BT2 is an Arabidopsis thaliana protein with N-terminal BTB, central TAZ and a C-terminal calmodulin binding domain and associates with Cullin3 to form an E3 ubiquitin ligase. We have shown previously that BT2 regulates telomerase activity in mature vegetative organs and controls a variety of hormone, stress and metabolic responses in Arabidopsis thaliana. Loss of BT2 results in plants that are hypersensitive to inhibition of germination by ABA and sugars. Conversely, overexpression of BT2 results in resistance to ABA and sugars, suggesting that BT2 is a negative regulator of ABA and sugar responses. Here, we report the roles of BT2-interacting partners GTE9 and GTE11, bromodomain and extraterminal-domain proteins of Global Transcription Factor Group E, in BT2-mediated responses to sugars and hormones. Loss-of-function mutants gte9-1 and gte11-1 phenocopy the bt2-1-null mutant responses; germination in all three mutants is hypersensitive to inhibition by glucose and ABA. Loss of either GTE9 or GTE11 in a BT2 over-expressing background blocks resistance to sugars and ABA, indicating that both GTE9 and GTE11 are required for BT2 function. Additionally, loss of GTE9 or GTE11, similar to loss of BT2, suppresses transcriptional gene activation mediated by CaMV 35S enhancers in Arabidopsis. The suppressed phenotype was accompanied by decreased transcription and hypermethylation of the 35S enhancers in the activation-tagged lines. We showed that BT2 and GTE9 co-immunoprecipitate and physically interact in vivo to mediate diverse responses to biotic and abiotic signals and 35S enhancer activity. Our working model is that the GTE9 and GTE11 function as chromatin adaptors that localize the BT2-CULLIN3 E3 ubiquitin ligase complex to acetylated chromatin on transcriptionally competent promoters in response to calcium signals detected by BT2’s calmodulin-binding domain.

Le virus de l'hépatite Delta (HDV) est un agent infectieux transmissible satellite du virus de l'hépatite B (HBV), induisant des maladies du foie plus sévères que la mono–infection par le HBV. Aucun traitement totalement efficace n'est disponible contre l'HDV et les 15 millions de personnes infectées par le HDV dans le monde sont exposées a un risque élevé de cirrhose et de carcinome hépatocellulaire. HDV est un virus unique qui ne code pas pour une polymérase virale contrairement aux autres virus a ARN. La réplication de l'ARN HDV s'effectue par un double mécanisme de cercle roulant générant des brins d'ARN de longueur génomique ou antigénomiques unitaires. La synthèse de l'ARN génomique est sensible à de faibles concentrations d'alpha–amanitine, ce qui suggère qu'elle soit médiée par l'ARN polymérase II (ARN Pol II) classiquement ADN dépendante. Ce processus repose sur la petite protéine du HDV (S–HDAg), qui doit être acétylée sur l'acide amine K72 pour activer la synthèse de l'ARN génomique. Nous avons récemment identifié la protéine BAZ2B (Bromodomain Associated Zinc finger protein 2B) comme un interactant majeur de S–HDAg par capture par affinité, couplée à la spectrométrie de masse à partir de l'expression de S– HDAg étiqueté par un double motif Strep–TagR dans les cellules HepaRG différentiées. La fonction biologique de BAZ2B est inconnue. Cependant, en comparant avec des protéines apparentées BAZ (BAZ–1A/1B/2A), on postule que BAZ2B représente la sous–unité accessoire d'un nouveau complexe de remodelage de chromatine de type ISWI, qui régule le positionnement des nucléosomes par hydrolyse de l'ATP. Des études récentes ont révélé que le bromodomaine de BAZ2B (BRD) reconnait la signature épigénétique spécifique K14ac–X–X–R sur l'histone H3. Cela pourrait impliquer le mode d'action du complexe de remodelage de la chromatine dont BAZ2B représente l'unité régulatrice reconnaissant des marques spécifiques d'acétylation des histones propagées séquentiellement modifiant la dynamique de la chromatine et favorisant le recrutement de l'ARN Pol II pour activer la transcription. Nous émettons l'hypothèse que l'acétylation, médiée par p300, du motif K72–X–X–R conserve dans les S–HDAg interagissant avec l'ARN antigénomique pseudo double brin, mimerait l'acétylation des histone H3 en K14 permettant de recruter le complexe de remodelage de la chromatine BAZ2B associée et de lancer la réplication HDV. Brièvement, pour confirmer la pertinence fonctionnelle du recrutement BAZ2B pour la réplication HDV, nous avons transfecté des lignées cellulaire Huh–7 exprimant de façon stable, soit la protéine sauvage S–HDAg ou le mutant R75A pour étudier la réplication HDV à partir plasmide pSVLD2m défectif pour l'expression de S–HDAg. Nos résultats indiquent que la synthèse de l'ARN génomique est fortement réduite dans les cellules exprimant le mutant R75A S–HDAg par rapport aux cellules exprimant le type sauvage S–HDAg, alors que la quantité d'ARN antigénomique est restée le même dans les deux cas. Des expériences de co–cristallisation et de siRNA sont actuellement menées afin de mieux caractériser au niveau moléculaire l'association entre BAZ2B BRD et des peptides dérivés de la séquence de S–HDAg et d'étudier les conséquences de l'inhibition par siRNA de BAZ2B. L'implication des BAZ2B dans la réplication de HDV pourra ouvrir des possibilités de développement de médicaments anti–HDV, basées sur l'optimisation des inhibiteurs émergents de BAZ2B–BRD / Hepatitis Delta Virus (HDV) is a satellite of Hepatitis B Virus (HBV), leading to more severe life threatening liver diseases than HBV mono–infection. No efficient therapy is available against HDV and the estimated 15 million HDV infected individuals worldwide are at a high risk of cirrhosis and hepatocellular carcinoma. HDV is a unique RNA virus as it does not encode a viral polymerase. HDV RNA replication occurs via a double rolling circle mechanism generating unit–length genomic or antigenomic RNA strands. The synthesis of the genomic RNA is sensitive to low concentrations of α–amanitin, suggesting that the RNA–dependent RNA synthesis is mediated by DNA–dependent RNA polymerase II (RNA Pol II). This process relies on the HDV encoded Small Hepatitis Delta antigen (S–HDAg), which must be acetylated at K72 to activate the synthesis of the genomic RNA. We recently identified BAZ2B (Bromodomain Associated to Zinc finger protein 2B) as a major interactant of S–HDAg by affinity capture coupled to mass spectrometry in differentiated HepaRG cells. The biological function of BAZ2B is however unknown. In comparison with related BAZ proteins (BAZ–1A/1B/2A), it is postulated that BAZ2B is the accessory subunit of a new chromatin remodeling complex of ISWI–type, which regulates nucleosome positioning through ATP hydrolysis. Recent studies revealed that the BAZ2B bromodomain (BRD) recognizes the distinct epigenetic signature K14ac–X–X–R on histone H3. This suggests that the mode of action of BAZ2B associated chromatin remodeling complex involves recognizing propagated specific histone acetylation marks to subsequently alter the chromatin dynamic and recruit the RNA Pol II for transcriptional activation. We hypothesized that the p300–mediated acetylation of the conserved K72–X–X–R motif in S–HDAg mimics acetylated histones on the pseudo–double stranded antigenomic RNA, to recruit the BAZ2B associated chromatin remodeling complex to initiate RNA Pol II mediated synthesis of HDV genome. To confirm the functional relevance of BAZ2B recruitment for HDV replication, we transfected Huh 7 cells stably expressing either wild–type S–HDAg or R75A mutant S–HDAg with the HDV replication defective plasmid pSVLD2m. Our results indicate that the synthesis of genomic RNA was greatly reduced in cells expressing the R75A mutant S–HDAg in comparison to cells expressing wild–type S–HDAg, whereas the amount of antigenomic RNA remained the same in both cases. Co–crystallization experiments are currently being carried out to better characterize at the molecular level the association between BAZ2B BRD and S–HDAg derived peptides. Furthermore, siRNA experiments directed against the BAZ2B gene are expected to reveal the consequences of BAZ2B inhibition on HDV viral replication. The involvement of BAZ2B in HDV replication may open anti–HDV drug development opportunities, based on the optimization of emerging BAZ2B–BRD inhibitors

Malaria is a major public health problem that affects millions of lives globally. The increased burden of malaria requires new interventions that will address the eradication of the disease. Current interventions include vector control by using insecticide-treated bed nets and indoor residual spraying, and antimalarial drugs to control the parasite. Parasite resistance has been reported for the currently used effective antimalarial drugs. To pre-empt the impact of parasite resistance a continued development of new antimalarial drugs that have novel mechanisms of action should be pursued. Antimalarial drug discovery requires that potential antimalarial drugs should have different drug targets to those already targeted, to lower the chances of resistance. Potential antimalarial drugs should preferably provide a single radical cure to prevent reproduction at all life cycle stages. This study tested the effects of in silico designed compounds targeting plasmodial Ca2+- dependent protein kinases (CDPK) 1 & 4, FIKK kinases and bromodomain proteins on the Plasmodium parasite. These enzymes are involved in gene regulation and are important factors during gene transcription. In P. falciparum the gatekeeper kinases contain small hydrophobic pockets near the ATP-binding site. These hydrophobic pockets allow for selective inhibition of these proteins at the ATP-binding site. The compounds were tested in vitro to determine their antiplasmodial activity. These compounds are shown to be potential inhibitors of the intra-erythrocytic P. falciparum parasites as three of the compounds showed selective cytotoxic activity at less than 1 μM against the chloroquine sensitive laboratory strains (3D7 and NF54). Even though the proteins targeted by these compounds have been previously indicated to play a role at specific stages during the parasite’s life cycle, the compounds tested here were not able to target the sexual gametocyte stages of the Plasmodium parasite. Further optimisation of these compounds should be performed to improve activity against both the asexual and sexual stages of the parasites. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted