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International audience; GnRH is a neuropeptide known to regulate reproduction in vertebrates. The purpose of this study was to design and produce recombinant gonadotropin-releasing hormone associated peptide (rGnRH/GAP) as an alternative of the previous GnRHs and native extracted hormone from tissue, to induce final maturation in fish. Decapeptide as well as GAP area sequences were compared between GnRH1, GnRH2, and mGnRH from Acipenser sp and Huso huso, respectively. Considering the conserved amino acids and the replacement of un-stable amino acids with those that were more stable against proteolytic digestion as well as had a longer half-life, the sequence was designed. The sequences of decapeptide and GAP region were synthesized and then cloned on pET28a expression vector and transformed into expression host Escherichia coli BL21(DE3). The supernatant of cultured recombinant bacteria was used for purification using TALON Metal affinity resin. The purity of the GnRH/GAP was confirmed by single 8 kDa band on SDS-PAGE and Western blot. Bioinformatics studies were performed for evaluation of homology between GnRH protein sequences and prediction of 3D protein structure using Swiss Model. The result showed that the structure prediction of the recombinant GnRH decapeptide was relatively similar to decapeptide of GnRH2 from Beluga (Huso huso). The GAP structure was similar to GAP1 of Nile tilapia (Oreochromis niloticus) and sturgeon and GnRH2 of Chinese sturgeon (Acipenser sinensis). The mass analysis showed that the sequence was exactly the same as designated sequence. Biology activity of rGnRH/GAP was tested in mature goldfish (Carassius auratus) and results showed that rGnRH/GAP had a positive effect in final maturation. Indeed 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) was increased 17 h and 24 h after injection with rGnRH/GAP and spawning stemmed from that injection. These novel findings introduce the potential of utilizing rGnRH/GAP in aquaculture.

The Renin-Angiotensin System (RAS) plays a crucial role in numerous pathological conditions. Two of the critical RAS players, the angiotensin receptors AT1R and AT2R, possess differential functional profiles, although they share high sequence similarity. Although the main focus has been placed on AT1R, several epidemiological studies have evidenced that activation of AT2R could operate as a multimodal therapeutic target for different diseases. Thus, the development of selective AT2R ligands could have a high clinical potential for different therapeutic directions. Furthermore, they could serve as a powerful tool to interrogate the molecular mechanisms that are mediated by AT2R. Based on our recently established high affinity and AT2R selective compound [Y]6-AII we developed several analogues through modifying aminoacids located at positions 6 and 7 with various conformationally constrained analogues to enhance both the selectivity and stability. We report the development of high-affinity AT2R binders, which displayed high selectivity for AT2R versus AT1R. Furthermore, all analogues presented enhanced stability in human plasma with respect to the parent hormone Angiotensin II as also [Y]6-AII.

The pathological role of zinc in Alzheimer's disease (AD) is not yet fully elucidated, but there is strong evidence that zinc homeostasis is impaired in the AD brain and that this contributes to disease pathogenesis. In this study we examined the effects of zinc on the proteolysis of synthetic Apolipoprotein E (ApoE), a protein whose allelic variants differentially contribute to the onset/progression of disease. We have demonstrated that zinc promotes the proteolysis (using plasma kallikrein, thrombin and chymotrypsin) of synthetic ApoE in an isoform-specific way (E4>E2 and E3), resulting in more ApoE fragments, particularly for ApoE4. In the absence of exogenous proteases there was no effect of metal modulation on either lipidated or non-lipidated ApoE isoforms. Thus, increased zinc in the complex milieu of the ageing and AD brain could reduce the level of normal full-length ApoE and increase other forms that are involved in neurodegeneration. We further examined human plasma samples from people with different ApoE genotypes. Consistent with previous studies, plasma ApoE levels varied according to different genotypes, with ApoE2 carriers showing the highest total ApoE levels and ApoE4 carriers the lowest. The levels of plasma ApoE were not affected by either the addition of exogenous metals (copper, zinc or iron) or by chelation. Taken together, our study reveals that zinc may contribute to the pathogenesis of AD by affecting the proteolysis of ApoE, which to some extent explains why APOE4 carriers are more susceptible to AD.

Development of antimicrobial peptides has attracted considerable attention in recent years due to the excessive use of antibiotics, which has led to multiresislant bacteria. Cationic amphiphilic Alb-containing peptide models Ac-(Aib-ArgAib-Leu)(n)-NH2, n = 1-4, and sequential cationic polypeptides (Arg-X-Gly)(n), X = Ala, Val, Leu, were prepared and studied for their antimicrobial and hemolytic activity, as well as for their proteolytic stability. Ac-(Aib-Arg-Aib-Leu)(n)-NH2, n = 2, 3 and the polypeptide (Arg-Leu-Gly)(n) exhibited significant antimicrobial activity, and they were nontoxic at their MIC values and resistant, in particular the Aib-peptide models, to enzymatic degradation. The conformational characteristics of the peptide models were studied by circular dichroism (CD). Structure-activity relationship studies revealed the importance of the amphipathic a-helical conformation of the reported peptides in inducing antimicrobial effects. It is concluded that peptide models comprising cationic amino acids (Arg), helicogenic and noncoding residues (Aib) and/or hydrophobic and helix-promoting components (Leu) may lead to the development of antimicrobial therapeutics. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd. Journal of Peptide Science

A set of designed internally quenched fluorescence peptide substrates has been used to probe the effects of insertion of beta-peptide bonds into peptide sequences. The test sequence chosen corresponds to a proteolytically susceptible site in hemoglobin alpha-chain, residues 32-37. Fluorescence and mass spectral measurements demonstrate that the insertion of an beta-residues at the potential cleavage sites completely abolishes the action of proteases; in addition, the rate of cleavage of the peptide bond preceding the site of modification is also considerably reduced.

High performance liquid chromatography and high performance liquid chromatography/electrospray ionization-mass spectrometry were used to study the effect of N-terminal acetylation and the inhibition activity of acetylated enkephalins on the aminopeptidase M (EC 3.4.11.2)-catalyzed hydrolysis of methionine (Met-enk) and leucine enkephalins (Leu-enk). Acetylation imparts a significant enhancement in the proteolytic stability of these two peptides. After 30 min of the reaction,10% of both acetylated enkephalins was hydrolyzed. In an 8-h incubation period, only a maximum of 54% acetylated (Ac)-Met-enk and 38% Ac-Leu-enk was hydrolyzed. Vmax and Km [infil] for the degradation of Ac-Met-enk were 1.4 nmol/min/50 ng and 2.2 mM, respectively. The corresponding values for the reaction of Ac-Leu-enk were 0.5 nmol/min/50 ng and 0.9 mM. Also, the aminopeptidase M activity on Met-enk can be inhibited in the presence of Ac-Met-enk, which acts as a mixed-type inhibitor with the inhibition constant (K(i)) of I x 10(-3) M.

La durée d'activité très limitée dans le temps des protéases du raisin est confirmée en vinification en blanc, mais une nouvelle activité protéolytique peut être caractérisée à 37 °C et à pH 3,7 dans le moût au cours de la fermentation alcoolique.Cette activité doit être attribuée à des protéases exocellulaires libérées dans le milieu des levures. Parmi les souches essayées S. bayanus a une activité protéolytique plus importante que S. cerevisiae, ce qui se traduit par l'obtention de vins plus riches en azote soluble, donc plus stables du point de vue protéique et vraisemblablement d'une meilleure fermentescibilité malolactique.+++The short time of activity of grape proteolytic enzymes is confirmed in fermentation off skins. However, a new proteolytic activity has been characterized at 37 °C and at pH 3.7, occuring in alcoholic fermentation of the must.This activity comes from exocellular proteolytic enzymes released by the yeasts in the medium. Among tested strains S. bayanus has a proteolytic activity greater than S. cerevisiae; so the soluble nitrogen content of wines is higher. According to this, proteolytic stability is better as, probably, malolactic fermentation capabilities.

A zymographic assay of diamine oxidase (DAO, histaminase, EC 1.4.3.6), based on a coupled peroxidase reaction, and its behavior at proteolysis in simulated gastric and intestinal conditions, are described. The DAO activity from a vegetal extract of Lathyrus sativus seedlings was directly determined on sodium dodecyl sulfate polyacrylamide electrophoretic gels containing entrapped horseradish peroxidase, with putrescine as substrate of histaminase and ortho-phenylenediamine as co-substrate of peroxidase. The accumulation of azo-aniline, as peroxidase-catalyzed oxidation product, led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of DAO. After image analysis of gels, a linear dependency of DAO content (Coomassie-stained protein bands) and of its enzymatic activity (zymographic bands) with the concentration of the vegetal extract was obtained. In simulated gastric conditions (pH 1.2, 37 °C), the DAO from the vegetal extract lost its enzymatic activity before 15 min of incubation, either in the presence or absence of pepsin. The protein pattern (Coomassie-stained) revealed that the DAO content from the vegetal extract was kept almost constant in the simulated intestinal fluid (containing pancreatin or not), with a slight diminution in the presence of pancreatic proteases. After 10 h of incubation at 37 °C, the DAO enzymatic activity from the vegetal extract was 44.7% in media without pancreatin and 13.6% in the presence of pancreatin, whereas the purified DAO retained only 4.65% of its initial enzymatic activity in the presence of pancreatin. Figure Figure Zymographic assay of DAO with peroxidase included in polyacrylamide gels

A custom-designed, highly hydrophilic gelatin was produced in Pichia pastoris. Secreted production levels in single-copy transformants were in the range 3-6 g/l of clarified broth and purification to near homogeneity could be accomplished by differential ammonium sulfate precipitation. Despite the fact that gelatins are highly susceptible to proteolysis because of their unfolded structure, the recombinant protein was shown to be fully intact by SDS-PAGE, N-terminal sequencing, gel filtration chromatography and mass spectrometry. Owing to its highly hydrophilic nature, the migration of the synthetic gelatin in SDS-PAGE was severely delayed. Esterification of the carboxylic amino acid side chains resulted in normal migration. The high polarity of the synthetic gelatin also accounts for its negligible surface activity in water at concentrations up to 5% (w/v), as determined by tensiometry. Circular dichroism spectrometry showed that the non-hydroxylated gelatin did not form triple helices at 4 degrees C. The spectrum was even more representative of the random coil conformation than the spectrum of natural non-hydroxylated gelatins.

The oral delivery of protein and peptide drugs is limited by their proteolytic degradation and the poor absorption across the intestinal epithelia. In this work, we exposed a phage library of small bicyclic peptides (