Les parasites apicomplexes sont ubiquitaires et ont une forte incidence en médecine humaine et vétérinaire. Certains de ces parasites, comme Plasmodium falciparum, l’agent du paludisme, ou Toxoplasma gondii, l’agent de la toxoplasmose, posent des problèmes de santé publique. Les thérapies existantes montrent parfois une efficacité limitée, une forte toxicité et entraînent des résistances, d’où la nécessité de nouvelles approches plus spécifiques. Dans ce contexte, nous avons développé deux approches d’inhibition des apicomplexes : -la synthèse de biphénylimidazoazines à large spectre efficaces au micromolaire sur cinq parasites apicomplexes différents in vitro. -la synthèse d’imidazo[1,2-b]pyridazines ciblant spécifiquement une protéine kinase (CDPK1) de T. gondii et efficaces au submicromolaire sur le parasite in vitro. Une diminution de plus de 90 % de la charge parasitaire chez la souris et une innocuité à court terme font de ces imidazo[1,2-b]pyridazines de bons candidats thérapeutiques. / Apicomplexan parasites are ubiquitous and have a strong incidence in veterinary and human medicine. Some of them, like Plasmodium falciparum, causing malaria, or Toxoplasma gondii, causing toxoplasmosis, are matter of public health concern. The existing therapies may have limited efficiency, high toxicity, and may lead to resistance, highlighting the necessity of new more specific approaches. In this context, we have developed two approaches to inhibit Apicomplexa: -the synthesis of biphenylimidazoazines with broad-spectrum and efficient at the micromolar range on five different apicomplexan parasites in vitro. -the synthesis of imidazo[1,2-b]pyridazines specifically targeting a kinase protein (CDPK1) of T. gondii and efficient at the submicromolar range on the parasite in vitro. More than 90% diminution of parasite burden in mice and short term safety make these imidazo[1,2-b]pyridazines good therapeutic candidates.

Phosphoenolpyruvate (PEP) carboxylase (PEPC) is a tightly controlled enzyme situated at a pivotal branchpoint of plant C-metabolism. Two physically and kinetically distinct oligomeric classes of PEPC exist in the endosperm of developing castor oil seeds (COS). Class-1 PEPC is a typical homotetramer composed of 107-kDa plant-type PEPC (PTPC) subunits, whereas the 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between Class-1 PEPC and distantly related 118-kDa bacterial-type PEPC (BTPC) subunits. BTPC functions as both a catalytic and regulatory subunit of the allosterically-desensitized Class-2 PEPC, which has been hypothesized to support massive PEP-flux to malate for leucoplast fatty acid synthesis. Previous studies established that BTPC: (i) subunits of COS Class-2 PEPC are subject to inhibitory phosphorylation in vivo, and (ii) at Ser425 and Ser451 within an intrinsically disordered region. This study focuses on characterization of the COS protein kinase (BTPC-K) that phosphorylates BTPC at Ser451. BTPC-K, having a native molecular mass of 63 kDa, was purified ~500-fold from developing COS endosperm. Its activity was absolutely dependent upon the presence of Ca2+ (Ka= 2.7 μM) and millimolar Mg2+. BTPC-K phosphorylated BTPC subunits of Class-2 PEPC strictly at Ser451 (Km= 1.1 μM), as well as histone type III-S (Km= 1.7 μM), but did not phosphorylate a BTPC S451D phosphomimetic mutant, native COS PTPC or sucrose synthase, or α-casein. BTPC-K displayed a broad pH-activity optima of pH 7.3, a Km for Mg2+-ATP of 6.6 μM, and marked inhibition by 3-P-glycerate and PEP. The possible control of BTPC-K by disulfide-dithiol interconversion was suggested by its rapid inactivation and subsequent reactivation when incubated with oxidized glutathione and then dithiothreitol. BTPC-K activity was insensitive to exogenous calmodulin, but potently inhibited by 100 µM trifluoperazine (a calmodulin antagonist). BTPC-K-mediated Ser451 phosphorylation of BTPC subunits of Class-2 PEPC inhibited BTPC activity by ~50% when assayed under suboptimal conditions (pH 7.3, 1 mM PEP with 10 mM L-malate). Overall the results of this study have led to the hypothesis that in vivo phosphorylation of COS BTPC at Ser451 is mediated by a dedicated calcium-dependent protein kinase (CDPK). / Thesis (Master, Biology) -- Queen's University, 2013-08-30 14:23:39.648

In plants, calcium is a ubiquitous signaling molecule and changes in cytosolic calcium levels reported in response to various abiotic and biotic stresses like salt stress, drought, pathogen attack and phytohormone signaling. Any calcium- mediated signal transduction process involves the establishment of a signal-specific change in the cytosolic calcium concentration termed as ‗calcium signature‘ which is decoded by the specific group of proteins called ‗calcium sensors‘ (eg: Calmodulin (CaM) and Ca2+ - regulated kinases). Plants have a novel group of kinases designated as Ca2+- dependent protein kinases (CDPK; EC 2.7.1.37). CDPKs are biochemically distinct from other Ca2+- dependent kinases, such as Ca2+- and phospholipid- dependent protein kinases, as they are activated directly by Ca2+-and are independent of CaM. They exist as monomeric serine/threonine protein kinases and consist of four domains namely an amino-terminal variable domain, a kinase domain, an autoinhibitory domain and a calmodulin-like domain (CaM-LD). CDPKs represent a unique class of Ca2+ sensors, having protein kinase as well as CaM-LD in a single polypeptide chain, enabling them to couple the calcium sensor directly to its responder (kinase). In the absence of calcium signature, CDPKs activity is inhibited by the autoinhibitory domain, which acts as a pseudo-substrate of kinase domain and thus blocks the active site of the enzyme. In the presence of calcium signature, CDPKs undergo conformational changes leading to removal of the inhibition. Besides plants, CDPKs are also reported in few protozoans viz Plasmodium falciparam, Paramecium and Taxoplasma. However, CDPKs are not found in the eukaryotic genome of yeast, nematodes, fruitflies and humans. In the current study, we have cloned CDPK1 gene from Cicer arietinum (CaCDPK1) in pRSET-A expression vector and expressed it in Escherichia coli BL21pLysS strain. However, while expressing the recombinant CaCDPK1 in E.coli, most of the recombinant CaCDPK1 protein was expressed as insoluble form. Therefore, we focused our efforts on optimizing the culture conditions for achieving the maximum yield of soluble recombinant CaCDPK1. Expression of the soluble CaCDPK1 was achieved by optimizing the different conditions like IPTG concentrations, temperature and growth time after induction. Maximum amount of soluble expression of recombinant CaCDPK1 was achieved by inducing the bacterial culture with 0.1 mM IPTG at 0.6 OD and growing it further for 4 h at 25°C. As with several other CDPKs, CaCDPK1 was found to get autophosphorylated in a calcium-dependent manner. To find the significance of autophosphorylation, we measured the substrate phosphorylation activity of the native and autophosphorylated CaCDPK1, which revealed that the autophosphorylation enhances the kinase activity of CaCDPK1 by 2-fold. Autophosphorylation was linearly dependant on concentrations of the enzyme suggesting that the autophosphorylation in CaCDPK1 occurs via an intra-molecular mechanism. Further analysis of autophosphorylation shows that autophosphorylation happens before substrate phosphorylation and provides calcium -independent substrate phosphorylation property. It also reduces the lag phase for activation of the enzyme and utilizes both ATP and GTP as phosphor-donor, but ATP is preferred over GTP. Autophosphorylation was found to occur at serine and threonine residues. The MALDI MS/MS analysis of the cold ATP autophosphorylated CaCDPK1 showed Thr- 339, Ser- 357, and Ser- 367 residues could be the potential autophosphorylation sites in CaCDPK1. Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress conditions. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1) from chickpea are reported in this study. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in Vmax of the enzyme activity by these phospholipids significantly decreased the Km indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K½ = 1.3 nM) when compared to PA (K½ = 56 nM). We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response. In the current study we also investigated CaCDPK1 interactions with calcium ions to address the Ca2+ -induced conformational changes in CaCDPK1 by using circular dichroism (CD), fluorescence spectroscopy and isothermal titration (ITC). Isothermal calorimetric analysis of calcium binding to CaCDPK1 shows a biphasic curve with two Kd of 27 nM and 1.72 µM respectively. The fluorescence measurements showed quenching in fluorescence intensity with a 5 nm red shift. The plot of changes in intensity against calcium concentrations again showed a biphasic curve, indicating that there may be more than one kind of Ca2+ binding sites. 8-anilinonaphthalene-1-sulfonic acid (ANS) binding showed that calcium bound form of CaCDPK1 exposes hydrophobic surfaces which may act as binding sites for other proteins. CD analysis of CaCDPK1 showed that it‘s an alpha helical rich protein and its helical content increases after binding to calcium. Taken all together this study describes the successful heterologous expression of Cicer arietinum CDPK isoform 1 in E.coli. and demonstrates that the autophoshorylation happens via an intra-molecular mechanism and it increases the kinase activity of CaCDPK1 at least by 2-fold. We also report here that CaCDPK1 prefers ATP as phosphodonor over GTP. The present study also shows the activation of CaCDPK1 by PC and PA, but not by PE or diacylglycerol. Both phospholipids were able to bind to CaCDPK1 and increased its Vmax and affinity towards the exogenous substrate, histone III-S. The current study also shows that calicum binding induces conformational changes in CaCDPK1 and the all four EF hand motifs of CaCDPK1 do not function in an equivalent manner.

Light and phytohormones, such as cytokinins, have been known to play a pivotal role in numerous physiological processes in plant cells. Previous work in our laboratory has revealed the light- and cytokinin- modulated changes both in the levels of specific tRNA species and their modified nucleotide contents, in addition to the characterization of specific tRNAs and tRNA genes from higher plants. The plant hormone cytokinin, which is of particular interest to us has been implicated to be involved in processes such as induction of cell division, plastid biogenesis and delay of senescence. Ongoing work in our laboratory also points towards the role of Ca2+ as a second messenger in cytokinin mediated gene expression. With the objective of isolation of specific tRNA genes which could then be used as probes to study the light- and phytohormone- induced changes in the levels of respective functional mature tRNAs, a previously isolated clone containing a 6.6kb insert that hybridized with 3 end labeled cucumber total cellular tRNA was sequenced by the dideoxy chain termination method. Sequence analysis of the 6.6 kb DNA fragment has revealed a chloroplast genome DNA fragment containing the trnNGUU and trnRACG genes in addition to the genes coding for the ribosomal RNAs 4.5S, 5S and 23S as well as the protein coding genes ccsA (cytochrome c-synthesis) and ndhD(NADH plastoquinone oxidoreductase).These genes were found to be arranged in the order-23S-4.5S-5S-trnRACG-trnNGUU-ccsA-ndhD. This shows a divergence from the gene organization in the completely sequenced chloroplast genomes of other higher plant species such as tobacco, maize, rice and Arabidopsis, especially with regard to the absence of a highly conserved trnLUAG gene that has been shown to be present in the trnNGUU-ndhD intergenic region. The cucumber chloroplast trnNGUU and trnRACG genes have shown very high homology (>90%) whereas ccsA and ndhD show 50-61% similarity to corresponding genes from chloroplast genomes of other plant species. The relative levels of tRNAArg and tRNAAsn were determined by Northern analysis using the tRNA gene probes, in etiolated excised cucumber cotyledons treated with light or phytohormones, such as cytokinin (BA) and auxin (2,4-D). Light and phytohormones were found to significantly increase the levels of tRNAArg unlike in the case tRNAAsn where no significant changes in the levels were observed. This result points towards the regulation of relative levels of specific tRNA species by light and cytokinin so as to match the codon usage of the mRNA population during light- and cytokinin- induced plant development in cucumber. Northern analyses were also performed to monitor the relative transcript levels of the plastid encoded ccsA and ndhD in etiolated excised cucumber cotyledons treated with light or phytohormones. ccsA transcript levels were found to be significantly reduced in auxin treated cucumber cotyledons where as exogenous application of cytokinin to either dark-grown or light exposed cotyledons did not seem to have any pronounced effect. ndhD transcripts were found to be up-regulated by cytokinin treatment or light exposure in comparison to un-treated controls probably indicating a point of overlap in the light/ cytokinin mediated signal transduction pathways. Auxin treatment on the other hand was found to down-regulate ndhD transcript levels also. Recent studies from our laboratory have demonstrated the involvement of a calcium-dependent protein kinase(CDPK) in the cytokinin-signal transduction pathway associated with the induction of pathogenesis-related proteins (chitinase and β 1-3 modulation of nuclear-encoded CDPK transcripts in response to light and exogenously added phytohormones such as cytokinins and auxin. Towards this end, partial CDPK cDNAs were generated from Cucumis Sativus by RT-PCR using degenerate primers designed based on the conserved regions of the known CDPK proteins available in the database, cloned in pGEM-T and sequenced. Sequence analysis of twenty partial cDNA clones revealed the presence of at least four CDPK isoforms in Cucumis sativus (CuCDPK 1-4). Of the four partial CDPK cDNAs, the tissue-specific expression level of CuCDPK3 was studied using the highly sensitive Taqman Analysis (Quantitative RT-PCR). The results obtained indicate that, in excised dark-grown cucumber cotyledons light and cytokinin were found to up-regulate the levels of CuCDPK3 unlike auxin, which was found to have no significant effect. In cucumber hypocotyls, which had the highest levels of CuCDPK3, light was found to have a down-regulatory effect whereas cytokinin and auxin did not bring about any significant changes in the levels of CuCDPK3. In cucumber root tissue, both light and cytokinin were found to have a down-regulatory effect on the levels of CuCDPK3, unlike auxin. The southern analysis of cucumber genomic DNA revealed a CDPK multi-gene family in cucumber. Since cytokinins have been known to play a role in both etioplast and chloroplast biogenesis and since various groups have recently reported the presence of higher plant homologues of bacterial cell-division protein FtsZ and the requirement of plant nuclear-encoded FtsZs for plastid division, efforts were also made to isolate and to study the expression of cucumber FtsZ in dark-grown cucumber cotyledon tissue treated exogenously with light/phytohormones. Towards this end, a partial FtsZ cDNA was generated from cucumber by RT-PCR using degenerate primers designed based on conserved regions of known plant FtsZ proteins. Results of the Taqman Analysis indicate that cytokinin, unlike auxin, mimics the action of light by increasing the levels of CuFtsZ transcripts in dark-grown cotyledon tissue suggesting the involvement of FtsZ in cytokinin-induced plastid-biogenesis.

Calcium ion plays a pivotal role as second messenger during signal/response coupling in plant cells (Trewavas, 1999). Elevations of cytosolic Ca2+ occur in plants as a consequence of abiotic and biotic stresses, environmental and hormonal stimuli. However, the molecular mechanism by which changes in cytosolic calcium are sensed and transduced in the plant cell has not been completely elucidated. The detection of Ca2+-binding proteins, especially Ca2+-dependent protein kinases (CDPKs) in plants led to drawing analogy with animal systems wherein the Ca2+-message is perceived and transduced by proteins that bind Ca2+. CDPKs are stimulated by the direct binding of Ca2+ to their endogenous calmodulin (CaM) -like domain (Harper et al, 1991). CDPKs exist as multiple isoforms in a single species, and show tissue-specific and developmentally regulated expression. Furthermore, the diversity among different CDPK isoforms with respect to Ca2+-binding properties, activation, substrate specificity, regulatory mechanisms and other kinetic properties suggest their specialization in the regulation of distinct signaling pathways. These observations therefore have led to the speculation that most of the Ca2+-mediated signal transduction in plants occurs via the mediation of CDPKs (Harmon et al, 2000). Over the last 15 years there has been a dramatic unfolding of information on Ca2+-mediated signaling in plants. Nevertheless, little is known about the environmental/hormonal signals and the signaling events that regulate early plant developmental processes such as embryogenesis, seed development and germination. The present investigation was initiated with the objectives 1) to determine the role of Ca2+ during embryogenesis, 2) to examine the involvement of a CDPK during early developmental processes in sandalwood plant (Santalum album L.) and 3) to purify and biochemically characterize this CDPK. The study initially investigated the possible involvement of calcium-mediated signaling in the induction/regulation of somatic embryogenesis from proembryogenic cells of sandalwood. 45 Ca + uptake studies and fura-2 fluorescence ratio photometry were used to measure changes in [Ca2+]cyt of proembryogenic cells in response to culture conditions conducive for embryo development. Sandalwood proembryogenic cell masses (PEMs) were obtained in the callus proliferation medium that contains the auxin 2,4-D. Subculture of PEMs into the embryo differentiation medium which lacks 2,4-D and has higher osmoticum resulted in a 4-fold higher 45Ca2+ incorporation into the symplast. Fura-2 based ratiometric analysis also showed a 10-16- fold increase in the [Ca2+]cyt of PEMs under identical culture conditions, increasing from a resting concentration of 30-50 nM to 650-800 nM. Chelation of exogenous Ca2+ with EGTA arrested such an elevation in [Ca2+]cyt. Exogenous Ca2+ when chelated or deprived also arrested embryo development and inhibited the accumulation of a Ca2+-dependent protein kinase (swCDPK) in embryogenic cultures. However, such culture conditions did not cause cell death as the PEMs continued to proliferate to form larger cell clumps. Culture treatment with W7 reduced embryogenic frequency by 85%, indicating that blockage of Ca2+-mediated signaling pathway(s) involving swCDPK and/or CaM caused inhibition of embryogenesis. These observations suggest a second messenger role for exogenous Ca2+ and the existence of Ca2+-mediated signaling pathway(s) during sandalwood somatic embryogenesis. The detection of a 55 kD protein showing cross reactivity with polyclonal antisoybean CDPK and the detection of Ca2+-dependent protein kinase activity in protein extracts from somatic embryos, prompted investigation on the spatio-temporal accumulation and activity of a CDPK in different developmental stages of sandalwood. Western blot analysis and protein kinase assays identified a Ca2+-dependent protein kinase (swCDPK) of 55 kD in soluble protein extracts of different developmental stages of sandalwood somatic embryos. However, swCDPK was not detected in plantlets regenerated from somatic embryos. swCDPK exhibited differential expression and activity in the developmental stages of sandalwood. Zygotic embryos, endosperm and seedlings showed high accumulation of swCDPK. However, the enzyme was not detected in the soluble proteins of shoots and flowers of sandalwood tree. swCDPK exhibited a temporal pattern of expression in endosperm, showing high accumulation and activity in mature fruit and germinating stages, the enzyme being localized strongly in the storage bodies of the endosperm cells. Interestingly, these storage bodies were thereafter identified as oil bodies, suggesting that a Ca2+-mediated regulation of oil hydrolysis and/or mobilization might be operative during seed germination. swCDPK in the zygotic embryo was found to be inactive during seed dormancy and early stages of germination, indicating a possible post-translational hibition/inactivation of the enzyme during these stages. The temporal expression of swCDPK during somatic/zygotic embryogenesis, seed maturation and germination thus suggests involvement of the enzyme in these early developmental processes. In view of the diversity exhibited by members of the CDPK family, characterization of swCDPK, the early development specific CDPK from sandalwood was undertaken. Purification of swCDPK was achieved by chromatography on DEAE-cellulose, hydroxyapatite and Blue-Sepharose. The purified enzyme resolved into a single band on 10 % polyacrylamide gels, both under denaturing and non-denaturing conditions. swCDPK was strictly dependent on Ca2+, K0.5 (apparent binding constant) for Ca2+-activation of substrate phosphorylation activity being 0.7 μM and for autophosphorylation activity —50 nM. Ca2+-dependence for activation, CaM-independence, inhibition by CaM-antagonist (IC50 for W7 = 6 μM, for W5 = 46 μM) and cross-reaction with polyclonal antibodies directed against the CaM-like domain of soybean CDPK, confirmed the presence of an endogenous CaM-like domain in the purified enzyme. Kinetic studies revealed a Km value of 13 mg/mL for histone III-S and a Vmax of 0.1 nmolmin-1rng-1. The enzyme exhibited high specificity for ATP with a Km value of 10 nM. Titration with Ca2+ resulted in enhancement of the intrinsic emission fluorescence of swCDPK and a shift in the λmax emission from tryptophan residues. A reduction in the efficiency of non-radiative energy transfer from tyrosine to tryptophan residues was also observed. These are taken as evidence for the occurrence of Ca2+-induced conformational change in swCDPK. The emission spectral properties of swCDPK in conjunction with Ca2+ levels required for autophosphorylation and substrate phosphorylation help elucidate the possible mode of Ca2+ activation of this enzyme.