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ACS Biomaterials Science & Engineering Oct 2021As a potential anticancer agent, azurin has attracted extensive attraction among chemists, physicists, and material scientists. Its structural and unfolding/folding...
As a potential anticancer agent, azurin has attracted extensive attraction among chemists, physicists, and material scientists. Its structural and unfolding/folding information has been partially understood, but some detailed information, such as the difference in the unfolding processes between apo-azurin and holo-azurin, the mechanical stability, and the role of the copper cluster in its stability, has not been addressed adequately, especially at the single-molecule level. Here, we employed AFM-based single-molecule force spectroscopy to investigate the unfolding process of azurin in the apo and holo forms under an external force. The results indicated that the unfolding processes of apo-azurin and holo-azurin are different, and holo-azurin requires a stronger force to unfold than does apo-azurin. The copper cluster exhibited a more significant impact on the stability and the folding process of holo-azurin: the copper cluster was completely broken, and the copper ion left the unfolded azurin during the unfolding process of azurin. We suspected that the presence of the disulfide bond in azurin made the unfolding of the copper cluster different from that in pseudoazurin, which is also a type I copper protein like azurin. Rarely reported in previous studies, the mechanical strength of the Cu-N(His) bond of the copper cluster was obtained in this study, which is weaker than that of most metal-S(Cys) bonds but higher than that of the Fe-N(His) bond. Altogether, our results offer a possible new scenario for azurin to widely extend its anticancer activity.
Topics: Azurin; Copper; Metals; Protein Folding
PubMed: 34558912
DOI: 10.1021/acsbiomaterials.1c00934 -
Journal of Gastrointestinal Cancer Sep 2022To discover new natural effective anticancer agents and new antibacterial agents against antibiotic-resistant bacteria which are the most serious public health concern....
OBJECTIVES
To discover new natural effective anticancer agents and new antibacterial agents against antibiotic-resistant bacteria which are the most serious public health concern. Another important concern is drug delivery which is the transport of pharmaceutical compounds to have a therapeutic effect in organisms having a disease. Azurin is a promising anticancer agent produced from Pseudomonas aeruginosa. This study tried to test the effectiveness of the immobilization of azurin on nano-chitosan to enhance its anticancer and antibacterial activity against gastrointestinal cancer and its related bacteria.
METHODS
We purified azurin protein from Pseudomonas aeruginosa and then immobilized it on nano-chitosan. The anticancer activity of the free and nano-azurin is tested against a gastric cancer cell line (CLS-145), pancreatic cancer cell line (AsPC-1), colon cancer cell line (HCT116), esophagus cancer cell line (KYSE-410), and liver cancer cell line (HepG2). The antibacterial activity of both free and immobilized azurin also is tested against bacterial species related to the gastrointestinal cancer biopsies: Helicobacter pylori, Bacteroides fragilis, Salmonella enterica, Fusobacterium nucleatum, and Porphyromonas gingivalis.
RESULTS
Both free and nano-azurin showed high anticancer and antibacterial activity. Immobilization significantly increased the anticancer and antibacterial activity of the azurin CONCLUSION: Nano-azurin can be used as an effective anticancer and antibacterial agent against gastrointestinal cancer and bacterial species related to these cancers.
Topics: Anti-Bacterial Agents; Antineoplastic Agents; Azurin; Bacteria; Chitosan; Gastrointestinal Neoplasms; Humans; Pseudomonas aeruginosa
PubMed: 34159520
DOI: 10.1007/s12029-021-00654-6 -
Chemical Communications (Cambridge,... Nov 2021Supramolecules, which are formed by assembling multiple molecules by noncovalent intermolecular interactions instead of covalent bonds, often show additional properties... (Review)
Review
Supramolecules, which are formed by assembling multiple molecules by noncovalent intermolecular interactions instead of covalent bonds, often show additional properties that cannot be exhibited by a single molecule. Supramolecules have evolved into molecular machines in the field of chemistry, and various supramolecular proteins are responsible for life activities in the field of biology. The design and creation of supramolecular proteins will lead to development of new enzymes, functional biomaterials, drug delivery systems, etc.; thus, the number of studies on the regulation of supramolecular proteins is increasing year by year. Several methods, including disulfide bond, metal coordination, and surface-surface interaction, have been utilized to construct supramolecular proteins. In nature, proteins have been shown to form oligomers by 3D domain swapping (3D-DS), a phenomenon in which a structural region is exchanged between molecules of the same protein. We have been studying the mechanism of 3D-DS and utilizing 3D-DS to construct supramolecular metalloproteins. Cytochrome forms cyclic oligomers and polymers by 3D-DS, whereas other metalloproteins, such as various -type cytochromes and azurin form small oligomers and myoglobin forms a compact dimer. We have also utilized 3D-DS to construct heterodimers with different active sites, a protein nanocage encapsulating a Zn-SO cluster in the internal cavity, and a tetrahedron with a designed building block protein. Protein oligomer formation was controlled for the 3D-DS dimer of a dimer-monomer transition protein. This article reviews our research on supramolecular metalloproteins.
Topics: Macromolecular Substances; Metalloproteins; Models, Molecular
PubMed: 34714300
DOI: 10.1039/d1cc04608j -
Alternative Therapies in Health and... Oct 2023Osteosarcoma (OS) is the most common bone malignancy, with a high mortality rate in adolescents. Despite advancements in therapeutic interventions, OS prognosis remains...
BACKGROUND
Osteosarcoma (OS) is the most common bone malignancy, with a high mortality rate in adolescents. Despite advancements in therapeutic interventions, OS prognosis remains poor due to drug resistance. P21, a cyclin-dependent kinase inhibitor, plays a critical role in cell cycle regulation and has been implicated in OS pathogenesis. Cisplatin (DDP) is a conventional chemotherapeutic agent for OS, but its efficacy is often limited due to drug resistance. Azurin, a bacterial redox protein, has been reported to exhibit antitumor activity. However, its interaction with P21 in OS remains unexplored. In this study, we sought to investigate the impact of azurin on the cytotoxic effect of DDP against OS cells in relation to P21 expression.
METHODS
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to determine the level of p21 and apoptosis-related factors in U2OS cells. A Cell Counting Kit-8 (CCK-8) was used to examine the effects of azurin-p21 on the U2OS cell proliferation rate. Flow cytometry (FCM)was used to analyze the impact of azurin-P21 on the apoptosis/cell cycle. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the effects of azurin-P21 on the secretion of oxygen free radicals, glutathione and glutathione peroxidase.
RESULTS
Azurin exhibited significant cytotoxic activity against U2OS cells expressing wild-type (WT) P21, with minimal impact on SAOS-2 and MG63 cells lacking endogenous P21. Azurin treatment resulted in increased expression of procaspase-3 and Bax, decreased expression of B-cell lymphoma-2 (Bcl-2) and a consequential increase in apoptosis. The depletion of P21 attenuated these effects, suggesting the crucial role of P21 in azurin-mediated cytotoxicity. Furthermore, azurin synergistically enhanced the cytotoxic effect of DDP against U2OS cells, which was mitigated by P21 depletion.
CONCLUSIONS
Our findings demonstrated that azurin selectively induces apoptosis and cell cycle arrest in U2OS cells, which is mediated via P21. This study highlights the potential of azurin as a sensitizer for DDP in the treatment of OS. Future studies on DDP-resistant OS cells may further elucidate the clinical relevance of our findings.
PubMed: 37442187
DOI: No ID Found -
Proteins May 2023Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order...
Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order of the secondary structure connectivity while retaining the three-dimensional structure, making it an elegant and powerful approach to altering native topology. Previous studies elucidated the influence of CP in proteins with different folds such as Greek key β-barrel, β-sandwich, β-α-β, and all α-Greek key. CP mainly affects the protein stability and unfolding kinetics, while folding kinetics remains mostly unaltered. However, the effect of CP on metalloproteins is yet to be elaborately studied. The active site of metalloproteins poses an additional complexity in studying protein folding. Here, we investigate a CP variant (cpN42) of azurin-in both metal-free and metal-bound (holo) forms. As observed earlier in other proteins, apo-forms of wild-type (WT) and cpN42 fold with similar rates. In contrast, zinc-binding accelerates the folding of WT but decelerates the folding of cpN42. On zinc-binding, the spontaneous folding rate of WT increases by >250 times that of cpN42, which is unprecedented and the highest for any CP to date. On the other hand, zinc-binding reduces the spontaneous unfolding rate of cpN42 by ~100 times, making the WT and CP azurins unfold at similar rates. Our study demonstrates metal binding as a novel way to modulate the unfolding and folding rates of CPs compared to their WT counterparts. We hope our study increases the understanding of the effect of CP on the folding mechanism and energy landscape of metalloproteins.
Topics: Azurin; Copper; Thermodynamics; Protein Folding; Zinc; Kinetics; Protein Denaturation
PubMed: 36511110
DOI: 10.1002/prot.26454 -
International Journal of Biological... May 2023To maintain life, charge transfer processes must be efficient to allow electrons to migrate across distances as large as 30-50 Å within a timescale from picoseconds to...
To maintain life, charge transfer processes must be efficient to allow electrons to migrate across distances as large as 30-50 Å within a timescale from picoseconds to milliseconds, and the free-energy cost should not exceed one electron volt. By employing local ionization and local affinity energies, we calculated the pathway for electron and electron-hole transport, respectively. The pathway is then used to calculate both the driving force and the activation energy. The electronic coupling is calculated using configuration interaction procedure. When the charge acceptor is not known, as in oxidative stress, the charge transport terminals are found using Monte-Carlo simulation. These parameters were used to calculate the rate described by Marcus theory. Our approach has been elaborately explained using the famous androstane example and then applied to two proteins: electron transport in azurin protein and hole-hopping migration route from the heme center of cytochrome c peroxidase to its surface. This model gives an effective method to calculate the charge transport pathway and the free-energy profile within 0.1 eV from the experimental measurements and electronic coupling within 3 meV.
Topics: Electron Transport; Proteins; Azurin; Computer Simulation
PubMed: 36948333
DOI: 10.1016/j.ijbiomac.2023.124065 -
Communications Biology Jan 2023Microorganisms living at many sites in the human body compose a complex and dynamic community. Accumulating evidence suggests a significant role for microorganisms in...
Microorganisms living at many sites in the human body compose a complex and dynamic community. Accumulating evidence suggests a significant role for microorganisms in cancer, and therapies that incorporate bacteria have been tried in various types of cancer. We previously demonstrated that cupredoxin azurin secreted by the opportunistic pathogen Pseudomonas aeruginosa, enters human cancer cells and induces apoptotic death. However, the physiological interactions between P. aeruginosa and humans and their role in tumor homeostasis are largely unknown. Here, we show that P. aeruginosa upregulated azurin secretion in response to increasing numbers of and proximity to cancer cells. Conversely, cancer cells upregulated aldolase A secretion in response to increasing proximity to P. aeruginosa, which also correlated with enhanced P. aeruginosa adherence to cancer cells. Additionally, we show that cancer patients had detectable P. aeruginosa and azurin in their tumors and exhibited increased overall survival when they did, and that azurin administration reduced tumor growth in transgenic mice. Our results suggest host-bacterial symbiotic mutualism acting as a diverse adjunct to the host defense system via inter-kingdom communication mediated by the evolutionarily conserved proteins azurin and human aldolase A. This improved understanding of the symbiotic relationship of bacteria with humans indicates the potential contribution to tumor homeostasis.
Topics: Mice; Animals; Humans; Azurin; Pseudomonas aeruginosa; Fructose-Bisphosphate Aldolase; Neoplasms; Cell Physiological Phenomena
PubMed: 36609683
DOI: 10.1038/s42003-022-04395-5 -
Biochemistry and Biophysics Reports Jul 2022The thermal unfolding of the copper redox protein azurin was studied in the presence of four different dipeptide-based ionic liquids (ILs) utilizing...
The thermal unfolding of the copper redox protein azurin was studied in the presence of four different dipeptide-based ionic liquids (ILs) utilizing tetramethylguanidinium as the cation. The four dipeptides have different sequences including the amino acids Ser and Asp: TMG-AspAsp, TMG-SerSer, TMG-SerAsp, and TMG-AspSer. Thermal unfolding curves generated from temperature-dependent fluorescence spectroscopy experiments showed that TMG-AspAsp and TMG-SerSer have minor destabilizing effects on the protein while TMG-AspSer and TMG-SerAsp strongly destabilize azurin. Red-shifted fluorescence signatures in the 25 °C correlate with the observed protein destabilization in the solutions with TMG-AspSer and TMG-SerAsp. These signals could correspond to interactions between the Asp residue in the dipeptide and the azurin Trp residue in the unfolded state. These results, supported by appropriate control experiments, suggest that dipeptide sequence-specific interactions lead to selective protein destabilization and motivate further studies of TMG-dipeptide ILs.
PubMed: 35280523
DOI: 10.1016/j.bbrep.2022.101242 -
Journal of Biological Inorganic... Sep 2022A large number of copper binding proteins coordinate metal ions using a shared three-dimensional fold called the cupredoxin domain. This domain was originally identified... (Review)
Review
A large number of copper binding proteins coordinate metal ions using a shared three-dimensional fold called the cupredoxin domain. This domain was originally identified in Type 1 "blue copper" centers but has since proven to be a common domain architecture within an increasingly large and diverse group of copper binding domains. The cupredoxin fold has a number of qualities that make it ideal for coordinating Cu ions for purposes including electron transfer, enzyme catalysis, assembly of other copper sites, and copper sequestration. The structural core does not undergo major conformational changes upon metal binding, but variations within the coordination environment of the metal site confer a range of Cu-binding affinities, reduction potentials, and spectroscopic properties. Here, we discuss these proteins from a structural perspective, examining how variations within the overall cupredoxin fold and metal binding sites are linked to distinct spectroscopic properties and biological functions. Expanding far beyond the blue copper proteins, cupredoxin domains are used by a growing number of proteins and enzymes as a means of binding copper ions, with many more likely remaining to be identified.
Topics: Azurin; Binding Sites; Copper; Ions; Metals
PubMed: 35994119
DOI: 10.1007/s00775-022-01955-2 -
Journal of Peptide Science : An... Oct 2014The penetration of polar or badly soluble compounds through a cell membrane into live cells requires mechanical support or chemical helpers. Cell-penetrating peptides... (Review)
Review
The penetration of polar or badly soluble compounds through a cell membrane into live cells requires mechanical support or chemical helpers. Cell-penetrating peptides (CPPs) are very promising chemical helpers. Because of their low cytotoxicity and final degradation to amino acids, they are particularly favored in in vivo studies and for clinical applications. Clearly, the future of CPP research is bright; however, the required optimization studies for each drug require considerable individualized attention. Thus, CPPs are not the philosopher's stone. As of today, a large number of such transporter peptides with very different sequences have been identified. These have different uptake mechanisms and can transport different cargos. Intracellular concentrations of cargos can reach a low micromole range and are able to influence intracellular reactions. Internalized ribonucleic acids such as small interfering RNA (siRNA) and mimics of RNA such as peptide nucleic acids, morpholino nucleic acids, and triesters of oligonucleotides can influence transcription and translation. Despite the highly efficient internalization of antibodies, enzymes, and other protein factors, as well as siRNA and RNA mimics, the uptake and stabile insertion of DNA into the genome of the host cells remain substantially challenging. This review describes a wide array of differing CPPs, cargos, cell lines, and tissues. The application of CPPs is compared with electroporation, magnetofection, lipofection, viral vectors, dendrimers, and nanoparticles, including commercially available products. The limitations of CPPs include low cell and tissue selectivity of the first generation and the necessity for formation of fusion proteins, conjugates, or noncovalent complexes to different cargos and of cargo release from intracellular vesicles. Furthermore, the noncovalent complexes require a strong molar excess of CPPs, and extensive experimentation is required to determine the most optimal CPP for any given cargo and cell type. Yet to predict which CPP is optimal for any given target remains a complex question. More recently, there have been promising developments: the enhancement of cell specificity using activatable CPPs, specific transport into cell organelles by insertion of corresponding localization sequences, and the transport of drugs through blood-brain barriers, through the conjunctiva of eyes, skin, and into nerve cells. Proteins, siRNA, and mimics of oligonucleotides can be efficiently transported into cells and have been tested for treatment of certain diseases. The recent state of the art in CPP research is discussed together with the overall scope, limitations, and some recommendations for future research directions.
Topics: Animals; Cell-Penetrating Peptides; Drug Delivery Systems; Endocytosis; Genetic Therapy; Humans; Models, Biological; Organ Specificity; Protein Transport; Translational Research, Biomedical
PubMed: 25112216
DOI: 10.1002/psc.2672