GLUT2 in pancreatic and extra-pancreatic gluco-detection (review).

Detection of variations in blood glucose concentrations by pancreatic beta-cells and a subsequent appropriate secretion of insulin are key events in the control of glucose homeostasis. Because a decreased capability to sense glycemic changes is a hallmark of type 2 diabetes, the glucose signalling pathway leading to insulin secretion in pancreatic beta-cells has been extensively studied. This signalling mechanism depends on glucose metabolism and requires the presence of specific molecules such as GLUT2, glucokinase and the K(ATP) channel subunits Kir6.2 and SUR1. Other cells are also able to sense variations in glycemia or in local glucose concentrations and to modulate different physiological functions participating in the general control of glucose and energy homeostasis. These include cells forming the hepatoportal vein glucose sensor, which controls glucose storage in the liver, counterregulation, food intake and glucose utilization by peripheral tissues and neurons in the hypothalamus and brainstem whose firing rates are modulated by local variations in glucose concentrations or, when not protected by a blood-brain barrier, directly by changes in blood glucose levels. These glucose-sensing neurons are involved in the control of insulin and glucagon secretion, food intake and energy expenditure. Here, recent physiological studies performed with GLUT2-/- mice will be described, which indicate that this transporter is essential for glucose sensing by pancreatic beta-cells, by the hepatoportal sensor and by sensors, probably located centrally, which control activity of the autonomic nervous system and stimulate glucagon secretion. These studies may pave the way to a fine dissection of the molecular and cellular components of extra-pancreatic glucose sensors involved in the control of glucose and energy homeostasis.

Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors.

The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon-gamma (IFN-gamma)-producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X(7) purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex ('inflammasome'), allowing for the secretion of interleukin-1beta (IL-1beta). The priming of IFN-gamma-producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3(-/-)) or caspase-1-deficient (Casp-1(-/-)) mice unless exogenous IL-1beta is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7(-/-) or Nlrp3(-/-) or Casp1(-/-) hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele. These results indicate that the NLRP3 inflammasome links the innate and adaptive immune responses against dying tumor cells.

Glucagon-like peptide-I and the control of insulin secretion in the normal state and in NIDDM.

Potentiation of glucose-induced insulin secretion by intestinal factors has been described for many years. Today, two major peptides with potent insulinotropic action have been recognized: gastric inhibitory peptide and truncated forms of glucagon-like peptide I, GLP-I(7-37) or the related GLP-I(7-36)amide. These hormones have specific beta-cell receptors that are coupled to production of cAMP and activation of cAMP-dependent protein kinase. Elevation in intracellular cAMP levels is required to mediate the glucoincretin effect of these hormones: the potentiation of insulin secretion in the presence of stimulatory concentrations of glucose. In addition, circulating glucoincretins maintain basal levels of cAMP, which are necessary to keep beta-cells in a glucose-competent state. Interactions between glucoincretin signaling and glucose-induced insulin secretion may result from the phosphorylation of key elements of the glucose signaling pathway by cAMP-dependent protein kinase. These include the ATP-dependent K+ channel, the Ca++ channel, or elements of the secretory machinery itself. In NIDDM, the glucoincretin effect is reduced. However, basal or stimulated gastric inhibitory peptide and glucagon-like peptide I levels are normal or even elevated, suggesting that signals induced by these hormones on the beta-cells are probably altered. At pharmacological doses, infusion of glucagon-like peptide I but not gastric inhibitory peptide, can ameliorate postprandial insulin secretory response in NIDDM patients. Agonists of the glucagon-like peptide I receptor have been proposed as new therapeutic agents in NIDDM.

Fanconi anemia and genome stability : the role of FANCM

RÉSUMÉ: Le génome de toute cellule est susceptible d'être attaqué par des agents endogènes et exogènes. Afin de préserver l'intégrité génomique, les cellules ont développé des multitudes de mécanismes. La réplication de l'ADN, une étape importante durant le cycle cellulaire, constitue un stress et présente un danger important pour l'intégrité du génome. L'anémie de Fanconi est une maladie héréditaire rare dont les protéines impliquées semblent jouer un rôle crucial dans la réponse au stress réplicatif. La maladie est associée à une instabilité chromosomique ainsi qu'à une forte probabilité de développer des cancers. Les cellules des patients souffrant de l'anémie de Fanconi sont sensibles à des agents interférant avec la réplication de l'ADN, et plus particulièrement àdes agents qui fient les deux brins d'ADN d'une manière covalente. L'anémie de Fanconi est une maladie génétiquement hétérogène. Treize protéines ont pu être identifiées. Elles semblent figurer dans une même voie de signalisation qui est aussi connue sous le nom de « FA/BRCA pathway », car un des gènes est identique au gène BRCA2 (breast cancer susceptibility gene 2). Huit protéines forment un complexe nucléaire dont l'intégrité est nécessaire à la monoubiquitination de deux autres protéines, FANCD2 et FANCI, en réponse à un stress réplicatif. A ce jour, la fonction moléculaire des protéines du « FA/BRCA pathway »reste encore mal décrite. Au début de mon travail de thèse, nous avons donc décidé de purifier les protéines du complexe nucléaire et d'étudier leurs propriétés biochimiques. Nous avons tout d'abord étudié les cinq protéines connues à l'époque qui sont FANCA, FANCC, FANCE, FANCF et FANCG. Par la suite, nous avons étendu notre étude à des protéines découvertes plus récemment, FANCL, FANCM et FAAP24, en concentrant finalement notre travail sur la caractérisation de FANCM. FANCM, contrairement aux autres protéines du complexe, est constituée de deux domaines conservés suggérant un rôle important dans le métabolisme de l'ADN. Il s'agit d'un domaine « DEAH box hélicase »situé dans la partie N-terminale et d'un domaine « ERCC4 nuclease »situé dans la partie C-terminale de la protéine. Dans cette étude, nous avons purifié avec succès la protéine FANCM entière à partir d'un système hétérologue. Nous montrons que FANCM s'attache de manière spécifique à des jonctions de Holliday et des fourches de réplication. De plus, nous démontrons que FANCM peut déplacer le point de jonction de ces structures via son domaine hélicase de manière dépendante de l'ATP. FANCM est aussi capable de dissocier de grands intermédiaires de la recombinaison, via la migration de jonctions de Holliday à travers une région d'homologie de 2.6 kb. Tous ces résultats suggèrent que FANCM peut s'attacher spécifiquement à des fourches de réplication et à des jonctions de Holliday in vitro et que son domaine hélicase est associé à une activité migratoire efficace. Nous pensons que FANCM peut avoir un rôle direct sur les intermédiaires de réplication. Ceci est en accord avec l'idée que les protéines de l'anémie de Fanconi coordonnent la réparation de l'ADN au niveau des fourches de réplication arrêtées. Nos résultats donnent une première indication quant au rôle de FANCM dans la cellule et peuvent contribuer à élucider la fonction de cette voie de signalisation peu comprise jusqu'à présent. SUMMARY: The genome of every cell is subject to a constant offence by endogenous and exogenous agents. Not surprisingly; cells have evolved a multitude of mechanisms which aim at preserving genomic integrity. A key step during the life cycle of a cell, DNA replication itself, constitutes a special danger to the integrity of the genome. The proteins defective in the rare hereditary disease Fanconi anemia (FA) are suspected to play a crucial role in the cellular response to DNA replication stress. The disease is associated with chromosomal instability and pronounced cancer susceptibility. Cells from Fanconi anemia patients are sensitive to a variety of agents which interfere with DNA replication, DNA interstrand cross-linking agents being particularly threatening to their survival. Fanconi anemia is a genetically heterogeneous disease with 13 different proteins identified, which seem to work together in a common pathway. Since one of the FA genes is identical to the breast cancer susceptibility gene BRCA2, it is also referred to as the FA/BRCA pathway. Eight proteins form a nuclear complex, whose integriry is required for the monoubiquitination of two other FA proteins, FANCD2 and FANCI, in response to DNA replication stress. Despite intensive research, the function of the FA/BRCA pathway at a molecular level has remained largely elusive so far. At the beginning of my thesis, we therefore decided to purify the proteins of the FA core complex and to investigate their biochemical properties. We started with the five proteins which were known at that time, FANCA, FANCC, FANCE, FANCF, and FACG. Later on, we extended our studies to the newly discovered proteins FANCL, FANCM, and FAAP24, and eventually focused our work on the characterisation of FANCM. In contrast to the other core complex proteins, FANCM contains two conserved domains, which point to a role in DNA metabolism: an N-terminal DEAH box helicase domain and a C-terminal ERCC4 nuclease domain. In this study, we have successfully purified full-length FANCM from a recombinant source. We show that purified FANCM binds to branched DNA molecules, such as Holliday junctions and replication forks, with high specificity and affinity. In addition, we demonstrate that FANCM can translocate the junction point of branched DNA molecules due to its helicase domain in an ATPase-dependent manner. FANCM can even dissociate large recombination intermediates, via branch migration of Holliday junctions through a 2.6 kb region of homology. Taken together, our data suggest that FANCM can specifically bind to replication forks and Holliday junctions in vitro, and that its DEAH box helicase domain is associated with a potent branch migration activity. We propose that FANCM might have a direct role in the processing of DNA replication intermediates. This is consistent with the current view that FA proteins coordinate DNA repair at stalled replication forks. Our findings provide a first hint as to the context in which FANCM might play a role in the cell. We are optimistic that they might be key to further elucidate the function of a pathway which is far from being understood.

Pattern-based sensing of nucleotides in aqueous solution with a multicomponent indicator displacement assay

A multicomponent indicator displacement assay ( MIDA) based on an organometallic receptor and three dyes can be used for the identification and quantification of nucleotides in aqueous solution at neutral pH.

In situ fluorescence imaging of glutamate-evoked mitochondrial Na+ responses in astrocytes.

Astrocytes can experience large intracellular Na+ changes following the activation of the Na+-coupled glutamate transport. The present study investigated whether cytosolic Na+ changes are transmitted to mitochondria, which could therefore influence their function and contribute to the overall intracellular Na+ regulation. Mitochondrial Na+ (Na+(mit)) changes were monitored using the Na+-sensitive fluorescent probe CoroNa Red (CR) in intact primary cortical astrocytes, as opposed to the classical isolated mitochondria preparation. The mitochondrial localization and Na+ sensitivity of the dye were first verified and indicated that it can be safely used as a selective Na+(mit) indicator. We found by simultaneously monitoring cytosolic and mitochondrial Na+ using sodium-binding benzofuran isophthalate and CR, respectively, that glutamate-evoked cytosolic Na+ elevations are transmitted to mitochondria. The resting Na+(mit) concentration was estimated at 19.0 +/- 0.8 mM, reaching 30.1 +/- 1.2 mM during 200 microM glutamate application. Blockers of conductances potentially mediating Na+ entry (calcium uniporter, monovalent cation conductances, K+(ATP) channels) were not able to prevent the Na+(mit) response to glutamate. However, Ca2+ and its exchange with Na+ appear to play an important role in mediating mitochondrial Na+ entry as chelating intracellular Ca2+ with BAPTA or inhibiting Na+/Ca2+ exchanger with CGP-37157 diminished the Na+(mit) response. Moreover, intracellular Ca2+ increase achieved by photoactivation of caged Ca2+ also induced a Na+(mit) elevation. Inhibition of mitochondrial Na/H antiporter using ethylisopropyl-amiloride caused a steady increase in Na+(mit) without increasing cytosolic Na+, indicating that Na+ extrusion from mitochondria is mediated by these exchangers. Thus, mitochondria in intact astrocytes are equipped to efficiently sense cellular Na+ signals and to dynamically regulate their Na+ content.

Blue light activates a specific protein kinase in higher plants.

Blue light mediates the phosphorylation of a membrane protein in seedlings from several plant species. When crude microsomal membrane proteins from dark-grown pea (Pisum sativum L.), sunflower (Helianthus annuus L.), zucchini (Cucurbita pepo L.), Arabidopsis (Arabidopsis thaliana L.), or tomato (Lycopersicon esculentum L.) stem segments, or from maize (Zea mays L.), barley (Hordeum vulgare L.), oat (Avena sativa L.), wheat (Triticum aestivum L.), or sorghum (Sorghum bicolor L.) coleoptiles are illuminated and incubated in vitro with [gamma-(32)P]ATP, a protein of apparent molecular mass from 114 to 130 kD is rapidly phosphorylated. Hence, this system is probably ubiquitous in higher plants. Solubilized maize membranes exposed to blue light and added to unirradiated solubilized maize membranes show a higher level of phosphorylation of the light-affected protein than irradiated membrane proteins alone, suggesting that an unirradiated substrate is phosphorylated by a light-activated kinase. This finding is further demonstrated with membrane proteins from two different species, where the phosphorylated proteins are of different sizes and, hence, unambiguously distinguishable on gel electrophoresis. When solubilized membrane proteins from one species are irradiated and added to unirradiated membrane proteins from another species, the unirradiated protein becomes phosphorylated. These experiments indicate that the irradiated fraction can store the light signal for subsequent phosphorylation in the dark. They also support the hypothesis that light activates a specific kinase and that the systems share a close functional homology among different higher plants.

Activation of human meiosis-specific recombinase Dmc1 by Ca2+.

Rad51 and its meiotic homolog Dmc1 are key proteins of homologous recombination in eukaryotes. These proteins form nucleoprotein complexes on single-stranded DNA that promote a search for homology and that perform DNA strand exchange, the two essential steps of genetic recombination. Previously, we demonstrated that Ca2+ greatly stimulates the DNA strand exchange activity of human (h) Rad51 protein (Bugreev, D. V., and Mazin, A. V. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 9988-9993). Here, we show that the DNA strand exchange activity of hDmc1 protein is also stimulated by Ca2+. However, the mechanism of stimulation of hDmc1 protein appears to be different from that of hRad51 protein. In the case of hRad51 protein, Ca2+ acts primarily by inhibiting its ATPase activity, thereby preventing self-conversion into an inactive ADP-bound complex. In contrast, we demonstrate that hDmc1 protein does not self-convert into a stable ADP-bound complex. The results indicate that activation of hDmc1 is mediated through conformational changes induced by free Ca2+ ion binding to a protein site that is distinct from the Mg2+.ATP-binding center. These conformational changes are manifested by formation of more stable filamentous hDmc1.single-stranded DNA complexes. Our results demonstrate a universal role of Ca2+ in stimulation of mammalian DNA strand exchange proteins and reveal diversity in the mechanisms of this stimulation.

Skeletal muscle mitochondrial energetics are associated with maximal aerobic capacity and walking speed in older adults.

BACKGROUND: Lower ambulatory performance with aging may be related to a reduced oxidative capacity within skeletal muscle. This study examined the associations between skeletal muscle mitochondrial capacity and efficiency with walking performance in a group of older adults. METHODS: Thirty-seven older adults (mean age 78 years; 21 men and 16 women) completed an aerobic capacity (VO peak) test and measurement of preferred walking speed over 400 m. Maximal coupled (State 3; St3) mitochondrial respiration was determined by high-resolution respirometry in saponin-permeabilized myofibers obtained from percutanous biopsies of vastus lateralis (n = 22). Maximal phosphorylation capacity (ATP) of vastus lateralis was determined in vivo by P magnetic resonance spectroscopy (n = 30). Quadriceps contractile volume was determined by magnetic resonance imaging. Mitochondrial efficiency (max ATP production/max O consumption) was characterized using ATP per St3 respiration (ATP/St3). RESULTS: In vitro St3 respiration was significantly correlated with in vivo ATP (r = .47, p = .004). Total oxidative capacity of the quadriceps (St3*quadriceps contractile volume) was a determinant of VO peak (r = .33, p = .006). ATP (r = .158, p = .03) and VO peak (r = .475, p < .0001) were correlated with preferred walking speed. Inclusion of both ATP/St3 and VO peak in a multiple linear regression model improved the prediction of preferred walking speed (r = .647, p < .0001), suggesting that mitochondrial efficiency is an important determinant for preferred walking speed. CONCLUSIONS: Lower mitochondrial capacity and efficiency were both associated with slower walking speed within a group of older participants with a wide range of function. In addition to aerobic capacity, lower mitochondrial capacity and efficiency likely play roles in slowing gait speed with age.

The peroxisome proliferator-activated receptor (PPAR) β/δ agonist GW501516 inhibits IL-6-induced signal transducer and activator of transcription 3 (STAT3) activation and insulin resistance in human liver cells.

AIM/HYPOTHESIS: IL-6 induces insulin resistance by activating signal transducer and activator of transcription 3 (STAT3) and upregulating the transcription of its target gene SOCS3. Here we examined whether the peroxisome proliferator-activated receptor (PPAR)β/δ agonist GW501516 prevented activation of the IL-6-STAT3-suppressor of cytokine signalling 3 (SOCS3) pathway and insulin resistance in human hepatic HepG2 cells. METHODS: Studies were conducted with human HepG2 cells and livers from mice null for Pparβ/δ (also known as Ppard) and wild-type mice. RESULTS: GW501516 prevented IL-6-dependent reduction in insulin-stimulated v-akt murine thymoma viral oncogene homologue 1 (AKT) phosphorylation and in IRS-1 and IRS-2 protein levels. In addition, treatment with this drug abolished IL-6-induced STAT3 phosphorylation of Tyr⁷⁰⁵ and Ser⁷²⁷ and prevented the increase in SOCS3 caused by this cytokine. Moreover, GW501516 prevented IL-6-dependent induction of extracellular-related kinase 1/2 (ERK1/2), a serine-threonine protein kinase involved in serine STAT3 phosphorylation; the livers of Pparβ/δ-null mice showed increased Tyr⁷⁰⁵- and Ser⁷²⁷-STAT3 as well as phospho-ERK1/2 levels. Furthermore, drug treatment prevented the IL-6-dependent reduction in phosphorylated AMP-activated protein kinase (AMPK), a kinase reported to inhibit STAT3 phosphorylation on Tyr⁷⁰⁵. In agreement with the recovery in phospho-AMPK levels observed following GW501516 treatment, this drug increased the AMP/ATP ratio and decreased the ATP/ADP ratio. CONCLUSIONS/INTERPRETATION: Overall, our findings show that the PPARβ/δ activator GW501516 prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 phosphorylation and preventing the reduction in phospho-AMPK levels. These effects of GW501516 may contribute to the prevention of cytokine-induced insulin resistance in hepatic cells.

Living with an imperfect cell wall: compensation of femAB inactivation in Staphylococcus aureus.

BACKGROUND: Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore beta-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. RESULTS: In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. CONCLUSION: Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors.

Mechanistic insights into cytosolic molecular chaperones in protein unfolding and disaggregation

Under optimal non-physiological conditions of low concentrations and low temperatures, proteins may spontaneously fold to the native state, as all the information for folding lies in the amino acid sequence of the polypeptide. However, under conditions of stress or high protein crowding as inside cells, a polypeptide may misfold and enter an aggregation pathway resulting in the formation of misfolded conformers and fibrils, which can be toxic and lead to neurodegenerative illnesses, such as Alzheimer's, Parkinson's or Huntington's diseases and aging in general. To avert and revert protein misfolding and aggregation, cells have evolved a set of proteins called molecular chaperones. Here, I focussed on the human cytosolic chaperones Hsp70 (DnaK) and HspllO, and co-chaperone Hsp40 (DnaJ), and the chaperonin CCT (GroEL). The cytosolic molecular chaperones Hsp70s/Hspll0s and the chaperonins are highly upregulated in bacterial and human cells under different stresses and are involved both in the prevention and the reversion of protein misfolding and aggregation. Hsp70 works in collaboration with Hsp40 to reactivate misfolded or aggregated proteins in a strict ATP dependent manner. Chaperonins (CCT and GroEL) also unfold and reactivate stably misfolded proteins but we found that it needed to use the energy of ATP hydrolysis in order to evict over- sticky misfolded intermediates that inhibited the unfoldase catalytic sites. Ill In this study, we initially characterized a particular type of inactive misfolded monomeric luciferase and rhodanese species that were obtained by repeated cycles of freeze-thawing (FT). These stable misfolded monomeric conformers (FT-luciferase and FT-rhodanese) had exposed hydrophobic residues and were enriched with wrong ß-sheet structures (Chapter 2). Using FT-luciferase as substrate, we found that the Hsp70 orthologs, called HspllO (Sse in yeast), acted similarly to Hsp70 as were bona fide ATP- fuelled polypeptide unfoldases and was much more than a mere nucleotide exchange factor, as generally thought. Moreover, we found that HspllO collaborated with Hsp70 in the disaggregation of stable protein aggregates in which Hsp70 and HspllO acted as equal partners that synergistically combined their individual ATP-consuming polypeptide unfoldase activities to reactivate the misfolded/aggregated proteins (Chapter 3). Using FT-rhodanese as substrate, we found that chaperonins (GroEL and CCT) could catalytically reactivate misfolded rhodanese monomers in the absence of ATP. Also, our results suggested that encaging of an unfolding polypeptide inside the GroEL cavity under a GroES cap was not an obligatory step as generally thought (Chapter 4). Further, we investigated the role of Hsp40, a J-protein co-chaperone of Hsp70, in targeting misfolded polypeptides substrates onto Hsp70 for unfolding. We found that even a large excess of monomeric unfolded a-synuclein did not inhibit DnaJ, whereas, in contrast, stable misfolded a-synuclein oligomers strongly inhibited the DnaK-mediated chaperone reaction by way of sequestering the DnaJ co-chaperone. This work revealed that DnaJ could specifically distinguish, and bind potentially toxic stably aggregated species, such as soluble a-synuclein oligomers involved in Parkinson's disease, and with the help of DnaK and ATP convert them into from harmless natively unfolded a-synuclein monomers (chapter 5). Finally, our meta-analysis of microarray data of plant and animal tissues treated with various chemicals and abiotic stresses, revealed possible co-expressions between core chaperone machineries and their co-chaperone regulators. It clearly showed that protein misfolding in the cytosol elicits a different response, consisting of upregulating the synthesis mainly of cytosolic chaperones, from protein misfolding in the endoplasmic reticulum (ER) that elicited a typical unfolded protein response (UPR), consisting of upregulating the synthesis mainly of ER chaperones. We proposed that drugs that best mimicked heat or UPR stress at increasing the chaperone load in the cytoplasm or ER respectively, may prove effective at combating protein misfolding diseases and aging (Chapter 6).  - Dans les conditions optimales de basse concentration et de basse température, les protéines vont spontanément adopter un repliement natif car toutes les informations nécessaires se trouvent dans la séquence des acides aminés du polypeptide. En revanche, dans des conditions de stress ou de forte concentration des protéines comme à l'intérieur d'une cellule, un polypeptide peu mal se replier et entrer dans un processus d'agrégation conduisant à la formation de conformères et de fibrilles qui peuvent être toxiques et causer des maladies neurodégénératives comme la maladie d'Alzheimer, la maladie de Parkinson ou la chorée de Huntington. Afin d'empêcher ou de rectifier le mauvais repliement des protéines, les cellules ont développé des protéines appelées chaperonnes. Dans ce travail, je me suis intéressé aux chaperonnes cytosoliques Hsp70 (DnaK) et HspllO, la co-chaperones Hsp40 (DnaJ), le complexe CCT/TRiC et GroEL. Chez les bactéries et les humains, les chaperonnes cytosoliques Hsp70s/Hspl 10s et les « chaperonines» sont fortement activées par différentes conditions de stress et sont toutes impliquées dans la prévention et la correction du mauvais repliement des protéines et de leur agrégation. Hsp70 collabore avec Hsp40 pour réactiver les protéines agrégées ou mal repliées et leur action nécessite de 1ATP. Les chaperonines (GroEL) déplient et réactivent aussi les protéines mal repliées de façon stable mais nous avons trouvé qu'elles utilisent l'ATP pour libérer les intermédiaires collant et mal repliés du site catalytique de dépliage. Nous avons initialement caractérisé un type particulier de formes stables de luciférase et de rhodanese monomériques mal repliées obtenues après plusieurs cycles de congélation / décongélation répétés (FT). Ces monomères exposaient des résidus hydrophobiques et étaient plus riches en feuillets ß anormaux. Ils pouvaient cependant être réactivés par les chaperonnes Hsp70+Hsp40 (DnaK+DnaJ) et de l'ATP, ou par Hsp60 (GroEL) sans ATP (Chapitre 2). En utilisant la FT-Luciferase comme substrat nous avons trouvé que HspllO (un orthologue de Hsp70) était une authentique dépliase, dépendante strictement de l'ATP. De plus, nous avons trouvé que HspllO collaborait avec Hsp70 dans la désagrégation d'agrégats stables de protéines en combinant leurs activités dépliase consommatrice d'ATP (Chapitre 3). En utilisant la FT-rhodanese, nous avons trouvé que les chaperonines (GroEL et CCT) pouvaient réactiver catalytiquement des monomères mal repliés en absence d'ATP. Nos résultats suggérèrent également que la capture d'un polypeptide en cours de dépliement dans la cavité de GroEL et sous un couvercle du complexe GroES ne serait pas une étape obligatoire du mécanisme, comme il est communément accepté dans la littérature (Chapitre 4). De plus, nous avons étudié le rôle de Hsp40, une co-chaperones de Hsp70, dans l'adressage de substrats polypeptidiques mal repliés vers Hsp70. Ce travail a révélé que DnaJ pouvait différencier et lier des polypeptide mal repliés (toxiques), comme des oligomères d'a-synucléine dans la maladie de Parkinson, et clairement les différencier des monomères inoffensifs d'a-synucléine (Chapitre 5). Finalement une méta-analyse de données de microarrays de tissus végétaux et animaux traités avec différents stress chimiques et abiotiques a révélé une possible co-expression de la machinerie des chaperonnes et des régulateurs de co- chaperonne. Cette meta-analyse montre aussi clairement que le mauvais repliement des protéines dans le cytosol entraîne la synthèse de chaperonnes principalement cytosoliques alors que le mauvais repliement de protéines dans le réticulum endoplasmique (ER) entraine une réponse typique de dépliement (UPR) qui consiste principalement en la synthèse de chaperonnes localisées dans l'ER. Nous émettons l'hypothèse que les drogues qui reproduisent le mieux les stress de chaleur ou les stress UPR pourraient se montrer efficaces dans la lutte contre le mauvais repliement des protéines et le vieillissement (Chapitre 6).

The human Rad52 protein exists as a heptameric ring.

The RAD52 epistasis group was identified in yeast as a group of genes required to repair DNA damaged by ionizing radiation [1]. Genetic evidence indicates that Rad52 functions in Rad51-dependent and Rad51-independent recombination pathways [2] [3] [4]. Consistent with this, purified yeast and human Rad52 proteins have been shown to promote single-strand DNA annealing [5] [6] [7] and to stimulate Rad51-mediated homologous pairing [8] [9] [10] [11]. Electron microscopic examinations of the yeast [12] and human [13] Rad52 proteins have revealed their assembly into ring-like structures in vitro. Using both conventional transmission electron microscopy and scanning transmission electron microscopy (STEM), we found that the human Rad52 protein forms heptameric rings. A three-dimensional (3D) reconstruction revealed that the heptamer has a large central channel. Like the hexameric helicases such as Escherichia coli DnaB [14] [15], bacteriophage T7 gp4b [16] [17], simian virus 40 (SV40) large T antigen [18] and papilloma virus E1 [19], the Rad52 rings show a distinctly chiral arrangement of subunits. Thus, the structures formed by the hexameric helicases may be a more general property of other proteins involved in DNA metabolism, including those, such as Rad52, that do not bind and hydrolyze ATP.

Functional analysis of cis- and trans-acting elements of the Candida albicans CDR2 promoter with a novel promoter reporter system.

Azole resistance in Candida albicans can be mediated by the upregulation of the ATP binding cassette transporter genes CDR1 and CDR2. Both genes are regulated by a cis-acting element called the drug-responsive element (DRE), with the consensus sequence 5'-CGGAWATCGGATATTTTTTT-3', and the transcription factor Tac1p. In order to analyze in detail the DRE sequence necessary for the regulation of CDR1 and CDR2 and properties of TAC1 alleles, a one-hybrid system was designed. This system is based on a P((CDR2))-HIS3 reporter system in which complementation of histidine auxotrophy can be monitored by activation of the reporter system by CDR2-inducing drugs such as estradiol. Our results show that most of the modifications within the DRE, but especially at the level of CGG triplets, strongly reduce CDR2 expression. The CDR2 DRE was replaced by putative DREs deduced from promoters of coregulated genes (CDR1, RTA3, and IFU5). Surprisingly, even if Tac1p was able to bind these putative DREs, as shown by chromatin immunoprecipitation, those from RTA3 and IFU5 did not functionally replace the CDR2 DRE. The one-hybrid system was also used for the identification of gain-of-function (GOF) mutations either in TAC1 alleles from clinical C. albicans isolates or inserted in TAC1 wild-type alleles by random mutagenesis. In all, 17 different GOF mutations were identified at 13 distinct positions. Five of them (G980E, N972D, A736V, T225A, and N977D) have already been described in clinical isolates, and four others (G980W, A736T, N972S, and N972I) occurred at already-described positions, thus suggesting that GOF mutations can occur in a limited number of positions in Tac1p. In conclusion, the one-hybrid system developed here is rapid and powerful and can be used for characterization of cis- and trans-acting elements in C. albicans.

Chaperones and proteases: cellular fold-controlling factors of proteins in neurodegenerative diseases and aging.

The formation of toxic protein aggregates is a common denominator to many neurodegenerative diseases and aging. Accumulation of toxic, possibly infectious protein aggregates induces a cascade of events, such as excessive inflammation, the production of reactive oxygen species, apoptosis and neuronal loss. A network of highly conserved molecular chaperones and of chaperone-related proteases controls the fold-quality of proteins in the cell. Most molecular chaperones can passively prevent protein aggregation by binding misfolding intermediates. Some molecular chaperones and chaperone-related proteases, such as the proteasome, can also hydrolyse ATP to forcefully convert stable harmful protein aggregates into harmless natively refoldable, or protease-degradable, polypeptides. Molecular chaperones and chaperone-related proteases thus control the delicate balance between natively folded functional proteins and aggregation-prone misfolded proteins, which may form during the lifetime and lead to cell death. Abundant data now point at the molecular chaperones and the proteases as major clearance mechanisms to remove toxic protein aggregates from cells, delaying the onset and the outcome of protein-misfolding diseases. Therapeutic approaches include treatments and drugs that can specifically induce and sustain a strong chaperone and protease activity in cells and tissues prone to toxic protein aggregations.