Two functionally distinctive phosphopantetheinyl transferases from amoeba Dictyostelium discoideum

The life cycle of Dictyostelium discoideum is proposed to be regulated by expression of small metabolites. Genome sequencing studies have revealed a remarkable array of genes homologous to polyketide synthases (PKSs) that are known to synthesize secondary metabolites in bacteria and fungi. A crucial step in functional activation of PKSs involves their post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases). PPTases have been recently characterized from several bacteria; however, their relevance in complex life cycle of protozoa remains largely unexplored. Here we have identified and characterized two phosphopantetheinyl transferases from D. discoideum that exhibit distinct functional specificity. DiAcpS specifically modifies a stand-alone acyl carrier protein (ACP) that possesses a mitochondrial import signal. DiSfp in contrast is specific to Type I multifunctional PKS/fatty acid synthase proteins and cannot modify the stand-alone ACP. The mRNA of two PPTases can be detected during the vegetative as well as starvation-induced developmental pathway and the disruption of either of these genes results in non-viable amoebae. Our studies show that both PPTases play an important role in Dictyostelium biology and provide insight into the importance of PPTases in lower eukaryotes

Dissecting the functional role of polyketide synthases in Dictyostelium discoideum

Dictyostelium discoideum exhibits the largest repository of polyketide synthase (PKS) proteins of all known genomes. However, the functional relevance of these proteins in the biology of this organism remains largely obscure. On the basis of computational, biochemical, and gene expression studies, we propose that the multifunctional Dictyostelium PKS (DiPKS) protein DiPKS1 could be involved in the biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol (MPBD). Our cell-free reconstitution studies of a novel acyl carrier protein Type III PKS didomain from DiPKS1 revealed a crucial role of protein-protein interactions in determining the final biosynthetic product. Whereas the Type III PKS domain by itself primarily produces acyl pyrones, the presence of the interacting acyl carrier protein domain modulates the catalytic activity to produce the alkyl resorcinol scaffold of MPBD. Furthermore, we have characterized an O-methyltransferase (OMT12) from Dictyostelium with the capability to modify this resorcinol ring to synthesize a variant of MPBD. We propose that such a modification in vivo could in fact provide subtle variations in biological function and specificity. In addition, we have performed systematic computational analysis of 45 multidomain PKSs, which revealed several unique features in DiPKS proteins. Our studies provide a new perspective in understanding mechanisms by which metabolic diversity could be generated by combining existing functional scaffolds

Adenylyl cyclase G is activated by an intramolecular osmosensor

Adenylyl cyclase G (ACG) is activated by high osmolality and mediates inhibition of spore germination by this stress factor. The catalytic domains of all eukaryote cyclases are active as dimers and dimerization often mediates activation. To investigate the role of dimerization in ACG activation, we coexpressed ACG with an ACG construct that lacked the catalytic domain (ACGΔcat) and was driven by a UV-inducible promoter. After UV induction of ACGΔcat, cAMP production by ACG was strongly inhibited, but osmostimulation was not reduced. Size fractionation of native ACG showed that dimers were formed between ACG molecules and between ACG and ACGΔcat. However, high osmolality did not alter the dimer/monomer ratio. This indicates that ACG activity requires dimerization via a region outside the catalytic domain but that dimer formation does not mediate activation by high osmolality. To establish whether ACG required auxiliary sensors for osmostimulation, we expressed ACG cDNA in a yeast adenylyl cyclase null mutant. In yeast, cAMP production by ACG was similarly activated by high osmolality as in Dictyostelium. This strongly suggests that the ACG osmosensor is intramolecular, which would define ACG as the first characterized primary osmosensor in eukaryotes

The determination of spatial pattern in Dictyostelium discoideum

Free-living amoebae of the cellular slime mould Dictyostelium discoideum aggregate when starved and give rise to a long and thin multicellular structure, the slug. The slug resembles a metazoan embryo, and as with other embryos it is possible to specify a fate map. In the case of Dictyostelium discoideum the map is especially simple: cells in the anterior fifth of the slug die and form a stalk while the majority of those in the posterior differentiate into spores. The genesis of this anterior-posterior distinction is the subject of our review. In particular, we ask: what are the relative roles of individual pre-aggregative predispositions and post-aggregative position in determining cell fate? We review the literature on the subject and conclude that both factors are important. Variations in nutritional status, or in cell cycle phase at starvation, can bias the probability that an amoeba differentiates into a stalk cell or a spore. On the other hand, isolates, or slug fragments, consisting of only prestalk cells or only prespore cells can regulate so as to result in a normal range of both cell types. We identify three levels of control, each being responsible for guiding patterning in normal development: (i) 'coin tossing', whereby a cell autonomously exhibits a preference for developing along either the stalk or the spore pathway with relative probabilities that can be influenced by the environment; (ii) 'chemical kinetics', whereby prestalk and prespore cells originate from undifferentiated amoebae on a probabilistic basis but, having originated, interact (e.g. via positive and negative feedbacks), and the interaction influences the possibility of conversion of one cell type into the other, and (iii) 'positional information', in which the spatial distribution of morphogens in the slug influences the pathway of differentiation. In the case of possibilities (i) and (ii), sorting out of like cell types leads to the final spatial pattern. In the case of possibility (iii), the pattern arises in situ

Peptide: N- glycanase is expressed in prestalk cells and plays a role in the differentiation of prespore cells during development of <i style="mso-bidi-font-style:normal">Dictyostelium discoideum</i>

197-206Peptide: N- glycanase (PNGase) enzyme is found throughout eukaryotes and plays an important role in the misfolded glycoprotein degradation pathway. This communication reports the expression patterns of the pngase transcript (as studied by the analysis of β- galactosidase reporter driven by the putative <i style="mso-bidi-font-style: normal">pngase promoter) and protein (as studied by the analysis of β- galactosidase reporter expressed under the putative pngase promoter as a fusion with the pngase ORF) during development and further elucidated the developmental defects of the cells lacking PNGase (png-). The results show that the DdPNGase is an essential protein expressed throughout development and <i style="mso-bidi-font-style: normal">β- galactosidase activity was present in the anterior part of the slug. In structures derived from a null mutant for <i style="mso-bidi-font-style: normal">pngase, the prestalk A and AO patterning was expanded and covered a large section of the prespore region of the slugs. When developed as chimeras with wild type, the png- cells preferentially populate the prestalk/stalk region. When the mutants were mixed in higher ratios, they also tend to form the prespore/spore cells. The results emphasize that the DdPNGase has an essential role during development and the mutants have defects in a system that changes the physiological dynamics in the prespore cells. DdPNGase play a role in development both during aggregation and in the differentiation of prespore cells

Spatial gradients of calcium in the slug of Dictyostelium discoideum

Starved amoebae of D. discoideum aggregate and give rise to a long and thin multicellular structure called the slug. The cells within the slug eventually differentiate according to a simple anterior/posterior dichotomy. This motivates a search for gradients of putative morphogens along its axis. Calcium may be one such morphogen. On the basis of observations made by using the calcium-sensitive fluorescent dyes fura-2 and chlortetracyline, we report that there are spatial gradients in cytoplasmic and sequestered calcium in the slug. Anteriorly located and genetically defined prestalk cells (ecmA/pstA, ecmB/pstAB) contain significantly higher levels of calcium than the prespore cells in the posterior. However, the proportion of 'calcium-rich' cells in the slug is greater than that of the subset of prestalk cells defined by the expression of the ecmA or ecmB genes

Spatial gradients of calcium in the slug of Dictyostelium discoideum

Starved amoebae of D. discoideum aggregate and give rise to a long and thin multicellular structure called the slug. The cells within the slug eventually differentiate according to a simple anterior/posterior dichotomy. This motivates a search for gradients of putative morphogens along its axis. Calcium may be one such morphogen. On the basis of observations made by using the calcium-sensitive fluorescent dyes fura-2 and chlortetracyline, we report that there are spatial gradients in cytoplasmic and sequestered calcium in the slug. Anteriorly located and genetically defined prestalk cells (ecmA/pstA, ecmB/pstAB) contain significantly higher levels of calcium than the prespore cells in the posterior. However, the proportion of 'calcium-rich' cells in the slug is greater than that of the subset of prestalk cells defined by the expression of the ecmA or ecmB genes

Identification and characterization of peptide: N- glycanase from <it>Dictyostelium discoideum</it>

<p>Abstract</p> <p>Background</p> <p>Peptide: N- glycanase (PNGase) enzyme cleaves oligosaccharides from the misfolded glycoproteins and prepares them for degradation. This enzyme plays a role in the endoplasmic reticulum associated degradation (ERAD) pathway in yeast and mice but its biological importance and role in multicellular development remain largely unknown.</p> <p>Results</p> <p>In this study, the PNGase from the cellular slime mold, <it>Dictyostelium discoideum</it> (<it>Dd</it>PNGase) was identified based on the presence of a common TG (transglutaminase) core domain and its sequence homology with the known PNGases. The domain architecture and the sequence comparison validated the presence of probable functional domains in <it>Dd</it>PNGase. The tertiary structure matched with the mouse PNGase. Here we show that <it>Dd</it>PNGase is an essential protein, required for aggregation during multicellular development and a knockout strain of it results in small sized aggregates, all of which did not form fruiting bodies. The <it>in situ</it> hybridization and RT-PCR results show higher level of expression during the aggregate stage. The expression gets restricted to the prestalk region during later developmental stages. <it>Dd</it>PNGase is a functional peptide:N-glycanase enzyme possessing deglycosylation activity, but does not possess any significant transamidation activity.</p> <p>Conclusions</p> <p>We have identified and characterized a novel PNGase from <it>D. discoideum</it> and confirmed its deglycosylation activity. The results emphasize the importance of PNGase in aggregation during multicellular development of this organism.</p

Introducing a simple model system for binding studies of known and novel inhibitors of AMPK: a therapeutic target for prostate cancer

<p>Prostate cancer (PC) is one of the leading cancers in men, raising a serious health issue worldwide. Due to lack of suitable biomarker, their inhibitors and the platform for testing those inhibitors result in poor prognosis of PC. AMP-activated protein kinase (AMPK) is a highly conserved protein kinase found in eukaryotes that is involved in growth and development, and also acts as a therapeutic target for PC. The aim of the present study is to identify novel potent inhibitors of AMPK and propose a simple cellular model system for understanding its biology. Structural modelling and MD simulations were performed to construct and refine the 3D models of <i>Dictyostelium</i> and human AMPK. Binding mechanisms of different drug compounds were studied by performing molecular docking, molecular dynamics and MM-PBSA methods. Two novel drugs were isolated having higher binding affinity over the known drugs and hydrophobic forces that played a key role during protein–ligand interactions. The study also explored the simple cellular model system for drug screening and understanding the biology of a therapeutic target by performing <i>in vitro</i> experiments.</p