scholarly journals Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase

2015 ◽  
Vol 290 (34) ◽  
pp. 21032-21041 ◽  
Author(s):  
Naman B. Shah ◽  
Thomas M. Duncan
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Intrinsic Rate ◽  
Synthesis Rate ◽  
Final Segment ◽  
Rotary Catalysis

F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.

Abstract MP247: Mitochondrial Complex V Is Regulated By GRK2 In The Heart

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Kimberly Ferrero ◽  
Jessica M Pfleger ◽  
Kurt Chuprun ◽  
Eric Barr ◽  
Erhe Gao ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Cardiac Injury ◽  
Atp Synthesis ◽  
Cardiac Metabolism ◽  
Neonatal Rat ◽  
Interacting Proteins ◽  
Atp Production

The GPCR kinase GRK2 is highly expressed the heart; importantly, during cardiac injury or heart failure (HF) both levels and activity of GRK2 increase. The role of GRK2 during HF is canonically studied upstream of β-adrenergic desensitization. However, GRK2 has a large interactome and noncanonical functions for this kinase are being uncovered. We have discovered that in the heart, GRK2 translocates to mitochondria ( mtGRK2 ) following injury and is associated with negative effects on cardiac metabolism. Thus, we have sought to identify the mechanism(s) by which GRK2 can regulate mitochondrial function. We hypothesize that mtGRK2 interacts with proteins which regulate bioenergetics and substrate utilization, and this never-before-described role may partially explain the altered mitochondrial phenotype seen following cardiac injury or HF. Stress-induced mitochondrial translocation of GRK2 was validated in neonatal rat ventricular myocytes, murine heart tissue and a cardiac-derived cell line. Consequently, the GRK2 interactome was mapped basally and under stress conditions in vitro, in vivo , and with tagged recombinant peptides. GRK2-interacting proteins were isolated via immunoprecipitation and analyzed via liquid chromatography-mass spectroscopy (LCMS). Proteomics analysis (IPA; Qiagen) identified mtGRK2 interacting proteins which were also involved in mitochondrial dysfunction. Excitingly, Complexes I, II, IV and V (ATP synthase) of the electron transport chain (ETC) were identified in the subset of mtGRK2-dysfunction partners. Several mtGRK2-ETC interactions were increased following stress, particularly those in Complex V. We further established that mtGRK2 phosphorylates some of the subunits of Complex V, particularly the ATP synthase barrel which is critical for ATP production in the heart. Specific amino acid residues on these subunits have been identified using PTM-LCMS and are currently being validated in a murine model of myocardial infarction. To support these data, we have also determined that alterations in either the levels or kinase activity of GRK2 appear to alter the enzymatic activity of Complex V in vitro , thus altering ATP production. In summary, the phosphorylation of the ATP synthesis machinery by mtGRK2 may be regulating some of the phenotypic effects of injured or failing hearts such as increased ROS production and reduced fatty acid metabolism. Research is ongoing in our lab to elucidate the novel role of GRK2 in regulating mitochondrial bioenergetics and cell death, thus uncovering an exciting, druggable novel target for rescuing cardiac function in patients with injured and/or failing hearts.

Assembly of the Escherichia coli FoF1 ATP synthase involves distinct subcomplex formation

2013 ◽  
Vol 41 (5) ◽  
pp. 1288-1293 ◽  
Author(s):  
Gabriele Deckers-Hebestreit
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Cytoplasmic Membrane ◽  
Atp Synthesis ◽  
E Coli ◽  
Assembly Pathway ◽  
Polypeptide Chains ◽  
Fof1 Atp Synthase

The ATP synthase (FoF1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane by Fo to ATP synthesis or hydrolysis in F1. Whereas good knowledge of the nanostructure and the rotary mechanism of the ATP synthase is at hand, the assembly pathway of the 22 polypeptide chains present in a stoichiometry of ab2c10α3β3γδϵ has so far not received sufficient attention. In our studies, mutants that synthesize different sets of FoF1 subunits allowed the characterization of individually formed stable subcomplexes. Furthermore, the development of a time-delayed in vivo assembly system enabled the subsequent synthesis of particular missing subunits to allow the formation of functional ATP synthase complexes. These observations form the basis for a model that describes the assembly pathway of the E. coli ATP synthase from pre-formed subcomplexes, thereby avoiding membrane proton permeability by a concomitant assembly of the open H+-translocating unit within a coupled FoF1 complex.

Functional analysis of membranous Fo-a subunit of F1Fo-ATP synthase by in vitro protein synthesis

2012 ◽  
Vol 442 (3) ◽  
pp. 631-638 ◽  
Author(s):  
Yutetsu Kuruma ◽  
Toshiharu Suzuki ◽  
Sakurako Ono ◽  
Masasuke Yoshida ◽  
Takuya Ueda
Keyword(s):  
Protein Synthesis ◽  
Atp Synthase ◽  
Transmembrane Helix ◽  
Atp Synthesis ◽  
Membrane Vesicles ◽  
F1fo Atp Synthase ◽  
Pure System ◽  
A Subunit

The a subunit of F1Fo (F1Fo-ATP synthase) is a highly hydrophobic protein with five putative transmembrane helices which plays a central role in H+-translocation coupled with ATP synthesis/hydrolysis. In the present paper, we show that the a subunit produced by the in vitro protease-free protein synthesis system (the PURE system) is integrated into a preformed Foa-less F1Fo complex in Escherichia coli membrane vesicles and liposomes. The resulting F1Fo has a H+-coupled ATP synthesis/hydrolysis activity that is approximately half that of the native F1Fo. By using this procedure, we analysed five mutations of F1Fo, where the conserved residues in the a subunit (Asn90, Asp112, Arg169, Asn173 and Gln217) were individually replaced with alanine. All of the mutant Foa subunits were successfully incorporated into F1Fo, showing the advantage over conventional expression in E. coli by which three (N90A, D112A, and Q217A) mutant a subunits were not found in F1Fo. The N173A mutant retained full activity and the mutants D112A and Q217A had weak, but detectable, activity. No activity was observed for the R169A and N90A mutants. Asn90 is located in the middle of putative second transmembrane helix and likely to play an important role in H+-translocation. The present study exemplifies that the PURE system provides an alternative approach when in vivo expression of membranous components in protein complexes turns out to be difficult.

scholarly journals ECS-based investigation of chloroplast ATP synthase regulation

2020 ◽  
Author(s):  
Felix Buchert ◽  
Benjamin Bailleul ◽  
Pierre Joliot
Keyword(s):  
Atp Synthase ◽  
Redox Regulation ◽  
Spectroscopic Method ◽  
Atp Synthesis ◽  
Redox States ◽  
Chloroplast Atp Synthase ◽  
Γ Subunit ◽  
Synthase Regulation

AbstractThe chloroplast ATP synthase (CF1Fo) contains a specific feature to the green lineage: a γ-subunit redox domain which contains a cysteine couple and interacts with the torque-generating βDELSEED-loop. Based on the recently solved structure of this domain, it was proposed to function as a chock. In vitro, γ-disulfide formation slows down the activity of the CF1Fo at low transmembrane electrochemical proton gradient . Here, we utilize in vivo absorption spectroscopy measurements for functional CF1Fo activity characterization in Arabidopsis leaves. The spectroscopic method allows us to measure the present in dark-adapted leaves, and to identify its mitochondrial sources. Furthermore, we follow the fate of the extra generated by an illumination, including its osmotic and electric components, and from there we estimate the lifetime of the light-generated ATP. In contrast with a previous report [Joliot and Joliot, Biochim. Biophys. Acta, 1777 (2008) 676-683], the CF1Fo γ-subunit exists mostly in an oxidized form in the dark-adapted state. To study the redox regulation of the CF1Fo, we used thiol agent infiltration in WT and a mutant that does not form the γ-disulfide. The obtained -dependent CF1Fo activity profile in the two γ-redox states in vivo reconciles with previous biochemical in vitro findings [Junesch and Gräber, Biochim. Biophys. Acta, 893 (1987) 275-288]. The highest rates of ATP synthesis we measured in the two γ-redox state were similar at high . In the presence of the γ-dithiol, similar rates were obtained at a ~45 mV lower value compared to the oxidized state, which closely resembled the energetic gap of 0.7 ΔpH units reported in vitro.

Безопасность соединений из группы терпенов ментанового ряда in vitro и in vivo

2020 ◽  
Vol 83 (4) ◽  
pp. 24-30
Author(s):  
Ирина Владимировна Акулина ◽  
Светлана Ивановна Павлова ◽  
Ирина Семеновна Степаненко ◽  
Назира Сунагатовна Карамова ◽  
Александр Владиславович Сергеев ◽  
...  
Keyword(s):  
Escherichia Coli

Проведено токсикологическое исследование соединений с антибактериальными свойствами из группы терпенов ментанового ряда в условиях in vitro и in vivo: лимонена (B34), его производного (+)-1,2-оксида лимонена (B60) и серосодержащего монотерпенового соединения 2-(1’-гидрокси-4’-изопренил-1’-метилциклогексил-2’-тио)метилэтаноата (B65). В условиях in vitro (культура опухолевых клеток HeLa) изучаемые монотерпены в диапазоне концентраций 2 – 200 мкг/мл обладали цитотоксичностью. Ингибирующая концентрация (ИК50) для B34 составила 231 (167 – 295) мкг/мл, для B60 – 181 (105 – 257) мкг/мл, ИК50 B65 – 229 (150 – 308) мкг/мл. Исследование генотоксичности показало, что B34 и B65 в диапазоне концентраций 50 – 1000 мкг/мл не индуцируют SOS мутагенез в клетках Escherichia coli PQ37, тогда как B60 в концентрациях 500 и 1000 мкг/мл проявляет генотоксичность. In vivo в остром эксперименте на беспородных мышах установлена низкая токсичность B34 и его производных при различных путях введения. Наименьший показатель острой токсичности имеет B65, в связи с чем дополнительно на крысах проведено изучение его хронической токсичности. Ежедневное внутрижелудочное введение B65 в разовых дозах, составляющих 1/10 и 1/20 ЛД50 (1000 мг/кг и 500 мг/кг), в течение 1 мес не вызывало гибели животных, значимых нарушений общего состояния, изменения динамики массы тела, морфопатологических изменений. Внутрижелудочное введение B65 крысам в высокой токсической дозе 2000 мг/кг (1/5 ЛД50) в течение месяца вызывает патоморфологические изменения структуры печени.

scholarly journals Antagonism of Ultraviolet-Light Mutagenesis by the Methyl-Directed Mismatch-Repair System of Escherichia coli

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 503-512 ◽  
Author(s):  
Hongbo Liu ◽  
Stephen R Hewitt ◽  
John B Hays
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Excision Repair ◽  
Spontaneous Mutation ◽  
Repair System ◽  
Uv Mutagenesis ◽  
Repair Protein ◽  
Mismatch Repair System

Abstract Previous studies have demonstrated that the Escherichia coli MutHLS mismatch-repair system can process UV-irradiated DNA in vivo and that the human MSH2·MSH6 mismatch-repair protein binds more strongly in vitro to photoproduct/base mismatches than to “matched” photoproducts in DNA. We tested the hypothesis that mismatch repair directed against incorrect bases opposite photoproducts might reduce UV mutagenesis, using two alleles at E. coli lacZ codon 461, which revert, respectively, via CCC → CTC and CTT → CTC transitions. F′ lacZ targets were mated from mut+ donors into mutH, mutL, or mutS recipients, once cells were at substantial densities, to minimize spontaneous mutation prior to irradiation. In umu+ mut+ recipients, a range of UV fluences induced lac+ revertant frequencies of 4–25 × 10−8; these frequencies were consistently 2-fold higher in mutH, mutL, or mutS recipients. Since this effect on mutation frequency was unaltered by an Mfd− defect, it appears not to involve transcription-coupled excision repair. In mut+ umuC122::Tn5 bacteria, UV mutagenesis (at 60 J/m2) was very low, but mutH or mutL or mutS mutations increased reversion of both lacZ alleles roughly 25-fold, to 5–10 × 10−8. Thus, at UV doses too low to induce SOS functions, such as Umu2′D, most incorrect bases opposite occasional photoproducts may be removed by mismatch repair, whereas in heavily irradiated (SOS-induced) cells, mismatch repair may only correct some photoproduct/base mismatches, so UV mutagenesis remains substantial.

Exploring Galleria mellonella larval model to evaluate antibacterial efficacy of Cecropin A (1-7)- Melittin against multi-drug resistant enteroaggregative Escherichia coli

2021 ◽  
Author(s):  
Jess Vergis ◽  
S V S Malik ◽  
Richa Pathak ◽  
Manesh Kumar ◽  
Nitin V Kurkure ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Galleria Mellonella ◽  
In Vivo Model ◽  
Drug Resistant ◽  
Physiological Concentration ◽  
Microbial Drug Resistance ◽  
Cecropin A ◽  
Screening And Identification

Abstract High throughput in vivo laboratory models is need for screening and identification of effective therapeutic agents to overcome microbial drug-resistance. This study was undertaken to evaluate in vivo antimicrobial efficacy of short-chain antimicrobial peptide- Cecropin A (1–7)-Melittin (CAMA) against three multi- drug resistant enteroaggregative Escherichia coli (MDR-EAEC) field isolates in a Galleria mellonella larval model. The minimum inhibitory concentration (MIC; 2.0 mg/L) and minimum bactericidal concentration (MBC; 4.0 mg/L) of CAMA were determined by microdilution assay. CAMA was found to be stable at high temperatures, physiological concentration of cationic salts and proteases; safe with sheep erythrocytes, secondary cell lines and commensal lactobacilli at lower MICs; and exhibited membrane permeabilisation. In vitro time-kill assay revealed concentration- and time-dependent clearance of MDR-EAEC in CAMA-treated groups at 30 min. CAMA- treated G. mellonella larvae exhibited an increased survival rate, reduced MDR-EAEC counts, immunomodulatory effect and proved non-toxic which concurred with histopathological findings. CAMA exhibited either an equal or better efficacy than the tested antibiotic control, meropenem. This study highlights the possibility of G. mellonella larvae as an excellent in vivo model for investigating the host-pathogen interaction, including the efficacy of antimicrobials against MDR-EAEC strains.

Regulation of RNA helicase activity: principles and examples

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Pascal Donsbach ◽  
Dagmar Klostermeier
Keyword(s):  
Atp Hydrolysis ◽  
Wide Spectrum ◽  
Mrna Translation ◽  
Rna Degradation ◽  
Rna Helicases ◽  
Interaction Partners ◽  
Rna Duplexes ◽  
Self Association

Abstract RNA helicases are a ubiquitous class of enzymes involved in virtually all processes of RNA metabolism, from transcription, mRNA splicing and export, mRNA translation and RNA transport to RNA degradation. Although ATP-dependent unwinding of RNA duplexes is their hallmark reaction, not all helicases catalyze unwinding in vitro, and some in vivo functions do not depend on duplex unwinding. RNA helicases are divided into different families that share a common helicase core with a set of helicase signature motives. The core provides the active site for ATP hydrolysis, a binding site for the non-sequence-specific interactions with RNA, and in many cases a basal unwinding activity. Its activity is often regulated by flanking domains, by interaction partners, or by self-association. In this review, we summarize the regulatory mechanisms that modulate the activities of the helicase core. Case studies on selected helicases with functions in translation, splicing, and RNA sensing illustrate the various modes and layers of regulation in time and space that harness the helicase core for a wide spectrum of cellular tasks.

scholarly journals DisA Limits RecG Activities at Stalled or Reversed Replication Forks

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1357
Author(s):  
Rubén Torres ◽  
Carolina Gándara ◽  
Begoña Carrasco ◽  
Ignacio Baquedano ◽  
Silvia Ayora ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Holliday Junctions ◽  
Genome Integrity ◽  
Stable Complex ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Dna Structures ◽  
Checkpoint Protein

The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3′, 5′-diadenosine monophosphate (c‑di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.

Sign in / Sign up

Export Citation Format

Share Document