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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 -
Journal of Biological Inorganic... Sep 2022A large number of copper binding proteins coordinate metal ions using a shared three-dimensional fold called the cupredoxin domain. This domain was originally identified... (Review)
Review
A large number of copper binding proteins coordinate metal ions using a shared three-dimensional fold called the cupredoxin domain. This domain was originally identified in Type 1 "blue copper" centers but has since proven to be a common domain architecture within an increasingly large and diverse group of copper binding domains. The cupredoxin fold has a number of qualities that make it ideal for coordinating Cu ions for purposes including electron transfer, enzyme catalysis, assembly of other copper sites, and copper sequestration. The structural core does not undergo major conformational changes upon metal binding, but variations within the coordination environment of the metal site confer a range of Cu-binding affinities, reduction potentials, and spectroscopic properties. Here, we discuss these proteins from a structural perspective, examining how variations within the overall cupredoxin fold and metal binding sites are linked to distinct spectroscopic properties and biological functions. Expanding far beyond the blue copper proteins, cupredoxin domains are used by a growing number of proteins and enzymes as a means of binding copper ions, with many more likely remaining to be identified.
Topics: Azurin; Binding Sites; Copper; Ions; Metals
PubMed: 35994119
DOI: 10.1007/s00775-022-01955-2 -
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 -
The Journal of Physical Chemistry. B Aug 2019Isotopologues are valuable vibrational probes that shift features in a vibrational spectrum while preserving the electronic structure of the molecule. We report the...
Isotopologues are valuable vibrational probes that shift features in a vibrational spectrum while preserving the electronic structure of the molecule. We report the vibrational and electronic spectra of perdeuterated tryptophan in solution (l-Trp-), as Trp48- in azurin, and as the photogenerated neutral tryptophan radical, Trp48-, in azurin. The UV resonance Raman bands of the perdeuterated closed-shell tryptophan in solution and in azurin are lower in frequency relative to the protiated counterpart. The observed decrease in frequencies of l-Trp- bands relative to l-Trp- enables the analysis of vibrational markers of other amino acids, e.g., phenylalanine, that overlap with some modes of l-Trp-. The Raman intensities vary between l-Trp- and l-Trp-; these differences likely reflect modifications in normal mode composition upon perdeuteration. Analysis of the W3, W6, and W17 modes suggests that the W3 mode retains its utility as a conformational marker; however, the H-bond markers W6 and W17 appear to be less sensitive upon perdeuteration. The neutral tryptophan radical, Trp48-, was generated in azurin with a slightly lower radical quantum yield than for Trp48-. The visible resonance Raman spectrum of Trp48- is different from that of Trp48-, especially in terms of relative intensities, and all assignable peaks decreased in frequency upon perdeuteration. The absorption and emission spectra of the perdeuterated closed-shell and radical species exhibited hypsochromic shifts of less than 1 nm relative to the protiated species. The data presented here indicate that l-Trp- is a valuable probe of vibrational structure, with minimal modification of photoreactivity and photophysics compared to l-Trp-.
Topics: Azurin; Models, Molecular; Protein Conformation; Spectrum Analysis, Raman; Tryptophan
PubMed: 31313925
DOI: 10.1021/acs.jpcb.9b04655 -
Journal of Peptide Science : An... May 2021Cell-penetrating peptides (CPPs) can transport various cargoes through membranes of live cells. Since the first generations of CPPs suffered from insufficient cell and... (Review)
Review
Cell-penetrating peptides (CPPs) can transport various cargoes through membranes of live cells. Since the first generations of CPPs suffered from insufficient cell and tissue selectivity, stability against proteases, and escape from endosomes, a new generation of peptides, with optimized properties, was developed. These are either derived from natural sources or created through the combination of multivalent structures. The second method allows achieving high internalization efficiency, high cell and tissue selectivity, and release from endosomes via hybrid structures, combining sequences for endosomal release, homing sequences, and sequences for activation at the target tissue and for local delivery of cargoes. CPPs with innate tumor selectivity include azurin, crotamine, maurocalcine, lycosin-I, buffalo cathelicidin, and peptide CB5005. Some of them can penetrate the membranes of live cells and influence intracellular signaling pathways, thereby exerting cytotoxic effects against tumor cells. To obtain multilayer penetration and stabilization against proteolytic degradation, as well as for better handling, CPPs are often conjugated to nanoparticles. A special problem for tumor treatment is the efficiency of drug transport through three-dimensional cell cultures. Therefore, the capability of CPPs to deliver the drug even to the innermost tissues is of crucial importance. Notably, the ability of certain CPPs to penetrate barriers such as skin, the blood-brain barrier (BBB), and cornea or conjunctiva of eyes enabled the replacement of dangerous and painful injections with soothing sprays, creams, and drops. However, it is difficult to rank the efficacy of CPPs because transport efficiency and tissue selectivity depend not only on the CPP itself but also on the target tissue or organ, as well as on the cargo and method of CPP-cargo coupling. Therefore, the present review describes some examples of new-generation CPPs and aims to provide advice on how to find or create the right CPP for a given task.
Topics: Animals; Antineoplastic Agents; Cell-Penetrating Peptides; Humans; Neoplasms
PubMed: 33615648
DOI: 10.1002/psc.3300 -
Inorganic Chemistry Jul 2021Metalloproteins are an important class of proteins involved in metal uptake, transport, and electron-transfer reactions. Mimicking the active sites of these proteins... (Comparative Study)
Comparative Study
Metalloproteins are an important class of proteins involved in metal uptake, transport, and electron-transfer reactions. Mimicking the active sites of these proteins through miniaturization is an active area of research with applications in biotechnology and medicine. Azurin is a 128-residue copper-binding cupredoxin protein involved in electron-transfer reactions. Previous studies have reported on the copper-binding-induced spectroscopic and structural properties of peptide loops (11 and 13 residues) from azurin. These azurin peptides exhibited novel stoichiometries. However, the underlying mechanism of fluorescence quenching upon copper binding remains to be understood, whether it is due to electron transfer, energy transfer, or both. Here, we report nickel-binding-associated spectroscopic and structural properties of the azurin peptides. They develop a β-turn upon nickel binding as seen in circular dichroism and exhibit electronic transitions centered at 270 and 450 nm. Unlike copper, which exhibited 1:1 and 1:2 peptide:metal stoichiometries, nickel exhibited only a 1:1 stoichiometry. Tryptophan-containing peptides showed fluorescence quenching upon nickel binding, which is due to electron transfer. These results further suggest that the quenching in copper-bound peptides is also due to electron transfer, which could not be ascertained in previous studies. Overall, azurin peptides provide a platform for studying metal-induced structural and spectroscopic properties using transition-metal ions.
Topics: Azurin; Binding Sites; Copper; Fluorescence; Metalloproteins; Nickel; Peptides
PubMed: 34137603
DOI: 10.1021/acs.inorgchem.1c01007 -
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 -
Biomedicines Sep 2021Peptides have been thoroughly studied as new therapeutic strategies for cancer treatment. In this work, we explored in vitro the anticancer potential of three novel...
Peptides have been thoroughly studied as new therapeutic strategies for cancer treatment. In this work, we explored in vitro the anticancer potential of three novel peptides derived from the C-terminal of azurin, an anticancer bacterial protein produced by . CT-p26, CT-p19 and CT-p19LC peptides were previously obtained through an in silico peptide design optimization process, CT-p19LC being the most promising as it presented higher hydrophobicity and solubility, positive total charge and, most importantly, greater propensity for anticancer activity. Therefore, in this study, through proliferation and apoptosis assays, CT-p19LC was tested in four cancer cell lines-A549, MCF-7, HeLa and HT-29-and in two non-cancer cell lines-16HBE14o- and MCF10A. Its membrane-targeting activity was further evaluated with zeta potential measurements and membrane order was assessed with the Laurdan probe. The results obtained demonstrated that CT-p19LC decreases cell viability through induction of cell death and binds to the plasma membrane of cancer cells, but not to non-cancer cells, making them less rigid. Overall, this study reveals that CT-p19LC is an auspicious selective anticancer peptide able to react with cancer cell membranes and cause effective action.
PubMed: 34572379
DOI: 10.3390/biomedicines9091194