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Bioengineering (Basel, Switzerland) Oct 2023Compared to chemical drugs, therapeutic proteins exhibit higher specificity and activity and are generally well-tolerated by the human body. However, the limitations,...
Compared to chemical drugs, therapeutic proteins exhibit higher specificity and activity and are generally well-tolerated by the human body. However, the limitations, such as poor stability both in vivo and in vitro as well as difficulties in penetrating cell membranes, hinder their widespread application. To overcome the challenges, a highly efficient protocol was developed and implemented for the recombinant expression of the therapeutic protein azurin and secretion into minicells derived from probiotic Nissle 1917. The novel coupled production with a delivery system of therapeutic proteins based on minicells was obtained through purification to enhance protein activity, circulation characteristics, and targeting specificity. This protein drug carrier integrates the production of carrier materials and the loading of expression proteins. The drug carrier also protects the encapsulated polypeptide drugs from enzymatic or gastric acid degradation until they are released. Nissle 1917-derived minicells have natural targeting to colon cancer cells, low toxicity, and can accumulate for a long time after penetrating tumor tissue. This self-produced protein drug delivery system simplified the process of protein preparation, and its inhibitory effect on different types of colon cancer cells was verified by CCK-8 cytotoxicity assay, cancer cell invasion, and migration assay. This work provided a simple method to prepare minicell drug delivery systems for protein drug production and a novel approach for the transport of recombinant protein drugs.
PubMed: 37892918
DOI: 10.3390/bioengineering10101188 -
Proteins Mar 2024
PubMed: 37881118
DOI: 10.1002/prot.26624 -
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 -
Proceedings of the National Academy of... Oct 2023The "Histidine-brace" (His-brace) copper-binding site, composed of Cu(His) with a backbone amine, is found in metalloproteins with diverse functions. A primary example...
The "Histidine-brace" (His-brace) copper-binding site, composed of Cu(His) with a backbone amine, is found in metalloproteins with diverse functions. A primary example is lytic polysaccharide monooxygenase (LPMO), a class of enzymes that catalyze the oxidative depolymerization of polysaccharides, providing not only an energy source for native microorganisms but also a route to more effective industrial biomass conversion. Despite its importance, how the Cu His-brace site performs this unique and challenging oxidative depolymerization reaction remains to be understood. To answer this question, we have designed a biosynthetic model of LPMO by incorporating the Cu His-brace motif into azurin, an electron transfer protein. Spectroscopic studies, including ultraviolet-visible (UV-Vis) absorption and electron paramagnetic resonance, confirm copper binding at the designed His-brace site. Moreover, the designed protein is catalytically active towards both cellulose and starch, the native substrates of LPMO, generating degraded oligosaccharides with multiturnovers by C1 oxidation. It also performs oxidative cleavage of the model substrate 4-nitrophenyl-D-glucopyranoside, achieving a turnover number ~9% of that of a native LPMO assayed under identical conditions. This work presents a rationally designed artificial metalloenzyme that acts as a structural and functional mimic of LPMO, which provides a promising system for understanding the role of the Cu His-brace site in LPMO activity and potential application in polysaccharide degradation.
Topics: Mixed Function Oxygenases; Copper; Histidine; Polysaccharides
PubMed: 37844252
DOI: 10.1073/pnas.2308286120 -
Physical Chemistry Chemical Physics :... Oct 2023Polarizability is a fundamental property of all molecular systems describing the deformation of the molecular electronic density in response to an applied electric...
Comment on "Applicability of perturbed matrix method for charge transfer studies at bio/metallic interfaces: a case of azurin" by O. Kontkanen, D. Biriukov and Z. Futera, , 2023, , 12479.
Polarizability is a fundamental property of all molecular systems describing the deformation of the molecular electronic density in response to an applied electric field. The question of whether polarizability of the active site needs to be included in theories of enzymatic activity remains open. Hybrid quantum mechanical/molecular mechanical calculations are hampered by difficulties faced by many quantum-chemistry algorithms to provide sufficiently accurate estimates of the anisotropic second-rank tensor of molecular polarizability. In this Comment, we provide general theoretical arguments for the values of polarizability of the quantum region or a molecule which have to be reproduced by electronic structure calculations.
PubMed: 37782532
DOI: 10.1039/d3cp03178k -
The Journal of Physical Chemistry. B Oct 2023In this study, we applied the concept of the "contribution factor of the first kind (CFFK)" to the original electron-transfer (ET) rate theory proposed by Marcus....
In this study, we applied the concept of the "contribution factor of the first kind (CFFK)" to the original electron-transfer (ET) rate theory proposed by Marcus. Mathematical derivations provided simple and convenient formulas for estimating the relative contributions of ten physical and chemical parameters involved in the Marcus ET rate formula: (1) the maximum strength of the electronic coupling energy between two molecules, (2) the exponential decay rate of the electronic coupling energy versus the distance between both molecules, (3) the distance between both molecules, (4) the equilibrium distance between both molecules, (5) the Gibbs free energy, (6) reorganization free energy in the prefactor of the Marcus ET rate equation, (7) reorganization free energy in the denominator of the exponential term, (8) reorganization free energy in the argument of the exponential term, (9) Boltzmann constant times absolute temperature in the prefactor of the rate equation, and (10) Boltzmann constant times absolute temperature in the denominator of the exponential term. We applied our theories to (i) ET reactions at bacterial photosynthesis reaction centers, PSI and PSII, and soluble ferredoxins (Fd); (ii) intraprotein ET reactions for designed azurin mutants; and (iii) ET reactions in flavodoxin (Fld). The formulas and calculations suggest that the theory behind the CFFK is useful for quantitatively identifying major and minor physical and chemical factors and corresponding trade-offs, all of which affect the magnitude of the Marcus ET rate.
PubMed: 37782079
DOI: 10.1021/acs.jpcb.3c03420 -
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 -
Pharmaceutics Jun 2023Azurin is a natural protein produced by that exhibits potential anti-tumor, anti-HIV, and anti-parasitic properties. The current study aimed to investigate the...
Azurin is a natural protein produced by that exhibits potential anti-tumor, anti-HIV, and anti-parasitic properties. The current study aimed to investigate the potential of azurin protein against breast cancer using both in silico and in vitro analyses. The amino acid sequence of Azurin was used to predict its secondary and tertiary structures, along with its physicochemical properties, using online software. The resulting structure was validated and confirmed using Ramachandran plots and ERRAT2. The mature azurin protein comprises 128 amino acids, and the top-ranked structure obtained from I-TASSER was shown to have a molecular weight of 14 kDa and a quality factor of 100% by ERRAT2, with 87.4% of residues in the favored region of the Ramachandran plot. Docking and simulation studies of azurin protein were conducted using HDOCK and Desmond servers, respectively. The resulting analysis revealed that Azurin docked against p53 and EphB2 receptors demonstrated maximum binding affinity, indicating its potential to cause apoptosis. The recombinant azurin gene was successfully cloned and expressed in a BL21 (DE3) strain using a pET20b expression vector under the control of the pelB ladder, followed by IPTG induction. The azurin protein was purified to high levels using affinity chromatography, yielding 70 mg/L. In vitro cytotoxicity assay was performed using MCF-7 cells, revealing the significant cytotoxicity of the azurin protein to be 105 µg/mL. These findings highlight the potential of azurin protein as an anticancer drug candidate.
PubMed: 37514012
DOI: 10.3390/pharmaceutics15071825 -
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 -
Inorganic Chemistry Jul 2023In order to investigate the effects of the secondary coordination sphere in fine-tuning redox potentials (°') of type 1 blue copper (T1Cu) in cupredoxins, we have...
In order to investigate the effects of the secondary coordination sphere in fine-tuning redox potentials (°') of type 1 blue copper (T1Cu) in cupredoxins, we have introduced M13F, M44F, and G116F mutations both individually and in combination in the secondary coordination sphere of the T1Cu center of azurin (Az) from . These variants were found to differentially influence the °' of T1Cu, with M13F Az decreasing °', M44F Az increasing °', and G116F Az showing a negligible effect. In addition, combining the M13F and M44F mutations increases °' by 26 mV relative to WT-Az, which is very close to the combined effect of °' by each mutation. Furthermore, combining G116F with either M13F or M44F mutation resulted in negative and positive cooperative effects, respectively. Crystal structures of M13F/M44F-Az, M13F/G116F-Az, and M44F/G116F-Az combined with that of G116F-Az reveal these changes arise from steric effects and fine-tuning of hydrogen bond networks around the copper-binding His117 residue. The insights gained from this study would provide another step toward the development of redox-active proteins with tunable redox properties for many biological and biotechnological applications.
Topics: Azurin; Copper; Phenylalanine; Models, Molecular; Mutation; Oxidation-Reduction; Pseudomonas aeruginosa
PubMed: 37424080
DOI: 10.1021/acs.inorgchem.3c01365