Long sequence duplications, repeats, and palindromes in HIV-1 gp120: length variation in V4 as the product of misalignment mechanism.

We have shown that indels in gp120 V4 are associated to the presence of duplicated and palindromic sequences, suggesting that they may be produced by strand-slippage misalignment mechanism. Indels in V4 involved region-specific duplications 9 to 15 bp long, and repeats of various lengths, associated to trinucleotides AAT. No duplications were found in V3 and C3. The frequency of palindromic sequences in individual genes was found to be significantly higher in gp120 (p < or = 3.00E-7), and significantly lower in Tat (p < or = 9.00E-7) than the average frequency calculated over the full genome. The finding of elements of misalignment in association with indels in V4 suggests that these mutations may occur in proviral DNA after integration of HIV into the host genome. It also implies that occurrence of large indels in gp120 is not random but is directed by the presence and distribution of elements of misalignment in the HIV genome.

Length variation in HIV-1 gp120 as the product of DNA misalignment mechanism

Background: To determine whether misalignment structures such as duplications, repeats, and palindromes are associated to insertions/deletions (indels) in gp120, indicating that indels are indeed frameshift mutations generated by DNA misalignment mechanism. Methods: Cloning and sequencing of a fragment of HIV-1 gp120 spanning C2-C4 derived from plasma RNA in 12 patients with early chronic disease and naïve to antiretroviral therapy. Results: Indels in V4 involved always insertion and deletion of duplicated nucleotide segments, and AAT repeats, and were associated to the presence of palindromic sequences. No duplications were detected in V3 and C3. Palindromic sequences occurred with similar frequencies in V3, C3 and V4; the frequency of palindromes in individual genes was found to be significantly higher in structural (gp120, p ≤ 3.00E-7) and significantly lower in regulatory (Tat, p ≤ 9.00E-7) genes, as compared to the average frequency calculated over the full genome. Discussion: Indels in V4 are associated to misalignment structures (i.e. duplications repeat and palindromes) indicating DNA misalignment as the mechanism underlying length variation in V4. The finding that indels in V4 are caused by DNA misalignment has some very important implications: 1) indels in V4 are likely to occur in proviral DNA (and not in RNA), after integration of HIV into the host genome; 2) they are likely to occur as progressive modifications of the early founder virus during chronic infection, as more and more cells get infected; 3) frameshift mutations involving any number of base pairs are likely to occur evenly across gp120; however, only those mutants carrying a functional gp120 (indels as multiples of three base pairs) will be able to perpetuate the virus cycle and to keep spreading through the population.

PAX5 activates the transcription of the human telomerase reverse transcriptase gene in B cells.

Telomerase is an RNA-dependent DNA polymerase that synthesizes telomeric DNA. Its activity is not detectable in most somatic cells but it is reactivated during tumorigenesis. In most cancers, the combination of hTERT hypermethylation and hypomethylation of a short promoter region is permissive for low-level hTERT transcription. Activated and malignant lymphocytes express high telomerase activity, through a mechanism that seems methylation-independent. The aim of this study was to determine which mechanism is involved in the enhanced expression of hTERT in lymphoid cells. Our data confirm that in B cells, some T cell lymphomas and non-neoplastic lymph nodes, the hTERT promoter is unmethylated. Binding sites for the B cell-specific transcription factor PAX5 were identified downstream of the ATG translational start site through EMSA and ChIP experiments. ChIP assays indicated that the transcriptional activation of hTERT by PAX5 does not involve repression of CTCF binding. In a B cell lymphoma cell line, siRNA-induced knockdown of PAX5 expression repressed hTERT transcription. Moreover, ectopic expression of PAX5 in a telomerase-negative normal fibroblast cell line was found to be sufficient to activate hTERT expression. These data show that activation of hTERT in telomerase-positive B cells is due to a methylation-independent mechanism in which PAX5 plays an important role.

Tumor sensitizing function and mechanism of action of a RasGAP derived peptide

Cancer is the second cause of death after cardio-vascular diseases in economically developed countries. Two of the most commonly used anti-cancer therapies are chemo and radiotherapy. Despite the remarkable advances made in term of delivery and specificity of these two anti-tumor regimens, their toxicity towards healthy tissue remains a limitation. A promising approach to overcome this obstacle would be the utilization of therapeutic peptides that specifically augment the sensitivity of tumoral cells to treatments. Lower therapeutical doses would then be required to kill malignant cells, limiting toxic effects on healthy tissues. It was previously shown in our laboratory that the caspase-3 generated fragment N2 of RasGAP is able to potentiate the genotoxin-induced apoptosis selectively in cancer cells. In this work we show that fragment N2 strictly requires a cytoplasmic localization to deliver its pro-apoptotic effect in genotoxin-treated cancer cells. The tumor sensitizing capacity of fragment N2 was found to reside within the 10 amino acid sequence 317-326. Our laboratory earlier demonstrated that a peptide corresponding to amino acids 317 to 326 of RasGAP fused to the TAT cell permeable moiety, called TAT-RasGAP317.326, is able to sensitize cancer cells, but not normal cells, to genotoxin-induced apoptosis. In the present study we describe the capacity of TAT-RasGAP 317.326 to sensitize tumors to both chemo and radiotherapy in an in vivo mouse model. The molecular mechanism underlying the TAT-RasGAP 317.326-mediated sensitization starts now to be elucidated. We demonstrate that G3BP1, an endoribonuclease binding to amino acids 317-326 of RasGAP, is not involved in the sensitization mechanism. We also provide evidence showing that TAT-RasGAP3 17-326 potentiates the genotoxin-mediated activation of Bax in a tBid-dependent manner. Altogether our results show that TAT-RasGAP 317.326 could be potentially used in cancer therapy as sensitizer, in order to improve the efficacy of chemo and radiotherapy and prolong the life expectancy of cancer patients. Moreover, the understanding of the TAT-RasGAP317.326 mode of action might help to unravel the mechanisms by which cancer cells resist to chemo and radiotherapy and therefore to design more targeted and efficient anti-tumoral strategies.

Implication of DNA methylation, CTCF and BORIS factors in the transcriptional regulation of the human telomerase catalytic subunit hTERT

RESUME La télomérase confère une durée de vie illimitée et est réactivée dans la plupart des cellules tumorales. Sa sous-unité catalytique hTERT est définie comme le facteur limitant pour son activation. De l'identification de facteurs liant la région régulatrice d'hTERT, au rôle de la méthylation de l'ADN et de la modification des histones, de nombreux modèles de régulation ont été suggérés. Cependant, aucun de ces modèles n'a pu expliquer l'inactivation de la télomérase dans la plupart des cellules somatiques et sa réactivation dans la majorité des cellules tumorales. De plus, les observations contradictoires entre le faible niveau d'expression d'ARN messager d'hTERT dans les cellules télomérase-positives et la très forte activité transcriptionnelle du promoteur d'hTERT en transfection restent incomprises. Dans cette étude, nous avons montré que la région proximale du gène hTERT (exon 1 et 2) était impliquée dans la répression de l'activité de son promoteur. Nous avons identifié le facteur CTCF comme étant un inhibiteur du promoteur d'hTERT, en se liant au niveau de son premier exon. La méthylation de l'exon 1 du gène hTERT, couramment observée dans les tumeurs mais pas dans les cellules normales, empêcherait la liaison de CTCF. L'étude du profil de méthylation du promoteur d'hTERT indique qu'une partie du promoteur reste déméthylée et qu'elle semble suffisante pour permettre une faible activité transcriptionnelle du gène hTERT. Ainsi, la méthylation particulière des régions régulatrices d'hTERT inhibe la liaison de CTCF tout en permettant une faible transcription du gène. Cependant, dans certaines cellules tumorales, le promoteur et la région proximale du gène hTERT ne sont pas méthylés. Dans les lignées cellulaires tumorales de tesitcules et d'ovaires, l'inhibition de CTCF est contrée par son paralogue BORIS, qui se lie aussi au niveau de l'exon 1 d'hTERT, mais permet ainsi l'activation du promoteur. L'étude de l'expression du gène BORIS montre qu'il est exclusivement exprimé dans les tissus normaux de testicules et d'ovaires jeunes, ainsi qu'à différents niveaux dans la plupart des tumeurs. Sa transcription est sous le contrôle de deux promoteurs. Le promoteur proximal est régulé par méthylation et un transcrit alternatif majoritaire, délété de l'exon 6, est trouvé lorsque ce promoteur est actif. Tous ces résultats conduisent à un modèle de régulation du gène hTERT qui tient compte du profil épigénétique du gène et qui permet d'expliquer le faible taux de transcription observé in vivo. De plus, l'expression de BORIS dans les cancers et son implication dans l'activation du gène hTERT pourrait permettre de comprendre les phénomènes de dérégulation épigénétique et d'immortalisation qui ont lieu durant la tumorigenèse. SUMMARY Telomerase confers an unlimited lifespan, and is reactivated in most tumor cells. The catalytic subunit of telomerase, hTERT, is defined as the limiting factor for telomerase activity. Between activators and repressors that bind to the hTERT 5' regulatory region, and the role of CpG methylation and histone acetylation, an abundance of regulatory models have been suggested. None of these models can explain the silence of telomerase in most somatic cells and its reactivation in tumor cells. Moreover, the contradictory observations of the low level of hTERT mRNA in telomerase-positive cells and the high transcriptional activity of the hTERT promoter in transfection experiments remain unresolved. In this study, we demonstrated that the proximal exonic region of the hTERT gene (exon 1 and 2) is involved in the inhibition of its promoter. We identified the protein CTCF as the inhibitor of the hTERT promoter, through its binding to the first exon. The methylation of the first exon region, which is often observed in cancer cells but not in noimal cells, represses CTCF binding. Study of hTERT promoter methylation shows a partial demethylation sufficient to activate the transcription of the hTERT gene. Therefore, we demonstrated that the particular methylation profile of the hTERT regulatory sequences inhibits the binding of CTCF, while it allows a low transcription of the gene. Nevertheless, in some tumor cells, the promoter and the proximal exonic region of hTERT are unmethylated. In testicular and ovarian cancer cell lines, CTCF inhibition is counteracted by its BORIS paralogue that also binds the hTERT first exon but allows the promoter activation. The study of BORIS gene regulation showed that this factor is exclusively expressed in normal tissue of testis and ovary of young woman, as well as in almost all tumors with different levels. Two promoters were found to induce its transcription. The proximal promoter was regulated by methylation. Moreover, a major alternative transcript, deleted of the exon 6, is detected when this promoter is active. All these results lead to a model for hTERT regulation that takes into account the epigenetic profile of the gene and provides an explanation for the low transcriptional level observed in vivo. BORIS expression in cancers and its implication in hTERT activation might also permit the understanding of epigenetic deregulation and immortalization phenomena that occur during tumorigenesis.

Correlations of glycogen synthase and phosphorylase activities with glycogen concentration in human muscle biopsies. Evidence for a double-feedback mechanism regulating glycogen synthesis and breakdown.

The purpose of this study was to verify in man the relationships of muscle glycogen synthase and phosphorylase activities with glycogen concentration that were reported in animal studies. The upper level of glycogen concentration in muscle is known to be tightly controlled, and glycogen concentration was reported to have an inhibitory effect on synthase activity and a stimulatory effect on phosphorylase activity. Glycogen synthase and phosphorylase activity and glycogen concentration were measured in muscle biopsies in a group of nine normal subjects after stimulating an increase of their muscle glycogen concentration through either an intravenous glucose-insulin infusion to stimulate glycogen synthesis, or an Intralipid (Vitrum, Stockholm, Sweden) infusion in the basal state to inhibit glycogen mobilization by favoring lipid oxidation at the expense of glucose oxidation. Phosphorylase activity increased from 71.3 +/- 21.0 to 152.8 +/- 20.0 nmol/min/mg protein (P < .005) after the glucose-insulin infusion. Phosphorylase activity was positively correlated with glycogen concentration (P = .005 and P = .0001) after the glucose-insulin and Intralipid infusions, respectively. Insulin-stimulated glycogen synthase activity was significantly negatively correlated with glycogen concentration at the end of the Intralipid infusion (P < .005). In conclusion, by demonstrating a negative correlation of glycogen concentration with glycogen synthase and a positive correlation with phosphorylase, this study might confirm in man the double-feedback mechanism by which changes in glycogen concentration regulate glycogen synthase and phosphorylase activities. It suggests that this mechanism might play an important role in the regulation of glucose storage.

Identification and mutational analyses of phosphorylation sites of the calcineurin-binding protein CbpA and the identification of domains required for calcineurin binding in Aspergillus fumigatus.

Calcineurin is a key protein phosphatase required for hyphal growth and virulence in Aspergillus fumigatus, making it an attractive antifungal target. However, currently available calcineurin inhibitors, FK506 and cyclosporine A, are immunosuppressive, limiting usage in the treatment of patients with invasive aspergillosis. Therefore, the identification of endogenous inhibitors of calcineurin belonging to the calcipressin family is an important parallel strategy. We previously identified the gene cbpA as the A. fumigatus calcipressin member and showed its involvement in hyphal growth and calcium homeostasis. However, the mechanism of its activation/inhibition through phosphorylation and its interaction with calcineurin remains unknown. Here we show that A. fumigatus CbpA is phosphorylated at three distinct domains, including the conserved SP repeat motif (phosphorylated domain-I; PD-I), a filamentous fungal-specific domain (PD-II), and the C-terminal CIC motif (Calcipressin Inhibitor of Calcineurin; PD-III). While mutation of three phosphorylated residues (Ser208, Ser217, Ser223) in the PD-II did not affect CbpA function in vivo, mutation of the two phosphorylated serines (Ser156, Ser160) in the SP repeat motif caused reduced hyphal growth and sensitivity to oxidative stress. Mutational analysis in the key domains in calcineurin A (CnaA) and proteomic interaction studies confirmed the requirement of PxIxIT motif-binding residues (352-NIR-354) and the calcineurin B (CnaB)-binding helix residue (V371) for the binding of CbpA to CnaA. Additionally, while the calmodulin-binding residues (442-RVF-444) did not affect CbpA binding to CnaA, three mutations (T359P, H361L, and L365S) clustered between the CnaA catalytic and the CnaB-binding helix were also required for CbpA binding. This is the first study to analyze the phosphorylation status of calcipressin in filamentous fungi and identify the domains required for binding to calcineurin.

The monocarboxylate transporter MCT4 : mechanism of regulation in astrocytes and its putative role in cerebral ischemia

Despite its small fraction of the total body weight (2%), the brain contributes for 20% and 25% respectively of the total oxygen and glucose consumption of the whole body. Indeed, glucose has been considered the energy substrate par excellence for the brain. However, evidence accumulated over the last half century revealed an important role for the monocarboxylate lactate in fulfilling the energy needs of neurons. This is particularly true during physiological neuronal activation and in pathological conditions. Lactate transport into and out of the cell is mediated by a family of proton-linked transporters called monocarboxylate transporters (MCTs). In the central nervous system, only three of them have been well characterized: MCT2 is the predominant neuronal isoform, while the other non¬neuronal cell types of the brain express the ubiquitous isoform MCT1. Quite recently, the MCT4 isoform has been described in astrocytes. Due to its high transport capacity compared to the other two isoforms, MCT4 is particularly adapted for glycolytic cells. Because of its recent discovery in the brain, nothing was known about its regulation in the central nervous system. Here we show that MCT4 is regulated by oxygen levels in primary cultures of astrocytes in a time- and concentration-dependent manner via the hypoxia inducible factor-la (HIF-la). Moreover, we showed that MCT4 expression is essential for astrocyte survival under low oxygen conditions. In parallel, we investigated the possible implication of the pyruvate kinase isoform Pkm2, a strong enhancer of glycolysis, in its regulation. Then we showed that MCT4 expression, as well as the expression of the other two MCT isoforms, is altered in a murine model of stroke. Surprisingly, neurons started to express MCT4, as well as MCT1, under such conditions. Altogether, these data suggest that MCT4, due to its high transport capacity for lactate, may be the isoform that enables cells to operate a major metabolic adaptation in response to pathological situations that alter metabolic homeostasis of the brain. -- Le cerveau représente 2% du poids corporel total, mais il contribue pour 20% de la consommation totale d'oxygène et 25% de celle de glucose au repos. Le glucose est considéré comme le substrat énergétique par excellence pour le cerveau. Néanmoins, depuis un demi- siècle maintenant, de plus en plus de travaux ont démontré que le lactate joue un rôle majeur dans le métabolisme cérébral et est capable du subvenir aux besoins énergétiques des neurones. Le lactate est tout particulièrement nécessaire pendant l'activation neuronale ainsi qu'en situation pathologique. Le transport du lactate à travers la barrière hématoencéphalique ainsi qu'à travers les membranes cellulaires est assuré par la famille des transporteurs aux monocarboxylates (MCTs). Dans le système nerveux central, uniquement trois d'entre eux ont été décrits: MCT2 est considéré comme le transporteur neuronal, alors que les autres types cellulaires qui constituent le cerveau expriment l'isoforme ubiquitaire MCT1. Récemment, l'isoforme MCT4 a été rapportée sur les astrocytes. Dû à sa grande capacité de transport pour le lactate, MCT4 est tout particulièrement adapté pour soutenir le métabolisme des cellules hautement glycolytiques, comme les astrocytes. En raison de sa toute récente découverte, les aspects comprenant sa régulation et son rôle dans le cerveau sont pour l'instant méconnus. Les résultats exposés dans ce travail démontrent dans un premier temps que l'expression de MCT4 est régulée par les niveaux d'oxygène dans les cultures d'astrocytes corticaux par le biais du facteur de transcription HIF-la. De plus, nous avons démontré que l'expression de MCT4 est essentielle à la survie des astrocytes quand le niveau d'oxygénation baisse. En parallèle, des résultats préliminaires suggèrent que l'isoforme 2 de la pyruvate kinase, un puissant régulateur de la glycolyse, pourrait jouer un rôle dans la régulation de MCT4. Dans la deuxième partie du travail nous avons démontré que l'expression de MCT4, ainsi que celle de MCT1 et MCT2, est altérée dans un modèle murin d'ischémie cérébrale. De façon surprenante, les neurones expriment MCT4 dans cette condition, alors que ce n'est pas le cas en condition physiologique. En tenant compte de ces résultats, nous suggérons que MCT4, dû à sa particulièrement grande capacité de transport pour le lactate, représente le MCT qui permet aux cellules du système nerveux central, notamment les astrocytes et les neurones, de s'adapter à de très fortes perturbations de l'homéostasie métabolique du cerveau qui surviennent en condition pathologique.

Diploid males support a two-step mechanism of endosymbiont-induced thelytoky in a parasitoid wasp.

BACKGROUND: Haplodiploidy, where females develop from diploid, fertilized eggs and males from haploid, unfertilized eggs, is abundant in some insect lineages. Some species in these lineages reproduce by thelytoky that is caused by infection with endosymbionts: infected females lay haploid eggs that undergo diploidization and develop into females, while males are very rare or absent. It is generally assumed that in thelytokous wasps, endosymbionts merely diploidize the unfertilized eggs, which would then trigger female development. RESULTS: We found that females in the parasitoid wasp Asobara japonica infected with thelytoky-inducing Wolbachia produce 0.7-1.2 % male offspring. Seven to 39 % of these males are diploid, indicating that diploidization and female development can be uncoupled in A. japonica. Wolbachia titer in adults was correlated with their ploidy and sex: diploids carried much higher Wolbachia titers than haploids, and diploid females carried more Wolbachia than diploid males. Data from introgression lines indicated that the development of diploid individuals into males instead of females is not caused by malfunction-mutations in the host genome but that diploid males are most likely produced when the endosymbiont fails to activate the female sex determination pathway. Our data therefore support a two-step mechanism by which endosymbionts induce thelytoky in A. japonica: diploidization of the unfertilized egg is followed by feminization, whereby each step correlates with a threshold of endosymbiont titer during wasp development. CONCLUSIONS: Our new model of endosymbiont-induced thelytoky overthrows the view that certain sex determination mechanisms constrain the evolution of endosymbiont-induced thelytoky in hymenopteran insects. Endosymbionts can cause parthenogenesis through feminization, even in groups in which endosymbiont-diploidized eggs would develop into males following the hosts' sex determination mechanism. In addition, our model broadens our understanding of the mechanisms by which endosymbionts induce thelytoky to enhance their transmission to the next generation. Importantly, it also provides a novel window to study the yet-poorly known haplodiploid sex determination mechanisms in haplodiploid insects.

Molecular mechanism of a green-shifted, pH-dependent red fluorescent protein mKate variant.

Fluorescent proteins that can switch between distinct colors have contributed significantly to modern biomedical imaging technologies and molecular cell biology. Here we report the identification and biochemical analysis of a green-shifted red fluorescent protein variant GmKate, produced by the introduction of two mutations into mKate. Although the mutations decrease the overall brightness of the protein, GmKate is subject to pH-dependent, reversible green-to-red color conversion. At physiological pH, GmKate absorbs blue light (445 nm) and emits green fluorescence (525 nm). At pH above 9.0, GmKate absorbs 598 nm light and emits 646 nm, far-red fluorescence, similar to its sequence homolog mNeptune. Based on optical spectra and crystal structures of GmKate in its green and red states, the reversible color transition is attributed to the different protonation states of the cis-chromophore, an interpretation that was confirmed by quantum chemical calculations. Crystal structures reveal potential hydrogen bond networks around the chromophore that may facilitate the protonation switch, and indicate a molecular basis for the unusual bathochromic shift observed at high pH. This study provides mechanistic insights into the color tuning of mKate variants, which may aid the development of green-to-red color-convertible fluorescent sensors, and suggests GmKate as a prototype of genetically encoded pH sensors for biological studies.