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Frontiers in Oncology 2020Cancer remains a major cause of morbidity and mortality irrespective of the type of conventional chemotherapy. Therefore, there is an urgent need for new and effective... (Review)
Review
Cancer remains a major cause of morbidity and mortality irrespective of the type of conventional chemotherapy. Therefore, there is an urgent need for new and effective anticancer therapeutic agents. Bacterial proteins and their derivative peptides appear as a promising approach for cancer treatment. Several, including an amphipathic, α-helical, 28-amino acid peptide derived from azurin, a 128-amino acid copper-containing redox protein secreted from , show clinical promise in the treatment of adult and pediatric solid tumors. The peptide, p28, is a post-translational, multi-target anticancer agent that preferentially enters a wide variety of solid tumor cells. Mechanistically, after entry, p28 has two major avenues of action. It binds to both wild-type and mutant p53 protein, inhibiting constitutional morphogenic protein 1 (Cop1)-mediated ubiquitination and proteasomal degradation of p53. This results in increased levels of p53, which induce cell-cycle arrest at G2/M and an eventual apoptosis that results in tumor cell shrinkage and death. In addition, p28 also preferentially enters nascent endothelial cells and decreases the phosphorylation of FAK and Akt inhibiting endothelial cell motility and migration. Here, we review the current basic and clinical evidence suggesting the potential of p28 as a cancer therapeutic peptide.
PubMed: 32850408
DOI: 10.3389/fonc.2020.01303 -
Journal of the American Chemical Society Sep 2023Much progress has been made in understanding the roles of the secondary coordination sphere (SCS) in tuning redox potentials of metalloproteins. In contrast, the impact...
Much progress has been made in understanding the roles of the secondary coordination sphere (SCS) in tuning redox potentials of metalloproteins. In contrast, the impact of SCS on reactivity is much less understood. A primary example is how copper proteins can promote -nitrosylation (SNO), which is one of the most important dynamic post-translational modifications, and is crucial in regulating nitric oxide storage and transportation. Specifically, the factors that instill Cu with -nitrosylating capabilities and modulate activity are not well understood. To address this issue, we investigated the influence of the primary and secondary coordination sphere on Cu-catalyzed -nitrosylation by developing a series of azurin variants with varying catalytic capabilities. We have employed a multidimensional approach involving electronic absorption, S and Cu K-edge XAS, EPR, and resonance Raman spectroscopies together with QM/MM computational analysis to examine the relationships between structure and molecular mechanism in this reaction. Our findings have revealed that kinetic competency is correlated with three balancing factors, namely Cu-S bond strength, Cu spin localization, and relative S(p) vs S(p) contributions to the ground state. Together, these results support a reaction pathway that proceeds through the attack of the Cu-S bond rather than electrophilic addition to Cu or radical attack of S. The insights gained from this work provide not only a deeper understanding of SNO in biology but also a basis for designing artificial and tunable SNO enzymes to regulate NO and prevent diseases due to SNO dysregulation.
Topics: Azurin; Copper; Metalloproteins; Catalysis; Electronics
PubMed: 37696009
DOI: 10.1021/jacs.3c07399 -
Microorganisms Dec 2021Azurin is a bacterial-derived cupredoxin, which is mainly involved in electron transport reactions. Interest in azurin protein has risen in recent years due to its...
Azurin is a bacterial-derived cupredoxin, which is mainly involved in electron transport reactions. Interest in azurin protein has risen in recent years due to its anticancer activity and its possible applications in anticancer therapies. Nevertheless, the attention of the scientific community only focused on the azurin protein found in (, ). In this work, we performed the first comprehensive screening of all the bacterial genomes available in online repositories to assess azurin distribution in the three domains of life. The Azurin coding gene was not detected in the domains Archaea and Eucarya, whereas it was detected in phyla other than , such as , and and a phylogenetic analysis of the retrieved sequences was performed. Observed patchy distribution and phylogenetic data suggest that once it appeared in the bacterial domain, the azurin coding gene was lost in several bacterial phyla and/or anciently horizontally transferred between different phyla, even though a vertical inheritance appeared to be the major force driving the transmission of this gene. Interestingly, a shared conserved domain has been found among azurin members of all the investigated phyla. This domain is already known in as p28 domain and its importance for azurin anticancer activity has been widely explored. These findings may open a new and intriguing perspective in deciphering the azurin anticancer mechanisms and to develop new tools for treating cancer diseases.
PubMed: 35056457
DOI: 10.3390/microorganisms10010009 -
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 -
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 -
Proceedings of the National Academy of... Mar 2021Hole hopping through tryptophan/tyrosine chains enables rapid unidirectional charge transport over long distances. We have elucidated structural and dynamical factors...
Hole hopping through tryptophan/tyrosine chains enables rapid unidirectional charge transport over long distances. We have elucidated structural and dynamical factors controlling hopping speed and efficiency in two modified azurin constructs that include a rhenium(I) sensitizer, Re(His)(CO)(dmp), and one or two tryptophans (W, W). Experimental kinetics investigations showed that the two closely spaced (3 to 4 Å) intervening tryptophans dramatically accelerated long-range electron transfer (ET) from Cu to the photoexcited sensitizer. In our theoretical work, we found that time-dependent density-functional theory (TDDFT) quantum mechanics/molecular mechanics/molecular dynamics (QM/MM/MD) trajectories of low-lying triplet excited states of Re(His)(CO)(dmp)-W(-W) exhibited crossings between sensitizer-localized (*Re) and charge-separated [Re(His)(CO)(dmp)/(W or W)] (CS1 or CS2) states. Our analysis revealed that the distances, angles, and mutual orientations of ET-active cofactors fluctuate in a relatively narrow range in which the cofactors are strongly coupled, enabling adiabatic ET. Water-dominated electrostatic field fluctuations bring *Re and CS1 states to a crossing where *Re(CO)(dmp)←W ET occurs, and CS1 becomes the lowest triplet state. ET is promoted by solvation dynamics around *Re(CO)(dmp)(W); and CS1 is stabilized by Re(dmp)/W electron/hole interaction and enhanced W solvation. The second hop, W←W, is facilitated by water fluctuations near the W/W unit, taking place when the electrostatic potential at W drops well below that at W Insufficient solvation and reorganization around W make W←W ET endergonic, shifting the equilibrium toward W and decreasing the charge-separation yield. We suggest that multiscale TDDFT/MM/MD is a suitable technique to model the simultaneous evolution of photogenerated excited-state manifolds.
Topics: Azurin; Electron Transport; Electrons; Molecular Dynamics Simulation; Oxidation-Reduction; Photochemistry; Pseudomonas aeruginosa; Quantum Theory; Rhenium; Static Electricity; Tryptophan; Water
PubMed: 33836608
DOI: 10.1073/pnas.2024627118 -
Bioinformation 2022Oral cancer is becoming more common, and it threatens to be a serious worldwide medical issue. Hence, it is of interest to elucidate the networks between proteins and...
Oral cancer is becoming more common, and it threatens to be a serious worldwide medical issue. Hence, it is of interest to elucidate the networks between proteins and biologically active compounds, as well as their functional annotations, and cell signaling pathways. The online STRING software was used to create a molecular genetics interaction network named AZURIN on oral bacterial proteins. We also used the cystoscope software to identify 11 nodes and 16 edges with an average node order of 2.91. Thus, we document data on the interaction of protein networks with other proteins for identifying potential therapeutic drug candidates linked to oral disease.
PubMed: 37323560
DOI: 10.6026/97320630018724 -
Sensors (Basel, Switzerland) Dec 2020The tumor suppressor p53 protein plays a crucial role in many biological processes. The presence of abnormal concentrations of wild-type p53, or some of its mutants, can... (Review)
Review
The tumor suppressor p53 protein plays a crucial role in many biological processes. The presence of abnormal concentrations of wild-type p53, or some of its mutants, can be indicative of a pathological cancer state. p53 represents therefore a valuable biomarker for tumor screening approaches and development of suitable biosensors for its detection deserves a high interest in early diagnostics. Here, we revisit our experimental approaches, combining Surface Enhanced Raman Spectroscopy (SERS) and nanotechnological materials, for ultrasensitive detection of wild-type and mutated p53, in the perspective to develop biosensors to be used in clinical diagnostics. The Raman marker is provided by a small molecule (4-ATP) acting as a bridge between gold nanoparticles (NPs) and a protein biomolecule. The Azurin copper protein and specific antibodies of p53 were used as a capture element for p53 (wild-type and its mutants). The developed approaches allowed us to reach a detection level of p53 down to 10 M in both buffer and serum. The implementation of the method in a biosensor device, together with some possible developments are discussed.
Topics: Gold; Humans; Metal Nanoparticles; Neoplasms; Spectrum Analysis, Raman; Tumor Suppressor Protein p53
PubMed: 33327383
DOI: 10.3390/s20247153 -
Saudi Journal of Biological Sciences Sep 2020Azurin protein of is an anti-tumor agent against breast cancer and mammaglobin-A (MAM-A) protein is a specific antigen on the surface of MCF-7 for induction of cellular...
Azurin protein of is an anti-tumor agent against breast cancer and mammaglobin-A (MAM-A) protein is a specific antigen on the surface of MCF-7 for induction of cellular immune. The purpose of the present study was to investigate the effects of simultaneous expression of and human genes on the mRNA expression level of apoptosis-related and cell cycle genes in MCF-7 breast cancer cell line. The recombinant or empty plasmids were separately transferred into MCF-7 cells using Lipofectamine reagent. Flow cytometry was done to detect cell death and apoptosis. The expression of genes were evaluated by IF assay, RT-PCR and western blot methods. Finally, apoptosis-related and cell cycle genes expression was examined in transformed and non-transformed MCF-7 cells by qPCR method. The successful expression of and genes in the MCF-7 cell were confirmed by RT-PCR, IF and western blotting. The apoptosis assay was showed a statistically significant ( < 0.05) difference after transfection. The expression of , , and genes in transformed cells compare with non-transformed and transformed MCF-7 by pBudCE4.1 were increased statistically significant ( < 0.05) increases. Although, the increase of and expressions in transformed cells were not statistically significant ( > 0.05). Co-expression of and genes could induce apoptosis and necrosis in human MCF-7 breast cancer cells by up-regulation of , and genes. In future researches, it must be better the immune stimulation of pBudCE4.1-azurin-MAM-A recombinant vector in animal models and therapeutic approaches will be evaluated.
PubMed: 32884412
DOI: 10.1016/j.sjbs.2020.04.007