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1.
Microb Cell Fact ; 19(1): 12, 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31973723

RESUMO

BACKGROUND: The ureolytic bacterium Sporosarcina pasteurii is well-known for its capability of microbially induced calcite precipitation (MICP), representing a great potential in constructional engineering and material applications. However, the molecular mechanism for its biomineralization remains unresolved, as few studies were carried out. RESULTS: The addition of urea into the culture medium provided an alkaline environment that is suitable for S. pasteurii. As compared to S. pasteurii cultivated without urea, S. pasteurii grown with urea showed faster growth and urease production, better shape, more negative surface charge and higher biomineralization ability. To survive the unfavorable growth environment due to the absence of urea, S. pasteurii up-regulated the expression of genes involved in urease production, ATPase synthesis and flagella, possibly occupying resources that can be deployed for MICP. As compared to non-mineralizing bacteria, S. pasteurii exhibited more negative cell surface charge for binding calcium ions and more robust cell structure as nucleation sites. During MICP process, the genes for ATPase synthesis in S. pasteurii was up-regulated while genes for urease production were unchanged. Interestingly, genes involved in flagella were down-regulated during MICP, which might lead to poor mobility of S. pasteurii. Meanwhile, genes in fatty acid degradation pathway were inhibited to maintain the intact cell structure found in calcite precipitation. Both weak mobility and intact cell structure are advantageous for S. pasteurii to serve as nucleation sites during MICP. CONCLUSIONS: Four factors are demonstrated to benefit the super performance of S. pasteurii in MICP. First, the good correlation of biomass growth and urease production of S. pasteurii provides sufficient biomass and urease simultaneously for improved biomineralization. Second, the highly negative cell surface charge of S. pasteurii is good for binding calcium ions. Third, the robust cell structure and fourth, the weak mobility, are key for S. pasteurii to be nucleation sites during MICP.


Assuntos
Complexos de ATP Sintetase/metabolismo , Biomineralização/fisiologia , Carbonato de Cálcio/metabolismo , Sporosarcina , Urease/genética , Meios de Cultura/química , Perfilação da Expressão Gênica , Genoma Bacteriano , Microscopia Eletrônica de Varredura , Sporosarcina/genética , Sporosarcina/metabolismo , Sporosarcina/ultraestrutura , Ureia
2.
Proc Natl Acad Sci U S A ; 117(2): 1167-1173, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879356

RESUMO

Chemiosmosis and substrate-level phosphorylation are the 2 mechanisms employed to form the biological energy currency adenosine triphosphate (ATP). During chemiosmosis, a transmembrane electrochemical ion gradient is harnessed by a rotary ATP synthase to phosphorylate adenosine diphosphate to ATP. In microorganisms, this ion gradient is usually composed of [Formula: see text], but it can also be composed of Na+ Here, we show that the strictly anaerobic rumen bacterium Pseudobutyrivibrio ruminis possesses 2 ATP synthases and 2 distinct respiratory enzymes, the ferredoxin:[Formula: see text] oxidoreductase (Rnf complex) and the energy-converting hydrogenase (Ech complex). In silico analyses revealed that 1 ATP synthase is [Formula: see text]-dependent and the other Na+-dependent, which was validated by biochemical analyses. Rnf and Ech activity was also biochemically identified and investigated in membranes of P. ruminis Furthermore, the physiology of the rumen bacterium and the role of the energy-conserving systems was investigated in dependence of 2 different catabolic pathways (the Embden-Meyerhof-Parnas or the pentose-phosphate pathway) and in dependence of Na+ availability. Growth of P. ruminis was greatly stimulated by Na+, and a combination of physiological, biochemical, and transcriptional analyses revealed the role of the energy conserving systems in P. ruminis under different metabolic scenarios. These data demonstrate the use of a 2-component ion circuit for [Formula: see text] bioenergetics and a 2nd 2-component ion circuit for Na+ bioenergetics in a strictly anaerobic rumen bacterium. In silico analyses infer that these 2 circuits are prevalent in a number of other strictly anaerobic microorganisms.


Assuntos
Complexos de ATP Sintetase/metabolismo , Trifosfato de Adenosina/metabolismo , Clostridiales/metabolismo , Metabolismo Energético/fisiologia , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Clostridiales/enzimologia , Clostridiales/genética , Clostridiales/crescimento & desenvolvimento , Metabolismo Energético/genética , Ferredoxinas/metabolismo , Hidrogenase/metabolismo , Transporte de Íons , Oxirredução , Oxirredutases/metabolismo , Sódio/metabolismo
3.
Genes (Basel) ; 10(11)2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31752425

RESUMO

Leaf premature senescence largely determines maize (Zea mays L.) grain yield and quality. A natural recessive premature-senescence mutant was selected from the breeding population, and near-isogenic lines were constructed using Jing24 as the recurrent parent. In the near-isogenic lines, the dominant homozygous material was wild-type (WT), and the recessive material of early leaf senescence was the premature-senescence-type ZmELS5. To identify major genes and regulatory mechanisms involved in leaf senescence, a transcriptome analysis of the ZmELS5 and WT near-isogenic lines (NILs) was performed. A total of 8,796 differentially expressed transcripts were identified between ZmELS5 and WT, including 3,811 up-regulated and 4,985 down-regulated transcripts. By combining gene ontology, Kyoto Encyclopedia of Genes and Genomes, gene set, and transcription factor enrichment analyses, key differentially expressed genes were screened. The senescence regulatory network was predicted based on these key differentially expressed genes, which indicated that the senescence process is mainly regulated by bHLH, WRKY, and AP2/EREBP family transcription factors, leading to the accumulations of jasmonic acid and ethylene. This causes stress responses and reductions in the chlorophyll a/b-binding protein activity level. Then, decreased ATP synthase activity leads to increased photosystem II photodamage, ultimately leading to leaf senescence.


Assuntos
Senescência Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Proteínas de Plantas/genética , Zea mays/fisiologia , Complexos de ATP Sintetase/genética , Complexos de ATP Sintetase/metabolismo , Albinismo Oculocutâneo , Clorofila A , Ciclopentanos/metabolismo , Etilenos/metabolismo , Perfilação da Expressão Gênica , Genes de Plantas/genética , Oxilipinas/metabolismo , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Acta Biochim Pol ; 66(3): 343-350, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31509370

RESUMO

Two winter triticale (x Triticosecale Wittmack) model cultivars: Hewo (tolerant to pink snow mould) and Magnat (sensitive) were used to test the effect of cold-hardening (4 weeks at 4°C) on soluble ≤50 kDa protein profiles of the seedling leaves. The presence and abundance of individual proteins were analysed via two-dimensional gel electrophoresis (2-DE) and Surface-Enhanced Laser Desorption/Ionization Time-of-Flight (SELDI-TOF). Up to now, no proteomics analysis of triticale response to hardening has been performed. Thus, the present paper is the first in the series describing the obtained results. In our experiments, the exposure to the low temperature-induced only quantitative changes in the leaves of both cultivars, causing either an increase or decrease of 4-50 kDa protein abundance. Among proteins which were cold-accumulated in cv. Hewo's leaves, we identified two thioredoxin peroxidases (chloroplastic thiol-specific antioxidant proteins) as well as mitochondrial- ß-ATP synthase subunit and ADP-binding resistance protein. On the contrary, in hardened seedlings of this genotype, we observed the decreased level of chloroplastic RuBisCO small subunit PW9 and epidermal peroxidase 10. Simultaneous SELDI-TOF analysis revealed several low mass proteins better represented in cold-hardened plants of tolerant genotype in comparison to the sensitive one and the impact of both genotype and temperature on their level. Based on those results, we suggest that indicated proteins might be potential candidates for molecular markers of cold-induced snow mould resistance of winter triticale and their role is worth to be investigated in the further inoculation experiments.


Assuntos
Temperatura Baixa , Resistência à Doença , Micoses/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Plântula/microbiologia , Triticale/microbiologia , Xylariales/isolamento & purificação , Complexos de ATP Sintetase , Proteínas de Arabidopsis , Grão Comestível/microbiologia , Eletroforese em Gel Bidimensional , Genótipo , Peroxirredoxinas , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Plântula/metabolismo , Neve/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triticale/metabolismo
5.
J Insect Sci ; 19(4)2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31346627

RESUMO

In insects, lipid transfer to the tissues is mediated by lipophorin, the major circulating lipoprotein, mainly through a nonendocytic pathway involving docking receptors. Currently, the role of such receptors in lipid metabolism remains poorly understood. In this work, we performed a histological characterization of the fat body of the Chagas' disease vector, Panstrongylus megistus (Burmeister), subjected to different nutritional conditions. In addition, we addressed the role of the ß-chain of ATP synthase (ß-ATPase) in the process of lipid transfer from lipophorin to the fat body. Fifth-instar nymphs in either fasting or fed condition were employed in the assays. Histological examination revealed that the fat body was composed by diverse trophocyte phenotypes. In the fasting condition, the cells were smaller and presented a homogeneous cytoplasmic content. The fat body of fed insects increased in size mainly due to the enlargement of lipid stores. In this condition, trophocytes contained abundant lipid droplets, and the rough endoplasmic reticulum was highly developed and mitochondria appeared elongated. Immunofluorescence assays showed that the ß-ATPase, a putative lipophorin receptor, was located on the surface of fat body cells colocalizing partially with lipophorin, which suggests their interaction. No changes in ß-ATPase expression were found in fasting and fed insects. Blocking the lipophorin-ß-ATPase interaction impaired the lipophorin-mediated lipid transfer to the fat body. The results showed that the nutritional status of the insect influenced the morphohistological features of the tissue. Besides, these findings suggest that ß-ATPase functions as a lipophorin docking receptor in the fat body.


Assuntos
Complexos de ATP Sintetase/metabolismo , Corpo Adiposo/citologia , Proteínas de Insetos/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Panstrongylus/citologia , Animais , Corpo Adiposo/enzimologia , Ninfa/citologia , Ninfa/enzimologia , Panstrongylus/enzimologia , Panstrongylus/crescimento & desenvolvimento
6.
Curr Top Med Chem ; 19(10): 847-860, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30977451

RESUMO

BACKGROUND: Due to the limited availability of antibiotics, Gram-negative bacteria (GNB) acquire different levels of drug resistance. It raised an urgent need to identify such agents, which can reverse the phenomenon of drug resistance. OBJECTIVE: To understand the mechanism of drug resistance reversal of glycosides; niaziridin and niazirin isolated from the pods of Moringa oleifera and ouabain (control) against the clinical isolates of multidrug-resistant Escherichia coli. METHODS: The MICs were determined following the CLSI guidelines for broth micro-dilution. In-vitro combination studies were performed by broth checkerboard method followed by Time-Kill studies, the efflux pump inhibition assay, ATPase inhibitory activity, mutation prevention concentration and in-silico studies. RESULTS: The results showed that both glycosides did not possess antibacterial activity of their own, but in combination, they reduced the MIC of tetracycline up to 16 folds. Both were found to inhibit efflux pumps, but niaziridin was the best. In real time expression pattern analysis, niaziridin was also found responsible for the down expression of the two important efflux pump acrB & yojI genes alone as well as in combination. Niaziridin was also able to over express the porin forming genes (ompA & ompX). These glycosides decreased the mutation prevention concentration of tetracycline. CONCLUSION: This is the first ever report on glycosides, niazirin and niaziridin acting as drug resistance reversal agent through efflux pump inhibition and modulation of expression pattern drug resistant genes. This study may be helpful in preparing an effective antibacterial combination against the drug-resistant GNB from a widely growing Moringa oleifera.


Assuntos
Complexos de ATP Sintetase/antagonistas & inibidores , Acetonitrilos/farmacologia , Antibacterianos/farmacologia , Derivados de Benzeno/farmacologia , Produtos Biológicos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Complexos de ATP Sintetase/metabolismo , Acetonitrilos/química , Acetonitrilos/isolamento & purificação , Antibacterianos/química , Antibacterianos/isolamento & purificação , Derivados de Benzeno/química , Derivados de Benzeno/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/genética , Sinergismo Farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Escherichia coli/genética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Moringa oleifera/química
7.
Chem Pharm Bull (Tokyo) ; 67(4): 341-344, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30930438

RESUMO

Type III secretion system (T3SS) is a protein injection nano-machine consisting of syringe and needle-like structure spanning bacterial inner and outer membranes. Bacteria insert the tip of T3SS needle to host cell membranes, and deliver effector proteins directly into host cells via T3SS to prime the host cell environment for infection. Thus inhibition of T3SS would be a potent strategy for suppressing bacterial infection. We previously demonstrated that T3SS needle rotates by proton-motive force (PMF) with the same mechanism as two evolutionally related rotary protein motors, flagellum and ATP synthase (FASEB J., 27, 2013, Ohgita et al.). Inhibition of needle rotation resulted in suppression of effector secretion, indicating the requirement of needle rotation for effector export. Simulation analysis of protein export by the T3SS needle suggests the importance of a hydrophobic helical groove formed by the conserved aromatic residue in the needle components. Based on these results, we have proposed a novel model of protein export by the T3SS needle, in which effector proteins are exported by PMF-dependent needle rotation oppositely to the hydrophobic helical groove in the needle. Quantitative examinations of the correlation between the speeds of T3SS rotation and the amount of effector export support this model. In this review, we summarize our current understanding of T3SS, and discuss our novel model of the protein export mechanism of T3SS based on the needle rotation.


Assuntos
Bactérias/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Complexos de ATP Sintetase/metabolismo , Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Transporte Proteico
8.
Phys Rev Lett ; 122(12): 128101, 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30978101

RESUMO

The more we learn about the cytoplasm of cells, the more we realize that the cytoplasm is not uniform but instead is highly inhomogeneous. In any inhomogeneous solution, there are concentration gradients, and particles move either up or down these gradients due to a mechanism called diffusiophoresis. I estimate that inside metabolically active cells, the dynamics of particles can be strongly accelerated by diffusiophoresis, provided that they are at least tens of nanometers across. The dynamics of smaller objects, such as single proteins, are largely unaffected.


Assuntos
Bactérias/metabolismo , Citoplasma/metabolismo , Células Eucarióticas/metabolismo , Modelos Biológicos , Complexos de ATP Sintetase/metabolismo , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Bactérias/citologia , Citoplasma/química , Difusão , Eletroforese , Células Eucarióticas/citologia
9.
Biomol Concepts ; 10(1): 1-10, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30888962

RESUMO

Oxidative phosphorylation is carried out by five complexes, which are the sites for electron transport and ATP synthesis. Among those, Complex V (also known as the F1F0 ATP Synthase or ATPase) is responsible for the generation of ATP through phosphorylation of ADP by using electrochemical energy generated by proton gradient across the inner membrane of mitochondria. A multi subunit structure that works like a pump functions along the proton gradient across the membranes which not only results in ATP synthesis and breakdown, but also facilitates electron transport. Since ATP is the major energy currency in all living cells, its synthesis and function have widely been studied over the last few decades uncovering several aspects of ATP synthase. This review intends to summarize the structure, function and inhibition of the ATP synthase.


Assuntos
Complexos de ATP Sintetase/metabolismo , Complexos de ATP Sintetase/antagonistas & inibidores , Complexos de ATP Sintetase/química , Complexos de ATP Sintetase/genética , Animais , Inibidores Enzimáticos/farmacologia , Humanos , Doença de Leigh/genética
10.
J Bacteriol ; 201(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30642991

RESUMO

In this study, the ATP synthase of Ignicoccus hospitalis was purified, characterized, and structurally compared to the respective enzymes of the other Ignicoccus species, to shed light on energy conservation in this unique group of archaea. The crenarchaeal genus Ignicoccus comprises three described species, i.e., I. hospitalis and Ignicoccus islandicus from hot marine sediments near Iceland and Ignicoccus pacificus from a hydrothermal vent system in the Pacific Ocean. This genus is unique among all archaea due to the unusual cell envelope, consisting of two membranes that enclose a large intermembrane compartment (IMC). I. hospitalis is the best studied member of this genus, mainly because it is the only known host for the potentially parasitic archaeon Nanoarchaeum equitans I. hospitalis grows chemolithoautotrophically, and its sole energy-yielding reaction is the reduction of elemental sulfur with molecular hydrogen, forming large amounts of hydrogen sulfide. This reaction generates an electrochemical gradient, which is used by the ATP synthase, located in the outer cellular membrane, to generate ATP inside the IMC. The genome of I. hospitalis encodes nine subunits of an A-type ATP synthase, which we could identify in the purified complex. Although the maximal in vitro activity of the I. hospitalis enzyme was measured around pH 6, the optimal stability of the A1AO complex seemed to be at pH 9. Interestingly, the soluble A1 subcomplexes of the different Ignicoccus species exhibited significant differences in their apparent molecular masses in native electrophoresis, although their behaviors in gel filtration and chromatography-mass spectrometry were very similar.IMPORTANCE The Crenarchaeota represent one of the major phyla within the Archaea domain. This study describes the successful purification of a crenarchaeal ATP synthase. To date, all information about A-type ATP synthases is from euryarchaeal enzymes. The fact that it has not been possible to purify this enzyme complex from a member of the Crenarchaeota until now points to significant differences in stability, possibly caused by structural alterations. Furthermore, the study subject I. hospitalis has a particular importance among crenarchaeotes, since it is the only known host of N. equitans The energy metabolism in this system is still poorly understood, and our results can help elucidate the unique relationship between these two microbes.


Assuntos
Complexos de ATP Sintetase/isolamento & purificação , Complexos de ATP Sintetase/metabolismo , Desulfurococcaceae/enzimologia , Complexos de ATP Sintetase/química , Desulfurococcaceae/isolamento & purificação , Estabilidade Enzimática , Sedimentos Geológicos , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo
11.
Proc Natl Acad Sci U S A ; 116(10): 4206-4211, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30683723

RESUMO

The crystal structure of the F1-catalytic domain of the adenosine triphosphate (ATP) synthase has been determined from Mycobacterium smegmatis which hydrolyzes ATP very poorly. The structure of the α3ß3-component of the catalytic domain is similar to those in active F1-ATPases in Escherichia coli and Geobacillus stearothermophilus However, its ε-subunit differs from those in these two active bacterial F1-ATPases as an ATP molecule is not bound to the two α-helices forming its C-terminal domain, probably because they are shorter than those in active enzymes and they lack an amino acid that contributes to the ATP binding site in active enzymes. In E. coli and G. stearothermophilus, the α-helices adopt an "up" state where the α-helices enter the α3ß3-domain and prevent the rotor from turning. The mycobacterial F1-ATPase is most similar to the F1-ATPase from Caldalkalibacillus thermarum, which also hydrolyzes ATP poorly. The ßE-subunits in both enzymes are in the usual "open" conformation but appear to be occupied uniquely by the combination of an adenosine 5'-diphosphate molecule with no magnesium ion plus phosphate. This occupation is consistent with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic products are probably the basis of the inhibition of ATP hydrolysis. It can be envisaged that specific as yet unidentified small molecules might bind to the F1 domain in Mycobacterium tuberculosis, prevent ATP synthesis, and inhibit the growth of the pathogen.


Assuntos
Complexos de ATP Sintetase/antagonistas & inibidores , Antituberculosos , Proteínas de Bactérias/antagonistas & inibidores , Diarilquinolinas/química , Farmacorresistência Bacteriana Múltipla , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/enzimologia , Complexos de ATP Sintetase/química , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/química , Humanos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico
12.
J Colloid Interface Sci ; 535: 325-330, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30316119

RESUMO

At present, photophosphorylation in natural or artificial systems is accomplished by the production of protons or their pumping across the biomembranes. Herein, different from this strategy above, we demonstrate a designed system which can effectively enhance photophosphorylation by photo-induced proton-scavenging through molecular assembly. Upon the introduction of photobase generators, a (photo-) chemical reaction occurs to produce hydroxyl ions. Accompanying the further extramembranous acid-base neutralization reaction, an outbound flow of protons is generated to drive the reconstituted adenosine triphosphate (ATP) synthase to produce ATP. That is, contrary to biochemistry, the proton gradient to drive photophosphorylation derives from the scavenging of protons present in the external medium by hydroxyl ions, produced by the partially photo-induced splitting of photobase generator. Such assembled system holds great potential in ATP-consuming bioapplications.


Assuntos
Complexos de ATP Sintetase/metabolismo , Trifosfato de Adenosina/biossíntese , Hidróxidos/síntese química , Nanoestruturas/química , Prótons , Trifosfato de Adenosina/química , Hidróxidos/química , Tamanho da Partícula , Fosforilação , Processos Fotoquímicos , Propriedades de Superfície
13.
J Biol Chem ; 294(4): 1152-1160, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30510135

RESUMO

ATP synthase uses a rotary mechanism to couple transmembrane proton translocation to ATP synthesis and hydrolysis, which occur at the catalytic sites in the ß subunits. In the presence of Mg2+, the three catalytic sites of ATP synthase have vastly different affinities for nucleotides, and the position of the central γ subunit determines which site has high, medium, or low affinity. Affinity differences and their changes as rotation progresses underpin the ATP synthase catalytic mechanism. Here, we used a series of variants with up to 45- and 60-residue-long truncations of the N- and C-terminal helices of the γ subunit, respectively, to identify the segment(s) responsible for the affinity differences of the catalytic sites. We found that each helix carries an affinity-determining segment of ∼10 residues. Our findings suggest that the affinity regulation by these segments is transmitted to the catalytic sites by the DELSEED loop in the C-terminal domain of the ß subunits. For the N-terminal truncation variants, presence of the affinity-determining segment and therefore emergence of a high-affinity binding site resulted in WT-like catalytic activity. At the C terminus, additional residues outside of the affinity-determining segment were required for optimal enzymatic activity. Alanine substitutions revealed that the affinity changes of the catalytic sites required no specific interactions between amino acid side chains in the γ and α3ß3 subunits but were caused by the presence of the helices themselves. Our findings help unravel the molecular basis for the affinity changes of the catalytic sites during ATP synthase rotation.


Assuntos
Complexos de ATP Sintetase/análise , Geobacillus stearothermophilus/enzimologia , Nucleotídeos/metabolismo , Complexos de ATP Sintetase/metabolismo , Sítios de Ligação , Biocatálise , Nucleotídeos/química , Subunidades Proteicas
14.
Biochim Biophys Acta Bioenerg ; 1860(3): 181-188, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30528692

RESUMO

ATPase activity of proton-translocating FOF1-ATP synthase (F-type ATPase or F-ATPase) is suppressed in the absence of protonmotive force by several regulatory mechanisms. The most conservative of these mechanisms found in all enzymes studied so far is allosteric inhibition of ATP hydrolysis by MgADP (ADP-inhibition). When MgADP is bound without phosphate in the catalytic site, the enzyme lapses into an inactive state with MgADP trapped. In chloroplasts and mitochondria, as well as in most bacteria, phosphate prevents MgADP inhibition. However, in Escherichia coli ATP synthase ADP-inhibition is relatively weak and phosphate does not prevent it but seems to enhance it. We found that a single amino acid residue in subunit ß is responsible for these features of E. coli enzyme. Mutation ßL249Q significantly enhanced ADP-inhibition in E. coli ATP synthase, increased the extent of ATP hydrolysis stimulation by sulfite, and rendered the ADP-inhibition sensitive to phosphate in the same manner as observed in FOF1 from mitochondria, chloroplasts, and most aerobic\photosynthetic bacteria.


Assuntos
Complexos de ATP Sintetase/antagonistas & inibidores , Substituição de Aminoácidos , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/enzimologia , Complexos de ATP Sintetase/genética , Complexos de ATP Sintetase/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Hidrólise , Fosfatos/metabolismo , Fosfatos/farmacologia , Ligação Proteica
15.
Int J Mol Sci ; 19(12)2018 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-30544843

RESUMO

Our previous studies suggested that both hydrogen gas (H2) and nitric oxide (NO) could enhance the postharvest freshness of cut flowers. However, the crosstalk of H2 and NO during that process is unknown. Here, cut lilies (Lilium "Manissa") were used to investigate the relationship between H2 and NO and to identify differentially accumulated proteins during postharvest freshness. The results revealed that 1% hydrogen-rich water (HRW) and 150 µM sodium nitroprusside (SNP) significantly extended the vase life and quality, while NO inhibitors suppressed the positive effects of HRW. Proteomics analysis found 50 differentially accumulated proteins in lilies leaves which were classified into seven functional categories. Among them, ATP synthase CF1 alpha subunit (chloroplast) (AtpA) was up-regulated by HRW and down-regulated by NO inhibitor. The expression level of LlatpA gene was consistent with the result of proteomics analysis. The positive effect of HRW and SNP on ATP synthase activity was inhibited by NO inhibitor. Meanwhile, the physiological-level analysis of chlorophyll fluorescence and photosynthetic parameters also agreed with the expression of AtpA regulated by HRW and SNP. Altogether, our results suggested that NO might be involved in H2-improved freshness of cut lilies, and AtpA protein may play important roles during that process.


Assuntos
Hidrogênio/metabolismo , Lilium/crescimento & desenvolvimento , Lilium/metabolismo , Óxido Nítrico/metabolismo , Proteômica/métodos , Complexos de ATP Sintetase/metabolismo , Biomassa , Clorofila/metabolismo , Eletroforese em Gel Bidimensional , Flores/anatomia & histologia , Flores/efeitos dos fármacos , Fluorescência , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Processamento de Imagem Assistida por Computador , Lilium/efeitos dos fármacos , Lilium/genética , Nitroprussiato/farmacologia , Fotossíntese/efeitos dos fármacos , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Azida Sódica/farmacologia , Compostos de Tungstênio/farmacologia
17.
Proc Natl Acad Sci U S A ; 115(50): E11761-E11770, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30409802

RESUMO

Cyanobacteria are emerging as attractive organisms for sustainable bioproduction. We previously described Synechococcus elongatus UTEX 2973 as the fastest growing cyanobacterium known. Synechococcus 2973 exhibits high light tolerance and an increased photosynthetic rate and produces biomass at three times the rate of its close relative, the model strain Synechococcus elongatus 7942. The two strains differ at 55 genetic loci, and some of these loci must contain the genetic determinants of rapid photoautotrophic growth and improved photosynthetic rate. Using CRISPR/Cpf1, we performed a comprehensive mutational analysis of Synechococcus 2973 and identified three specific genes, atpA, ppnK, and rpaA, with SNPs that confer rapid growth. The fast-growth-associated allele of each gene was then used to replace the wild-type alleles in Synechococcus 7942. Upon incorporation, each allele successively increased the growth rate of Synechococcus 7942; remarkably, inclusion of all three alleles drastically reduced the doubling time from 6.8 to 2.3 hours. Further analysis revealed that our engineering effort doubled the photosynthetic productivity of Synechococcus 7942. We also determined that the fast-growth-associated allele of atpA yielded an ATP synthase with higher specific activity, while that of ppnK encoded a NAD+ kinase with significantly improved kinetics. The rpaA SNPs cause broad changes in the transcriptional profile, as this gene is the master output regulator of the circadian clock. This pioneering study has revealed the molecular basis for rapid growth, demonstrating that limited genetic changes can dramatically improve the growth rate of a microbe by as much as threefold.


Assuntos
Synechococcus/crescimento & desenvolvimento , Synechococcus/genética , Complexos de ATP Sintetase/genética , Complexos de ATP Sintetase/metabolismo , Alelos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Cianobactérias/genética , Cianobactérias/crescimento & desenvolvimento , Cianobactérias/metabolismo , Genes Bacterianos , Engenharia Genética , Genômica , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fotossíntese/genética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de RNA , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Synechococcus/metabolismo , Transcriptoma
18.
Cell ; 175(4): 1088-1104.e23, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30318146

RESUMO

Despite the known causality of copy-number variations (CNVs) to human neurodevelopmental disorders, the mechanisms behind each gene's contribution to the constellation of neural phenotypes remain elusive. Here, we investigated the 7q11.23 CNV, whose hemideletion causes Williams syndrome (WS), and uncovered that mitochondrial dysfunction participates in WS pathogenesis. Dysfunction is facilitated in part by the 7q11.23 protein DNAJC30, which interacts with mitochondrial ATP-synthase machinery. Removal of Dnajc30 in mice resulted in hypofunctional mitochondria, diminished morphological features of neocortical pyramidal neurons, and altered behaviors reminiscent of WS. The mitochondrial features are consistent with our observations of decreased integrity of oxidative phosphorylation supercomplexes and ATP-synthase dimers in WS. Thus, we identify DNAJC30 as an auxiliary component of ATP-synthase machinery and reveal mitochondrial maladies as underlying certain defects in brain development and function associated with WS.


Assuntos
Complexos de ATP Sintetase/metabolismo , Encéfalo/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Mitocôndrias/metabolismo , Síndrome de Williams/genética , Animais , Encéfalo/crescimento & desenvolvimento , Células Cultivadas , Feminino , Células HEK293 , Proteínas de Choque Térmico HSP40/genética , Humanos , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação Oxidativa
19.
Curr Opin Struct Biol ; 52: 71-79, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30240940

RESUMO

ATP synthases are rotary enzymes found in bacteria, chloroplasts, and mitochondria. These complexes produce the majority of cellular ATP in aerobic cells using energy from the transmembrane proton motive force established by the electron transport chain. In mitochondria, dimeric ATP synthase is essential for formation of the inner membrane cristae. While rotary catalysis in the soluble F1 region has been studied extensively by X-ray crystallography, the structure of the membrane embedded FO region remained elusive until recently. In the past few years, electron cryomicroscopy structures of mitochondrial, chloroplast, and bacterial ATP synthases have revealed the architecture of the FO region, helping to explain the mechanisms of proton translocation, dimerization of the enzyme in mitochondria, and cristae formation. These structures also show that ATP synthases exist in different conformational states, illustrating the flexibility and dynamics of the complex.


Assuntos
Complexos de ATP Sintetase/química , Microscopia Crioeletrônica , Modelos Moleculares , Complexos de ATP Sintetase/metabolismo , Catálise , Microscopia Crioeletrônica/métodos , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade
20.
Metabolism ; 89: 18-26, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30253140

RESUMO

BACKGROUND: Skeletal muscle mitochondrial content and function appear to be altered in obesity. Mitochondria in muscle are found in well-defined regions within cells, and they are arranged in a way that form distinct subpopulations of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. We sought to investigate differences in the proteomes of SS and IMF mitochondria between lean subjects and subjects with obesity. METHODS: We performed comparative proteomic analyses on SS and IMF mitochondria isolated from muscle samples obtained from lean subjects and subjects with obesity. Mitochondria were isolated using differential centrifugation, and proteins were subjected to label-free quantitative tandem mass spectrometry analyses. Collected data were evaluated for abundance of mitochondrial proteins using spectral counting. The Reactome pathway database was used to determine metabolic pathways that are altered in obesity. RESULTS: Among proteins, 73 and 41 proteins showed different (mostly lower) expression in subjects with obesity in the SS and IMF mitochondria, respectively (false discovery rate-adjusted P ≤ 0.05). We specifically found an increase in proteins forming the tricarboxylic acid cycle and electron transport chain (ETC) complex II, but a decrease in proteins forming protein complexes I and III of the ETC and adenosine triphosphate (ATP) synthase in subjects with obesity in the IMF, but not SS, mitochondria. Obesity was associated with differential effects on metabolic pathways linked to protein translation in the SS mitochondria and ATP formation in the IMF mitochondria. CONCLUSIONS: Obesity alters the expression of mitochondrial proteins regulating key metabolic processes in skeletal muscle, and these effects are distinct to mitochondrial subpopulations located in different regions of the muscle fibers. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01824173).


Assuntos
Mitocôndrias Musculares/ultraestrutura , Proteínas Mitocondriais/metabolismo , Obesidade/metabolismo , Complexos de ATP Sintetase/metabolismo , Adulto , Feminino , Voluntários Saudáveis , Humanos , Masculino , Redes e Vias Metabólicas , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Obesidade/patologia , Proteômica , Sarcolema/metabolismo , Sarcolema/ultraestrutura , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Espectrometria de Massas em Tandem
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