Characterization of a cAMP-regulated enhancer-binding protein.

The transcription regulation of many hormone genes is modulated by intracellular second messengers such as cAMP. The cAMP response element binding protein, CREB, binds to the 8 base pair CRE enhancer, TGACGTCA, that is found in the 5'-flank of certain genes including those for somatostatin and the alpha-subunit of human chorionic gonadotropin. The recent characterization of CREB and CREB-related cDNA clones, combined with Southwesterns and Northern blot analyses, reveals a family of transcription factors that dimerize via a leucine zipper motif and bind to the CRE through positively charged basic regions. The CREB cDNA encoding a 327 residue protein is transcriptionally activated via phosphorylation by protein kinases, including the cAMP-dependent protein kinase-A.

Molecular basis of interaction between Na,K-ATPase and FXYD proteins.

Résumé au large public Notre corps est constitué de différents types de cellules. La condition minimale ou primordiale pour la survie des cellules est d'avoir de l'énergie. Cette tâche est assumée en partie par une protéine qui se situe dans la membrane de chaque cellule. Nommé Na, K¬ATPase ou pompe à sodium, c'est une protéine pressente dans toutes les cellules chez les mammifères est composée de deux sous-unités, α et β. En transportant 3 ions de sodium hors de la cellule et 2 ions de potassium à l'intérieur de la cellule, elle transforme l'énergie chimique sous forme de l'ATP en énergie motrice, qui permet aux cellules par la suite d'échanger des matériaux entre l'espace intracellulaire et extracellulaire ainsi que d'ingérer des nutriments provenant de son environnement. Le manque de cette protéine chez la souris entraîne la mort de l'embryon. Des défauts fonctionnels de cette protéine sont responsables de plusieurs maladies humaines comme par exemple, un type de migraine. En dehors de sa fonction vitale, cette protéine est également engagée dans diverses activités physiologiques comme la contractilité musculaire, l'activité nerveuse et la régulation du volume sanguin. Vue l'importance de cette protéine, sa découverte par Jens C. Skou en 1957 a été honorée d'un Prix Noble de chimie quarante ans plus tard. Depuis lors, nous connaissons de mieux en mieux les mécanismes de fonctionnement de la Na, K-ATPase. Entre autre, sa régulation par une famille de protéines appelées protéines FXYD. Cette famille contient 7 membres (FXYD 1-7). L'un d'entre eux nommé FXYD 2 est lié à une maladie héréditaire connue sous le nom de hypomagnesemia. Nous disposons actuellement d'informations concernant les conséquences de la régulation par les protéines FXYD sur activité de la Na, K-ATPase, mais nous savons très peu sur le mode d'interaction entre les protéines FXYD et la Na, K-ATPase. Dans ce travail de thèse, nous avons réussi à localiser des zones d'interaction dans la sous- unité a de la Na, K-ATPase et dans FXYD 7. En même temps, nous avons déterminé un 3ème site de liaison spécifique au sodium de la Na, K-ATPase. Une partie de ce site se situe à l'intérieur d'un domaine protéique qui interagit avec les protéines FXYD. De plus, ce site a été démontré comme responsable d'un mécanisme de transport de la Na, K-ATPase caractérisé par un influx ionique. En conclusion, les résultats de ce travail de thèse fournissent de nouvelles preuves sur les régions d'interaction entre la Na, K-ATPase et les protéines FXYD. La détermination d'un 3ème site spécifique au sodium et sa relation avec un influx ionique offrent la possibilité 1) d'explorer les mécanismes avec lesquels les protéines FXYD régulent l'activité de la Na, ATPase et 2) de localiser un site à sodium qui est essentielle pour mieux comprendre l'organisation et le fonctionnement de la Na, K-ATPase. Résumé Les gradients de concentration de Na+ et de K+ à travers la membrane plasmatique des cellules animales sont cruciaux pour la survie et l'homéostasie de cellules. De plus, des fonctions cellulaires spécifiques telles que la reabsorption de Na dans le rein et le côlon, la contraction musculaire et l'excitabilité nerveuse dépendent de ces gradients. La Na, K¬ATPase ou pompe à sodium est une protéine membranaire ubiquitaire. Elle crée et maintient ces gradients en utilisant l'énergie obtenu par l'hydrolyse de l'adénosine triphosphate. L'unité fonctionnelle minimale de cette protéine se compose d'une sous-unité catalytique α et d'une sous-unité régulatrice β. Récemment, il a été montré que des membres de la famille FXYD, sont des régulateurs tissu-spécifiques de la Na, K-ATPase qui influencent ses propriétés de transport. Cependant, on connaît peu de chose au sujet de la nature moléculaire de l'interaction entre les protéines FXYD et la Na, K-ATPase. Dans cette étude, nous fournissons, pour la première fois, l'évidence directe que des résidus du domaine transmembranaire (TM) 9 de la sous-unité α de la Na, K-ATPase sont impliqués dans l'interaction fonctionnelle et structurale avec les protéines FXYD. De plus nous avons identifié des régions dans le domaine transmembranaire de FXYD 7 qui sont importantes pour l'association stable avec la Na, K-ATPase et une série de résidus responsables des régulations fonctionnelles. Nous avons aussi montré les contributions fonctionnelles du TM 9 de la Na, K-ATPase à la translocation de Na + en déterminant un 3ème site spécifique au Na+. Ce site se situe probablement dans un espace entre TM 9, TM 6 et TM 5 de la sous-unité α de la pompe à sodium. De plus, nous avons constaté que le 3ème site de Na + est fonctionnellement lié à un courant entrant de la pompe sensible à l'ouabaïne et activé par le pH acide. En conclusion, ce travail donne de nouvelles perspectives de l'interaction structurale et fonctionnelle entre les protéines FXYD et la Na, K-ATPase. En outre, les contributions fonctionnelles de TM 9 offrent de nouvelles possibilités pour explorer le mécanisme par lequel les protéines FXYD régulent les propriétés fonctionnelles de la Na, K-ATPase. La détermination du 3ème site au Na + fournit une compréhension avancée du site spécifique au Na + de la Na, K-ATPase et du mécanisme de transport de la Na, K-ATPase. Summary The Na+ and K+ gradients across the plasma membrane of animal cells are crucial for cell survival and homeostasis. Moreover, specific tissue functions such as Na+ reabsorption in kidney and colon, muscle contraction and nerve excitability depend on the maintenance of these gradients. Na, K-ATPase or sodium pump, an ubiquitous membrane protein, creates and maintains these gradients by using the energy from the hydrolysis of ATP. The minimal functional unit of this protein is composed of a catalytic α subunit and a regulatory β subunit. Recently, members of the FXYD family, have been reported to be tissue-specific regulators of Na, K-ATPase by influencing its transport properties. However, little is known about the molecular nature of the interaction between FXYD proteins and Na, K-ATPase. In this study, we provide, for the first time, direct evidence that residues from the transmembrane (TM) domain 9 of the α subunit of Na, K-ATPase are implicated in the functional and structural interaction with FXYD proteins. Moreover, we have identified regions in the TM domain of FXYD 7 important for the stable association with Na, K-ATPase and a stretch of residues responsible for the functional regulations. We have further revealed the functional contributions of TM 9 of the Na, K-ATPase α subunit to the Na+ translocation by determining a 3rd Na+-specific cation binding site. This site is likely in a space between TM 9, TM 6 and TM 5 of the a subunit of the sodium pump. Moreover, we have found that the 3rd Na+ binding site is functionally linked to an acidic pH- activated ouabain-sensitive inward pump current. In conclusion, this work gives new insights into the structural and functional interaction between FXYD proteins and Na, K-ATPase. Functional contributions of TM 9 offer new possibilities to explore the mechanism by which FXYD proteins regulate functional properties of Na, K-ATPase. The determination of the 3rd Na+ binding site provides an advanced understanding concerning the Na+ -specific binding site of Na, K-ATPase and the 3rd Na+ site related transport mechanism.

Biochemical characterization of recombinant human telomerase

Résumé Les télomères sont les structures ADN-protéines des extrémités des chromosomes des eucaryotes. L'ADN télomérique est constitué de courtes séquences répétitives. L'intégrité des télomères est essentielle pour protéger les extrémités des chromosomes contre les systèmes de dégradations et pour les distinguer des cassures de l'ADN double brin. Parce que la machinerie de la réplication de l'ADN n'est pas capable de répliquer l'extrémité des chromosomes, les télomères raccourcissent au fur et à mesure des cycles de réplication. Dès que les télomères atteignent une longueur critique, leur structure protectrice est perdue. Cela induit un signal de dommage de l'ADN et l'arrêt du cycle cellulaire. Pour contrebalancer le raccourcissement des télomères, les cellules qui s'auto régénèrent, dont les cellules de la moelle osseuse, les lymphocytes activés et 80-90% des cellules cancéreuses, expriment la télomérase. C'est une ribonucléoprotéine qui a la capacité de synthétiser des séquences télomériques par transcription inverse d'une courte séquence contenue dans sa propre sous-unité ARN avec laquelle elle est associée. La télomérase humaine est une enzyme processive au niveau de l'addition des nucléotides et aussi des répétitions télomériques. La télomérase de levure et la télomérase humaine sont toutes deux dimériques et il a été montré que la télomérase humaine recombinante contient deux ARN qui coopèrent pour fonctionner ainsi que deux sous-unités catalytiques. Cependant, il n'a pas encore été montré quel est le rôle de la dimérisation dans l'activité de la télomérase. Afin d'élucider ce rôle, nous avons exprimé, reconstitué et purifié la télomérase humaine dimérique recombinante. Et pour étudier l'effet d'ARN mutants sur l'activité de la télomérase, nous avons développé une méthode pour reconstituer et enrichir en hétérodimères de télomérase. Les hétérodimères contiennent une sous-unité ARN sauvage et une sous-unité ARN mutée au niveau de la séquence de la matrice. Sur l'ARN muté nous avons introduit une étiquette aptamer ARN-S1 puis nous avons purifié la télomérase via l'etiquette Si. Nous avons montré que la dimérisation est essentielle pour l'activité de la télomérase. Nos données indiquent que chaque télomérase du dimère allonge leur substrat, l'ADN télomérique, indépendamment l'une de l'autre à chaque cycle d'élongation mais que l'addition itérative de répétitions télomériques nécessite une coopération entre les deux télomérases du dimère. Nous proposons donc un modèle dans lequel les deux télomérases du dimères se lient et allongent deux substrats télomères et que pendant l'élongation processive les deux enzymes subissent un changement de conformation de manière coordonnée, ce changement va permettre le repositionnement des substrats pour d'autres cycles d'additions de répétitions télomériques. Dyskeratosis congenita est une maladie mortelle due majoritairement au disfonctionnement de la moelle osseuse. Dans la forme autosomale de la maladie, l'ARN de la télomérase contient des mutations. En utilisant notre système de reconstitution, nous avons montré que ces ARN mutés, qui ont perdu leur activité enzymatique dans le cas d'un homodimère de mutants, sont dominant négatifs quand ils sont présents dans les hétérodimères sauvage/mutant. Cet effet trans-dominant négatif pourrait contribuer à la progression de la maladie. Abstract Telomeres are protein-DNA structures at the ends of linear eukaryotic chromosomes. The telomeric DNA consists of tandemly repeated sequences. Telomeric integrity is essential to protect chromosomal ends from nucleolytic degradation and to prevent their recognition as DNA double strand breaks. Due to the inability of the conventional DNA replication machinery to replicate terminal DNA stretches, telomeres shorten with continuous rounds of DNA replication. As soon as telomeres reach a critical length, their protective structure is lost and the deprotected telomeres will induce a DNA damage response leading to cell cycle arrest. To counteract telomere shortening, self-renewing cells, including bone marrow cells, activated lymphocytes and 80-90% of cancer cells express the cellular reverse transcriptase telomerase, which has the capacity to synthesize telomeric repeats by reverse transcription of a short template sequence encoded by its stably associated RNA subunit. Human telomerase is a processive enzyme for nucleotide as well as repeat addition. Both yeast and human telomerase are dimeric enzymes and recombinant human telomerase has been shown to contain two functionally cooperating RNAs and most probably also two protein subunits. However, it has remained unclear how dimerization may contribute to telomerase activity. To study the role of dimerization, we expressed, reconstituted and purified recombinant human telomerase. We also developed a new method to reconstitute and enrich for telomerase heterodimers containing wild-type (wt) and mutant telomerase RNA subunits. To this end we introduced an S1-RNA-aptamer tag into telomerase RNA and purified telomerase reconstituted with a mixture of untagged and tagged RNA via the S1-tag. Using this experimental system, we introduced template mutations in the tagged RNA subunit and examined the effect of mutant RNAs on wt telomerase activity in wt/mutant heterodimers. We obtained evidence that dimerization is essential for telomerase activity. Our data indicate that the two subunits elongate telomere substrates independently of each other during single rounds of elongation, but that iterative addition of telomeric repeats requires cooperation between the two subunits. We suggest a model, in which dimeric telomerases bind and elongate two telomere substrates and that the two subunits undergo coordinated conformational changes during processive elongation that enable repositioning the substrates for subsequent rounds of repeat addition. Dyskeratosis congenita is a multisystemic disease with bone marrow failure as the major cause of death. The autosomal form of this disease was found to harbor mutations in the telomerase RNA. Using our reconstitution system, we tested whether mutant dyskeratosis telomerase RNAs behaved in a dominant negative manner. We observed that dyskeratosis telomerase RNA mutants, which lacked enzymatic activity were dominant negative, when present in wt/ mutant heterodimers. The transdominant negative effect of these mutants may contribute to disease progression.

zyg-11and cul-2 promote the metaphase to anaphase transition and M phase exit of meiosis II and prevent polarity establishment in C.elegans

Résumé Les mécanismes qui coordonnent la progression du cycle cellulaire lors de la méiose avec les événements du développement embryonnaire précoce, y compris la formation des axes de polarité embryonnaire, sont peu compris. Dans le zygote du vers Caenorhabditis elegans, les premiers signes de polarité Antéro-Postérieur (A-P) embryonnaire apparaissent après que la méiose soit terminée. La nature des protéines et des mécanismes moléculaires qui cassent la symétrie du zygote n'est pas connue. Nous démontrons que zyg-11 et cul-2 promeuvent la transition métaphase - anaphase et la sortie de la phase M lors de la seconde division méiotique. Nos résultats indiquent que ZYG-11 agit comme unité recrutant le substrat d'une ligase E3 comprennant CUL-2. Nos résultats montrent aussi que le délai de sortie de la phase M dépend de l'accumulation de la Cyclin B, CYB-3. Nous démontrons que dans des embryons zyg-11(RNAi) ou cul-2(RNAi), une polarité inversée est établie lors du délai de méiosis II. Enfin nous montrons que les défauts de cycle cellulaire et ceux de polarité peuvent être séparés. De plus, nous faisons apparaitre que l'établissement d'une polarité inversée pendant le délai de méiose II des embryons zyg-11(RNAi), comme l'établissement de la A-P polarité des embryons sauvage ne semblent pas requérir les microtubules. Nous montrons également les premiers résultats d'un crible deux hybrides ainsi qu'un crible génomique qui vise à identifier des gènes dont l'inactivation augmente ou supprime les défauts de mutants pour le gène zyg-11, afin d'identifier les gènes qui intéragissent avec ZYG-11 pour assumer ses deux fonctions séparables. Par conséquent, nos trouvailles suggèrent un modèle selon lequel ZYG-11 est une sous-unité qui recrute les substrats d'une ligase E3 basée sur CUL-2 qui promeut la progression du cycle cellulaire et empêche l'établissement de la polarité pendant la méiose II, et où le centrosome agit comme la clé qui polarise l'embryon à la fin de la méiose. Summary The mechanisms that couple meiotic cell cycle progression to subsequent developmental events, including specification of embryonic axes, are poorly understood. In the one cell stage embryos of Caenorhabditis elegans, the first signs of Antero-Posterior (A-P) polarity appear after meiosis completion. A centrosome ¬derived component breaks symmetry of the embryo, but the molecular nature of this polarity signal is not known. We established that zyg-11 and cul-2 promote the metaphase to anaphase transition and M phase exit at meiosis II. Our results indicate that ZYG-11 acts as a substrate recruitment subunit of a CUL-2-based E3 ligase. Moreover, we find that the delayed meiosis II exit of embryos lacking zyg-11 is caused by accumulation of the B-type cyclin, CYB-3. We demonstrate that inverted A-P polarity is established during the meiosis II delay in zyg-11(RNAi) and cul¬2(RNAi) embryos. Importantly, we demonstrate that the polarity defects following zyg-11 or cul-2 inactivation can be uncoupled from the cell cycle defects. Furthermore, we found that microtubules appear dispensable for inverted polarity during the meiosis II delay in zyg-11(RNAi) embryos, as well as for A-P polarity during the first mitotic cell cycle in wild-type embryos. We also show the initial results from a comprehensive yeast two hybrid, as well as an RNAi-based functional genomic enhancer and suppressor screen, that may lead to identification of proteins that interact with zyg-11 to ensure the two functions. Our findings suggest a model in which ZYG-11 is a substrate recruitment subunit of an CUL-2-based E3 ligase that promotes cell cycle progression and prevents polarity establishment during meiosis II, and in which the centrosome acts as a cue to polarize the embryo along the AP axis after exit from the meiotic cell cycle.

Congenital ataxia and hemiplegic migraine with cerebral edema associated with a novel gain of function mutation in the calcium channel CACNA1A.

Mutations in the CACNA1A gene, encoding the α1 subunit of the voltage-gated calcium channel Ca(V)2.1 (P/Q-type), have been associated with three neurological phenotypes: familial and sporadic hemiplegic migraine type 1 (FHM1, SHM1), episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 (SCA6). We report a child with congenital ataxia, abnormal eye movements and developmental delay who presented severe attacks of hemiplegic migraine triggered by minor head traumas and associated with hemispheric swelling and seizures. Progressive cerebellar atrophy was also observed. Remission of the attacks was obtained with acetazolamide. A de novo 3 bp deletion was found in heterozygosity causing loss of a phenylalanine residue at position 1502, in one of the critical transmembrane domains of the protein contributing to the inner part of the pore. We characterized the electrophysiology of this mutant in a Xenopus oocyte in vitro system and showed that it causes gain of function of the channel. The mutant Ca(V)2.1 activates at lower voltage threshold than the wild type. These findings provide further evidence of this molecular mechanism as causative of FHM1 and expand the phenotypic spectrum of CACNA1A mutations with a child exhibiting severe SHM1 and non-episodic ataxia of congenital onset.

Remodelage des canaux potassiques dépendants de l'ATP lors de l'hypertrophie et de l'insuffisance cardiaque

RESUME :Introduction. Les maladies cardiovasculaires représentent la première cause de mortalité dans les pays développés et l'insuffisance cardiaque (IC) est la plus fréquente. Suite à un infarctus, le coeur des patients subit un remodelage ventriculaire pouvant évoluer vers un état d'IC. L'IC se définit comme un état dans lequel le coeur n'est plus capable d'approvisionner suffisamment les organes et cet état s'accompagne souvent de troubles du rythme cardiaque. Le remodelage ventriculaire touche de nombreux gènes codant à la fois pour les voies métaboliques et pour des canaux ioniques favorisant ainsi l'apparition des arythmies responsables de la mort subite des patients atteints d'IC. Comprendre ce passage entre remodelage et IC est crucial afin de pouvoir un jour prévenir l'IC et les complications médicales qui l'accompagnent. Nous nous sommes intéressés aux canaux potassiques dépendants de l'ATP (KATP) car ces canaux ont la capacité de coupler le métabolisme de la cellule à son activité électrique. En effet, les canaux KATP s'ouvrent quand la charge énergétique (rapport ATP/ ADP) de la cellule chute. Dans les cardiomyocytes, l'ouverture des KATP induit une hyperpolarisation de la membrane cellulaire ce qui diminue indirectement la surcharge calcique et de ce fait préserve la cellule. Les canaux KATp sont formés de 4 sous-unités Kir6.x (Kir6.1 ou Kir6.2) formant le pore du canal associées à 4 sous-unités régulatrices SUR. Les propriétés électrophysiologiques ainsi que la sensibilité pharmacologique des canaux KATP dépendent de leur composition et seuls les canaux KATP formés par la sous-unité Kirô.l sont activés par le diazoxyde.Méthodes et résultats. Nous avons d'abord montré dans un modèle in vivo d'IC chez le rat adulte que les sous-unités Kir6.1 et SUR sont surexprimées dans ces conditions pathologiques. Par ailleurs, les cardiomyocytes issus des coeurs infarcis deviennent sensibles au diazoxyde reflétant la surexpression de Kir6.1. Les potentiels d'action qui sont prolongés dans l'IC et qui sont à l'origine d'arythmies majeures sont normalisés par l'ouverture des canaux KATp induite par le diazoxyde. Ainsi, l'ouverture pharmacologique des canaux KATp contribuerait à la cardio-protection. Dans une seconde partie, nous avons déterminé quels étaient les facteurs de transcription responsables de ce changement d'expression des sous-unités formant les KATP. Dans notre modèle, nous avons pu montrer que la surexpression de Kirô.l est due aux facteurs de transcription Fox03 et FoxF2 qui est aussi responsable de la surexpression des sous-unités SUR. Dans la dernière partie de ce travail, nous avons mis au point un modèle d'IC in vitro en cultivant les cardiomyocytes de rats adultes en présence d'angiotensine II (Angll) ou de TNFa. Ce modèle expérimental nous a non seulement permis de mettre en relation l'importance de L'AnglI et du TNFa sur le remodelage des canaux KATP mais aussi de développer un modèle in vitro présentant les mêmes caractéristiques que le modèle in vivo concernant le remodelage des KATP lors de l'IC. Ce dernier modèle expérimental ouvre des perspectives afin de mieux caractériser les voies de signalisation impliquées dans le remodelage des canaux KATp lors de l'IC.Conclusion. Les canaux KATp subissent un remodelage lors de l'IC et les résultats obtenus montrent le potentiel cardio-protecteur de ces canaux.ABSTRACT :Background and aim. Cardiovascular disease is the leading cause of death in developed countries and heart failure (HF) is the most common. Following myocardial infarction, the heart of the patient undergoes ventricular remodeling which may evolve toward a state of HF. HF is defined as a state in which heart is unable to supply enough blood to organs and this state is often accompanied by cardiac arrhythmias. Ventricular remodeling involves many genes coding for both metabolic enzymes and ion channels. Changes in ion channel expression can promote arrhythmias responsible for sudden death in patients with HF. A better understanding of the transition between remodeling and HF is crucial in order to prevent the complications associated to HF We were interested in ATP-dependent potassium channels (KATp) because they couple cell metabolism to electrical activity of the cell. Indeed, KATP channels open when the energy charge (ratio of ATP / ADP) of the cell collapses. In cardiomyocytes, the opening of KATP channels induces hyper- polanzation of the cell membrane which reduces calcium overload and thereby protects the cell. KATp channels are composed by 4 Kir6.x subumts (Kir6.1 or Kir6.2) forming the pore channel associated with 4 regulatory subunits SUR. The electrophysiological properties as well as pharmacological sensitivity of KATp channels depend on their composition and only KATP channels formed by Kir6.1 subunit are activated by diazoxide.Methods and results. Firstly, using an in vivo model of HF in adult rats, we showed that Kir6.1 and SUR subunits are overexpressed in HF. In addition, cardiomyocytes from post-infarction hearts became sensitive to diazoxide reflecting the overexpression of the Kir6.1 subunit. The opening of KATP by diazoxide tended to reduce the action potential duration (APD) which is extended in HF. This increase in APD is known to be a major source of arrhythmias during HF. Therefore, the opening of KATP channels by diazoxide would be cardio-protective. Secondly, we wanted to determine which transcription factors were responsible for this KATP remodeling. In our model of HF, we showed that overexpression of Kir6.1 is due to the transcription factors Fox03 and FOXF2 which is also responsible for SUR subunits overexpression. Thirdly, we developed an in vitro model of HF by cultivation of adult rat cardiomyocytes in the presence of angiotensin II (Angll) or TNFa. This model is very interesting not only because it underlines the importance of Angll and TNFa in KATp remodeling but also because this in vitro model presents the same KATP remodeling as the in vivo model of HF. These findings show that our in vitro model of HF opens up many possibilities to investigate more precisely the signaling pathways involved in remodeling of the KATP channels in HF.Conclusion. KATP channels undergo remodeling during HF and our results show the cardio¬protective potential of KATP channels in this disease.

Genetic dysregulation of glutathione synthesis predicts alteration of plasma thiol redox status in schizophrenia.

Abstract Genetic studies have shown an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL), the key enzyme for glutathione (GSH) synthesis. The present study was aimed at analyzing the influence of a GSH dysregulation of genetic origin on plasma thiols (total cysteine, homocysteine, and cysteine-glycine) and other free amino acid levels as well as fibroblast cultures GSH levels. Plasma thiols levels were also compared between patients and controls. As compared with patients with a low-risk GCLC GAG TNR genotype, patients with a high-risk genotype, having an impaired GSH synthesis, displayed a decrease of fibroblast GSH and plasma total cysteine levels, and an increase of the oxidized form of cysteine (cystine) content. Increased levels of plasma free serine, glutamine, citrulline, and arginine were also observed in the high-risk genotype. Taken together, the high-risk genotypes were associated with a subgroup of schizophrenia characterized by altered plasma thiols and free amino acid levels that reflect a dysregulation of redox control and an increased susceptibility to oxidative stress. This altered pattern potentially contributes to the development of a biomarker profile useful for early diagnosis and monitoring the effectiveness of novel drugs targeting redox dysregulation in schizophrenia. Antioxid. Redox Signal. 15, 2003-2010.

Neurofilament triplet proteins are restricted to a subset of neurons in the rat neocortex.

The cellular localisation of neurofilament triplet subunits was investigated in the rat neocortex. A subset of mainly pyramidal neurons showed colocalisation of subunit immunolabelling throughout the neocortex, including labelling with the antibody SMI32, which has been used extensively in other studies of the primate cortex as a selective cellular marker. Neurofilament-labelled neurons were principally localised to two or three cell layers in most cortical regions, but dramatically reduced labelling was present in areas such as the perirhinal cortex, anterior cingulate and a strip of cortex extending from caudal motor regions through the medial parietal region to secondary visual areas. However, quantitative analysis demonstrated a similar proportion (10-20%) of cells with neurofilament triplet labelling in regions of high or low labelling. Combining retrograde tracing with immunolabelling showed that cellular content of the neurofilament proteins was not correlated with the length of projection. Double labelling immunohistochemistry demonstrated that neurofilament content in axons was closely associated with myelination. Analysis of SMI32 labelling in development indicated that content of this epitope within cell bodies was associated with relatively late maturation, between postnatal days 14 and 21. This study is further evidence of a cell type-specific regulation of neurofilament proteins within neocortical neurons. Neurofilament triplet content may be more closely related to the degree of myelination, rather than the absolute length, of the projecting axon.

Airway surface liquid volume regulation determines different airway phenotypes in liddle compared with betaENaC-overexpressing mice.

Studies in cystic fibrosis patients and mice overexpressing the epithelial Na(+) channel beta-subunit (betaENaC-Tg) suggest that raised airway Na(+) transport and airway surface liquid (ASL) depletion are central to the pathogenesis of cystic fibrosis lung disease. However, patients or mice with Liddle gain-of-function betaENaC mutations exhibit hypertension but no lung disease. To investigate this apparent paradox, we compared the airway phenotype (nasal versus tracheal) of Liddle with CFTR-null, betaENaC-Tg, and double mutant mice. In mouse nasal epithelium, the region that functionally mimics human airways, high levels of CFTR expression inhibited Liddle epithelial Nat channel (ENaC) hyperfunction. Conversely, in mouse trachea, low levels of CFTR failed to suppress Liddle ENaC hyperfunction. Indeed, Na(+) transport measured in Ussing chambers ("flooded" conditions) was raised in both Liddle and betaENaC-Tg mice. Because enhanced Na(+) transport did not correlate with lung disease in these mutant mice, measurements in tracheal cultures under physiologic "thin film" conditions and in vivo were performed. Regulation of ASL volume and ENaC-mediated Na(+) absorption were intact in Liddle but defective in betaENaC-Tg mice. We conclude that the capacity to regulate Na(+) transport and ASL volume, not absolute Na(+) transport rates in Ussing chambers, is the key physiologic function protecting airways from dehydration-induced lung disease.

Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant beta(1) subunits.

Voltage-dependent calcium channel (Ca(v)) pores are modulated by cytosolic beta subunits. Four beta-subunit genes and their splice variants offer a wide structural array for tissue- or disease-specific biophysical gating phenotypes. For instance, the length of the N terminus of beta(2) subunits has major effects on activation and inactivation rates. We tested whether a similar mechanism principally operates in a beta(1) subunit. Wild-type beta(1a) subunit (N terminus length 60 aa) and its newly generated N-terminal deletion mutants (51, 27 and 18 aa) were examined within recombinant L-type calcium channel complexes (Ca(v)1.2 and alpha(2)delta2) in HEK293 cells at the whole-cell and single-channel level. Whole-cell currents were enhanced by co-transfection of the full-length beta(1a) subunit and by all truncated constructs. Voltage dependence of steady-state activation and inactivation did not depend on N terminus length, but inactivation rate was diminished by N terminus truncation. This was confirmed at the single-channel level, using ensemble average currents. Additionally, gating properties were estimated by Markov modeling. In confirmation of the descriptive analysis, inactivation rate, but none of the other transition rates, was reduced by shortening of the beta(1a) subunit N terminus. Our study shows that the length-dependent mechanism of modulating inactivation kinetics of beta(2) calcium channel subunits can be confirmed and extended to the beta(1) calcium channel subunit.

Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures.

The epithelial sodium channel (ENaC) is responsible for Na(+) and fluid absorption across colon, kidney, and airway epithelia. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its NH2-terminal region lacks structure. Here we found that an 18 amino acid peptide, S18, which corresponded to residues G22-A39 of the SPLUNC1 NH2 terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold), while SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the βENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid (ASL) height was abnormally low (4.2 ± 0.6 μm), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 ± 0.6 μm, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease.

Specific processing of tenascin-C by the metalloprotease meprin beta neutralizes its inhibition of cell spreading

The metalloprotease meprin has been implicated in tissue remodelling due to its capability to degrade extracellular matrix components. Here, we investigated the susceptibility of tenascin-C to cleavage by meprin beta and the functional properties of its proteolytic fragments. A set of monoclonal antibodies against chicken and human tenascin-C allowed the mapping of proteolytic fragments generated by meprin beta. In chicken tenascin-C, meprin beta processed all three major splicing variants by removal of 10 kDa N-terminal and 38 kDa C-terminal peptides, leaving a large central part of subunits intact. IN similar cleavage pattern was found for large human tenascin-C variant where two N-terminal peptides (10 or 15 kDa) and two C-terminal fragments (40 and 55 kDa) were removed from the intact subunit. N-terminal sequencing revealed the exact amino acid positions of cleavage sites. In both chicken and human tenascin-C N-terminal cleavages occurred just before and/or after the heptad repeats involved in subunit oligomerization. In the human protein, an additional cleavage site was identified in the alternative fibronectin type III repeat D. Whereas all these sites are known to be attacked by several other proteases, a unique cleavage by meprin beta was located to the 7th constant fibronectin type III repeat in both chicken and human tenascin-C, thereby removing the C-terminal domain involved in its anti-adhesive activity. In cell adhesion assays meprin beta-digested human tenascin-C was not able to interfere with fibronectin-mediated cell spreading, confirming cleavage in the anti-adhesive domain. Whereas the expression of meprin beta and tenascin-C does not overlap in normal colon tissue, inflamed lesions of the mucosa from patients with Crohn's disease exhibited many meprin beta-positive leukocytes in regions where tenascin-C was strongly induced. Our data indicate that, at least under pathological conditions, meprin beta might attack specific functional sites in tenascin-C that are important for its oligomerization and anti-adhesive activity. (C) 2009 Elsevier B.V. All rights reserved.

Genomic acute myeloid leukemia-associated inv(16)(p13q22) breakpoints are tightly clustered.

The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor beta (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2 Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.

Decreased specific CD8+ T cell cross-reactivity of antigen recognition following vaccination with Melan-A peptide.

The aim of T cell vaccines is the expansion of antigen-specific T cells able to confer immune protection against pathogens or tumors. Although increase in absolute cell numbers, effector functions and TCR repertoire of vaccine-induced T cells are often evaluated, their reactivity for the cognate antigen versus their cross-reactive potential is rarely considered. In fact, little information is available regarding the influence of vaccines on T cell fine specificity of antigen recognition despite the impact that this feature may have in protective immunity. To shed light on the cross-reactive potential of vaccine-induced cells, we analyzed the reactivity of CD8(+) T cells following vaccination of HLA-A2(+) melanoma patients with Melan-A peptide, incomplete Freund's adjuvant and CpG-oligodeoxynucleotide adjuvant, which was shown to induce strong expansion of Melan-A-reactive CD8(+) T cells in vivo. A collection of predicted Melan-A cross-reactive peptides, identified from a combinatorial peptide library, was used to probe functional antigen recognition of PBMC ex vivo and Melan-A-reactive CD8(+) T cell clones. While Melan-A-reactive CD8(+) T cells prior to vaccination are usually constituted of widely cross-reactive naive cells, we show that peptide vaccination resulted in expansion of memory T cells displaying a reactivity predominantly restricted to the antigen of interest. Importantly, these cells are tumor-reactive.

Differential effects of selective HDAC inhibitors on macrophage inflammatory responses to the Toll-like receptor 4 agonist LPS.

Broad-spectrum inhibitors of HDACs are therapeutic in many inflammatory disease models but exacerbated disease in a mouse model of atherosclerosis. HDAC inhibitors have anti- and proinflammatory effects on macrophages in vitro. We report here that several broad-spectrum HDAC inhibitors, including TSA and SAHA, suppressed the LPS-induced mRNA expression of the proinflammatory mediators Edn-1, Ccl-7/MCP-3, and Il-12p40 but amplified the expression of the proatherogenic factors Cox-2 and Pai-1/serpine1 in primary mouse BMM. Similar effects were also apparent in LPS-stimulated TEPM and HMDM. The pro- and anti-inflammatory effects of TSA were separable over a concentration range, implying that individual HDACs have differential effects on macrophage inflammatory responses. The HDAC1-selective inhibitor, MS-275, retained proinflammatory effects (amplification of LPS-induced expression of Cox-2 and Pai-1 in BMM) but suppressed only some inflammatory responses. In contrast, 17a (a reportedly HDAC6-selective inhibitor) retained anti-inflammatory but not proinflammatory properties. Despite this, HDAC6(-/-) macrophages showed normal LPS-induced expression of HDAC-dependent inflammatory genes, arguing that the anti-inflammatory effects of 17a are not a result of inhibition of HDAC6 alone. Thus, 17a provides a tool to identify individual HDACs with proinflammatory properties.