Tyr Pro Research Articles

Molecular docking and molecular dynamics simulation of wheat gluten-derived antioxidant peptides acting through the Keap1-Nrf2 pathway.

In our previous study, we successfully identified five peptides from wheat gluten: Ala-Pro-Ser-Tyr (APSY), Leu-Tyr (LY), Pro-Tyr (PY), Arg-Gly-Gly-Tyr (RGGY) and Tyr-Gln (YQ). Molecular docking and molecular dynamics simulation methods were employed to investigate the interaction between these antioxidant peptides and the Kelch-like ECH-associated protein 1 (Keap1 protein), revealing the molecular mechanism of their non-competitive binding. In addition, the total antioxidant capacity of the five peptides was determined using the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) method. The affinities of APSY, LY, PY, RGGY and YQ were -8.9, -8.3, -8.5, -9.1 and - 7.9 kcal mol-1, respectively. The five peptides effectively bound to Keap1 protein through hydrogen, π-σ, π-alkyl and alkyl interactions. Significant roles were observed for the P1 pocket residue ARG-415 and the P3 pocket residue ALA-556 in the interactions of the Keap1-peptide complexes. Molecular dynamics simulations further elucidated the dynamic process of peptide binding to the Keap1 protein. All five peptides formed stable complexes with Keap1 protein, with van der Waals forces playing crucial roles in these complex systems, indicative of the peptides' strong binding ability to Keap1 protein. The van der Waals forces were -178.74, -123.11, -134.36, -132.59, and -121.44 kJ mol-1 for the Keap1-APSY, Keap1-LY, Keap1-PY, Keap1-RGGY and Keap1-YQ complexes, respectively. These peptides exhibited excellent antioxidant effects. Among them, the YQ peptide exhibited the highest total antioxidant capacity, with an activity value of 1.18 ± 0.06 mmol Trolox equivalent (TE) L-1 at a concentration of 0.10 mg mL-1. The RGGY, PY, LY and APSY peptides followed in descending order, with activity values of 0.91 ± 0.05, 0.72 ± 0.06, 0.62 ± 0.04 and 0.60 ± 0.05 mmol TE L-1, respectively. These results unveiled the molecular mechanism by which the five antioxidant peptides act on active pockets through the Keap1-Nrf2 signaling pathway, providing a theoretical basis for the development of antioxidants. © 2024 Society of Chemical Industry.

Inhibitory effects and action mechanism of five antioxidant peptides derived from wheat gluten on cells oxidative stress injury

Our previous study identified five peptides Leu-Tyr (LY), Pro-Tyr (PY), Tyr-Gln (YQ), Ala-Pro-Ser-Tyr (APSY), and Arg-Gly-Gly-Tyr (RGGY) from wheat gluten. However, the mechanism of antioxidant action and structure-function relationship of these five peptides were still unclear. In this study, we found that five peptides induced by AAPH in human embryonic kidney cells (HEK-293) suppressed the intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) production. Moreover, they also upregulated the antioxidant activity-related enzymes (SOD, CAT and GSH-Px) and GSH activities. Additionally, five peptides treatment decreased Keap1 protein level and increased Nrf2 and HO-1proteins levels. It was concluded that five peptides showed promise in activating the Keap1-Nrf2-HO-1 signaling pathway and enhancing the antioxidant defense system. Quantum chemical calculation and molecular docking technologies were used to explore the action mechanism of five wheat peptides. Five antioxidant peptides were able to effectively bind Keap1, and the peptides predominantly formed hydrogen and π bonds during their binding with the active pocket region of Keap1.

A novel epitope tag from rabies virus has versatile in vitro applications.

Fusion tag technology is an important tool for rapid separation, purification, and characterization of proteins. Combined with monoclonal antibodies, tag epitope systems can be rapidly adapted to many assay systems. A monoclonal antibody that reacts with the matrix protein of the rabies virus CVS-11 strain was reported. The epitope (termed M) targeted by this antibody contains only six amino acids. We examine whether this specific sequence epitope can be applied as a protein tag. We show ectopic expression of M-tagged proteins has little impact on cell viability or major signaling pathways. The M tag system can be used for western blotting, immunoprecipitation, immunofluorescence staining, and flow cytometry assays. The results indicate the specificity, sensitivity, and versatility of this novel epitope tag system are comparable to the widely used FLAG tag system, providing researchers with an additional tool for molecular analysis. KEY POINTS: • A short peptide (Pro Pro Tyr Asp Asp Asp) can be applied as a new tag. • The new epitope-tagging fusion system has no effect on the main cellular signaling pathway. • The epitope-tagging fusion system can be widely used for western blotting, immunoprecipitation, immunofluorescence, flow cytometry, etc.

The C-terminal pentapeptide acein analogue (JMV3315) stimulates dopamine release in the brain

We have previously reported the synthesis and biological activity of a newly identified peptide of sequence H–Pro–Pro–Thr–Thr–Thr–Lys–Phe–Ala–Ala–OH called acein that is able to stimulate dopamine release in the brain of rodents in vivo and ex vivo by interacting with angiotensin converting enzyme (ACE). In the present piece of work, we studied the structure–activity relationships of acein using displacement experiments of the labelled ligand [125I]Tyr–Pro–Pro–Thr–Thr–Thr–Lys–Phe–Ala–Ala–OH on guinea pig brain membranes, known to have high-affinity acein binding sites. We determined that the C-terminal pentapeptide H–Thr–Lys–Phe–Ala–Ala–OH is the minimal structure able to interact with high affinity (Ki (inhibitory constant) 13 ± 2 nM) with acein binding sites. Among the analogues of the pentapeptide that were synthesized, the pentapeptide H–Thr–Lys–Tyr–Ala–Ala–OH showed the highest affinity (Ki 3.7 ± 1.0 nM). Accordingly, this pentapeptide was able to stimulate dopamine release from striatal slices taken from the sensorimotor territory of rats.

PH-induced changes in β-casomorphin 7 structure studied by 1H nuclear magnetic resonance and Fourier-transform infrared spectroscopy

β-Casomorphins are opioid peptides that can be liberated from β-casein during digestion, with some suggested effects on human physiology linked to their properties. In this study, the structural behaviour of β-casomorphin 7 (BCM7; Tyr–Pro–Phe–Pro–Gly–Pro–Ile) was studied at pH 2.3 and 6.7 using 1H nuclear magnetic resonance (1H NMR) and Fourier-transform infrared (FTIR) spectroscopy. Cis–trans isomerism involving the Tyr1–Pro2, Phe3–Pro4 and Gly5–Pro6 bonds of BCM7 was observed. Cis and trans isomers of Tyr1–Pro2 and Phe3–Pro4 and trans isomers for Gly5–Pro6 were observed at both pH 6.7 and 2.3 but cis isomers of Gly5–Pro6 were only observed at pH 2.3 and not at pH 6.7. These pH-induced conformational changes at the Gly5–Pro6 bond of BCM7 may play important role in enzymatic release of this peptide from the parent protein β-casein under gastro-intestinal conditions, as well as further cleavage of BCM7 into smaller peptides.

Reactivity of Tyrosyl–Proline toward Benzoylation in Aqueous 1,4-Dioxane

The kinetics of the reaction of L-tyrosyl-L-proline (Tyr–Pro) with di- and trinitrophenyl benzoates in aqueous 1,4-dioxane (40 wt % of water) were studied in the temperature range 298–313 K. The reaction rate constant k298 was found to vary in the range from 0.035 to 0.564 L·mol–1·s–1, and the activation barrier changed from 39 to 51 kJ/mol. The neutral and anionic forms of the dipeptide were simulated by the DFT/B3LYP/ cc-pVTZ method. Natural bond orbital analysis of the electron density distribution in these forms showed that the preferred nucleophilic center in the dipeptide molecule is the nitrogen atom of the primary amino group rather than the oxygen atom of the phenolic hydroxy group.

Praline metabolism by germinating Lilium longiflorum pollen. I. Labelling of cytoplasmic, wall and culture medium molecules

Radioactivity occurs in trithloroacetic acid (TCA)-soluble and precipitable, cytoplasm and salt-washed walls following germination of <em>Lilium longiflorum</em>, cv. 'Ace' pollen in medium containing [<sup>14</sup>C]-proline (Pro). Sephadex gel filtration on G-25 through G-100 was employed to determine whether radioactivity in cytoplasm, wall and growth medium from pollen fed [<sup>14</sup>C]-Pro or [<sup>3</sup>H]=Pro plus [<sup>14</sup>C]-arafbinose (Ara) was contained within molecules possessing molecular weights of 5,000 to 100,000 daltones or greater. G-25 elution profiles of a crude cytoplasmic fraction (15,000 X g supernatant) from [<sup>14</sup>C]-Pro labelled pollen yielded a radioctive void volume peak and a retarded peak. The void volume peak contained hydroxyproline (Hyp), and exhibited a coincidence of [<sup>3</sup>H]-Pro and [<sup>14</sup>C] -Ara labelling when pollen was double labelled with the two isotopes. This peak also contained radioactivity when pollen was germinated in 2-[<sup>3</sup>H]-myo-inositol. Germination in medium supplemented with 100 µM 2,2'-dipyridyl eliminated radioactivity from 2-[<sup>3</sup>H]-myo-inositol or [<sup>14</sup>C]-,Pro in the peak. Filtratian on G-25 of aTCA-soluble fraction of a salt-extract of walls from [<sup>14</sup>C]-Pro labelled pollen resulted in void volume and two retarded peaks. Void volume and two retarded peaks were also obtained upon G-25 filtration of a cellulase-digest of walls from [M]-Pro labeled pollen. The void volume peak contained Hyp, Lys, Gly, Ala, Ser, Glu and Asp acids, Val, Tyr, Leu or lieu and Pro. Sephadex G-90, 75, and 100 elution profiles of cellulasedigests of walls from [<sup>3</sup>H]-,Pro and [<sup>14</sup>C]-Ara labelled pollen yielded radioactive retarded and Hyp-containing void volume peaks with a coincidence of [<sup>3</sup>H] and [<sup>14</sup>C] labelling. Label in the void volume was obtained when either rhozyme P11- or pepsin-digests of walls from [<sup>14</sup>C]-Pro labelled pollen were gel filtered on G-50. Paper electrophoresis coupled with paper chromatography of acid hydrolyzates of salt-washed wall fractions demonstrated 15 of the common amino acids. Gel filtration on G-25 of growth medium in which pollen was germinated resulted in two peaks, one of which eluted in the void volume. contained Hyp and excluded during subsequent gel filtration on G-100.

Effect of potent endomorphin degradation blockers on analgesic and antidepressant-like responses in mice

The biological effects of endomorphins (EMs) are short-lasting due to their rapid degradation by endogenous enzymes. Competing enzymatic degradation is an approach to prolong EM bioavailability. In the present study, a series of tetra- and tripeptides of similar to EMs structure was synthesized and tested in vitro and in vivo for their ability to inhibit degradation of EMs. The obtained results indicated that, among the series of analogs, the tetrapeptide Tyr–Pro–d–ClPhe–Phe–NH2 and the tripeptide Tyr–Pro–Ala–NH2, which did not bind to the μ-opioid receptors, were potent inhibitors of EM catabolism in rat brain homogenate. In vivo, these two peptides significantly prolonged the analgesic and antidepressant-like effects, induced by exogenous EMs, by blocking EM degrading enzymes. These new potent inhibitors may therefore increase the level and the half life of endogenous EMs and could be used in a new therapeutic strategy against pain and mood disorders, based on increasing of EM bioavailability.

Conformational analysis and vibrational spectroscopic investigation of l-proline–tyrosine ( l-Pro–Tyr) dipeptide

In this study the conformational properties of the drug based dipeptide l-proline– l-tyrosine (Pro–Tyr) in its monomeric and dimeric forms, have been investigated by molecular mechanic and ab initio calculations. The energy calculations on Pro–Tyr dipeptide as a function of side chains torsion angles, enable us to determine their energetically preferred conformations. One-hundred and eight possible conformations of Pro–Tyr dipeptide have been investigated by conformational analysis and the low energy conformations of dipeptide have been determined by using the Ramachandran maps. Afterwards, the geometrical parameters of obtained stable conformations were used as starting parameters for quantum chemical calculations. The molecular structure of Pro–Tyr dipeptide, in the ground electronic state (in vacuum) was optimized by density functional theory method with B3LYP functional and using 6-31G(d,p) and 6-31++G(d,p) basis sets. The dimeric forms of the dipeptide were also formed and energetically preferred conformations of dimers were investigated using the same method and the same level of theory by using 6-31G(d,p) basic set. The fundamental vibrational wavenumbers, IR intensities and Raman activities of the global conformation of monomeric and dimeric forms of the dipeptide were calculated and compared with the experimental vibrational spectra of solid Pro–Tyr dipeptide. The total energy distributions (TED) of the vibrational modes were calculated by using Scaled Quantum Mechanical (SQM) analysis. Vibrational assignment was performed on the basis of calculated total energy distribution (TED) of the modes.

Synthesis and activity of endomorphin-2 and morphiceptin analogues with proline surrogates in position 2

The opioid agonists endomorphins (Tyr–Pro–Trp–Phe–NH 2; EM1 and Tyr–Pro–Phe–Phe–NH 2; EM2) and morphiceptin (Tyr–Pro–Phe–Pro–NH 2) exhibit an extremely high selectivity for μ-opioid receptor. Here a series of novel EM2 and morphiceptin analogues containing in place of the proline at position 2 the S and R residues of β-homologues of proline (HPro), of 2-pyrrolidinemethanesulphonic acid (HPrs) and of 3-pyrrolidinesulphonic acid (βPrs) have been synthesized and their binding affinity and functional activity have been investigated. The highest μ-receptor affinity is shown by [(S)βPrs 2]EM2 analogue ( 6e) which represents the first example of a β-sulphonamido analogue in the field of opioid peptides.

Two chemical derivatives of bacterial metabolites suppress cellular immune responses and enhance pathogenicity of Bacillus thuringiensis against the diamondback moth, Plutella xylostella

Eicosanoids mediate insect immune responses, especially against bacterial infection. Phospholipase A 2 (PLA 2) catalyzes the committed step of the eicosanoid biosynthesis pathway. Three PLA 2 inhibitors have been identified from metabolites of an entomopathogenic bacterium, Xenorhabdus nematophila: benzylideneacetone (BZA), Pro-Tyr (PY), and acetylated Phe-Gly-Val (Ac-FGV). Interestingly, they share benzenepropane as a core chemical structure. We analyzed the functional significance of the core structure using structural derivatives. Removing a phenyl ring from PY resulted in significant loss of the PLA 2 inhibitory activity, as seen in a Pro-Ala derivative. Though the p-hydroxyl group was not critical in PY as seen in Pro-Phe derivative, its addition to BZA resulted in significant loss of inhibitory activity. Some alterations of structures other than the core structure increased PLA 2-inhibitory activity in some derivatives, including Ala-Tyr (AY) and Phe-Gly-Val (FGV) derivatives. Using these selected derivatives, we further analyzed synergistic effects on pathogenicity of Bacillus thuringiensis (Bt) against the second instar larvae of Plutella xylostella. These two derivatives significantly enhanced the Bt pathogenicity. This study introduces two novel compounds that inhibit PLA 2 and suggests their application in combination with Bt to control P. xylostella.

Derivatives of human β-Casein fragments (54–59) exhibit highly potent immunosuppressant activity

Human β-casein fragment (54–59) having the amino acid sequence Val–Glu–Pro–Ile–Pro–Tyr, has shown potent immunostimulant activity. Several analogs of this hexapeptide have been synthesized with modification at the N-terminal region and two analogs, viz. peptide I and peptide II have shown significant immunosuppressant activity in-vivo mouse model. Effect on cell mediated immunity (CMI) and humoral immunity was studied in mouse/SRBC model. Both the peptides failed to stimulate immune response in vivo and showed inhibition of CMI and humoral response to sheep red blood cells (SRBC). Peptides showed inhibition in alloantigen induced lymphocyte proliferation, i.e., mixed lymphocyte reaction (MLR) in vitro. Treatment with peptides inhibited the production of interferon-γ (IFN-γ), and increased the production of interleukin-4 (IL-4) as well as improved the skin graft survival. Cyclosporine a known immunosuppressant showed similar effect on mouse model. Present study thus provides a lead for the development of safe and effective immunosuppressant.

The inositol phosphatase MTMR4 is a novel target of the ubiquitin ligase Nedd4

The inositol phosphatase, MTMR4 (myotubularin-related protein 4), was identified as a novel interactor of the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). hMTMR4 (human MTMR4) and Nedd4 co-immunoprecipitated and co-localized to late endosomes. The PY (Pro-Tyr) motif of hMTMR4 binds to WW (Trp-Trp) domains of hNedd4. MTMR4 expression was decreased in atrophying muscle, whereas Nedd4 expression was increased and hMTMR4 was ubiquitinated by hNedd4, suggesting that this novel interaction may underlie the biological process of muscle breakdown.

A novel TRH analog, Glp–Asn–Pro– d-Tyr– d-TrpNH 2, binds to [ 3H][3-Me-His 2]TRH-labelled sites in rat hippocampus and cortex but not pituitary or heterologous cells expressing TRHR1 or TRHR2

Glp–Asn–Pro– d-Tyr– d-TrpNH 2 is a novel synthetic peptide that mimics and amplifies central actions of thyrotropin-releasing hormone (TRH) in rat without releasing TSH. The aim of this study was to compare the binding properties of this pentapeptide and its all- l counterpart (Glp–Asn–Pro–Tyr–TrpNH 2) to TRH receptors in native rat brain tissue and cells expressing the two TRH receptor subtypes identified in rat to date, namely TRHR1 and TRHR2. Radioligand binding studies were carried out using [ 3H][3-Me-His 2]TRH to label receptors in hippocampal, cortical and pituitary tissue, GH4 pituitary cells, as well as CHO cells expressing TRHR1 and/or TRHR2. In situ hybridization studies suggest that cortex expresses primarily TRHR2 mRNA, hippocampus primarily TRHR1 mRNA and pituitary exclusively TRHR1 mRNA. Competition experiments showed [3-Me-His 2]TRH potently displaced [ 3H][3-Me-His 2]TRH binding from all tissues/cells investigated. Glp–Asn–Pro– d-Tyr– d–TrpNH 2 in concentrations up to 10 −5 M did not displace [ 3H][3-Me-His 2]TRH binding to membranes derived from GH4 cells or CHO-TRHR1 cells, consistent with its lack of binding to pituitary membranes and TSH-releasing activity. Similar results were obtained for the corresponding all- l peptide. In contrast, both pentapeptides displaced binding from rat hippocampal membranes (pIC 50 Glp–Asn–Pro– d-Tyr– d-TrpNH 2: 7.7 ± 0.2; pIC 50 Glp–Asn–Pro–Tyr–TrpNH 2: 6.6 ± 0.2), analogous to cortical membranes (pIC 50 Glp–Asn–Pro– d-Tyr– d-TrpNH 2: 7.8 ± 0.2; pIC 50 Glp–Asn–Pro–Tyr–TrpNH 2: 6.6 ± 0.2). Neither peptide, however, displaced [ 3H][3-Me-His 2]TRH binding to CHO-TRHR2. Thus, this study reveals for the first time significant differences in the binding properties of native and heterologously expressed TRH receptors. Also, the results raise the possibility that Glp–Asn–Pro– d-Tyr– d-TrpNH 2 is not displacing [ 3H][3-Me-His 2]TRH from a known TRH receptor in rat cortex, but rather a hitherto unidentified TRH receptor.

Domain architecture and structure of the bacterial cell division protein DivIB

Bacterial cytokinesis requires the coordinated assembly of a complex of proteins, collectively known as the divisome, at the incipient division site. DivIB/FtsQ is a conserved component of the divisome in bacteria with cell walls, suggesting that it plays a role in synthesis and/or remodeling of septal peptidoglycan. We demonstrate that the extracytoplasmic region of DivIB comprises three discrete domains that we designate alpha, beta, and gamma from the N to C terminus. The alpha-domain is proximal to the cytoplasmic membrane and coincident with the polypeptide transport-associated domain that was proposed previously to function as a molecular chaperone. The beta-domain has a unique 3D fold, with no eukaryotic counterpart, and we show that it interconverts between two discrete conformations via cis-trans isomerization of a Tyr-Pro peptide bond. We propose that this isomerization might modulate protein-protein interactions of the flanking alpha- and gamma-domains. The C-terminal gamma-domain is unstructured in the absence of other divisomal proteins, but we show that it is critical for DivIB function.

Crystal structure of dimeric FabZ of Plasmodium falciparum reveals conformational switching to active hexamers by peptide flips

The crystal structure of β-hydroxyacyl acyl carrier protein dehydratase of Plasmodium falciparum (PfFabZ) has been determined at a resolution of 2.4 Å. PfFabZ has been found to exist as a homodimer (d-PfFabZ) in the crystals of the present study in contrast to the reported hexameric form (h-PfFabZ) which is a trimer of dimers crystallized in a different condition. The catalytic sites of this enzyme are located in deep narrow tunnel-shaped pockets formed at the dimer interface. A histidine residue from one subunit of the dimer and a glutamate residue from the other subunit lining the tunnel form the catalytic dyad in the reported crystal structures. While the position of glutamate remains unaltered in the crystal structure of d-PfFabZ compared to that in h-PfFabZ, the histidine residue takes up an entirely different conformation and moves away from the tunnel leading to a His-Phe cis–trans peptide flip at the histidine residue. In addition, a loop in the vicinity has been observed to undergo a similar flip at a Tyr–Pro peptide bond. These alterations not only prevent the formation of a hexamer but also distort the active site geometry resulting in a dimeric form of FabZ that is incapable of substrate binding. The dimeric state and an altered catalytic site architecture make d-PfFabZ distinctly different from the FabZ structures described so far. Dynamic light scattering and size exclusion chromatographic studies clearly indicate a pH-related switching of the dimers to active hexamers.

D-1-Nal 4]endomorphin-2 is a potent μ-opioid receptor antagonist in the aequorin luminescence-based calcium assay

A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca 2+ levels, was used to examine relative potency and efficacy of the μ-opioid receptor antagonists. A series of position 3- and 4-substituted endomorphin-2 (Tyr–Pro–Phe–Phe–NH 2) analogues containing d-3-(1-naphthyl)-alanine ( d-1-Nal) or d-3-(2-naphthyl)-alanine ( d-2-Nal), which were previously shown to reverse antinociception induced by endomorphin-2 in the in vivo hot-plate test in mice, was tested in the aequorin luminescence-based calcium assay to examine their μ-opioid antagonist potency in vitro. A recombinant mammalian cell line expressing the μ-opioid receptor together with a luminescent reporter protein, apoaequorin, was used in the study. The results obtained in this functional assay indicated that analogues with d-1-Nal or d-2-Nal substitutions in position 4 of endomorphin-2 are strong μ-opioid receptor antagonists, while those substituted in position 3 are partial agonists. Exceptional antagonist potency in the calcium assay was observed for [ d-1-Nal 4]endomorphin-2. The pA 2 value for this analogue was 7.95, compared to the value of 8.68 obtained for the universal, non-selective opioid antagonist of the alkaloid structure, naloxone. The obtained results were compared with the data from the hot-plate test in mice. In that in vivo assay [ d-1-Nal 4]endomorphin-2 was also the most potent analogue of the series.

Divergent effects of Aβ1–42 on ionotropic glutamate receptor-mediated responses in CA1 neurons in vivo

Aggregated Aβ1–42 is hypothesized to be the central cause of Alzheimer's disease. However, early changes in synaptic activity may be detected in the disease long before a significant cell loss is manifested. Despite the fact that Aβ1–42 interference with long-term potentiation (LTP) and the field excitatory postsynaptic potential (fEPSP) is well documented, the exact mechanism of these events remains to be clarified. Here we studied the effects of iontophoretically applied Aβ1–42 on the neuronal firing evoked in vivo on the CA1 hippocampal neurons of Wistar rats by different agonists of the ionotropic glutamate receptors: N-methyl- d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainic acid (KA). NMDA elicited firing enhanced in all of the measured cells; in contrast, the AMPA-mediated responses decreased significantly after Aβ1–42 ejection. The changes in KA-evoked responses to Aβ1–42 revealed two types of cells. In the first type, the KA-mediated firing remained at the control level, while in the second type, Aβ1–42 attenuated the KA-evoked responses. A protective pentapeptide, Leu–Pro–Tyr–Phe–Asp–amide, was used to verify the specificity of these β-amyloid-elicited effects. The pentapeptide protected against the modulatory effects of Aβ1–42 on the NMDA and AMPA responses. In conclusion, we have shown that Aβ1–42 exerts divergent effects on the activity of the ionotropic glutamate receptors in vivo. These results suggest that the LTP disruption and fEPSP attenuation seen after Aβ1–42 application are in part due to the altered function of these receptors.

Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue

Prolyl endopeptidases have potential for treating coeliac sprue, a disease of the intestine caused by proteolytically resistant peptides from proline-rich prolamins of wheat, barley and rye. We compared the properties of three similar bacterial prolyl endopeptidases, including the known enzymes from Flavobacterium meningosepticum (FM) and Sphingomonas capsulate (SC) and a novel enzyme from Myxococcus xanthus (MX). These enzymes were interrogated with reference chromogenic substrates, as well as two related gluten peptides (PQPQLPYPQPQLP and LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), believed to play a key role in coeliac sprue pathogenesis. In vitro and in vivo studies were conducted to evaluate the activity, specificity and acid/protease stability of the enzymes. All peptidases were relatively resistant to acid, pancreatic proteases and membrane peptidases of the small intestinal mucosa. Although their activities against reference substrates were similar, the enzymes exhibited substantial differences with respect to chain length and subsite specificity. SC hydrolysed PQPQLPYPQPQLP well, but had negligible activity against LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. In contrast, the FM and MX peptidases cleaved both substrates, although the FM enzyme acted more rapidly on LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF than MX. Whereas the FM enzyme showed a preference for Pro-Gln bonds, SC cleaved both Pro-Gln and Pro-Tyr bonds with comparable efficiency, and MX had a modest preference for Pro-(Tyr/Phe) sites over Pro-Gln sites. While a more comprehensive understanding of sequence and chain-length specificity may be needed to assess the relative utility of alternative prolyl endopeptidases for treating coeliac sprue, our present work has illustrated the diverse nature of this class of enzymes from the standpoint of proteolysing complex substrates such as gluten.

Characterization of a peptide adduct formed by N-acetoxy-2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), a reactive intermediate of the food carcinogen PhIP

2-Amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) is a member of a class of compounds known as the heterocyclic amines (HCAs) that are formed in meat during cooking. It is a multi-organ carcinogen in rodents forms adducts and with DNA and protein. Although protein adducts are not thought to be involved in cancer development, they may be useful as internal dosimeters of PhIP exposure and bioactivation. Towards the goals of characterizing the adducts formed in humans and the development of an assay for quantitation of adduct levels, we have characterized a peptide adduct formed by the putative genotoxic metabolite, N-acetoxy-PhIP. A model peptide with the internal sequence Leu–Gln–Lys–Cys–Pro–Tyr, which is homologous to a potential target sequence for HCAs in human serum albumin, was reacted with N-acetoxy-PhIP and an adduct was identified and further characterized by LC–ESI-MS/MS. N-acetoxy-PhIP is covalently bound to the peptide via cysteine and the exocyclic amino group of PhIP. Future work is needed to establish if this adduct is formed and is stable in vivo in humans following exposure to PhIP.