Two open states of P2X receptor channels

The occupancy of the orthosteric ligand binding sites of P2X receptor (P2XR) channels causes the rapid opening of a small cation-permeable pore, followed by a gradual dilation that renders the pore permeable to large organic cations. Electrophysiologically, this phenomenon was shown using whole-cell current recording on P2X2R-, P2X2/X5R-, P2X4R- and P2X7R-expressing cells that were bathed in N-methyl-D-glucamine (NMDG(+))-containing buffers in the presence and/or absence of small monovalent and divalent cations. The pore dilation of P2X4R and P2X7R caused a secondary current growth, whereas that of P2X2R showed a sustained kinetic coupling of dilation and desensitization, leading to receptor channel closure. The pore size of the P2X7R open and dilated states was estimated to be approximately 0.85 nm and greater than 1 nm, respectively. The P2XR pore dilation was also observed in intact cells by measurement of fluorescent dye uptake/release, application of polyethylene glycols of different sizes, and atomic force microscopy. However, pore dilation was not observed at the single channel level. Structural data describing the dilated state are not available, and the relevance of orthosteric and allosteric ligand interactions to pore dilation was not studied

Structural and functional properties of the rat P2X4 purinoreceptor extracellular vestibule during gating

P2X receptors are ATP-gated cation channels consisting of three subunits that are mutually intertwined and form an upper, central, and extracellular vestibule with three lateral portals and the channel pore. Here we used cysteine and alanine scanning mutagenesis of the rat P2X4R receptor V47 V61 and K326 N338 sequences to study structural and functional properties of extracellular vestibule during gating. Cysteine mutants were used to test the accessibility of these residue side chains to cadmium during closedopen-desensitized transitions, whereas alanine mutants served as controls. This study revealed the accessibility of residues E51, T57, S59, V61, K326, and M336 to cadmium in channels undergoing a transition from a closed-to-open state and the accessibility of residues V47, G53, D331, 1332, 1333, T335, 1337, and N338 in channels undergoing a transition from an open-to-desensitized state; residues E56 and K329 were accessible during both transitions. The effect of cadmium on channel gating was stimulatory in all reactive V47 V61 mutants and inhibitory in the majority of reactive K326 N338 mutants. The rat P2X4 receptor homology model suggests that residues affected by cadmium in the closed-to-open transition were located within the lumen of the extracellular vestibule and toward the central vestibule; however, the residues affected by cadmium in the open-to-desensitized state were located at the bottom of the vestibule near the pore. Analysis of the model assumed that there is ion access to extracellular and central vestibules through lateral ports when the channel is closed, with residues above the first transmembrane domain being predominantly responsible for ion uptake. Upon receptor activation, there is passage of ions toward the residues located on the upper region of the second transmembrane domain, followed by permeation through the gate region

Structural and functional properties of the rat P2X4 purinoreceptor extracellular vestibule during gating

P2X receptors are ATP-gated cation channels consisting of three subunits that are mutually intertwined and form an upper, central, and extracellular vestibule with three lateral portals and the channel pore. Here we used cysteine and alanine scanning mutagenesis of the rat P2X4R receptor V47 V61 and K326 N338 sequences to study structural and functional properties of extracellular vestibule during gating. Cysteine mutants were used to test the accessibility of these residue side chains to cadmium during closedopen-desensitized transitions, whereas alanine mutants served as controls. This study revealed the accessibility of residues E51, T57, S59, V61, K326, and M336 to cadmium in channels undergoing a transition from a closed-to-open state and the accessibility of residues V47, G53, D331, 1332, 1333, T335, 1337, and N338 in channels undergoing a transition from an open-to-desensitized state; residues E56 and K329 were accessible during both transitions. The effect of cadmium on channel gating was stimulatory in all reactive V47 V61 mutants and inhibitory in the majority of reactive K326 N338 mutants. The rat P2X4 receptor homology model suggests that residues affected by cadmium in the closed-to-open transition were located within the lumen of the extracellular vestibule and toward the central vestibule; however, the residues affected by cadmium in the open-to-desensitized state were located at the bottom of the vestibule near the pore. Analysis of the model assumed that there is ion access to extracellular and central vestibules through lateral ports when the channel is closed, with residues above the first transmembrane domain being predominantly responsible for ion uptake. Upon receptor activation, there is passage of ions toward the residues located on the upper region of the second transmembrane domain, followed by permeation through the gate region

Identification of Functionally Important Residues of the Rat P2X4 Receptor by Alanine Scanning Mutagenesis of the Dorsal Fin and Left Flipper Domains

Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. Here, we focused on the role of these domains in receptor activation, responsiveness to orthosteric ATP analogue agonists, and desensitization. Alanine scanning mutagenesis of the R203-L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor showed that ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists 2-(methylthio) adenosine 5'-triphosphate, adenosine 5'-(gamma-thio) triphosphate, 2'(3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate, and alpha,beta-methyleneadenosine 5'-triphosphate. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. Among these mutants, D280A, T281A, R282A, H286A, G291A, and Y292A also exhibited increased time-constant of the desensitizing current response. These experiments, together with homology modeling, indicate that residues located in the upper part of the dorsal fin and left flipper domains, relative to distance from the channel pore, contribute to the organization of the ATP binding pocket and to the initiation of signal transmission towards residues in the lower part of both domains. The R203 and N204 residues, deeply buried in the protein, may integrate the output signal from these two domains towards the gate. In addition, the left flipper residues predominantly account for the control of transition of channels from an open to a desensitized state

NEW APPROACHES IN THE TREATMENT OF CHRONIC BACTERIAL INFECTIONS

The rapid emergence and spread of multidrug- resistant pathogens present a global healthcare challenge. One common cause of resistance and/or tolerance to antibiotics is biofilms, a complex communities of bacteria embedded in a self-produced matrix. Biofilm formation and maturation are regulated by quorum sensing, a cell density-dependent communication system that relies on the synthesis, diffusion, and detection of small signaling molecules - autoinducers (AIs). Quorum quenching (QQ) enzymes that cut Ais emerged as a promising strategy for persistent bacterial infections. However, a significant drawback for the use of QQ enzymes as therapeutics is their poor stability and efficacy in vivo. Since one of the major health issues linked to biofilm development is persistent wound infections, our goal was to improve enzyme properties by immobilizing it on a natural biopolymer to make it suitable for use as a wound dressing. The best candidate for immobilization was YtnP lactonase from Bacillus paralicheniformis ZP1, as in concentrations higher than 25 μg/mL it improved the survival of Pseudomonas aeruginosa PAO1-infected zebrafish, rescuing 80% of embryos. When combined with tobramycin or gentamicin, the survival rate of zebrafish embryos increased to 100%. Purified YtnP lactonase at a concentration of 1 mg was immobilized on 10 mg of polymer disks by crosslinking with glutaraldehyde. Specific modifications of the polymer were also made to eliminate the use of glutaraldehyde, which is a skin irritant. In in vivo experiments on a murine chronic wound model, immobilized enzyme inhibited biofilm development, cleared already formed biofilms, and overall improved wound healing. These results provide a foundation for the development of advanced wound dressings that will prevent infection development in wounds and enable proper therapy for infected chronic wounds.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi

In silico pre-selection of β-glucosidase gene for heterologous recombinant expression

Biofilms are ubiquitous in nature, and the food industry is vulnerable to the risks posed by biofilm formation. Not only do they interfere with the food production process, but they also pose a public health threat. However, complete elimination of biofilms on food and food contact surfaces cannot be achieved by conventional methods (cleaning and disinfection) alone. New biofilm control strategies must be developed to prevent its formation and/or persistence. Novel approaches may be based on enzymes that depolymerize components of the biofilm matrix, making bacterial cells accessible to antimicrobial agents. Environmental microorganisms are an inexhaustible source of new enzymes. In Salmonella Enteritidis and Escherichia coli, known foodborne pathogens, cellulose is an important component of the biofilm matrix, so our isolates from untapped environments were tested for cellulolytic activity. Of the more than 70 isolates examined, isolate BG28 was selected as the most promising. Its genome was sequenced, annotated, and it was identified as Gram-positive Microbacterium sp. Genome mining revealed the presence of four complete genes for different β-glucosidases, one of three enzyme types of cellulase complexes. To select the best candidate for heterologous expression DeepTMHMM, ProtParam, and SoluProt were used to predict the presence/absence of signal peptide and transmembrane domains, instability index, aliphatic index, hydrophilicity, and soluble expression in E. coli. Based on the prediction results, the gene annotated as β-glucosidase B was selected for recombinant expression. In addition, I-TASSER was used to model the tertiary structure of the selected enzyme. The β-glucosidase B was recombinantly expressed, purified, and tested for its anti-biofilm activity. It was active and showed a 50% inhibitory effect on S. Enteritidis and E. coli biofilm formation at a concentration of 100 μg/ml. To further evaluate this in silico approach in the preselection of candidate enzymes for recombinant expression and purification, we will use it to identify other enzymes of the cellulase complex.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor

The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47-V61 and F324-N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a beta-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening

Roles of Conserved Ectodomain Cysteines of the Rat P2X4 Purinoreceptor in Agonist Binding and Channel Gating

Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating

GLYCOSIDE HYDROLASES FROM FRESHWATER FISH GILL MICROBIOTA AS BIOFILM INHIBITORS FOR ENHANCED FOOD SAFETY

The formation of biofilms by foodborne pathogens is a constant challenge in the food industry, leading to an increased risk of contamination and compromising food safety. Many of the chemicals commonly used for sanitation in the food industry are unable to remove biofilms, are harmful to surfaces and can be toxic. The effectiveness of disinfectants can be improved using enzymes that specifically target biofilm components such as exopolysaccharides, extracellular DNA, or proteins. In this study we investigated the potential of glycoside hydrolases originating from the gill microbiota of freshwater fish to control biofilm formation in the most common foodborne pathogens. We demonstrated that β-glucosidase from Microbacterium sp. BG28 (BglB-BG28) effectively inhibits cellulose-rich biofilms formed by Salmonella enteritidis, S. typhimurium, S. infantis, and Escherichia coli. When these bacteria were cultivated overnight with 200 μL/mL enzyme, up to 80% less biofilm was formed. By fluorescence microscopy, we visualised the inhibition of biofilms on plastic, glass and aluminium, materials commonly used in the food industry. When used as a pre-treatment, BglB-BG28 increased the bactericidal efficacy of Oxicid®S, a commercially available surface disinfectant. Its effectiveness at temperatures up to 50 °C and in a pH range from 4 to 8 together with compatibility with non-ionic detergents and high tolerance to sodium chloride and glucose give BglB-BG28 advantages in harsh and diverse industrial environments. Importantly, no toxicity to Caenorhabditis elegans was observed at enzyme concentrations of up to 1 mg/ml. Overall, these results demonstrate the suitability of the β-glucosidase BglB-BG28 for the formulation of a novel enzyme-based disinfectant to be used in food processing facilities.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi