New GroEL-like chaperonin of bacteriophage OBP Pseudomonas fluorescens suppresses thermal protein aggregation in an ATP-dependent manner

2016 ◽  
Vol 473 (15) ◽  
pp. 2383-2393 ◽  
Author(s):  
Pavel I. Semenyuk ◽  
Victor N. Orlov ◽  
Olga S. Sokolova ◽  
Lidia P. Kurochkina
Keyword(s):  
Pseudomonas Fluorescens ◽  
Functional Characterization ◽  
Dependent Manner ◽  
Double Ring ◽  
Protein Inactivation ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Ring Architecture

Recently, we discovered and studied the first virus-encoded chaperonin of bacteriophage EL Pseudomonas aeruginosa, gene product (gp) 146. In the present study, we performed bioinformatics analysis of currently predicted GroEL-like proteins encoded by phage genomes in comparison with cellular and mitochondrial chaperonins. Putative phage chaperonins share a low similarity and do not form a monophyletic group; nevertheless, they are closer to bacterial chaperonins in the phylogenetic tree. Experimental investigation of putative GroEL-like chaperonin proteins has been continued by physicochemical and functional characterization of gp246 encoded by the genome of Pseudomonas fluorescens bacteriophage OBP. Unlike the more usual double-ring architecture of chaperonins, including the EL gp146, the recombinant gp246 produced by Escherichia coli cells has been purified as a single heptameric ring. It possesses ATPase activity and does not require a co-chaperonin for its function. In vitro experiments demonstrated that gp246 is able to suppress the thermal protein inactivation and aggregation in an ATP-dependent manner, thus indicating chaperonin function. Single-particle electron microscopy analysis revealed the different conformational states of OBP chaperonin, depending on the bound nucleotide.

scholarly journals Structural and Functional Characterization of Sapovirus RNA-Dependent RNA Polymerase

2006 ◽  
Vol 81 (4) ◽  
pp. 1858-1871 ◽  
Author(s):  
Stephen W. B. Fullerton ◽  
Martina Blaschke ◽  
Bruno Coutard ◽  
Julia Gebhardt ◽  
Alexander Gorbalenya ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Functional Characterization ◽  
Transferase Activity ◽  
Dependent Manner ◽  
Rna Dependent Rna Polymerase ◽  
Key Enzyme

ABSTRACT Sapoviruses are one of the major agents of acute gastroenteritis in childhood. They form a tight genetic cluster (genus) in the Caliciviridae family that regroups both animal and human pathogenic strains. No permissive tissue culture has been developed for human sapovirus, limiting its characterization to surrogate systems. We report here on the first extensive characterization of the key enzyme of replication, the RNA-dependent RNA polymerase (RdRp) associated with the 3Dpol-like protein. Enzymatically active sapovirus 3Dpol and its defective mutant were expressed in Escherichia coli and purified. The overall structure of the sapovirus 3Dpol was determined by X-ray crystallography to 2.32-Å resolution. It revealed a right hand fold typical for template-dependent polynucleotide polymerases. The carboxyl terminus is located within the active site cleft, as observed in the RdRp of some (norovirus) but not other (lagovirus) caliciviruses. Sapovirus 3Dpol prefers Mn2+ over Mg2+ but may utilize either as a cofactor in vitro. In a synthetic RNA template-dependent reaction, sapovirus 3Dpol synthesizes a double-stranded RNA or labels the template 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurs de novo on heteropolymeric templates or in a primer-dependent manner on polyadenylated templates. Strikingly, this mode of initiation of RNA synthesis was also described for norovirus, but not for lagovirus, suggesting structural and functional homologies in the RNA-dependent RNA polymerase of human pathogenic caliciviruses. This first experimental evidence makes sapovirus 3Dpol an attractive target for developing drugs to control calicivirus infection in humans.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

Effects of Various Antioxidant Pretreatment Modalities on Adhesion to Sound and Caries-Affected Dentin: An In Vitro Study

2021 ◽  
pp. 232020682199798
Author(s):  
Beyza Unalan Degirmenci ◽  
Alperen Degirmenci ◽  
Emine Kara
Keyword(s):  
Bond Strength ◽  
In Vitro Study ◽  
Natural Antioxidants ◽  
Microtensile Bond Strength ◽  
Mandibular Molar ◽  
Electron Microscopy Analysis ◽  
Molar Teeth ◽  
Microscopy Analysis ◽  
Two Parameters

Aim: Natural antioxidants were offered as the answer of dentin adhesion issue. The aim of this study is to investigate the effects of proanthocyanidin and lycopene as pretreatment agents on the sound and caries-affected dentin surface on microtensile bond strength and microleakage. Materials and Methods: This study was designed as in vitro because of that 84 mandibular molar teeth were collected. Forty-two of the included teeth were carious teeth, while the other 42 were without caries. Sixty of them were used for microleakage and 24 for microtensile bond strength testing and scanning electron microscopy analysis. The samples were divided into six subgroups randomly according to dentin pretreatments: 5% proanthocyanidin, 5% lycopene, and no antioxidant application. After the restorative procedures, samples were attached to the microtensile tester. Samples were subjected to tensile stress in the load cell until they broke at a speed of 0.5 mm per min. Microtensile bond strength (µTBS) and microleakage test data were analyzed with two-way analysis of variance, Bonferroni correction, and Tamhane’s T2 tests. Results: Two-way variance analysis showed that dentin pretreatment applications, dentin substrate, and the interaction between these two parameters had statistically significant effects on µTBS values ( P < .001). There was no difference between dentin pretreatment applications in terms of microleakage scores ( P > .05). Conclusion: The application of dentin pretreatment with proanthocyanidin is a successful procedure that increases the bond strength in both dentin substrate, while pretreatment with lycopene in caries-affected dentin reduces it.

scholarly journals Antiparasitic Properties of Cardiovascular Agents against Human Intravascular Parasite Schistosoma mansoni

2021 ◽  
Vol 14 (7) ◽  
pp. 686
Author(s):  
Raquel Porto ◽  
Ana C. Mengarda ◽  
Rayssa A. Cajas ◽  
Maria C. Salvadori ◽  
Fernanda S. Teixeira ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Screening Strategy ◽  
Early Infection ◽  
Global Public Health ◽  
Parasitic Worm ◽  
Electron Microscopy Analysis ◽  
Health Significance ◽  
Microscopy Analysis

The intravascular parasitic worm Schistosoma mansoni is a causative agent of schistosomiasis, a disease of great global public health significance. Praziquantel is the only drug available to treat schistosomiasis and there is an urgent demand for new anthelmintic agents. Adopting a phenotypic drug screening strategy, here, we evaluated the antiparasitic properties of 46 commercially available cardiovascular drugs against S. mansoni. From these screenings, we found that amiodarone, telmisartan, propafenone, methyldopa, and doxazosin affected the viability of schistosomes in vitro, with effective concentrations of 50% (EC50) and 90% (EC90) values ranging from 8 to 50 µM. These results were further supported by scanning electron microscopy analysis. Subsequently, the most effective drug (amiodarone) was further tested in a murine model of schistosomiasis for both early and chronic S. mansoni infections using a single oral dose of 400 mg/kg or 100 mg/kg daily for five consecutive days. Amiodarone had a low efficacy in chronic infection, with the worm and egg burden reduction ranging from 10 to 30%. In contrast, amiodarone caused a significant reduction in worm and egg burden in early infection (>50%). Comparatively, treatment with amiodarone is more effective in early infection than praziquantel, demonstrating the potential role of this cardiovascular drug as an antischistosomal agent.

scholarly journals Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila

2006 ◽  
Vol 74 (7) ◽  
pp. 3742-3755 ◽  
Author(s):  
Lakshmi Pillai ◽  
Jian Sha ◽  
Tatiana E. Erova ◽  
Amin A. Fadl ◽  
Bijay K. Khajanchi ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Aeromonas Hydrophila ◽  
Functional Characterization ◽  
Affinity Column ◽  
Serum Resistance ◽  
Classical Pathway ◽  
Dependent Manner ◽  
T7 Promoter

ABSTRACT Human diseases caused by species of Aeromonas have been classified into two major groups: septicemia and gastroenteritis. In this study, we reported the molecular and functional characterization of a new virulence factor, ToxR-regulated lipoprotein, or TagA, from a diarrheal isolate, SSU, of Aeromonas hydrophila. The tagA gene of A. hydrophila exhibited 60% identity with that of a recently identified stcE gene from Escherichia coli O157:H7, which encoded a protein (StcE) that provided serum resistance to the bacterium and prevented erythrocyte lysis by controlling classical pathway of complement activation by cleaving the complement C1-esterase inhibitor (C1-INH). We purified A. hydrophila TagA as a histidine-tagged fusion protein (rTagA) from E. coli DE3 strain using a T7 promoter-based pET30 expression vector and nickel affinity column chromatography. rTagA cleaved C1-INH in a time-dependent manner. The tagA isogenic mutant of A. hydrophila, unlike its corresponding wild-type (WT) or the complemented strain, was unable to cleave C1-INH, which is required to potentiate the C1-INH-mediated lysis of host and bacterial cells. We indeed demonstrated colocalization of C1-INH and TagA on the bacterial surface by confocal fluorescence microscopy, which ultimately resulted in increased serum resistance of the WT bacterium. Likewise, we delineated the role of TagA in contributing to the enhanced ability of C1-INH to inhibit the classical complement-mediated lysis of erythrocytes. Importantly, we provided evidence that the tagA mutant was significantly less virulent in a mouse model of infection (60%) than the WT bacterium at two 50% lethal doses, which resulted in 100% mortality within 48 h. Taken together, our data provided new information on the role of TagA as a virulence factor in bacterial pathogenesis. This is the first report of TagA characterization from any species of Aeromonas.

scholarly journals NFB-01. FUNCTIONAL CHARACTERIZATION OF ATRX LOSS IN NF1-ASSOCIATED GLIOMA AND MPNST

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii417-iii418
Author(s):  
Ming Yuan ◽  
Karlyne Reilly ◽  
Christine Pratilas ◽  
Christopher Heaphy ◽  
Fausto Rodriguez
Keyword(s):  
Cell Lines ◽  
Functional Characterization ◽  
Tert Promoter ◽  
Aggressive Tumors ◽  
Sirna Knockdown ◽  
Nih 3T3 ◽  
Vitro Effect ◽  
Murine Glioma

Abstract To identify the biologic relevance of ATRX loss in NF1-associated gliomagenesis, we studied the effects of Atrx loss using four previously characterized Nf1+/-Trp53+/- murine glioma lines. Lines 130G#3 and 158D#8 (corresponding to grade IV and III gliomas, respectively) displayed preserved ATRX protein expression compared to NIH-3T3 cells. We studied the effects of Atrx knockdown in these two lines in the presence and absence of the TERT inhibitor, BIRBR1532. Using a telomere-specific FISH assay, we identified increased signal intensity after Atrx knockdown, only in the presence of the TERT inhibitor. These features are reminiscent of ALT, although there were no significant alterations in cell growth. Next, we studied the effect of ATRX loss in MPNST lines ST88-14, NF90-8, STS-26T. These cell lines all expressed ATRX and DAXX. However, STS-26T contained a TERT promoter mutation and ST88-14 had a known SNP in the TERT promoter, while NF90-8 had no alterations. ATRX siRNA knockdown showed no significant effects in cell proliferation or apoptosis. However, ATRX knockdown resulted in rare ultra-bright foci, indicative of ALT. Next, we studied the in vitro effect of the ATR inhibitor VE-821 in MPNST cell lines. Only NF90-8 (lacking TERT alterations) demonstrated a decrease in growth after ATRX knockdown and VE-821 treatment. However, ATRX knockdown alone did not affect sensitivity to carboplatin. Our findings further support a role for ATRX loss with subsequent ALT activation in a biologic subset of NF1-associated malignancies, thereby opening an opportunity for therapeutic targeting of these aggressive tumors using specific classes of drugs.

Functional analysis of a stable transcription arrest site in the first intron of the murine adenosine deaminase gene

1993 ◽  
Vol 13 (5) ◽  
pp. 2718-2729
Author(s):  
S F Kash ◽  
J W Innis ◽  
A U Jackson ◽  
R E Kellems
Keyword(s):  
Rna Polymerase Ii ◽  
Adenosine Deaminase ◽  
Functional Characterization ◽  
Gel Filtration ◽  
Point Mutations ◽  
Elongation Factor ◽  
Dependent Manner ◽  
First Intron ◽  
Intron 1

Transcription arrest plays a role in regulating the expression of a number of genes, including the murine adenosine deaminase (ADA) gene. We have previously identified two prominent arrest sites at the 5' end of the ADA gene: one in the first exon and one in the first intron (J. W. Innis and R. E. Kellems, Mol. Cell. Biol. 11:5398-5409, 1991). Here we report the functional characterization of the intron 1 arrest site, located 137 to 145 nucleotides downstream of the cap site. We have determined, using gel filtration, that the intron 1 arrest site is a stable RNA polymerase II pause site and that the transcription elongation factor SII promotes read-through at this site. Additionally, the sequence determinants for the pause are located within a 37-bp fragment encompassing this site (+123 to +158) and can direct transcription arrest in an orientation-dependent manner in the context of the ADA and adenovirus major late promoters. Specific point mutations in this region increase or decrease the relative pausing efficiency. We also show that the sequence determinants for transcription arrest can function when placed an additional 104 bp downstream of their natural position.

Functional Characterization of GABAA Receptor Ligands In Vitro

2004 ◽  
pp. 85-94
Author(s):  
Bjarke Ebert ◽  
Sally Anne Thompson ◽  
Signe Í. Stórustovu ◽  
Keith A. Wafford
Keyword(s):  
Gabaa Receptor ◽  
Functional Characterization ◽  
Receptor Ligands

scholarly journals Biochemical and Functional Characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L.

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2876 ◽  
Author(s):  
Lin Tan ◽  
Mei Wang ◽  
Youfa Kang ◽  
Farrukh Azeem ◽  
Zhaoxi Zhou ◽  
...  
Keyword(s):  
Enzyme Assay ◽  
Molecular Mechanisms ◽  
Mangifera Indica ◽  
Functional Characterization ◽  
Citrate Buffer ◽  
Anthocyanidin Reductase ◽  
High Amino Acid ◽  
Optimum Ph

Mango (Mangifera indica L.) is abundant in proanthocyanidins (PAs) that are important for human health and plant response to abiotic stresses. However, the molecular mechanisms involved in PA biosynthesis still need to be elucidated. Anthocyanidin reductase (ANR) catalyzes a key step in PA biosynthesis. In this study, three ANR cDNAs (MiANR1-1,1-2,1-3) were isolated from mango, and expressed in Escherichia coli. In vitro enzyme assay showed MiANR proteins convert cyanidin to their corresponding flavan-3-ols, such as (−)-catechin and (−)-epicatechin. Despite high amino acid similarity, the recombinant ANR proteins exhibited differences in enzyme kinetics and cosubstrate preference. MiANR1-2 and MiANR1-3 have the same optimum pH of 4.0 in citrate buffer, while the optimum pH for MiANR1-1 is pH 3.0 in phosphate buffer. MiANR1-1 does not use either NADPH or NADH as co-substrate while MiANR1-2/1-3 use only NADPH as co-substrate. MiANR1-2 has the highest Km and Vmax for cyanidin, followed by MiANR1-3 and MiANR1-1. The overexpression of MiANRs in ban mutant reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate MiANRs can form the ANR pathway, leading to the formation of two types of isomeric flavan-3-ols and PAs in mango.

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