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1.
mBio ; 12(1)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531402

RESUMO

Fatty acid biosynthesis (FASII) enzymes are considered valid targets for antimicrobial drug development against the human pathogen Staphylococcus aureus However, incorporation of host fatty acids confers FASII antibiotic adaptation that compromises prospective treatments. S. aureus adapts to FASII inhibitors by first entering a nonreplicative latency period, followed by outgrowth. Here, we used transcriptional fusions and direct metabolite measurements to investigate the factors that dictate the duration of latency prior to outgrowth. We show that stringent response induction leads to repression of FASII and phospholipid synthesis genes. (p)ppGpp induction inhibits synthesis of malonyl-CoA, a molecule that derepresses FapR, a key regulator of FASII and phospholipid synthesis. Anti-FASII treatment also triggers transient expression of (p)ppGpp-regulated genes during the anti-FASII latency phase, with concomitant repression of FapR regulon expression. These effects are reversed upon outgrowth. GTP depletion, a known consequence of the stringent response, also occurs during FASII latency, and is proposed as the common signal linking these responses. We next showed that anti-FASII treatment shifts malonyl-CoA distribution between its interactants FapR and FabD, toward FapR, increasing expression of the phospholipid synthesis genes plsX and plsC during outgrowth. We conclude that components of the stringent response dictate malonyl-CoA availability in S. aureus FASII regulation, and contribute to latency prior to anti-FASII-adapted outgrowth. A combinatory approach, coupling a (p)ppGpp inducer and an anti-FASII, blocks S. aureus outgrowth, opening perspectives for bi-therapy treatment.IMPORTANCE Staphylococcus aureus is a major human bacterial pathogen for which new inhibitors are urgently needed. Antibiotic development has centered on the fatty acid synthesis (FASII) pathway, which provides the building blocks for bacterial membrane phospholipids. However, S. aureus overcomes FASII inhibition and adapts to anti-FASII by using exogenous fatty acids that are abundant in host environments. This adaptation mechanism comprises a transient latency period followed by bacterial outgrowth. Here, we use metabolite sensors and promoter reporters to show that responses to stringent conditions and to FASII inhibition intersect, in that both involve GTP and malonyl-CoA. These two signaling molecules contribute to modulating the duration of latency prior to S. aureus adaptation outgrowth. We exploit these novel findings to propose a bi-therapy treatment against staphylococcal infections.


Assuntos
Antibacterianos/farmacologia , Ácidos Graxos/antagonistas & inibidores , Guanosina Pentafosfato/fisiologia , Guanosina Trifosfato/fisiologia , Malonil Coenzima A/fisiologia , Staphylococcus aureus/efeitos dos fármacos , Adaptação Fisiológica/efeitos dos fármacos , Ácidos Graxos/biossíntese , Humanos , Malonil Coenzima A/análise , Mupirocina/farmacologia , Fosfolipídeos/biossíntese , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/fisiologia
2.
Shock ; 53(4): 468-475, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31090681

RESUMO

INTRODUCTION: Rats subjected to polytrauma and hemorrhage develop a coagulopathy that is similar to acute coagulopathy of trauma in humans, and is associated with a rise in prothrombin time and a fall in clot strength. Because platelet aggregation accounts for a major proportion of clot strength, we set out to characterize the effects of polytrauma on platelet function. METHODS: Sprague-Dawley rats were anesthetized with isoflurane. Polytrauma included laparotomy and damage to 10 cm of the small intestines, right and medial liver lobes, right leg skeletal muscle, femur fracture, and hemorrhage (40% of blood volume). No resuscitation was given. Blood samples were taken before and after trauma for the measurement of impedance electrode aggregometry, and intracellular levels of cyclic adenosine and guanosine monophosphate (cAMP, cGMP), inositol trisphosphate (IP3), and adenosine and guanosine triphosphates (ATP, GTP). RESULTS: Polytrauma significantly increased the response of collagen (24%) and thrombin (12%) to stimulate platelet aggregation. However, aggregation to adenosine diphosphate (ADP) or arachidonic acid (AA) was significantly decreased at 2 (52% and 46%, respectively) and 4 h (45% and 39%). Polytrauma and hemorrhage also led to a significant early rise in cAMP (101 ±â€Š11 to 202 ±â€Š29 pg/mL per 1,000 platelets), mirrored by a decrease in cGMP (7.8 ±â€Š0.9 to 0.6 ±â€Š0.5). In addition, there was a late fall in ATP (8.1 ±â€Š0.7 to 2.2 ±â€Š0.6 ng/mL per 1,000 platelets) and GTP (1.5 ±â€Š0.2 to 0.3 ±â€Š0.1). IP3 rose initially, and then fell back to baseline. CONCLUSIONS: Polytrauma and hemorrhage led to a deficit in the platelet aggregation response to ADP and AA after trauma, likely due to the early rise in cAMP, and a later fall in energy substrates, and may explain the decrease in clot strength and impaired hemostasis observed after severe trauma.


Assuntos
Trifosfato de Adenosina/fisiologia , Transtornos da Coagulação Sanguínea/etiologia , Guanosina Trifosfato/fisiologia , Hemorragia/complicações , Traumatismo Múltiplo/complicações , Agregação Plaquetária/fisiologia , Animais , Modelos Animais de Doenças , Hemorragia/sangue , Masculino , Traumatismo Múltiplo/sangue , Ratos , Ratos Sprague-Dawley
3.
EMBO J ; 35(21): 2270-2284, 2016 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-27670760

RESUMO

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.


Assuntos
Membrana Celular/fisiologia , Dinaminas/fisiologia , Animais , Guanosina Trifosfato/fisiologia , Humanos
4.
Cell Signal ; 27(12): 2363-70, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26341143

RESUMO

Phospholipase D (PLD) is one of the key enzymes to mediate a variety of cellular phenomena including endocytosis, actin rearrangement, proliferation, differentiation, and migration. Dynamin as a PLD-interacting partner is a large GTP binding protein that has been considered a mechanochemical enzyme involved in endocytosis by hydrolyzing GTP. Although both PLD and dynamin have been implicated in the regulation of actin cytoskeleton, it is not known how they have a link to regulate fibronectin (FN)-induced cell spreading. Furthermore, it is unknown whether dynamin can work as a GTP-dependent regulator through its interaction with other proteins. Here, we demonstrate that PLD can be regulated by dynamin in a GTP-dependent manner and that this is critical for FN-mediated cell spreading. First, we verified that GTP-loaded dynamin can mediate the cell spreading by FN by using dynamin's GTP binding deficient mutant (K44A). Also, we confirmed that blocking the PLD activity inhibited FN-induced cell spreading, not cell adhesion. Moreover, PLD interacted with dynamin in a GTP-dependent manner in FN signaling, and this interaction was crucial for FN-induced PLD activation and cell spreading. Also, we found that PLD mutant (R128K) that didn't have GAP activity increased the GTP-dependent interaction between PLD and dynamin; it also increased PLD activity and cell spreading. These findings suggest that the observed increase in PLD activity was through boosting the binding of PLD with dynamin and it facilitated FN-induced cell spreading. These results imply that GTP-loaded dynamin, like a small GTPase could mediate a "switch on" signaling via interaction with PLD that has a role as an effector.


Assuntos
Dinaminas/metabolismo , Fibronectinas/fisiologia , Guanosina Trifosfato/fisiologia , Fosfolipase D/metabolismo , Adesão Celular , Forma Celular , Ativação Enzimática , Células HEK293 , Humanos
5.
J Biol Chem ; 290(33): 20325-35, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26124275

RESUMO

The effects of Kil peptide from bacteriophage λ on the assembly of Escherichia coli FtsZ into one subunit thick protofilaments were studied using combined biophysical and biochemical methods. Kil peptide has recently been identified as the factor from bacteriophage λ responsible for the inhibition of bacterial cell division during lytic cycle, targeting FtsZ polymerization. Here, we show that this antagonist blocks FtsZ assembly into GTP-dependent protofilaments, producing a wide distribution of smaller oligomers compared with the average size of the intact protofilaments. The shortening of FtsZ protofilaments by Kil is detectable at concentrations of the peptide in the low micromolar range, the mid-point of the inhibition being close to its apparent affinity for GDP-bound FtsZ. This antagonist not only interferes with FtsZ assembly but also reverses the polymerization reaction. The negative regulation by Kil significantly reduces the GTPase activity of FtsZ protofilaments, and FtsZ polymers assembled in guanosine-5'-[(α,ß)-methyleno]triphosphate are considerably less sensitive to Kil. Our results suggest that, at high concentrations, Kil may use an inhibition mechanism involving the sequestration of FtsZ subunits, similar to that described for other inhibitors like the SOS response protein SulA or the moonlighting enzyme OpgH. This mechanism is different from those employed by the division site selection antagonists MinC and SlmA. This work provides new insight into the inhibition of FtsZ assembly by phages, considered potential tools against bacterial infection.


Assuntos
Bactérias/citologia , Proteínas de Bactérias/fisiologia , Bacteriófago lambda/química , Divisão Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Peptídeos/fisiologia , Proteínas Virais/química , Proteínas de Bactérias/química , Biopolímeros/química , Proteínas do Citoesqueleto/química , Guanosina Trifosfato/fisiologia
6.
J Biol Chem ; 289(44): 30645-30656, 2014 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-25237193

RESUMO

Drp1 is a dynamin-like GTPase that mediates mitochondrial and peroxisomal division in a process dependent on self-assembly and coupled to GTP hydrolysis. Despite the link between Drp1 malfunction and human disease, the molecular details of its membrane activity remain poorly understood. Here we reconstituted and directly visualized Drp1 activity in giant unilamellar vesicles. We quantified the effect of lipid composition and GTP on membrane binding and remodeling activity by fluorescence confocal microscopy and flow cytometry. In contrast to other dynamin relatives, Drp1 bound to both curved and flat membranes even in the absence of nucleotides. We also found that Drp1 induced membrane tubulation that was stimulated by cardiolipin. Moreover, Drp1 promoted membrane tethering dependent on the intrinsic curvature of the membrane lipids and on GTP. Interestingly, Drp1 concentrated at membrane contact surfaces and, in the presence of GTP, formed discrete clusters on the vesicles. Our findings support a role of Drp1 not only in the formation of lipid tubes but also on the stabilization of tightly apposed membranes, which are intermediate states in the process of mitochondrial fission.


Assuntos
Dinaminas/fisiologia , Dinâmica Mitocondrial , Membranas Mitocondriais/fisiologia , Animais , Cardiolipinas/fisiologia , Células Cultivadas , Dinaminas/química , Guanosina Trifosfato/fisiologia , Humanos , Lipossomos/química , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Camundongos , Ligação Proteica , Estrutura Quaternária de Proteína , Transporte Proteico
8.
J Inherit Metab Dis ; 37(3): 353-7, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24154984

RESUMO

BACKGROUND: Valproic acid (VPA) is an effective antiepileptic drug that may induce progressive microvesicular steatosis. The impairment of mitochondrial function may be an important metabolic effect of VPA treatment with potential adverse consequences. OBJECTIVE: To investigate the influence of VPA on the activity of GTP- and ATP-specific succinate:CoA ligases (G-SUCL and A-SUCL). METHODS: The GTP- and ATP-specific SUCL activities were measured in human fibroblasts in the reverse direction, i.e. the formation of succinyl-CoA. These were assessed at different concentrations of succinate in the presence of VPA, valproyl-CoA and zinc chloride, an established inhibitor of the enzymes. Activities were measured using an optimized HPLC procedure. RESULTS: Valproyl-CoA (1 mM) inhibited the activity of A-SUCL and G-SUCL by 45-55% and 25-50%, respectively. VPA (1 mM) had no influence on the activity of the two enzymes. DISCUSSION: Valproyl-CoA appears to affect the activity of SUCL, especially with the ATP-specific enzyme. Considering the key role of SUCL in the Krebs cycle, interference with its activity might impair the cellular energy status. Moreover, A-SUCL is bound to the nucleoside diphosphate kinase (NDPK), which is responsible for the mitochondrial (deoxy)nucleotide synthesis. An inhibition of A-SUCL might influence the activity of NDPK inducing an imbalance of nucleotides in the mitochondria and eventually mitochondrial DNA depletion. This may account for the potential liver failure associated with valproate therapy, reported in patients with deficiencies within the mitochondrial DNA replicase system such as polymerase gamma 1.


Assuntos
Acil Coenzima A/farmacologia , Trifosfato de Adenosina/fisiologia , Guanosina Trifosfato/fisiologia , Succinato-CoA Ligases/antagonistas & inibidores , DNA Mitocondrial/metabolismo , Humanos , Falência Hepática/induzido quimicamente , Núcleosídeo-Difosfato Quinase/fisiologia , Ácido Valproico/efeitos adversos , Ácido Valproico/farmacologia
9.
Nat Cell Biol ; 15(11): 1317-27, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24161932

RESUMO

Both subunits of αß-tubulin that comprise the core components of microtubules bind GTP. GTP binding to α-tubulin has a structural role, whereas ß-tubulin binds and hydrolyses GTP to regulate microtubule dynamics. γ-tubulin, another member of the tubulin superfamily that seeds microtubule nucleation at microtubule-organizing centres, also binds GTP; however, the importance of this association remains elusive. To address the role of GTP binding to γ-tubulin, we systematically mutagenized the GTP contact residues in the yeast γ-tubulin Tub4. Tub4(GTP)-mutant proteins that exhibited greatly reduced GTP affinity still assembled into the small γ-tubulin complex. However, tub4(GTP) mutants were no longer viable, and had defects in interaction between γ-tubulin and αß-tubulin, decreased microtubule nucleation and defects in microtubule organization. In vitro and in vivo data show that only γ-tubulin loaded with GTP nucleates microtubules. Our results suggest that GTP recruitment to γ-tubulin enhances its interaction with αß-tubulin similarly to GTP recruitment to ß-tubulin.


Assuntos
Guanosina Trifosfato/fisiologia , Microtúbulos/fisiologia , Tubulina (Proteína)/fisiologia , Modelos Moleculares , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Tubulina (Proteína)/química
10.
Biochemistry ; 52(40): 7071-81, 2013 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-24007276

RESUMO

FtsZ polymerizes to form a cytokinetic ring at the center of a bacterial cell, which engineers bacterial cell division. FtsZ consists of N-terminal and C-terminal core domains followed by a C-terminal spacer and a conserved C-terminal tail region. Though it has been reported that both N- and C-domains can fold independently, the assembly behaviors of the N- and C-domains are not clear. In this study, we created five truncated constructs of Bacillus subtilis FtsZ, two N-domain and three C-domain constructs, and expressed and purified them. We determined their assembly properties and their effect on the assembly of full-length FtsZ to gain insight into the mechanism of FtsZ polymerization. We found that the N-domain of B. subtilis FtsZ can polymerize on its own in a GTP-dependent manner. Further, we obtained evidence indicating that the N-domain could bind to GTP but could not hydrolyze GTP by itself. In addition, the N-domain was found to inhibit the assembly of full-length FtsZ. Interestingly, the N-domain was found to enhance the GTPase activity of full-length FtsZ. An analysis of the effects of the N- and C-domains on FtsZ assembly indicated that the assembly of FtsZ might be directional. The work has provided new insight into the assembly characteristics of FtsZ domains and the mechanism of FtsZ polymerization.


Assuntos
Proteínas de Bactérias/biossíntese , Proteínas do Citoesqueleto/biossíntese , Bacillus subtilis/citologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/isolamento & purificação , Divisão Celular/fisiologia , Clonagem Molecular , Proteínas do Citoesqueleto/isolamento & purificação , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/fisiologia , Multimerização Proteica , Estrutura Terciária de Proteína
11.
Open Biol ; 2(5): 120076, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22724071

RESUMO

The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface ß1 integrin, and ß1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating ß1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.


Assuntos
Movimento Celular/fisiologia , Adesões Focais/fisiologia , Proteínas de Neoplasias/fisiologia , Proteína rhoB de Ligação ao GTP/fisiologia , Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Forma Celular , Feminino , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Guanosina Trifosfato/fisiologia , Humanos , Integrina beta1/metabolismo , Masculino , Microtúbulos/metabolismo , Invasividade Neoplásica , Neoplasias da Próstata/patologia , Pseudópodes/fisiologia , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho , Proteínas rac1 de Ligação ao GTP/fisiologia
12.
EMBO J ; 31(4): 1014-27, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22157747

RESUMO

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Membrana/química , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Técnicas do Sistema de Duplo-Híbrido
13.
Proc Natl Acad Sci U S A ; 108(39): 16283-8, 2011 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-21930898

RESUMO

The mechanisms governing atlastin-mediated membrane fusion are unknown. Here we demonstrate that a three-helix bundle (3HB) within the middle domain is required for oligomerization. Mutation of core hydrophobic residues within these helices inactivates atlastin function by preventing membrane tethering and the subsequent fusion. GTP binding induces a conformational change that reorients the GTPase domain relative to the 3HB to permit self-association, but the ability to hydrolyze GTP is required for full fusion, indicating that nucleotide binding and hydrolysis play distinct roles. Oligomerization of atlastin stimulates its ability to hydrolyze GTP, and the energy released drives lipid bilayer merger. Mutations that prevent atlastin self-association also abolish oligomerization-dependent stimulation of GTPase activity. Furthermore, increasing the distance of atlastin complex formation from the membrane inhibits fusion, suggesting that this distance is crucial for atlastin to promote fusion.


Assuntos
Proteínas de Ligação ao GTP/fisiologia , Guanosina Trifosfato/fisiologia , Proteínas de Membrana/fisiologia , Animais , Sequência de Bases , Primers do DNA , Drosophila , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Hidrólise
14.
J Neurosci ; 30(40): 13441-53, 2010 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-20926670

RESUMO

Rab GTPases are molecular switches that orchestrate protein complexes before membrane fusion reactions. In synapses, Rab3 and Rab5 proteins have been implicated in the exo-endocytic cycling of synaptic vesicles (SVs), but an involvement of additional Rabs cannot be excluded. Here, combining high-resolution mass spectrometry and chemical labeling (iTRAQ) together with quantitative immunoblotting and fluorescence microscopy, we have determined the exocytotic (Rab3a, Rab3b, Rab3c, and Rab27b) and endocytic (Rab4b, Rab5a/b, Rab10, Rab11b, and Rab14) Rab machinery of SVs. Analysis of two closely related proteins, Rab3a and Rab27b, revealed colocalization in synaptic nerve terminals, where they reside on distinct but overlapping SV pools. Moreover, whereas Rab3a readily dissociates from SVs during Ca(2+)-triggered exocytosis, and is susceptible to membrane extraction by Rab-GDI, Rab27b persists on SV membranes upon stimulation and is resistant to GDI-coupled Rab retrieval. Finally, we demonstrate that selective modulation of the GTP/GDP switch mechanism of Rab27b impairs SV recycling, suggesting that Rab27b, probably in concert with Rab3s, is involved in SV exocytosis.


Assuntos
Cálcio/fisiologia , Exocitose/fisiologia , Homologia de Genes , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Proteína rab3A de Ligação ao GTP/fisiologia , Animais , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Células Cultivadas , Exocitose/genética , Guanosina Difosfato/genética , Guanosina Difosfato/fisiologia , Guanosina Trifosfato/genética , Guanosina Trifosfato/fisiologia , Hipocampo/metabolismo , Proteoma/genética , Proteoma/fisiologia , Ratos , Frações Subcelulares/metabolismo , Vesículas Sinápticas/genética , Proteínas rab de Ligação ao GTP/genética , Proteína rab3A de Ligação ao GTP/genética
15.
ACS Chem Neurosci ; 1(6): 463-73, 2010 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-22778838

RESUMO

The molecular modes of action of antipsychotic drugs are poorly understood beyond their effects at the dopamine D2 receptor. Previous studies have placed Akt signaling downstream of D2 dopamine receptors, and recent data have suggested an association between psychotic illnesses and defective Akt signaling. To characterize the effect of antipsychotic drugs on the Akt pathway, we used the model organism C. elegans, a simple system where the Akt/forkhead box O transcription factor (FOXO) pathway has been well characterized. All major classes of antipsychotic drugs increased signaling through the insulin/Akt/FOXO pathway, whereas four other drugs that are known to affect the central nervous system did not. The antipsychotic drugs inhibited dauer formation, dauer recovery, and shortened lifespan, three biological processes affected by Akt signaling. Genetic analysis showed that AKT-1 and the insulin and insulin-like growth factor receptor, DAF-2, were required for the antipsychotic drugs to increase signaling. Serotonin synthesis was partially involved, whereas the mitogen activated protein kinase (MAPK), SEK-1 is a MAP kinase kinase (MAPKK), and calcineurin were not involved. This is the first example of a common but specific molecular effect produced by all presently known antipsychotic drugs in any biological system. Because untreated schizophrenics have been reported to have low levels of Akt signaling, increased Akt signaling might contribute to the therapeutic actions of antipsychotic drugs.


Assuntos
Antipsicóticos/farmacologia , Proteínas de Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptor IGF Tipo 1/efeitos dos fármacos , Receptor de Insulina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Benzodiazepinas/farmacologia , Caenorhabditis elegans/crescimento & desenvolvimento , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Clozapina/farmacologia , Guanosina Trifosfato/fisiologia , Larva/efeitos dos fármacos , Olanzapina , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Trifluoperazina/farmacologia
16.
Mol Biol Cell ; 20(20): 4400-11, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19710422

RESUMO

Deleted in Liver Cancer 1 (DLC1) is a GTPase-activating protein (GAP) with specificity for RhoA, RhoB, and RhoC that is frequently deleted in various tumor types. By inactivating these small GTPases, DLC1 controls actin cytoskeletal remodeling and biological processes such as cell migration and proliferation. Here we provide evidence that DLC1 binds to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) through a previously unrecognized polybasic region (PBR) adjacent to its RhoGAP domain. Importantly, PI(4,5)P(2)-containing membranes are shown to stimulate DLC1 GAP activity in vitro. In living cells, a DLC1 mutant lacking an intact PBR inactivated Rho signaling less efficiently and was severely compromised in suppressing cell spreading, directed migration, and proliferation. We therefore propose that PI(4,5)P(2) is an important cofactor in DLC1 regulation in vivo and that the PBR is essential for the cellular functions of the protein.


Assuntos
Lipídeos de Membrana/fisiologia , Fosfatidilinositol 4,5-Difosfato/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Linhagem Celular , Sequência Conservada , Ensaio de Imunoadsorção Enzimática , Proteínas Ativadoras de GTPase , Guanosina Trifosfato/fisiologia , Humanos , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína rhoA de Ligação ao GTP/metabolismo
17.
Mol Biol Cell ; 20(20): 4390-9, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19710424

RESUMO

Rho GTPases regulate the actin cytoskeleton in all eukaryotes. Fission yeast Cdc42 is involved in actin cable assembly and formin For3 regulation. We isolated cdc42-879 as a thermosensitive strain with actin cable and For3 localization defects. In a multicopy suppressor screening, we identified pob1(+) as suppressor of cdc42-879 thermosensitivity. Pob1 overexpression also partially restores actin cables and localization of For3 in the mutant strain. Pob1 interacts with Cdc42 and this GTPase regulates Pob1 localization and/or stability. The C-terminal pleckstrin homology (PH) domain of Pob1 is required for Cdc42 binding. Pob1 also binds to For3 through its N-terminal sterile alpha motif (SAM) domain and contributes to the formin localization at the cell tips. The previously described pob1-664 mutant strain (Mol. Biol. Cell. 10, 2745-2757, 1999), which carries a mutation in the PH domain, as well as pob1 mutant strains in which Pob1 lacks the N-terminal region (pob1DeltaN) or the SAM domain (pob1DeltaSAM), have cytoskeletal defects similar to that of cdc42-879 cells. Expression of constitutively active For3DAD* partially restores actin organization in cdc42-879, pob1-664, pob1DeltaN, and pob1DeltaSAM. Therefore, we propose that Pob1 is required for For3 localization to the tips and facilitates Cdc42-mediated relief of For3 autoinhibition to stimulate actin cable formation.


Assuntos
Citoesqueleto de Actina/ultraestrutura , Citoesqueleto/ultraestrutura , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/metabolismo , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Substituição de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiologia , Polaridade Celular , Forminas , Regulação Fúngica da Expressão Gênica , Guanosina Trifosfato/fisiologia , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Schizosaccharomyces/ultraestrutura , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/química
18.
Mol Biol Cell ; 20(20): 4303-12, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19692570

RESUMO

Shiga-toxin-producing Escherichia coli remain a food-borne health threat. Shiga toxin is endocytosed by intestinal epithelial cells and transported retrogradely through the secretory pathway. It is ultimately translocated to the cytosol where it inhibits protein translation. We found that Shiga toxin transport through the secretory pathway was dependent on the cytoskeleton. Recent studies reveal that Shiga toxin activates signaling pathways that affect microtubule reassembly and dynein-dependent motility. We propose that Shiga toxin alters cytoskeletal dynamics in a way that facilitates its transport through the secretory pathway. We have now found that Rho GTPases regulate the endocytosis and retrograde motility of Shiga toxin. The expression of RhoA mutants inhibited endocytosis of Shiga toxin. Constitutively active Cdc42 or knockdown of the Cdc42-specific GAP, ARHGAP21, inhibited the transport of Shiga toxin to the juxtanuclear Golgi apparatus. The ability of Shiga toxin to stimulate microtubule-based transferrin transport also required Cdc42 and ARHGAP21 function. Shiga toxin addition greatly decreases the levels of active Cdc42-GTP in an ARHGAP21-dependent manner. We conclude that ARHGAP21 and Cdc42-based signaling regulates the dynein-dependent retrograde transport of Shiga toxin to the Golgi apparatus.


Assuntos
Citoesqueleto/fisiologia , Proteínas Ativadoras de GTPase/fisiologia , Toxinas Shiga/metabolismo , Proteína cdc42 de Ligação ao GTP/fisiologia , Animais , Chlorocebus aethiops , Endocitose/fisiologia , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/genética , Técnicas de Silenciamento de Genes , Complexo de Golgi/metabolismo , Guanosina Trifosfato/fisiologia , Microscopia Confocal , Microtúbulos/fisiologia , Transporte Proteico/fisiologia , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Toxinas Shiga/farmacologia , Transferrina/fisiologia , Células Vero/metabolismo , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/fisiologia
19.
Biochemistry ; 48(31): 7519-24, 2009 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-19527054

RESUMO

Soluble guanylate cyclase (sGC) serves as a receptor for the signaling agent nitric oxide (NO). sGC synthesis of cGMP is regulated by NO, GTP, ATP, and allosteric activators such as YC-1. The guanylate cyclase activity and adenylate cyclase activity of full-length sGC and the sGC catalytic domain constructs (alpha1(cat)beta1(cat)) are reported here. ATP is a mixed-type inhibitor of cGMP production for both sGC and alpha1(cat)beta1(cat), indicating that the C-terminus of sGC contains an allosteric nucleotide binding site. YC-1 did not activate alpha1(cat)beta1(cat) or compete with ATP inhibition of cGMP synthesis, which suggests that YC-1 and ATP bind to distinct sites. alpha1(cat)beta1(cat) and NO-stimulated sGC also synthesize cAMP, but this activity is inhibited by ATP via noncompetitive substrate inhibition and by GTP via mixed-type inhibition. Additionally, the adenylate cyclase activity of purified sGC was inhibited by PC12 lysate, suggesting that an intracellular small molecule or protein regulates this activity in vivo.


Assuntos
Trifosfato de Adenosina/química , Guanosina Trifosfato/química , Guanilato Ciclase/metabolismo , Subunidades Proteicas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/fisiologia , Adenilil Ciclases/metabolismo , Regulação Alostérica/fisiologia , Sítio Alostérico/fisiologia , Animais , Domínio Catalítico , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/fisiologia , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/química , Células PC12 , Conformação Proteica , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Ratos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/química , Guanilil Ciclase Solúvel , Especificidade por Substrato
20.
DNA Cell Biol ; 28(6): 277-84, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19348590

RESUMO

Apicomplexa is a phylum within the kingdom Protista that contains some of the most significant threats to public health. One of the members of this phylum, Toxoplasma gondii, is amenable to molecular genetic analyses allowing for the identification of factors critical for colonization and disease. A pathway found to be important for T. gondii pathogenesis is the Ran network of nuclear trafficking. Bioinformatics analysis of apicomplexan genomes shows that while Ran is well conserved, the key regulators of Ran--Regulator of Chromosome Condensation 1 and Ran GTPase activating protein--are either highly divergent or absent. Likewise, several import and export receptor molecules that are crucial for nuclear transport are either not present or have experienced genetic drift such that they are no longer recognizable by bioinformatics tools. In this minireview we describe the basics of nuclear trafficking and compare components within apicomplexans to defined systems in humans and yeast. A detailed analysis of the nuclear trafficking network in these eukaryotes is required to understand how this potentially unique cellular biological pathway contributes to host-parasite interactions.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Apicomplexa/citologia , Proteínas de Protozoários/fisiologia , Animais , Proteínas de Ciclo Celular/fisiologia , Núcleo Celular/metabolismo , Cryptosporidium parvum/citologia , Proteínas Ativadoras de GTPase/fisiologia , Guanosina Trifosfato/fisiologia , Humanos , Carioferinas/fisiologia , Modelos Biológicos , Proteínas Nucleares/fisiologia , Plasmodium falciparum/citologia , Especificidade da Espécie , Toxoplasma/citologia , beta Carioferinas/fisiologia , Proteína ran de Ligação ao GTP/fisiologia
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