scholarly journals Molecular insights into the copper-sensitive operon repressor in Acidithiobacillus caldus

2021 ◽  
Author(s):  
Shaoxiang Hou ◽  
Yanjun Tong ◽  
Hailin Yang ◽  
Shoushuai Feng
Keyword(s):  
Binding Affinity ◽  
Dissociation Constants ◽  
Copper Ions ◽  
Copper Resistance ◽  
Size Exclusion ◽  
Transcriptional Analysis ◽  
Copper Stress ◽  
Acidithiobacillus Caldus ◽  
Competition Assays

The copper-sensitive operon repressor (CsoR) family is the main Cu(I)-sensing family, which is widely distributed, and regulates regulons involved in detoxification in response to extreme copper stress (a general range of ≥ 3 g/L copper ions). Here, we identified CsoR Ac in hyper-copper-resistant Acidithiobacillus caldus , a type strain used in the bioleaching process of copper ores. CsoR Ac possesses highly conserved Cu(I) ligands and structures within the CsoR family members. Transcriptional analysis assays indicated that the promoter (PIII) of csoR was active but weakly responsive to copper in Escherichia coli . Copper titration assays gave a stoichiometry of 0.8 mol Cu(I) per apo-CsoR Ac monomer in vitro combined with atomic absorption spectroscopy analysis. Cu I -CsoR Ac and apo-CsoR Ac share essentially identical secondary structures and assembly states, as demonstrated by circular dichroism spectra and size exclusion chromatography profiles. The average dissociation constants ( K D = 2.26 × 10 −18 M and 0.53 × 10 −15 M) and Cu(I) binding affinity of apo-CsoR Ac were estimated by bathocuproine disulfonate (BCS) and bicinchoninic acid (BCA) competition assays, respectively. Site-directed mutations of conserved Cu(I) ligands in CsoR Ac did not significantly alter the secondary structure or assembly state. Competition assays showed that mutants shared the same order of magnitude of Cu(I) binding affinity with apo-CsoR Ac . Moreover, apo-CsoR Ac could bind to the DNA fragment P08430 in vitro , although with low affinity. Finally, a working model was proposed to illustrate putative copper resistance mechanisms in A. caldus . Importance Research on copper resistance among various species has attracted considerable interest. However, due to the lack of effective and reproducible genetic tools, few studies regarding copper resistance have been reported for A. caldus . Here, we characterized a major Cu(I)-sensing family protein, CsoR Ac , which binds Cu(I) with an attomolar affinity higher than that of the Cu(I)-specific chelator, bathocuproine disulfonate. In particular, CsoR family proteins were only identified in A. caldus , rather than A. ferrooxidans and A. thiooxidans , which are both type strains used for bioleaching. Meanwhile, A. caldus harbored more copper resistance determinants and a relatively full-scale regulatory system involved in copper homeostasis. These observations suggested that A. caldus may play an essential role in the application of engineered strains with higher copper resistance in the near future.

scholarly journals Copper Stress Induces a Global Stress Response in Staphylococcus aureus and Represses sae and agr Expression and Biofilm Formation

2009 ◽  
Vol 76 (1) ◽  
pp. 150-160 ◽  
Author(s):  
Jonathan Baker ◽  
Sutthirat Sitthisak ◽  
Mrittika Sengupta ◽  
Miranda Johnson ◽  
R. K. Jayaswal ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Transcriptional Profiling ◽  
Resistance Mechanism ◽  
Growth Conditions ◽  
Copper Resistance ◽  
Transcriptional Analysis ◽  
Copper Stress ◽  
High Copper

ABSTRACT Copper is an important cofactor for many enzymes; however, high levels of copper are toxic. Therefore, bacteria must ensure there is sufficient copper for use as a cofactor but, more importantly, must limit free intracellular levels to prevent toxicity. In this study, we have used DNA microarray to identify Staphylococcus aureus copper-responsive genes. Transcriptional profiling of S. aureus SH1000 grown in excess copper identified a number of genes which fall into four groups, suggesting that S. aureus has four main mechanisms for adapting to high levels of environmental copper, as follows: (i) induction of direct copper homeostasis mechanisms; (ii) increased oxidative stress resistance; (iii) expression of the misfolded protein response; and (iv) repression of a number of transporters and global regulators such as Agr and Sae. Our experimental data confirm that resistance to oxidative stress and particularly to H2O2 scavenging is an important S. aureus copper resistance mechanism. Our previous studies have demonstrated that Eap and Emp proteins, which are positively regulated by Agr and Sae, are required for biofilm formation under low-iron growth conditions. Our transcriptional analysis has confirmed that sae, agr, and eap are repressed under high-copper conditions and that biofilm formation is indeed repressed under high-copper conditions. Therefore, our results may provide an explanation for how copper films can prevent biofilm formation on catheters.

scholarly journals Functional and Expression Analyses of the cop Operon, Required for Copper Resistance in Agrobacterium tumefaciens

2009 ◽  
Vol 191 (16) ◽  
pp. 5159-5168 ◽  
Author(s):  
Sirikan Nawapan ◽  
Nisanart Charoenlap ◽  
Anchalee Charoenwuttitam ◽  
Panatda Saenkham ◽  
Skorn Mongkolsuk ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Copper Ions ◽  
Protein A ◽  
Copper Resistance ◽  
Copper Chaperone ◽  
Phytopathogenic Bacterium ◽  
Ion Concentrations ◽  
Increased Sensitivity ◽  
Cop Operon

ABSTRACT The copper resistance determinant copARZ, which encodes a CPx-type copper ATPase efflux protein, a transcriptional regulator, and a putative intracellular copper chaperone, was functionally characterized for the phytopathogenic bacterium Agrobacterium tumefaciens. These genes are transcribed as an operon, and their expression is induced in response to increasing copper and silver ion concentrations in a copR-dependent fashion. Analysis of the copARZ promoter revealed a putative CopR binding box located within the spacer of the −35 and −10 promoter motifs. In vitro, purified CopR could specifically bind to the box. The inactivation of the copARZ operon or copZ reduces the level of resistance to copper but not to other metal ions. Also, the copARZ operon mutant shows increased sensitivity to the superoxide generators menadione and plumbagin. In addition, the loss of functional copZ does not affect the ability of copper ions to induce the copARZ promoter, indicating that CopZ is not involved in the copper-sensing mechanism of CopR. Altogether, the results demonstrate a crucial role for the copARZ operon as a component of the copper resistance machinery in A. tumefaciens.

scholarly journals Transcriptional Repressor CopR: Use of SELEX To Study the copR Operator Indicates that Evolution Was Directed at Maximal Binding Affinity

2004 ◽  
Vol 186 (18) ◽  
pp. 6254-6264 ◽  
Author(s):  
Peggy Freede ◽  
Sabine Brantl
Keyword(s):  
Binding Affinity ◽  
Copy Number ◽  
Dissociation Constants ◽  
Mobility Shift ◽  
Copy Numbers ◽  
Maximal Binding ◽  
Equilibrium Dissociation Constants ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT CopR is one of the two copy number control elements of the streptococcal plasmid pIP501. It represses transcription of the repR mRNA encoding the essential replication initiator protein about 10- to 20-fold by binding to its operator region upstream of the repR promoter pII. CopR binds at two consecutive sites in the major groove of the DNA that share the consensus motif 5′-CGTG. Previously, the minimal operator was narrowed down to 17 bp, and equilibrium dissociation constants for DNA binding and dimerization were determined to be 0.4 nM and 1.4 μM, respectively. In this work, we used a SELEX procedure to study copR operator sequences of different lengths in combination with electrophoretic mobility shift assays of mutated copR operators as well as copy number determinations to assess the sequence requirements for CopR binding. The results suggest that in vivo evolution was directed at maximal binding affinity. Three simultaneous nucleotide exchanges outside the bases directly contacted by CopR only slightly affected CopR binding in vitro or copy numbers in vivo. Furthermore, the optimal spacer sequence was found to comprise 7 bp, to be AT rich, and to need an A/T and a T at the 3′ positions, whereas broad variations in the sequences flanking the minimal 17-bp operator were well tolerated.

scholarly journals Determination of intracellular protein–ligand binding affinity by competition binding in-cell NMR

2021 ◽  
Vol 77 (10) ◽  
Author(s):  
Enrico Luchinat ◽  
Letizia Barbieri ◽  
Matteo Cremonini ◽  
Matteo Pennestri ◽  
Alessio Nocentini ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Affinity ◽  
Dissociation Constants ◽  
Attrition Rates ◽  
Cellular Environment ◽  
Competition Binding ◽  
Intracellular Target ◽  
Intracellular Binding ◽  
Target Binding

Structure-based drug development suffers from high attrition rates due to the poor activity of lead compounds in cellular and animal models caused by low cell penetrance, off-target binding or changes in the conformation of the target protein in the cellular environment. The latter two effects cause a change in the apparent binding affinity of the compound, which is indirectly assessed by cellular activity assays. To date, direct measurement of the intracellular binding affinity remains a challenging task. In this work, in-cell NMR spectroscopy was applied to measure intracellular dissociation constants in the nanomolar range by means of protein-observed competition binding experiments. Competition binding curves relative to a reference compound could be retrieved either from a series of independent cell samples or from a single real-time NMR bioreactor run. The method was validated using a set of sulfonamide-based inhibitors of human carbonic anhydrase II with known activity in the subnanomolar to submicromolar range. The intracellular affinities were similar to those obtained in vitro, indicating that these compounds selectively bind to the intracellular target. In principle, the approach can be applied to any soluble intracellular target that gives rise to measurable chemical shift changes upon ligand binding.

Microfluidic Molecular Trap: Probing Extracellular Signaling by Selectively Blocking Exchange of Specific Molecules in Cell-Cell Interactions

2013 ◽  
Author(s):  
Bryson M. Brewer ◽  
Yandong Gao ◽  
Rebecca M. Sappington ◽  
Deyu Li
Keyword(s):  
Cell Culture ◽  
Signaling Pathway ◽  
Binding Affinity ◽  
Dissociation Constants ◽  
Gene Knockout ◽  
In Vitro Cell Culture ◽  
Extracellular Signaling ◽  
Time Periods ◽  
Avidin Biotin Complex

Communication among cell populations is achieved via a wide variety of soluble, extracellular signaling molecules [1]. In order to investigate the role of specific molecules in a cellular process, researchers often utilize in vitro cell culture techniques in which the molecule under question has been removed from the signaling pathway. Traditionally, this has been accomplished by eliminating the gene in the cell that is responsible for coding the targeted ligand/receptor by using modern DNA technology such as gene knockout; however, this process is expensive, time-consuming, and labor intensive. Previously, we have demonstrated a microfluidic platform that uses a semi-permeable barrier with embedded receptor-coated nanoparticles to selectively remove a specific molecule or ligand from the extracellular signaling pathway in a cell co-culture environment [2]. This initial proof-of-principle was conducted using biotinylated nanoparticles and fluorescently tagged avidin molecules, as the avidin/biotin complex is the strongest known non-covalent interaction between a protein and a ligand (Dissociation constant kd = 10−15 M). Also, the trap was only effective for short time periods (<15 min) because the high concentration of fluorescently tagged avidin molecules required for visualization quickly saturated the barrier. However, nearly all biologically relevant ligand-receptor interactions have lower binding affinities than the avidin-biotin complex, with dissociation constants that are larger by several orders of magnitude. In addition, many in vitro cell culture experiments are conducted over multiple hours or days. Thus, a practically useful molecular trap device must be able to operate in a lower binding affinity regime while also lasting for extended time periods. Here we present results in which a biotinylated-particle barrier was used to successfully block lower concentrations of fluorescently tagged avidin for multiple days, showcasing the applicability of the device for long term experiments. In addition, we introduce a modified molecular trap in which the protein A/goat IgG complex was used to demonstrate the effectiveness of the platform for lower binding affinity protein-ligand interactions. These results indicate the potential usefulness of the microfluidic molecular trap platform for probing extracellular signaling pathways.

scholarly journals The Helicobacter pylori CrdRS Two-Component Regulation System (HP1364/HP1365) Is Required for Copper-Mediated Induction of the Copper Resistance Determinant CrdA

2005 ◽  
Vol 187 (13) ◽  
pp. 4683-4688 ◽  
Author(s):  
Barbara Waidner ◽  
Klaus Melchers ◽  
Frank Nils Stähler ◽  
Manfred Kist ◽  
Stefan Bereswill
Keyword(s):  
Helicobacter Pylori ◽  
Copper Ions ◽  
Response Regulator ◽  
Copper Resistance ◽  
Resistance Determinant ◽  
Upstream Region ◽  
Regulation System ◽  
Direct Role ◽  
H Pylori

ABSTRACT Here we describe that the Helicobacter pylori sensor kinase produced by HP1364 and the response regulator produced by HP1365 and designated CrdS and CrdR, respectively, are both required for transcriptional induction of the H. pylori copper resistance determinant CrdA by copper ions. CrdRS-deficient mutants lacked copper induction of crdA expression and were copper sensitive. A direct role of CrdR in transcriptional regulation of crdA was confirmed by in vitro binding of CrdR to the crdA upstream region. A 21-nucleotide sequence located near the crdA promoter was shown to be required for CrdR binding.

Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity

2019 ◽  
Vol 476 (16) ◽  
pp. 2297-2319 ◽  
Author(s):  
Marta Grzechowiak ◽  
Milosz Ruszkowski ◽  
Joanna Sliwiak ◽  
Kamil Szpotkowski ◽  
Michal Sikorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Inorganic Pyrophosphatase ◽  
Prolonged Storage ◽  
Mitochondrial Targeting ◽  
Cleavage Rate ◽  
Inorganic Pyrophosphate ◽  
Putative Signal Peptide ◽  
Targeting Peptide

Abstract Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31–Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  
Keyword(s):  
Modifier Gene ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Lipoprotein Profile ◽  
Exclusion Chromatography ◽  
Low Hdl ◽  
Lcat Deficiency ◽  
Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

Computational Evaluation and In Vitro Validation of New Epidermal Growth Factor Receptor Inhibitors

2020 ◽  
Vol 20 (18) ◽  
pp. 1628-1639
Author(s):  
Sergi Gómez-Ganau ◽  
Josefa Castillo ◽  
Andrés Cervantes ◽  
Jesus Vicente de Julián-Ortiz ◽  
Rafael Gozalbes
Keyword(s):  
Cell Proliferation ◽  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Affinity ◽  
Cell Lines ◽  
Growth Factor Receptor ◽  
Significant Activity ◽  
Epidermal Growth

Background: The Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein that acts as a receptor of extracellular protein ligands of the epidermal growth factor (EGF/ErbB) family. It has been shown that EGFR is overexpressed by many tumours and correlates with poor prognosis. Therefore, EGFR can be considered as a very interesting therapeutic target for the treatment of a large variety of cancers such as lung, ovarian, endometrial, gastric, bladder and breast cancers, cervical adenocarcinoma, malignant melanoma and glioblastoma. Methods: We have followed a structure-based virtual screening (SBVS) procedure with a library composed of several commercial collections of chemicals (615,462 compounds in total) and the 3D structure of EGFR obtained from the Protein Data Bank (PDB code: 1M17). The docking results from this campaign were then ranked according to the theoretical binding affinity of these molecules to EGFR, and compared with the binding affinity of erlotinib, a well-known EGFR inhibitor. A total of 23 top-rated commercial compounds displaying potential binding affinities similar or even better than erlotinib were selected for experimental evaluation. In vitro assays in different cell lines were performed. A preliminary test was carried out with a simple and standard quick cell proliferation assay kit, and six compounds showed significant activity when compared to positive control. Then, viability and cell proliferation of these compounds were further tested using a protocol based on propidium iodide (PI) and flow cytometry in HCT116, Caco-2 and H358 cell lines. Results: The whole six compounds displayed good effects when compared with erlotinib at 30 μM. When reducing the concentration to 10μM, the activity of the 6 compounds depends on the cell line used: the six compounds showed inhibitory activity with HCT116, two compounds showed inhibition with Caco-2, and three compounds showed inhibitory effects with H358. At 2 μM, one compound showed inhibiting effects close to those from erlotinib. Conclusion: Therefore, these compounds could be considered as potential primary hits, acting as promising starting points to expand the therapeutic options against a wide range of cancers.

Synthesis, Antimicrobial Evaluation and Docking Study of Novel Thiosemicarbazone clubbed with 1,2,3-Triazoles

2020 ◽  
Vol 16 ◽  
Author(s):  
Adinath D. Badar ◽  
Shubham M. Sulakhe ◽  
Mahesh B. Muluk ◽  
Naziya N. M. A. Rehman ◽  
Prashant P. Dixit ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Biological Activities ◽  
Dna Gyrase ◽  
Docking Study ◽  
Antimicrobial Activities ◽  
Diffusion Method ◽  
Molecular Docking Study ◽  
Triazole Derivatives ◽  
Antibacterial And Antifungal

Background: Thiosemicarbazone, 1,2,3-triazole and their derivatives received great pharmaceutical importance due to their prominent biological activities. In the present study, the molecular hybrid thiosemicarbazone-1,2,3-triazoles derivatives were synthesized and screened for their antimicrobial activities. Methods: A series of thiosemicarbazone clubbed with 1,2,3-triazole derivatives were synthesized via click chemistry approach in good yields. The structures of synthesized compounds were assigned by their spectral data. The in vitro antimicrobial activity was performed by the agar well diffusion method. A molecular docking study was performed to identify the possible mode of action of synthesized derivatives. Results: The compounds 5d, 5h, 5i and 5k have exhibited excellent antimicrobial activities against both antibacterial and antifungal pathogens. The active thiosemicarbazone-1,2,3-triazole derivatives have shown excellent binding affinity towards DNA gyrase. Conclusion: The molecular hybrid thiosemicarbazone-1,2,3-triazole derivatives were synthesized. The newly synthesized compounds were evaluated for their antimicrobial activities. Few of the thiosemicarbazone-1,2,3-triazoles derivatives have exhibited good antimicrobial activities. They have been shown excellent binding affinity towards DNA gyrase.

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