scholarly journals Insights into the regulatory function of the ɛ subunit from bacterial F-type ATP synthases: a comparison of structural, biochemical and biophysical data

Open Biology ◽  
2018 ◽  
Vol 8 (5) ◽  
pp. 170275 ◽  
Author(s):  
Alexander Krah ◽  
Mariel Zarco-Zavala ◽  
Duncan G. G. McMillan
Keyword(s):  
Atp Hydrolysis ◽  
Structural Data ◽  
Regulatory Function ◽  
Storage Unit ◽  
Energy Storage Unit ◽  
Change Mechanism ◽  
Hairpin Conformation ◽  
Atp Synthases

ATP synthases catalyse the formation of ATP, the most common chemical energy storage unit found in living cells. These enzymes are driven by an electrochemical ion gradient, which allows the catalytic evolution of ATP by a binding change mechanism. Most ATP synthases are capable of catalysing ATP hydrolysis to varying degrees, and to prevent wasteful ATP hydrolysis, bacteria and mitochondria have regulatory mechanisms such as ADP inhibition. Additionally, ɛ subunit inhibition has also been described in three bacterial systems, Escherichia coli , Bacillus PS3 and Caldalkalibacillus thermarum TA2.A1. Previous studies suggest that the ɛ subunit is capable of undergoing an ATP-dependent conformational change from the ATP hydrolytic inhibitory ‘extended’ conformation to the ATP-induced non-inhibitory ‘hairpin’ conformation. A recently published crystal structure of the F 1 domain of the C. thermarum TA2.A1 F 1 F o ATP synthase revealed a mutant ɛ subunit lacking the ability to bind ATP in a hairpin conformation. This is a surprising observation considering it is an organism that performs no ATP hydrolysis in vivo , and appears to challenge the current dogma on the regulatory role of the ɛ subunit. This has prompted a re-examination of present knowledge of the ɛ subunits role in different organisms. Here, we compare published biochemical, biophysical and structural data involving ɛ subunit-mediated ATP hydrolysis regulation in a variety of organisms, concluding that the ɛ subunit from the bacterial F-type ATP synthases is indeed capable of regulating ATP hydrolysis activity in a wide variety of bacteria, making it a potentially valuable drug target, but its exact role is still under debate.

scholarly journals Bacterial F-type ATP synthases follow a well-choreographed assembly pathway

2021 ◽  
Author(s):  
Khanh Vu Huu ◽  
René Zangl ◽  
Jan Hoffmann ◽  
Nina Morgner
Keyword(s):  
Self Assembly ◽  
Atp Hydrolysis ◽  
Complex Activity ◽  
The Past ◽  
Assembly Pathway ◽  
Major Energy Source ◽  
Atp Synthases

Abstract F-type ATP synthases are multiprotein complexes composed of two separate coupled motors (F1 and FO) generating adenosine triphosphate (ATP) as the universal major energy source in a variety of relevant biological processes in mitochondria, bacteria and chloroplasts. In the past decades, ATP synthases have become a subject of high interest, as a target for therapeutic use in the treatment of a variety of diseases. While the structure of many ATPases is solved today, the precise assembly pathway of F1FO-ATP synthases is mostly still unclear. To probe the bacterial F1 assembly of Acetobacterium woodii, we studied the self-assembly of purified proteins under different environments. We report assembly requirements, important assembly intermediates in vitro and in vivo, the crucial role of nucleotide binding (as opposed to ATP hydrolysis) and correlate results with complex activity. Finally, we propose a model for the assembly pathway for the formation of a functional F1 complex.

scholarly journals Circ_0000215 Exerts Oncogenic Function in Nasopharyngeal Carcinoma by Targeting miR-512-5p

2021 ◽  
Vol 9 ◽  
Author(s):  
Xinping Chen ◽  
Weihua Xu ◽  
Zhichao Ma ◽  
Juan Zhu ◽  
Junjie Hu ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Cell Lines ◽  
Cell Counting ◽  
Regulatory Subunit ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Migration And Invasion ◽  
Circular Rnas

Background: Increasing circular RNAs (circRNAs) are reported to participate in cancer progression. Nonetheless, the role of circRNAs in nasopharyngeal carcinoma (NPC) has not been fully clarified. This work is aimed to probe the role of circ_0000215 in NPC.Methods: Circ_0000215 expression in NPC tissues and cell lines was examined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8) assay, 5-bromo-2′-deoxyuridine (BrdU) assay, scratch healing assay and Transwell experiment were executed to investigate the regulatory function of circ_0000215 on the proliferation, migration and invasion of NPC cells. RNA immunoprecipitation (RIP), pull-down and dual-luciferase reporter experiments were utilized to determine the binding relationship between circ_0000215 and miR-512-5p, and between miR-512-5p and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) 3′UTR. The effects of circ_0000215 on NPC growth and metastasis in vivo were examined with nude mice model. Western blot was applied to detect the regulatory effects of circ_0000215 and miR-512-5p on PIK3R1 expression.Results: Circ_0000215 was overexpressed in NPC tissues and cell lines. The functional experiments confirmed that knockdown of circ_0000215 impeded the growth and metastasis of NPC cells in vitro and in vivo. Additionally, circ_0000215 could also work as a molecular sponge to repress miR-512-5p expression. PIK3R1 was validated as a target gene of miR-512-5p, and circ_0000215 could increase the expression level of PIK3R1 in NPC cells via suppressing miR-512-5p.Conclusion: Circ_0000215 is overexpressed in NPC and exerts oncogenic effects in NPC through regulating miR-512-5p/PIK3R1 axis.

scholarly journals In vivo FRET analyses reveal a role of ATP hydrolysis–associated conformational changes in human P-glycoprotein

2020 ◽  
Vol 295 (15) ◽  
pp. 5002-5011 ◽  
Author(s):  
Ryota Futamata ◽  
Fumihiko Ogasawara ◽  
Takafumi Ichikawa ◽  
Atsushi Kodan ◽  
Yasuhisa Kimura ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Hek293 Cells ◽  
Atp Binding ◽  
Binding Domains ◽  
P Glycoprotein ◽  
Atp Binding And Hydrolysis

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

scholarly journals Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
FuJung Chang ◽  
Alberto Riera ◽  
Cecile Evrin ◽  
Jingchuan Sun ◽  
Huilin Li ◽  
...  
Keyword(s):  
Dna Replication ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
S Phase ◽  
G1 Phase ◽  
Helicase Loading ◽  
Mcm Helicase

To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading. Using purified proteins, an ATPase-defective Cdc6 mutant ‘Cdc6-E224Q’ promoted MCM loading on DNA. Cdc6-E224Q also promoted MCM binding at origins in vivo but cells remained blocked in G1-phase. If after loading MCM, Cdc6-E224Q was degraded, cells entered an apparently normal S-phase and replicated DNA, a phenotype seen with two additional Cdc6 ATPase-defective mutants. Cdc6 ATP hydrolysis is therefore required for Cdc6 disengagement from the pre-RC after helicase loading to advance subsequent steps in helicase activation in vivo.

scholarly journals Role of HrcA and CIRCE in the Heat Shock Regulatory Network of Bradyrhizobium japonicum

2000 ◽  
Vol 182 (1) ◽  
pp. 14-22 ◽  
Author(s):  
Alexander C. Minder ◽  
Hans-Martin Fischer ◽  
Hauke Hennecke ◽  
Franz Narberhaus
Keyword(s):  
Heat Shock ◽  
Bradyrhizobium Japonicum ◽  
Target Genes ◽  
Specific Binding ◽  
Regulatory Function ◽  
In The Wild ◽  
Number Of Bacteria

ABSTRACT A large number of bacteria regulate chaperone gene expression by the CIRCE-HrcA system in which a DNA element called CIRCE serves as binding site for the repressor protein HrcA under non-heat-shock conditions. We have cloned the two consecutive genes hrcAand grpE of Bradyrhizobium japonicum by using a complementation approach that screened for GrpE function. In vivo and in vitro transcript mapping demonstrated that both genes are transcribed separately from RpoH (ς32)-dependent promoters. To investigate the supposed negative regulatory function of HrcA, we compared the expression of putative target genes in the wild type with that in an hrcA mutant. Transcription of the CIRCE-associated chaperonin operons groESL 4 andgroESL 5, as well as the β-galactosidase activity derived from corresponding groE-lacZ fusions, was strongly elevated in the hrcA mutant even at physiological temperatures. Expression of other heat shock regulons (RpoH or ROSE dependent) was not affected. To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein under nondenaturing conditions. Specific binding to the CIRCE element was obtained with a soluble fraction of HrcA in gel retardation experiments.

scholarly journals Single mutations in the ε subunit from thermophilic Bacillus PS3 generate a high binding affinity site for ATP

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5505 ◽  
Author(s):  
Alexander Krah ◽  
Peter J. Bond
Keyword(s):  
Binding Affinity ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Evolutionary Analysis ◽  
Binding Affinities ◽  
Wild Type ◽  
Dynamics Simulations ◽  
High Binding Affinity ◽  
Atp Synthases

The ε subunit from ATP synthases acts as an ATP sensor in the bacterial cell to prevent ATP hydrolysis and thus the waste of ATP under conditions of low ATP concentration. However, the ATP binding affinities from various bacterial organisms differ markedly, over several orders of magnitude. For example, the ATP synthases from thermophilic Bacillus PS3 and Escherichia coli exhibit affinities of 4 µM and 22 mM, respectively. The recently reported R103A/R115A double mutant of Bacillus PS3 ATP synthase demonstrated an increased binding affinity by two orders of magnitude with respect to the wild type. Here, we used atomic-resolution molecular dynamics simulations to determine the role of the R103A and R115A single mutations. These lead us to predict that both single mutations also cause an increased ATP binding affinity. Evolutionary analysis reveals R103 and R115 substitutions in the ε subunit from other bacillic organisms, leading us to predict they likely have a higher ATP binding affinity than previously expected.

scholarly journals c-di-AMP, a likely master regulator of bacterial K+ homeostasis machinery, activates a K+ exporter

2021 ◽  
Vol 118 (14) ◽  
pp. e2020653118
Author(s):  
Tatiana B. Cereija ◽  
João P. L. Guerra ◽  
João M. P. Jorge ◽  
João H. Morais-Cabral
Keyword(s):  
Bacterial Species ◽  
Structural Data ◽  
Regulatory Function ◽  
Dna Integrity ◽  
Master Regulator ◽  
Quantitative Evidence ◽  
Physiological Properties ◽  
The Impact

bis-(3′,5′)-cyclic diadenosine monophosphate (c-di-AMP) is a second messenger with roles in virulence, cell wall and biofilm formation, and surveillance of DNA integrity in many bacterial species, including pathogens. Strikingly, it has also been proposed to coordinate the activity of the components of K+ homeostasis machinery, inhibiting K+ import, and activating K+ export. However, there is a lack of quantitative evidence supporting the direct functional impact of c-di-AMP on K+ transporters. To gain a detailed understanding of the role of c-di-AMP on the activity of a component of the K+ homeostasis machinery in B. subtilis, we have characterized the impact of c-di-AMP on the functional, biochemical, and physiological properties of KhtTU, a K+/H+ antiporter composed of the membrane protein KhtU and the cytosolic protein KhtT. We have confirmed c-di-AMP binding to KhtT and determined the crystal structure of this complex. We have characterized in vitro the functional properties of KhtTU and KhtU alone and quantified the impact of c-di-AMP and of pH on their activity, demonstrating that c-di-AMP activates KhtTU and that pH increases its sensitivity to this nucleotide. Based on our functional and structural data, we were able to propose a mechanism for the activation of KhtTU by c-di-AMP. In addition, we have analyzed the impact of KhtTU in its native bacterium, providing a physiological context for the regulatory function of c-di-AMP and pH. Overall, we provide unique information that supports the proposal that c-di-AMP is a master regulator of K+ homeostasis machinery.

Is dimerization a common feature in thioredoxins? The case of thioredoxin fromLitopenaeus vannamei

2017 ◽  
Vol 73 (4) ◽  
pp. 326-339 ◽  
Author(s):  
Adam A. Campos-Acevedo ◽  
Rogerio R. Sotelo-Mundo ◽  
Javier Pérez ◽  
Enrique Rudiño-Piñera
Keyword(s):  
Quaternary Structure ◽  
Hydrophobic Interactions ◽  
Cysteine Residue ◽  
Redox Activity ◽  
Regulatory Function ◽  
Wild Type ◽  
Biochemical Assays

The quaternary structure of the redox protein thioredoxin (Trx) has been debated. For bacterial Trx, there is no question regarding its monomeric state. In humans and other eukaryotes, the presence of a cysteine residue at the crystallographic symmetry axis points to the relevance of dimer formation in solution andin vivo. Crystallographic data for shrimp thioredoxin (LvTrx) obtained under different redox conditions reveal a dimeric arrangement mediated by a disulfide bond through residue Cys73 and other hydrophobic interactions located in the crystallographic interface, as reported for human Trx. Through the analysis of five mutants located at the crystallographic interface, this study provides structural and biochemical evidence for the existence in solution of monomeric and dimeric populations of wild-typeLvTrx and five mutants. Based on the results of biochemical assays, SAXS studies and the crystallographic structures of three of the studied mutants (Cys73Ser, Asp60Ser and Trp31Ala), it is clear that the Cys73 residue is essential for dimerization. However, its mutation to Ser produces an enzyme which has similar redox activityin vitroto the wild type. A putative regulatory function of dimerization is proposed based on structural analysis. Nonetheless, the biological role ofLvTrx dimerization needs to be experimentally unveiled. Additionally, the findings of this work reopen the discussion regarding the existence of similar behaviour in human thioredoxin, which shares a Cys at position 73 withLvTrx, a structural feature that is also present in some Trxs from vertebrates and crustaceans.

scholarly journals A Potential Probiotic for Diarrhea: Clostridium tyrobutyricum Protects Against LPS-Induced Epithelial Dysfunction via IL-22 Produced By Th17 Cells in the Ileum

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhiping Xiao ◽  
Lujie Liu ◽  
Xun Pei ◽  
Wanjing Sun ◽  
Yuyue Jin ◽  
...  
Keyword(s):  
Th17 Cells ◽  
Intestinal Inflammation ◽  
Flow Cytometric Analysis ◽  
Protective Role ◽  
Regulatory Function ◽  
Clostridium Tyrobutyricum ◽  
Dependent Manner ◽  
Protective Function

Probiotics are clinically used for diarrhea and inflammatory bowel diseases in both humans and animals. Previous studies have shown that Clostridium tyrobutyricum (Ct) protects against intestinal dysfunction, while its regulatory function in the gut needs further investigation and the related mechanisms are still not fully elucidated. This study aims to further verify the protective function of Ct and reveal its underlying mechanisms in alleviating diarrhea and intestinal inflammation. Ct inhibited LPS-induced diarrhea and intestinal inflammation in the ileum. IL-22 was identified and the protective role of Ct in the ileum presented an IL-22-dependent manner according to the transcriptomic analysis and in vivo interference mice experiments. The flow cytometric analysis of immune cells in the ileum showed that Ct enhanced the proportions of Th17 cells in response to LPS. The results of in situ hybridization further verified that Ct triggered Th17 cells to produce IL-22, which combined with IL-22RA1 expressed in the epithelial cells. Moreover, Ct was unable to enhance the levels of short-chain fatty acids (SCFAs) in the ileum, suggesting that the protective role of Ct in the ileum was independent of SCFAs. This study uncovered the role of Ct in alleviating diarrhea and inflammation with the mechanism of stimulating Th17 cells in the lamina propria to produce IL-22, highlighting its potential application as a probiotic for diarrhea and inflammation in the ileum.

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