécanismes d’action et de régulation du complexe BAP1 restent très peu connus. Dans cette étude nous nous sommes intéressés à la caractérisation moléculaire et fonctionnelle des partenaires protéiques de BAP1. De manière significative nous avons caractérisé un mécanisme unique de régulation entre deux composants majeurs du complexe BAP1 à savoir, HCF-1 et OGT. En effet, nous avons démontré que HCF-1 est requis pour maintenir le niveau protéique de OGT et que cette dernière est indispensable pour la maturation protéolytique de HCF-1 en promouvant son clivage par O-GlcNAcylation, une signalisation cellulaire nécessaire au bon fonctionnement de HCF-1. Également, nous avons découvert un nouveau mécanisme de régulation de BAP1 par l’ubiquitine ligase atypique UBE2O. En effet, UBE2O agit comme un régulateur négatif de BAP1 puisque l’ubiquitination de ce dernier induit sa séquestration dans le cytoplasme et l’inhibition de sa fonction suppressive de tumeurs. D’autre part nous nous sommes penchés sur la caractérisation de l’association de BAP1 avec deux facteurs de la famille des protéines Polycombes nommés ASXL1 et ASXL2 (ASXL1/2). Nous avons investigué le rôle de BAP1/ASXL1/2, particulièrement dans les mécanismes de déubiquitination et suppression de tumeurs. Nous avons démontré que BAP1 interagit directement iii via son domaine C-terminale avec le même domaine ASXM de ASXL1/2 formant ainsi deux complexes mutuellement exclusifs indispensables pour induire l’activité déubiquitinase de BAP1. De manière significative, ASXM s’associe avec BAP1 pour créer un nouveau domaine composite de liaison à l’ubiquitine. Ces interactions BAP1/ASXL1/2 régulent la progression harmonieuse du cycle cellulaire. De plus, la surexpression de BAP1 et de ASXL2 au niveau des fibroblastes induit la sénescence de manière dépendante de leurs interactions. D’autre part, nous avons identifié des mutations de cancers au niveau de BAP1 le rendant incapable de lier ASXL1/2, d’exercer sa fonction d’autodéubiquitination et de ce fait d’agir comme suppresseur de tumeurs. Ainsi nous avons révélé un lien étroit entre le gène suppresseur de tumeurs BAP1, son activité déubiquitinase et le contrôle de la prolifération cellulaire.

Structural characterization of interaction of K11 and K63 mixed-linkage polyubiquitin chains with the tUIMs of epsin1

Ubiquitylation or covalent attachment of ubiquitin (Ub) to a variety of substrate proteins in cells is a versatile post-translational modification involved in the regulation of numerous cellular processes. The distinct messages that polyubiquitylation encodes are attributed to the multitude of conformations possible through attachment of ubiquitin monomers within a polyubiquitin chain via a specific lysine residue. Thus the hypothesis is that linkage defines polyubiquitin conformation which in turn determines specific recognition by cellular receptors. Ubiquitylation of membrane surface receptor proteins plays a very important role in regulating receptor-mediated endocytosis as well as endosomal sorting for lysosomal degradation. Epsin1 is an endocytic adaptor protein with three tandem UIMs (Ubiquitin Interacting Motifs) which are responsible for the highly specific interaction between epsin and ubiquitylated receptors. Epsin1 is also an oncogenic protein and its expression is upregulated in some types of cancer. Recently it has been shown that novel K11 and K63 mixed-linkage polyubiquitin chains serve as internalization signal for MHC I (Major Histocompatibility Complex I) molecule through their association with the tUIMs of epsin1. However the molecular mode of action and structural details of the interaction between polyubiquitin chains on receptors and tUIMs of epsin1 is yet to be determined. This information is crucial for the development of anticancer therapeutics targeting epsin1. The molecular basis for the linkage-specific recognition of K11 and K63 mixed-linkage polyubiquitin chains by the tandem UIMs of the endocytic adaptor protein epsin1 is investigated using a combination of NMR methods.

Investigation of the role of long non-coding RNAs in oncogene induced cellular senescence

Cellular senescence is a stable arrest of cell proliferation induced by several factors such as activated oncogenes, oxidative stress and shortening of telomeres. Senescence acts as a tumour suppression mechanism to halt the progression of cancer. However, senescence may also impact negatively upon tissue regeneration, thus contributing to the effects of ageing. The eukaryotic genome is controlled by various modes of transcriptional and translational regulation. Focus has therefore centred on the role of long non- coding RNAs (lncRNAs) in regulating the genome. Accordingly, understanding how lncRNAs function to regulate the senescent genome is integral to improving our knowledge and understanding of tumour suppression and ageing. Within this study, I set out to investigate the expression of lncRNAs’ expression within models of senescence. Through a custom expression array, I have shown that expression of multiple different lncRNAs is up-regulated and down regulated in IMR90 replicative senescent fibroblasts and oncogene-induced senescent melanocytes. LncRNA expression was determined to be specific to stable senescence-associated cell arrest and predominantly within the nucleus of senescent cells. In order to examine the function of lncRNA expression in senescence, I selected lncRNA transcript ENST0000430998 (lncRNA_98) to focus my investigations upon. LncRNA_98 was robustly upregulated within multiple models of senescence and efficiently depleted using anti-sense oligonucleotide technology. Characterisation and unbiased RNA-sequencing of lncRNA_98 deficient senescent cells highlighted a list of genes that are regulated by lncRNA_98 expression in senescent cells and may regulate aspects of the senescence program. Specifically, the formation of SAHF was impeded upon depletion of lncRNA_98 expression and levels of total pRB protein expression severely decreased. Validation and recapitulation of consequences of pRB depletion was confirmed through lncRNA_98 knock-out cells generated using CRISPR technology. Surprisingly, inhibition of ATM kinase functions permitted the restoration of pRB protein levels within lncRNA_98 deficient cells. I propose that lncRNA_98 antagonizes the ability of ATM kinase to downregulate pRB expression at a post-transcriptional level, thereby potentiating senescence. Furthermore, lncRNA expression was detected within fibroblasts of old individuals and visualised within senescent melanocytes in human benign nevi, a barrier to melanoma progression. Conversely, mining of 337 TCGA primary melanoma data sets highlighted that the lncRNA_98 gene and its expression was lost from a significant proportion of melanoma samples, consistent with lncRNA_98 having a tumour suppressor functions. The data presented in this study illustrates that lncRNA_98 expression has a regulatory role over pRB expression in senescence and may regulate aspects of tumourigenesis and ageing.

Phospholipases and the network of auxin signal transduction with ABP1 and TIR1 as two receptors: a comprehensive and provocative model

Three types of phospholipases, phospholipase D, secreted phospholipase A2, and patatin-related phospholipase A (pPLA) have functions in auxin signal transduction. Potential linkage to auxin receptors ABP1 or TIR1, their rapid activation or post-translational activation mechanisms, and downstream functions regulated by these phospholipases is reviewed and discussed. Only for pPLA all aspects are known at least to some detail. Evidence is gathered that all these signal reactions are located in the cytosol and seem to merge on regulation of PIN-catalyzed auxin efflux transport proteins. As a consequence, auxin concentration in the nucleus is also affected and this regulates the E3 activity of this auxin receptor. We showed that ABP1, PIN2, and pPLA, all outside the nucleus, have an impact on regulation of auxin-induced genes within 30 min. We propose that regulation of PIN protein activities and of auxin efflux transport are the means to coordinate ABP1 and TIR1 activity and that no physical contact between components of the ABP1-triggered cytosolic pathways and TIR1-triggered nuclear pathways of signaling is necessary to perform this.

Influência do estímulo por PAF no perfil de abundância proteica em neutrófilos : uma abordagem proteômica

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2015.

Biosynthesis and mode of action of ribosomally synthesized and post-translationally modified antimicrobial peptides

One of the greatest sources of biologically active compounds is natural products. Often these compounds serve as platforms for the design and development of novel drugs and therapeutics. The overwhelming amount of genomic information acquired in recent years has revealed that ribosomally synthesized and post-translationally modified natural products are much more widespread than originally anticipated. Identified in nearly all forms of life, these natural products display incredible structural diversity and possess a wide range of biological functions that include antimicrobial, antiviral, anti-inflammatory, antitumor, and antiallodynic activities. The unique pathways taken to biosynthesize these compounds offer exciting opportunities for the bioengineering of these complex molecules. The studies described herein focus on both the mode of action and biosynthesis of antimicrobial peptides. In Chapter 2, it is demonstrated that haloduracin, a recently discovered two-peptide lantibiotic, possesses nanomolar antimicrobial activity against a panel of bacteria strains. The potency of haloduracin rivals that of nisin, an economically and therapeutically relevant lantibiotic, which can be attributed to a similar dual mode of action. Moreover, it was demonstrated that this lantibiotic of alkaliphile origin has better stability at physiological pH than nisin. The molecular target of haloduracin was identified as the cell wall peptidoglycan precursor lipid II. Through the in vitro biosynthesis of haloduracin, several analogues of Halα were prepared and evaluated for their ability to inhibit peptidoglycan biosynthesis as well as bacterial cell growth. In an effort to overcome the limitations of in vitro biosynthesis strategies, a novel strategy was developed resulting in a constitutively active lantibiotic synthetase enzyme. This methodology, described in Chapter 3, enabled the production of fully-modified lacticin 481 products with proteinogenic and non-proteinogenic amino acid substitutions. A number of lacticin 481 analogues were prepared and their antimicrobial activity and ability to bind lipid II was assessed. Moreover, site-directed mutagenesis of the constitutively active synthetase resulted in a kinase-like enzyme with the ability to phosphorylate a number of peptide substrates. The hunt for a lantibiotic synthetase enzyme responsible for installing the presumed dehydro amino acids and a thioether ring in the natural product sublancin, led to the identification and characterization of a unique post-translational modification. The studies described in Chapter 4, demonstrate that sublancin is not a lantibiotic, but rather an unusual S-linked glycopeptide. Its structure was revised based on extensive chemical, biochemical, and spectroscopic characterization. In addition to structural investigation, bioinformatic analysis of the sublancin gene cluster led to the identification of an S-glycosyltransferase predicted to be responsible for the post-translational modification of the sublancin precursor peptide. The unprecedented glycosyltransferase was reconstituted in vitro and demonstrated remarkable substrate promiscuity for both the NDP-sugar co-substrate as well as the precursor peptide itself. An in vitro method was developed for the production of sublancin and analogues which were subsequently evaluated in bioactivity assays. Finally, a number of putative biosynthetic gene clusters were identified that appear to harbor the necessary genes for production of an S-glycopeptide. An additional S-glycosyltransferase with more favorable intrinsic properties including better expression, stability, and solubility was reconstituted in vitro and demonstrated robust catalytic abilities.

Étude fonctionnelle d’un nouveau complexe multi-enzymatique régulant l’épigénome

L’ubiquitination, une modification post-traductionnelle importante pour le contrôle de nombreux processus cellulaires, est une réaction réversible. La réaction inverse, nommée déubiquitination est catalysée par les déubiquitinases (DUB). Nous nous sommes intéressés dans nos travaux à étudier l’ubiquitination de l’histone H2A (H2Aub), au niveau des résidus lysines 118 et 119 (K118/K119), une marque épigénétique impliquée dans la régulation de la prolifération cellulaire et la réparation de l’ADN. Le régulateur transcriptionnel BAP1, une déubiquitinase nucléaire, a été initialement identifié pour sa capacité à promouvoir la fonction suppressive de tumeurs de BRCA1. BAP1 forme un complexe multi-protéique avec plusieurs facteurs transcriptionnels et sa fonction principale est la déubiquitination de H2Aub. Plusieurs études ont démontré que BAP1 est un gène suppresseur de tumeurs majeur et qu’il est largement muté et inactivé dans une multitude de cancers. En effet, BAP1 émerge comme étant la DUB la plus mutée au niveau des cancers. Cependant, le ou les mécanismes d’action et de régulation du complexe BAP1 restent très peu connus. Dans cette étude nous nous sommes intéressés à la caractérisation moléculaire et fonctionnelle des partenaires protéiques de BAP1. De manière significative nous avons caractérisé un mécanisme unique de régulation entre deux composants majeurs du complexe BAP1 à savoir, HCF-1 et OGT. En effet, nous avons démontré que HCF-1 est requis pour maintenir le niveau protéique de OGT et que cette dernière est indispensable pour la maturation protéolytique de HCF-1 en promouvant son clivage par O-GlcNAcylation, une signalisation cellulaire nécessaire au bon fonctionnement de HCF-1. Également, nous avons découvert un nouveau mécanisme de régulation de BAP1 par l’ubiquitine ligase atypique UBE2O. En effet, UBE2O agit comme un régulateur négatif de BAP1 puisque l’ubiquitination de ce dernier induit sa séquestration dans le cytoplasme et l’inhibition de sa fonction suppressive de tumeurs. D’autre part nous nous sommes penchés sur la caractérisation de l’association de BAP1 avec deux facteurs de la famille des protéines Polycombes nommés ASXL1 et ASXL2 (ASXL1/2). Nous avons investigué le rôle de BAP1/ASXL1/2, particulièrement dans les mécanismes de déubiquitination et suppression de tumeurs. Nous avons démontré que BAP1 interagit directement iii via son domaine C-terminale avec le même domaine ASXM de ASXL1/2 formant ainsi deux complexes mutuellement exclusifs indispensables pour induire l’activité déubiquitinase de BAP1. De manière significative, ASXM s’associe avec BAP1 pour créer un nouveau domaine composite de liaison à l’ubiquitine. Ces interactions BAP1/ASXL1/2 régulent la progression harmonieuse du cycle cellulaire. De plus, la surexpression de BAP1 et de ASXL2 au niveau des fibroblastes induit la sénescence de manière dépendante de leurs interactions. D’autre part, nous avons identifié des mutations de cancers au niveau de BAP1 le rendant incapable de lier ASXL1/2, d’exercer sa fonction d’autodéubiquitination et de ce fait d’agir comme suppresseur de tumeurs. Ainsi nous avons révélé un lien étroit entre le gène suppresseur de tumeurs BAP1, son activité déubiquitinase et le contrôle de la prolifération cellulaire.

Investigating the Role of O-GlcNAc Glycosylation in Neurodegeneration

O-GlcNAc glycosylation of nuclear and cytosolic proteins is an essential post-translational modification implicated in many diseases, from cancer to diabetes. Importantly, many important neuronal proteins are also O-GlcNAc modified, and aberrant O-GlcNAcylation of these proteins may contribute to the pathology of neurodegenerative diseases although these mechanisms have not been well defined. Here we investigated the role of O-GlcNAc glycosylation in the brain, utilizing both chemistry and molecular biology to study O-GlcNAc transferase (OGT), the enzyme that adds the sugar modification. To evaluate the role of OGT in adult neurons, we generated a forebrain-specific conditional knockout of OGT (OGT cKO) in mice. Although indistinguishable from wild-type littermates at birth, after three weeks we observe progressive neurodegeneration in OGT cKO mice. Hallmarks of Alzheimer’s disease, including neuronal loss, neuroinflammation, behavioral deficits, hyperphosphorylated tau, and amyloid beta peptide accumulation, are observed. Furthermore, decreases in OGT protein levels were found in human AD brain tissue, suggesting that altered O-GlcNAcylation likely contributes to neurodegenerative diseases in humans. This model is one of a few mouse models that recapitulate AD phenotypes without mutating and overexpressing human tau, amyloid precursor protein, or presenilin, highlighting the essential role of OGT in neurodegenerative pathways.

Given the importance of OGT in the brain, we further investigated the regulation of the OGT enzyme by phosphorylation. We found that phosphorylation of OGT near its C-terminus reduces its activity in cancer cells, and have developed phosphorylation-specific antibodies to aid mechanistic studies. Furthermore, mutation of this phosphorylation site on OGT, followed by overexpression in neurons was shown to enhance neurite outgrowth, demonstrating a functional consequence for this site. Thus phosphorylation of OGT inhibits its activity and enhances neurite outgrowth, and current studies aim to characterize the signaling pathway that regulates OGT phosphorylation in neurons.

Hormone-Mediated Gene-Specific Translation Regulation

The central role of translation regulation in the control of critical cellular processes has long been recognized. Yet the systematic exploration of quantitative changes in translation at a genome-wide scale in response to specific stimuli has only recently become technically feasible. Using a genetic approach, we have identified new Arabidopsis weak-ethylene insensitive mutants that also display defects in translation, which suggested the existence of a previously unknown molecular module involved in ethylene-mediated translation regulation of components of this signaling pathway. To explore this link in detail, we implemented for Arabidopsis the ribosome-footprinting technology, which enables the study of translation at a whole-genome level at single codon resolution[1]. Using ribosome-footprinting we examined the effects of short exposure to ethylene on the Arabidopsis translatome looking for ethylene-triggered changes in translation rates that could not be explained by changes in transcript levels. The results of this research, in combination with the characterization of a subset of the aforementioned weak-ethylene insensitive mutants that are defective in the UPF genes (core-components of the nonsense-mediated mRNA decay machinery), uncovered a translation-based branch of the ethylene signaling pathway[2]. In the presence of ethylene, translation of a negative regulator of ethylene signaling EBF2 is repressed, despite induced transcription of this gene. These translational effects of ethylene require the long 3´UTR of EBF2 (3´EBF2), which is recognized by the C-terminal end of the key ethylene-signaling protein EIN2 (EIN2C) in the cytoplasm once EIN2C is released from the ER-membrane by proteolytic cleavage. EIN2C binds the 3´EBF2, recruits the UPF proteins and moves to P-bodies, where the translation of EBF2 in inhibited despite its mRNA accumulation. Once the ethylene signal is withdrawn, the translation of the stored EBF2 mRNAs is resumed, thus rapidly dampening the ethylene response. These findings represent a mechanistic paradigm of gene-specific regulation of translation in response to a key growth regulator. Translation regulatory elements can be located in both 3′ and 5′ UTRs. We are now focusing on the ead1 and ead2 mutants, another set of ethylene-signaling mutants defective in translational regulation. Ribosome-footprinting on the ead1 mutant revealed an accumulation of translating ribosomes in the 5´UTRs of uORF-containing genes and reduction in the levels of ribosomes in the main ORF. The mutant is also impaired in the translation of GFP when this reporter is fused to WT 5´UTR of potential EAD1 targets but not when GFP is fused to the uORF-less versions of the same 5´UTRs. Our hypothesis is that EAD1/2 work as a complex that is required for the efficient translation of mRNAs that have common structural (complex 5´UTR with uORFs) and functional (regulation of key cellular processes) features. We are working towards the identification of the conditions where the EAD1 regulation of translation is required. [1] Ingolia, N. et al. (2009) Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling. Science, 324; 218-222 [2] Merchante, C. et al. (2015) Gene-Specific Translation Regulation Mediated by the Hormone-Signaling Molecule EIN2. Cell, 163(3): 684-697

Past, present and future of epigenetics applied to livestock breeding

This article reviews the concept of Lamarckian inheritance and the use of the term epigenetics in the field of animal genetics. Epigenetics was first coined by Conrad Hal Waddington (1905–1975), who derived the term from the Aristotelian word epigenesis. There exists some controversy around the word epigenetics and its broad definition. It includes any modification of the expression of genes due to factors other than mutation in the DNA sequence. This involves DNA methylation, post-translational modification of histones, but also linked to regulation of gene expression by non-coding RNAs, genome instabilities or any other force that could modify a phenotype. There is little evidence of the existence of transgenerational epigenetic inheritance in mammals, which may commonly be confounded with environmental forces acting simultaneously on an individual, her developing fetus and the germ cell lines of the latter, although it could have an important role in the cellular energetic status of cells. Finally, we review some of the scarce literature on the use of epigenetics in animal breeding programs.

L'acide valproïque inhibe la progression dans le cycle cellulaire chez Saccharomyces cerevisiae

L’acétylation est une modification post-traductionnelle des protéines essentielles. Elle est impliquée dans bon nombre de processus cellulaires importants comme la régulation de la structure de la chromatine et le recrutement de protéines. Deux groupes d’enzymes, soient les lysines acétyltransférases et les lysines désacétylases, régulent cette modification, autant sur les histones que sur les autres protéines. Au cours des dernières années, de petites molécules inhibitrices des désacétylases ont été découvertes. Certaines d’entre elles semblent prometteuses contre diverses maladies telles le cancer. L’acide valproïque, un inhibiteur de deux des trois classes des désacétylases, a un effet antiprolifératif chez plusieurs organismes modèles. Toutefois, les mécanismes cellulaires sous-jacents à cet effet restent encore méconnus. Ce mémoire met en lumière l’effet pH dépendant de l’acide valproïque sur différentes voies cellulaires importantes chez la levure Saccharomyces cerevisiae. Il démontre que ce composé a la capacité d’inhiber la transition entre les phases G1 et S par son action sur l’expression des cyclines de la phase G1. De plus, il inhibe l’activation de la kinase principale de la voie activée suite à un stress à la paroi cellulaire. L’acide valproïque occasionne également un arrêt dans la réplication de l’ADN sans y causer de dommage. Il s’agit là d’un effet unique qui, à notre connaissance, n’est pas observable avec d’autres agents qui inhibent la progression en phase S.