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1.
J Microbiol ; 59(9): 848-853, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34449058

RESUMO

Rap small GTPases are involved in diverse signaling pathways associated with cell growth, proliferation, and cell migration. There are three Rap proteins in Dictyostelium, RapA, RapB, and RapC. RapA is a key regulator in the control of cell adhesion and migration. Recently RapA and RapC have been reported to have opposite functions in the regulation of cellular processes. In this study, we demonstrate that the C-terminus of RapC, which is not found in RapA, is essential for the opposite functions of RapC and is able to reverse the functions of RapA when fused to the tail of RapA. Cells lacking RapC displayed several defective phenotypes, including spread morphology, strong adhesion, and decreased cell migration compared to wild-type cells. These phenotypes were rescued by full-length RapC, but not by RapC missing the C-terminus. Furthermore, recombinant RapA fused with the C-terminus of RapC completely recovered the phenotypes of rapC null cells, indicating that the functions of RapA were modified to become similar to those of RapC by the C-terminus of RapC with respect to cell morphology, cell adhesion and migration, cytokinesis, and development. These results suggest that the C-terminal residues of RapC are able to suppress and change the functions of other Ras proteins in Ras oncogenic signaling pathways.


Assuntos
Dictyostelium/enzimologia , Proteínas de Protozoários/metabolismo , Proteínas ras/metabolismo , Motivos de Aminoácidos , Dictyostelium/química , Dictyostelium/genética , Regulação da Expressão Gênica , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas ras/genética
2.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200865

RESUMO

A novel cytoplasmic dye-decolorizing peroxidase from Dictyostelium discoideum was investigated that oxidizes anthraquinone dyes, lignin model compounds, and general peroxidase substrates such as ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the proximal axial position and either an activated oxygen or CN- molecule at the distal axial position. Asp149 is in an optimal conformation to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate-binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long-range electron transfer pathways associated with a hydrogen-bonding network that connects the substrate-binding sites with the heme moiety are described.


Assuntos
Corantes/química , Dictyostelium/enzimologia , Heme/química , Peróxido de Hidrogênio/química , Peroxidase/química , Peroxidase/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , Heme/metabolismo , Ligação de Hidrogênio , Oxirredução
3.
PLoS One ; 16(4): e0250704, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33909675

RESUMO

In this report, we extend our previous characterization of Dictyostelium discoideum glutathione S-transferase (DdGST) enzymes that are expressed in the eukaryotic model organism. Transcript profiling of gstA1-gstA5 (alpha class) genes in vegetative, log phase cells identified gstA2 and gstA3 with highest expression (6-7.5-fold, respectively) when compared to other gstA transcripts. Marked reductions in all gstA transcripts occurred under starvation conditions, with gstA2 and gstA3 exhibiting the largest decreases (-96% and -86.6%, respectively). When compared to their pre-starvation levels, there was also a 60 percent reduction in total GST activity. Glutathione (GSH) pull-down assay and mass spectroscopy detected three isozymes (DdGSTA1, DdGSTA2 and DdGSTA3) that were predominantly expressed in vegetative cells. Biochemical and kinetic comparisons between rDdGSTA2 and rDdGSTA3 shows higher activity of rDdGSTA2 to the CDNB (1-chloro-2,4-dinitrobenzene) substrate. RNAi-mediated knockdown of endogenous DdGSTA2 caused a 60 percent reduction in proliferation, delayed development, and altered morphogenesis of fruiting bodies, whereas overexpression of rDdGSTA2 enzyme had no effect. These findings corroborate previous studies that implicate a role for phase II GST enzymes in cell proliferation, homeostasis, and development in eukaryotic cells.


Assuntos
Dictyostelium/enzimologia , Glutationa Transferase/metabolismo , Proliferação de Células , Dictyostelium/crescimento & desenvolvimento , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/genética , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de Proteína
4.
Arch Biochem Biophys ; 699: 108733, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33388313

RESUMO

Muscle myosins are molecular motors that hydrolyze ATP and generate force through coordinated interactions with actin filaments, known as cross-bridge cycling. During the cross-bridge cycle, functional sites in myosin 'sense' changes in interactions with actin filaments and the nucleotide binding region, resulting in allosteric transmission of information throughout the structure. We investigated whether the dynamics of the post-powerstroke state of the cross-bridge cycle are modulated in a nucleotide-dependent fashion. We compared molecular dynamics simulations of the myosin II motor domain (M) from Dictyostelium discoideum in the presence of ADP (M.ADP) versus 2'-deoxy-ADP bound myosin (M.dADP). We found that dADP was more flexible than ADP and the two nucleotides interacted with myosin in different ways. Replacement of ADP with dADP in the post-powerstroke state also altered the conformation of the actin binding region in myosin heads. Our results provide atomic level insights into allosteric communication networks in myosin that provide insight into the nucleotide-dependent dynamics of the cross-bridge cycle.


Assuntos
Nucleotídeos de Desoxiadenina/metabolismo , Miosina Tipo II/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Sítios de Ligação , Nucleotídeos de Desoxiadenina/química , Dictyostelium/enzimologia , Simulação de Dinâmica Molecular , Miosina Tipo II/química , Maleabilidade , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Domínios Proteicos
5.
Org Biomol Chem ; 19(2): 370-374, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33337456

RESUMO

The main product of DpTPS9 from the social amoeba Dictyostelium purpureum was identified as (4S,7R)-germacra-(1(10)E,5E)-dien-11-ol that is also known as an intermediate of bacterial geosmin synthase, but the experimentally verified cyclisation mechanisms differ. Together with the low sequence identity this points to convergent evolution. The functionality of selected residues in DpTPS9 was investigated via site-directed mutagenesis experiments.


Assuntos
Dictyostelium/enzimologia , Ligases/metabolismo , Sesquiterpenos de Germacrano/metabolismo , Streptomyces coelicolor/enzimologia , Ciclização , Ligases/genética , Mutagênese Sítio-Dirigida , Especificidade da Espécie
6.
J Biol Chem ; 295(49): 16545-16561, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-32934009

RESUMO

In animals, the response to chronic hypoxia is mediated by prolyl hydroxylases (PHDs) that regulate the levels of hypoxia-inducible transcription factor α (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the cellular slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-phase kinase-associated protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full-length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies, TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.


Assuntos
Dictyostelium/enzimologia , Prolil Hidroxilases/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Humanos , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Cinética , Simulação de Dinâmica Molecular , Oxigênio/metabolismo , Prolil Hidroxilases/química , Prolil Hidroxilases/genética , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Quinases Associadas a Fase S/química , Proteínas Quinases Associadas a Fase S/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
7.
Int J Mol Sci ; 21(15)2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32717856

RESUMO

Dictyostelium discoideum, the model organism for the evolutionary supergroup of Amoebozoa, is a social amoeba that, upon starvation, undergoes transition from a unicellular to a multicellular organism. In its genome, we identified two genes encoding for tRNA nucleotidyltransferases. Such pairs of tRNA nucleotidyltransferases usually represent collaborating partial activities catalyzing CC- and A-addition to the tRNA 3'-end, respectively. In D. discoideum, however, both enzymes exhibit identical activities, representing bona-fide CCA-adding enzymes. Detailed characterization of the corresponding activities revealed that both enzymes seem to be essential and are regulated inversely during different developmental stages of D. discoideum. Intriguingly, this is the first description of two functionally equivalent CCA-adding enzymes using the same set of tRNAs and showing a similar distribution within the cell. This situation seems to be a common feature in Dictyostelia, as other members of this phylum carry similar pairs of tRNA nucleotidyltransferase genes in their genome.


Assuntos
Dictyostelium , Genoma de Protozoário , Proteínas de Protozoários , RNA Nucleotidiltransferases , Dictyostelium/enzimologia , Dictyostelium/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo
8.
Biochim Biophys Acta Proteins Proteom ; 1868(9): 140460, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32474107

RESUMO

Eukaryotic serine racemase (SR) is a pyridoxal 5'-phosphate enzyme belonging to the Fold-type II group, which catalyzes serine racemization and is responsible for the synthesis of D-Ser, a co-agonist of the N-methyl-d-aspartate receptor. In addition to racemization, SR catalyzes the dehydration of D- and L-Ser to pyruvate and ammonia. The bifuctionality of SR is thought to be important for D-Ser homeostasis. SR catalyzes the racemization of D- and L-Ser with almost the same efficiency. In contrast, the rate of L-Ser dehydration catalyzed by SR is much higher than that of D-Ser dehydration. This has caused the argument that SR does not catalyze the direct D-Ser dehydration and that D-Ser is first converted to L-Ser, then dehydrated. In this study, we investigated the substrate and solvent isotope effect of dehydration of D- and L-Ser catalyzed by SR from Dictyostelium discoideum (DdSR) and demonstrated that the enzyme catalyzes direct D-Ser dehydration. Kinetic studies of dehydration of four Thr isomers catalyzed by D. discoideum and mouse SRs suggest that SR discriminates the substrate configuration at C3 but not at C2. This is probably the reason for the difference in efficiency between L- and D-Ser dehydration catalyzed by SR.


Assuntos
Desidratação , Eucariotos/enzimologia , Racemases e Epimerases/química , Racemases e Epimerases/metabolismo , Serina/metabolismo , Animais , Catálise , Cristalização , Dictyostelium/enzimologia , Cinética , Camundongos , Modelos Moleculares , Racemases e Epimerases/genética , Receptores de N-Metil-D-Aspartato , Especificidade por Substrato
9.
Biol Cell ; 112(8): 222-237, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32324907

RESUMO

BACKGROUND INFORMATION: Poly(ADP-ribose) Polymerase-1 (PARP-1) is predominantly a nuclear protein and involved in various cellular processes like DNA repair, cell death, development, chromatin modulation etc. PARP-1 utilizes NAD+ and adds negatively charged PAR moieties on the target proteins. Over-activation of PARP-1 has been shown to cause energy crisis mediated cell death in which mitochondrial homeostasis is also affected. Moreover, the presence of mitochondrial NAD+ pools highlights the role of PARP-1 in mitochondria. The aim of present study is to understand the physiological role of PARP-1 in regulating mitochondrial functioning by varying the levels of PARP-1 in Dictyostelium discoideum. Intra-mitochondrial PARylation was analyzed by indirect immunofluorescence. Further, the effect of altered levels of PARP-1 i.e. overexpression, downregulation, knockout and its chemical inhibition was studied on mitochondrial respiration, reactive oxygen species (ROS) levels, ATP production, mitochondrial fission-fusion, mitochondrial morphology and mitochondrial DNA (mtDNA) content of D. discoideum. RESULTS: Our results show intra-mitochondrial PARylation under oxidative stress. Altered levels of PARP-1 caused impairment in the mitochondrial respiratory capacity, leading to elevated ROS levels and reduced ATP production. Moreover, PARP-1 affects the mitochondrial morphology and mtDNA content, alters the mitochondrial fission-fusion processes in lieu of preventing cell death under physiological conditions. CONCLUSION: The current study highlights the physiological role of PARP-1 in mitochondrial respiration, its morphology, fission-fusion processes and mtDNA maintenance in D. discoideum. SIGNIFICANCE: This study would provide new clues on the PARP-1's crucial role in mitochondrial homeostasis, exploring the therapeutic potential of PARP-1 in various mitochondrial diseases.


Assuntos
Dictyostelium/enzimologia , Homeostase/efeitos dos fármacos , Mitocôndrias/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Trifosfato de Adenosina/biossíntese , Benzamidas/farmacologia , Morte Celular/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Dictyostelium/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Dinâmica Mitocondrial/genética , Estresse Oxidativo/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
BMC Res Notes ; 13(1): 16, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31910901

RESUMO

OBJECTIVE: The nuclear and mitochondrial genomes of Dictyostelium discoideum, a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectively. To begin to investigate how the pyrimidine biosynthetic pathway fulfills the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. RESULTS: The annotated genome of D. discoideum identifies a gene encoding a polypeptide containing the five conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the shortened N-termini that arose from the loss of residues 1-37. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.


Assuntos
Dictyostelium/enzimologia , Mitocôndrias/enzimologia , Pirofosfatases/química , Pirofosfatases/metabolismo , Sequência de Aminoácidos , Cinética , Família Multigênica , Multimerização Proteica , Transporte Proteico , Pirofosfatases/genética , Relação Estrutura-Atividade
11.
Nucleic Acids Res ; 48(5): 2209-2219, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31943056

RESUMO

Ongoing large-scale genome sequencing projects are forecasting a data deluge that will almost certainly overwhelm current analytical capabilities of evolutionary genomics. In contrast to population genomics, there are no standardized methods in evolutionary genomics for extracting evolutionary and functional (e.g. gene-trait association) signal from genomic data. Here, we examine how current practices of multi-species comparative genomics perform in this aspect and point out that many genomic datasets are under-utilized due to the lack of powerful methodologies. As a result, many current analyses emphasize gene families for which some functional data is already available, resulting in a growing gap between functionally well-characterized genes/organisms and the universe of unknowns. This leaves unknown genes on the 'dark side' of genomes, a problem that will not be mitigated by sequencing more and more genomes, unless we develop tools to infer functional hypotheses for unknown genes in a systematic manner. We provide an inventory of recently developed methods capable of predicting gene-gene and gene-trait associations based on comparative data, then argue that realizing the full potential of whole genome datasets requires the integration of phylogenetic comparative methods into genomics, a rich but underutilized toolbox for looking into the past.


Assuntos
Biologia Computacional/métodos , Epistasia Genética , Genoma , Família Multigênica , Filogenia , Animais , Celulase/classificação , Celulase/genética , Celulase/metabolismo , Sistema Enzimático do Citocromo P-450/classificação , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Dictyostelium/enzimologia , Dictyostelium/genética , Fungos/classificação , Fungos/enzimologia , Fungos/genética , Dosagem de Genes , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Phascolarctidae/genética , Phascolarctidae/metabolismo , Plantas/classificação , Plantas/genética , Plantas/metabolismo
12.
Cell Microbiol ; 22(1): e13129, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31652367

RESUMO

Phagocytic cells ingest bacteria by phagocytosis and kill them efficiently inside phagolysosomes. The molecular mechanisms involved in intracellular killing and their regulation are complex and still incompletely understood. Dictyostelium discoideum has been used as a model to discover and to study new gene products involved in intracellular killing of ingested bacteria. In this study, we performed random mutagenesis of Dictyostelium cells and isolated a mutant defective for growth on bacteria. This mutant is characterized by the genetic inactivation of the lrrkA gene, which encodes a protein with a kinase domain and leucine-rich repeats. LrrkA knockout (KO) cells kill ingested Klebsiella pneumoniae bacteria inefficiently. This defect is not additive to the killing defect observed in kil2 KO cells, suggesting that the function of Kil2 is partially controlled by LrrkA. Indeed, lrrkA KO cells exhibit a phenotype similar to that of kil2 KO cells: Intraphagosomal proteolysis is inefficient, and both intraphagosomal killing and proteolysis are restored upon exogenous supplementation with magnesium ions. Bacterially secreted folate stimulates intracellular killing in Dictyostelium cells, but this stimulation is lost in cells with genetic inactivation of kil2, lrrkA, or far1. Together, these results indicate that the stimulation of intracellular killing by folate involves Far1 (the cell surface receptor for folate), LrrkA, and Kil2. This study is the first identification of a signalling pathway regulating intraphagosomal bacterial killing in Dictyostelium cells.


Assuntos
Dictyostelium/enzimologia , Ácido Fólico/metabolismo , Fagossomos/microbiologia , Fosfotransferases/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Dictyostelium/genética , Dictyostelium/microbiologia , Regulação Bacteriana da Expressão Gênica , Espaço Intracelular/microbiologia , Klebsiella pneumoniae/metabolismo , Leucina/química , Fagocitose , Fosfotransferases/genética , Domínios Proteicos , Proteínas de Protozoários/genética
13.
J Exp Zool B Mol Dev Evol ; 332(8): 339-348, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31617664

RESUMO

John Bonner pointed out that microorganisms differ in several ways, some of which may reflect neutral phenotypic evolution. For making his case, Bonner referred to interspecies differences and morphological traits. Here we consider intraspecies differences and physiological traits. As a case-study, we examine the production of an extracellular cyclic 3 ' ,5 ' monophosphate phosphodiesterase in the cellular slime mold Dictyostelium discoideum. Temporal profiles of phosphodiesterase activity differ significantly between wild-type strains. From that we argue that the inference drawn initially from studies on a single wild-type, namely that the profile displayed by it pointed to an adaptive role, was mistaken. We generalize the conclusion to suggest that physiological differences exhibited by microorganisms of the same species may, but need not, reflect adaptations to different environments. Rather, the differences could be related to the fact that microorganisms live in groups whose composition can vary between homogeneous (clonal) and heterogeneous (polyclonal). More than one physiological profile is consistent with the normal development of the group in a given environment; the alternatives are neutral. When studying microbial physiology and behavior, it is expected that the observations are made on a clonal population; genetic (and so phenotypic) heterogeneity is carefully guarded against. As the example from D. discoideum shows, an unintended consequence of overlooking phenotypic heterogeneity is that one can fall into the trap of accepting a seemingly plausible, but possibly erroneous, adaptive explanation for a "normal" wild-type phenotype.


Assuntos
Evolução Biológica , Dictyostelium/fisiologia , Fenótipo , 3',5'-AMP Cíclico Fosfodiesterases , Adaptação Biológica , Dictyostelium/enzimologia
14.
Chem Commun (Camb) ; 55(88): 13255-13258, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31621716

RESUMO

The products of three terpene synthases from two social amoebae, Dictyostelium discoideum and Dictyostelium purpureum, were identified, showing sesquiterpene synthase activity for one and diterpene synthase activity for the other two enzymes. Site-directed mutagenesis experiments revealed the importance of a newly identfied highly conserved residue for catalytic activity. For one of the enzyme products, ß-araneosene, a bromonium ion induced transannular cyclisation was investigated, yielding the first brominated derivatives of this diterpene.


Assuntos
Alquil e Aril Transferases/metabolismo , Dictyostelium/enzimologia , Terpenos/metabolismo , Alquil e Aril Transferases/biossíntese , Biocatálise , Estrutura Molecular , Terpenos/química
15.
Elife ; 82019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31063135

RESUMO

Terpenoids are enormously diverse, but our knowledge of their biosynthesis and functions is limited. Here we report on a terpene synthase (DdTPS8)-cytochrome P450 (CYP521A1) gene cluster that produces a novel C12 trisnorsesquiterpene and affects the development of Dictyostelium discoideum. DdTPS8 catalyzes the formation of a sesquiterpene discoidol, which is undetectable from the volatile bouquet of wild type D. discoideum. Interestingly, a DdTPS8 knockout mutant lacks not only discoidol, but also a putative trisnorsesquiterpene. This compound was hypothesized to be derived from discoidol via cytochrome P450 (CYP)-catalyzed oxidative cleavage. CYP521A1, which is clustered with DdTPS8, was identified as a top candidate. Biochemical assays demonstrated that CYP521A1 catalyzes the conversion of discoidol to a novel trisnorsesquiterpene named discodiene. The DdTPS8 knockout mutant exhibited slow progression in development. This study points to the untapped diversity of natural products made by D. discoideum, which may have diverse roles in its development and chemical ecology.


Assuntos
Alquil e Aril Transferases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Dictyostelium/enzimologia , Dictyostelium/crescimento & desenvolvimento , Sesquiterpenos/metabolismo , Alquil e Aril Transferases/genética , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/genética , Dictyostelium/genética , Dictyostelium/metabolismo , Família Multigênica
16.
J Cell Sci ; 132(2)2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30617109

RESUMO

Macropinocytosis is an actin-driven process of large-scale and non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum In Dictyostelium, macropinocytic cups are organized by 'macropinocytic patches' in the plasma membrane. These contain activated Ras, Rac and phospholipid PIP3, and direct actin polymerization to their periphery. We show that a Dictyostelium Akt (PkbA) and an SGK (PkbR1) protein kinase act downstream of PIP3 and, together, are nearly essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Through phosphoproteomics, we identify a RhoGAP, GacG, as a PkbA and PkbR1 target, and show that it is required for efficient macropinocytosis and expansion of macropinocytic patches. The function of Akt and SGK in cell feeding through control of macropinosome size has implications for cancer cell biology.


Assuntos
Dictyostelium/enzimologia , Pinocitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Protozoários/metabolismo , Dictyostelium/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas de Protozoários/genética
17.
Nat Commun ; 9(1): 4481, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30367048

RESUMO

Phosphatidylinositol 3,4,5-trisphosphate (PIP3) and PIP3 phosphatase (PTEN) are enriched mutually exclusively on the anterior and posterior membranes of eukaryotic motile cells. However, the mechanism that causes this spatial separation between the two molecules is unknown. Here we develop a method to manipulate PIP3 levels in living cells and used it to show PIP3 suppresses the membrane localization of PTEN. Single-molecule measurements of membrane-association and -dissociation kinetics and of lateral diffusion reveal that PIP3 suppresses the PTEN binding site required for stable PTEN membrane binding. Mutual inhibition between PIP3 and PTEN provides a mechanistic basis for bistability that creates a PIP3-enriched/PTEN-excluded state and a PTEN-enriched/PIP3-excluded state underlying the strict spatial separation between PIP3 and PTEN. The PTEN binding site also mediates the suppression of PTEN membrane localization in chemotactic signaling. These results illustrate that the PIP3-PTEN bistable system underlies a cell's decision-making for directional movement irrespective of the environment.


Assuntos
Compartimento Celular/fisiologia , Polaridade Celular/fisiologia , Dictyostelium/citologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Protozoários/metabolismo , Sítios de Ligação/genética , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Quimiotaxia/fisiologia , AMP Cíclico/farmacologia , Dictyostelium/enzimologia , Dictyostelium/genética , Cinética , Modelos Biológicos , Mutação , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/genética , Ligação Proteica/efeitos dos fármacos , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Transdução de Sinais/fisiologia
18.
Sci Rep ; 8(1): 14361, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30254228

RESUMO

Dictyostelids, or social amoebae, have a unique life style in forming multicellular fruiting bodies from unicellular amoeboids upon starvation. Recently, dictyostelids were found to contain terpene synthase (TPS) genes, a gene type of secondary metabolism previously known to occur only in plants, fungi and bacteria. Here we report an evolutionary functional study of dictyostelid TPS genes. The number of TPS genes in six species of dictyostelids examined ranges from 1 to 19; and the model species Dictyostelium purpureum contains 12 genes. Using in vitro enzyme assays, the 12 TPS genes from D. purpureum were shown to encode functional enzymes with distinct product profiles. The expression of the 12 TPS genes in D. purpureum is developmentally regulated. During multicellular development, D. purpureum releases a mixture of volatile terpenes dominated by sesquiterpenes that are the in vitro products of a subset of the 12 TPS genes. The quality and quantity of the terpenes released from D. purpureum, however, bear little resemblance to those of D. discoideum, a closely related dictyostelid. Despite these variations, the conserved clade of dictyostelid TPSs, which have an evolutionary distance of more than 600 million years, has the same biochemical function, catalyzing the formation of a sesquiterpene protoillud-7-ene. Taken together, our results indicate that the dynamic evolution of dictyostelid TPS genes includes both purifying selection of an orthologous group and species-specific expansion with functional divergence. Consequently, the terpenes produced by these TPSs most likely have conserved as well as species-adaptive biological functions as chemical languages in dictyostelids.


Assuntos
Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Dictyostelium/enzimologia , Dictyostelium/genética , Evolução Molecular , Biocatálise , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Filogenia , Especificidade da Espécie , Terpenos/química , Terpenos/metabolismo , Volatilização
19.
Dis Model Mech ; 11(9)2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30135067

RESUMO

Valproic acid (VPA) provides a common treatment for both epilepsy and bipolar disorder; however, common cellular mechanisms relating to both disorders have yet to be proposed. Here, we explore the possibility of a diacylglycerol kinase (DGK) playing a role in regulating the effect of VPA relating to the treatment of both disorders, using the biomedical model Dictyostelium discoideum DGK enzymes provide the first step in the phosphoinositide recycling pathway, implicated in seizure activity. They also regulate levels of diacylglycerol (DAG), thereby regulating the protein kinase C (PKC) activity that is linked to bipolar disorder-related signalling. Here, we show that ablation of the single Dictyostelium dgkA gene results in reduced sensitivity to the acute effects of VPA on cell behaviour. Loss of dgkA also provides reduced sensitivity to VPA in extended exposure during development. To differentiate a potential role for this DGKA-dependent mechanism in epilepsy and bipolar disorder treatment, we further show that the dgkA null mutant is resistant to the developmental effects of a range of structurally distinct branched medium-chain fatty acids with seizure control activity and to the bipolar disorder treatment lithium. Finally, we show that VPA, lithium and novel epilepsy treatments function through DAG regulation, and the presence of DGKA is necessary for compound-specific increases in DAG levels following treatment. Thus, these experiments suggest that, in Dictyostelium, loss of DGKA attenuates a common cellular effect of VPA relating to both epilepsy and bipolar disorder treatments, and that a range of new compounds with this effect should be investigated as alternative therapeutic agents.This article has an associated First Person interview with the first author of the paper.


Assuntos
Transtorno Bipolar/tratamento farmacológico , Transtorno Bipolar/enzimologia , Diacilglicerol Quinase/metabolismo , Dictyostelium/enzimologia , Epilepsia/tratamento farmacológico , Epilepsia/enzimologia , Ácido Valproico/uso terapêutico , Sequência de Aminoácidos , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Transtorno Bipolar/patologia , Diacilglicerol Quinase/química , Dictyostelium/efeitos dos fármacos , Diglicerídeos/metabolismo , Epilepsia/patologia , Proteínas de Fluorescência Verde/metabolismo , Lítio/farmacologia , Lítio/uso terapêutico , Modelos Biológicos , Mutação/genética , Ácido Valproico/farmacologia
20.
Methods Mol Biol ; 1821: 371-392, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30062425

RESUMO

The model organism D. discoideum is well suited to investigate basic questions of molecular and cell biology, particularly those related to the structure, regulation, and dynamics of the cytoskeleton, signal transduction, cell-cell adhesion, and development. D. discoideum cells make use of Rho-regulated signaling pathways to reorganize the actin cytoskeleton during chemotaxis, endocytosis, and cytokinesis. In this organism the Rho family encompasses 20 members, several belonging to the Rac subfamily, but there are no representatives of the Cdc42 and Rho subfamilies. Here we present protocols suitable for monitoring the actin polymerization response and the activation of Rac upon stimulation of aggregation-competent cells with the chemoattractant cAMP, and for monitoring the localization and dynamics of Rac activity in live cells.


Assuntos
Quimiotaxia/fisiologia , Dictyostelium/enzimologia , Proteínas de Protozoários/metabolismo , Transdução de Sinais/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Fatores Quimiotáticos/metabolismo , AMP Cíclico/metabolismo , Dictyostelium/citologia , Dictyostelium/genética , Endocitose/fisiologia , Proteínas de Protozoários/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genética
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