-
Archives of Biochemistry and Biophysics Jul 2020The active sites of metalloproteins may be mimicked by designing peptides that bind to their respective metal ions. Studying the binding of protein ligands to metal ions...
The active sites of metalloproteins may be mimicked by designing peptides that bind to their respective metal ions. Studying the binding of protein ligands to metal ions along with the associated structural changes is important in understanding metal uptake, transport and electron transfer functions of proteins. Copper-binding metalloprotein azurin is a 128-residue electron transfer protein with a redox-active copper cofactor. Here, we report the copper-binding associated spectroscopic and structural properties of peptide loops (11 and 13 residues) from the copper-binding site of azurin. These peptides develop a β-turn upon copper-binding with a 1:1 Cu:peptide stoichiometry as seen in circular dichroism and exhibit electronic transitions centered at 340 nm and 540 nm. Further addition of copper develops a helical feature along with a shift in the absorption maxima to ~360 nm and ~580 nm at 2:1 Cu:peptide stoichiometry, indicating stoichiometric dependence of copper-binding geometry. Mass spectrometry indicates the copper-binding to cysteine, histidine and methionine in the peptide with 1:1 stoichiometry, and interestingly, dimerization through a disulfide linkage at 2:1 stoichiometry, as observed previously for denatured azurin. Fluorescence quenching studies on peptides with tryptophan further confirm the copper-binding induced changes in the two peptides are bi-phasic.
Topics: Azurin; Catalytic Domain; Copper; Fluorescence; Fluorescence Resonance Energy Transfer; Peptide Fragments; Protein Binding; Protein Conformation; Spectrometry, Mass, Electrospray Ionization; Tryptophan
PubMed: 32343975
DOI: 10.1016/j.abb.2020.108388 -
Physical Chemistry Chemical Physics :... Nov 2020Electron Paramagnetic Resonance (EPR) spectroscopy is a powerful method for unraveling structures and dynamics of biomolecules. Out of the EPR tool box, Pulsed...
Electron Paramagnetic Resonance (EPR) spectroscopy is a powerful method for unraveling structures and dynamics of biomolecules. Out of the EPR tool box, Pulsed Electron-Electron Double Resonance spectroscopy (PELDOR or DEER) enables one to resolve such structures by providing distances between spin centers on the nanometer scale. Most commonly, both spin centers are spin labels or one is a spin label and the other is a paramagnetic metal ion, cluster, amino acid or cofactor radical. Often, the translation of the measured distances into structures is complicated by the long and flexible linker connecting the spin center of the spin label with the biomolecule. Nowadays, this challenge is overcome by computational methods but the currently available approaches have a rather large mean error of roughly 2-3 Å. Here, the new GFN-FF general force-field is combined with the fully automated Conformer Rotamer Ensemble Search Tool (CREST) [P. Pracht et al., Phys. Chem. Chem. Phys., 2020, 22, 7169-7192] to generate conformer ensembles of the R1 side chain (methanthiosulfonate spin label (MTSL) covalently bound to a cysteine) in several cysteine mutants of azurin and T4 lysozyme. In order to determine the Cu2+-R1 and R1-R1 distance distributions, GFN-FF based MD simulations were carried out starting from the most probable R1 conformers found by CREST. The deviation between theory and experiment in mean inter-spin distances was 0.98 Å on average for the mutants of azurin (1.84 Å for T4 lysozyme) and the right modality was obtained. The error of the most probable distances for each mode was only 0.76 Å in the case of azurin. This CREST/MD procedure does thus enable precise distance-to-structure translations and provides a means to disentangle label from protein conformers.
Topics: Azurin; Macromolecular Substances; Models, Molecular; Muramidase; Mutation; Nitrogen Oxides; Protein Conformation; Spin Labels
PubMed: 33107523
DOI: 10.1039/d0cp04920d -
Cell Cycle (Georgetown, Tex.) 2018Membrane lipid rafts are highly ordered microdomains and essential components of plasma membranes. In this work, we demonstrate that azurin uptake by cancer cells is, in...
Membrane lipid rafts are highly ordered microdomains and essential components of plasma membranes. In this work, we demonstrate that azurin uptake by cancer cells is, in part, mediated by caveolin-1 and GM-1, lipid rafts' markers. This recognition is mediated by a surface exposed hydrophobic core displayed by azurin since the substitution of a phenylalanine residue in position 114 facing the hydrophobic cavity by alanine impacts such interactions, debilitating the uptake of azurin by cancer cells. Treating of cancer cells with azurin leads to a sequence of events: alters the lipid raft exposure at plasma membranes, causes a decrease in the plasma membrane order as examined by Laurdan two-photon imaging and leads to a decrease in the levels of caveolin-1. Caveolae, a subset of lipid rafts characterized by the presence of caveolin-1, are gaining increasing recognition as mediators in tumor progression and resistance to standard therapies. We show that azurin inhibits growth of cancer cells expressing caveolin-1, and this inhibition is only partially observed with mutant azurin. Finally, the simultaneous administration of azurin with anticancer therapeutic drugs (paclitaxel and doxorubicin) results in an enhancement in their activity, contrary to the mutated protein.
Topics: Amino Acid Sequence; Antineoplastic Agents; Azurin; Caveolin 1; Cell Line, Tumor; G(M1) Ganglioside; Humans; Membrane Fluidity; Membrane Microdomains; Mutant Proteins; Point Mutation; Protein Domains
PubMed: 29963969
DOI: 10.1080/15384101.2018.1489178 -
Infection and Drug Resistance 2020The purpose of this study was to analyze the sequence of gene in relation to its expression in strains isolated from different clinical specimens of burn patients....
AIM
The purpose of this study was to analyze the sequence of gene in relation to its expression in strains isolated from different clinical specimens of burn patients. Moreover, in silico sequence analysis of gene using globally reported sequences was intended.
MATERIALS AND METHODS
Fifty-nine multidrug-resistant isolates were selected from different clinical specimens of patients suffering from burn wound infections in two university hospitals and subjected to antibacterial susceptibility testing. The frequency and genetic diversity of the gene was determined by polymerase chain reaction (PCR) and Sanger sequencing. The gene sequences were compared with the sequence data from other countries. The expression level of gene in isolates with different sequences from different clinical specimens was evaluated by real-time PCR.
RESULTS AND CONCLUSION
About 98%-100% of the isolates were resistant to gentamicin, tobramycin, cefoxitin, ciprofloxacin, amikacin, and imipenem, while 100% and 23.9% of the isolates were susceptible to colistin and ceftazidime, respectively. Only eight point mutations were detected with amino acid substitutions in only two positions (81 and 102). In global analysis, 93% of strains showed missense mutation at positions 81 (alanine to threonine). The majority (81%) of Iranian strains were allocated to two major clusters distinct from the rest of world, which may suggest that strains from Iran have made a distinct genetic stockpile through point mutations which has established them separate from the other counties. However, 19% were distributed in different clusters together with the strains from different countries of North and South America, Europe, South and East Asia. The expression level of the gene was statistically higher in the isolates collected from the blood of burns patients with systemic infection compared to the isolates collected from other specimens (wound, catheter and tissue), which shows a positive correlation between gene expression and increased pathogenicity and capability for dissemination. This study may open new insight about genetic variation and significance in pathogenesis.
PubMed: 32765002
DOI: 10.2147/IDR.S248043 -
Communications Biology Jan 2023Tumor suppressor p53 prevents tumorigenesis by promoting cell cycle arrest and apoptosis through transcriptional regulation. Dysfunction of p53 occurs frequently in...
Tumor suppressor p53 prevents tumorigenesis by promoting cell cycle arrest and apoptosis through transcriptional regulation. Dysfunction of p53 occurs frequently in human cancers. Thus, p53 becomes one of the most promising targets for anticancer treatment. A bacterial effector protein azurin triggers tumor suppression by stabilizing p53 and elevating its basal level. However, the structural and mechanistic basis of azurin-mediated tumor suppression remains elusive. Here we report the atomic details of azurin-mediated p53 stabilization by combining X-ray crystallography with nuclear magnetic resonance. Structural and mutagenic analysis reveals that the p28 region of azurin, which corresponds to a therapeutic peptide, significantly contributes to p53 binding. This binding stabilizes p53 by disrupting COP1-mediated p53 ubiquitination and degradation. Using the structure-based design, we obtain several affinity-enhancing mutants that enable amplifying the effect of azurin-induced apoptosis. Our findings highlight how the structure of the azurin-p53 complex can be leveraged to design azurin derivatives for cancer therapy.
Topics: Humans; Azurin; Bacterial Proteins; Peptides; Tumor Suppressor Protein p53; Ubiquitination
PubMed: 36650277
DOI: 10.1038/s42003-023-04458-1 -
Bioengineered 2015Azurin and Laz (lipidated azurin) are 2 bacterial proteins with anticancer, anti-viral and anti-parasitic activities. Azurin, isolated from the bacterium Pseudomonas...
Azurin and Laz (lipidated azurin) are 2 bacterial proteins with anticancer, anti-viral and anti-parasitic activities. Azurin, isolated from the bacterium Pseudomonas aeruginosa, termed Paz, demonstrates anticancer activity against a range of cancers but not against brain tumors. In contrast, Laz is produced by members of Gonococci/Meningococci, including Neisseria meningitides which can cross the blood-brain barrier to infect brain meninges. It has been previously reported that Laz has an additional 39 amino acid moiety, called an H.8 epitope, in the N-terminal part of the azurin moiety that allows Laz to cross the entry barrier to brain tumors such as glioblastomas. Exactly, how the H.8 epitope helps the azurin moiety of Laz to cross the entry barriers to attack glioblastoma cells is unknown. In this paper, we describe the structural features of the H.8 moiety in Laz using X-ray crystallography and demonstrate that while the azurin moiety of Laz adopts a β-sandwich fold with 2 β-sheets arranged in the Greek key motif, the H.8 epitope was present as a disordered structure outside the Greek key motif. Structures of Paz and H.8 epitope-deficient Laz are well superimposed. The structural flexibility of the H.8 motif in Laz explains the extracellular location of Laz in Neisseria where it can bind the key components of brain tumor cells to disrupt their tight junctions and allow entry of Laz inside the tumors to exert cytotoxicity.
Topics: Amino Acid Sequence; Antineoplastic Agents; Azurin; Bacterial Proteins; Brain; Cell Line, Tumor; Escherichia coli; Humans; Molecular Sequence Data; Neisseria; Protein Conformation; Pseudomonas aeruginosa; Tight Junctions; X-Ray Diffraction
PubMed: 25714335
DOI: 10.1080/21655979.2015.1022303 -
Metallomics : Integrated Biometal... Mar 2020Protein oligomers have gained interest, owing to their increased knowledge in cells and promising utilization for future materials. Various proteins have been shown to...
Protein oligomers have gained interest, owing to their increased knowledge in cells and promising utilization for future materials. Various proteins have been shown to 3D domain swap, but there has been no domain swapping report on a blue copper protein. Here, we found that azurin from Alcaligenes xylosoxidans oligomerizes by the procedure of 2,2,2-trifluoroethanol addition to Cu(i)-azurin at pH 5.0, lyophilization, and dissolution at pH 7.0, whereas it slightly oligomerizes when using Cu(ii)-azurin. The amount of high order oligomers increased with the addition of Cu(ii) ions to the dissolution process of a similar procedure for apoazurin, indicating that Cu(ii) ions enhance azurin oligomerization. The ratio of the absorbance at 460 nm to that at ∼620 nm of the azurin dimer (Abs460/Abs618 = 0.113) was higher than that of the monomer (Abs460/Abs622 = 0.067) and the EPR A‖ value of the dimer (5.85 mT) was slightly smaller than that of the monomer (5.95 mT), indicating a slightly more rhombic copper coordination for the dimer. The redox potential of the azurin dimer was 342 ± 5 mV vs. NHE, which was 50 mV higher than that of the monomer. According to X-ray crystal analysis, the azurin dimer exhibited a domain-swapped structure, where the N-terminal region containing three β-strands was exchanged between protomers. The copper coordination structure was tetrahedrally distorted in the azurin dimer, similar to that in the monomer; however, the Cu-O(Gly45) bond length was longer for the dimer (monomer, 2.46-2.59 Å; dimer, 2.98-3.25 Å). These results open the door for designing oligomers of blue copper proteins by domain swapping.
Topics: Alcaligenes; Azurin; Bacterial Proteins; Copper; Crystallography, X-Ray; Models, Molecular; Protein Conformation; Protein Domains; Protein Multimerization
PubMed: 31956880
DOI: 10.1039/c9mt00255c -
Biomedicines Sep 2021Peptides have been thoroughly studied as new therapeutic strategies for cancer treatment. In this work, we explored in vitro the anticancer potential of three novel...
Peptides have been thoroughly studied as new therapeutic strategies for cancer treatment. In this work, we explored in vitro the anticancer potential of three novel peptides derived from the C-terminal of azurin, an anticancer bacterial protein produced by . CT-p26, CT-p19 and CT-p19LC peptides were previously obtained through an in silico peptide design optimization process, CT-p19LC being the most promising as it presented higher hydrophobicity and solubility, positive total charge and, most importantly, greater propensity for anticancer activity. Therefore, in this study, through proliferation and apoptosis assays, CT-p19LC was tested in four cancer cell lines-A549, MCF-7, HeLa and HT-29-and in two non-cancer cell lines-16HBE14o- and MCF10A. Its membrane-targeting activity was further evaluated with zeta potential measurements and membrane order was assessed with the Laurdan probe. The results obtained demonstrated that CT-p19LC decreases cell viability through induction of cell death and binds to the plasma membrane of cancer cells, but not to non-cancer cells, making them less rigid. Overall, this study reveals that CT-p19LC is an auspicious selective anticancer peptide able to react with cancer cell membranes and cause effective action.
PubMed: 34572379
DOI: 10.3390/biomedicines9091194 -
Proteins Oct 2020Multicopper oxidases (MCOs) use copper ions as cofactors to oxidize a variety of substrates while reducing oxygen to water. MCOs have been identified in various taxa,...
Multicopper oxidases (MCOs) use copper ions as cofactors to oxidize a variety of substrates while reducing oxygen to water. MCOs have been identified in various taxa, with notable occurrences in fungi. The role of these fungal MCOs in lignin degradation sparked an interest due to their potential for application in biofuel production and various other industries. MCOs consist of different protein domains, which led to their classification into two-, three-, and six-domain MCOs. The previously established Laccase and Multicopper Oxidase Engineering Database (https://lcced.biocatnet.de) was updated and now includes 51 058 sequences and 229 structures of MCOs. Sequences and structures of all MCOs were systematically compared. All MCOs consist of cupredoxin-like domains. Two-domain MCOs are formed by the N- and C-terminal domain (domain N and C), while three-domain MCOs have an additional domain (M) in between, homologous to domain C. The six-domain MCOs consist of alternating domains N and C, each three times. Two standard numbering schemes were developed for the copper-binding domains N and C, which facilitated the identification of conserved positions and a comparison to previously reported results from mutagenesis studies. Two sequence motifs for the copper binding sites were identified per domain. Their modularity, depending on the placement of the T1-copper binding site, was demonstrated. Protein sequence networks showed relationships between two- and three-domain MCOs, allowing for family-specific annotation and inference of evolutionary relationships.
Topics: Amino Acid Sequence; Azurin; Binding Sites; Coenzymes; Copper; Data Mining; Databases, Protein; Evolution, Molecular; Fungal Proteins; Fungi; Models, Molecular; Oxidation-Reduction; Oxidoreductases; Oxygen; Protein Binding; Protein Engineering; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Substrate Specificity; Water
PubMed: 32447824
DOI: 10.1002/prot.25952 -
Bioprocess and Biosystems Engineering Mar 2022Azurin which is a bacterial secondary metabolite has attracted much attention as potential anticancer agent in recent years. This copper-containing periplasmic redox...
Azurin which is a bacterial secondary metabolite has attracted much attention as potential anticancer agent in recent years. This copper-containing periplasmic redox protein supresses the tumor growth selectively. High-level secretion of proteins into the culture medium offers a significant advantage over periplasmic or cytoplasmic expression. The aim of this study was to investigate the effect of nonionic surfactants on the expression of the Pseudomonas aeruginosa azurin. Different concentrations of Triton X-100 and Tween 80 were used as supplements in growth media and extracellular azurin production was stimulated by both surfactants. According to western blot analysis results, in the presence of Triton X-100, maximum azurin expression level was achieved with 96 h of incubation at 1% concentration, and 48 h at 2% concentration. On the other hand, maximum azurin expression level was achieved in the presence of 1% Tween 80 at 72 h incubation. This study suggested for the first time a high level of azurin secretion from P. aeruginosa in the presence of Triton X-100 or Tween 80, which would be advantageous for the purification procedure.
Topics: Azurin; Bacterial Proteins; Copper; Octoxynol; Polysorbates; Pseudomonas aeruginosa
PubMed: 35039942
DOI: 10.1007/s00449-021-02678-5