RESUMEN
BACKGROUND INFORMATION: Two pore channels (TPCs) are voltage-gated ion channel superfamily members that release Ca2+ from acidic intracellular stores and are ubiquitously present in both animals and plants. Starvation initiates multicellular development in Dictyostelium discoideum. Increased intracellular calcium levels bias Dictyostelium cells towards the stalk pathway and thus we decided to analyze the role of TPC2 in development, differentiation, and autophagy. RESULTS: We showed TPC2 protein localizes in lysosome-like acidic vesicles and the in situ data showed stalk cell biasness. Deletion of tpc2 showed defective and delayed development with formation of multi-tipped structures attached to a common base, while tpc2OE cells showed faster development with numerous small-sized aggregates and wiry fruiting bodies. The tpc2OE cells showed higher intracellular cAMP levels as compared to the tpc2- cells while pinocytosis was found to be higher in the tpc2- cells. Also, TPC2 regulates cell-substrate adhesion and cellular morphology. Under nutrient starvation, deletion of tpc2 reduced autophagic flux as compared to Ax2. During chimera formation, tpc2- cells showed a bias towards the prestalk/stalk region while tpc2OE cells showed a bias towards the prespore/spore region. tpc2 deficient strain exhibits aberrant cell-type patterning and loss of distinct boundary between the prestalk/prespore regions. CONCLUSION: TPC2 is required for effective development and differentiation in Dictyostelium and supports autophagic cell death and cell-type patterning. SIGNIFICANCE: Decreased calcium due to deletion of tpc2 inhibit autophagic flux.
Asunto(s)
Autofagia , Dictyostelium , Proteínas Protozoarias , Dictyostelium/genética , Dictyostelium/metabolismo , Dictyostelium/citología , Dictyostelium/crecimiento & desarrollo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Eliminación de Gen , Canales de Calcio/metabolismo , Canales de Calcio/genética , Calcio/metabolismo , Diferenciación CelularRESUMEN
Nutrient-sensing plays a crucial role in maintaining cellular energy and metabolic homeostasis. Perturbations in sensing pathways are associated with a wide variety of pathologies, especially metabolic diseases. Very little is understood about sensing fluctuations in nutrients and how this information is integrated into physiological and metabolic adaptation that could further affect cell-fate decisions during differentiation in Dictyostelium discoideum (henceafter, Dictyostelium). Glucose is the primary metabolic fuel among all nutrients. Carbohydrates, lipids and proteins ultimately breakdown into glucose, which is further used for providing energy. The maintenance of optimum glucose levels is important for efficient cell-survival. Glucose is not only a nutrient, but also a signaling molecule influencing cell growth and differentiation in Dictyostelium. Modulation of endogenous glucose levels either by varying exogenous glucose levels or genetic overexpression or deletion of genes involved in glucose signaling lead to changes in endogenous metabolite levels such as ADP/ATP ratio, NAD+ /NADH ratio, cAMP and ROS levels which further influence cell-fate decisions. Here, we show that AMPKα and Sir2D are components of glucose-signaling pathway in Dictyostelium which adjust cell metabolism interdependently in response to nutrient-status and promote cell-fate decisions.
Asunto(s)
Dictyostelium , Dictyostelium/genética , Dictyostelium/metabolismo , Transducción de Señal , Diferenciación Celular , Ciclo Celular , Glucosa/metabolismoRESUMEN
Autophagy is a degradation process, wherein long-lived proteins, damaged organelles, and protein aggregates are degraded to maintain cellular homeostasis. Upon starvation, 5'-AMP-activated protein kinase (AMPK) initiates autophagy. We show that ampkα- cells exhibit 50% reduction in pinocytosis and display defective phagocytosis. Re-expression of AMPKα in ampkα- cells co-localizes with red fluorescence protein-tagged bacteria. The ampkα- cells show reduced cell survival and autophagic flux under basal and starvation conditions. Co-immunoprecipitation studies show conservation of the AMPK-ATG1 axis in basal autophagy. Computational analyses suggest that the N-terminal region of DdATG1 is amenable for interaction with AMPK. Furthermore, ß-actin was found to be a novel interacting partner of AMPK, attributed to the alteration in macropinocytosis and phagocytosis in the absence of AMPK. Additionally, ampkα- cells exhibit enhanced poly-ubiquitinated protein levels and allied large ubiquitin-positive protein aggregates. Our findings suggest that AMPK provides links among pinocytosis, phagocytosis, autophagy, and is a requisite for basal autophagy in Dictyostelium.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia , Proteínas Protozoarias/metabolismo , Proteínas Quinasas Activadas por AMP/deficiencia , Proteínas Quinasas Activadas por AMP/genética , Actinas/genética , Actinas/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Dictyostelium , Regulación Enzimológica de la Expresión Génica , Simulación de Dinámica Molecular , Fagocitosis , Pinocitosis , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/genética , Transducción de SeñalRESUMEN
In the present study, we have analyzed in detail the functions of Htt during growth and development of the protist, Dictyostelium discoideum by creating mutants (both overexpressor and knockout). The mRNA was present at all stages of growth and development. Overexpression of htt did not show any major anomaly, while deletion resulted in delayed aggregation territory formation and showed asynchronous development especially after slug stage. The slugs formed by htt - cells showed aberration in anterior-posterior boundary, showing increased prestalk region. DdHtt regulates STAT transcription factors in the tip organizer region that help maintain patterning and culmination. In chimeras with the wild-type, htt - cells preferentially localized to the tip of the slug and basal disc regions of the fruiting body showing prestalk/stalk bias, while the overexpressing cells majorly populated the prespore/spore region showing spore bias. These differences could be attributed to protein kinase A (PKA)-regulated cyclic adenosine monophosphate (cAMP) signaling.
Asunto(s)
AMP Cíclico/metabolismo , Dictyostelium/metabolismo , Eliminación de Gen , Proteínas Protozoarias/metabolismo , Transducción de Señal , Linaje de la Célula , Proliferación Celular/genética , Dictyostelium/citología , Dictyostelium/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Agregado de Proteínas , Proteínas Protozoarias/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción STAT/metabolismo , Esporas/citología , Esporas/metabolismoRESUMEN
The unicellular, as well as multicellular stages of Dictyostelium discoideum's life cycle, make it an excellent model system for cell type determination, differentiation, development, and cell death studies. Our preliminary results show the involvement of poly (ADP-ribose) polymerase-1 (PARP-1) during D. discoideum growth by its constitutive downregulation as well as by its ortholog overexpression. The current study now analyzes and strengthens the role of the PARP-1 ortholog in cellular proliferation of D. discoideum. ADPRT1A was knocked out (KO) from D. discoideum and studied for its effect on cell growth, cell cycle, morphology, and oxidative stress. The present findings show that ADPRT1A KO ( A KO) cells exhibited reduced cellular proliferation, stressed phenotype, and cell cycle arrest in G2-M phase. Under oxidative stress, A KO cells exhibited slower growth and DNA damage. This is the first report where the involvement of ADPRT1A in growth in D. discoideum is established.
Asunto(s)
Proliferación Celular/fisiología , Dictyostelium/metabolismo , Dictyostelium/fisiología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/fisiología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Daño del ADN/fisiología , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/fisiología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiologíaRESUMEN
The Serine Threonine kinase Receptor Associated Protein (STRAP) is a WD40 containing protein that provides a platform for protein interactions during cell proliferation and development. Overexpression and misregulation of STRAP contributes to various carcinomas that are now recognized as therapeutic targets especially for colorectal and lung cancers. The present study was undertaken to find an effective drug against this molecule using a simple system like Dictyostelium discoideum; which shares close homology to humans. Using techniques like structural modeling, molecular dynamics (MD) simulation and molecular docking, we found similar structure and dynamic behaviors in both, except for the presence of dissimilar numbers of ß-sheets and loop segments. We identified a novel and potential drug targeted to STRAP. The results obtained allow us to use Dictyostelium as a model system for further in vivo studies. Finally, the results of protein-protein interactions using molecular docking and essential dynamics studies show STRAP to participate in TGF-ß signaling in humans. Further, we show some structural units that govern the interaction of TGFß-RI with STRAP and Smad7 proteins in TGF-ß signaling pathway. In conclusion, we propose that D. discoideum can be used for enhancing our knowledge about STRAP protein.
Asunto(s)
Dictyostelium/química , Descubrimiento de Drogas/métodos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas de Neoplasias/química , Antineoplásicos , Sitios de Unión , Humanos , Enlace de Hidrógeno , Ligandos , Proteínas de Neoplasias/antagonistas & inhibidores , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas de Unión al ARN , Receptor Tipo I de Factor de Crecimiento Transformador beta/química , Proteína smad7/química , Homología Estructural de ProteínaRESUMEN
We have earlier classified the homeobox containing genes from Dictyostelium discoideum and identified 5 genes belonging to the TALE class. Here, we have characterized hbx9, a member of TALE class. In situ hybridization results show it to be preferentially expressed in the prestalkA (pstA) cells but not in the prestalkO (pstO) cells. Disruption of the hbx9 gene in Ax2 cells delay initiation of development and also slows cell proliferation. There was decreased cadA expression during the development of hbx9- cells, which results in inappropriate cell contacts, altered cell motility and patterning properties. ecmA and ecmB were mis-expressed in the pstO and pstA regions resulting in an effectively smaller pstO region. Surprisingly, the total ecmAO expression in pstAO cells was comparable to the wild type, suggesting that in hbx9- cells, ecmA was expressed in the pstO region. In hbx9-, proportioning and patterning was altered in favour of pstA cell-type. Moreover, precocious expression of ecmB and pspA was also observed in the vegetative cells. Expression profiling of components of cAMP signalling suggest that Hbx9 controls cell proportioning of prestalk cells by modulating cAMP signalling during growth-to-development transition.
Asunto(s)
Diferenciación Celular/genética , Dictyostelium/genética , Proteínas de Homeodominio/genética , Morfogénesis/genética , Proteínas Protozoarias/genética , Animales , Dictyostelium/crecimiento & desarrollo , Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Poly (ADP-ribose) polymerase (PARP)-1 regulates various biological processes like DNA repair, cell death etc. However, the role of PARP-1 in growth and differentiation still remains elusive. The present study has been undertaken to understand the role of PARP-1 in growth and development of a unicellular eukaryote, Dictyostelium discoideum. In silico analysis demonstrates ADPRT1A as the ortholog of human PARP-1 in D. discoideum. The present study shows that ADPRT1A overexpression (A OE) led to slow growth of D. discoideum and significant population of AOE cells were in S and G2/M phase. Also, AOE cells exhibited high endogenous PARP activity, significant NAD(+) depletion and also significantly lower ADPRT1B and ADPRT2 transcript levels. Moreover, AOE cells are intrinsically stressed and also exhibited susceptibility to oxidative stress. AOE also affected development of D. discoideum predominantly streaming, aggregation and formation of early culminant which are concomitant with reports on PARP's role in D. discoideum development. In addition, under developmental stimuli, increased PARP activity was seen along with developmentally regulated transcript levels of ADPRT1A during D. discoideum multicellularity. Thus the present study suggests that PARP-1 regulates growth as well as the developmental morphogenesis of D. discoideum, thereby opening new avenues to understand the same in higher eukaryotes.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Diferenciación Celular , Dictyostelium/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Estrés Oxidativo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , ADP Ribosa Transferasas/genética , Secuencia de Aminoácidos , Animales , Apoptosis , Western Blotting , Ciclo Celular , Células Cultivadas , Dictyostelium/enzimología , Dictyostelium/metabolismo , Potencial de la Membrana Mitocondrial , Filogenia , Poli(ADP-Ribosa) Polimerasa-1/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
TOR (target of rapamycin) protein kinase acts as a central controller of cell growth and development of an organism. Present study was undertaken to find the expression pattern and role of TOR during growth and development of Dictyostelium discoideum. Failures to generate either knockout and/or knockdown mutants indicate that interference with its levels led to cellular defects. Thus, the effects of TOR (DDB_G0281569) overexpression specifically, cells expressing Dd(Δ211-TOR)-Eyfp mutant was analyzed. Elevated expression of (Δ211-TOR)-Eyfp reduced both cell size and cell proliferation. DdTOR was found to be closer to fungus. mRNA level of TOR was found maximally in the freshly starved/aggregate cells that gradually declined. This was also strengthened by the expression patterns observed by in situ and the analysis of ß-galactosidase reporter driven by the putative TOR promoter. The TOR protein was found to be highest at the aggregate stage. The fusion protein, (Δ211-TOR)-Eyfp was localized to the cell membrane, cytosol, and the nucleus. We suggest, DdTOR to be an essential protein and high TOR expression inhibits cell proliferation.
Asunto(s)
Proliferación Celular/fisiología , Tamaño de la Célula , Dictyostelium/crecimiento & desarrollo , Dictyostelium/metabolismo , Serina-Treonina Quinasas TOR/genética , Proteínas Bacterianas/genética , Dictyostelium/genética , Proteínas Luminiscentes/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/biosíntesisRESUMEN
Peptide: 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 beta-galactosidase reporter driven by the putative pngase promoter) and protein (as studied by the analysis of beta-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 beta-galactosidase activity was present in the anterior part of the slug. In structures derived from a null mutant for 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.
Asunto(s)
Diferenciación Celular/genética , Dictyostelium/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Quimera , Dictyostelium/crecimiento & desarrollo , Galactosidasas/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/biosíntesis , Esporas/citología , Esporas/genéticaRESUMEN
Natural autophagy and autophagic cell death is being studied in the model system, D. discoideum, which has well known genetic and experimental advantages over the other known systems. There is no apoptotic machinery present in this organism which could interfere with the non-apoptotic cell death. The target of rapamycin (TOR) pathway is a major nutrient-sensing pathway which when inhibited by the drug rapamycin induces autophagy. Rapamycin was originally discovered as an anti-fungal agent but its use was abandoned when it was discovered to have potent immunosuppressive and anti-proliferative properties. It is a known drug used today for various cancer treatments and also for increasing longevity in many model organisms. It has a wide usage but its effects on other pathways or molecules are not known. This model system was used to study the action of rapamycin on autophagy induction. Using the GFP-Atg8, an autophagosome marker, it was shown that rapamycin treatment can induce autophagy by an accumulation of reactive oxygen species and intracellular free calcium. Rapamycin suppresses proliferation by induction of cell cycle arrest in the G1 phase. Taken together, the results suggest that the core machinery for autophagy is conserved in D. discoideum and it can serve as a good model system to delineate the action of rapamycin induced autophagy.
Asunto(s)
Autofagia/efectos de los fármacos , Dictyostelium/efectos de los fármacos , Dictyostelium/fisiología , Sirolimus/farmacología , Antioxidantes/metabolismo , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Fase G1/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: 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. RESULTS: In this study, the PNGase from the cellular slime mold, Dictyostelium discoideum (DdPNGase) 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 DdPNGase. The tertiary structure matched with the mouse PNGase. Here we show that DdPNGase 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 in situ 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. DdPNGase is a functional peptide:N-glycanase enzyme possessing deglycosylation activity, but does not possess any significant transamidation activity. CONCLUSIONS: We have identified and characterized a novel PNGase from D. discoideum and confirmed its deglycosylation activity. The results emphasize the importance of PNGase in aggregation during multicellular development of this organism.
Asunto(s)
Dictyostelium/enzimología , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Técnicas de Inactivación de Genes , Glicosilación , Ratones , Datos de Secuencia Molecular , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , ARN/metabolismo , Alineación de Secuencia , Homología de SecuenciaRESUMEN
Comparative modelling helps compare the structure and functions of a given protein, to track the path of its origin and evolution and also guide in structure-based drug discovery. Presently, this has been applied for modelling the tertiary structure of highly conserved eukaryotic TCTP (translationally controlled tumour protein) which is involved in a plethora of functions during growth and development and also acts as a biomarker for many cancers like lung, breast, and prostate cancer. The modelled TCTP structures of different organisms belonging to the eukaryotic group showed similar spatial arrangement of structural units except loops and similar patterns of root mean square deviation (RMSD), root mean square fluctuation, and radius of gyration (Rg) inspected through molecular dynamics simulations. Essential dynamics (ED) analyses revealed different domains that exhibited different motions for the assistance in its multifunctional properties. Construction of a free-energy landscape (FEL) based on Rg versus RMSD was employed to characterize the folding behaviours of structures and observe that all proteins had nearly similar conformation and topologies, indicating common thermodynamic/kinetic pathways. A physico-chemical interaction study demonstrated the helices and sheets were well stabilized with ample amounts of bonding compared to turns or loops and charged residues were more accessible to solvent molecules. Hence, the current study reveals the important structural features of TCTP that aid in diverse functions in a wide range of organisms, thus extending our knowledge of TCTP and also providing a venue for designing the potent inhibitors against it.
RESUMEN
Simple organisms are preferred for understanding the molecular and cellular function(s) of complex processes. Dictyostelium discoideum is a lower eukaryote, a protist and a cellular slime mould, which has been in recent times used for various studies such as cell differentiation, development, cell death, stress responses etc. It is a soil amoeba (unicellular) that undertakes a remarkable, facultative shift to multicellularity when exposed to starvation and requires signal pathways that result in alteration of gene expression and finally show cell differentiation. The amoebae aggregate, differentiate and form fruiting bodies with two terminally differentiated cells: the dead stalk (non-viable) and dormant spores (viable). In India, starting from the isolation of Dictyostelium species to morphogenesis, cell signalling and social evolution has been studied with many more new research additions. Advances in molecular genetics make Dictyostelium an attractive model system to study cell biology, biochemistry, signal transduction and many more.
Asunto(s)
Evolución Biológica , Investigación Biomédica/tendencias , Dictyostelium/fisiología , Regulación de la Expresión Génica , Morfogénesis , Animales , Diferenciación Celular , India , Modelos BiológicosRESUMEN
Translationally controlled tumor protein (TCTP) is a multifunctional protein implicated in various types of cellular processes involving growth and development of an organism. Here, we identified tctp gene in Dictyostelium discoideum and unraveled its function. The sequence analysis of D. discoideum TCTP (DdTCTP) showed its conservation among eukaryotes. Transcript of DdTCTP was highly expressed at the initial time points of development and protein is localized both in the cytoplasm and nucleus. Disruption of tctp was achieved by BSR cassette using double homologous recombination method. Abrogation of tctp resulted in reduced cell proliferation but increased cell size. Additionally, development was delayed by 4 h wherein small-sized aggregates and fruiting bodies were produced by tctp - cells while larger aggregates and fruiting bodies were produced by tctp OE cells concordant with the fact that TCTP regulates prestalk/prespore ratio and cell-type differentiation. tctp - cells produced round spores with reduced viability and stalk cells are arranged in septate pattern as compared to polyhedral manner of wild type. Abrogation of tctp resulted in aberrant localization of cell type specific markers and show low proclivity toward prespore/spore region, in presence of wild type cells.
RESUMEN
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 Dictyostelium 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 in vitro experiments.
Asunto(s)
Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Modelos Biológicos , Terapia Molecular Dirigida , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Humanos , Ligandos , Masculino , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Proteínas Protozoarias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/metabolismo , TermodinámicaRESUMEN
The etoposide-induced 2.4 kb transcript (ei24) gene is induced both by p53 and etoposide, an anti-cancer tumour drug. There is no p53 gene present in Dictyostelium discoideum. Thus, the functions of ei24 in the absence of p53 were analysed. Both overexpressor (ei24OE) and knockout (ei24-) mutants were made to study its role during growth, development and differentiation. Additionally, cell cycle and its response to DNA-damage were also analysed. We identified, characterized and elucidated the functions of the ei24 gene in Dictyostelium. In silico analyses demonstrated the conservation across eukaryotes and in situ hybridization showed it to be prestalk-specific. ei24- cells showed reduced cell proliferation and cell-cohesive properties, ultimately forming small-sized aggregates that developed into miniature and stalky fruiting bodies. The ei24OE cells formed fruiting bodies with engorged or double-decker type sori with short stalks. The ei24- cells showed reduced cAMP signalling with lower intracellular cAMP levels resulting in diminished migration of cells along cAMP gradients. Deletion of ei24 resulted in mis-expression of prestalk-specific markers. Cell cycle analysis revealed an increased bias towards the stalk-pathway by ei24- cells and vice-versa for ei24OE cells. EI24 in Dictyostelium functions even in the absence of p53 and is induced in response to both UV-radiation and etoposide treatments. ei24OE cells showed enhanced DNA-damage repair mechanisms. Also, etoposide treatment and overexpression of ei24 caused G2/M arrest in the cell cycle. Our results indicate that EI24 is important for the growth, development and differentiation of Dictyostelium apart from being a DNA-damage response gene.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Proliferación Celular/genética , Dictyostelium/genética , Proteínas Nucleares/genética , Antineoplásicos Fitogénicos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Dictyostelium/citología , Dictyostelium/efectos de los fármacos , Dictyostelium/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Etopósido/farmacología , Proteínas Nucleares/metabolismo , Organismos Modificados Genéticamente , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
The polyamines putrescine, spermidine and spermine are essential polycations involved in the regulation of cellular proliferation. They exert dynamic effects on nucleic acids and macromolecular synthesis in vitro but their specific functions in vivo are poorly understood. Here, we have modulated the spermidine levels either by overexpressing the S-adenosylmethionine decarboxylase (samdc) gene or treating the cells with methylglyoxal-bis (guanylhydrazone) (MGBG), an inhibitor of SAMDC. In Dictyostelium, overexpression of SAMDC slowed cell proliferation, delayed development and arrested cells in the S-phase of the cell cycle. Treatment with MGBG reduced cell proliferation and stimulated development, but in samdcOE cells, it increased cell proliferation suggesting critical levels of spermidine to be important. In samdcOE cells, spermidine levels remained high throughout development but only small changes in the spermine levels were observed. Initial putrescine levels did increase but reverted to wild-type levels after the mound stage. As tight regulation of polyamine homeostasis is required, we identified genes that could be involved in its maintenance. In conclusion, we characterised samdcOE cells and observed the maintenance of polyamine homeostasis during the development of Dictyostelium cells.
Asunto(s)
Adenosilmetionina Descarboxilasa/metabolismo , Dictyostelium/crecimiento & desarrollo , Dictyostelium/metabolismo , Homeostasis , Poliaminas/metabolismo , Adenosilmetionina Descarboxilasa/antagonistas & inhibidores , Animales , Dictyostelium/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Mitoguazona/farmacologíaRESUMEN
Sirtuins are a family of deacetylases (Class III histone deacetylases) with evolutionarily conserved functions in cellular metabolism and chromatin regulation. Out of the seven human Sirtuins, the function of Sirt2 is the least understood. The purpose of the present study was to investigate the role of Sir2A, a homolog of human Sirt2 in Dictyostelium discoideum (Dd), a lower eukaryote. We created both overexpressing and deletion strains of Ddsir2A to analyse its functions. We observed sir2A mRNA expression throughout development and the transcript was present in the prespore/spore region of multicellular structures developed. They show a preference towards prestalk/stalk pathway when co-developed with wildtype cells during chimera formation. Deletion strain showed a multi-tipped phenotype, decrease in cell proliferation and inhibition of autophagy. In conclusion, our results show low cAMP levels, reduced cell-adhesion, weak cell migration and impaired autophagy to be responsible for the phenotype shown by the null cells. This study provides new insights into the functions of Ddsir2A.
Asunto(s)
Autofagia/genética , Diferenciación Celular/genética , Proliferación Celular/genética , Sirtuinas/genética , Adhesión Celular/genética , AMP Cíclico/química , Dictyostelium/genética , Eliminación de Secuencia/genéticaRESUMEN
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 (ACGDeltacat) and was driven by a UV-inducible promoter. After UV induction of ACGDeltacat, 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 ACGDeltacat. 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.