Biochemical and functional characterization of the tumor suppressors BRCA1 and BAP1

L’ubiquitination est une modification post-traductionnelle qui joue un rôle majeur dans la régulation d’une multitude de processus cellulaires. Dans cette thèse, je discuterai de la caractérisation de deux protéines, BRCA1 et BAP1, soit deux suppresseurs de tumeurs fonctionnellement reliés. BRCA1, une ubiquitine ligase qui catalyse la liaison de l’ubiquitine à une protéine cible, est mutée dans les cancers du sein et de l'ovaire. Il est bien établi que cette protéine aide à maintenir la stabilité génomique suite à un bris double brin de l’ADN (BDB), et ce, à l’aide d’un mécanisme de réparation bien caractérisé appelé recombinaison homologue. Cependant, les mécanismes de régulation de BRCA1 suite à des stresses génotoxiques n’impliquant pas directement un BDB ne sont pas pleinement élucidés. Nous avons démontré que BRCA1 est régulée par dégradation protéasomale suite à une exposition des cellules à deux agents génotoxiques reconnus pour ne pas directement générer des BDBs, soit les rayons UV, qui provoquent la distorsion de l’hélice d’ADN, et le méthyle méthanesulfonate (MMS), qui entraîne l’alkylation de l’ADN. La dégradation de BRCA1 est réversible et indépendante des kinases associées à la voie des PI3 kinase, soit ATM, ATR et DNA-PK, protéines qui sont rapidement activées par les dommages à l’ADN. Nous proposons que la dégradation de BRCA1 prévienne son recrutement intempestif, ainsi que celui des facteurs qui lui sont associés, à des sites de dommages d’ADN qui ne sont pas des BDBs, et que cette régulation coordonne la réparation de l’ADN. L’enzyme de déubiquitination BAP1 a initialement été identifiée comme une protéine capable d’interagir avec BRCA1 et de réguler sa fonction. Elle est également connue pour sa capacité à se lier avec les protéines du groupe Polycomb, ASXL1 et ASXL2. Cependant, l’importance de ces interactions n’a toujours pas été établie. Nous avons démontré que BAP1 forme deux complexes protéiques mutuellement exclusifs avec ASXL1 et ASXL2. Ces interactions sont critiques pour la liaison de BAP1 à l’ubiquitine ainsi que pour la stimulation de son activité enzymatique envers l’histone H2A. Nous avons également identifié des mutations de BAP1 dérivées de cancers qui empêchent à la fois son interaction avec ASXL1 et AXSL2, et son activité de déubiquitinase, ce qui fournit un lien mécanistique direct entre la déubiquitination de H2A et la tumorigenèse. Élucider les mécanismes de régulation de BRCA1 et BAP1 menera à une meilleure compréhension de leurs rôles de suppresseurs de tumeurs, permettant ainsi d’établir de nouvelles stratégies de diagnostic et traitement du cancer.

Convergencia de vías de señalización en un modelo de apoptosis

La sepsis es un evento inflamatorio generalizado del organismo inducido por un daño causado generalmente por un agente infeccioso. El patógeno más frecuentemente asociado con esta entidad es el Staphylococcus aureus, responsable de la inducción de apoptosis en células endoteliales debida a la producción de ceramida. Se ha descrito el efecto protector de la proteína C activada (PCA) en sepsis y su relación con la disminución de la apoptosis de las células endoteliales. En este trabajo se analizó la activación de las quinasas AKT, ASK1, SAPK/JNK y p38 en un modelo de apoptosis endotelial usando las técnicas de Western Blotting y ELISA. Las células endoteliales (EA.hy926), se trataron con C2-ceramida (130μM) en presencia de inhibidores químicos de cada una de estas quinasas y PCA. La supervivencia de las células en presencia de inhibidores químicos y PCA fue evaluada por medio de ensayos de activación de las caspasas 3, 7 y 9, que verificaban la muerte celular por apoptosis. Los resultados evidencian que la ceramida reduce la activación de AKT y aumenta la activación de las quinasas ASK, SAPK/JNK y p38, en tanto que PCA ejerce el efecto contrario. Adicionalmente se encontró que la tiorredoxina incrementa la activación/fosforilación de AKT, mientras que la quinasa p38 induce la defosforilación de AKT.

PDGF regulates the actin cytoskeleton through hnRNP-K-mediated activation of the ubiquitin E-3-ligase MIR

PDGF is a potent chemotactic mitogen and a strong inductor of fibroblast motility. In Swiss 3T3 fibroblasts, exposure to PDGF but not EGF or IGF-1 causes a rapid loss of actin stress fibers (SFs) and focal adhesions (FAs), which is followed by the development of retractile dendritic protrusions and induction of motility. The PDGF-specific actin reorganization was blocked by inhibition of Src-kinase and the 26S proteasome. PDGF induced Src-dependent association between the multifunctional transcription/translation regulator hnRNP-K and the mRNA-encoding myosin regulatory light-chain (MRLC)-interacting protein (MIR), a E3-ubiquitin ligase that is MRLC specific. This in turn rapidly increased MIR expression, and led to ubiquitination and proteasome-mediated degradation of MRLC. Downregulation of MIR by RNA muting prevented the reorganization of actin structures and severely reduced the migratory and wound-healing potential of PDGF-treated cells. The results show that activation of MIR and the resulting removal of diphosphorylated MRLC are essential for PDGF to instigate and maintain control over the actin-myosin-based contractile system in Swiss 3T3 fibroblasts. The PDGF induced protein destabilization through the regulation of hnRNP-K controlled ubiquitin-ligase translation identifies a novel pathway by which external stimuli can regulate phenotypic development through rapid, organelle-specific changes in the activity and stability of cytoskeletal regulators.

Preliminary X-ray diffraction analysis of YqjH from Escherichia coli: a putative cytoplasmic ferri-siderophore reductase

YqjH is a cytoplasmic FAD-containing protein from Escherichia coli; based on homology to ViuB of Vibrio cholerae, it potentially acts as a ferri-siderophore reductase. This work describes its overexpression, purification, crystallization and structure solution at 3.0 A resolution. YqjH shares high sequence similarity with a number of known siderophore-interacting proteins and its structure was solved by molecular replacement using the siderophore-interacting protein from Shewanella putrefaciens as the search model. The YqjH structure resembles those of other members of the NAD(P)H:flavin oxidoreductase superfamily.

An interaction between two RNA binding proteins, Nab2 and Pub1, links mRNA Processing/Export and mRNA stability

mRNA stability is modulated by elements in the mRNA transcript and their cognate RNA binding proteins. Poly(U) binding protein 1 (Pub1) is a cytoplasmic Saccharomyces cerevisiae mRNA binding protein that stabilizes transcripts containing AU-rich elements (AREs) or stabilizer elements (STEs). In a yeast two-hybrid screen, we identified nuclear poly(A) binding protein 2 (Nab2) as being a Pub1-interacting protein. Nab2 is an essential nucleocytoplasmic shuttling mRNA binding protein that regulates poly(A) tail length and mRNA export. The interaction between Pub1 and Nab2 was confirmed by copurification and in vitro binding assays. The interaction is mediated by the Nab2 zinc finger domain. Analysis of the functional link between these proteins reveals that Nab2, like Pub1, can modulate the stability of specific mRNA transcripts. The half-life of the RPS16B transcript, an ARE-like sequence-containing Pub1 target, is decreased in both nab2-1 and nab2-67 mutants. In contrast, GCN4, an STE-containing Pub1 target, is not affected. Similar results were obtained for other ARE- and STE-containing Pub1 target transcripts. Further analysis reveals that the ARE-like sequence is necessary for Nab2-mediated transcript stabilization. These results suggest that Nab2 functions together with Pub1 to modulate mRNA stability and strengthen a model where nuclear events are coupled to the control of mRNA turnover in the cytoplasm.

Genome-Wide Analysis of Differentially Expressed Genes During the Early Stages of Tomato Infection by a Potyvirus

Plant responses against pathogens cause up-and downward shifts in gene expression. To identify differentially expressed genes in a plant-virus interaction, susceptible tomato plants were inoculated with the potyvirus Pepper yellow mosaic virus (PepYMV) and a subtractive library was constructed from inoculated leaves at 72 h after inoculation. Several genes were identified as upregulated, including genes involved in plant defense responses (e. g., pathogenesis-related protein 5), regulation of the cell cycle (e. g., cytokinin-repressed proteins), signal transduction (e. g., CAX-interacting protein 4, SNF1 kinase), transcriptional regulators (e. g., WRKY and SCARECROW transcription factors), stress response proteins (e. g., Hsp90, DNA-J, 20S proteasome alpha subunit B, translationally controlled tumor protein), ubiquitins (e. g., polyubiquitin, ubiquitin activating enzyme 2), among others. Downregulated genes were also identified, which likewise display identity with genes involved in several metabolic pathways. Differential expression of selected genes was validated by macroarray analysis and quantitative real-time polymerase chain reaction. The possible roles played by some of these genes in the viral infection cycle are discussed.

Genes expressed in dental enamel development are associated with molar-incisor hypomineralization

Genetic disturbances during dental development influence variation of number and shape of the dentition. In this study, we tested if genetic variation in enamel formation genes is associated with molar-incisor hypomineralization (MIH), also taking into consideration caries experience. DNA samples from 163 cases with MIH and 82 unaffected controls from Turkey, and 71 cases with MIH and 89 unaffected controls from Brazil were studied. Eleven markers in five genes [ameloblastin (AMBN), amelogenin (AMELX), enamelin (ENAM), tuftelin (TUFT1), and tuftelin-interacting protein 11 (TFIP11)] were genotyped by the TaqMan method. Chi-square was used to compare allele and genotype frequencies between cases with MIH and controls. In the Brazilian data, distinct caries experience within the MIH group was also tested for association with genetic variation in enamel formation genes. The ENAM rs3796704 marker was associated with MIH in both populations (Brazil: p = 0.03; OR = 0.28; 95% C.I. = 0.06-1.0; Turkey: p = 1.22e-012; OR = 17.36; 95% C.I. = 5.98-56.78). Associations between TFIP11 (p = 0.02), ENAM (p = 0.00001), and AMELX (p = 0.01) could be seen with caries independent of having MIH or genomic DNA copies of Streptococcus mutans detected by real time PCR in the Brazilian sample. Several genes involved in enamel formation appear to contribute to MIH. © 2013.

Análise funcional e estrutural da proteína Pub 1 de Saccharomyces cerevisiae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação genética da hipomineralização molar-incisivo

Pós-graduação em Ciências Odontológicas - FOAR

Influência da suplementação da ração com diferentes doses de vitamina D sobre a proteína de interação com a tiorredoxina (TXNIP) no coração e suas vias de sinalização

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Associations among genotype, clinical phenotype, and intracellular localization of trafficking proteins in ARC syndrome

Arthrogryposisrenal dysfunctioncholestasis (ARC) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in vacuolar protein sorting 33 homologue B (VPS33B) and VPS33B interacting protein, apicalbasolateral polarity regulator (VIPAR). Cardinal features of ARC include congenital joint contractures, renal tubular dysfunction, cholestasis, severe failure to thrive, ichthyosis, and a defect in platelet alpha-granule biogenesis. Most patients with ARC do not survive past the first year of life. We report two patients presenting with a mild ARC phenotype, now 5.5 and 3.5 years old. Both patients were compound heterozygotes with the novel VPS33B donor splice-site mutation c.1225+5G>C in common. Immunoblotting and complementary DNA analysis suggest expression of a shorter VPS33B transcript, and cell-based assays show that c.1225+5G>C VPS33B mutant retains some ability to interact with VIPAR (and thus partial wild-type function). This study provides the first evidence of genotypephenotype correlation in ARC and suggests that VPS33B c.1225+5G>C mutation predicts a mild ARC phenotype. We have established an interactive online database for ARC (https://grenada.lumc.nl/LOVD2/ARC) comprising all known variants in VPS33B and VIPAR. Also included in the database are 15 novel pathogenic variants in VPS33B and five in VIPAR. Hum Mutat 33:16561664, 2012. (c) 2012 Wiley Periodicals, Inc.

DEPTOR Cell-Autonomously Promotes Adipogenesis, and Its Expression Is Associated with Obesity

DEP domain-containing mTOR-interacting protein (DEPTOR) inhibits the mechanistic target of rapamycin (mTOR), but its in vivo functions are unknown. Previous work indicates that Deptor is part of the Fob3a quantitative trait locus (QTL) linked to obesity/leanness in mice, with Deptor expression being elevated in white adipose tissue (WAT) of obese animals. This relation is unexpected, considering the positive role of mTOR in adipogenesis. Here, we dissected the Fob3a QTL and show that Deptor is the highest-priority candidate promoting WAT expansion in this model. Consistently, transgenic mice overexpressing DEPTOR accumulate more WAT. Furthermore, in humans, DEPTOR expression in WAT correlates with the degree of obesity. We show that DEPTOR is induced by glucocorticoids during adipogenesis and that its overexpression promotes, while its suppression blocks, adipogenesis. DEPTOR activates the proadipogenic Akt/PKB-PPAR-gamma axis by dampening mTORC1-mediated feedback inhibition of insulin signaling. These results establish DEPTOR as a new regulator of adipogenesis.

Resistance to octreotide LAR in acromegalic patients with high SSTR2 expression: analysis of AIP expression

We present here the clinical and molecular data of two patients with acromegaly treated with octreotide LAR after non-curative surgery, and who presented different responses to therapy. Somatostatin receptor type 2 and 5 (SSTR2 and SSTR5), and aryl hydrocarbon receptor-interacting protein (AIP) expression levels were analyzed by qPCR. In both cases, high SSTR2 and low SSTR5 expression levels were detected; however, only one of the patients achieved disease control after octreotide LAR therapy. When we analyzed AIP expression levels of both cases, the patient whose disease was controlled after therapy exhibited AIP expression levels that were two times higher than the patient whose disease was still active. These two cases illustrate that, although the currently available somatostatin analogs bind preferentially to SSTR2, some patients are not responsive to therapy despite high expression of this receptor. This difference could be explained by differences in post-receptor signaling pathways, including the recently described involvement of AIP. Arq Bras Endocrinol Metab. 2012;56(8):501-6

Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C (q)) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (beta-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.

Computational investigation of the Plasmodium falciparum fatty acid biosynthetic pathway toward the discovery of novel antimalarials

The structural peculiarities of a protein are related to its biological function. In the fatty acid elongation cycle, one small carrier protein shuttles and delivers the acyl intermediates from one enzyme to the other. The carrier has to recognize several enzymatic counterparts, specifically interact with each of them, and finally transiently deliver the carried substrate to the active site. Carry out such a complex game requires the players to be flexible and efficiently adapt their structure to the interacting protein or substrate. In a drug discovery effort, the structure-function relationships of a target system should be taken into account to optimistically interfere with its biological function. In this doctoral work, the essential role of structural plasticity in key steps of fatty acid biosynthesis in Plasmodium falciparum is investigated by means of molecular simulations. The key steps considered include the delivery of acyl substrates and the structural rearrangements of catalytic pockets upon ligand binding. The ground-level bases for carrier/enzyme recognition and interaction are also put forward. The structural features of the target have driven the selection of proper drug discovery tools, which captured the dynamics of biological processes and could allow the rational design of novel inhibitors. The model may be perspectively used for the identification of novel pathway-based antimalarial compounds.