Peptidase activities in Crotalus durissus terrificus semen

To understand the role of peptidases in seminal physiology of Crotalus durissus terrificus, activity levels of representative enzymes in semen and their sensitivities to inhibitors, cofactors, and peptide hormones were evaluated. The existence of seminal fractions and the association of peptidases with these fractions were also characterized for the first time in snakes. The prominent inhibitors of aminopeptidases (APs) were amastatin for acid, basic, and neutral; bestatin for basic; and diprotin A for dipeptidyl-IV. Cystyl and prolylimino AN were similarly susceptible to the majority of these inhibitors. The basic and neutral were characterized as metallo-peptidases, acid AP was activated by MnCl(2), and cystyl, prolyl-imino, and type I pyroglutamyl were characterized as sulphydryl-dependent APs. Angiotensin II, vasotocin, bradykinin, fertilization-promoting peptide, and TRH altered the majority of these peptidase activities; these peptides are possible substrates and/or modulators of these peptidases. Peptidase activities were found in all seminal fractions: seminal plasma (SP), prostasome-like (PR) structures, and soluble (S-) and membrane-bound fractions (MFs) of spermatozoa. The levels of activity of each peptidase varied among different seminal fractions. In SP, the higher activities were puromycin-insensitive neutral and basic APs. in PR, the higher activity was puromycin-insensitive neutral AP. In spermatozoa, the higher activity in subcellular SF was puromycin-sensitive neutral, while in MF both puromycin-sensitive and -insensitive neutral AN were equally higher than the other examined peptidases. Data suggested that these peptidases, mainly basic and neutral, have a high relevance in regulating seminal functions of C. d. terrificus.

Peptidase activities in the semen from the ductus deferens and uterus of the neotropical rattlesnake Crotalus durissus terrificus

To understand the role of peptidases in seminal physiology of Crotalus durissus terrificus, intra- and inter-seasonal activity levels of acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive neutral (APN-PI), cystyl (CAP), dipeptidyl-IV (DPPIV), type-1 pyroglutamyl (PAP-I) and prolyl-imino (PIP) aminopeptidases as well as prolyl endopeptidase (POP) were evaluated in soluble (SF) and/or membrane-bound (MF) fractions of semen collected from the ductus deferens of the male reproductive tract and from the posterior portion of the uterus. Seminal APB, PIP and POP were detected in SF, while other peptidases were detected in SF and MF. Only the convoluted posterior uterus in winter and autumn had semen. Relative to other examined peptidases, in general, APN-PI, APN-PS and APB activities were predominant in the semen from the uterus and throughout the year in the semen from the ductus deferens, suggesting their great relevance in the seminal physiology of C. d. terrificus. The levels of peptidase activities in the ductus deferens semen varied seasonally and were different from those of semen in the uterus, suggesting that their modulatory actions on susceptible peptides are integrated to the male reproductive cycle events and spermatozoa viability of this snake.

Seasonal variation of peptidase activities in the reproductive tract of Crotalus durissus terrificus

Seasonal quantitative patterns of acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive neutral (APN-PI), cystyl (CAP), dipeptidyl IV (DPPIV), type-1 pyroglutamyl (PAP-I) and prolylimino (PIP) aminopeptidases and prolyl oligopeptidase (POP) activities in soluble (SF) and solubilized membrane-bound (MF) fractions from ductus deferens, vagina and uterus were studied to evaluate their relationships with the reproductive cycle and the extensive long-term spermatozoa storage (LTSS) of the Neotropical rattlesnake Crotalus durissus terrificus. APB, PIP and POP were detected only in SF, while other peptidases were detected in SF and MF. APB, APN-PI and APN-PS were predominant in most tissues in all seasons. Peptidase activities had a common pattern of increment during the dry season (winter/autumn), which coincides with the mating period (autumn) and LTSS in the female (winter), as well as the reduction of spermatozoa motility and maintenance of fertilization capacity of spermatozoa. The high CAP activity in the soluble fraction of the vagina during winter, compared to summer (time of parturition) and spring, coincides with the relaxation of this tissue. In the soluble fraction, the low PAP-1 activity of the ductus deferens coincided with its high activity in the vagina during the winter; and the inverse occurred in summer, which is consistent with the physiological process of preserving spermatozoon viability. In conclusion, the studied peptidase activities had seasonal and tissue-specific characteristics, which suggest a relevant role in the reproductive physiology of C. d. terrificus. (C) 2008 Elsevier Inc. All rights reserved.

N-terminal His7-modification of glucagon-like peptide-1(7-36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity

Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid inactivation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His7-modified analogue of GLP-1, N-pyroglutamyl-GLP-1 as well as N-acetyl-GLP-1 were synthesised and tested for DPP IV stability and biological activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degradation of native GLP-1 to GLP-1(9-36)amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degradation. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC50 values 32.9 and 6.7 nM, respectively) compared with native GLP-1 (IC50-37 nM). Similarly, both analogues stimulated cAMP production with EC50 values of 16.3 and 27 nM respectively compared with GLP-1 (EC50 4.7 nM). However, N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5.6 mM glucose (P< 0.05 to P< 0.001) similar to native GLP-1. Both analogues (25 nM/kg body weight) lowered plasma glucose and increased plasma insulin levels when administered in conjunction with glucose (18 nM/kg body weight) to adult obese diabetic (ob/ob) mice. N-pyroglutamyl-GLP-1 was substantially better at lowering plasma glucose compared with the native peptide, while N-acetyl-GLP-1 was significantly more potent at stimulating insulin secretion. These studies indicate that N-terminal modification of GLP-1 results in DPP IV-resistant and biologically potent forms of GLP-1. The particularly powerful antihyperglycaemic action of N-pyroglutamyl-GLP-1 shows potential for the treatment of type 2 diabetes. © 2004 Society for Endocrinology.

Digestive Peptidase Evolution In Holometabolous Insects Led To A Divergent Group Of Enzymes In Lepidoptera.

Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic l-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

Dipeptidyl-peptidase II and cathepsin B activities in amelogenesis of the rat incisor

A body of published evidence suggests that a significant portion of enamel matrix protein synthesized by ameloblasts localises in the lysosomal-endosomal organelles of these enamel organ cells. Little is known regarding the lysosomal proteolytic activities during amelogenesis. The aims of this study were to detect and measure the activities of lysosomal peptidases cathepsin B (E.C. 3.4.22.1) and dipeptidyl-peptidase II (E.C. 3.4.14.2) in the enamel organ of the rat incisor and to ascertain whether rat enamel matrix proteins are degraded by these peptidases in vitro. Whole enamel organs were dissected from rat mandibular incisors. Enamel protein was also collected from the rat teeth. Analysis indicated that the rat incisor enamel organs contained specific activities of both dipeptidyl-peptidase II and cathepsin B at levels comparable with those of kidney which is rich in both these lysosomal peptidases. Gel electrophoresis and immunoblotting demonstrated that both cathepsin B and dipeptidyl-peptidase II were able to substantially degrade the rat enamel proteins in vitro. Based on these observations, we propose that lysosomal proteases have roles in amelogenesis in the intracellular degradation of amelogenins.

Dipeptidyl peptidase IV is a target for covalent adduct formation with the acyl glucuronide metabolite of the anti-inflammatory drug zomepirac

The nonsteroidal anti-inflammatory drug zomepirac (ZP) is metabolised to a chemically reactive acyl glucuronide conjugate (ZAG) which can form covalent adducts with proteins. In vivo, such adducts could initiate immune or toxic responses. In rats given ZP, the major band detected in liver homogenates by immunoblotting with a polyclonal ZP antiserum was at 110 kDa. This adduct was identified as ZP-modified dipeptidyl peptidase IV (DPP IV) by immunoblotting using the polyclonal ZP antiserum and monoclonal DPP IV antibodies OX-61 and 236.3. In vitro, ZAG, but not ZP itself, covalently modified recombinant human and rat DPP IV. Both monoclonal antibodies recognized DPP IV in livers from ZP- and vehicle-dosed rats. Confirmation that the 110 kDa bands which were immunoreactive with the ZP and DPP IV antibodies represented the same molecule was obtained from a rat liver extract reciprocally immunodepleted of antigens reactive with these two antibodies. Furthermore, immunoprecipitations with OX-61 antibody followed by immunolotting with ZP antiserum, and the reciprocal experiment, showed that both these antibodies recognised the same 110 kDa molecule in extracts of ZP-dosed rat liver. The results verify that DPP IV is one of the protein targets for covalent modification during hepatic transport and biliary excretion of ZAG in rats. (C) 2001 Elsevier Science Inc. All rights reserved.

Evolutionary histories of expanded peptidase families in Schistosoma mansoni

Schistosoma mansoni is one of the three main causative agents of human schistosomiasis, a major health problem with a vast socio-economic impact. Recent advances in the proteomic analysis of schistosomes have revealed that peptidases are the main virulence factors involved in the pathogenesis of this disease. In this context, evolutionary studies can be applied to identify peptidase families that have been expanded in genomes over time in response to different selection pressures. Using a phylogenomic approach, we searched for expanded endopeptidase families in the S. mansoni predicted proteome with the aim of contributing to the knowledge of such enzymes as potential therapeutic targets. We found three endopeptidase families that comprise leishmanolysins (metallopeptidase M8 family), cercarial elastases (serine peptidase S1 family) and cathepsin D proteins (aspartic peptidase A1 family). Our results suggest that the Schistosoma members of these families originated from successive gene duplication events in the parasite lineage after its diversification from other metazoans. Overall, critical residues are conserved among the duplicated genes/proteins. Furthermore, each protein family displays a distinct evolutionary history. Altogether, this work provides an evolutionary view of three S. mansoni peptidase families, which allows for a deeper understanding of the genomic complexity and lineage-specific adaptations potentially related to the parasitic lifestyle.

Dipeptidyl peptidase IV and its inhibitors: therapeutics for type 2 diabetes and what else?

The proline-specific dipeptidyl aminopeptidase IV (DPP IV, DPP-4, CD26), widely expressed in mammalians, releases X-Pro/Ala dipeptides from the N-terminus of peptides. DPP IV is responsible of the degradation of the incretin peptide hormones regulating blood glucose levels. Several families of DPP IV inhibitors have been synthesized and evaluated. Their positive effects on the degradation of the incretins and the control of blood glucose levels have been demonstrated in biological models and in clinical trials. Presently, several DPP IV inhibitors, the "gliptins", are approved for type 2 diabetes or are under clinical evaluation. However, the gliptins may also be of therapeutic interest for other diseases beyond the inhibition of incretin degradation. In this Perspective, the biological functions and potential substrates of DPP IV enzymes are reviewed and the characteristics of the DPP IV inhibitors are discussed in view of type 2 diabetes and further therapeutic interest.

Leishmania major metacaspase can replace yeast metacaspase in programmed cell death and has arginine-specific cysteine peptidase activity.

The human protozoan parasite Leishmania major has been shown to exhibit several morphological and biochemical features characteristic of a cell death program when differentiating into infectious stages and under a variety of stress conditions. Although some caspase-like peptidase activity has been reported in dying parasites, no caspase gene is present in the genome. However, a single metacaspase gene is present in L. major whose encoded protein harbors the predicted secondary structure and the catalytic dyad histidine/cysteine described for caspases and other metacaspases identified in plants and yeast. The Saccharomyces cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In this study, we describe the functional heterologous complementation of a S. cerevisiae yca1 null mutant with the L. major metacaspase (LmjMCA) in cell death induced by oxidative stress. We show that LmjMCA is involved in yeast cell death, similar to YCA1, and that this function depends on its catalytic activity. LmjMCA was found to be auto-processed as occurs for caspases, however LmjMCA did not exhibit any activity with caspase substrates. In contrast and similarly to Arabidopsis thaliana metacaspases, LmjMCA was active towards substrates with arginine in the P1 position, with the activity being abolished following H147A and C202A catalytic site mutations. These results suggest that metacaspases are members of a family of peptidases with a role in cell death conserved in evolution notwithstanding possible differences in their catalytic activity.

Type I signal peptidase from Leishmania is a target of the immune response in human cutaneous and visceral leishmaniasis.

The gene encoding type I signal peptidase (Lmjsp) has been cloned from Leishmania major. Lmjsp encodes a protein of 180 amino residues with a predicted molecular mass of 20.5 kDa. Comparison of the protein sequence with those of known type I signal peptidases indicates homology in five conserved domains A-E which are known to be important, or essential, for catalytic activity. Southern blot hybridisation analysis indicates that there is a single copy of the Lmjsp gene. A recombinant SPase protein and a synthetic peptide of the L. major signal peptidase were used to examine the presence of specific antibodies in sera from either recovered or active individuals of both cutaneous and visceral leishmaniasis. This evaluation demonstrated that sera from cutaneous and visceral forms of leishmaniasis are highly reactive to both the recombinant and synthetic signal peptidase antigens. Therefore, the Leishmania signal peptidase, albeit localised intracellularly, is a significant target of the Leishmania specific immune response and highlights its potential use for serodiagnosis of cutaneous and visceral leishmaniasis.

Dipeptidyl-peptidase-IV by cleaving neuropeptide Y induces lipid accumulation and PPAR-γ expression.

We evaluated the effects of dipeptidyl peptidase-IV (DPPIV), and its inhibitor, vildagliptin, on adipogenesis and lipolysis in a pre-adipocyte murine cell line (3T3-L1). The exogenous rDPPIV increased lipid accumulation and PPAR-γ expression, whereas an inhibitor of DPPIV, the anti-diabetic drug vildagliptin, suppresses the stimulatory role of DPPIV on adipogenesis and lipid accumulation, but had no effect on lipolysis. NPY immunoneutralization or NPY Y(2) receptor blockage inhibited DPPIV stimulatory effects on lipid accumulation, collectively, indicating that DPPIV has an adipogenic effect through NPY cleavage and subsequent NPY Y(2) activation. Vildagliptin inhibits PPAR-γ expression and lipid accumulation without changing lipolysis, suggesting that this does not impair the ability of adipose tissue to store triglycerides inside lipid droplets. These data indicate that DPPIV and NPY interact on lipid metabolism to promote adipose tissue depot.

Leishmania metacaspase: an arginine-specific peptidase.

The purpose of this chapter is to give insights into metacaspase of Leishmania protozoan parasites as arginine-specific cysteine peptidase. The physiological role of metacaspase in Leishmania is still a matter of debate, whereas its peptidase enzymatic activity has been well characterized. Among the different possible expression systems, metacaspase-deficient yeast cells (Δyca1) have been instrumental in studying the activity of Leishmania major metacaspase (LmjMCA). Here, we describe techniques for purification of LmjMCA and its activity measurement, providing a platform for further identification of LmjMCA substrates.

Long-lasting antidiabetic effect of a dipeptidyl peptidase IV-resistant analog of glucagon-like peptide-1.

Glucagon-like peptide-1(7-37) (GLP-1) is the most potent insulinotropic hormone characterized thus far. Because its activity is preserved in non-insulin-dependent diabetes mellitus (NIDDM) patients, it is considered a potential new drug for the treatment of this disease. One limitation in its therapeutic use is a short half-life in vivo (5 minutes), due in part to a fast degradation by the endoprotease dipeptidylpeptidase IV (DPPIV). Recently, it was reported that GLP-1 became resistant to DPPIV when the alanine residue at position 8 was replaced by a glycine (GLP-1-Gly8). We report here that this change slightly decreased the affinity of the peptide for its receptor (IC50, 0.41 +/- 0.14 and 1.39 +/- 0.61 nmol/L for GLP-1 and GLP-1-Gly8, respectively) but did not change the efficiency to stimulate accumulation of intracellular cyclic adenosine monophosphate (cAMP) (EC50, 0.25 +/- 0.05 and 0.36 +/- 0.06 nmol/L for GLP-1 and GLP-1-Gly8, respectively). Second, we demonstrate for the first time that this mutant has an improved insulinotropic activity compared with the wild-type peptide when tested in vivo in an animal model of diabetes. A single injection of 0.1 nmol GLP-1-Gly8 in diabetic mice fed a high-fat diet can correct fasting hyperglycemia and glucose intolerance for several hours, whereas the activity of 1 nmol GLP-1 vanishes a few minutes after injection. These actions were correlated with increased insulin and decreased glucagon levels. Interestingly, normoglycemia was maintained over a period that was longer than the predicted peptide half-life, suggesting a yet undescribed long-term effect of GLP-1-Gly8. GLP-1-Gly8 thus has a markedly improved therapeutic potential compared with GLP-1, since it can be used at much lower doses and with a more flexible schedule of administration.