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Chemical Communications (Cambridge,... Mar 2021Fluorinated 5-hydroxytryptophans (F-5HOWs) were synthesized in gram scale quantities and incorporated into a β-hairpin peptide and the protein azurin. The redox-active...
Fluorinated 5-hydroxytryptophans (F-5HOWs) were synthesized in gram scale quantities and incorporated into a β-hairpin peptide and the protein azurin. The redox-active F-5HOWs exhibit unique radical spectroscopic signatures that expand the function of as probes for biological electron transfer.
Topics: 5-Hydroxytryptophan; Chemistry Techniques, Synthetic; Electron Transport; Halogenation; Models, Molecular; Molecular Conformation
PubMed: 33626126
DOI: 10.1039/d1cc00187f -
International Journal of Biological... Jun 2021The thermal unfolding of the copper redox protein azurin was studied in the presence of four different amino acid-based ionic liquids (ILs), all of which have...
The thermal unfolding of the copper redox protein azurin was studied in the presence of four different amino acid-based ionic liquids (ILs), all of which have tetramethylguanidium as cation. The anionic amino acid includes two with alcohol side chains, serine and threonine, and two with carboxylic acids, aspartate and glutamate. Control experiments showed that amino acids alone do not significantly change protein stability and pH changes anticipated by the amino acid nature have only minor effects on the protein. With the ILs, the protein is destabilized and the melting temperature is decreased. The two ILs with alcohol side chains strongly destabilize the protein while the two ILs with acid side chains have weaker effects. Unfolding enthalpy (ΔH) and entropy (ΔS) values, derived from fits of the unfolding data, show that some ILs increase ΔHwhile others do not significantly change this value. All ILs, however, increase ΔS. MD simulations of both the folded and unfolded protein conformations in the presence of the ILs provide insight into the different IL-protein interactions and how they affect the ΔH values. The simulations also confirm that the ILs increase the unfolded state entropies which can explain the increased ΔS values.
Topics: Amino Acids; Anions; Azurin; Cations; Entropy; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Imidazoles; Ionic Liquids; Methylguanidine; Molecular Dynamics Simulation; Protein Stability; Protein Structure, Secondary; Protein Unfolding; Transition Temperature
PubMed: 33744247
DOI: 10.1016/j.ijbiomac.2021.03.090 -
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 -
Sarcoma 2022We report a retrospective case series analysis of clinical outcomes of patients with soft tissue sarcoma around the elbow.
BACKGROUND
We report a retrospective case series analysis of clinical outcomes of patients with soft tissue sarcoma around the elbow.
METHODS
Twenty-two patients underwent surgical tumor excision between January 1999 and May 2017, with a mean follow-up of 85.2 months.
RESULTS
Six tumors were localized in the upper arm, nine in the elbow, and seven in the forearm. Sixteen tumors were deep-seated, and six were superficially located. Fifteen patients underwent wide excision, including one amputation, and 18 achieved (81.8%) negative margins histologically. Two local recurrences and four distant metastases developed. The mean Musculoskeletal Tumor Society score was 92.0% (range, 33.3-100). The 5-year local recurrence-free survival rate, metastasis-free survival rate, and overall survival rate were 90.0%, 77.0%, and 79.7%, respectively.
CONCLUSIONS
Local control and limb function can have favorable outcomes when the tumor excised has a histologically negative margin without sacrificing the major structure.
PubMed: 36573098
DOI: 10.1155/2022/1087726 -
The FEBS Journal Jan 2021Cu-containing nitrite reductases that convert NO to NO are critical enzymes in nitrogen-based energy metabolism. Among organisms in the order Rhizobiales, we have...
Cu-containing nitrite reductases that convert NO to NO are critical enzymes in nitrogen-based energy metabolism. Among organisms in the order Rhizobiales, we have identified two copies of nirK, one encoding a new class of 4-domain CuNiR that has both cytochrome and cupredoxin domains fused at the N terminus and the other, a classical 2-domain CuNiR (Br NiR). We report the first enzymatic studies of a novel 4-domain CuNiR from Bradyrhizobium sp. ORS 375 (BrNiR), its genetically engineered 3- and 2-domain variants, and Br NiR revealing up to ~ 500-fold difference in catalytic efficiency in comparison with classical 2-domain CuNiRs. Contrary to the expectation that tethering would enhance electron delivery by restricting the conformational search by having a self-contained donor-acceptor system, we demonstrate that 4-domain BrNiR utilizes N-terminal tethering for downregulating enzymatic activity instead. Both Br NiR and an engineered 2-domain variant of BrNiR (Δ(Cytc-Cup) BrNiR) have 3 to 5% NiR activity compared to the well-characterized 2-domain CuNiRs from Alcaligenes xylosoxidans (AxNiR) and Achromobacter cycloclastes (AcNiR). Structural comparison of Δ(Cytc-Cup) BrNiR and Br NiR with classical 2-domain AxNiR and AcNiR reveals structural differences of the proton transfer pathway that could be responsible for the lowering of activity. Our study provides insights into unique structural and functional characteristics of naturally occurring 4-domain CuNiR and its engineered 3- and 2-domain variants. The reverse protein engineering approach utilized here has shed light onto the broader question of the evolution of transient encounter complexes and tethered electron transfer complexes. ENZYME: Copper-containing nitrite reductase (CuNiR) (EC 1.7.2.1). DATABASE: The atomic coordinate and structure factor of Δ(Cytc-Cup) BrNiR and Br NiR have been deposited in the Protein Data Bank (http://www.rcsb.org/) under the accession code 6THE and 6THF, respectively.
Topics: Achromobacter cycloclastes; Alcaligenes; Amino Acid Sequence; Azurin; Bacterial Proteins; Bradyrhizobium; Catalytic Domain; Cloning, Molecular; Copper; Crystallography, X-Ray; Cytochromes c; Electrons; Escherichia coli; Gene Expression; Genetic Vectors; Models, Molecular; Nitrite Reductases; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Engineering; Protein Interaction Domains and Motifs; Protons; Recombinant Proteins; Reverse Genetics; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity
PubMed: 32255260
DOI: 10.1111/febs.15324 -
Molecular Diversity Aug 2021The H1N1 influenza virus causes a severe disease that affects the human respiratory tract leading to millions of deaths every year. At present, certain vaccines and few...
The H1N1 influenza virus causes a severe disease that affects the human respiratory tract leading to millions of deaths every year. At present, certain vaccines and few drugs are used to control the virus during seasonal outbreaks. However, high mutation rates and genetic reassortment make it challenging to prevent and mitigate outbreaks, leading to pandemics. Thus, alternate therapies are required for its management and control. Here, we report that a bacterial protein, azurin, and its peptide derivatives p18 and p28 target critical proteins of the influenza virus in an effective manner. The molecular docking studies show that the p28 peptide could target C-PB1, NS1-ED, PB2-CBD, PB2-RBD, NP, and PA proteins. These complexes were further subjected to the simulation of molecular dynamics and binding free energy calculations. The data indicate that p28 has an unusually high affinity and forms stable complexes with the viral proteins C-PB1, PB2-CBD, PB2-RBD, and NP. We suggest that the azurin derivative p28 peptide can act as an anti-influenza agent as it can bind to multiple targets and neutralize the virus. Additional experimental studies need to be conducted to evaluate its safety and efficacy as an anti-H1N1 molecule.
Topics: Antiviral Agents; Azurin; Binding Sites; Catalytic Domain; Drug Discovery; Influenza A Virus, H1N1 Subtype; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Peptide Fragments; Protein Binding; Structure-Activity Relationship; Viral Proteins
PubMed: 33575983
DOI: 10.1007/s11030-021-10193-8 -
Cell Reports. Medicine May 2024Bacteria-based therapies are powerful strategies for cancer therapy, yet their clinical application is limited by a lack of tunable genetic switches to safely regulate...
Bacteria-based therapies are powerful strategies for cancer therapy, yet their clinical application is limited by a lack of tunable genetic switches to safely regulate the local expression and release of therapeutic cargoes. Rapid advances in remote-control technologies have enabled precise control of biological processes in time and space. We developed therapeutically active engineered bacteria mediated by a sono-activatable integrated gene circuit based on the thermosensitive transcriptional repressor TlpA. Through promoter engineering and ribosome binding site screening, we achieved ultrasound (US)-induced protein expression and secretion in engineered bacteria with minimal noise and high induction efficiency. Specifically, delivered either intratumorally or intravenously, engineered bacteria colonizing tumors suppressed tumor growth through US-irradiation-induced release of the apoptotic protein azurin and an immune checkpoint inhibitor, a nanobody targeting programmed death-ligand 1, in different tumor mouse models. Beyond developing safe and high-performance designer bacteria for tumor therapy, our study illustrates a sonogenetics-controlled therapeutic platform that can be harnessed for bacteria-based precision medicine.
Topics: Animals; Mice; Humans; Neoplasms; Disease Models, Animal; Cell Line, Tumor; Female; B7-H1 Antigen; Immune Checkpoint Inhibitors; Escherichia coli
PubMed: 38608697
DOI: 10.1016/j.xcrm.2024.101513 -
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 -
The Journal of Physical Chemistry. B Apr 2021apoazurin (apo, without the copper cofactor) has a single disulfide bond between residues 3 and 26 and unfolds in a two-state reaction . The disulfide bond covalently...
apoazurin (apo, without the copper cofactor) has a single disulfide bond between residues 3 and 26 and unfolds in a two-state reaction . The disulfide bond covalently connects the N-termini of β-strands 1 and 3; thereby, it creates a zero-order loop or a "cinch" that restricts conformational space. Covalent loops and threaded topologies are emerging as important structural elements in folded proteins and may be important for function. In order to understand the role of a zero-order loop in the folding process of a protein, here we used coarse-grained molecular dynamics (CGMD) simulations to compare two variants of apoazurin: one named "loop" which contained the disulfide, and another named "open" in which the disulfide bond between residues 3 and 26 was removed. CGMD simulations were performed to probe the stability and unfolding pathway of the two apoazurin variants at different urea concentrations and temperatures. Our results show that the covalent loop plays a prominent role in the unfolding mechanism of apoazurin; its removal alters both the folding-transition state and the unfolded-state ensemble of conformations. We propose that modulation of azurin's folding landscape by the disulfide bridge may be related to both copper capturing and redox sensing.
Topics: Apoproteins; Azurin; Kinetics; Protein Conformation; Protein Folding
PubMed: 33818090
DOI: 10.1021/acs.jpcb.1c00219 -
Nature Communications Feb 2021The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but...
The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.
Topics: Animals; Bacterial Proteins; Carrier Proteins; Cell Death; Complement System Proteins; Humans; Mice; Microbial Viability; Mixed Function Oxygenases; Oxidation-Reduction; Polysaccharides; Protein Domains; Proteome; Proteomics; Pseudomonas Infections; Pseudomonas aeruginosa; Substrate Specificity; Transcription, Genetic; Virulence; Virulence Factors
PubMed: 33623002
DOI: 10.1038/s41467-021-21473-0