Search

While temperature has been shown to affect the survival and growth of bacteria and their phage parasites, it is unclear if trade-offs between phage resistance and other bacterial traits depend on the temperature. Here, we experimentally compared the evolution of phage resistance–virulence trade-offs and underlying molecular mechanisms in phytopathogenic Ralstonia solanacearum bacterium at 25 °C and 35 °C temperature environments. We found that while phages reduced R. solanacearum densities relatively more at 25 °C, no difference in the final level of phage resistance was observed between temperature treatments. Instead, small colony variants (SCVs) with increased growth rate and mutations in the quorum-sensing (QS) signaling receptor gene, phcS , evolved in both temperature treatments. Interestingly, SCVs were also phage-resistant and reached higher frequencies in the presence of phages. Evolving phage resistance was costly, resulting in reduced carrying capacity, biofilm formation, and virulence in planta, possibly due to loss of QS-mediated expression of key virulence genes. We also observed mucoid phage-resistant colonies that showed loss of virulence and reduced twitching motility likely due to parallel mutations in prepilin peptidase gene, pilD. Moreover, phage-resistant SCVs from 35 °C-phage treatment had parallel mutations in type II secretion system (T2SS) genes (gspE and gspF). Adsorption assays confirmed the role of pilD as a phage receptor, while no loss of adsorption was found with phcS or T2SS mutants, indicative of other downstream phage resistance mechanisms. Additional transcriptomic analysis revealed upregulation of CBASS and type I restriction-modification phage defense systems in response to phage exposure, which coincided with reduced expression of motility and virulence-associated genes, including pilD and type II and III secretion systems. Together, these results suggest that while phage resistance–virulence trade-offs are not affected by the growth temperature, they could be mediated through both pre- and postinfection phage resistance mechanisms. [ABSTRACT FROM AUTHOR]

Among the C1A cysteine proteases, the plant cathepsin F-like group has been poorly studied. This paper describes the molecular and functional characterization of the HvPap-1 cathepsin F-like protein from barley. This peptidase is N-glycosylated and has to be processed to become active by its own propeptide being an important modulator of the peptidase activity. The expression pattern of its mRNA and protein suggest that it is involved in different proteolytic processes in the barley plant. HvPap-1 peptidase has been purified in Escherichia coli and the recombinant protein is able to degrade different substrates, including barley grain proteins (hordeins, albumins, and globulins) stored in the barley endosperm. It has been localized in protein bodies and vesicles of the embryo and it is induced in aleurones by gibberellin treatment. These three features support the implication of HvPap-1 in storage protein mobilization during grain germination. In addition, a complex regulation exerted by the barley cystatins, which are cysteine protease inhibitors, and by its own propeptide, is also described [ABSTRACT FROM AUTHOR]

Most mitochondrial proteins contain an N-terminal targeting signal that is removed by specific proteases following import. In plant mitochondria, only mitochondrial processing peptidase (MPP) has been characterized to date. Therefore, we sought to determine the substrates and cleavage sites of the Arabidopsis thaliana homologues to the yeast Icp55 and Oct1 proteins, using the newly developed ChaFRADIC method for N-terminal protein sequencing. We identified 88 and seven putative substrates for Arabidopsis ICP55 and OCT1, respectively. It was determined that the Arabidopsis ICP55 contains an almost identical cleavage site to that of Icp55 from yeast. However, it can also remove a far greater range of amino acids. The OCT1 substrates from Arabidopsis displayed no consensus cleavage motif, and do not contain the classical -10R motif identified in other eukaryotes. Arabidopsis OCT1 can also cleave presequences independently, without the prior cleavage of MPP. It was concluded that while both OCT1 and ICP55 were probably acquired early on in the evolution of mitochondria, their substrate profiles and cleavage sites have either remained very similar or diverged completely. [ABSTRACT FROM AUTHOR]

Most of the proteins present in mitochondria and chloroplasts, the organelles acquired via endosymbiotic events, are encoded in the nucleus and translated into the cytosol. Most of such nuclear-encoded proteins are specifically recognized via an N-terminal-encoded targeting peptide (TP) and imported into the organelles via a translocon machinery. Once imported, the TP is degraded by a succession of cleavage steps ensured by dedicated peptidases. Here, we retrace the evolution of the families of the mitochondrial processing peptidase (MPP), stromal processing peptidase (SPP), presequence protease (PreP), and organellar oligo-peptidase (OOP) that play a central role in TP processing and degradation across the tree of life. Their bacterial distributions are widespread but patchy, revealing unsurprisingly complex history of lateral transfers among bacteria. We provide evidence for the eukaryotic acquisition of MPP, OOP, and PreP by lateral gene transfers from bacteria at the time of the mitochondrial endosymbiosis. We show that the acquisition of SPP and of a second copy of OOP and PreP at the time of the chloroplast endosymbiosis was followed by a differential loss of one PreP paralog in photosynthetic eukaryotes. We identified some contrasting sequence conservations between bacterial and eukaryotic homologs that could reflect differences in the functional context of their peptidase activity. The close vicinity of the eukaryotic peptidases MPP and OOP to those of several bacterial pathogens, showing antimicrobial resistance, supports a scenario where such bacteria were instrumental in the establishment of the proteolytic pathway for TP degradation in organelles. The evidence for their role in the acquisition of PreP is weaker, and none is observed for SPP, although it cannot be excluded by the present study. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to demonstrate the mechanistic basis of Heritiera fomes, which has traditionally been used to treat diabetes. Methods: Clonal pancreatic β-cells and primary islets were used to measure insulin release. 3T3-L1 cells were used to analyse insulin action, and in vitro systems were used to measure further glucose-lowering activity. In vivo assessment was performed on streptozotocin (STZ)-induced type-2 diabetic rats and reversed-phase-HPLC followed by liquid chromatography mass spectrometry (LC-MS) to detect bioactive molecules. Key findings: Ethanol extract of Heritiera fomes (EEHF) significantly increased insulin release with stimulatory effects comparable to 1 µM glucagon-like peptide 1, which were somewhat reduced by diazoxide, verapamil and calcium-free conditions. Insulin release was stimulated by tolbutamide, isobutyl methylxanthine and KCl. EEHF induced membrane depolarization and increased intracellular Ca2+ levels. EEHF enhanced glucose uptake in 3T3L1 cells and decreased protein glycation. EEHF significantly inhibited postprandial hyperglycaemia following sucrose loading and inversely elevated unabsorbed sucrose concentration in the gut. It suppressed glucose absorption during in situ gut perfusion. Furthermore, EEHF improved glucose tolerance, plasma insulin and gut motility, and decreased plasma dipeptidyl peptidase IV activity. Procyanidins, epicatechin and proanthocyanidins were some of the identified bioactive constituents that may involve in β-cell actions. Conclusions: This study provides some evidence to support the use of H. fomes as an antidiabetic traditional remedy. [ABSTRACT FROM AUTHOR]

Objective To provide the scientific basis for the utility of rhizome of Trillium govanianum as nutraceutical supplements in managing physiological glycemic levels. Methods The in vitro enzyme inhibitory activity of the extract, fractions, and the isolated steroidal saponins from the rhizome part of T. govanianum was carried out against a-amylase, a-glucosidase, and dipeptidyl peptidase IV. The molecular interactions, binding score, and pharmacokinetic parameters (absorption, distribution metabolism, and excretion) of steroidal saponins were analyzed by the Schrodinger molecular docking software. Key findings Current study explained that the extract, fractions, and isolated steroidal saponins from T. govanianum possess good a-amylase and a-glucosidase inhibitory activity while moderate dipeptidyl peptidase IV inhibitory activity. Moreover, in vitro results revealed that borassoside E (IC50 7.15 ± 1.78 µM), protodioscin (IC50 6.72 ± 0.04 µM), and diosgenin (IC50 12.75 ± 2.70 µM) are most effective in inhibiting the activity of a-amylase, a-glucosidase, and dipeptidyl peptidase IV, respectively. Current in silico and in vitro studies established an association between the steroidal saponins from T. govanianum and their molecular interactions with a-amylase, a-glucosidase, and dipeptidyl peptidase IV. Conclusion The results of this investigation suggest that fractions and steroidal saponins from T. govanianum exhibit good antidiabetic activity which could be used as nutraceutical supplements for the management of systemic glucose level. [ABSTRACT FROM AUTHOR]

Background: The 4Kscore Test determines a personalized risk score for aggressive prostate cancer by combining the blood sample measurements of total prostate-specific antigen (tPSA), free PSA (fPSA), intact PSA (iPSA), and human kallikrein-related peptidase 2 (hK2) with patient clinical information to generate the patient risk's score; thus, accuracy and precision of the 4Kscore depend on the reliability of these measurements. Although tPSA and fPSA are measured on a Food and Drug Administration (FDA)-approved platform, the performance of the iPSA and hK2 assays in the clinical setting has not previously been reported. Methods: Analytical performance was determined for the iPSA and hK2 assays in both serum and EDTA plasma, according to Clinical and Laboratory Standards Institute guidelines. Equivalence of the 4Kscore in both sample matrices was demonstrated in a 353-patient clinical cohort, and the stability of endogenous iPSA and hK2 for at least 3 days was demonstrated in a smaller subset. Results: Intralaboratory and interlaboratory precision of the iPSA and hK2 assays in both matrices was comparable with that of FDA-approved tPSA and fPSA assays (<18% for iPSA; <8% for hK2). The picogram per milliliter sensitivity and wide dynamic range of the iPSA and hK2 assays allowed for accurate measurements in the target population. The 4Kscore generated in either matrix up to 3 days after collection is equivalent to that measured within 24 h of collection (Passing--Bablok slope 95% CI: plasma, 0.999-1.034; serum, 0.997-1.040). Conclusions: The robust performance of component assays and reliable stability of the endogenous analytes in clinical samples proven here ensures an accurate 4Kscore Test result. [ABSTRACT FROM AUTHOR]

Background Cutaneous microdialysis ( CM) is an ex vivo technique that allows study of tissue chemistry, including bioavailability of actual tissue concentration of unbound drug in the interstitial fluid of the body. Aim To test the penetration and dermal bioavailability of galenic formulations of the small-molecule IP10.C8, a dual-protease inhibitor of the dipeptidyl peptidase and aminopeptidase families. Methods Using CM, we tested the penetration and dermal bioavailability of IP10.C8 into the dermis and subcutis of pigs, and determined the tissue concentration of IP10.C8 enzymatically, using an enzyme activity assay (substrate Gly-Pro- pNA) and high performance liquid chromatography. Results Dermal bioavailability was enhanced by using microemulsion or the addition of the penetration enhancer oleic acid to a hydroxyethylcellulose ( HEC) gel formulation. Dermal bioavailability was also enhanced when galenic formulations were prepared with higher pH (7.5 vs. 6.5) or higher drug concentration (5% vs. 1%) in HEC gel. Conclusion It seems possible, using CM for topical skin penetration testing in anaesthetized domestic pigs, to test the bioavailability of newly designed drugs. However, the experimental time is limited due to the anaesthesia, and is dependent on drug recovery. Validation of this technique for routine use is challenging, and more experiments are needed to validate this preclinical set-up. [ABSTRACT FROM AUTHOR]

Arabinogalactan-proteins (AGPs) are highly diverse plant proteoglycans found on the plant cell surface. AGPs have large arabinogalactan (AG) moieties attached to a core-protein rich in hydroxyproline (Hyp). The AG undergoes hydrolysis by various glycoside hydrolases, most of which have been identified, whereas the core-proteins is presumably degraded by unknown proteases/peptidases secreted from fungi and bacteria in nature. Although several enzymes hydrolyzing other Hyp-rich proteins are known, the enzymes acting on the core-proteins of AGPs remain to be identified. The present study describes the detection of protease/peptidase activity toward AGP core-proteins in the culture medium of winter mushroom (Flammulina velutipes) and partial purification of the enzyme by several conventional chromatography steps. The enzyme showed higher activity toward Hyp residues than toward proline and alanine residues and acted on core-proteins prepared from gum arabic. Since the activity was inhibited in the presence of Pefabloc SC, the enzyme is probably a serine protease. The degradation of the core-protein of AGPs by a protease/peptidase from winter mushroom. [ABSTRACT FROM PUBLISHER]

Background. Aberrancies in gene expression in immune effector cells and in end-organs are implicated in lupus pathogenesis. To gain insights into the mechanisms of tissue injury, we profiled the expression of micro-RNAs in inflammatory kidney lesions of human lupus nephritis (LN). Methods. Kidney specimens were from patients with active proliferative, membranous or mixed LN and unaffected control tissue. Micro-RNAs were quantified by TaqMan Low Density Arrays. Bioinformatics was employed to predict gene targets, gene networks and perturbed signaling pathways. Results were validated by transfection studies (luciferase assay, realtime PCR) and in murine LN. Protein expression was determined by immunoblotting and immunohistochemistry. Results. Twenty-four micro-RNAs were dysregulated (9 upregulated, 15 down-regulated) in human LN compared with control renal tissue. Their predicted gene targets participated in pathways associated with TGF-β, kinases, NF-κB, HNF4A, Wnt/β-catenin, STAT3 and IL-4. miR-422a showed the highest upregulation (17-fold) in active LN and correlated with fibrinoid necrosis lesions (β = 0.63, P = 0.002). In transfection studies, miR-422a was found to directly target kallikrein-related peptidase 4 (KLK4) mRNA. Concordantly, KLK4 mRNA was significantly reduced in the kidneys of human and murine LN and correlated inversely with miR-422a levels. Immunohistochemistry confirmed reduced KLK4 protein expression in renal mesangial and tubular epithelial cells in human and murine LN. Conclusions. KLK4, a serine esterase with putative renoprotective properties, is down-regulated by miR-422a in LN kidney suggesting that, in addition to immune activation, local factors may be implicated in the disease. [ABSTRACT FROM AUTHOR]

CD26/DPP4 (dipeptidyl peptidase 4/DP4/DPPIV) is a surface T cell activation antigen and has been shown to have DPP4 enzymatic activity, cleaving-off amino-terminal dipeptides with either L-proline or L-alanine at the penultimate position. It plays a major role in glucose metabolism by N-terminal truncation and inactivation of the incretins glucagon-like peptide-1 (GLP) and gastric inhibitory protein (GIP). In 2006, DPP4 inhibitors have been introduced to clinics and have been demonstrated to efficiently enhance the endogenous insulin secretion via prolongation of the half-life of GLP-1 and GIP in patients. However, a large number of studies demonstrate clearly that CD26/DPP4 also plays an integral role in the immune system, particularly in T cell activation. Therefore, inhibition of DPP4 might represent a double-edged sword. Apart from the metabolic benefit, the associated immunological effects of long term DPP4 inhibition on regulatory processes such as T cell homeostasis, maturation and activation are not understood fully at this stage. The current data point to an important role for CD26/DPP4 in maintaining lymphocyte composition and function, T cell activation and co-stimulation, memory T cell generation and thymic emigration patterns during immune-senescence. In rodents, critical immune changes occur at baseline levels as well as after in-vitro and in-vivo challenge. In patients receiving DPP4 inhibitors, evidence of immunological side effects also became apparent. The scope of this review is to recapitulate the role of CD26/DPP4 in the immune system regarding its pharmacological inhibition and T cell-dependent immune regulation. [ABSTRACT FROM AUTHOR]

A method to express, purify and modify the Peptidyl- Lys metallopeptidase (LysN) of Armillaria mellea in Pichia pastoris was developed to enable functional studies of the protease. Based on prior work, we propose a mechanism of action of LysN. Catalytic residues were investigated by site-directed mutagenesis. As anticipated, these mutations resulted in significantly reduced catalytic rates. Additionally, based on molecular modelling eleven mutants were designed to have altered substrate specificity. The S1' binding pocket of LysN is quite narrow and lined with negative charge to specifically accommodate lysine. To allow for arginine specificity in S1', it was proposed to extend the S1' binding pocket by mutagenesis, however the resulting mutant did not show any activity with arginine in P1'. Two mutants, A101D and T105D, showed increased specificity towards arginine in subsites S2'-S4' compared to the wild type protease. We speculate that the increased specificity to result from the additional negative charge which attract and interact with positively charged residues better than the wild type. [ABSTRACT FROM AUTHOR]

Legumains or vacuolar processing enzymes are cysteine peptidases (C13 family, clan CD) with increasingly recognized physiological significance in plants. They have previously been classified as seed and vegetative legumains. In this work, the entire barley legumain family is described. The eight members of this family belong to the two phylogenetic clades in which the angiosperm legumains are distributed. An in-depth molecular and functional characterization of a barley legumain from each group, HvLeg-2 and HvLeg-4, was performed. Both legumains contained a signal peptide and were located in the endoplasmic reticulum, were expressed in seeds and vegetative tissues, and when expressed as recombinant proteins showed legumain and caspase proteolytic activities. However, the role of each protein seemed to be different in their target tissues. HvLeg-2 responded in leaves to biotic and abiotic stimuli, such as salicylic acid, jasmonic acid, nitric oxide, abscisic acid, and aphid infestation, and was induced by gibberellic acid in seeds, where the protein is able to degrade storage globulins. HvLeg-4 responded in leaves to wounding, nitric oxide, and abscisic acid treatments, and had an unknown role in the germinating seed. From these results, a multifunctional role was assumed for these two phylogenetically distant legumains, achieving different physiological functions in both seed and vegetative tissues. [ABSTRACT FROM AUTHOR]

The article focuses on a study related to the effect of conversion of zinc-binding motif sequence HEXXH to HEXXXH on activity and stability of thermolysin. The study mentions that thermolysin variants were constructed by site-directed mutagenesis. The study further informs that thermolysin variants were measured by casein-hydrolyzing activities.

Aims Thimet oligopeptidase (TOP, endopeptidase EC3.4.24.15) is a soluble metallopeptidase known to be expressed within the mammalian vasculature. We examine for the first time the relationship between TOP expression and laminar shear stress, a haemodynamic force associated with endothelium-mediated vascular homeostasis. Methods and results Human and bovine aortic endothelial cells were exposed to physiological levels of laminar shear (0–10 dynes/cm2, 24–48 h) and monitored for TOP expression using promoter activity assay, qRT–PCR, western blotting, and immunocytochemistry. Using a luciferase reporter encoding the full-length rat TOP promoter, initial studies demonstrated shear-dependent promoter activation (∼five-fold). TOP mRNA and protein were also consistently up-regulated by shear, events which could be completely prevented by pre-treatment of cells with either N-acetylcysteine, superoxide dismutase, or catalase, confirming ROS involvement. Consistent with this, targeted inhibition of NADPH oxidase (apocynin, NSC23766, NOX4 siRNA) had a similar blocking effect. Finally, in view of its pivotal role in cellular antigen presentation and major histocompatibility complex (MHC) class-1 regulation, we hypothesized that the shear-dependent induction of TOP may lower MHC1 expression. In this respect, we observed that recombinant TOP over-expression in static HAECs dose-dependently depleted MHC1 (>60%), while siRNA-mediated blockade of TOP induction in sheared HAECs led to substantially elevated MHC1 (∼66%). Conclusion Our findings demonstrate that laminar shear positively regulates endothelial TOP expression. Moreover, a role for ROS production by NADPH oxidase is indicated. Finally, our studies suggest that shear-dependent TOP induction down-regulates MHC1 levels, pointing to a role for TOP in the flow-mediated regulation of endothelial immunogenicity. [ABSTRACT FROM PUBLISHER]

Phytomonas serpens, a phytoflagellate trypanosomatid, shares common antigens with Trypanosoma cruzi. In the present work, we compared the hydrolytic capability of cysteine peptidases in both trypanosomatids. Trypanosoma cruzi epimastigotes presented a 10-fold higher efficiency in hydrolyzing the cysteine peptidase substrate Z-Phe-Arg-AMC than P. serpens promastigotes. Moreover, two weak cysteine-type gelatinolytic activities were detected in P. serpens, while a strong 50-kDa cysteine peptidase was observed in T. cruzi. Cysteine peptidase activities were detected at twofold higher levels in the cytoplasmic fraction when compared with the membrane-rich or the content released from P. serpens. The cysteine peptidase secreted by P. serpens cleaved several proteinaceous substrates. Corroborating these findings, the cellular distribution of the cruzipain-like molecules in P. serpens was attested through immunocytochemistry analysis. Gold particles were observed in all cellular compartments, including the cytoplasm, plasma membrane, flagellum, flagellar membrane and flagellar pocket. Interestingly, some gold particles were visualized free in the flagellar pocket, suggesting the release of the cruzipain-like molecule. The antigenic properties of the cruzipain-like molecules of P. serpens were also analyzed. Interestingly, sera from chagasic patients recognized both cellular and extracellular antigens of P. serpens, including the cruzipain-like molecule. These results point to the use of P. serpens antigens, especially the cruzipain-like cysteine-peptidases, as an alternative vaccination approach to T. cruzi infection. [ABSTRACT FROM AUTHOR]

Possible promoter regions preceding 14 genes belonging to the proteolytic system of Streptococcus thermophilus KLDS 3.0503 were predicted by a promoter analysis softwarennpp. The 14 genes included an extracellular protease gene prtS, an oligopeptide ABC transport system gene amiA1, and 12 genes, respectively, encoding peptidases pepA, pepS, pepN, pepC, pepB, pepQ, pepV, pepT, pepM, pepXP, pepP, and pepO. These predicted promoter sequences were cloned and inserted into the upstream of a promoterless Escherichia coli gusA (β-glucuronidase) gene in a promoter probe vector pNZ273. The resulting vectors were, respectively, introduced into S. thermophilus KLDS 3.0503 and all 14 predicted promoter sequences were able to drive gusA expression, which indicated that these sequences were functional promoters. These promoters were able to interact with the S. thermophilus CodY homolog in an in vitro DNA binding assay but they did not contain a conserved CodY-box sequence identified in Lactococcus lactis. These results were useful for further studies on the regulation of protein metabolism in S. thermophilus. [ABSTRACT FROM AUTHOR]

An aminopeptidase specific for the N-terminal acidic residue (BcepAP) was purified from the cell extract of Burkholderia cepacia svr as a homotrimeric (subunit mass 66 kDa) molecule. It was identified as an unassigned peptidase of family M61. The only other member characterized so far from this family is a broad-specificity aminopeptidase of Sphingomonas capsulata (ScapAP) with preference for Gly or Ala residues. However, BcepAP exhibited narrow specificity and the preferred substrate was a peptide with an N-terminal Asp or Glu residue, which is quite unusual. The proteins assigned to this family were grouped separately on the basis of their homology to either BcepAP or ScapAP. It led the conclusion that BcepAP is a prototype of a new PepM61 subfamily, with a representative in other Proteobacteria, and to the prediction that members of the family share the ability to cleave N-terminal acidic residues of peptide substrates. [ABSTRACT FROM AUTHOR]

Effects of salts on the activity and stability of actinidain were examined. With increasing salt concentration up to 0.5 M, the activity (kcat/Km) for N-α-Cbz-l-lysine p-nitrophenyl ester decreased to 40% of that in the absence of salt. The inhibitor constant Ki of LiCl, NaCl, and KCl was 0.16–0.43 M. With 3 M KCl and NaCl, the specificity constant kcat/Km recovered to 110 and 75%, respectively. No re-activation was observed with LiCl. The inhibition and re-activation were dependent on the changes in both Km and kcat, whereas no CD change was observed. The tryptophan fluorescence of actinidain was not affected by 0–0.5 M salt, but a considerable decrease in its intensity was observed with increasing salt concentration from 0.5 to 3.0 M. These results suggest that the inhibition observed with the lower salt concentration (<0.5 M) is due to attenuation of the electrostatic interaction between the enzyme and substrate, and the higher concentration (0.5–3.0 M) induces structural change in the states of tryptophan residues, which is associated with the re-activation. Actinidain keeps considerably high activity and stability even in the presence of 3 M salts. [ABSTRACT FROM PUBLISHER]

The cerebrospinal fluid (CSF) provides a ready access into the health state of the central nervous system, and alterations in some CSF proteins have been documented in brain disease. However, the complete variety of proteins is not known and methods to identify protein components are still being developed. The goal of this study was to examine the sequence coverage obtained from human CSF digests produced with different proteases. Enzymatic digests of CSF proteins were obtained with arginine-C endopeptidase (ArgC), glutamic acid endopeptidase (GluC), chymotrypsin, trypsin and their combinations, and then examined using reverse phase chromatography and a Finnigan™ LTQ™ linear ion trap mass spectrometer. Peptide sequences were identified with BioWorks 3.1 and sequence coverage calculated for the 38 most confidently identified proteins. Trypsin and GluCyielded greater coverage than chymotrypsin, while ArgC had the least sequence coverage. Protein sequence coverage was affected only slightly over four orders of magnitude dynamic range of abundance. Combining the peptides derived from different proteases further increased the coverage. Maximal sequence coverage was achieved by combining digest results from both GluC and trypsin. These results have implications for future studies to identify CSF proteins and their post-translational modifications. [ABSTRACT FROM AUTHOR]

Candida albicans expresses a vast number of hydrolytic enzymes, playing roles in several phases of yeast–host interactions. Here, we identified two novel extracellular peptidase classes in C. albicans. Using gelatin–sodium dodecyl sulfate polyacrylamide gel electrophoresis two gelatinolytic activities were detected at physiological pH: a 60-kDa metallopeptidase, completely blocked by 1,10-phenanthroline, and a 50-kDa serine peptidase inhibited by phenylmethylsulfonyl fluoride. In an effort to establish a probable functional implication for these novel peptidase classes, we demonstrated that the 50-kDa secretory serine peptidase was active over a broad pH range (5.0–7.2) and was capable to hydrolyze some soluble human serum proteins and extracellular matrix components. Conversely, when this isolate was grown in yeast carbon base supplemented with bovine serum albumin, a secretory aspartyl peptidase activity was measured, instead of metallo- and serine peptidases, suggesting that distinct medium composition induces different expression of released peptidases in C. albicans. Additionally, we showed by quantitative proteolytic measurement, flow cytometry and immunoblotting assays that the brain heart infusion medium might repress the Sap1–3 production. Collectively, our results showed for the first time the capability of an extracellular proteolytic enzyme other than aspartic-type peptidases to cleave a broad spectrum of relevant host proteinaceous substrates by the human pathogen C. albicans. [ABSTRACT FROM AUTHOR]

Proteolytic enzymes in general, and cysteine proteases in particular, play key roles in seed germination and early seedling growth. However, the precise mechanism by which the serine proteases are regulated remains unclear. Trypsin-like activity was detected in wheat germ (quiescent embryo) and this activity increased in the germinating embryo. In this work, a trypsin-like serine protease expressed in wheat germ was purified to homogeneity by chromatography through DEAE-cellulose, phenyl-Sepharose, Ultrogel AcA-34 and Blue-Sepharose. The molecular mass of the enzyme was estimated to be 81 kDa by SDS-PAGE under reducing conditions. Amino acid analysis of the peptides generated following digestion of the enzyme with lysyl endopeptidase indicated that the enzyme is a plant homologue of Escherichia. coli oligopeptidase B. The subsite specificity of the enzymes differ, although both enzymes hydrolyze synthetic substrates and model peptides at the carboxyl side of basic amino acids. The wheat enzyme is more sensitive to leupeptin and antipain than the E. coli emzyme. These results provide the basis for characterizing plant oligopeptidase B and contribute to our understanding of its role in the early development of seedlings. [ABSTRACT FROM PUBLISHER]

x. ma, x. zhou and t. yoshimoto. 2004.To study the properties and show the potential application of a glycine aminopeptidase fromActinomucor elegans.The enzyme was estimated to have molecular mass of 320 kDa by gel filtration and the subunit size of 56·5 kDa by SDS-PAGE. It hydrolysed glycine from substrate more efficiently than other amino acids. The optimal temperature for this enzyme was 40°C and at pH 8·0 it showed its highest activity. TheKm andKcat of the enzyme for glycine-β-naphthylamine was 0·24 mmand 100·8 s−1, respectively. Zinc, copper, cadmium ando-phenanthrolin suppressed almost all enzyme activities at the concentration of 1·0 mm. In the process of hydrolysing proteins, it could improve the protease activity considerably.It was a hexamer metalloenzyme which was specific for the substrates with glycinse residue at the N-terminal and some metal cations were needed to maintain its activity.This study demonstrates the properties of a novel aminopeptidase and shows its potential application in the process of the food industry. [ABSTRACT FROM AUTHOR]

Mitochondrial processing peptidase (MPP) specifically cleaves off the N-terminal presequence of the mitochondrial protein precursor. Previous studies demonstrated that Arg at position —2 from the cleavage site, which is found among many precursors, plays a critical role in recognition by MPP. We analyzed the structural elements of bovine cytochrome P450 side-chain cleavage enzyme precursor [pre-P450(SCO], which has Ala at position —2, for recognition by MPP. Replacement of Ala position —2 of pre-P450(SCC) with Arg resulted in an increase in the cleavage rate. Replacement with Gly caused a reduction in the cleavage rate and the appearance of an additional cleavage site downstream of the authentic site. A pre-P450(SCC) mutant with Met at position —2 retained cleavage efficiency equal to that of the wild type. These results indicate that —2 Ala of pre-P450(SCC) is recognized by MPP as a determinant for precise cleavage, and that the amino acid at —2 is required to have a straight methylene chain for interaction with the S2 site. The preference for distal basic residues, a hydrophobic residue at +1, and hy-droxyl residues at +2 and +3, was almost the same as those of the precursors with Arg at —2, indicating that the recognition mechanism of pre-P450(SCC) by MPP is essentially the same as that of the precursors with Arg at position —2. [ABSTRACT FROM AUTHOR]

A gene for a pyrrolidone carboxyl peptidase (Pcp: EC 3.4.19.3, pyroglutamyl peptidase), which removes amino-terminal pyroglutamyl residues from peptides and proteins, has been cloned from the hyperthermophilic Archaeon Pyrococcus furiosus using its cosmid protein library, sequenced, and expressed in Escherichia coli. The DNA sequence encodes a protein containing 208 amino acid residues with methionine at the N-terminus. Analysis of the recombinant protein expressed in E. coli, including amino acid sequence analysis from the N-terminus by automated Edman degradation and ionspray mass spectrometric analysis of the peptides generated by enzymatic digestions with lysylendopeptidase and Staphylococcus aureus V8 protease, showed its primary structure to be completely identical with that deduced from its cDNA sequence. Comparison of the amino acid sequence of P. furiosus Pcp (P.f.Pcp) with those of bacterial Pcps revealed that a high degree of sequence identity (more than 40%) and conservation of the amino acid residues comprising the catalytic triad, Cys142, His166, and G1u79. On the other hand, a unique short stretch sequence (positions around 175–185) that is absent in bacterial Pcps was found in P.f.Pcp. A similar stretch has also been reported recently in the amino acid sequence of Pcp from the hyperthermophilic Archaeon Thermococcus litoralis [Littlechild et al., in abstracts of the “International Congress on Exthermophiles '98” p. 58 (1998)]. To elucidate their contribution to the hyperthermostability of these enzymes, further structural studies are required. [ABSTRACT FROM AUTHOR]

The Euglena light harvesting chlorophyll a/b binding protein of photosystem II (LHCPII) is synthesized as a polyprotein precursors composed of 8 LHCPIIs covalently joined by a decapeptide. A soluble chloroplast protease releases LHCPII from the polyprotein. The polyprotein processing peptidase has a pH optima between 8.0 and 9.0. It is inhibited by Zn2+, Cu2+, phenylmethylsulfonyl fluoride and E64 suggesting it is a novel thiol protease. [ABSTRACT FROM AUTHOR]

Endopeptidase activity was characterized in plasma membranes prepared from an antigen-presenting B cell lymphoma, A20. Activity was detected at both neutral and acid pH, and studies with inhibitors confirmed that a number of different enzymes, most probably cysteine, metallo, and aspartic endopeptidases, were associated with the A20 cell surface. [ABSTRACT FROM AUTHOR]

Peptidases are enzymes that hydrolyse peptide bonds in proteins and peptides. Peptidases are important in pathological conditions such as Alzheimer's disease, tumour and parasite invasion, and for processing viral polyproteins. The MEROPS database is an Internet resource containing information on peptidases, their substrates and inhibitors. The database now includes details of cleavage positions in substrates, both physiological and non-physiological, natural and synthetic. There are 39 118 cleavages in the collection; including 34 606 from a total of 10 513 different proteins and 2677 cleavages in synthetic substrates. The number of cleavages designated as ‘physiological’ is 13 307. The data are derived from 6095 publications. At least one substrate cleavage is known for 45% of the 2415 different peptidases recognized in the MEROPS database. The website now has three new displays: two showing peptidase specificity as a logo and a frequency matrix, the third showing a dynamically generated alignment between each protein substrate and its most closely related homologues. Many of the proteins described in the literature as peptidase substrates have been studied only in vitro. On the assumption that a physiologically relevant cleavage site would be conserved between species, the conservation of every site in terms of peptidase preference has been examined and a number have been identified that are not conserved. There are a number of cogent reasons why a site might not be conserved. Each poorly conserved site has been examined and a reason postulated. Some sites are identified that are very poorly conserved where cleavage is more likely to be fortuitous than of physiological relevance. This data-set is freely available via the Internet and is a useful training set for algorithms to predict substrates for peptidases and cleavage positions within those substrates. The data may also be useful for the design of inhibitors and for engineering novel specificities into peptidases. [ABSTRACT FROM PUBLISHER]