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/genéticaRESUMO
In many phenomena of biological systems, not a majority, but a minority of cells act on the entire multicellular system causing drastic changes in the system properties. To understand the mechanisms underlying such phenomena, it is essential to observe the spatiotemporal dynamics of a huge population of cells at sub-cellular resolution, which is difficult with conventional tools such as microscopy and flow cytometry. Here, we describe an imaging system named AMATERAS that enables optical imaging with an over-one-centimeter field-of-view and a-few-micrometer spatial resolution. This trans-scale-scope has a simple configuration, composed of a low-power lens for machine vision and a hundred-megapixel image sensor. We demonstrated its high cell-throughput, capable of simultaneously observing more than one million cells. We applied it to dynamic imaging of calcium ions in HeLa cells and cyclic-adenosine-monophosphate in Dictyostelium discoideum, and successfully detected less than 0.01% of rare cells and observed multicellular events induced by these cells.
Assuntos
Células/citologia , Microscopia de Fluorescência/métodos , Animais , Encéfalo/citologia , Cálcio/análise , AMP Cíclico/análise , Dictyostelium/química , Dictyostelium/ultraestrutura , Cães , Entose , Células Epiteliais/ultraestrutura , Desenho de Equipamento , Proteínas de Fluorescência Verde , Células HeLa/química , Células HeLa/ultraestrutura , Humanos , Interneurônios/ultraestrutura , Proteínas Luminescentes , Células Madin Darby de Rim Canino , Camundongos , Microscopia de Fluorescência/instrumentação , Neurônios/ultraestrutura , Semicondutores , Proteína Vermelha FluorescenteRESUMO
Ceramides are a special class of sphingolipids and play a central role in sphingolipid metabolism, and have diverse structures. In this book chapter, tandem quadrupole mass spectrometric approaches applying multiple linked scannings including various constant neutral loss scan (NLS) and precursor ion scan (PIS), the unique applicable feature of a triple-stage quadrupole (TSQ) instrument for analysis of ceramides desorbed as [M-H]- and [M+Li]+ ions are described. These multiple dimensional tandem mass spectrometric approaches are fully adapted to the conventional shotgun lipidomics workflow with minimal or without prior chromatographic separation to profile ceramide molecules, and thus detection of a whole class of ceramide or various specific ceramide subclasses in crude lipid extract can be achieved. With addition of internal standard(s), semi-quantitation of ceramide in the lipid extract of biological origin is possible. Examples have shown promise in ceramide profiling of several whole lipid extracts from porcine brain, the model Dictyostelium Discoideum cells for cancer study, and skin.
Assuntos
Ceramidas/análise , Dictyostelium/química , Lipidômica/métodos , Pele/química , Animais , Química Encefálica , Humanos , Espectrometria de Massas por Ionização por Electrospray , Suínos , Espectrometria de Massas em TandemRESUMO
The trajectory of moving eukaryotic cells depends on the kinetics and direction of extending pseudopods. The direction of pseudopods has been well studied to unravel mechanisms for chemotaxis, wound healing and inflammation. However, the kinetics of pseudopod extension-when and why do pseudopods start and stop- is equally important, but is largely unknown. Here the START and STOP of about 4000 pseudopods was determined in four different species, at four conditions and in nine mutants (fast amoeboids Dictyostelium and neutrophils, slow mesenchymal stem cells, and fungus B.d. chytrid with pseudopod and a flagellum). The START of a first pseudopod is a random event with a probability that is species-specific (23%/s for neutrophils). In all species and conditions, the START of a second pseudopod is strongly inhibited by the extending first pseudopod, which depends on parallel filamentous actin/myosin in the cell cortex. Pseudopods extend at a constant rate by polymerization of branched F-actin at the pseudopod tip, which requires the Scar complex. The STOP of pseudopod extension is induced by multiple inhibitory processes that evolve during pseudopod extension and mainly depend on the increasing size of the pseudopod. Surprisingly, no differences in pseudopod kinetics are detectable between polarized, unpolarized or chemotactic cells, and also not between different species except for small differences in numerical values. This suggests that the analysis has uncovered the fundament of cell movement with distinct roles for stimulatory branched F-actin in the protrusion and inhibitory parallel F-actin in the contractile cortex.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Miosinas/metabolismo , Pseudópodes/fisiologia , Citoesqueleto de Actina/química , Citoesqueleto de Actina/fisiologia , Actinas/química , Animais , Movimento Celular/fisiologia , Quimiotaxia/fisiologia , Dictyostelium/química , Dictyostelium/fisiologia , Fungos/química , Fungos/fisiologia , Cinética , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/fisiologia , Miosinas/química , Neutrófilos/química , Neutrófilos/fisiologia , Pseudópodes/metabolismoRESUMO
The actomyosin system generates mechanical work with the execution of the power stroke, an ATP-driven, two-step rotational swing of the myosin-neck that occurs post ATP hydrolysis during the transition from weakly to strongly actin-bound myosin states concomitant with Pi release and prior to ADP dissociation. The activating role of actin on product release and force generation is well documented; however, the communication paths associated with weak-to-strong transitions are poorly characterized. With the aid of mutant analyses based on kinetic investigations and simulations, we identified the W-helix as an important hub coupling the structural changes of switch elements during ATP hydrolysis to temporally controlled interactions with actin that are passed to the central transducer and converter. Disturbing the W-helix/transducer pathway increased actin-activated ATP turnover and reduced motor performance as a consequence of prolonged duration of the strongly actin-attached states. Actin-triggered Pi release was accelerated, while ADP release considerably decelerated, both limiting maximum ATPase, thus transforming myosin-2 into a high-duty-ratio motor. This kinetic signature of the mutant allowed us to define the fractional occupancies of intermediate states during the ATPase cycle providing evidence that myosin populates a cleft-closure state of strong actin interaction during the weak-to-strong transition with bound hydrolysis products before accomplishing the power stroke.
Assuntos
Actomiosina/química , Difosfato de Adenosina/química , Dictyostelium/química , Fosfatos/química , Proteínas de Protozoários/química , Actomiosina/genética , Trifosfato de Adenosina/química , Regulação Alostérica , Dictyostelium/genética , Proteínas de Protozoários/genéticaRESUMO
We report a protoilludane-type sesquiterpene, mucoroidiol, and a geranylated bicyclogermacranol, firmibasiol, isolated from Dictyostelium cellular slime molds. The methanol extracts of the fruiting bodies of cellular slime molds were separated by chromatographic methods to give these compounds. Their structures have been established by several spectral means. Mucoroidiol and firmibasiol are the first examples of more modified and oxidized terpenoids isolated from cellular slime molds. Mucoroidiol showed moderate osteoclast-differentiation inhibitory activity despite demonstrating very weak cell-proliferation inhibitory activity. Therefore, cellular slime molds produce considerably diverse secondary metabolites, and they are promising sources of new natural product chemistry.
Assuntos
Dictyostelium/química , Terpenos/isolamento & purificação , Animais , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Dictyostelium/metabolismo , Escherichia coli/efeitos dos fármacos , Células HeLa , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Camundongos , Testes de Sensibilidade Microbiana , Osteogênese/efeitos dos fármacos , Células RAW 264.7 , Staphylococcus aureus/efeitos dos fármacos , Terpenos/química , Terpenos/farmacologiaRESUMO
PTEN, a 3-phosphatase of phosphoinositide, regulates asymmetric PI(3,4,5)P3 signaling for the anterior-posterior polarization and migration of motile cells. PTEN acts through posterior localization on the plasma membrane, but the mechanism for this accumulation is poorly understood. Here we developed an in vitro single-molecule imaging assay with various lipid compositions and use it to demonstrate that the enzymatic product, PI(4,5)P2, stabilizes PTEN's membrane-binding. The dissociation kinetics and lateral mobility of PTEN depended on the PI(4,5)P2 density on artificial lipid bilayers. The basic residues of PTEN were responsible for electrostatic interactions with anionic PI(4,5)P2 and thus the PI(4,5)P2-dependent stabilization. Single-molecule imaging in living Dictyostelium cells revealed that these interactions were indispensable for the stabilization in vivo, which enabled efficient cell migration by accumulating PTEN posteriorly to restrict PI(3,4,5)P3 distribution to the anterior. These results suggest that PI(4,5)P2-mediated positive feedback and PTEN-induced PI(4,5)P2 clustering may be important for anterior-posterior polarization.
Assuntos
Membranas/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Polaridade Celular , Células Cultivadas , Dictyostelium/química , Dictyostelium/metabolismo , Retroalimentação Fisiológica/fisiologia , PTEN Fosfo-Hidrolase/análise , Fosfatidilinositol 4,5-Difosfato/análise , Ligação Proteica , Imagem Individual de Molécula/métodosRESUMO
Dictyostelium discoideum is gaining increasing attention as a model organism for the study of calcium binding and calmodulin function in basic biological events as well as human diseases. After a short overview of calcium-binding proteins, the structure of Dictyostelium calmodulin and the conformational changes effected by calcium ion binding to its four EF hands are compared to its human counterpart, emphasizing the highly conserved nature of this central regulatory protein. The calcium-dependent and -independent motifs involved in calmodulin binding to target proteins are discussed with examples of the diversity of calmodulin binding proteins that have been studied in this amoebozoan. The methods used to identify and characterize calmodulin binding proteins is covered followed by the ways Dictyostelium is currently being used as a system to study several neurodegenerative diseases and how it could serve as a model for studying calmodulinopathies such as those associated with specific types of heart arrythmia. Because of its rapid developmental cycles, its genetic tractability, and a richly endowed stock center, Dictyostelium is in a position to become a leader in the field of calmodulin research.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Dictyostelium/metabolismo , Proteínas de Protozoários/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Sinalização do Cálcio , Calmodulina/química , Proteínas de Ligação a Calmodulina/química , Dictyostelium/química , Motivos EF Hand , Humanos , Modelos Moleculares , Ligação Proteica , Infecções por Protozoários/parasitologia , Proteínas de Protozoários/químicaRESUMO
Cytoplasmic dynein is a dimeric motor protein which processively moves along microtubule. Its motor domain (head) hydrolyzes ATP and induces conformational changes of linker, stalk, and microtubule binding domain (MTBD) to trigger stepping motion. Here we applied scattering imaging of gold nanoparticle (AuNP) to visualize load-free stepping motion of processive dynein. We observed artificially-dimerized chimeric dynein, which has the head, linker, and stalk from Dictyostelium discoideum cytoplasmic dynein and the MTBD from human axonemal dynein, whose structure has been well-studied by cryo-electron microscopy. One head of a dimer was labeled with 30 nm AuNP, and stepping motions were observed with 100 µs time resolution and sub-nanometer localization precision at physiologically-relevant 1 mM ATP. We found 8 nm forward and backward steps and 5 nm side steps, consistent with on- and off-axes pitches of binding cleft between αß-tubulin dimers on the microtubule. Probability of the forward step was 1.8 times higher than that of the backward step, and similar to those of the side steps. One-head bound states were not clearly observed, and the steps were limited by a single rate constant. Our results indicate dynein mainly moves with biased small stepping motion in which only backward steps are slightly suppressed.
Assuntos
Dineínas do Axonema/química , Dineínas do Citoplasma/química , Dictyostelium/química , Proteínas de Protozoários/química , Dineínas do Axonema/metabolismo , Fenômenos Bioquímicos , Microscopia Crioeletrônica , Dictyostelium/metabolismo , Ouro/química , Humanos , Nanopartículas Metálicas/química , Microtúbulos/química , Microtúbulos/metabolismo , Ligação Proteica , Proteínas de Protozoários/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMO
Blebs and pseudopods can both power cell migration, with blebs often favored in tissues, where cells encounter increased mechanical resistance. To investigate how migrating cells detect and respond to mechanical forces, we used a "cell squasher" to apply uniaxial pressure to Dictyostelium cells chemotaxing under soft agarose. As little as 100 Pa causes a rapid (<10 s), sustained shift to movement with blebs rather than pseudopods. Cells are flattened under load and lose volume; the actin cytoskeleton is reorganized, with myosin II recruited to the cortex, which may pressurize the cytoplasm for blebbing. The transition to bleb-driven motility requires extracellular calcium and is accompanied by increased cytosolic calcium. It is largely abrogated in cells lacking the Piezo stretch-operated channel; under load, these cells persist in using pseudopods and chemotax poorly. We propose that migrating cells sense pressure through Piezo, which mediates calcium influx, directing movement with blebs instead of pseudopods.
Assuntos
Dictyostelium/citologia , Dictyostelium/metabolismo , Canais Iônicos/metabolismo , Proteínas de Protozoários/metabolismo , Pseudópodes/metabolismo , Fenômenos Biomecânicos , Movimento Celular , Citoplasma/química , Citoplasma/genética , Citoplasma/metabolismo , Dictyostelium/química , Dictyostelium/genética , Canais Iônicos/genética , Mecanotransdução Celular , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Pressão , Proteínas de Protozoários/genética , Pseudópodes/genéticaRESUMO
Myosin II molecules in the thick filaments of striated muscle form a structure in which the heads interact with each other and fold back onto the tail. This structure, the "interacting heads motif" (IHM), provides a mechanistic basis for the auto-inhibition of myosin in relaxed thick filaments. Similar IHM interactions occur in single myosin molecules of smooth and nonmuscle cells in the switched-off state. In addition to the interaction between the two heads, which inhibits their activity, the IHM also contains an interaction between the motor domain of one head and the initial part (subfragment 2, S2) of the tail. This is thought to be a crucial anchoring interaction that holds the IHM in place on the thick filament. S2 appears to cross the head at a specific location within a broader region of the motor domain known as the myosin mesa. Here, we show that the positive and negative charge distribution in this part of the mesa is complementary to the charge distribution on S2. We have designated this the "mesa trail" owing to its linear path across the mesa. We studied the structural sequence alignment, the location of charged residues on the surface of myosin head atomic models, and the distribution of surface charge potential along the mesa trail in different types of myosin II and in different species. The charge distribution in both the mesa trail and the adjacent S2 is relatively conserved. This suggests a common basis for IHM formation across different myosin IIs, dependent on attraction between complementary charged patches on S2 and the myosin head. Conservation from mammals to insects suggests that the mesa trail/S2 interaction plays a key role in the inhibitory function of the IHM.
Assuntos
Miosina Tipo II/metabolismo , Animais , Aracnídeos/química , Aracnídeos/metabolismo , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Dictyostelium/química , Dictyostelium/metabolismo , Insetos , Mamíferos , Modelos Moleculares , Miosina Tipo II/química , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Especificidade da EspécieRESUMO
The yeast Saccharomyces cerevisiae has given us much information on the metabolism and function of inositol polyphosphates and inorganic polyphosphate. To expand our knowledge of the metabolic as well as functional connections between inositol polyphosphates and inorganic polyphosphate, we have refined and developed techniques to extract and analyze these molecules in a second eukaryotic experimental model, the amoeba Dictyostelium discoideum. This amoeba, possessing a well-defined developmental program, is ideal to study physiological changes in the levels of inositol polyphosphates and inorganic polyphosphate, since levels of both molecules increase at late stages of development. We detail here the methods used to extract inositol polyphosphates using perchloric acid and inorganic polyphosphate using acidic phenol. We also present the postextraction procedures to visualize and quantify these molecules by polyacrylamide gel electrophoresis and by malachite green assay.
Assuntos
Dictyostelium/crescimento & desenvolvimento , Polifosfatos/análise , Fracionamento Químico , Dictyostelium/química , Eletroforese em Gel de Poliacrilamida , Modelos Biológicos , Percloratos/química , Fenóis/química , Corantes de Rosanilina/químicaRESUMO
In this study, a two-photon fluorescence microscopic imaging technique is reported for assessment the effect of dynamic hypertonic environment on the overall energy metabolism alteration and adaptation of soil-living amoeba Dictyostelium discoideum. For that purpose the fluorescence intensity of mitochondrial reduced nicotinamide adenine dinucleotide (NADH) was monitored and quantified in order to evaluate the corresponded metabolic state of monolayer cultured cells. The two-photon excitation of NADH with 720 nm near infrared irradiation produced blue fluorescence emission with maximum wavelength centered at 460 nm. The benefits of reported noninvasive microscopic technique are the significantly less cellular damage and avoiding the excitation of other biomolecules except of NADH. It enabled to acquire data for NADH levels of the observed cells on agar plate specimen and hypertonic nutrition media in a Petri dish. The method demonstrated also good sensitivity, reproducibility and the obtained results revealed that D. discoideum species form aggregation in hypertonic environment within several minutes with aim to survive. The formed aggregate had amorphous shape and it consisted from dozen amoeba cells, which kept their NADH amount in constant level for few hours. The reported imaging method might be applicable in various studies for characterization of metabolic events and assessment of the cell energy balance in hypertonic environment.
Assuntos
Dictyostelium/metabolismo , Metabolismo Energético/efeitos dos fármacos , Exposição Ambiental , Microscopia de Fluorescência , Pressão Osmótica , Meios de Cultura/química , Dictyostelium/química , Dictyostelium/efeitos dos fármacos , NAD/análise , Solução Salina Hipertônica/metabolismoRESUMO
The eye arose during the Cambrian explosion from pre-existing proteins that would have been recruited for the formation of the specialized components of this organ, such as the transparent lens. Proteins suitable for the role of lens crystallins would need to possess unusual physical properties and the study of such earliest analogs of ocular crystallins would add to our understanding of the nature of recruitment of proteins as lens/corneal crystallins. We show that the Abundant Perithecial Protein (APP) of the fungi Neurospora and Sordaria fulfils the criteria for an early crystallin analog. The perithecia in these fungal species are phototropic, and APP accumulates at a high concentration in the neck of the pitcher-shaped perithecium. Spores are formed at the base of the perithecium, and light contributes to their maturation. The hydrodynamic properties of APP appear to exclude dimer formation or aggregation at high protein concentrations. APP is also deficient in Ca2+ binding, a property seen in its close homolog, the calcium-binding cell adhesion molecule (DdCAD-1) from Dictyostelium discoidum. Comparable to crystallins, APP occurs in high concentrations and seems to have dispensed with Ca2+ binding in exchange for greater stability. These crystallin-like attributes of APP lead us to demonstrate that it is a primitive form of ocular crystallins.
Assuntos
Proteínas de Ligação ao Cálcio/química , Cristalinas/química , Proteínas Fúngicas/química , Neurospora/química , Esporos Fúngicos/química , Animais , Sítios de Ligação , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Clonagem Molecular , Cristalinas/genética , Cristalinas/metabolismo , Dictyostelium/química , Dictyostelium/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cristalino/química , Cristalino/metabolismo , Luz , Modelos Moleculares , Neurospora/metabolismo , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sordariales/química , Sordariales/metabolismo , Esporos Fúngicos/metabolismo , Homologia Estrutural de ProteínaRESUMO
Triple-negative breast cancer (TNBC) is highly proliferative and metastatic, and because it lacks three major molecular targets for chemotherapy (estrogen receptor, progesterone receptor, and human epidermal receptor 2), it is extremely refractory. Differentiation-inducing factor 1 (DIF-1) and DIF-3, which are chlorinated alkylphenones, are lead anticancer compounds found in the cellular slime mold Dictyostelium discoideum. Here, we examined the in vitro effects of DIF-1, DIF-3, and 25 DIF derivatives on cell proliferation and serum-induced cell migration in human MDA-MB-231 cells, a model TNBC cell line. We found that Br-DIF-1, a chlorine-to-bromine-substituted derivative of DIF-1, strongly suppressed cell migration (IC50, 3.8 ïM) with negligible effects on cell proliferation (IC50, >20 ïM). We then synthesized 18 derivatives of Br-DIF-1 and examined the in vitro effects of these derivatives on cell proliferation and serum-induced cell migration in MDA-MB-231 cells. Among the derivatives, Br-DIF-1(+1), Br-DIF-1(+2), and Br-DIF-3(+2) exhibited strong anti-cell migration activities with IC50 values of 1.5, 1.0, and 3.1 ïM, respectively, without affecting cell proliferation (IC50, >20 ïM). These results suggest that these Br-DIF derivatives are good lead compounds for the development of anti-metastatic drugs against TNBC.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Dictyostelium/química , Halogênios/farmacologia , Hexanonas/farmacologia , Hidrocarbonetos Clorados/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Halogênios/química , Hexanonas/síntese química , Hexanonas/química , Humanos , Hidrocarbonetos Clorados/síntese química , Hidrocarbonetos Clorados/química , Relação Estrutura-Atividade , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Naturstoff reagent A (diphenylboric acid 2-aminoethyl ester [DPBA]) has been used historically in plant science to observe polyphenolic pigments, such as flavonoids, whose fluorescence requires enhancement to be visible by microscopy. Flavonoids are common dietary constituents and are the focus of considerable attention because of their potential as novel therapies for numerous diseases. The molecular basis of therapeutic activity is only gradually being established, and one strand of such research is making use of the social amoeba Dictyostelium discoideum. We extended the application of DPBA to flavonoid imaging in these preclinical studies, and report the first method for use of DPBA in this eukaryotic model microbe and its applicability alongside subcellular markers. This in vivo fluorescence imaging provided a useful adjunct to parallel chemical and genetic studies.
Assuntos
Flavonoides/isolamento & purificação , Imagem Molecular/métodos , Imagem Óptica/métodos , Compostos de Boro/química , Compostos de Boro/farmacologia , Dictyostelium/química , Éter/química , Éter/farmacologia , Flavonoides/química , Fluorescência , HumanosRESUMO
Slime mold species in the genus Dictyostelium are considered to have a close relationship with non-parasitic nematodes; they are sympatric in soils and can exhibit interspecific competition for food. We investigated whether this relationship extends to a plant-parasitic nematode that is active in the rhizosphere and has broad host specificity, damaging crops worldwide. Using a novel assay to examine the interaction between the cellular slime mold, Dictyostelium discoideum, and the plant-parasitic nematodes, Meloidogyne spp., we found that cellular slime molds can repel plant parasitic nematodes. Specifically, the repulsion activity was in response to chemical compounds released by cellular slime mold fruiting bodies. Under laboratory conditions, these soluble chemical extracts from fruiting bodies of D. discoideum showed repulsion activity strong enough to protect plant roots. The fruiting body cell extracts repelled but were not toxic to the plant-parasitic nematodes.
Assuntos
Antiparasitários/química , Antiparasitários/farmacologia , Dictyostelium/química , Dictyostelium/fisiologia , Doenças das Plantas/parasitologia , Tylenchoidea/efeitos dos fármacos , Tylenchoidea/patogenicidade , Animais , Dictyostelium/crescimento & desenvolvimento , Carpóforos/química , Carpóforos/fisiologia , Lotus/efeitos dos fármacos , Lotus/parasitologia , Doenças das Plantas/prevenção & controle , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/parasitologia , Microbiologia do Solo , Simpatria/fisiologiaRESUMO
Actin bundling protein 34 (ABP34) is the one of 11 actin-crosslinking proteins identified in Dictyostelium discoideum, a novel model organism for the study of actin-associated neurodegenerative disorders such as Alzheimer's disease and Huntington's disease. ABP34 localizes at the leading and trailing edges of locomotory cells, i.e., at the cell cortex, filopodia, and pseudopodia. Functionally, it serves to stabilize membrane-associated actin at sites of cell-cell contact. In addition, this small crosslinking protein is involved in actin bundle formation, and its bundling activity is regulated by the concentration of calcium ion. Several studies have sought to determine the mechanism underlying the calcium-regulated actin bundling activity of ABP34, but it remains unclear. Using several mutational and structural analyses, we revealed that calcium binding to the EF2 motif disrupts the inter-domain interaction between the N- and C-domains, thereby inhibiting the actin bundling activity of ABP34. This finding provides clues about the pathogenesis of neurodegenerative disorders related to actin bundling.
Assuntos
Actinas/metabolismo , Cálcio/metabolismo , Proteínas dos Microfilamentos/antagonistas & inibidores , Fator 2 de Elongação de Peptídeos/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Sítios de Ligação , Cromatografia em Gel , Dictyostelium/química , Dictyostelium/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fator 2 de Elongação de Peptídeos/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
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.
Assuntos
Dictyostelium/química , Descoberta de Drogas/métodos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas de Neoplasias/química , Antineoplásicos , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Ligantes , Proteínas de Neoplasias/antagonistas & inibidores , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas de Ligação a RNA , Receptor do Fator de Crescimento Transformador beta Tipo I/química , Proteína Smad7/química , Homologia Estrutural de ProteínaRESUMO
Chemical and biochemical analyses of one of the most basic nonribosomal peptide synthetases (NRPS) from a Pseudomonas fluorescens strain revealed its striking plasticity. Determination of the potential substrate scope enabled us to anticipate novel secondary metabolites that could subsequently be isolated and tested for their bioactivities. Detailed analyses of the monomodular pyreudione synthetase showed that the biosynthesis of the bacterial pyreudione alkaloids does not require additional biosynthetic enzymes. Heterologous expression of a similar and functional, yet cryptic, NRPS of Pseudomonas entomophila was successful and allowed us to perform a phylogenetic analysis of their thioesterase domains.