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Biomacromolecules Dec 2020Azulitox as a new fusion polypeptide with cancer cell specificity and phototoxicity was generated and is composed of a photosensitizer domain and the cell-penetrating...
Azulitox as a new fusion polypeptide with cancer cell specificity and phototoxicity was generated and is composed of a photosensitizer domain and the cell-penetrating peptide P28. The photosensitizer domain (EcFbFP) was derived from a bacterial blue-light receptor, which belongs to the family of light-oxygen-voltage proteins and produces reactive oxygen species (ROS) upon excitation. P28 is derived from the cupredoxin protein azurin that is known to specifically penetrate cancer cells and bind to the tumor suppressor protein p53. We show that the P28 domain specifically directs and translocates the fused photosensitizer into cancer cells. Under blue-light illumination, Azulitox significantly induced cytotoxicity. Compared to the extracellular application of EcFbFP, Azulitox caused death to about 90% of cells, as monitored by flow cytometry, which also directly correlated with the amount of ROS produced in the cells. Azulitox may open new avenues toward targeted polypeptide-photosensitizer-based photodynamic therapies with reduced systemic toxicity compared to conventional photosensitizers.
Topics: Antineoplastic Agents; Neoplasms; Peptide Fragments; Photochemotherapy; Photosensitizing Agents; Pseudomonas aeruginosa; Tumor Suppressor Protein p53
PubMed: 33140635
DOI: 10.1021/acs.biomac.0c01216 -
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 -
Journal of Magnetic Resonance (San... Jan 2023A new deep neural network based on the WaveNet architecture (WNN) is presented, which is designed to grasp specific patterns in the NMR spectra. When trained at a fixed...
A new deep neural network based on the WaveNet architecture (WNN) is presented, which is designed to grasp specific patterns in the NMR spectra. When trained at a fixed non-uniform sampling (NUS) schedule, the WNN benefits from pattern recognition of the corresponding point spread function (PSF) pattern produced by each spectral peak resulting in the highest quality and robust reconstruction of the NUS spectra as demonstrated in simulations and exemplified in this work on 2D H-N correlation spectra of three representative globular proteins with different sizes: Ubiquitin (8.6 kDa), Azurin (14 kDa), and Malt1 (44 kDa). The pattern recognition by WNN is also demonstrated for successful virtual homo-decoupling in a 2D methyl H-C - HMQC spectrum of MALT1. We demonstrate using WNN that prior knowledge about the NUS schedule, which so far was not been fully exploited, can be used for designing new powerful NMR processing techniques that surpass the existing algorithmic methods.
Topics: Magnetic Resonance Spectroscopy; Magnetic Resonance Imaging; Neural Networks, Computer; Ubiquitin; Nuclear Magnetic Resonance, Biomolecular
PubMed: 36459916
DOI: 10.1016/j.jmr.2022.107342 -
Life Sciences Jul 2023Preliminary studies have identified the use of probiotics as a potential treatment strategy against colorectal cancer (CRC). However, natural probiotics lack direct...
AIMS
Preliminary studies have identified the use of probiotics as a potential treatment strategy against colorectal cancer (CRC). However, natural probiotics lack direct tumor-targeting and tumor-killing activity in the intestine. This study aimed to construct a tumor-targeting engineered probiotic to combat CRC.
MAIN METHODS
Standard adhesion assay was performed to analyze the adherence ability of tumor-binding protein HlpA to CT26 cells. CCK-8 assay, Hoechst 33258 staining and flow cytometry analysis were used for examining cytotoxicity of tumoricidal protein azurin toward CT26 cells. An engineered probiotic Ep-AH harboring azurin and hlpA genes was developed using Escherichia coli Nissle 1917 (EcN) chassis. Antitumor effects of Ep-AH were evaluated in the azoxymethane (AOM) and dextran sodium sulfate salt (DSS)-induced CRC mice. Moreover, analysis of gut microbiota was conducted via fecal 16S rRNA gene sequencing and shotgun metagenomic sequencing.
KEY FINDINGS
Azurin caused a dose-dependent increase of apoptosis in CT26 cells. Ep-AH treatment reversed weight loss (p < 0.001), fecal occult blood (p < 0.01), and shortening of colon length (p < 0.001) than model group, as well as reducing tumorigenesis by 36 % (p < 0.001). Both Ep-H and Ep-A (EcN expressing HlpA or azurin) were less effective than Ep-AH. Furthermore, Ep-AH enriched the members of beneficial bacteria (e.g., Blautia and Bifidobacterium) and reversed abnormal changes of genes associated with several metabolic pathways (e.g., lipopolysaccharide biosynthesis).
SIGNIFICANCE
These results demonstrated that Ep-AH had excellent therapeutic benefits on cancer remission and gut microbiota modulation. Our study provides an effective strategy for anti-CRC treatment.
Topics: Animals; Mice; Gastrointestinal Microbiome; RNA, Ribosomal, 16S; Azurin; Carcinogenesis; Cell Transformation, Neoplastic; Probiotics; Colorectal Neoplasms; Escherichia coli; Dextran Sulfate; Disease Models, Animal; Colitis
PubMed: 37100380
DOI: 10.1016/j.lfs.2023.121709 -
Microbial Pathogenesis Dec 2023Pseudomonas aeruginosa is a Gram-negative bacteria and it has been demonstrated that immunization with the outer membrane proteins of the microbe produces most of the... (Review)
Review
Pseudomonas aeruginosa is a Gram-negative bacteria and it has been demonstrated that immunization with the outer membrane proteins of the microbe produces most of the relevant human antibodies. The peritrichous P. aeruginosa strain with MSHA fimbriae (PA-MSHA strain) has been found to be effective in the inhibition of growth and proliferation of different types of cancer cells. Furthermore, it has been revealed that PA-MSHA exhibits cytotoxicity because of the presence of MSHA and therefore it possesses anti-carcinogenic ability against different types of human cancer cell lines including, gastric, breast, hepatocarcinoma and nasopharyngeal cells. Studies have revealed that PA-MSHA exhibits therapeutic potential against cancer growth by induction of apoptosis, arrest of cell cycle, activating NF-κB/TLR5 pathway, etc. In China, PA-MSHA injections have been approved for the treatment of malignant tumor patients from very long back. The present review article demonstrates the therapeutic potential of PA-MSHA against various types of human cancers and explains the underlying mechanism.
Topics: Humans; Signal Transduction; Pseudomonas aeruginosa; Hemagglutinins; Mannose; Cell Proliferation; Liver Neoplasms
PubMed: 37871855
DOI: 10.1016/j.micpath.2023.106422 -
Journal of Biomolecular Structure &... Sep 2021The current pandemic SARS-CoV-2 has wreaked havoc in the world, and neither drugs nor vaccine is available for the treatment of this disease. Thus, there is an immediate...
The current pandemic SARS-CoV-2 has wreaked havoc in the world, and neither drugs nor vaccine is available for the treatment of this disease. Thus, there is an immediate need for novel therapeutics that can combat this deadly infection. In this study, we report the therapeutic assessment of azurin and its peptides: p18 and p28 against the viral structural S-protein and non-structural 3CL and PL proteins. Among the analyzed complexes, azurin docked relatively well with the S2 domain of S-protein compared to the other viral proteins. The derived peptide p18 bound to the active site domain of the PL protein; however, in other complexes, lesser interactions were recorded. The second azurin derived peptide p28, fared the best among the docked proteins. p28 interacted with all the three viral proteins and the host ACE-2 receptor by forming several electrostatic and hydrogen bonds with the S-protein, 3CL, and PL. MD simulations indicated that p28 exhibited a strong affinity to S-protein and ACE-2 receptor, indicating a possibility of p28 as a protein-protein interaction inhibitor. Our data suggest that the p28 has potential as an anti-SARS-CoV-2 agent and can be further exploited to establish its validity in the treatment of current and future SARS-CoV crisis.Communicated by Ramaswamy H. Sarma.
Topics: Azurin; Bacterial Proteins; COVID-19; Humans; Molecular Docking Simulation; Molecular Dynamics Simulation; Peptides; SARS-CoV-2
PubMed: 32619162
DOI: 10.1080/07391102.2020.1787864 -
Microbial Pathogenesis Oct 2020Anti-adhesion therapy and anti-adhesin immunity are meant to diminish the interaction between pathogens and host tissues, either by prevention or by exclusion of...
Anti-adhesion therapy and anti-adhesin immunity are meant to diminish the interaction between pathogens and host tissues, either by prevention or by exclusion of bacterial adhesion and entrance to cells. Azurin is a scaffold protein possessing antiviral, antiparasitic, and anticancer activities. The purpose of the present study was to determine the effect of recombinant Azurin (rAzurin) on the adhesion and invasion capacity of invasive (Shigella sonnei, Shigella flexneri, Campylobacter jejuni) and non-invasive (Vibrio cholerae) enteric bacteria to cells. The non-toxic dose of rAzurin and the best MOI (Multiplicity of Infection) of bacterial species was assessed by MTT assay. Bacterial species were used at MOIs of 20:1 and Azurin was applied at the concentrations of 5 and 25 μg/mL and added to Caco-2 cells in competition and replacement assay to assess the anti-adhesion and anti-invasion properties of rAzurin. The protein caused significant decrease in the adhesion rate of S. sonnei, S. flexneri, C. jejuni, and V. cholerae strains to Caco-2 cells by 43, 39, 72, and 38% in competition and 45, 46, 75, and 48% in replacement assays, respectively. Also, S. sonnei, S. flexneri, and C. jejuni strains invasion rate was reduced to 50, 50, and 70% in anti-invasion assay, respectively. The inhibitory effect of Azurin against C. jejuni and V. cholerae strains adhesion was more significant (p < .001) compared to Shigella spp. (p < .05) which may be due to smaller size of the former bacteria. On the contrary, in invasion assay, rAzurin showed a greater inhibitory effect against Shigella spp. (p < .001) compared to C. jejuni (p < .05), which may probably be due to the interaction of rAzurin with several effectors or ligands, involved in Shigella invasion and internalization. The findings of the present study opens new insights of rAzurin as a new and potent candidate for reducing or probably preventing enteric bacterial attachment, invasion, and pathogenesis.
Topics: Azurin; Bacterial Adhesion; Caco-2 Cells; Diarrhea; Humans; Recombinant Proteins; Shigella
PubMed: 32562811
DOI: 10.1016/j.micpath.2020.104246 -
The Journal of Physical Chemistry. B May 2023Metal cofactors are critical centers for different biochemical processes of metalloproteins, and often, this metal coordination renders additional structural stability....
Metal cofactors are critical centers for different biochemical processes of metalloproteins, and often, this metal coordination renders additional structural stability. In this study, we explore the additional stability conferred by the copper ion on azurin by analyzing both the and forms using temperature replica exchange molecular dynamics (REMD) data. We find a 14 K decrease in denaturation temperature for (406 K) azurin relative to that of (420 K), indicating a copper ion-induced additional thermal stability for azurin. The unfolding of azurin begins with the melting of α-helix and β-sheet V, similar to that of form. β-Sheets IV, VII, and VIII are comparatively more stable than other β-strands and melt at higher temperatures. Similar to azurin, the strong hydrophobic interactions among the apolar residues in the protein core is the key factor that renders high stability to protein as well. We construct free energy surfaces at different temperatures to capture the major conformations along the unfolding basins of the protein. Using contact maps from different basins we show the changes in the interaction between different residues along the unfolding pathway. Furthermore, we compare the Cα root-mean-square fluctuations (Cα-RMSF) and B-factor of all residues of and forms to understand the flexibility of different regions. The concerted displacement of α-helix and β-sheets V and VI from the protein core is another distinction we observe for compared to the form, where β-sheet VI was relatively stable.
Topics: Azurin; Copper; Temperature; Hot Temperature; Molecular Dynamics Simulation; Protein Denaturation; Protein Folding
PubMed: 37183371
DOI: 10.1021/acs.jpcb.3c00318 -
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 -
Journal of the American Chemical Society Mar 2022Long-range electron tunneling through metalloproteins is facilitated by evolutionary tuning of donor-acceptor electronic couplings, formal electrochemical potentials,...
Long-range electron tunneling through metalloproteins is facilitated by evolutionary tuning of donor-acceptor electronic couplings, formal electrochemical potentials, and active-site reorganization energies. Although the minimal frustration of the folding landscape enables this tuning, residual frustration in the vicinity of the metallocofactor can allow conformational fluctuations required for protein function. We show here that the constrained copper site in wild-type azurin is governed by an intricate pattern of minimally frustrated local and distant interactions that together enable rapid electron flow to and from the protein. In contrast, sluggish electron transfer reactions (unfavorable reorganization energies) of active-site azurin variants are attributable to increased frustration near to as well as distant from the copper site, along with an exaggerated oxidation-state dependence of both minimally and highly frustrated interaction patterns.
Topics: Azurin; Copper; Electron Transport; Electrons; Pseudomonas aeruginosa
PubMed: 35171591
DOI: 10.1021/jacs.1c13454