Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.570
Filtrar
1.
Life Sci ; 248: 117477, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32119961

RESUMO

AIMS: A newly emerged Human Coronavirus (HCoV) is reported two months ago in Wuhan, China (COVID-19). Until today >2700 deaths from the 80,000 confirmed cases reported mainly in China and 40 other countries. Human to human transmission is confirmed for COVID-19 by China a month ago. Based on the World Health Organization (WHO) reports, SARS HCoV is responsible for >8000 cases with confirmed 774 deaths. Additionally, MERS HCoV is responsible for 858 deaths out of about 2500 reported cases. The current study aims to test anti-HCV drugs against COVID-19 RNA dependent RNA polymerase (RdRp). MATERIALS AND METHODS: In this study, sequence analysis, modeling, and docking are used to build a model for Wuhan COVID-19 RdRp. Additionally, the newly emerged Wuhan HCoV RdRp model is targeted by anti-polymerase drugs, including the approved drugs Sofosbuvir and Ribavirin. KEY FINDINGS: The results suggest the effectiveness of Sofosbuvir, IDX-184, Ribavirin, and Remidisvir as potent drugs against the newly emerged HCoV disease. SIGNIFICANCE: The present study presents a perfect model for COVID-19 RdRp enabling its testing in silico against anti-polymerase drugs. Besides, the study presents some drugs that previously proved its efficiency against the newly emerged viral infection.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/química , Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Guanosina Monofosfato/análogos & derivados , Pneumonia Viral/tratamento farmacológico , RNA Replicase/antagonistas & inibidores , Ribavirina/química , Sofosbuvir/química , Proteínas Virais/antagonistas & inibidores , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Alanina/química , Alanina/metabolismo , Alphacoronavirus/enzimologia , Alphacoronavirus/genética , Sequência de Aminoácidos , Antivirais/metabolismo , Betacoronavirus/genética , Domínio Catalítico , Biologia Computacional/métodos , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos/métodos , Guanosina Monofosfato/química , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Simulação de Acoplamento Molecular , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Replicase/química , RNA Replicase/metabolismo , Ribavirina/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sofosbuvir/metabolismo , Termodinâmica , Uridina Trifosfato/química , Uridina Trifosfato/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(6): 2770-2778, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31988132

RESUMO

Organelle-specific nanocarriers (NCs) are highly sought after for delivering therapeutic agents into the cell nucleus. This necessitates nucleocytoplasmic transport (NCT) to bypass nuclear pore complexes (NPCs). However, little is known as to how comparably large NCs infiltrate this vital intracellular barrier to enter the nuclear interior. Here, we developed nuclear localization signal (NLS)-conjugated polymersome nanocarriers (NLS-NCs) and studied the NCT mechanism underlying their selective nuclear uptake. Detailed chemical, biophysical, and cellular analyses show that karyopherin receptors are required to authenticate, bind, and escort NLS-NCs through NPCs while Ran guanosine triphosphate (RanGTP) promotes their release from NPCs into the nuclear interior. Ultrastructural analysis by regressive staining transmission electron microscopy further resolves the NLS-NCs on transit in NPCs and inside the nucleus. By elucidating their ability to utilize NCT, these findings demonstrate the efficacy of polymersomes to deliver encapsulated payloads directly into cell nuclei.


Assuntos
Núcleo Celular/metabolismo , Nanopartículas/química , Polímeros/química , Transporte Ativo do Núcleo Celular , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Núcleo Celular/genética , Sistemas de Liberação de Medicamentos , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Carioferinas , Nanopartículas/metabolismo , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Poro Nuclear/metabolismo , Polímeros/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(3): 1429-1437, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31900355

RESUMO

Translation initiation controls protein synthesis by regulating the delivery of the first aminoacyl-tRNA to messenger RNAs (mRNAs). In eukaryotes, initiation is sophisticated, requiring dozens of protein factors and 2 GTP-regulated steps. The GTPase eIF5B gates progression to elongation during the second GTP-regulated step. Using electron cryomicroscopy (cryo-EM), we imaged an in vitro initiation reaction which is set up with purified yeast components and designed to stall with eIF5B and a nonhydrolyzable GTP analog. A high-resolution reconstruction of a "dead-end" intermediate at 3.6 Šallowed us to visualize eIF5B in its ribosome-bound conformation. We identified a stretch of residues in eIF5B, located close to the γ-phosphate of GTP and centered around the universally conserved tyrosine 837 (Saccharomyces cerevisiae numbering), that contacts the catalytic histidine of eIF5B (H480). Site-directed mutagenesis confirmed the essential role that these residues play in regulating ribosome binding, GTP hydrolysis, and translation initiation both in vitro and in vivo. Our results illustrate how eIF5B transmits the presence of a properly delivered initiator aminoacyl-tRNA at the P site to the distant GTPase center through interdomain communications and underscore the importance of the multidomain architecture in translation factors to sense and communicate ribosomal states.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Guanosina Trifosfato/metabolismo , Iniciação Traducional da Cadeia Peptídica , Sítios de Ligação , Microscopia Crioeletrônica , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Hidrólise , Mutagênese Sítio-Dirigida , Ligação Proteica , Ribossomos/metabolismo , Saccharomyces cerevisiae
4.
PLoS Biol ; 18(1): e3000593, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31995552

RESUMO

During host colonization, bacteria use the alarmones (p)ppGpp to reshape their proteome by acting pleiotropically on DNA, RNA, and protein synthesis. Here, we elucidate how the initiating ribosome senses the cellular pool of guanosine nucleotides and regulates the progression towards protein synthesis. Our results show that the affinity of guanosine triphosphate (GTP) and the inhibitory concentration of ppGpp for the 30S-bound initiation factor IF2 vary depending on the programmed mRNA. The TufA mRNA enhanced GTP affinity for 30S complexes, resulting in improved ppGpp tolerance and allowing efficient protein synthesis. Conversely, the InfA mRNA allowed ppGpp to compete with GTP for IF2, thus stalling 30S complexes. Structural modeling and biochemical analysis of the TufA mRNA unveiled a structured enhancer of translation initiation (SETI) composed of two consecutive hairpins proximal to the translation initiation region (TIR) that largely account for ppGpp tolerance under physiological concentrations of guanosine nucleotides. Furthermore, our results show that the mechanism enhancing ppGpp tolerance is not restricted to the TufA mRNA, as similar ppGpp tolerance was found for the SETI-containing Rnr mRNA. Finally, we show that IF2 can use pppGpp to promote the formation of 30S initiation complexes (ICs), albeit requiring higher factor concentration and resulting in slower transitions to translation elongation. Altogether, our data unveil a novel regulatory mechanism at the onset of protein synthesis that tolerates physiological concentrations of ppGpp and that bacteria can exploit to modulate their proteome as a function of the nutritional shift happening during stringent response and infection.


Assuntos
Guanosina Tetrafosfato/farmacologia , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Ligação Competitiva , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Tetrafosfato/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Interações Hospedeiro-Patógeno/fisiologia , Cinética , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Iniciação Traducional da Cadeia Peptídica/fisiologia , Fator Tu de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/química , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética
5.
Int J Mol Sci ; 21(1)2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31861427

RESUMO

Even though the Obg protein is essential for bacterial viability, the cellular functions of this universally conserved GTPase remain enigmatic. Moreover, the influence of GTP and GDP binding on the activity of this protein is largely unknown. Previously, we identified a mutant isoform of ObgE (the Obg protein of Escherichia coli) that triggers cell death. In this research we explore the biochemical requirements for the toxic effect of this mutant ObgE* isoform, using cell death as a readily accessible read-out for protein activity. Both the absence of the N-terminal domain and a decreased GTP binding affinity neutralize ObgE*-mediated toxicity. Moreover, a deletion in the region that connects the N-terminal domain to the G domain likewise abolishes toxicity. Taken together, these data indicate that GTP binding by ObgE* triggers a conformational change that is transmitted to the N-terminal domain to confer toxicity. We therefore conclude that ObgE*-GTP, but not ObgE*-GDP, is the active form of ObgE* that is detrimental to cell viability. Based on these data, we speculate that also for wild-type ObgE, GTP binding triggers conformational changes that affect the N-terminal domain and thereby control ObgE function.


Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Escherichia coli/química , Guanosina Trifosfato/química , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Mutantes , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Relação Estrutura-Atividade
6.
PLoS Comput Biol ; 15(10): e1007193, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589600

RESUMO

Guanylate binding proteins (GBPs) belong to the dynamin-related superfamily and exhibit various functions in the fight against infections. The functions of the human guanylate binding protein 1 (hGBP1) are tightly coupled to GTP hydrolysis and dimerization. Despite known crystal structures of the hGBP1 monomer and GTPase domain dimer, little is known about the dynamics of hGBP1. To gain a mechanistic understanding of hGBP1, we performed sub-millisecond multi-resolution molecular dynamics simulations of both the hGBP1 monomer and dimer. We found that hGBP1 is a highly flexible protein that undergoes a hinge motion similar to the movements observed for other dynamin-like proteins. Another large-scale motion was observed for the C-terminal helix α13, providing a molecular view for the α13-α13 distances previously reported for the hGBP1 dimer. Most of the loops of the GTPase domain were found to be flexible, revealing why GTP binding is needed for hGBP1 dimerization to occur.


Assuntos
Biologia Computacional/métodos , Proteínas de Ligação ao GTP/fisiologia , Algoritmos , Sítios de Ligação , Simulação por Computador , Dimerização , Dinaminas , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Simulação de Dinâmica Molecular , Movimento (Física) , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/fisiologia , Software
7.
Nat Commun ; 10(1): 4914, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31664033

RESUMO

Mitofusin-2 (MFN2) is a dynamin-like GTPase that plays a central role in regulating mitochondrial fusion and cell metabolism. Mutations in MFN2 cause the neurodegenerative disease Charcot-Marie-Tooth type 2A (CMT2A). The molecular basis underlying the physiological and pathological relevance of MFN2 is unclear. Here, we present crystal structures of truncated human MFN2 in different nucleotide-loading states. Unlike other dynamin superfamily members including MFN1, MFN2 forms sustained dimers even after GTP hydrolysis via the GTPase domain (G) interface, which accounts for its high membrane-tethering efficiency. The biochemical discrepancy between human MFN2 and MFN1 largely derives from a primate-only single amino acid variance. MFN2 and MFN1 can form heterodimers via the G interface in a nucleotide-dependent manner. CMT2A-related mutations, mapping to different functional zones of MFN2, lead to changes in GTP hydrolysis and homo/hetero-association ability. Our study provides fundamental insight into how mitofusins mediate mitochondrial fusion and the ways their disruptions cause disease.


Assuntos
Doença de Charcot-Marie-Tooth/enzimologia , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/fisiopatologia , Dimerização , GTP Fosfo-Hidrolases/genética , Guanosina Trifosfato/metabolismo , Humanos , Mitocôndrias/química , Mitocôndrias/enzimologia , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/genética , Mutação , Domínios Proteicos
8.
Int J Mol Med ; 44(4): 1523-1530, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31364723

RESUMO

Non­alcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, insulin resistance and inflammation; however, the exact pathogenesis of NAFLD is not fully understood. Green tea polyphenols (GTP) exhibit beneficial effects against metabolic syndrome. However, the effect of GTP on NAFLD remains largely unknown. The aim of the present study was to investigate the effects of GTP on NAFLD in high­fat diet (HFD)­induced rats. The NAFLD rat model was induced with a HFD for 8 weeks. A total of 30 adult male Sprague Dawley rats were randomly divided into three groups: i) Normal control group; ii) HFD group; and iii) HFD with GTP group. Hematoxylin and eosin and Oil Red O analyses were performed. The levels of alanine aminotransferase (ALT), aspartate amino-transferase (AST) and inflammatory cytokines in the serum, as well as oxidative stress markers and hepatic lipids in the liver were measured. In addition, parameters associated with glucose metabolism were also assessed. Western blotting and RT­qPCR were used to determine the expression levels of 5' adenosine monophosphate­activated protein kinase (AMPK). HFD­induced rats exhibited features associated with NAFLD. GTP intervention significantly reduced serum ALT and AST levels. Fasting serum glucose, insulin resistance and hepatic lipid levels were all decreased in the GTP­treated rats. GTP also significantly decreased the levels of TNF­α, IL­6 and malondialdehyde. In contrast, superoxide dismutase levels were increased in the liver. Furthermore, GTP also significantly increased phosphorylation of AMPK and attenuated histopathological changes indicative of injury in liver tissue. GTP has a protective effect on HFD­induced hepatic steatosis, insulin resistance and inflammation, and the underlying mechanism may involve the AMPK pathway.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Resistência à Insulina , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Chá/química , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Glicemia/efeitos dos fármacos , Peso Corporal , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/patologia , Guanosina Trifosfato/metabolismo , Insulina/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Testes de Função Hepática , Masculino , Extratos Vegetais/química , Polifenóis/química , Ratos
9.
PLoS Comput Biol ; 15(8): e1007327, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31469822

RESUMO

Thirteen tubulin protofilaments, made of αß-tubulin heterodimers, interact laterally to produce cytoskeletal microtubules. Microtubules exhibit the striking property of dynamic instability, manifested in their intermittent growth and shrinkage at both ends. This behavior is key to many cellular processes, such as cell division, migration, maintenance of cell shape, etc. Although assembly and disassembly of microtubules is known to be linked to hydrolysis of a guanosine triphosphate molecule in the pocket of ß-tubulin, detailed mechanistic understanding of corresponding conformational changes is still lacking. Here we take advantage of the recent generation of in-microtubule structures of tubulin to examine the properties of protofilaments, which serve as important microtubule assembly and disassembly intermediates. We find that initially straight tubulin protofilaments, relax to similar non-radially curved and slightly twisted conformations. Our analysis further suggests that guanosine triphosphate hydrolysis primarily affects the flexibility and conformation of the inter-dimer interface, without a strong impact on the shape or flexibility of αß-heterodimer. Inter-dimer interfaces are significantly more flexible compared to intra-dimer interfaces. We argue that such a difference in flexibility could be key for distinct stability of the plus and minus microtubule ends. The higher flexibility of the inter-dimer interface may have implications for development of pulling force by curving tubulin protofilaments during microtubule disassembly, a process of major importance for chromosome motions in mitosis.


Assuntos
Tubulina (Proteína)/química , Fenômenos Biomecânicos , Biologia Computacional , Microscopia Crioeletrônica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Microtúbulos/química , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Simulação de Dinâmica Molecular , Análise de Componente Principal , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestrutura
10.
Nat Commun ; 10(1): 3530, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387998

RESUMO

Microtubules are a vital component of the cell's cytoskeleton and their organization is crucial for healthy cell functioning. The use of label-free SH imaging of microtubules remains limited, as sensitive detection is required and the true molecular origin and main determinants required to generate SH from microtubules are not fully understood. Using advanced correlative imaging techniques, we identified the determinants of the microtubule-dependent SH signal. Microtubule polarity, number and organization determine SH signal intensity in biological samples. At the molecular level, we show that the GTP-bound tubulin dimer conformation is fundamental for microtubules to generate detectable SH signals. We show that SH imaging can be used to study the effects of microtubule-targeting drugs and proteins and to detect changes in tubulin conformations during neuronal maturation. Our data provide a means to interpret and use SH imaging to monitor changes in the microtubule network in a label-free manner.


Assuntos
Microscopia Intravital/métodos , Microtúbulos/ultraestrutura , Imagem Molecular/métodos , Microscopia de Geração do Segundo Harmônico , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Células Cultivadas , Colchicina/farmacologia , Estudos de Viabilidade , Guanosina Trifosfato/metabolismo , Camundongos , Microscopia Eletrônica , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neurogênese , Cultura Primária de Células , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestrutura
11.
Nat Commun ; 10(1): 3058, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296862

RESUMO

The AAA+ GTPase McrB powers DNA cleavage by the endonuclease McrC. The GTPase itself is activated by McrC. The architecture of the GTPase and nuclease complex, and the mechanism of their activation remained unknown. Here, we report a 3.6 Å structure of a GTPase-active and DNA-binding deficient construct of McrBC. Two hexameric rings of McrB are bridged by McrC dimer. McrC interacts asymmetrically with McrB protomers and inserts a stalk into the pore of the ring, reminiscent of the γ subunit complexed to α3ß3 of F1-ATPase. Activation of the GTPase involves conformational changes of residues essential for hydrolysis. Three consecutive nucleotide-binding pockets are occupied by the GTP analogue 5'-guanylyl imidodiphosphate and the next three by GDP, which is suggestive of sequential GTP hydrolysis.


Assuntos
Domínio AAA , Enzimas de Restrição do DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Microscopia Crioeletrônica , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/isolamento & purificação , Enzimas de Restrição do DNA/ultraestrutura , DNA Bacteriano/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Guanosina Trifosfato/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Relação Estrutura-Atividade
12.
Elife ; 82019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31355745

RESUMO

Plasma membrane-located transport proteins are key adaptations for obligate intracellular Microsporidia parasites, because they can use them to steal host metabolites the parasites need to grow and replicate. However, despite their importance, the functions and substrate specificities of most Microsporidia transporters are unknown. Here, we provide functional data for a family of transporters conserved in all microsporidian genomes and also in the genomes of related endoparasites. The universal retention among otherwise highly reduced genomes indicates an important role for these transporters for intracellular parasites. Using Trachipleistophora hominis, a Microsporidia isolated from an HIV/AIDS patient, as our experimental model, we show that the proteins are ATP and GTP transporters located on the surface of parasites during their intracellular growth and replication. Our work identifies a new route for the acquisition of essential energy and nucleotides for a major group of intracellular parasites that infect most animal species including humans.


Assuntos
Trifosfato de Adenosina/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Microsporídios/genética , Microsporídios/metabolismo , Animais , Linhagem Celular , Biologia Computacional , Sequência Conservada , Genoma Fúngico , Microsporídios/crescimento & desenvolvimento , Coelhos
13.
Microbiol Res ; 223-225: 88-98, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178056

RESUMO

CodY and (p)ppGpp synthetases are two important global regulators of bacteria. In some pathogens, such as Listeria monocytogenes, the GTP pool links these two regulatory systems, and introducing a codY mutant into the ΔrelA strain restored the pathogenicity of the attenuated ΔrelA mutant. In previous studies, we identified the (p)ppGpp synthetases (RelA and RelQ) and CodY of Streptococcus suis. To understand the interrelationships between these two regulators in S. suis, a ΔrelAΔrelQΔcodY mutant was constructed, and its growth, morphology, and pathogenicity were evaluated. Compared with ΔrelAΔrelQ, ΔcodY, its growth was very slow, but its chain length was partly restored to the wild-type length and its capsule became thick and rough. The adherence, invasion ability, and resistance to whole-blood killing in vitro of ΔrelAΔrelQΔcodY and its lethality and colonization ability in mice were clearly reduced, which differs from the effects of these mutations in L. monocytogenes. An analysis of gene expression showed that CodY interacted with the relA promoter in a GTP-independent manner to positively regulate the expression of relA. The introduction of a codY mutant into the ΔrelAΔrelQ strain further reduced the expression of virulence factors, which suggests a novel interaction between the (p)ppGpp synthetases and CodY. This study extends our understanding of the relationship between the (p)ppGpp-mediated stringent response and the regulation of CodY in S. suis.


Assuntos
Regulação Bacteriana da Expressão Gênica , Ligases/metabolismo , Streptococcus suis/citologia , Streptococcus suis/metabolismo , Streptococcus suis/patogenicidade , Fatores de Transcrição/metabolismo , Transcriptoma , Adesinas Bacterianas/genética , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Guanosina Trifosfato/metabolismo , Ligases/genética , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Camundongos , Mutação , Regiões Promotoras Genéticas , Infecções Estreptocócicas/microbiologia , Streptococcus suis/genética , Fatores de Transcrição/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
14.
Mol Biol (Mosk) ; 53(3): 446-455, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184610

RESUMO

Ran is an evolutionarily conserved GTPase crucial in regulating various cell divisions, including mitosis and meiosis. A previous study showed that the knockdown of RAN1 inhibited macronuclear amitosis with the abnormal organization of intramacronuclear microtubules in Tetrahymena thermophila. This study aimed to further investigate the effects of the inducible expression of wild-type Ran1 (Ran1WT), GTP-bound Ran1-mimetic (Ran1Q70L), and GDP-bound Ran1-mimetic (Ran1T25N) on cytoplasmic microtubule assembly during amitosis of T. thermophila, based on previous studies about their effects on intramacronuclear microtubule. The mutant strains of T. thermophila for inducible expression of Ran1WT/T25N/Q70L by Cd^(2+) were constructed. The inducibly expressed HA-Ran1Q70L/T25N distributed asymmetrically across the macronuclear envelope during amitosis. At the lower level of inducible expression, only Ran1T25N showed a significant decreasing effect on T. thermophila reproduction, macronuclear amitosis and cytokinesis. At the higher level of inducible expression, Ran1WT/Q70L/T25N inhibited T. thermophila reproduction, macronuclear amitosis and cytokinesis, and the inhibitive effect of Ran1T25N was the most significant. The inducible expression of Ran1WT/Q70L/T25N led to defects in amitosis and cytokinesis with abnormal cytoplasmic microtubule assembly. These results further confirmed the regulatory function of Ran1 on amitosis and suggested a novel role of Ran1 in cytokinesis and the alignment of cytoplasmic microtubules in T. thermophila.


Assuntos
Citocinese , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Microtúbulos/metabolismo , Mutação , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila , Proteína ran de Ligação ao GTP/metabolismo , Microtúbulos/patologia , Mitose , Proteínas de Protozoários/genética , Tetrahymena thermophila/citologia , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Proteína ran de Ligação ao GTP/genética
15.
Nat Cell Biol ; 21(7): 845-855, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209295

RESUMO

RPEL proteins, which contain the G-actin-binding RPEL motif, coordinate cytoskeletal processes with actin dynamics. We show that the ArhGAP12- and ArhGAP32-family GTPase-activating proteins (GAPs) are RPEL proteins. We determine the structure of the ArhGAP12/G-actin complex, and show that G-actin contacts the RPEL motif and GAP domain sequences. G-actin inhibits ArhGAP12 GAP activity, and this requires the G-actin contacts identified in the structure. In B16 melanoma cells, ArhGAP12 suppresses basal Rac and Cdc42 activity, F-actin assembly, invadopodia formation and experimental metastasis. In this setting, ArhGAP12 mutants defective for G-actin binding exhibit more effective downregulation of Rac GTP loading following HGF stimulation and enhanced inhibition of Rac-dependent processes, including invadopodia formation. Potentiation or disruption of the G-actin/ArhGAP12 interaction, by treatment with the actin-binding drugs latrunculin B or cytochalasin D, has corresponding effects on Rac GTP loading. The interaction of G-actin with RPEL-family rhoGAPs thus provides a negative feedback loop that couples Rac activity to actin dynamics.


Assuntos
Actinas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/efeitos dos fármacos , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Citocalasina D/farmacologia , Proteínas Ativadoras de GTPase/efeitos dos fármacos , Proteínas Ativadoras de GTPase/genética , Guanosina Trifosfato/metabolismo , Humanos , Camundongos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Coelhos , Tiazolidinas/farmacologia , Proteína cdc42 de Ligação ao GTP/efeitos dos fármacos , Proteínas rac de Ligação ao GTP/metabolismo
16.
Nucleic Acids Res ; 47(11): 5761-5776, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31216040

RESUMO

Giant viruses have extraordinarily large dsDNA genomes, and exceptionally, they encode various components of the translation apparatus, including tRNAs, aminoacyl-tRNA synthetases and translation factors. Here, we focused on the elongation factor 1 (EF1) family of viral translational GTPases (trGTPases), using computational and functional approaches to shed light on their functions. Multiple sequence alignment indicated that these trGTPases clustered into two groups epitomized by members of Mimiviridae and Marseilleviridae, respectively. trGTPases in the first group were more closely related to GTP-binding protein 1 (GTPBP1), whereas trGTPases in the second group were closer to eEF1A, eRF3 and Hbs1. Functional characterization of representative GTPBP1-like trGTPases (encoded by Hirudovirus, Catovirus and Moumouvirus) using in vitro reconstitution revealed that they possess eEF1A-like activity and can deliver cognate aa-tRNAs to the ribosomal A site during translation elongation. By contrast, representative eEF1A/eRF3/Hbs1-like viral trGTPases, encoded by Marseillevirus and Lausannevirus, have eRF3-like termination activity and stimulate peptide release by eRF1. Our analysis identified specific aspects of the functioning of these viral trGTPases with eRF1 of human, amoebal and Marseillevirus origin.


Assuntos
Acanthamoeba castellanii/metabolismo , Amoeba/metabolismo , GTP Fosfo-Hidrolases/química , Vírus Gigantes/metabolismo , Fator 1 de Elongação de Peptídeos/química , Análise por Conglomerados , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Proteínas Monoméricas de Ligação ao GTP/química , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Filogenia , Ligação Proteica , Biossíntese de Proteínas , Ribossomos/metabolismo
17.
Int J Biol Macromol ; 136: 1258-1272, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31242450

RESUMO

Myxovirus resistance (Mx) proteins represents the subclass of the dynamin superfamily of large Guanosine triphosphates (GTPases), play esential role in intracellular vesicle trafficking, endocytosis, organelle homeostasis and mitochondria distribution. These proteins are key players of the vertebrate immune system, induced by type-I and type-III interferons (IFN) of infected host and inhibit viral replication by sequestering its nucleoprotein. In the present study, we report the sequencing and characterization of Cirrhinus mrigala Mx protein (CmMx) for the first time and observed its constitutive expression in different tissues for a period of fourteen days. The synthetic peptide, LSGVALPRGTGI, was dissolved in PBS and injected into a rabbit and the antibody raised against CmMx was used to study the level of its expression. The full length of the CmMx cDNA is 2244 bp with a molecular mass of 70.9 kDa and a predicted isoelectric point of 8.25. The 627 amino acids polypeptide formed of three main functional domains: N-terminal GTPase domain (GD), a middle domain (MD) and GTPase effector domain (GED) with carboxy terminal leucine zipper motif. The 3D models of CmMx protein was modeled based on available close structural homologs and further validated through molecular dynamics (MD) simulations. MD study revealed the importance of G-domain responsible for recognition of GTP, which perfectly corroborate with earlier studies. MM/PBSA binding free energy analysis displayed that van der Waals and electrostatic energy were the key driving force behind molecular recognition of GTP by CmMx protein. The results from this study will illuminate more lights into the ongoing research on myxovirus resistance protein and its role in inhibition of viral replication in other eukaryotic system as well.


Assuntos
Cipriniformes/genética , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Guanosina Trifosfato/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/genética , Proteínas de Peixes/química , Cinética , Simulação de Dinâmica Molecular , Proteínas de Resistência a Myxovirus/química , Filogenia , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , RNA Mensageiro/genética , Termodinâmica
18.
Dev Cell ; 49(6): 894-906.e12, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31105010

RESUMO

RhoA stimulates cell contractility by recruiting downstream effectors to the cortical plasma membrane. We now show that direct binding by anillin is required for effective signaling: this antagonizes the otherwise labile membrane association of GTP-RhoA to promote effector recruitment. However, since its binding to RhoA blocks access by other effectors, we demonstrate that anillin must also concentrate membrane phosphoinositide-4,5-P2 (PIP2) to promote signaling. We propose and test a sequential pathway where GTP-RhoA first binds to anillin and then is retained at the membrane by PIP2 after it disengages from anillin. Importantly, re-binding of membrane GTP-RhoA to anillin, regulated by the cortical density of anillin, creates cycles through this pathway. These cycles repeatedly reset the dissociation kinetics of GTP-RhoA, substantially increasing its dwell time to recruit effectors. Thus, anillin regulates RhoA signaling by a paradigm of kinetic scaffolding that may apply to other signals whose efficacy depends on their cortical dwell times.


Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular/efeitos dos fármacos , Proteínas Contráteis/farmacologia , Citocinese/fisiologia , Guanosina Trifosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Citocinese/efeitos dos fármacos , Feminino , Humanos , Cinética , Células MCF-7 , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/genética
19.
Org Biomol Chem ; 17(19): 4879-4891, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31041977

RESUMO

The mechanism of the deceptively simple reaction of guanosine triphosphate (GTP) hydrolysis catalyzed by the cellular protein Ras in complex with the activating protein GAP is an important issue because of the significance of this reaction in cancer research. We show that molecular modeling of GTP hydrolysis in the Ras-GAP active site reveals a diversity of mechanisms of the intrinsic chemical reaction depending on molecular groups at position 61 in Ras occupied by glutamine in the wild-type enzyme. First, a comparison of reaction energy profiles computed at the quantum mechanics/molecular mechanics (QM/MM) level shows that an assignment of the Gln61 side chain in the wild-type Ras either to QM or to MM parts leads to different scenarios corresponding to the glutamine-assisted or the substrate-assisted mechanisms. Second, replacement of Gln61 by the nitro-analog of glutamine (NGln) or by Glu, applied in experimental studies, results in two more scenarios featuring the so-called two-water and the concerted-type mechanisms. The glutamine-assisted mechanism in the wild-type Ras-GAP, in which the conserved Gln61 plays a decisive role, switching between the amide and imide tautomer forms, is consistent with the known experimental results of structural, kinetic and spectroscopy studies. The results emphasize the role of the Ras residue Gln61 in Ras-GAP catalysis and explain the retained catalytic activity of the Ras-GAP complex towards GTP hydrolysis in the Gln61NGln and Gln61Glu mutants of Ras.


Assuntos
Biocatálise , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Proteínas Ativadoras de ras GTPase/metabolismo , Sítios de Ligação , Guanosina Trifosfato/química , Hidrólise , Conformação Molecular , Mutação , Teoria Quântica , Proteínas Ativadoras de ras GTPase/química , Proteínas Ativadoras de ras GTPase/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA