Prediction of CD44 Structure by Deep Learning-Based Protein Modeling.

CD44 is a cell surface glycoprotein transmembrane receptor that is involved in cell-cell and cell-matrix interactions. It crucially associates with several molecules composing the extracellular matrix, the main one of which is hyaluronic acid. It is ubiquitously expressed in various types of cells and is involved in the regulation of important signaling pathways, thus playing a key role in several physiological and pathological processes. Structural information about CD44 is, therefore, fundamental for understanding the mechanism of action of this receptor and developing effective treatments against its aberrant expression and dysregulation frequently associated with pathological conditions. To date, only the structure of the hyaluronan-binding domain (HABD) of CD44 has been experimentally determined. To elucidate the nature of CD44s, the most frequently expressed isoform, we employed the recently developed deep-learning-based tools D-I-TASSER, AlphaFold2, and RoseTTAFold for an initial structural prediction of the full-length receptor, accompanied by molecular dynamics simulations on the most promising model. All three approaches correctly predicted the HABD, with AlphaFold2 outperforming D-I-TASSER and RoseTTAFold in the structural comparison with the crystallographic HABD structure and confidence in predicting the transmembrane helix. Low confidence regions were also predicted, which largely corresponded to the disordered regions of CD44s. These regions allow the receptor to perform its unconventional activity.

Non-synonymous variation and protein structure of candidate genes associated with selection in farm and wild populations of turbot (Scophthalmus maximus).

Non-synonymous variation (NSV) of protein coding genes represents raw material for selection to improve adaptation to the diverse environmental scenarios in wild and livestock populations. Many aquatic species face variations in temperature, salinity and biological factors throughout their distribution range that is reflected by the presence of allelic clines or local adaptation. The turbot (Scophthalmus maximus) is a flatfish of great commercial value with a flourishing aquaculture which has promoted the development of genomic resources. In this study, we developed the first atlas of NSVs in the turbot genome by resequencing 10 individuals from Northeast Atlantic Ocean. More than 50,000 NSVs where detected in the ~ 21,500 coding genes of the turbot genome, and we selected 18 NSVs to be genotyped using a single Mass ARRAY multiplex on 13 wild populations and three turbot farms. We detected signals of divergent selection on several genes related to growth, circadian rhythms, osmoregulation and oxygen binding in the different scenarios evaluated. Furthermore, we explored the impact of NSVs identified on the 3D structure and functional relationship of the correspondent proteins. In summary, our study provides a strategy to identify NSVs in species with consistently annotated and assembled genomes to ascertain their role in adaptation.

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis.

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.

Virtual screening and molecular dynamics simulations provide insight into repurposing drugs against SARS-CoV-2 variants Spike protein/ACE2 interface.

After over two years of living with Covid-19 and hundreds of million cases worldwide there is still an unmet need to find proper treatments for the novel coronavirus, due also to the rapid mutation of its genome. In this context, a drug repositioning study has been performed, using in silico tools targeting Delta Spike protein/ACE2 interface. To this aim, it has been virtually screened a library composed by 4388 approved drugs through a deep learning-based QSAR model to identify protein-protein interactions modulators for molecular docking against Spike receptor binding domain (RBD). Binding energies of predicted complexes were calculated by Molecular Mechanics/Generalized Born Surface Area from docking and molecular dynamics simulations. Four out of the top twenty ranking compounds showed stable binding modes on Delta Spike RBD and were evaluated also for their effectiveness against Omicron. Among them an antihistaminic drug, fexofenadine, revealed very low binding energy, stable complex, and interesting interactions with Delta Spike RBD. Several antihistaminic drugs were found to exhibit direct antiviral activity against SARS-CoV-2 in vitro, and their mechanisms of action is still debated. This study not only highlights the potential of our computational methodology for a rapid screening of variant-specific drugs, but also represents a further tool for investigating properties and mechanisms of selected drugs.

Restricted T-Cell Repertoire in the Epicardial Adipose Tissue of Non-ST Segment Elevation Myocardial Infarction Patients.

Aims: Human epicardial adipose tissue, a dynamic source of multiple bioactive factors, holds a close functional and anatomic relationship with the epicardial coronary arteries and communicates with the coronary artery wall through paracrine and vasocrine secretions. We explored the hypothesis that T-cell recruitment into epicardial adipose tissue (EAT) in patients with non-ST segment elevation myocardial infarction (NSTEMI) could be part of a specific antigen-driven response implicated in acute coronary syndrome onset and progression. Methods and Results: We enrolled 32 NSTEMI patients and 34 chronic coronary syndrome (CCS) patients undergoing coronary artery bypass grafting (CABG) and 12 mitral valve disease (MVD) patients undergoing surgery. We performed EAT proteome profiling on pooled specimens from three NSTEMI and three CCS patients. We performed T-cell receptor (TCR) spectratyping and CDR3 sequencing in EAT and peripheral blood mononuclear cells of 29 NSTEMI, 31 CCS, and 12 MVD patients. We then used computational modeling studies to predict interactions of the TCR beta chain variable region (TRBV) and explore sequence alignments. The EAT proteome profiling displayed a higher content of pro-inflammatory molecules (CD31, CHI3L1, CRP, EMPRINN, ENG, IL-17, IL-33, MMP-9, MPO, NGAL, RBP-4, RETN, VDB) in NSTEMI as compared to CCS (P < 0.0001). CDR3-beta spectratyping showed a TRBV21 enrichment in EAT of NSTEMI (12/29 patients; 41%) as compared with CCS (1/31 patients; 3%) and MVD (none) (ANOVA for trend P < 0.001). Of note, 11/12 (92%) NSTEMI patients with TRBV21 perturbation were at their first manifestation of ACS. Four patients with the first event shared a distinctive TRBV21-CDR3 sequence of 178 bp length and 2/4 were carriers of the human leukocyte antigen (HLA)-A*03:01 allele. A 3D analysis predicted the most likely epitope able to bind HLA-A3*01 and interact with the TRBV21-CDR3 sequence of 178 bp length, while the alignment results were consistent with microbial DNA sequences. Conclusions: Our study revealed a unique immune signature of the epicardial adipose tissue, which led to a 3D modeling of the TCRBV/peptide/HLA-A3 complex, in acute coronary syndrome patients at their first event, paving the way for epitope-driven therapeutic strategies.

A TLR/CD44 axis regulates T cell trafficking in experimental and human multiple sclerosis.

In the pathogenesis of autoimmune disorders, the modulation of leukocytes' trafficking plays a central role, still poorly understood. Here, we focused on the effect of TLR2 ligands in trafficking of T helper cells through reshuffling of CD44 isoforms repertoire. Concurrently, strain background and TLR2 haplotype affected Wnt/ß-catenin signaling pathway and expression of splicing factors. During EAE, mCD44 v9- v 10 was specifically enriched in the forebrain and showed an increased ability to bind stably to osteopontin. Similarly, we observed that hCD44 v7 was highly enriched in cells of cerebrospinal fluid from MS patients with active lesions. Moreover, TLRs engagement modulated the composition of CD44 variants also in human T helper cells, supporting the hypothesis that pathogens or commensals, through TLRs, in turn modulate the repertoire of CD44 isoforms, thereby controlling the distribution of lesions in the CNS. The interference with this mechanism(s) represents a potential tool for prevention and treatment of autoimmune relapses and exacerbations.

Targeting SARS-CoV-2 Spike Protein/ACE2 Protein-Protein Interactions: a Computational Study.

The spike glycoprotein (S) of the SARS-CoV-2 virus surface plays a key role in receptor binding and virus entry. The S protein uses the angiotensin converting enzyme (ACE2) for entry into the host cell and binding to ACE2 occurs at the receptor binding domain (RBD) of the S protein. Therefore, the protein-protein interactions (PPIs) between the SARS-CoV-2 RBD and human ACE2, could be attractive therapeutic targets for drug discovery approaches designed to inhibit the entry of SARS-CoV-2 into the host cells. Herein, with the support of machine learning approaches, we report structure-based virtual screening as an effective strategy to discover PPIs inhibitors from ZINC database. The proposed computational protocol led to the identification of a promising scaffold which was selected for subsequent binding mode analysis and that can represent a useful starting point for the development of new treatments of the SARS-CoV-2 infection.

Identification and Modeling of a GT-A Fold in the α-Dystroglycan Glycosylating Enzyme LARGE1.

The acetylglucosaminyltransferase-like protein LARGE1 is an enzyme that is responsible for the final steps of the post-translational modifications of dystroglycan (DG), a membrane receptor that links the cytoskeleton with the extracellular matrix in the skeletal muscle and in a variety of other tissues. LARGE1 acts by adding the repeating disaccharide unit [-3Xyl-α1,3GlcAß1-] to the extracellular portion of the DG complex (α-DG); defects in the LARGE1 gene result in an aberrant glycosylation of α-DG and consequent impairment of its binding to laminin, eventually affecting the connection between the cell and the extracellular environment. In the skeletal muscle, this leads to degeneration of the muscular tissue and muscular dystrophy. So far, a few missense mutations have been identified within the LARGE1 protein and linked to congenital muscular dystrophy, and because no structural information is available on this enzyme, our understanding of the molecular mechanisms underlying these pathologies is still very limited. Here, we generated a 3D model structure of the two catalytic domains of LARGE1, combining different molecular modeling approaches. Furthermore, by using molecular dynamics simulations, we analyzed the effect on the structure and stability of the first catalytic domain of the pathological missense mutation S331F that gives rise to a severe form of muscle-eye-brain disease.

Selective Inhibitors of T Cell Receptor Recognition of Antigen-MHC Complexes for Rheumatoid Arthritis.

Autoreactive T cells specific to human collagen type II have a crucial role in the development of rheumatoid arthritis (RA) in the context of MHC class II allele HLA-DRB1-*04. The protein-protein interactions between the T cell receptor (TCR) and the type II collagen bound to the allele MHC of class II may thus represent the target for the development of new drugs against RA. In this study, a structure-based pharmacophore model for potential small molecule inhibitors was developed from protein-protein interface structure. The 3D model obtained was used for a virtual screening workflow, which resulted in three hits for experimental follow up. Three compounds have been identified that interfere with the TCR/collagenII-MHCII (K i values below 10 µM) and open up new possibilities in the treatment of RA.

Identification and antitumor activity of a novel inhibitor of the NIMA-related kinase NEK6.

The NIMA (never in mitosis, gene A)-related kinase-6 (NEK6), which is implicated in cell cycle control and plays significant roles in tumorigenesis, is an attractive target for the development of novel anti-cancer drugs. Here we describe the discovery of a potent ATP site-directed inhibitor of NEK6 identified by virtual screening, adopting both structure- and ligand-based techniques. Using a homology-built model of NEK6 as well as the pharmacophoric features of known NEK6 inhibitors we identified novel binding scaffolds. Twenty-five compounds from the top ranking hits were subjected to in vitro kinase assays. The best compound, i.e. compound 8 ((5Z)-2-hydroxy-4-methyl-6-oxo-5-[(5-phenylfuran-2-yl)methylidene]-5,6-dihydropyridine-3-carbonitrile), was able to inhibit NEK6 with low micromolar IC50 value, also displaying antiproliferative activity against a panel of human cancer cell lines. Our results suggest that the identified inhibitor can be used as lead candidate for the development of novel anti-cancer agents, thus opening the possibility of new therapeutic strategies.

In silico investigation of the molecular effects caused by R123H variant in secretory phospholipase A2-IIA associated with ARDS.

Phospholipase A2-IIA catalyzes the hydrolysis of the sn-2 ester of glycerophospholipids. A rare c.428G > A (p.Arg143His) variant in PLA2G2A gene was found in two infants affected by acute respiratory distress syndrome (ARDS) by whole coding region and exon/intron boundaries sequencing. To obtain insights into the possible molecular effects of the rare R123H mutation in secretory PLA2-IIA (sPLA2-IIA), molecular modelling, molecular dynamics (MD) using principal component analysis (PCA) and continuum electrostatic calculations were conducted on the crystal structure of the wild type protein and on a generated model structure of the R123H mutant. Analysis of MD trajectories indicate that the overall stability of the protein is not affected by this mutation but nevertheless the catalytically crucial H-bond between Tyr51 and Asp91 as well as main electrostatic interactions in the region close to the mutation site are altered. PCA results indicate that the R123H replacement alter the internal molecular motions of the enzyme and that collective motions are increased. Electrostatic surface potential studies suggest that after mutation the interfacial binding to anionic phospholipid membranes and anionic proteins may be changed. The strengthening of electrostatic interactions may be propagated into the active site region thus potentially affecting the substrate recognition and enzymatic activity. Our findings provide the basis for further investigation and advances our understanding of the effects of mutations on sPLA2 structure and function.

Structural model of the full-length Ser/Thr protein kinase StkP from S. pneumoniae and its recognition of peptidoglycan fragments.

The unique eukaryotic-like Ser/Thr protein kinases of Streptococcus pneumoniae, StkP, plays a primary role in the cell division process. It is composed of an intracellular kinase domain, a transmembrane helix and four extracellular PASTA subunits. PASTA domains were shown to interact with cell wall fragments but the key questions related to the molecular mechanism governing ligand recognition remain unclear. To address this issue, the full-length structural model of StkP was generated by combining small-angle X-ray scattering data with the results of computer simulations. Docking and molecular dynamics studies on the generated three-dimensional model structure reveal the possibility of peptidoglycan fragment binding at the hinge regions between PASTA subunits with a preference for a bent hinge between PASTA3 and PASTA4.

Structural studies and SH3 domain binding properties of a human antiviral salivary proline-rich peptide.

Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016.

Insights into the properties of the two enantiomers of trans-δ-viniferin, a resveratrol derivative: antioxidant activity, biochemical and molecular modeling studies of its interactions with hemoglobin.

Resveratrol is widely known as an antioxidant and anti-inflammatory molecule. The present study first reports the effects of trans-δ-viniferin (TVN), a dimer of resveratrol, on human erythrocytes. The antioxidant activity of TVN was tested using in vitro model systems such as hydroxy radical scavenging, DPPH and lipid peroxidation. In addition we have examined the influence of the 15R,22R- and 15S,22S-enantiomers (abbreviated R,R-TVN, and S,S-TVN, respectively) on anion transport, ATP release, caspase 3 activation. Given that hemoglobin (Hb) redox reactions are the major source of RBC oxidative stress, we also explored the effects of TVN on hemoglobin function. TVN showed moderate antioxidant properties and good protective activity from hemoglobin oxidation. Potential binding sites of R,R-TVN and S,S-TVN with oxy- and deoxy-Hb were also investigated through an extensive in silico docking approach and molecular dynamics calculations. The whole molecular modeling studies indicate that binding of R,R-TVN and S,S-TVN to Hb lacks of specific ligand-target interactions. This is the first report on the biological activity of the individual enantiomers of a resveratrol-related dimer.

Characterization of the cell penetrating properties of a human salivary proline-rich peptide.

Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary proline-rich proteins. Among these peptides we found that a 20 residue proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblast cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-ß-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms highlights their possible application as novel drug delivery agents.

Evolutionary history and adaptive significance of the polymorphic Pan I in migratory and stationary populations of Atlantic cod (Gadus morhua).

The synaptophysin (SYP) family comprises integral membrane proteins involved in vesicle-trafficking events, but the physiological function of several members has been enigmatic for decades. The presynaptic SYP protein controls neurotransmitter release, while SYP-like 2 (SYPL2) contributes to maintain normal Ca(2+)-signaling in the skeletal muscles. The polymorphic pantophysin (Pan I) of Atlantic cod shows strong genetic divergence between stationary and migratory populations, which seem to be adapted to local environmental conditions. We have investigated the functional involvement of Pan I in the different ecotypes by analyzing the 1) phylogeny, 2) spatio-temporal gene expression, 3) structure-function relationship of the Pan I(A) and I(B) protein variants, and 4) linkage to rhodopsin (rho) recently proposed to be associated with different light sensitivities in Icelandic populations of Atlantic cod. We searched for SYP family genes in phylogenetic key species and identified a single syp-related gene in three invertebrate chordates, while four members, Syp, Sypl1, Sypl2 and synaptoporin (Synpr), were found in tetrapods, Comoran coelacanth and spotted gar. Teleost fish were shown to possess duplicated syp, sypl2 and synpr genes of which the sypl2b paralog is identical to Pan I. The ubiquitously expressed cod Pan I codes for a tetra-spanning membrane protein possessing five amino acid substitutions in the first intravesicular loop, but only minor structural differences were shown between the allelic variants. Despite sizable genomic distance (>2.5 Mb) between Pan I and rho, highly significant linkage disequilibrium was found by genotyping shallow and deep water juvenile settlers predominated by the Pan I(A)-rho(A) and Pan I(B)-rho(B) haplotypes, respectively. However, the predicted rhodopsin protein showed no amino acid changes, while multiple polymorphic sites in the upstream region might affect the gene expression and pigment levels in stationary and migratory cod. Alternatively, other strongly linked genes might be responsible for the sharp settling stratification of juveniles and the different vertical behavior patterns of adult Atlantic cod.

Chrono-proteomics of human saliva: variations of the salivary proteome during human development.

An important contribution to the variability of any proteome is given by the time dimension that should be carefully considered to define physiological modifications. To this purpose, whole saliva proteome was investigated in a wide age range. Whole saliva was collected from 17 preterm newborns with a postconceptional age at birth of 178-217 days. In these subjects sample collection was performed serially starting immediately after birth and within about 1 year follow-up, gathering a total of 111 specimens. Furthermore, whole saliva was collected from 182 subjects aged between 0 and 17 years and from 23 adults aged between 27 and 57 years. The naturally occurring intact salivary proteome of the 316 samples was analyzed by low- and high-resolution HPLC-ESI-MS platforms. Proteins peculiar of the adults appeared in saliva with different time courses during human development. Acidic proline-rich proteins encoded by PRH2 locus and glycosylated basic proline-rich proteins encoded by PRB3 locus appeared following 180 days of postconceptional age, followed at 7 months (±2 weeks) by histatin 1, statherin, and P-B peptide. The other histatins and acidic proline-rich proteins encoded by PRH1 locus appeared in whole saliva of babies from 1 to 3 weeks after the normal term of delivery, S-type cystatins appeared at 1 year (±3 months), and basic proline-rich proteins appeared at 4 years (±1 year) of age. All of the proteinases involved in the maturation of salivary proteins were more active in preterm than in at-term newborns, on the basis of the truncated forms detected. The activity of the Fam20C kinase, involved in the phosphorylation of various proteins, started around 180 days of postconceptional age, slowly increased reaching values comparable to adults at about 2 years (±6 months) of age. Instead, MAPK14 involved in the phosphorylation of S100A9 was fully active since birth also in preterm newborns.

Protein kinase C mediates caspase 3 activation: A role for erythrocyte morphology changes.

We have previously showed that morphological alterations in Red Blood Cells (RBCs) are correlated to an impaired eNOS enzymatic activity and a concomitant reduced NO derived metabolites formation. Here we extend our previous observations, reporting that RBC morphology is regulated by a series of specific cell signaling events linked to Protein Kinase C (PKC)-mediated activation of caspase 3. Pretreatment of RBCs with the PKC inhibitor chelerythrine, prior to the addition of phorbol-12-myristate-13-acetate (PMA), an activator of PKC, blocks the appearance of the morphology alterations and the sustained decrease in nitrates and nitrites levels induced by PMA. Inhibition of PKC also completely inhibits PMA mediated caspase-3 activation. On the other hand, caspase 3 inhibition, lessens the PMA induced-effects on the appearance of RBC morphology alterations, although it enhances PMA-mediated effects on nitric oxide (NO) derived metabolites levels. These data demonstrate that PKC-mediated activation of caspase 3 is an integral and essential part of signaling pathway in RBCs, that may be a regulatory factor of RBC mechanical properties, through regulation of NO metabolism.

NO Metabolites Levels in Human Red Blood Cells are Affected by Palytoxin, an Inhibitor of Na(+)/K(+)-ATPase Pump.

Palytoxin (PTX), a marine toxin, represents an increasing hazard for human health. Despite its high toxicity for biological systems, the mechanisms triggered by PTX, are not well understood. The high affinity of PTX for erythrocyte Na(+)/K(+)-ATPase pump is largely known, and it indicates PTX as a sensitive tool to characterize the signal transducer role for Na(+)/K(+)-ATPase pump. Previously, it has been reported that in red blood cells (RBC), probably via a signal transduction generated by the formation of a PTX-Na(+)/K(+)-ATPase complex, PTX alters band 3 functions and glucose metabolism. The present study addresses the question of which other signaling pathways are regulated by Na(+)/K(+)-ATPase in RBC. Here it has been evidenced that PTX following its interaction with Na(+)/K(+)-ATPase pump, alters RBC morphology and this event is correlated to decreases by 30% in nitrites and nitrates levels, known as markers of plasma membrane eNOS activity. Orthovanadate (OV), an antagonist of PTX binding to Na(+)/K(+)-ATPase pump, was able to reverse the effects elicited by PTX. Finally, current investigation firstly suggests that Na(+)/K(+)-ATPase pump, following its interaction with PTX, triggers a signal transduction involved in NO metabolism regulation.