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1.
Dev Cell ; 57(3): 361-372.e5, 2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-35045336

RESUMO

The symbiotic relationship between commensal microbes and host animals predicts unidentified beneficial impacts of individual bacterial metabolites on animal physiology. Peptidoglycan fragments (muropeptides) from the bacterial cell wall are known for their roles in pathogenicity and for inducing host immune responses. However, the potential beneficial usage of muropeptides from commensal bacteria by the host needs exploration. We identified a striking role for muropeptides in supporting mitochondrial homeostasis, development, and behaviors in Caenorhabditis elegans. We determined that the beneficial molecules are disaccharide muropeptides containing a short AA chain, and they enter intestinal-cell mitochondria to repress oxidative stress. Further analyses indicate that muropeptides execute this role by binding to and promoting the activity of ATP synthase. Therefore, given the exceptional structural conservation of ATP synthase, the role of muropeptides as a rare agonist of the ATP synthase presents a major conceptual modification regarding the impact of bacterial cell metabolites on animal physiology.


Assuntos
Complexos de ATP Sintetase/metabolismo , Caenorhabditis elegans/fisiologia , Homeostase , Mitocôndrias/metabolismo , Peptídeos/metabolismo , Peptidoglicano/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Comportamento Alimentar/efeitos dos fármacos , Células HEK293 , Humanos , Intestinos/metabolismo , Metaboloma/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos
2.
Open Biol ; 11(12): 210177, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34905702

RESUMO

There is a surprisingly high morphological similarity between multilamellar concentric thylakoids in cyanobacteria and the myelin sheath that wraps the nerve axons. Thylakoids are multilamellar structures, which express photosystems I and II, cytochromes and ATP synthase necessary for the light-dependent reaction of photosynthesis. Myelin is a multilamellar structure that surrounds many axons in the nervous system and has long been believed to act simply as an insulator. However, it has been shown that myelin has a trophic role, conveying nutrients to the axons and producing ATP through oxidative phosphorylation. Therefore, it is tempting to presume that both membranous structures, although distant in the evolution tree, share not only a morphological but also a functional similarity, acting in feeding ATP synthesized by the ATP synthase to the centre of the multilamellar structure. Therefore, both molecular structures may represent a convergent evolution of life on Earth to fulfill fundamentally similar functions.


Assuntos
Trifosfato de Adenosina/metabolismo , Cianobactérias/ultraestrutura , Bainha de Mielina/ultraestrutura , Tilacoides/ultraestrutura , Complexos de ATP Sintetase/metabolismo , Animais , Evolução Biológica , Cianobactérias/metabolismo , Metabolismo Energético , Humanos , Bainha de Mielina/metabolismo , Fosforilação Oxidativa , Tilacoides/metabolismo
3.
Protein Sci ; 30(9): 1974-1982, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34191368

RESUMO

Membrane proteins play key roles in cellular signaling and transport, represent the majority of drug targets, and are implicated in many diseases. Their relevance renders them important subjects for structural, biophysical, and functional investigations. However, obtaining membrane proteins in high purities is often challenging with conventional purification steps alone. To address this issue, we present here an approach to increase the purity of α-helical transmembrane proteins. Our approach exploits the Thioredoxin (Trx) tag system, which is able to confer some of its favorable properties, such as high solubility and thermostability, to its fusion partners. Using Trx fusions of transmembrane helical hairpin constructs derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) and a bacterial ATP synthase, we establish conditions for the successful implementation of the selective heat treatment procedure to increase sample purity. We further examine systematically its efficacy with respect to different incubation times and temperatures using quantitative gel electrophoresis. We find that minute-timescale heat treatment of Trx-tagged fusion constructs with temperatures ranging from 50 to 90°C increases the purity of the membrane protein samples from ~60 to 98% even after affinity purification. We show that this single-step approach is even applicable in cases where regular selective heat purification from crude extracts, as reported for Trx fusions to soluble proteins, fails. Overall, our approach is easy to integrate into existing purification strategies and provides a facile route for increasing the purity of membrane protein constructs after purification by standard chromatography approaches.


Assuntos
Complexos de ATP Sintetase/química , Proteínas de Bactérias/química , Regulador de Condutância Transmembrana em Fibrose Cística/química , Subunidades Proteicas/química , Proteínas Recombinantes de Fusão/química , Tiorredoxinas/química , Complexos de ATP Sintetase/genética , Complexos de ATP Sintetase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fusobactérias/química , Fusobactérias/enzimologia , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Temperatura Alta , Humanos , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
4.
Nat Commun ; 12(1): 3669, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135327

RESUMO

Human rhinoviruses (HRV) are common cold viruses associated with exacerbations of lower airways diseases. Although viral induced epithelial damage mediates inflammation, the molecular mechanisms responsible for airway epithelial damage and dysfunction remain undefined. Using experimental HRV infection studies in highly differentiated human bronchial epithelial cells grown at air-liquid interface (ALI), we examine the links between viral host defense, cellular metabolism, and epithelial barrier function. We observe that early HRV-C15 infection induces a transitory barrier-protective metabolic state characterized by glycolysis that ultimately becomes exhausted as the infection progresses and leads to cellular damage. Pharmacological promotion of glycolysis induces ROS-dependent upregulation of the mitochondrial metabolic regulator, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), thereby restoring epithelial barrier function, improving viral defense, and attenuating disease pathology. Therefore, PGC-1α regulates a metabolic pathway essential to host defense that can be therapeutically targeted to rescue airway epithelial barrier dysfunction and potentially prevent severe respiratory complications or secondary bacterial infections.


Assuntos
Antivirais/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Mucosa Respiratória/metabolismo , Rhinovirus/fisiologia , Complexos de ATP Sintetase/antagonistas & inibidores , Complexos de ATP Sintetase/metabolismo , Antivirais/farmacologia , Técnicas de Cultura de Células , Citoesqueleto/metabolismo , Células Epiteliais , Ácidos Graxos/biossíntese , Glicólise , Humanos , Redes e Vias Metabólicas , Mitocôndrias/metabolismo , Oligomicinas/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Infecções por Picornaviridae/virologia , Espécies Reativas de Oxigênio/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Replicação Viral/efeitos dos fármacos
5.
Sci Rep ; 11(1): 4506, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627709

RESUMO

Upon endoplasmic-reticulum (ER) stress, the ER-located transmembrane protein, Ire1, is autophosphorylated and acts as an endoribonuclease to trigger the unfolded protein response (UPR). Previous biochemical studies have shown that Ire1 exhibits strong endoribonuclease activity when its cytosolic kinase region captures ADP. Here, we asked how this event contributes to the regulation of Ire1 activity. At the beginning of this study, we obtained a luminal-domain mutant of Saccharomyces cerevisiae Ire1, deltaIdeltaIIIdeltaV/Y225H Ire1, which is deduced to be controlled by none of the luminal-side regulatory events. ER-stress responsiveness of deltaIdeltaIIIdeltaV/Y225H Ire1 was largely compromised by a further mutation on the kinase region, D797N/K799N, which allows Ire1 to be activated without capturing ADP. Therefore, in addition to the ER-luminal domain of Ire1, which monitors ER conditions, the kinase region is directly involved in the ER-stress responsiveness of Ire1. We propose that potent ER stress harms cells' "vividness", increasing the cytosolic ADP/ATP ratio, and eventually strongly activates Ire1. This mechanism seems to contribute to the suppression of inappropriately potent UPR under weak ER-stress conditions.


Assuntos
Complexos de ATP Sintetase/metabolismo , Difosfato de Adenosina/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Citosol/metabolismo , Endorribonucleases/metabolismo , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/fisiologia
6.
Exp Cell Res ; 401(1): 112520, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33639177

RESUMO

We previously found that ATP synthases localize to male-specific sensory cilia and control the ciliary response by regulating polycystin signalling in Caenorhabditis elegans. Herein, we discovered that the ciliary localization of ATP synthase is evolutionarily conserved in mammals. We showed that the ATP synthase subunit F1ß is colocalized with the cilia marker acetylated α-tubulin in both mammalian renal epithelial cells (MDCK) and normal mouse cholangiocytes (NMCs). Treatment with ATP synthase inhibitor oligomycin impaired ciliogenesis in MDCK cells, and F1ß was co-immunoprecipitated with PKD2 in mammalian cells. Our study provides evidence for the evolutionarily conserved localization of ATP synthase in cilia from worm to mammals. Defects in ATP synthase can lead to ciliary dysfunction, which may be a potential mechanism of polycystic kidney disease.


Assuntos
Cílios/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Chaperonas Moleculares/genética , Canais de Cátion TRPP/genética , Complexos de ATP Sintetase/química , Complexos de ATP Sintetase/genética , Trifosfato de Adenosina/genética , Animais , Caenorhabditis elegans/genética , Cílios/metabolismo , Cães , Cinesinas/genética , Células Madin Darby de Rim Canino , Mamíferos , Camundongos , Oligomicinas/farmacologia , Doenças Renais Policísticas/enzimologia , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Processamento de Proteína Pós-Traducional/genética
7.
Hum Mol Genet ; 30(5): 381-392, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33600551

RESUMO

The human ATP synthase is an assembly of 29 subunits of 18 different types, of which only two (a and 8) are encoded in the mitochondrial genome. Subunit a, together with an oligomeric ring of c-subunit (c-ring), forms the proton pathway responsible for the transport of protons through the mitochondrial inner membrane, coupled to rotation of the c-ring and ATP synthesis. Neuromuscular diseases have been associated to a number of mutations in the gene encoding subunit a, ATP6. The most common, m.8993 T > G, leads to replacement of a strictly conserved leucine residue with arginine (aL156R). We previously showed that the equivalent mutation (aL173R) dramatically compromises respiratory growth of Saccharomyces cerevisiae and causes a 90% drop in the rate of mitochondrial ATP synthesis. Here, we isolated revertants from the aL173R strain that show improved respiratory growth. Four first-site reversions at codon 173 (aL173M, aL173S, aL173K and aL173W) and five second-site reversions at another codon (aR169M, aR169S, aA170P, aA170G and aI216S) were identified. Based on the atomic structures of yeast ATP synthase and the biochemical properties of the revertant strains, we propose that the aL173R mutation is responsible for unfavorable electrostatic interactions that prevent the release of protons from the c-ring into a channel from which protons move from the c-ring to the mitochondrial matrix. The results provide further evidence that yeast aL173 (and thus human aL156) optimizes the exit of protons from ATP synthase, but is not essential despite its strict evolutionary conservation.


Assuntos
Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Subunidades Proteicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Complexos de ATP Sintetase/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , DNA Mitocondrial , Genes Mitocondriais , Humanos , Modelos Moleculares , Mutação , Domínios Proteicos , Subunidades Proteicas/metabolismo , Prótons
8.
Proc Natl Acad Sci U S A ; 118(7)2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33526592

RESUMO

The construction of energetically autonomous artificial protocells is one of the most ambitious goals in bottom-up synthetic biology. Here, we show an efficient manner to build adenosine 5'-triphosphate (ATP) synthesizing hybrid multicompartment protocells. Bacterial chromatophores from Rhodobacter sphaeroides accomplish the photophosphorylation of adenosine 5'-diphosphate (ADP) to ATP, functioning as nanosized photosynthetic organellae when encapsulated inside artificial giant phospholipid vesicles (ATP production rate up to ∼100 ATP∙s-1 per ATP synthase). The chromatophore morphology and the orientation of the photophosphorylation proteins were characterized by cryo-electron microscopy (cryo-EM) and time-resolved spectroscopy. The freshly synthesized ATP has been employed for sustaining the transcription of a DNA gene, following the RNA biosynthesis inside individual vesicles by confocal microscopy. The hybrid multicompartment approach here proposed is very promising for the construction of full-fledged artificial protocells because it relies on easy-to-obtain and ready-to-use chromatophores, paving the way for artificial simplified-autotroph protocells (ASAPs).


Assuntos
Trifosfato de Adenosina/biossíntese , Células Artificiais/metabolismo , Cromatóforos Bacterianos/metabolismo , Transcrição Gênica , Complexos de ATP Sintetase/genética , Complexos de ATP Sintetase/metabolismo , Células Artificiais/química , Cromatóforos Bacterianos/ultraestrutura , Fotossíntese , Rhodobacter sphaeroides/metabolismo , Luz Solar , Biologia Sintética/métodos
9.
Curr Drug Targets ; 22(11): 1207-1221, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33480344

RESUMO

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is one of the leading causes of mortality worldwide, with an estimated 1.5 million deaths annually. The majority of infection cases are reported from the Southeast Asian region, including India. After the discovery of Streptomycin in 1943 and its anti-tubercular activity in 1945, drug discovery efforts identified Isoniazid, Ethambutol, and Rifampin as TB-actives. However, over the years, these drugs have been rendered ineffective due to genetic mutations in mycobacterial strains. This has shifted drug discovery efforts towards identifying new targets and drugs for drug-resistant forms of bacteria. ATP synthase was identified as one of the key targets of MDR-TB. This review provides key insights into the ATP synthase target, structure activity relationship studies (SAR) of diarylquinoline class of inhibitors and their clinical relevance for treating MDR-TB.


Assuntos
Complexos de ATP Sintetase/antagonistas & inibidores , Antituberculosos , Diarilquinolinas/farmacologia , Descoberta de Drogas , Tuberculose , Trifosfato de Adenosina , Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico
10.
Biochim Biophys Acta Bioenerg ; 1862(4): 148378, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33460587

RESUMO

ATP synthases are the key elements of cellular bioenergetics and present in any life form and the overall structure and function of this rotary energy converter is conserved in all domains of life. However, ancestral microbes, the archaea, have a unique and huge diversity in the size and number of ion-binding sites in their membrane-embedded rotor subunit c. Due to the harsh conditions for ATP synthesis in these life forms it has never been possible to address the consequences of these unusual c subunits for ATP synthesis. Recently, we have found a Na+-dependent archaeal ATP synthase with a V-type c subunit in a mesophilic bacterium and here, we have cloned and expressed the genes in the ATP synthase-negative strain Escherichia coli DK8. The enzyme was present in membranes of E. coli DK8 and catalyzed ATP hydrolysis with a rate of 35 nmol·min-1·mg protein-1. Inverted membrane vesicles of this strain were then checked for their ability to synthesize ATP. Indeed, ATP was synthesized driven by NADH oxidation despite the V-type c subunit. ATP synthesis was dependent on Na+ and inhibited by ionophores. Most importantly, ATPase activity was inhibited by DCCD and this inhibition was relieved by addition of Na+, indicating a functional coupling of the F1 and FO domains, a prerequisite for studies on structure-function relationship. A first step in this direction was the exchange of a conserved arginine (Arg530) in the FO motor subunit a which led to loss of ATP synthesis whereas ATP hydrolysis was retained.


Assuntos
Complexos de ATP Sintetase , Archaea/enzimologia , Proteínas Arqueais , Proteínas de Bactérias , Escherichia coli , Eubacterium/genética , Microrganismos Geneticamente Modificados , Complexos de ATP Sintetase/genética , Complexos de ATP Sintetase/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Archaea/genética , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Eubacterium/enzimologia , Microrganismos Geneticamente Modificados/enzimologia , Microrganismos Geneticamente Modificados/genética
11.
Mol Cell Biochem ; 476(1): 493-506, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33000352

RESUMO

Mitochondria have various cellular functions, including ATP synthesis, calcium homeostasis, cell senescence, and death. Mitochondrial dysfunction has been identified in a variety of disorders correlated with human health. Among the many underlying mechanisms of mitochondrial dysfunction, the opening up of the mitochondrial permeability transition pore (mPTP) is one that has drawn increasing interest in recent years. It plays an important role in apoptosis and necrosis; however, the molecular structure and function of the mPTP have still not been fully elucidated. In recent years, the abnormal opening up of the mPTP has been implicated in the development and pathogenesis of diverse diseases including ischemia/reperfusion injury (IRI), neurodegenerative disorders, tumors, and chronic obstructive pulmonary disease (COPD). This review provides a systematic introduction to the possible molecular makeup of the mPTP and summarizes the mitochondrial dysfunction-correlated diseases and highlights possible underlying mechanisms. Since the mPTP is an important target in mitochondrial dysfunction, this review also summarizes potential treatments, which may be used to inhibit pore opening up via the molecules composing mPTP complexes, thus suppressing the progression of mitochondrial dysfunction-related diseases.


Assuntos
Trifosfato de Adenosina/metabolismo , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/terapia , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Complexos de ATP Sintetase/metabolismo , Animais , Ânions , Apoptose , Transporte Biológico , Humanos , Mitocôndrias Cardíacas/metabolismo , Membranas Mitocondriais/metabolismo , Necrose , Doenças Neurodegenerativas/metabolismo , Fosfatos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptores de GABA/metabolismo , Traumatismo por Reperfusão
12.
Nature ; 589(7840): 143-147, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33299175

RESUMO

Tuberculosis-the world's leading cause of death by infectious disease-is increasingly resistant to current first-line antibiotics1. The bacterium Mycobacterium tuberculosis (which causes tuberculosis) can survive low-energy conditions, allowing infections to remain dormant and decreasing their susceptibility to many antibiotics2. Bedaquiline was developed in 2005 from a lead compound identified in a phenotypic screen against Mycobacterium smegmatis3. This drug can sterilize even latent M. tuberculosis infections4 and has become a cornerstone of treatment for multidrug-resistant and extensively drug-resistant tuberculosis1,5,6. Bedaquiline targets the mycobacterial ATP synthase3, which is an essential enzyme in the obligate aerobic Mycobacterium genus3,7, but how it binds the intact enzyme is unknown. Here we determined cryo-electron microscopy structures of M. smegmatis ATP synthase alone and in complex with bedaquiline. The drug-free structure suggests that hook-like extensions from the α-subunits prevent the enzyme from running in reverse, inhibiting ATP hydrolysis and preserving energy in hypoxic conditions. Bedaquiline binding induces large conformational changes in the ATP synthase, creating tight binding pockets at the interface of subunits a and c that explain the potency of this drug as an antibiotic for tuberculosis.


Assuntos
Complexos de ATP Sintetase/química , Antituberculosos/química , Microscopia Crioeletrônica , Diarilquinolinas/química , Mycobacterium smegmatis/enzimologia , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Complexos de ATP Sintetase/antagonistas & inibidores , Complexos de ATP Sintetase/metabolismo , Trifosfato de Adenosina/metabolismo , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Diarilquinolinas/metabolismo , Diarilquinolinas/farmacologia , Hidrólise/efeitos dos fármacos , Modelos Moleculares , Mycobacterium smegmatis/efeitos dos fármacos , Rotação
13.
mBio ; 11(5)2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900803

RESUMO

Staphylococcus aureus is a major cause of prosthetic joint infection (PJI), which is characterized by biofilm formation. S. aureus biofilm skews the host immune response toward an anti-inflammatory profile by the increased recruitment of myeloid-derived suppressor cells (MDSCs) that attenuate macrophage proinflammatory activity, leading to chronic infection. A screen of the Nebraska Transposon Mutant Library identified several hits in the ATP synthase operon that elicited a heightened inflammatory response in macrophages and MDSCs, including atpA, which encodes the alpha subunit of ATP synthase. An atpA transposon mutant (ΔatpA) had altered growth kinetics under both planktonic and biofilm conditions, along with a diffuse biofilm architecture that was permissive for leukocyte infiltration, as observed by confocal laser scanning microscopy. Coculture of MDSCs and macrophages with ΔatpA biofilm elicited significant increases in the proinflammatory cytokines interleukin 12p70 (IL-12p70), tumor necrosis factor alpha (TNF-α), and IL-6. This was attributed to increased leukocyte survival resulting from less toxin and protease production by ΔatpA biofilm as determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The enhanced inflammatory response elicited by ΔatpA biofilm was cell lysis-dependent since it was negated by polyanethole sodium sulfanate treatment or deletion of the major autolysin, Atl. In a mouse model of PJI, ΔatpA-infected mice had decreased MDSCs concomitant with increased monocyte/macrophage infiltrates and proinflammatory cytokine production, which resulted in biofilm clearance. These studies identify S. aureus ATP synthase as an important factor in influencing the immune response during biofilm-associated infection and bacterial persistence.IMPORTANCE Medical device-associated biofilm infections are a therapeutic challenge based on their antibiotic tolerance and ability to evade immune-mediated clearance. The virulence determinants responsible for bacterial biofilm to induce a maladaptive immune response remain largely unknown. This study identified a critical role for S. aureus ATP synthase in influencing the host immune response to biofilm infection. An S. aureus ATP synthase alpha subunit mutant (ΔatpA) elicited heightened proinflammatory cytokine production by leukocytes in vitro and in vivo, which coincided with improved biofilm clearance in a mouse model of prosthetic joint infection. The ability of S. aureus ΔatpA to augment host proinflammatory responses was cell lysis-dependent, as inhibition of bacterial lysis by polyanethole sodium sulfanate or a ΔatpAΔatl biofilm did not elicit heightened cytokine production. These studies reveal a critical role for AtpA in shaping the host immune response to S. aureus biofilm.


Assuntos
Complexos de ATP Sintetase/genética , Complexos de ATP Sintetase/imunologia , Biofilmes/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Staphylococcus aureus/enzimologia , Staphylococcus aureus/imunologia , Complexos de ATP Sintetase/metabolismo , Animais , Citocinas/imunologia , Modelos Animais de Doenças , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade
14.
Curr Top Med Chem ; 20(29): 2723-2734, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32885753

RESUMO

BACKGROUND: Tuberculosis (TB) is a major infectious disease caused by Mycobacterium Tuberculosis. As per the World Health Organization (WHO) report of 2019, there were 1.5 million deaths in the year 2018, mainly because of multi- and extensively drug-resistant tuberculosis (MDR & XDR-TB). Among several antitubercular drugs in clinical trials, bedaquiline (TMC207) is a highly promising drug that was approved by the FDA in 2012 and marketed in 2016 for the treatment of multidrug resistant TB in combination with other drugs. Bedaquiline acts on mycobacterial ATP synthase and is highly effective in replicating as well as on dormant mycobacteria. Several series of substituted quinolines have been reported with their antitubercular and ATP synthase inhibitory activity. METHODS: To understand the role of physicochemical parameters like hydrophobicity, electronic and steric factors in eliciting the biological response, the Quantitative structure-activity relationship (QSAR) studies have been carried out using the computed parameters as independent variable and activity (-log IC50/MIC) as the dependent variable. RESULTS: The developed QSAR models in terms of positively contributing Molar Refractivity (MR) and negatively contributing Partition Coefficient (PC) and Connolly Molecular Area (CMA) parameters have high predictivity as also shown on external data set and the mean value of the computed 3D parameters of enantiomers may be used in QSAR analysis for racemic compounds. CONCLUSION: These results are also substantiated by pharmacophore modeling. The similar dependence of antitubercular activity against whole-cell M.Tb.H37Rv on MR and CMA suggests ATP synthase as the main target for antitubercular activity and the QSAR models may be useful in the identification of novel antitubercular agents.


Assuntos
Complexos de ATP Sintetase/antagonistas & inibidores , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Quantitativa Estrutura-Atividade , Quinolinas/farmacologia , Complexos de ATP Sintetase/metabolismo , Antituberculosos/química , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Quinolinas/química , Tuberculose/tratamento farmacológico , Tuberculose/metabolismo
15.
Mol Cell Proteomics ; 19(11): 1805-1825, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32788343

RESUMO

The EGFR tyrosine kinase inhibitor gefitinib is commonly used for lung cancer patients. However, some patients eventually become resistant to gefitinib and develop progressive disease. Here, we indicate that ecto-ATP synthase, which ectopically translocated from mitochondrial inner membrane to plasma membrane, is considered as a potential therapeutic target for drug-resistant cells. Quantitative multi-omics profiling reveals that ecto-ATP synthase inhibitor mediates CK2-dependent phosphorylation of DNA topoisomerase IIα (topo IIα) at serine 1106 and subsequently increases the expression of long noncoding RNA, GAS5. Additionally, we also determine that downstream of GAS5, p53 pathway, is activated by ecto-ATP synthase inhibitor for regulation of programed cell death. Interestingly, GAS5-proteins interactomic profiling elucidates that GAS5 associates with topo IIα and subsequently enhancing the phosphorylation level of topo IIα. Taken together, our findings suggest that ecto-ATP synthase blockade is an effective therapeutic strategy via regulation of CK2/phospho-topo IIα/GAS5 network in gefitinib-resistant lung cancer cells.


Assuntos
Complexos de ATP Sintetase/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/metabolismo , RNA Longo não Codificante/metabolismo , Complexos de ATP Sintetase/genética , Complexos de ATP Sintetase/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Membrana Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , DNA Topoisomerases Tipo II/metabolismo , Gefitinibe/farmacologia , Ontologia Genética , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteômica , RNA Longo não Codificante/genética , RNA Interferente Pequeno , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Espectrometria de Massas em Tandem , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
17.
Sci Rep ; 10(1): 8134, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424122

RESUMO

Despite the remarkable advances due to the discovery and development of antimicrobials agents, infectious diseases remain the second leading cause of death worldwide. This fact underlines the importance of developing new therapeutic strategies to address the widespread antibiotic resistance, which is the major contributing factor for clinical failures of the current therapeutics. In a screen for antibiotic adjuvants, we identified a natural product from actinomycetes, venturicidin A (VentA), that potentiates the aminoglycoside antibiotic gentamicin against multidrug-resistant clinical isolates of Staphylococcus, Enterococcus, and Pseudomonas aeruginosa. Furthermore, the combination of gentamicin and VentA was bactericidal and rapidly eradicated methicillin-resistant S. aureus (MRSA). The molecular mechanism of gentamicin potentiation activity is attributed to uncoupling of ATP synthesis by VentA from electron transport presumably by blocking the proton flow through ATP synthase, which results in an elevated concentration of extracellular protons and subsequent anticipated raise in gentamicin uptake. The disruption of the proton flux was characterized by perturbed membrane potential in MRSA. These results demonstrate that inhibition of ATP synthase along with the subsequent membrane dysregulation, as shown here with VentA, complements aminoglycoside antibiotics against MDR bacteria, and that this approach may be employed to combat bacterial resistance.


Assuntos
Complexos de ATP Sintetase/antagonistas & inibidores , Actinobacteria/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Venturicidinas/farmacologia , Complexos de ATP Sintetase/genética , Complexos de ATP Sintetase/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoglicosídeos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/enzimologia , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética
18.
Protein J ; 39(2): 152-159, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32112190

RESUMO

The segmented annelid worm, Mesenchytraeus solifugus, is a permanent resident of temperate, maritime glaciers in the Pacific northwestern region of North America, displaying atypically high intracellular ATP levels which have been linked to its unusual ability to thrive in hydrated glacier ice. We have shown previously that ice worms contain a highly basic, carboxy terminal extension on their ATP6 regulatory subunit, likely acquired by horizontal gene transfer from a microbial dietary source. Here we examine the full complement of F1F0 ATP synthase structural subunits with attention to non-conservative, ice worm-specific structural modifications. Our genomics analyses and molecular models identify putative proton shuttling domains on either side of the F0 hemichannel, which predictably function to enhance proton flow across the mitochondrial membrane. Other components of the ice worm ATP synthase complex have remained largely unchanged in the context of Metazoan evolution.


Assuntos
Complexos de ATP Sintetase/química , Evolução Molecular , Oligoquetos/enzimologia , Complexos de ATP Sintetase/genética , Adaptação Biológica , Animais , Temperatura Baixa , Metabolismo Energético , Oligoquetos/genética , Domínios Proteicos
19.
Electromagn Biol Med ; 39(2): 45-48, 2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-32160792

RESUMO

The Warburg observation concerning ATP generation in cancer cells is analyzed with regard to the likely involvement of H+ resonance effects on the angular velocity of the ATP synthase rotor. It is reasonable to expect that the variety of diseases associated with mitochondrial dysfunction may in part be related to the ATP synthase rate of rotation. Experimental measurements of ATP synthase rotational rates, as found in the literature, are consistent with what might be expected from the ion cyclotron resonance (ICR) frequencies of protons moving under a Lorentz force determined by the approximate surface intensity of the geomagnetic field (~26-65 ?T). One research approach proposes that applying the electronic sum of two critical multiple resonance frequencies simultaneously may serve to more closely resemble real world biochemical changes as compared to applying these frequencies sequentially. Accordingly, it is suggested that applying the sum of the two individual resonance frequencies corresponding to H+ and H3O+ has the capacity to both increase proton density as well as couple to ATP synthase rotation. Not only will this tend to stabilize the F0 rotational rate but also perhaps make it feasible to control this rate, thereby providing a new and potentially efficacious means of treating diseases connected to mitochondrial dysfunction electromagnetically.


Assuntos
Trifosfato de Adenosina/metabolismo , Ondas de Rádio , Complexos de ATP Sintetase/metabolismo , Ciclotrons , Rotação
20.
Plant Sci ; 292: 110387, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32005392

RESUMO

The cyclic electron flow (CEF) around photosystem I (PSI) plays a crucial role in photosynthesis and also functions in plant tolerance of abiotic environmental stress. However, the role of PGR5/PGRL1- and NDH-dependent CEF in tomato under hightemperature (HT) is poorly understood. Here, we assessed the photoprotective effect of these pathways in tomato leaves under HT by using antimycin A (AA) and rotenone (R), which are chemical inhibitors of PGR5/PGRL1- and NDH-dependent CEF, respectively. The results showed that AA treatment caused significantly greater inhibition of CEF under HT compared to R treatment. Moreover, AA treatment caused a greater decrease in maximal photochemistry efficiency (Fv/Fm) and increased damage to the donor and acceptor side of photosystem II (PSII); however, the limitation of the acceptor side in PSI [Y(NA)] was significantly increased. In addition, thylakoid membrane integrity was compromised and reactive oxygen species, proton gradient (ΔpH), antioxidant enzyme activity, and the expression of photosystem core subunit genes were significantly decreased under AA treatment. These findings indicate that PGR5/PGRL1-dependent CEF protects PSII and PSI from photooxidative damage through the formation of ΔpH while maintaining thylakoid membrane integrity and normal gene expression levels of core photosystem components. This study demonstrates that PGR5/PGRL1-dependent CEF plays a major role in HT response in tomato.


Assuntos
Elétrons , Temperatura Alta , Proteínas de Plantas/genética , Espécies Reativas de Oxigênio/metabolismo , Solanum lycopersicum/genética , Complexos de ATP Sintetase/genética , Complexos de ATP Sintetase/metabolismo , Expressão Gênica , Solanum lycopersicum/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Força Próton-Motriz
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