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Iranian Journal of Basic Medical... 2023Colon cancer is well-known as a life-threatening disease. Since the current treatment modalities for this type of cancer are powerful yet face some limitations, finding...
OBJECTIVES
Colon cancer is well-known as a life-threatening disease. Since the current treatment modalities for this type of cancer are powerful yet face some limitations, finding novel treatments is required to achieve better outcomes with fewer side effects. Here we investigated the therapeutic potential of Azurin-p28 alone or along with iRGD (Ac-CRGDKGPDC-amide) as a tumor-penetrating peptide and 5-fluorouracil (5-FU) for colon cancer.
MATERIALS AND METHODS
Inhibitory effect of p28 with or without iRGD/5-FU was studied in CT26 and HT29, as well as the xenograft animal model of cancer. The effect of p28 alone or along with iRGD/5-FU on cell migration, apoptotic activity, and cell cycle of the cell lines was assessed. Level of the BAX and BCL2 genes, tumor suppressor genes [(p53 and collagen type-Iα1 (COL1A1), collagen type-Iα2 (COL1A2)] were assessed by quantitative RT-PCR.
RESULTS
These findings show that using p28 with or without iRGD and 5-FU raised the level of p53 and BAX but decreased BCL2, compared with control and 5-FU groups in tissues of the tumor, which result in raising the apoptosis.
CONCLUSION
It seems that p28 may be used as a new therapeutic approach in colon cancer therapy that can enhance the anti-tumor effect of 5-FU.
PubMed: 37396945
DOI: 10.22038/IJBMS.2023.68331.14913 -
Molecular Biotechnology Jun 2024Transfection efficiency of the immortalized human breast epithelial cell line MCF-10A remains an issue that needs to be resolved. In this study, it was aimed to deliver...
Transfection efficiency of the immortalized human breast epithelial cell line MCF-10A remains an issue that needs to be resolved. In this study, it was aimed to deliver a recombinant DNA (pCMV-Azu-GFP) to the MCF-10A cells by the magnetofection method using magnetic nanoparticles (MNPs) and a simple magnet to accelerate the DNA delivery. Surface positively modified silica-coated iron oxide MNPs (MSNP-NH) were produced and characterized via TEM, FTIR, and DLS analyses. The recombinant DNA (rDNA) was obtained by the integration of codon-optimized azurin to produce a fusion protein. Then, rDNA cloned in Escherichia coli cells was validated by sequence analysis. The electrostatically conjugated rDNA on MSNP-NH with an enhancer polyethyleneimine (PEI) was studied by agarose gel electrophoresis and the optimum conditions were determined to apply to the cell. A dose-dependent statistical difference was observed on treated cells based on the MTS test. The expression of the fusion protein after magnetofection was determined using laser scanning confocal microscope imaging and western blot analysis. It was observed that the azurin gene could be transferred to MCF-10A cells by magnetofection. Thus, when the azurin gene is used as a breast cancer treatment agent, it can be expressed in healthy cells without toxic effects.
Topics: Female; Humans; Azurin; Cell Line, Tumor; Codon; DNA, Ribosomal; Escherichia coli; Magnetic Iron Oxide Nanoparticles; Magnetite Nanoparticles; Polyethyleneimine; Recombinant Fusion Proteins; Transfection
PubMed: 37378861
DOI: 10.1007/s12033-023-00798-9 -
Journal of Inorganic Biochemistry Sep 2023The rational structural and computational studies of a blue copper protein, pseudoazurin (PAz), and its Met16X (X = Phe, Leu, Val, Ile) variants gave clear functional...
The rational structural and computational studies of a blue copper protein, pseudoazurin (PAz), and its Met16X (X = Phe, Leu, Val, Ile) variants gave clear functional meanings of the noncovalent interaction (NCI) through the second coordination sphere. The high-resolution X-ray crystal structures of Met16X PAz demonstrated that the active site geometry is significantly affected by the substitution of Met16, which is located within the NCI distance from the His81 imidazole ring at the copper active site. The computational chemistry calculations based on the crystal structure analyses confirmed that the NCI of S-π/CH-π (wild-type), π-π (Met16Phe), double CH-π (Met16Leu), and single CH-π (Met16Val and Met16Ile). The estimated interaction energies for the NCI demonstrated that the fine-tuning of the protein stability and Cu site properties form the second coordination sphere of PAz.
Topics: Copper; Models, Molecular; Azurin; Catalytic Domain; Crystallography, X-Ray
PubMed: 37354604
DOI: 10.1016/j.jinorgbio.2023.112292 -
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 -
ACS Omega Jun 2023Redox-active amino acid residues are at the heart of biological electron-transfer reactions. They play important roles in natural protein functions and are implicated in...
Redox-active amino acid residues are at the heart of biological electron-transfer reactions. They play important roles in natural protein functions and are implicated in disease states (e.g., oxidative-stress-associated disorders). Tryptophan (Trp) is one such redox-active amino acid residue, and it has long been known to serve a functional role in proteins. Broadly speaking, there is still much to learn about the local features that make some Trp redox active and others inactive. Herein, we describe a new protein model system where we investigate how a methionine (Met) residue proximal to a redox-active Trp affects its reactivity and spectroscopy. We use an artificial variant of azurin from to produce these models. We employ a series of UV-visible spectroscopy, electrochemistry, electron paramagnetic resonance, and density functional theory experiments to demonstrate the effect that placing Met near Trp radicals has in the context of redox proteins. The introduction of Met proximal to Trp lowers its reduction potential by ca. 30 mV and causes clear shifts in the optical spectra of the corresponding radicals. While the effect may be small, it is significant enough to be a way for natural systems to tune Trp reactivity.
PubMed: 37305310
DOI: 10.1021/acsomega.3c01589 -
Preventing Chronic Disease Jun 2023A transformative change grounded in a commitment to antiracism and racial and health equity is underway at the University of California, Berkeley, School of Public...
A transformative change grounded in a commitment to antiracism and racial and health equity is underway at the University of California, Berkeley, School of Public Health. Responding to a confluence of national, state, and local circumstances, bold leadership, and a moral and disciplinary imperative to name and address racism as a root cause of health inequities, our community united around a common vision of becoming an antiracist institution. Berkeley Public Health has a long history of efforts supporting diversity, equity, inclusion, belonging, and justice. Building upon those efforts, we pursued an institution-wide initiative, one that creates a more equitable and inclusive school of public health that models and supports the development of future public health leaders, practitioners, scholars, and educators. Grounded in the principles of cultural humility, we recognized that our vision was a journey, not a destination. This article describes our efforts from June 2020 through June 2022 in developing and implementing ARC4JSTC (Anti-racist Community for Justice and Social Transformative Change), a comprehensive, multiyear antiracist change initiative encompassing faculty and workforce development, student experience, curriculum and pedagogy, community engagement outreach, and business processes. Our work is data informed, grounded in principles of change management, and focused on building internal capacity to promote long-term change. Our discussion of lessons learned and next steps helps to inform our ongoing work and antiracist institutional change efforts at other schools and programs of public health.
Topics: Humans; Public Health; Racism; Curriculum; Health Equity; Social Justice
PubMed: 37290006
DOI: 10.5888/pcd20.220370 -
Infection, Genetics and Evolution :... Aug 2023Gonorrhea is an urgent antimicrobial resistance threat and its therapeutic options are continuously getting restricted. Moreover, no vaccine has been approved against it...
Reverse vaccinology approaches to introduce promising immunogenic and drug targets against antibiotic-resistant Neisseria gonorrhoeae: Thinking outside the box in current prevention and treatment.
Gonorrhea is an urgent antimicrobial resistance threat and its therapeutic options are continuously getting restricted. Moreover, no vaccine has been approved against it so far. Hence, the present study aimed to introduce novel immunogenic and drug targets against antibiotic-resistant Neisseria gonorrhoeae strains. In the first step, the core proteins of 79 complete genomes of N. gonorrhoeae were retrieved. Next, the surface-exposed proteins were evaluated from different aspects such as antigenicity, allergenicity, conservancy, and B-cell and T-cell epitopes to introduce promising immunogenic candidates. Then, the interactions with human Toll-like receptors (TLR-1, 2, and 4), and immunoreactivity to elicit humoral and cellular immune responses were simulated. On the other hand, to identify novel broad-spectrum drug targets, the cytoplasmic and essential proteins were detected. Then, the N. gonorrhoeae metabolome-specific proteins were compared to the drug targets of the DrugBank, and novel drug targets were retrieved. Finally, the protein data bank (PDB) file availability and prevalence among the ESKAPE group and common sexually transmitted infection (STI) agents were assessed. Our analyses resulted in the recognition of ten novel and putative immunogenic targets including murein transglycosylase A, PBP1A, Opa, NlpD, Azurin, MtrE, RmpM, LptD, NspA, and TamA. Moreover, four potential and broad-spectrum drug targets were identified including UMP kinase, GlyQ, HU family DNA-binding protein, and IF-1. Some of the shortlisted immunogenic and drug targets have confirmed roles in adhesion, immune evasion, and antibiotic resistance that can induce bactericidal antibodies. Other immunogenic and drug targets might be associated with the virulence of N. gonorrhoeae as well. Thus, further experimental studies and site-directed mutations are recommended to investigate the role of potential vaccine and drug targets in the pathogenesis of N. gonorrhoeae. It seems that the efforts for proposing novel vaccines and drug targets appear to be paving the way for a prevention-treatment strategy against this bacterium. Additionally, a combination of bactericidal monoclonal antibodies and antibiotics is a promising approach to curing N. gonorrhoeae.
Topics: Humans; Neisseria gonorrhoeae; Anti-Bacterial Agents; Vaccinology; Gonorrhea; Membrane Proteins
PubMed: 37225067
DOI: 10.1016/j.meegid.2023.105449 -
Biomolecules Apr 2023Due to the similarity in the basic coordination behavior of their mono-charged cations, silver biochemistry is known to be linked to that of copper in biological...
Due to the similarity in the basic coordination behavior of their mono-charged cations, silver biochemistry is known to be linked to that of copper in biological systems. Still, Cu/ is an essential micronutrient in many organisms, while no known biological process requires silver. In human cells, copper regulation and trafficking is strictly controlled by complex systems including many cytosolic copper chaperones, whereas some bacteria exploit the so-called "blue copper" proteins. Therefore, evaluating the controlling factors of the competition between these two metal cations is of enormous interest. By employing the tools of computational chemistry, we aim to delineate the extent to which Ag might be able to compete with the endogenous copper in its Type I (T1Cu) proteins, and where and if, alternatively, it is handled uniquely. The effect of the surrounding media (dielectric constant) and the type, number, and composition of amino acid residues are taken into account when modelling the reactions in the present study. The obtained results clearly indicate the susceptibility of the T1Cu proteins to a silver attack due to the favorable composition and geometry of the metal-binding centers, along with the similarity between the Ag/Cu-containing structures. Furthermore, by exploring intriguing questions of both metals' coordination chemistry, an important background for understanding the metabolism and biotransformation of silver in organisms is provided.
Topics: Humans; Copper; Silver
PubMed: 37189429
DOI: 10.3390/biom13040681 -
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 -
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