Anti-Leukemia Activity of MS-275 Histone Deacetylase Inhibitor Implicates 4-1BBL/4-1BB Immunomodulatory Functions

Histone deacetylase inhibitors (HDACi) have demonstrated promising therapeutic potential in clinical trials for hematological malignancies. HDACi, such as SAHA/Vorinostat, Trichostatin A, and MS-275 were found to induce apoptosis of leukemic blasts through activation of the death receptor pathway and transcriptional induction of the Tumor Necrosis Factor (TNF)-related pro-apoptotic family members, TRAIL and FasL. The impact of HDACi on TNF-related costimulatory molecules such as 4-1BB ligand (4-1BBL/TNFSF9) is however not known. Following exposure to SAHA/Vorinostat, Trichostatin A, and MS-275, transcript levels were determined by real time PCR in Jurkat, Raji and U937 cells. Treatment with HDACi up-regulated TNFSF9 gene expression in the three leukemia cell lines, yet to different extend and with distinct kinetics, which did not require de novo protein synthesis and was not associated with DNAse I hypersensitive chromatin remodeling. Transcriptional activity of TNFSF9 promoter-luciferase constructs was induced up to 12 fold by HDACi, and implication of Sp1/Sp3 transcription factors binding to functional GC-box elements was evidenced by reporter gene assays, site-directed mutagenesis, and electrophoretic mobility shift assays. Functionality of modulated target genes was assessed in allogeneic mixed leukocyte reaction experiments. MS-275- and to a lesser extent Trichostatin A- and SAHA-treated Raji cells significantly up regulated T lymphocytes proliferation which was reduced by about 50% by a 4-1BB blocking recombinant protein, while MS-275- but neither Trichostatin A- nor SAHA-treated cells up-regulated IFNγ secretion by T lymphocytes. Our results identify 4-1BBL/4-1BB as a downstream target of HDACi, especially of MS-275 anti-leukemia action in vitro. Thus, HDACi such as MS-275 displaying dual TNF-dependent proapoptotic and costimulatory activities might be favored for inclusion in HDACi-based anti-cancer therapeutic strategies

Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity

We have previously identified in the pol gene of human immunodeficiency virus type 1 (HIV-1) a new positive transcriptional regulatory element (nt 4481–4982) containing recognition sites for nuclear proteins (sites B, C, D and a GC-box) [C. Van Lint, J. Ghysdael, P. Paras, Jr, A. Burny and E. Verdin (1994) J. Virol. 68, 2632–2648]. In this study, we have further physically characterized each binding site and have shown that the transcription factors Oct-1, Oct-2, PU.1, Sp1 and Sp3 interact in vitro with the pol region. Chromatin immunoprecipitation assays using HIV-infected cell lines demonstrated in the context of chromatin that Sp1, Sp3, Oct-1 and PU.1 are recruited to the HS7 region in vivo. For each site, we have identified mutations abolishing factor binding to their cognate DNA sequences without altering the underlying amino acid sequence of the integrase. By transient transfection assays, we have demonstrated the involvement of the pol binding sites in the transcriptional enhancing activity of the intragenic region. Our functional results with multimerized wild-type and mutated pol binding sites separately (i.e. in the absence of the other sites) have demonstrated that the PU.1, Sp1, Sp3 and Oct-1 transcription factors regulate the transcriptional activity of a heterologous promoter through their respective HS7 binding sites. Finally, we have investigated the physiological role of the HS7 binding sites in HIV-1 replication and have shown that these sites are important for viral infectivity

Re-Imagining School Feeding : A High-Return Investment in Human Capital and Local Economies

Analysis shows that a quality education, combined with a guaranteed package of health and nutrition interventions at school, such as school feeding, can contribute to child and adolescent development and build human capital. School feeding programs can help get children into school and help them stay there, increasing enrollment and reducing absenteeism. Once children are in the classroom, these programs can contribute to their learning by avoiding hunger and enhancing cognitive abilities. The benefits are especially great for the poorest and most disadvantaged children. As highlighted in the World Bank’s 2018 World Development Report (World Bank 2018), countries need to prioritize learning, not just schooling. Children must be healthy, not hungry, if they are to match learning opportunities with the ability to learn. In the most vulnerable communities, nutrition-sensitive school meals can offer children a regular source of nutrients that are essential for their mental and physical development. And for the growing number of countries with a “double burden” of undernutrition and emerging obesity problems, well-designed school meals can help set children on the path toward more healthy diets. In Latin America, for example, where there is a growing burden of noncommunicable diseases (NCDs), school feeding programs are a key intervention in reducing undernutrition and promoting healthy diet choices. Mexico’s experience reducing sugary beverages in school cafeterias, for example, was found to be beneficial in advancing a healthy lifestyle. A large trial of school-based interventions in China also found that nutritional or physical activity interventions alone are not as effective as a joint program that combines nutritional and educational interventions. In poor communities, economic benefits from school feeding programs are also evident—reducing poverty by boosting income for households and communities as a whole. For families, the value of meals in school is equivalent to about 10 percent of a household’s income. For families with several children, that can mean substantial savings. As a result, school feeding programs are often part of social safety nets in poor countries, and they can be a stable way to reliably target pro-poor investments into communities, as well as a system that can be scaled up rapidly to respond to crises. There are also direct economic benefits for smallholder farmers in the community. Buying local food creates stable markets, boosting local agriculture, impacting rural transformation, and strengthening local food systems. In Brazil, for example, 30 percent of all purchases for school feeding come from smallholder agriculture (Drake and others 2016). These farmers are oftentimes parents with schoolchildren, helping them break intergenerational cycles of hunger and poverty. Notably, benefits to households and communities offer important synergies. The economic growth in poor communities helps provide stability and better-quality education and health systems that promote human capital. At the same time, children and adolescents grow up to enjoy better employment and social opportunities as their communities grow

Signals and proteins involved in sorting of glycolytic enzymes to glycosomes in trypanosomatidae

Chez le trypanosome africain Trypanosoma brucei, comme chez tous les autres membres de la famille des Trypanosomatidae, la majorité des enzymes de la voie glycolytique est compartimentée dans une organelle appelée glycosome. Les glycosomes sont apparentés aux peroxysomes que l’on trouve dans la plupart des cellules eucaryotes et qui ont été particulièrement bien étudiés chez les mammifères, les plantes et chez les champignons. Les signaux qui dirigent les enzymes glycolytiques vers les glycosomes et une des protéines impliquées dans le processus d’importation ont fait l’objet des recherches présentées dans cette thèse. La majorité des enzymes compartimentées dans les glycosomes sont importées via un signal C-terminal appelé Signal de Ciblage Peroxisomal de type 1 (PTS-1 si on utilise l’acronyme anglais). Il a déjà été montré, chez les levures et les humains, que les protéines qui portent ce signal sont reconnues par le récepteur PTS-1, également appelé PEX5. Nous avons identifié chez les T. brucei l’homologie d’un gène PEX5. La protéine encodée par ce gène possède toutes les caractéristiques d’un récepteur PTS-1. L’analyse de la séquence protéique déduite révèle sept répétitions d’un motif fortement dégénéré de 34 acides aminés qui constituent un domaine dit TPR (pour TetratricoPeptide Repeat) localisé dans la moitié C-terminale de la protéine. La moitié N-terminale de la protéine contient trois répétitions du pentapeptide W-X-X-X-F/Y qui pourraient être responsables de l’interaction avec la protéine PEX14, associée à la face externe de la membrane peroxysomiale. La protéine a été produite chez Escherichia coli et purifiée par chromatographie d’affinité pour un métal. Le récepteur est capable in vitro d’interagir fortement avec une protéine contenant un PTS-1. En utilisant des techniques biochimiques et cytochimiques, nous avons montré que, chez T. brucei, PEX5 est essentiellement présente dans le cytosol, avec une fraction minoritaire associée aux glycosomes. Cette distribution est en accord avec le modèle généralement admis pour l’importation des protéines possédant un PTS-A dans la matrice des peroxysomes. le triosephosphate isomérase (TIM) de T. brucei est une des rares enzymes glycosomiales qui ne contienne pas de PTS reconnaissable. A fin de délimiter le signal responsable du ciblage de TIM vers es glycosomes, nous avons réalisé plusieurs protéines de fusion. Chacune de ces protéines de fusion contient un fragment de la séquence de TIM fusionné à une protéine rapporteuse, la chloramphénicol acétyl transférase (CAT) de E. coli. Des trypanosomes au stade procyclique ont été transfectés transitoirement par les plasmides encodant les différentes protéines de fusion. Après une titration à la digitonine, la quantité de protéine rapporteuse présente dans le cytosole et les organelles a été mesurée par ELISA. Nous avons identifié un peptide interne de 22 acides aminés capable de diriger la protéine rapporteuse vers les glycosomes. En accord avec son rôle possible, ce peptide est localisé à la surface de la structure de TIM de T. brucei. Plusieurs résidus du peptide ont été modifiés par mutagénèse mais ceci ne nous a pas permis d’identifier les résidus qui jouent un rôle crucial dans le ciblage de la protéine de fusion vers le glycosomes. Nous avons montré que le PTS N-termina, ou PTS-2, présent dans la séquence de l’aldolase (ALD) de T. brucei est capable de diriger la protéine vers les peroxysomes d’un hôte hétérologue, la levure Hansenula polymorpha. Un PTS-2 est également présent dans la séquence de l’ALD du parasite apparenté Leishmania mexicana. Cette protéine a été produite chez E. coli et purifiée. Sa caractérisation biochimique a montré que les enzymes de T. brucei et de L. mexicana sont très similaires. Les structures cristallines des deux ALD ont été résolues dans le laboratoire du Prof W. Hol (Université de Washington). La résolution de ces structures révèle des perspectives étonnantes sur le fonctionnement du PTS-2. Dans la discussion, nous proposons que le récepteur PTS-1, le signal de ciblage de TIM et le PTS-2 de l’aldolase pourraient tous trois constituer des cibles valables pour le développement de nouveaux médicaments contre la maladie du sommeilIn the African trypanosome Trypanosoma brucei, like in all members of the Trypanosomatidae family, the majority of the enzymes of the glycolytic pathways are compartmentalized in an organelle called the glycosome. Glycosomes are related to peroxisomes, found in most other eukaryotic cells, and which have been well studied in mammals, plants, and fungi. The signals that target the glycolytic enzymes to the glycosomes and one of the proteins involved in the routing process were the subject of the research described in this thesis. The majority of the enzymes found in glycosomes are imported via a C-terminal signal called Peroxisome-Targeting Signal of type-1 (PTS-1). In yeast and human, it has previously been shown that proteins bearing this signal are recognized by a PTS-1 receptor, also called PEX5. We identified in T. brucei a PEX5 gene homologue. The protein encoded by this gene has all the characteristics of the PTS-1 receptor. Analysis if the deduced protein sequence showed seven repeats of a loosely conserved 34 amino-acid motif, constituting a so called tetracopeptide repeat (TPR) domain at the C-terminal half of the protein. The N-terminal half contains three repeats of the pentapeptide W-X-X-X-F/Y that may interact with the protein PEX14, associated with the peroxisomal membrane. The protein was produced in Escherichia coli and purified using immobilized metal affinity chromatography. The receptor was shown in vitro to strongly binf a PTS-1 containing protein. Using biochemical and cytochemical techniques PEX-5 was predominantly found in the cytosol of T. brucei with only a small amount associated with glycosomes. This distribution is consistent with the current model for the import of PTS-1 proteins into the peroxisomal matrix. The triosephospate isomerase (TIM) of T. brucei is one of the few glycosomal enzymes that does not contain a recognizable PTS. To delineate the signal responsible for TIM targeting to glycosomes we prepared different fusion proteins between parts of the TIM polypeptide and, as a reporter protein, E. coli chloramphenicol acetyl transferase (CAT). Procyclic trypanosomes were transiently transfected with plasmids encoding the different fusion proteins. Following digitonin titration, the amount of CAT present in the cytosol or in the organelles was quantified using an ELISA system. We could identify an internal 22 amino-acid peptide able to target the reporter protein to the glycosomes. The localization of the peptide at the surface of the T. brucei TIM structure is consistent with its putative role. The mutagenesis of specific residues in the peptide did not allow us to identify the amino acids essential for the glycosomal targeting of the fusion protein. We have shown that the N-terminal, or PTS-2, found in the T. brucei aldolase (ALD) sequence is able to direct the import of the protein into the peroxisomes of an heterologous host, the yeast Hansenula polymorpha. A PTS-2 was also found in the ALD sequence of the related parasite Leishmania Mexicana. This protein has been produced in E. coli and purified. Biochemical characterization showed that the T. brucei and L. Mexicana enzymes are very similar. The crystal structures of the two ALDs have been solved in the laboratory of Prof. W. Hol (University of Washington). The elucidation of the structure reveals surprising insight as to how the PTS-2 might function. In the discussion we argue that the PTS-1 receptor, the targeting signal of TIM, and the PTS-2 of ALD might each constitute a good target for the development of, urgently needed, new drugs against sleeping sicknessThèse de doctorat en sciences biomédicales -- UCL, 200

Chromatin-associated regulation of HIV-1 transcription: implications for the development of therapeutic strategies.

Human Immunodeficiency Virus type 1 (HIV-1) infection can now be treated effectively in many patients in the developed world, using combinations of antiretroviral therapeutics, called Highly Active Anti-Retroviral Therapy (HAART). However, despite prolonged treatment with HAART, the persistence of latently HIV-1-infected cellular reservoirs harboring transcriptionally silent but replication-competent proviruses represents the major hurdle to virus eradication. These latently infected cells are a permanent source for virus reactivation and lead to a rebound of the viral load after interruption of HAART. Therefore, a greater understanding of the molecular mechanisms regulating proviral latency and reactivation should lead to rational strategies aimed at purging these cellular reservoirs of HIV-1. This review summarizes our current knowledge and understanding of the elements involved in HIV-1 transcriptional reactivation: (1) the site of integration; (2) the transcription factor NF-kappaB, which is induced by proinflammatory cytokines (such as TNFalpha) and binds to two kappaB sites in the HIV-1 promoter region; (3) the specific remodeling of a single nucleosome (called nuc-1 and located immediately downstream of the HIV-1 transcription start site under latency conditions) upon activation of the HIV-1 promoter; (4) post-translational acetylation of histones and of non-histone proteins (following treatment with deacetylases inhibitors, which induce viral transcription and nuc-1 remodeling); and (5) the viral trans-activator Tat, which promotes transcription by mediating the recruitment to the HIV-1 promoter of histone-modifying enzymes and ATP-dependent chromatin remodeling complexes required for nucleosome disruption and transcriptional processivity. Finally, this review highlights experimental therapies aimed at administrating HIV-1 gene expression activators (such as HDAC inhibitors) combined with an effective HAART in order to reactivate and decrease/eliminate the pool of latently HIV-1-infected cellular reservoirsJournal ArticleResearch Support, Non-U.S. Gov'tReviewSCOPUS: re.jinfo:eu-repo/semantics/publishe

Cloning and analysis of the PTS-1 receptor in Trypanosoma brucei

Kinetoplastid organisms, such as the protozoan parasite Trypanosoma brucei, compartmentalise several important metabolic pathways in organelles called glycosomes. Glycosomes are related to peroxisomes of yeast and mammalian cells. A subset of glycosomal matrix proteins is routed to the organelles via the peroxisome-targeting signal type 1 (PTS-1). The PEX 5 gene homologue has been cloned from T. brucei coding for a protein of the translocation machinery, the PTS-1 receptor. The gene codes for a polypeptide of 654 amino acids with a calculated molecular mass of 70 kDa. Like its homologue in other organisms T. brucei PTS-1 receptor protein (TbPEX5) is a member of the tetratricopeptide repeat (TPR) protein family and contains several copies of the pentapeptide W-X-X-X-F:Y. Northern and Western blot analysis showed that the protein is expressed at different stages of the life cycle of the parasite. The protein has been overproduced in Escherichia coli and purified using immobilized metal affinity chromatography. The purified protein specifically interacts in vitro with glycosomal phosphoglycerate kinase-C (PGK-C) of T. brucei, a PTS-1 containing protein. The equilibrium dissociation constant (Kd) of PGK-C for purified TbPEX5 is 40 nM. Using biochemical and cytochemical techniques a predominantly cytosolic localization was found for TbPEX5. This is consistent with the idea of receptor cycling between the glycosomes and the cytosol.

Transcription factor binding sites in the pol gene intragenic region of HIV-1 are important for virus infectivity

info:eu-repo/semantics/publishe