Pharmaceuticals and endocrine disrupting compounds modulate adverse effects of climate change on resource quality in freshwater food webs.

Freshwater biodiversity, ecosystem functions and services are changing at an unprecedented rate due to the impacts of vast number of stressors overlapping in time and space. Our study aimed at characterizing individual and combined impacts of pollution with pharmaceuticals (PhACs) and endocrine disrupting compounds (EDCs) and increased water temperature (as a proxy for climate change) on primary producers and first level consumers in freshwaters. We conducted a microcosm experiment with a simplified freshwater food web containing moss (Bryophyta) and shredding caddisfly larvae of Micropterna nycterobia (Trichoptera). The experiment was conducted with four treatments; control (C), increased water temperature + 4 °C (T2), emerging contaminants' mix (EC = 15 PhACs & 5 EDCs), and multiple stressor treatment (MS = EC + T2). Moss exhibited an overall mild response to selected stressors and their combination. Higher water temperature negatively affected development of M. nycterobia through causing earlier emergence of adults and changes in their lipidome profiles. Pollution with PhACs and EDCs had higher impact on metabolism of all life stages of M. nycterobia than warming. Multiple stressor effect was recorded in M. nycterobia adults in metabolic response, lipidome profiles and as a decrease in total lipid content. Sex specific response to stressor effects was observed in adults, with impacts on metabolome generally more pronounced in females, and on lipidome in males. Thus, our study highlights the variability of both single and multiple stressor impacts on different traits, different life stages and sexes of a single insect species. Furthermore, our research suggests that the combined impacts of warming, linked to climate change, and contamination with PhACs and EDCs could have adverse consequences on the population dynamics of aquatic insects. Additionally, these findings point to a potential decrease in the quality of resources available for both aquatic and potentially terrestrial food webs.

Identification of an Additional Metal-Binding Site in Human Dipeptidyl Peptidase III.

Dipeptidyl peptidase III (DPP III, EC 3.4.14.4) is a monozinc metalloexopeptidase that hydrolyzes dipeptides from the N-terminus of peptides consisting of three or more amino acids. Recently, DPP III has attracted great interest from scientists, and numerous studies have been conducted showing that it is involved in the regulation of various physiological processes. Since it is the only metalloenzyme among the dipeptidyl peptidases, we considered it important to study the process of binding and exchange of physiologically relevant metal dications in DPP III. Using fluorimetry, we measured the Kd values for the binding of Zn2+, Cu2+, and Co2+ to the catalytic site, and using isothermal titration calorimetry (ITC), we measured the Kd values for the binding of these metals to an additional binding site. The structure of the catalytic metal's binding site is known from previous studies, and in this work, the affinities for this site were calculated for Zn2+, Cu2+, Co2+, and Mn2+ using the QM approach. The structures of the additional binding sites for the Zn2+ and Cu2+ were also identified, and MD simulations showed that two Cu2+ ions bound to the catalytic and inhibitory sites exchanged less frequently than the Zn2+ ions bound to these sites.

Phenanthridine-pyrene conjugates as fluorescent probes for DNA/RNA and an inactive mutant of dipeptidyl peptidase enzyme.

Two novel conjugate molecules were designed: pyrene and phenanthridine-amino acid units with a different linker length between the aromatic fragments. Molecular modelling combined with spectrophotometric experiments revealed that in neutral and acidic buffered water solutions conjugates predominantly exist in intramolecularly stacked conformations because of the π-π stacking interaction between pyrene and phenanthridine moieties. The investigated systems exhibited a pH-dependent excimer formation that is significantly red-shifted relative to the pyrene and phenanthridine fluorescence. While the conjugate with a short linker showed negligible spectrophotometric changes due to the polynucleotide addition, the conjugate with a longer and more flexible linker exhibited a micromolar and submicromolar binding affinity for ds-polynucleotides and inactivated a mutant of dipeptidyl peptidase enzyme E451A. Confocal microscopy revealed that the conjugate with the longer linker entered the HeLa cell membranes and blue fluorescence was visualized as the dye accumulated in the cell membrane.

Influence of Mutations of Conserved Arginines on Neuropeptide Binding in the DPP III Active Site.

Dipeptidyl peptidase III (DPP III), a zinc exopeptidase, is involved in the final steps of intercellular protein degradation and has a marked affinity for opioid peptides such as enkephalins and endomorphins. Recently, we characterized a number of neuropeptides as potential substrates and inhibitors of human DPP III and provided an explanation for their differential behavior. These studies prompted us to investigate the influence of the conserved R399 and R669 on neuropeptides binding to DPP III. Measuring kinetic parameters in inhibitory assays, we found that mutation of R669 to Ala or Met significantly reduced the inhibitory properties of the slow substrates tynorphin and valorphin, whereas the effects on binding of the good substrates Arg2-2NA and Leu-enkephalin were small. Molecular dynamics simulations of wild-type (WT) and mutant DPP III complexes with Leu-enkephalin, tynorphin, valorphin, and Arg2-2NA in conjunction with calculations of binding free energies revealed that the lower inhibitory potency of slow substrates in the R669A mutant can be explained by the lower binding affinity of tynorphin and the higher propensity of valorphin to hydrolyze in the mutant than in WT. The R399A mutation was shown to affect the binding and/or hydrolysis of both good and slow substrates, with the effects on Leu-enkephalin being the most pronounced.

Fate and effects of microplastics in combination with pharmaceuticals and endocrine disruptors in freshwaters: Insights from a microcosm experiment.

Microplastic contamination of freshwater ecosystems has become an increasing environmental concern. To advance the hazard assessment of microplastics, we conducted a microcosm experiment in which we exposed a simplified aquatic ecosystem consisting of moss and caddisflies to microplastics (polyethylene, polystyrene and polypropylene) and pharmaceuticals and personal care products (1H-benzotriazole, bisphenol A, caffeine, gemfibrozil, ketoprofen, methylparaben, estriol, diphenhydramine, tris (1-chloro-2-propyl) phosphate) over the course of 60 days. We monitored the flux of microplastics within the microcosm, as well as the metabolic and total protein variation of organisms. This study offers evidence highlighting the capacity of moss to act as a sink for free-floating microplastics in freshwater environments. Moss is also shown to serve as a source and pathway for microplastic particles to enter aquatic food webs via caddisflies feeding off of the moss. Although most ingested microparticles were eliminated between caddisflies life stages, a small fraction of microplastics was transferred from aquatic to terrestrial ecosystem by emergence. While moss exhibited a mild response to microplastic stress, caddisflies ingesting microplastics showed stress comparable to that caused by exposure to pharmaceuticals. The molecular responses that the stressors triggered were tentatively identified and related to phenotypic responses, such as the delayed development manifested through the delayed emergence of caddisflies exposed to stress. Overall, our study provides valuable insights into the adverse effects of microplastics on aquatic species, compares the impacts of microplastics on freshwater biota to those of pharmaceuticals and endocrine disrupting compounds, and demonstrates the role aquatic organisms have in redistributing microplastics between aquatic and terrestrial ecosystems.

Experimental and computational evaluation of dipeptidyl peptidase III inhibitors based on quinazolinone-Schiff's bases.

Dipeptidyl peptidase III (DPP III) is a zinc-dependent enzyme that sequentially hydrolyzes biologically active peptides by cleaving dipeptides from their N-termini. Although its fundamental role is not been fully elucidated, human DPP III (hDPP III) has been recognized in several pathophysiological processes of interest for drug development. In this article 27 quinazolinone-Schiff's bases were studied for their inhibitory activity against hDPP III combining an in vitro experiment with a computational approach. The biochemical assay showed that most compounds exhibited inhibitory activity at the 100 µM concentration. The best QSAR model included descriptors from the following 2D descriptor groups: information content indices, 2D autocorrelations, and edge adjacency indices. Five compounds were found to be the most potent inhibitors with IC50 values below 10 µM, while molecular docking predicted that these compounds bind to the central enzyme cleft and interact with residues of the substrate binding subsites. Molecular dynamics simulations of the most potent inhibitor (IC50=0.96 µM) provided valuable information explaining the role of PHE109, ARG319, GLU327, GLU329, and ILE386 in the mechanism of the inhibitor binding and stabilization. This is the first study that gives insight into quinazolinone-Schiff's bases binding to this metalloenzyme.Communicated by Ramaswamy H. Sarma.

Neuropeptides, substrates and inhibitors of human dipeptidyl peptidase III, experimental and computational study - A new substrate identified.

Dipeptidyl peptidase III (DPP III) is a cytosolic, two-domain zinc-exopeptidase. It is widely distributed in mammalian tissues, where it's involved in the final steps of normal intracellular protein degradation. However, its pronounced affinity for some bioactive peptides (angiotensins, enkephalins, and endomorphins) suggests more specific functions such as blood pressure regulation and involvement in pain regulation. We have investigated several different neuropeptides as potential substrates and inhibitors of human DPP III. The binding affinities and kinetic data determined by isothermal titration calorimetry, in combination with measurements of enzyme inhibition identified the hemorphin-related valorphin, tynorphin, S-tynorphin, and I-tynorphin as the most potent inhibitors of DPP III (actually slow substrates), whereas hemorphin-4 proved to be the best substrate of all neuropeptides examined. In addition, we have shown that the neuropeptides valorphin, Leu-valorphin-Arg, and the opioid peptide ß-casomorphin, are DPP III substrates. The molecular modelling of selected peptides shows uniform binding to the lower domain ß-strand residues of DPP III via peptide backbone atoms, but also previously unrecognized stabilizing interactions with conserved residues of the metal-binding site and catalytic machinery in the upper domain. The computational data helped explain the differences between substrates that are hydrolyzed effectively and those hydrolysed slowly by DPP III.

Triarylborane Dyes as a Novel Non-Covalent and Non-Inhibitive Fluorimetric Markers for DPP III Enzyme.

Novel dyes were prepared by simple "click CuAAC" attachment of a triarylborane-alkyne to the azide side chain of an amino acid yielding triarylborane dye 1 which was conjugated with pyrene (dye 2) forming a triarylborane-pyrene FRET pair. In contrast to previous cationic triarylboranes, the novel neutral dyes interact only with proteins, while their affinity to DNA/RNA is completely abolished. Both the reference triarylborane amino acid and triarylborane-pyrene conjugate bind to BSA and the hDPP III enzyme with high affinities, exhibiting a strong (up to 100-fold) fluorescence increase, whereby the triarylborane-pyrene conjugate additionally retained FRET upon binding to the protein. Furthermore, the triarylborane dyes, upon binding to the hDPP III enzyme, did not impair its enzymatic activity under a wide range of experimental conditions, thus being the first non-covalent fluorimetric markers for hDPP III, also applicable during enzymatic reactions with hDPP III substrates.

Coumarin Derivatives Act as Novel Inhibitors of Human Dipeptidyl Peptidase III: Combined In Vitro and In Silico Study.

Dipeptidyl peptidase III (DPP III), a zinc-dependent exopeptidase, is a member of the metalloproteinase family M49 with distribution detected in almost all forms of life. Although the physiological role of human DPP III (hDPP III) is not yet fully elucidated, its involvement in pathophysiological processes such as mammalian pain modulation, blood pressure regulation, and cancer processes, underscores the need to find new hDPP III inhibitors. In this research, five series of structurally different coumarin derivatives were studied to provide a relationship between their inhibitory profile toward hDPP III combining an in vitro assay with an in silico molecular modeling study. The experimental results showed that 26 of the 40 tested compounds exhibited hDPP III inhibitory activity at a concentration of 10 µM. Compound 12 (3-benzoyl-7-hydroxy-2H-chromen-2-one) proved to be the most potent inhibitor with IC50 value of 1.10 µM. QSAR modeling indicates that the presence of larger substituents with double and triple bonds and aromatic hydroxyl groups on coumarin derivatives increases their inhibitory activity. Docking predicts that 12 binds to the region of inter-domain cleft of hDPP III while binding mode analysis obtained by MD simulations revealed the importance of 7-OH group on the coumarin core as well as enzyme residues Ile315, Ser317, Glu329, Phe381, Pro387, and Ile390 for the mechanism of the binding pattern and compound 12 stabilization. The present investigation, for the first time, provides an insight into the inhibitory effect of coumarin derivatives on this human metalloproteinase.

The guanidiniocarbonylpyrrole-fluorophore conjugates as theragnostic tools for dipeptidyl peptidase III monitoring and inhibition.

Study of seven new guanidiniocarbonylpyrrole (GCP)-fluorophore conjugates interactions with dipeptidyl peptidase III (DPP III) showed that all compounds bind strongly (Ks ≈ µM) to enzyme active site, but with very different fluorimetric response (varying from quenching to strong increase), dependent on the fluorophore type and intramolecular pre-organisation of molecule. Positively charged lysine side chain improved significantly compound solubility but diminished fluorescence increase upon DPP III binding and completely abolished inhibitory effect on DPP III activity, whereas linker-neutral analogues showed stronger emission increase and were efficient enzyme inhibitors. By far the best fluorimetric response and inhibitive properties showed cyanine-GCP analogue, thus being promising lead compound for both enzyme sensing and bio-activity inhibiting (theragnostic) studies of DPP III in the future.Communicated by Ramaswamy H. Sarma.

Conservation of the conformational dynamics and ligand binding within M49 enzyme family.

The hydrogen deuterium exchange (HDX) mass spectrometry combined with molecular dynamics (MD) simulations was employed to investigate conformational dynamics and ligand binding within the M49 family (dipeptidyl peptidase III family). Six dipeptidyl peptidase III (DPP III) orthologues, human, yeast, three bacterial and one plant (moss) were studied. According to the results, all orthologues seem to be quite compact wherein DPP III from the thermophile Caldithrix abyssi seems to be the most compact. The protected regions are located within the two domains core and the overall flexibility profile consistent with semi-closed conformation as the dominant protein form in solution. Besides conservation of conformational dynamics within the M49 family, we also investigated the ligand, pentapeptide tynorphin, binding. By comparing HDX data obtained for unliganded protein with those obtained for its complex with tynorphin it was found that the ligand binding mode is conserved within the family. Tynorphin binds within inter-domain cleft, close to the lower domain ß-core and induces its stabilization in all orthologues. Docking combined with MD simulations revealed details of the protein flexibility as well as of the enzyme-ligand interactions.

A novel Porphyromonas gingivalis enzyme: An atypical dipeptidyl peptidase III with an ARM repeat domain.

Porphyromonas gingivalis, an asaccharolytic Gram-negative oral anaerobe, is a major pathogen associated with adult periodontitis, a chronic infective disease that a significant percentage of the human population suffers from. It preferentially utilizes dipeptides as its carbon source, suggesting the importance of dipeptidyl peptidase (DPP) types of enzyme for its growth. Until now DPP IV, DPP5, 7 and 11 have been extensively investigated. Here, we report the characterization of DPP III using molecular biology, biochemical, biophysical and computational chemistry methods. In addition to the expected evolutionarily conserved regions of all DPP III family members, PgDPP III possesses a C-terminal extension containing an Armadillo (ARM) type fold similar to the AlkD family of bacterial DNA glycosylases, implicating it in alkylation repair functions. However, complementation assays in a DNA repair-deficient Escherichia coli strain indicated the absence of alkylation repair function for PgDPP III. Biochemical analyses of recombinant PgDPP III revealed activity similar to that of DPP III from Bacteroides thetaiotaomicron, and in the range between activities of human and yeast counterparts. However, the catalytic efficiency of the separately expressed DPP III domain is ~1000-fold weaker. The structure and dynamics of the ligand-free enzyme and its complex with two different diarginyl arylamide substrates was investigated using small angle X-ray scattering, homology modeling, MD simulations and hydrogen/deuterium exchange (HDX). The correlation between the experimental HDX and MD data improved with simulation time, suggesting that the DPP III domain adopts a semi-closed or closed form in solution, similar to that reported for human DPP III. The obtained results reveal an atypical DPP III with increased structural complexity: its superhelical C-terminal domain contributes to peptidase activity and influences DPP III interdomain dynamics. Overall, this research reveals multifunctionality of PgDPP III and opens direction for future research of DPP III family proteins.

A novel plant enzyme with dual activity: an atypical Nudix hydrolase and a dipeptidyl peptidase III.

In a search for plant homologues of dipeptidyl peptidase III (DPP III) family, we found a predicted protein from the moss Physcomitrella patens (UniProt entry: A9TLP4), which shared 61% sequence identity with the Arabidopsis thaliana uncharacterized protein, designated Nudix hydrolase 3. Both proteins contained all conserved regions of the DPP III family, but instead of the characteristic hexapeptide HEXXGH zinc-binding motif, they possessed a pentapeptide HEXXH, and at the N-terminus, a Nudix box, a hallmark of Nudix hydrolases, known to act upon a variety of nucleoside diphosphate derivatives. To investigate their biochemical properties, we expressed heterologously and purified Physcomitrella (PpND) and Arabidopsis (AtND) protein. Both hydrolyzed, with comparable catalytic efficiency, the isopentenyl diphosphate (IPP), a universal precursor for the biosynthesis of isoprenoid compounds. In addition, PpND dephosphorylated four purine nucleotides (ADP, dGDP, dGTP, and 8-oxo-dATP) with strong preference for oxidized dATP. Furthermore, PpND and AtND showed DPP III activity against dipeptidyl-2-arylamide substrates, which they cleaved with different specificity. This is the first report of a dual activity enzyme, highly conserved in land plants, which catalyzes the hydrolysis of a peptide bond and of a phosphate bond, acting both as a dipeptidyl peptidase III and an atypical Nudix hydrolase.

Validation of flavonoids as potential dipeptidyl peptidase III inhibitors: Experimental and computational approach.

Fifteen flavonoids were studied for their inhibitory activity against human dipeptidyl peptidase III (hDPP III) combining an in vitro assay with an in silico molecular modeling study. All analyzed flavonoids showed inhibitory effects against hDPP III with the IC50 values ranging from 22.0 to 437.2 µm. Our 3D QSAR studies indicate that the presence of hydrophilic regions at a flavonoid molecule increases its inhibitory activity, while the higher percentage of hydrophobic surfaces has negative impact on enzyme inhibition. Furthermore, molecular dynamics (MD) simulations of the complex of hDPP III with one of the most potent inhibitors, luteolin, were performed, and binding mode analysis revealed that the 3' and 4' hydroxyl group on B-ring as well as 5 and 7 hydroxyl group on A-ring helps luteolin to interact with the Asn391, Asn406, Tyr417, His450, Glu451, Val447, Glu512, Asn545, Gln566, and Arg572 residues. The MD results clearly provide valuable information explaining the importance of flavonoid hydroxyl groups in the mechanism for the binding pattern at the active site of hDPP III.

Aspartate 496 from the subsite S2 drives specificity of human dipeptidyl peptidase III.

Human dipeptidyl peptidase III (hDPP III) is a member of the M49 metallopeptidase family, which is involved in intracellular protein catabolism and oxidative stress response. To investigate the structural basis of hDPP III preference for diarginyl arylamide, using site-directed mutagenesis, we altered its S2 subsite to mimic the counterpart in yeast enzyme. Kinetic studies revealed that the single mutant D496G lost selectivity due to the increase of the Km value. The D496G, but not S504G, showed significantly decreased binding of peptides with N-terminal arginine, and of tynorphin. The results obtained identify Asp496 as an important determinant of human DPP III substrate specificity.

Molecular determinants of human dipeptidyl peptidase III sensitivity to thiol modifying reagents.

Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49, involved in protein metabolism and oxidative stress response. DPPIII crystal structure shows the two lobe-like domains separated by a wide cleft. The human enzyme has a total of six cysteines, three in the lower (Cys19, Cys147,and Cys176) and three in the upper (Cys509, Cys519,and Cys654), catalytic, domain containing the active site zinc ion. To elucidate the molecular basis of this enzyme ' s susceptibility to sulfhydryl reagents, biochemical analysis of a set of Cys to Ala mutants was used, supported by mass spectrometry. Cys176, a residue 44 A apart from the catalytic center of the ligand-free enzyme, was found responsible for the inactivation with the submicromolar concentration of an organomercurial compound, and three additional cysteines contributed to sensitivity to aromatic disulfides. Upon treatment with oxidized glutathione [glutathione disulfide(GSSG)], cysteine residues at positions 147, 176, and 654 were found glutathionylated. The mutational analysis confirmed the involvement of Cys176 and Cys654 inhuman DPP III inactivation by GSSG. Observation that Cys176, a residue quite distant from the active center,contributes to enzyme inactivation, indicates that the substrate-binding site of human DPP III comprises both lower and upper protein domain.