Cross-talk between cancer and Pseudomonas aeruginosa mediates tumor suppression.

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 death1-4. 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.

Microbial Azurin Immobilized on Nano-Chitosan as Anticancer and Antibacterial Agent Against Gastrointestinal Cancers and Related Bacteria.

OBJECTIVES: To discover new natural effective anticancer agents and new antibacterial agents against antibiotic-resistant bacteria which are the most serious public health concern. Another important concern is drug delivery which is the transport of pharmaceutical compounds to have a therapeutic effect in organisms having a disease. Azurin is a promising anticancer agent produced from Pseudomonas aeruginosa. This study tried to test the effectiveness of the immobilization of azurin on nano-chitosan to enhance its anticancer and antibacterial activity against gastrointestinal cancer and its related bacteria. METHODS: We purified azurin protein from Pseudomonas aeruginosa and then immobilized it on nano-chitosan. The anticancer activity of the free and nano-azurin is tested against a gastric cancer cell line (CLS-145), pancreatic cancer cell line (AsPC-1), colon cancer cell line (HCT116), esophagus cancer cell line (KYSE-410), and liver cancer cell line (HepG2). The antibacterial activity of both free and immobilized azurin also is tested against bacterial species related to the gastrointestinal cancer biopsies: Helicobacter pylori, Bacteroides fragilis, Salmonella enterica, Fusobacterium nucleatum, and Porphyromonas gingivalis. RESULTS: Both free and nano-azurin showed high anticancer and antibacterial activity. Immobilization significantly increased the anticancer and antibacterial activity of the azurin CONCLUSION: Nano-azurin can be used as an effective anticancer and antibacterial agent against gastrointestinal cancer and bacterial species related to these cancers.

Therapeutic p28 peptide targets essential H1N1 influenza virus proteins: insights from docking and molecular dynamics simulations.

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.

The anti-adhesive and anti-invasive effects of recombinant azurin on the interaction between enteric pathogens (invasive/non-invasive) and Caco-2 cells.

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.

Influence of Heterologously Expressed azurin from Pseudomonas aeruginosa on the Adhesion and Invasion of Pathogenic Bacteria to the Caco-2 Cell Line.

This study proposed to investigate the effect of azurin on the major stages of pathogenesis (adhesion and invasion) of intestinal bacterial pathogens (Salmonella spp. and Escherichia coli) and epithelial pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) on the human colorectal adenocarcinoma (Caco-2) cell line. Azurin protein was produced by cloning the azurin gene into pET21a and heterologous expression in E. coli BL21. The protein was purified using affinity chromatography and confirmed by Western blotting. The purified protein was evaluated by three experiments of adhesion and invasion assays, including exclusion, competition, and replacement. Azurin was observed to significantly inhibit the attachment and invasion of S. aureus, Salmonella spp., and E. coli, while no such inhibitory effects were observed on P. aeruginosa. In fact, the protein increased the adhesion of P. aeruginosa to the cell. In conclusion, our study proposes that azurin is a potential prophylactic or preventive helper candidate to inhibit the attachment and invasion of pathogenic bacteria to host cells and reduce the progression of the infection process. Our study also reveals the involvement of azurin in bacteria-host cell interactions, providing novel and important insights toward the elucidation of its biological function in this field. Thus, this study provides new opportunities to use azurin as an adjunct therapy against critical stages of infection by a wide range of pathogenic bacteria.

Solochrome cyanine: A histological stain for cobalt-chromium wear particles in metal-on-metal periprosthetic tissues.

Metal-on-metal (MoM) hip arthroplasties produce abundant implant-derived wear debris composed mainly of cobalt (Co) and chromium (Cr). Cobalt-chromium (Co-Cr) wear particles are difficult to identify histologically and need to be distinguished from other wear particle types and endogenous components (e.g., haemosiderin, fibrin) which may be present in MoM periprosthetic tissues. In this study we sought to determine whether histological stains that have an affinity for metals are useful in identifying Co-Cr wear debris in MoM periprosthetic tissues. Histological sections of periprosthetic tissue from 30 failed MoM hip arthroplasties were stained with haematoxylin-eosin (HE), Solochrome Cyanine (SC), Solochrome Azurine (SA) and Perls' Prussian Blue (PB). Sections of periprosthetic tissue from 10 cases of non-MoM arthroplasties using other implant biomaterials, including titanium, ceramic, polymethylmethacrylate (PMMA) and ultra-high molecular weight polyethylene (UHMWP) were similarly analysed. Sections of 10 cases of haemosiderin-containing knee tenosynovial giant cell tumour (TSGCT) were also stained with HE, SC, SA and PB. In MoM periprosthetic tissues, SC stained metal debris in phagocytic macrophages and in the superficial necrotic zone which exhibited little or no trichrome staining for fibrin. In non-MoM periprosthetic tissues, UHMWP, PMMA, ceramic and titanium particles were not stained by SC. Prussian Blue, but not SC or SA, stained haemosiderin deposits in MoM periprosthetic tissues and TSGT. Our findings show that SC staining (most likely Cr-associated) is useful in distinguishing Co-Cr wear particles from other metal/non-metal wear particles types in histological preparations of periprosthetic tissue and that SC reliably distinguishes haemosiderin from Co-Cr wear debris.

Heterologous expression of azurin from Pseudomonas aeruginosa in food-grade Lactococcus lactis.

In this study, azurin, a bacteriocin with anticancer property, was produced by food-grade Lactococcus lactis using the Nisin Controlled Gene Expression (NICE) System. In addition, the antibacterial and cytotoxic properties of recombinant azurin in the culture supernatant were also investigated. Azurin gene from Pseudomonas aeruginosa was cloned into the pNZ8149 vector and the resulting recombinant DNA was transformed into food grade L. lactis NZ3900. The expression of azurin protein was induced by the optimum concentration of nisin for 3 h. Inhibition zones for Escherichia coli and Bacillus cereus were observed at 5.0 and 10 mg/mL concentrations of lyophilized supernatants containing azurin, but no inhibition zone at azurin-free lyophilized supernatants. When HUVEC, HT29, HCT116, and MCF7 cell lines were treated with lyophilized culture supernatants with azurin or without azurin, cell viability decreased with increasing concentrations of the supernatant. Furthermore, the supernatants containing azurin showed more anti-proliferative effect than the azurin-free supernatants. This work provides a practicable method to produce recombinant azurin in the food grade L. lactis strain. As a result, the recombinant L. lactis strain, producing azurin, can be used in the investigation of food biopreservatives and in the development of a therapeutic probiotic.

Bacteriocins: perspective for the development of novel anticancer drugs.

Antimicrobial peptides (AMPs) from prokaryotic source also known as bacteriocins are ribosomally synthesized by bacteria belonging to different eubacterial taxonomic branches. Most of these AMPs are low molecular weight cationic membrane active peptides that disrupt membrane by forming pores in target cell membranes resulting in cell death. While these peptides known to exhibit broad-spectrum antimicrobial activity, including antibacterial and antifungal, they displayed minimal cytotoxicity to the host cells. Their antimicrobial efficacy has been demonstrated in vivo using diverse animal infection models. Therefore, we have discussed some of the promising peptides for their ability towards potential therapeutic applications. Further, some of these bacteriocins have also been reported to exhibit significant biological activity against various types of cancer cells in different experimental studies. In fact, differential cytotoxicity towards cancer cells as compared to normal cells by certain bacteriocins directs for a much focused research to utilize these compounds as novel therapeutic agents. In this review, bacteriocins that demonstrated antitumor activity against diverse cancer cell lines have been discussed emphasizing their biochemical features, selectivity against extra targets and molecular mechanisms of action.

Bacterial cupredoxin azurin hijacks cellular signaling networks: Protein-protein interactions and cancer therapy.

Azurin secreted by Pseudomonas aeruginosa is an anticancer bacteriocin, which preferentially enters human cancer cells and induces apoptosis or growth inhibition. It turns out that azurin is a multi-target anticancer agent interfering in the p53 signaling pathway and the non-receptor tyrosine kinases signaling pathway. This suggests that azurin exerts its anticancer activity by interacting with multiple targets and interfering in multiple steps in disease progression. Therefore, azurin could overcome resistance to therapy. Besides azurin, putative bacteriocins that possess functional properties similar to those of azurin have been identified in more bacteria species. A systematic investigation on the anticancer mechanisms of azurin and the azurin-like bacteriocins will provide more and better options in cancer therapy. In this review, we summarize how azurin and the derived peptides hijack key cellular regulators or cell surface receptors to remodel the cellular signaling networks. In particular, we highlight the necessity of determining the structure of azurin/p53 complex and investigating the influence of post-translational modifications on interactions between azurin and p53. Therapeutic applications of azurin and derived peptides are also discussed.

An anti-cancer WxxxE-containing azurin polypeptide inhibits Rac1-dependent STAT3 and ERK/GSK-3ß signaling in breast cancer cells.

In our previous study, we characterized a mycoplasmal small GTPase-like polypeptide of 240 amino acids that possesses an N-terminal WVLGE sequence. The N-terminal WVLGE sequence promotes activation of Rac1 and subsequent host cancer cell proliferation. To investigate the function of the WxxxE motif in the interaction with Rac1 and host tumor progression, we synthesized a 35-amino acid WVLGE-containing polypeptide derived from a cell-penetrating peptide derived from the azurin protein. We verified that the WVLGE-containing polypeptide targeted MCF-7 cells rather than MCF-10A cells. However, the WVLGE-containing polypeptide inhibited activation of Rac1 and induced cellular phenotypes that resulted from inhibition of Rac1. In addition, the WVLGE-containing polypeptide down-regulated phosphorylation of the STAT3 and ERK/GSK-3ß signaling pathways, and this effect was abolished by either stimulation or inhibition of Rac1 activity. We also found that the WVLGE-containing polypeptide has a Rac1-dependent potential to suppress breast cancer growth in vitro and in vivo. We suggest that by acting as a Rac1 inhibitor, this novel polypeptide may be useful for the treatment of breast cancer.

Discovery of Azurin-Like Anticancer Bacteriocins from Human Gut Microbiome through Homology Modeling and Molecular Docking against the Tumor Suppressor p53.

Azurin from Pseudomonas aeruginosa is known anticancer bacteriocin, which can specifically penetrate human cancer cells and induce apoptosis. We hypothesized that pathogenic and commensal bacteria with long term residence in human body can produce azurin-like bacteriocins as a weapon against the invasion of cancers. In our previous work, putative bacteriocins have been screened from complete genomes of 66 dominant bacteria species in human gut microbiota and subsequently characterized by subjecting them as functional annotation algorithms with azurin as control. We have qualitatively predicted 14 putative bacteriocins that possessed functional properties very similar to those of azurin. In this work, we perform a number of quantitative and structure-based analyses including hydrophobic percentage calculation, structural modeling, and molecular docking study of bacteriocins of interest against protein p53, a cancer target. Finally, we have identified 8 putative bacteriocins that bind p53 in a same manner as p28-azurin and azurin, in which 3 peptides (p1seq16, p2seq20, and p3seq24) shared with our previous study and 5 novel ones (p1seq09, p2seq05, p2seq08, p3seq02, and p3seq17) discovered in the first time. These bacteriocins are suggested for further in vitro tests in different neoplastic line cells.

Modulation of membrane properties of lung cancer cells by azurin enhances the sensitivity to EGFR-targeted therapy and decreased ß1 integrin-mediated adhesion.

In lung cancer, the Epidermal Growth Factor Receptor (EGFR) is one of the main targets for clinical management of this disease. The effectiveness of therapies toward this receptor has already been linked to the expression of integrin receptor subunit ß1 in NSCLC A549 cells. In this work we demonstrate that azurin, an anticancer therapeutic protein originated from bacterial cells, controls the levels of integrin ß1 and its appropriate membrane localization, impairing the intracellular signaling cascades downstream these receptors and the invasiveness of cells. We show evidences that azurin when combined with gefitinib and erlotinib, tyrosine kinase inhibitors which targets specifically the EGFR, enhances the sensitivity of these lung cancer cells to these molecules. The broad effect of azurin at the cell surface level was examined by Atomic Force Microscopy. The Young 's module (E) shows that the stiffness of A549 lung cancer cells decreased with exposure to azurin and also gefitinib, suggesting that the alterations in the membrane properties may be the basis of the broad anticancer activity of this protein. Overall, these results show that azurin may be relevant as an adjuvant to improve the effects of other anticancer agents already in clinical use, to which patients often develop resistance hampering its full therapeutic response.

Structural studies on Laz, a promiscuous anticancer Neisserial protein.

Azurin and Laz (lipidated azurin) are 2 bacterial proteins with anticancer, anti-viral and anti-parasitic activities. Azurin, isolated from the bacterium Pseudomonas aeruginosa, termed Paz, demonstrates anticancer activity against a range of cancers but not against brain tumors. In contrast, Laz is produced by members of Gonococci/Meningococci, including Neisseria meningitides which can cross the blood-brain barrier to infect brain meninges. It has been previously reported that Laz has an additional 39 amino acid moiety, called an H.8 epitope, in the N-terminal part of the azurin moiety that allows Laz to cross the entry barrier to brain tumors such as glioblastomas. Exactly, how the H.8 epitope helps the azurin moiety of Laz to cross the entry barriers to attack glioblastoma cells is unknown. In this paper, we describe the structural features of the H.8 moiety in Laz using X-ray crystallography and demonstrate that while the azurin moiety of Laz adopts a ß-sandwich fold with 2 ß-sheets arranged in the Greek key motif, the H.8 epitope was present as a disordered structure outside the Greek key motif. Structures of Paz and H.8 epitope-deficient Laz are well superimposed. The structural flexibility of the H.8 motif in Laz explains the extracellular location of Laz in Neisseria where it can bind the key components of brain tumor cells to disrupt their tight junctions and allow entry of Laz inside the tumors to exert cytotoxicity.

Bacterial proteins and peptides in cancer therapy: today and tomorrow.

Cancer is one of the most deadly diseases worldwide. In the last three decades many efforts have been made focused on understanding how cancer grows and responds to drugs. The dominant drug-development paradigm has been the "one drug, one target." Based on that, the two main targeted therapies developed to combat cancer include the use of tyrosine kinase inhibitors and monoclonal antibodies. Development of drug resistance and side effects represent the major limiting factors for their use in cancer treatment. Nowadays, a new paradigm for cancer drug discovery is emerging wherein multi-targeted approaches gain ground in cancer therapy. Therefore, to overcome resistance to therapy, it is clear that a new generation of drugs is urgently needed. Here, regarding the concept of multi-targeted therapy, we discuss the challenges of using bacterial proteins and peptides as a new generation of effective anti-cancer drugs.

High-throughput molecular profiling of a P-cadherin overexpressing breast cancer model reveals new targets for the anti-cancer bacterial protein azurin.

Azurin is a bacterial protein from Pseudomonas aeruginosa which exerts an inhibitory activity in cancer cells. In P-cadherin-overexpressing models, a bad prognosis marker in breast cancer increasing invasion and other malignant features, azurin decreases the invasion of cancer cells. We performed a microarray analysis to compare the expression profile of azurin treated cells with different P-cadherin expression levels. Azurin up-regulated apoptosis mediated by p53 protein, endocytosis and vesicle-mediated transport. In the contrary, in invasive MCF-7/AZ.Pcad cells, azurin decreased the expression of genes associated with cell surface receptors and signal transduction, as well as biological adhesion. Further, azurin decreased adhesion of cells to proteins from the extracellular matrix (ECM) and altered protein expression of integrins α6, ß4 and ß1 and interfered with the ability of these cells to form mammospheres. Altogether, our results further enlighten the anti-cancer effects mediated by azurin in P-cadherin overexpression breast cancer models.

Nanoscale biomemory composed of recombinant azurin on a nanogap electrode.

We fabricate a nanoscale biomemory device composed of recombinant azurin on nanogap electrodes. For this, size-controllable nanogap electrodes are fabricated by photolithography, electron beam lithography, and surface catalyzed chemical deposition. Moreover, we investigate the effect of gap distance to optimize the size of electrodes for a biomemory device and explore the mechanism of electron transfer from immobilized protein to a nanogap counter-electrode. As the distance of the nanogap electrode is decreased in the nanoscale, the absolute current intensity decreases according to the distance decrement between the electrodes due to direct electron transfer, in contrast with the diffusion phenomenon of a micro-electrode. The biomemory function is achieved on the optimized nanogap electrode. These results demonstrate that the fabricated nanodevice composed of a nanogap electrode and biomaterials provides various advantages such as quantitative control of signals and exclusion of environmental effects such as noise. The proposed bioelectronics device, which could be mass-produced easily, could be applied to construct a nanoscale bioelectronics system composed of a single biomolecule.

The bacterial protein azurin impairs invasion and FAK/Src signaling in P-cadherin-overexpressing breast cancer cell models.

P-cadherin overexpression occurs in about 30% of all breast carcinomas, being a poor prognostic factor for breast cancer patients. In a cellular background of wild-type E-cadherin, we have previously shown that its expression promotes invasion, motility and migration of breast cancer cells due to the induced secretion of metalloproteases (MMPs) to the extracellular medium and to the concomitant shedding of a pro-invasive soluble form of this protein (sP-cad). Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human cancer cells relative to normal cells. Three different breast cancer cell lines, MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 were treated with sub-killing doses of azurin. Invasion of these cells was measured using Matrigel Invasion Assays and MTT assays were performed to determine cell viability upon treatment and the effects on cadherins expression was determined by Western blot and Immunofluorescence. Gelatin Zymography was used to determine activity of MMP2 in the conditioned media of azurin treated and untreated cells and the phosphorylation levels of intracellular signaling proteins were determined by Western blot. The invasive phenotype of these breast cancer cells was significantly reduced by azurin. Azurin (50-100 µM) also caused a specific decrease on P-cadherin protein levels from 30-50% in MCF-7/AZ.Pcad and SUM149 breast cancer cell lines, but the levels of E-cadherin remain unaltered. More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins. Our data show that azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling. Therefore, azurin could possibly be considered a therapeutic tool to treat poor-prognosis breast carcinomas overexpressing P-cadherin in a wild type E-cadherin context.

Antimalarial activity of cupredoxins: the interaction of Plasmodium merozoite surface protein 119 (MSP119) and rusticyanin.

The discovery of effective new antimalarial agents is urgently needed. One of the most frequently studied molecules anchored to the parasite surface is the merozoite surface protein-1 (MSP1). At red blood cell invasion MSP1 is proteolytically processed, and the 19-kDa C-terminal fragment (MSP119) remains on the surface and is taken into the red blood cell, where it is transferred to the food vacuole and persists until the end of the intracellular cycle. Because a number of specific antibodies inhibit erythrocyte invasion and parasite growth, MSP119 is therefore a promising target against malaria. Given the structural homology of cupredoxins with the Fab domain of monoclonal antibodies, an approach combining NMR and isothermal titration calorimetry (ITC) measurements with docking calculations based on BiGGER is employed on MSP119-cupredoxin complexes. Among the cupredoxins tested, rusticyanin forms a well defined complex with MSP119 at a site that overlaps with the surface recognized by the inhibitory antibodies. The addition of holo-rusticyanin to infected cells results in parasitemia inhibition, but negligible effects on parasite growth can be observed for apo-rusticyanin and other proteins of the cupredoxin family. These findings point to rusticyanin as an excellent therapeutic tool for malaria treatment and provide valuable information for drug design.

p28, a first in class peptide inhibitor of cop1 binding to p53.

BACKGROUND: A 28 amino-acid (aa) cell-penetrating peptide (p28) derived from azurin, a redox protein secreted from the opportunistic pathogen Pseudomonas aeruginosa, produces a post-translational increase in p53 in cancer cells by inhibiting its ubiquitination. METHODS: In silico computational simulations were used to predict motifs within the p53 DNA-binding domain (DBD) as potential sites for p28 binding. In vitro direct and competitive pull-down studies as well as western blot and RT-PCR analyses were used to validate predictions. RESULTS: The L1 loop (aa 112-124), a region within the S7-S8 loop (aa 214-236) and T140, P142, Q144, W146, R282 and L289 of the p53DBD were identified as potential sites for p28 binding. p28 decreased the level of the E3 ligase COP1 >80%, in p53wt and p53mut cells with no decrease in COP1 in p53dom/neg or p53null cells. Brief increases in the expression of the E3 ligases, TOPORS, Pirh2 and HDM2 (human double minute 2) in p53wt and p53mut cells were in response to sustained increases in p53. CONCLUSION: These data identify the specific motifs within the DBD of p53 that bind p28 and suggest that p28 inhibition of COP1 binding results in the sustained, post-translational increase in p53 levels and subsequent inhibition of cancer cell growth independent of an HDM2 pathway.

Does azurin bind to the transactivation domain of p53? A Trp phosphorescence study.

The bacterial redox protein azurin has been shown to be able to enter into cancer cells and induce apoptosis by stabilizing p53. Although the formation of a complex between the two proteins has been demonstrated, little is known about their binding features. We investigated the interaction between the transcription activation domain of p53 (p53(1-63)) and Pseudomonas aeruginosa azurin using fluorescence and phosphorescence spectroscopic techniques. Trp phosphorescence lifetime measurements revealed conformational changes in azurin induced by the interaction with p53(1-63). Acrylamide quenching of Trp phosphorescence also indicated a significant increase in the overall flexibility of azurin upon binding to p53(1-63). We show that azurin binds to the N-terminal region of p53 with a dissociation constant in the 5-10 µM range. No change in the fluorescence and phosphorescence emission of p53(1-63) was detected in the presence of azurin. This result indicated that no Trp residue of p53(1-63) is located in the interaction site with azurin and therefore suggested that the azurin binding site does not overlap that of MDM2, the protein that plays a crucial role in the p53 regulation. The present results may assist in the design of novel cancer treatments based on p53 stabilization by azurin.