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Nascent chains undergo co-translational enzymatic processing as soon as their N-terminus becomes accessible at the ribosomal polypeptide tunnel exit (PTE). In eukaryotes, N-terminal methionine excision (NME) by Methionine Aminopeptidases (MAP1 and MAP2), and N-terminal acetylation (NTA) by N-Acetyl-Transferase A (NatA), is the most common combination of subsequent modifications carried out on the 80S ribosome. How these enzymatic processes are coordinated in the context of a rapidly translating ribosome has remained elusive. Here, we report two cryo-EM structures of multi-enzyme complexes assembled on vacant human 80S ribosomes, indicating two routes for NME-NTA. Both assemblies form on the 80S independent of nascent chain substrates. Irrespective of the route, NatA occupies a non-intrusive 'distal' binding site on the ribosome which does not interfere with MAP1 or MAP2 binding nor with most other ribosome-associated factors (RAFs). NatA can partake in a coordinated, dynamic assembly with MAP1 through the hydra-like chaperoning function of the abundant Nascent Polypeptide-Associated Complex (NAC). In contrast to MAP1, MAP2 completely covers the PTE and is thus incompatible with NAC and MAP1 recruitment. Together, our data provide the structural framework for the coordinated orchestration of NME and NTA in protein biogenesis. How methionine excision and acetylation are co-translationally coordinated at the ribosome has remained elusive. Here, the authors report cryo-EM structures of two different multi-protein assemblies capable of both successive enzymatic functions. [ABSTRACT FROM AUTHOR]

Aminopeptidases are metal co-factor-dependent hydrolases releasing N-terminal amino acid residues from peptides. Many of these enzymes, particularly the M24 methionine aminopeptidases (MetAPs), are considered valid drug targets in the fight against many parasitic and non-parasitic diseases. Targeting MetAPs has shown promising results against the malarial parasite, Plasmodium, which is regarded as potential anti-cancer targets. While targeting these essential enzymes represents a potentially promising approach, many challenges are often ignored by scientists when designing drugs or inhibitory scaffolds against the MetAPs. One such aspect is the metal co-factor, with inadequate attention paid to its role in catalysis, folding and remodeling of the catalytic site, and its role in inhibitor binding or potency. Knowing that a metal co-factor is essential for aminopeptidase enzyme activity and active site remodeling, it is intriguing that most computational biologists often ignore the metal ion while screening millions of potential inhibitors to find hits. Ironically, a similar trend is followed by biologists who avoid metal promiscuity of these enzymes while screening inhibitor libraries in vitro which may lead to false positives. This review highlights the importance of considering a physiologically relevant metal co-factor during the drug discovery processes targeting metal-dependent aminopeptidases. [ABSTRACT FROM AUTHOR]

Excision of the initiator methionine is among the first co-translational processes that occur at the ribosome. While this crucial step in protein maturation is executed by two types of methionine aminopeptidases in eukaryotes (MAP1 and MAP2), additional roles in disease and translational regulation have drawn more attention to MAP2. Here, we report several cryo-EM structures of human and fungal MAP2 at the 80S ribosome. Irrespective of nascent chains, MAP2 can occupy the tunnel exit. On nascent chain displaying ribosomes, the MAP2-80S interaction is highly dynamic and the MAP2-specific N-terminal extension engages in stabilizing interactions with the long rRNA expansion segment ES27L. Loss of this extension by autoproteolytic cleavage impedes interactions at the tunnel, while promoting MAP2 to enter the ribosomal A-site, where it engages with crucial functional centers of translation. These findings reveal that proteolytic remodeling of MAP2 severely affects ribosome binding, and set the stage for targeted functional studies. The role of methionine aminopeptidase 2 (MAP2) at the ribosome goes beyond N-terminal methionine excision. Klein et al. use cryo-EM to identify a second MAP2 binding site on the ribosome, and describe the dynamic interactions of MAP2 at the ribosome. [ABSTRACT FROM AUTHOR]

Protein hydrolysates are easily digested and utilized by humans and animals, and are less likely to cause allergies. Protein hydrolysis caused by endopeptidases often leads to the exposure of hydrophobic amino acids at the ends of peptides, which consequently causes bitter taste. Microbial aminopeptidases remove the exposed hydrophobic amino acids at the ends of aminopeptides, which improves taste, allowing for easier production. This processe is attacking significant attention from industry and laboratories. Aminopeptidases selectively hydrolyze peptide bonds from the N-terminal of proteins or peptides to produce free amino acids. Aminopeptidases can be classified into leucine, lysine, methionine and proline aminopeptidases by hydrolyzed N-terminal residues; metallo-, serine- and cysteine- aminopeptidases by the reaction mechanisms; dipeptide and triphoptide enzymes by the released number of amino acid residues at the end of hydrolyzed peptides; or acidic, neutral and basic aminopeptidases by their optimal hydrolysis pH. Commercial aminopeptidases are generally produced by microbial fermentation, and are mainly applied in the debittering of protein hydrolysates, the deep hydrolysis of protein, and the production of condiments, cheese, and bioactive peptides, as well as for disease detection in the medical industry. [ABSTRACT FROM AUTHOR]

Feather biodegradation is an important premise for efficient resource development and utilization, in which keratinase plays an important role. However, there are few keratinases that combine the high activity, thermal stability, and organic solvent tolerance required for industrialization. This paper reported an efficient feather-degrading Pseudomonas aeruginosa 4–3 isolated from slaughterhouses. After 48 h of fermentation by P. aeruginosa 4–3 in a feather medium at 40 °C, pH 8.0, keratinase was efficiently produced (295.28 ± 5.42 U/mL) with complete feather degradation (95.3 ± 1.5%). Moreover, the keratinase from P. aeruginosa 4–3 showed high optimal temperature (55 °C), good thermal stability, wide pH tolerance, and excellent organic solvent resistance. In addition, P. aeruginosa 4–3–derived aminopeptidases also exhibit excellent thermal stability and organic solvent tolerance. Encouragingly, the reaction of crude keratinase and aminopeptidase with feathers for 8 h resulted in a 78% degradation rate of feathers. These properties make P. aeruginosa 4–3 keratinase and aminopeptidase ideal proteases for potential applications in keratin degradation, as well as provide ideas for the synergistic degradation of keratin by multiple enzymes. [ABSTRACT FROM AUTHOR]

This study aims to explore the potential aminopeptidases of Lysinibacillus sphaericus based on the unique metabolic characteristics of this species which cannot metabolize carbohydrates and may have a strong ability to metabolize amino acids. Fifteen peptidase-encoding genes predicted in L. sphaericus C3-41 have been heterologously expressed in Escherichia coli BL21, and of these genes, only Amp0279 shows a high ability to hydrolyze L-leucine-4-nitroanilide (Leu-pNA). Phylogenetic analysis, 3D-structure modeling, and enzyme assays indicated that Amp0279 should be a novel Co2+-dependent aminopeptidase belonging to the M29 family. The optimal conditions of Amp0279 were determined to be 50 °C and pH 8.0 with the addition of 100 μM Co2+, and under this condition, the specific activity of Amp0279 matched that of Flavourzyme® (3.54 × 104 vs. 3.37 × 104 U/mg for the protein ingredient of Flavourzyme®). Amp0279 is mainly expressed in the middle sporulation phase in wild-type L. sphaericus or in Bacillus subtilis under the control of the sporulation-dependent strong promoter pcry8E, which is carried by the recombinant vector pHT315-8E21b. Furthermore, the secretory expression systems based on B. subtilis and Corynebacterium glutamicum were used to enhance the soluble expression of Amp0279. Obvious expression and enzymatic activity were detected from the crude supernatant media of both host bacteria without further concentration and purification. Moreover, expression can occur in the vegetative phase in B. subtilis under the control of the Pgrac promoter. Key points: • A novel Co2+-dependent leucyl aminopeptidase Amp0279 originating from L. sphaericus was characterized. • The activity of Amp0279 as a leucyl aminopeptidase matches that of Flavourzyme® under optimal conditions. • B. subtilis– and C. glutamicum–based expression systems are built to promote secretory (soluble) Amp0279 expression. [ABSTRACT FROM AUTHOR]

Processing of N-terminally elongated antigenic peptide precursors by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) is a key step in antigen presentation and the adaptive immune response. Although ERAP1 can efficiently process long peptides in solution, it has been proposed that it can also process peptides bound onto Major Histocompatibility Complex I molecules (MHCI). In a previous study, we suggested that the occasionally observed "ontο MHCI" trimming by ERAP1 is likely due to fast peptide dissociation followed by solution trimming, rather than direct action of ERAP1 onto the MHCI complex. However, other groups have proposed that ERAP1 can trim peptides covalently bound onto MHCI, which would preclude peptide dissociation. To explore this interaction, we constructed disulfide-linked MHCI-peptide complexes using HLA-B*08 and a 12mer kinetically labile peptide, or a 16mer carrying a phosphinic transition-state analogue N-terminus with high-affinity for ERAP1. Kinetic and biochemical analyses suggested that while both peptides could access the ERAP1 active site when free in solution, they were unable to do so when tethered in the MHCI binding groove. Our results suggest that MHCI binding protects, rather than promotes, antigenic peptide precursor trimming by ERAP1 and thus solution trimming is the more likely model of antigenic peptide processing. [ABSTRACT FROM AUTHOR]

We report structural characterization of proteases mined in silico from solid tannery waste (STW) metagenome. The physico-chemical analysis revealed the molecular weight of selected query proteases in the range of 34–43 kDa except 52.46 kDa for Cp-6. Secondary structure analysis suggested the dominance of α-helices (26–51 %) followed by β-sheets (13–34 %). Conserved regions in the selected proteases were identified using multiple sequence alignment. Diversity analysis of the proteases was performed by aligning with their best hits and already characterized proteases of similar families. The 3D structures of 19 selected amino acid sequences were deduced using homology modeling and evaluated following Ramachandran plots (R-plots), stability of the 3D conformation, and overall quality factor (G factor). The R-plots of all structures had 96.4–98.9 % residues in the favoured region, whereas 0-0.8 % lied in the outlier region. Fifteen modeled protein structures passed the quality assessment criteria and were subsequently used for molecular docking. All models showed two domains except Cp-6, which had an extra domain. Ligand binding sites and active sites were identified using sequence and structural homologs of the respective protease molecules. Serine, lysine, and serine of different conserved motifs were specified as the active site residues in carboxypeptidases, whereas serine, aspartate, and histidine were specific to aminopeptidases Ap-1, Ap-2, and Ap-4. Further, Ap-8 and Ap-9 required Mn2+ for enzyme activity along with histidine and glutamate in their active sites. These 3D structures were used for molecular docking with their specific peptide ligands to obtain stable docked conformations. Successfully docked complexes indicated the catalytic activity of these enzymes in hydrolyzing the peptide ligand. The structural and functional insights gained from our study may help in the identification of novel industrially important proteases after validation through experimental studies. [ABSTRACT FROM AUTHOR]

The transmembrane aminopeptidase CD13 is highly expressed in cells of the myeloid lineage, regulates dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. Here, we show that CD13-deficient mice present a low bone density phenotype with increased numbers of osteoclasts per bone surface, but display a normal distribution of osteoclast progenitor populations in the bone marrow and periphery. In addition, the bone formation and mineral apposition rates are similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Lack of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells containing remarkably high numbers of nuclei. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP1 must be downregulated for fusion to proceed, these are aberrantly sustained at high levels even in CD13-deficient mature multi-nucleated osteoclasts. Further, the stability of fusion-promoting proteins is maintained in the absence of CD13, implicating CD13 in protein turnover mechanisms. Together, we conclude that CD13 may regulate cell–cell fusion by controlling the expression and localization of key fusion regulatory proteins that are critical for osteoclast fusion. [ABSTRACT FROM AUTHOR]

Malaria transmission-blocking vaccines (TBVs) prevent the completion of the developmental lifecycle of malarial parasites within the mosquito vector, effectively blocking subsequent infections. The mosquito midgut protein Anopheline alanyl aminopeptidase N (AnAPN1) is the leading, mosquito-based TBV antigen. Structure-function studies identified two Class II epitopes that can induce potent transmission-blocking (T-B) antibodies, informing the design of the next-generation AnAPN1. Here, we functionally screened new immunogens and down-selected to the UF6b construct that has two glycine-linked copies of the T-B epitopes. We then established a process for manufacturing UF6b and evaluated in outbred female CD1 mice the immunogenicity of the preclinical product with the human-safe adjuvant Glucopyranosyl Lipid Adjuvant in a liposomal formulation with saponin QS21 (GLA-LSQ). UF6b:GLA-LSQ effectively immunofocused the humoral response to one of the key T-B epitopes resulting in potent T-B activity, underscoring UF6b as a prime TBV candidate to aid in malaria elimination and eradication efforts. [ABSTRACT FROM AUTHOR]

The clinical spectrum of hypertensive disorders of pregnancy (HDP) is determined by the interplay between environmental and genetic factors, most of which remains unknown. ERAP1, ERAP2 and LNPEP genes code for multifunctional aminopeptidases involved with antigen processing and degradation of small peptides such as angiotensin II (Ang II), vasopressin and oxytocin. We aimed to test for associations between genetic variants in aminopeptidases and HDP. A total of 1282 pregnant women (normotensive controls, n = 693; preeclampsia, n = 342; chronic hypertension with superimposed preeclampsia, n = 61; eclampsia, n = 74; and HELLP syndrome, n = 112) were genotyped for variants in LNPEP (rs27300, rs38034, rs2303138), ERAP1 (rs27044, rs30187) and ERAP2 (rs2549796 rs2927609 rs11135484). We also evaluated the effect of ERAP1 rs30187 on plasma Ang II levels in an additional cohort of 65 pregnant women. The genotype C/C, in ERAP1 rs30187 variant (c.1583 T > C, p.Lys528Arg), was associated with increased risk of eclampsia (OR = 1.85, p = 0.019) whereas ERAP2 haplotype rs2549796(C)–rs2927609(C)–rs11135484(G) was associated with preeclampsia (OR = 1.96, corrected p-value = 0.01). Ang II plasma levels did not differ across rs30187 genotypic groups (p = 0.895). In conclusion, ERAP1 gene is associated with eclampsia whereas ERAP2 is associated with preeclampsia, although the mechanism by which genetic variants in ERAPs influence the risk of preeclampsia and eclampsia remain to be elucidated. [ABSTRACT FROM AUTHOR]

The Plasmodium falciparum M1 alanyl aminopeptidase and M17 leucyl aminopeptidase, PfM1AAP and PfM17LAP, are potential targets for novel anti-malarial drug development. Inhibitors of these aminopeptidases have been shown to kill malaria parasites in culture and reduce parasite growth in murine models. The two enzymes may function in the terminal stages of haemoglobin digestion, providing free amino acids for protein synthesis by the rapidly growing intra-erythrocytic parasites. Here we have performed a comparative cellular and biochemical characterisation of the two enzymes. Cell fractionation and immunolocalisation studies reveal that both enzymes are associated with the soluble cytosolic fraction of the parasite, with no evidence that they are present within other compartments, such as the digestive vacuole (DV). Enzyme kinetic studies show that the optimal pH of both enzymes is in the neutral range (pH 7.0–8.0), although PfM1AAP also possesses some activity (< 20%) at the lower pH range of 5.0–5.5. The data supports the proposal that PfM1AAP and PfM17LAP function in the cytoplasm of the parasite, likely in the degradation of haemoglobin-derived peptides generated in the DV and transported to the cytosol. [ABSTRACT FROM AUTHOR]

A novel protease-producing Bacillus sp. CN2 isolated from chicken manure composts exhibited a relatively high proteolytic specific activity. The strain CN2 degradome consisted of at least 149 proteases and homolog candidates, which were distributed into 4 aspartic, 30 cysteine, 55 metallo, 56 serine, and 4 threonine proteases. Extracellular proteolytic activity was almost completely inhibited by PMSF (phenylmethylsulfonyl fluoride) rather than o-P, E-64, or pepstatin A, suggesting that strain CN2 primarily secreted serine protease. More importantly, analysis of the extracellular proteome of strain CN2 revealed the presence of a highly efficient protein degradation system. Three serine proteases of the S8 family with different active site architectures firstly fragmented protein substrates which were then degraded to smaller peptides by a M4 metalloendopeptidase that prefers to degrade hydrophobic peptides and by a S13 carboxypeptidase. Those enzymes acted synergistically to degrade intact substrate proteins outside the cell. Furthermore, highly expressed sequence-specific intracellular aminopeptidases from multiple families (M20, M29, and M42) accurately degraded peptides into oligopeptides or amino acids, thus realizing the rapid acquisition and utilization of nitrogen sources. In this paper, a systematic study of the functional-degradome provided a new perspective for understanding the complexity of the protease hydrolysis system of Bacillus, and laid a solid foundation for further studying the precise degradation of proteins with the cooperative action of different family proteases. Key points: • Bacillus sp. CN2 has relatively high proteolytic specific activity. • Bacillus sp. CN2 harbors a highly efficient protein degradation system. • The site-specific endopeptidases were secreted extracellular, while the sequence-specific aminopeptidases played a role in the cell. [ABSTRACT FROM AUTHOR]

Inborn errors of metabolism are often associated with neurodevelopmental disorders and brain injury. A deficiency of aminopeptidase P1, a proline-specific endopeptidase encoded by the Xpnpep1 gene, causes neurological complications in both humans and mice. In addition, aminopeptidase P1-deficient mice exhibit hippocampal neurodegeneration and impaired hippocampus-dependent learning and memory. However, the molecular and cellular changes associated with hippocampal pathology in aminopeptidase P1 deficiency are unclear. We show here that a deficiency of aminopeptidase P1 modifies the glial population and neuronal excitability in the hippocampus. Microarray and real-time quantitative reverse transcription-polymerase chain reaction analyses identified 14 differentially expressed genes (Casp1, Ccnd1, Myoc, Opalin, Aldh1a2, Aspa, Spp1, Gstm6, Serpinb1a, Pdlim1, Dsp, Tnfaip6, Slc6a20a, Slc22a2) in the Xpnpep1−/− hippocampus. In the hippocampus, aminopeptidase P1-expression signals were mainly detected in neurons. However, deficiency of aminopeptidase P1 resulted in fewer hippocampal astrocytes and increased density of microglia in the hippocampal CA3 area. In addition, Xpnpep1−/− CA3b pyramidal neurons were more excitable than wild-type neurons. These results indicate that insufficient astrocytic neuroprotection and enhanced neuronal excitability may underlie neurodegeneration and hippocampal dysfunction in aminopeptidase P1 deficiency. [ABSTRACT FROM AUTHOR]

Activated allyl acetates (the Morita–Baylis–Hillman acetates) were confirmed as convenient electrophilic precursors of the P1′ fragment of phosphinic dehydrodipeptides, and then, their selected saturated analogs were obtained in the subsequent reduction. This approach is particularly appropriate for structurally complex side chains and thus is an alternative to the addition of α-substituted acrylates with aminoalkylphosphinic acids. While phosphinic dehydrodipeptides were found to be good inhibitors of two mammalian alanyl metalloaminopeptidases, the saturated compounds showed higher activities than their structurally constrained counterparts. Docking calculations and molecular modeling showed that conformational freedom allowed for the favorable binding of distinct stereoisomers of phosphinates, while the presence of the double bond was more restrictive. [ABSTRACT FROM AUTHOR]

The halophilic archaeal strain ZS-3T (= CGMCC 1.12866T = JCM 30239T) was isolated from a sediment sample of Zhoushan marine solar saltern, P. R. China. Phylogenetic analyses based on 16S rRNA, rpoB′ genes and the concatenation of 738 protein sequences reveal that strain ZS-3T was related to members of the genus Halorussus. The OrthoANI and in silico DDH values between strain ZS-3T and the current Halorussus members are much lower than the threshold values proposed as the species boundary (ANI 95–96% and in silico DDH 70%), suggesting that strain ZS-3T represents a novel species of Halorussus (Halorussus halophilus sp. nov.). Diverse phenotypic characteristics differentiate strain ZS-3T from current Halorussus members. Since the strain expressed diverse hydrolyzing enzyme activity, its complete genome was sequenced. The genome of strain ZS-3T was found to be 4,450,731 bp with total GC content of 61.51%, and comprises one chromosome and three plasmids. A total of 4694 protein coding genes, 43 tRNA genes and 6 rRNA genes were predicted. A CRISPR–Cas system was also detected. The genome encodes sixteen putative glycoside hydrolases, nine extracellular proteases, seventeen aminopeptidases, seven carboxypeptidases, one esterase and one nitrite reductase. The exploration of the hydrolase genes may expand our understanding of adapted mechanism of halophilic archaea surviving optimally in hypersaline environments where containing organic matter. Meanwhile, various hydrolyzing enzymes may extend this microorganism for further applications in salt-based fermentation. [ABSTRACT FROM AUTHOR]

Aminopeptidases (EC 3.4.11.) belongs to exoprotease family, which can catalyze the cleavage of peptide bond which connects the N-terminal amino acid to the penultimate residue in a protein. Aminopeptidases catalyze the process of removal of the N-terminal amino acids of target substrates by sequential cleavage of one amino acid residue at a time. Microbial aminopeptidase are of great acceptance as industrial enzymes with varying applications in food and pharma industry since these enzymes possess unique characteristics than aminopeptidases from other sources. This review describes the various applications of microbial aminopeptidases in different industrial sectors. These enzymes are widely used in food industry as a debittering agent as well as in the preparation of protein hydrolysates. In baking, brewing, and cheese making aminopeptidases are extensively used for removing the bitterness of peptides. The inhibitors of these enzymes are found great clinical applications against various diseases such as cancer, diabetes, and viral infections. Aminopeptidases are widely used for the synthesis of biopeptides and amino acids, and found to be efficient than chemical synthesis. These enzymes are capable of hydrolyzing organophosphate compounds, thus having biological as well as environmental significance. Key Points • Cleaves the amino-terminal amino acid residues from proteins and peptides. • Microbial aminopeptidase are of great acceptance as both therapeutic and industrial enzyme. • Review describes the potential applications of microbial aminopeptidases. [ABSTRACT FROM AUTHOR]

Acanthamoeba is a free-living pathogenic protozoan that is distributed in different environmental reservoirs, including lakes and soil. Pathogenic Acanthamoeba can cause severe human diseases, such as blinding keratitis and granulomatous encephalitis. Therefore, it is important to understand the pathogenic relationship between humans and Acanthamoeba. By comparison of systemic analysis results for Acanthamoeba isolates, we identified a novel secreted protein of Acanthamoeba, an M28 aminopeptidase (M28AP), which targets of the human innate immune defense. We investigated the molecular functions and characteristics of the M28AP protein by anti-M28 antibodies and a M28AP mutant strain generated by the CRISPR/Cas9 system. Human complement proteins such as C3b and iC3b were degraded by Acanthamoeba M28AP. We believe that M28AP is an important factor in human innate immunity. This study provides new insight for the development of more efficient medicines to treat Acanthamoeba infection. [ABSTRACT FROM AUTHOR]

Because of its competitive inhibitor activity against aminopeptidase B, bestatin isolated from the broth of Streptomyces olivoreticuli ATCC 31159 is famous and currently used as an approved therapeutic agent for cancer and bacterial infections. It can be used alone or in combination with other antibiotics or anticancer drugs as adjuvant therapy drug for chemotherapy and radiotherapy. Due to the therapeutic importance of bestatin, mining of its biosynthetic mechanism is imperative. Genome mining, one of the bioinformatics-based approaches for the discovery of novel natural product, has been developed and applied widely. Herein, we reported the complete genome of Streptomyces olivoreticuli ATCC 31159 obtained from American Type Culture Collection (ATCC). It consists of 8,809,793 base pairs with a linear chromosome, GC content of 71.1%, 7520 protein-coding genes, 75 tRNA operons, 21 rRNA operons, 63 sRNAs. In addition, predictive analysis showed that at least 37 putative biosynthetic gene clusters (BGCs) of the secondary metabolites were obtained, 18 new BGCs with low similarity (< 25%) were included. The availability of novel and abundant gene clusters not only will provide clues for cracking the biosynthetic mechanism of bestatin, but also will provide valuable insight for mining the diverse bioactive compounds based on rational strategies. [ABSTRACT FROM AUTHOR]

A casein solution was hydrolyzed with Alcalase 2.4 L (EC 3.4.21.62) and the recombinantly produced aminopeptidases PepX (EC 3.4.14.11) and PepN (EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 in various combinations to analyze the synergistic effect of these peptidases during casein hydrolysis. The sequential application of PepX or PepN after prehydrolysis with Alcalase resulted in an relative degree of hydrolysis (rDH) increase of 1.12- or 2.00-fold, respectively, compared to only using Alcalase. By a combined application of PepX and PepN the rDH increased ~ 2.32-fold. Using Alcalase, PepX and PepN simultaneously from the beginning the rDH increased ~ 2.42-fold. Compared to the single application of PepX or PepN after an Alcalase treatment, the combined usage led to an increased amount of small peptides (< 1.1 kDa) and free amino acids. After the sequential application of first Alcalase and then PepX and PepN, only 14 peptides, which originated mainly from the C-terminal end of the β-casein chain remained. Even the opioid precursor peptide VYPFPGPIPN [β-casein, ƒ(59-68); V-β-casomorphine-9], generated by the Alcalase treatment was fully hydrolyzed after adding PepX and PepN. Therefore, the synergistic effect of PepX and PepN during casein hydrolysis was confirmed. The simultaneous application of Alcalase, PepX and PepN from the beginning showed similar results as the sequential application, but only three remaining peptides were observed by the mass spectrometric analysis. Additionally, the hydrolysis time was reduced from 16 h (sequential approach) to 6.5 h (simultaneous approach). This indicated a further synergism between Alcalase and the two aminopeptidases. [ABSTRACT FROM AUTHOR]

Three strains of haloalkaliphilic bacteria were isolated from microbial mats of soda-saline lakes of the Badain Jaran Desert, Inner Mongolia (China). Based on the data on ribosomal phylogeny, they were identified as members of the genus Halomonas. These bacteria were moderate alkaliphiles and extreme halophiles. The peptidases secreted by these bacteria were shown to have narrow substrate specificity. They hydrolyzed proteins and para-nitroanilide substrates and showed maximal activity in the hydrolysis of L-leucyl-p-nitroanilides (LpNA). The maximum activity of the peptidases occurred at alkaline pH values (8-10) and elevated salinity (50-100 g/L); the enzymes were thermostable (up to 50°С). The results of inhibitor analysis and substrate specificity examination of extracellular enzymes indicated them to belong to the class of aminopeptidase-like metallopeptidases. [ABSTRACT FROM AUTHOR]

The PepP protein has been purified in vitro and characterized for the first time. It is encoded by the sll0136 gene of the unicellular cyanobacterium Synechocystis sp. PCC6803. It is established that the PepP protein is a Mn2+-dependent Xaa-Pro-specific aminopeptidase. The protein in the reaction of hydrolysis of the fluorescent peptide Lys(N-Abz)-Pro-Pro-pNA has a maximal activity at pH 7.6 and 32°C. [ABSTRACT FROM AUTHOR]

Chemical reactor theory (CRT) suggests that the digestive tract functions as a chemical reactor for processing food. Presumably, gut structure and function should match diet to ensure adequate nutrient and energy uptake to maintain performance. Within CRT, dietary biochemical composition is the most important factor affecting gut structure and function in vertebrates. We fed Danio rerio (zebrafish) diets ranging from high- to moderate- to low-quality (i.e., ranging from high-protein, low-fiber to low-protein, high-fiber), and observed how gut length and surface area, as well as the activity levels of digestive enzymes (amylase, maltase, trypsin, aminopeptidase, and lipase) shifted in response to these dietary changes. Fish on the low-quality diet had the longest guts with the largest intestinal epithelial surface area and enterocyte cellular volumes. Fish on the moderate-quality diet had intermediate values of most of these parameters, and fish on the high-quality diet, the lowest. These data largely support CRT. Digestive enzyme activity levels were generally elevated in fish fed the moderate- and low-quality diets, but were highest in the fish fed the moderate-quality diet, suggesting that a diet with protein levels closest to that of the natural diet of D. rerio (they are omnivorous in nature) may elicit the best gut performance. However, fish fed the carnivore diet reached the largest terminal body size. Our results support CRT in terms of gut structure; however, our enzyme results do not necessarily agree with CRT and largely depend on which enzyme is discussed. In particular, the evidence for lipase activities being elevated in the fish fed the low-protein, high-fiber diet perhaps reflects a lipid-scavenging mechanism in fish consuming high-fiber foods rather than CRT. [ABSTRACT FROM AUTHOR]

Several works have shown that triterpenes induce peripheral antinociception by activation of cannabinoid receptors and endocannabinoids; besides, several research groups have reported activation of cannabinoid receptors in peripheral antinociception. The aim of this study was to assess the involvement of the cannabinoid system in the antinociceptive effect induced by tingenone against hyperalgesia evoked by prostaglandin E (PGE) at peripheral level. The paw pressure test was used and the hyperalgesia was induced by intraplantar injection of PGE (2 μg/paw). All drugs were injected subcutaneously in the hind paws of male Swiss mice. Tingenone (200 µg/paw) administered into the right hind paw induced a local antinociceptive effect, that was antagonized by AM630, a selective antagonist to CB cannabinoid receptor. AM251, a selective antagonist to CB cannabinoid receptor, did not alter the peripheral antinociceptive effect of tingenone. MAFP, a fatty acid amide hydrolase (FAAH) inhibitor; VDM11, an anandamide reuptake inhibitor; and JZL184, monoacylglycerol lipase (MAGL) inhibitor did not potentiate the peripheral antinociceptive effect of the lower dose of tingenone (50 µg/paw). The results suggest that tingenone induced a peripheral antinociceptive effect via cannabinoid receptor activation. Therefore, this study suggests a pharmacological potential for a new analgesic drug. [ABSTRACT FROM AUTHOR]

Antigen cross-presentation by dendritic cells (DC) stimulates cytotoxic T cell activation to promote immunity to intracellular pathogens, viruses and cancer. Phagocytosed antigens generate potent T cell responses, but the signalling and trafficking pathways regulating their cross-presentation are unclear. Here, we show that ablation of the store-operated-Ca2+-entry regulator STIM1 in mouse myeloid cells impairs cross-presentation and DC migration in vivo and in vitro. Stim1 ablation reduces Ca2+ signals, cross-presentation, and chemotaxis in mouse bone-marrow-derived DCs without altering cell differentiation, maturation or phagocytic capacity. Phagosomal pH homoeostasis and ROS production are unaffected by STIM1 deficiency, but phagosomal proteolysis and leucyl aminopeptidase activity, IRAP recruitment, as well as fusion of phagosomes with endosomes and lysosomes are all impaired. These data suggest that STIM1-dependent Ca2+ signalling promotes the delivery of endolysosomal enzymes to phagosomes to enable efficient cross-presentation. [ABSTRACT FROM AUTHOR]

The vasopressin- and oxytocin-degrading enzyme insulin-regulated aminopeptidase (IRAP) is expressed in various organs including the brain. However, knowledge about its presence in human hypothalamus is fragmentary. Functionally, for a number of reasons (genetic linkage, hydrolysis of oxytocin and vasopressin, its role as angiotensin IV receptor in learning and memory and others) IRAP might play a role in schizophrenia. We studied the regional and cellular localization of IRAP in normal human brain with special emphasis on the hypothalamus and determined numerical densities of IRAP-expressing cells in the paraventricular, supraoptic and suprachiasmatic nuclei in schizophrenia patients and controls. By using immunohistochemistry and Western blot analysis, IRAP was immunolocalized in postmortem human brains. Cell countings were performed to estimate numbers and numerical densities of IRAP immunoreactive hypothalamic neurons in schizophrenia patients and control cases. Shape, size and regional distribution of IRAP-expressing cells, as well the lack of co-localization with the glia marker glutamine synthetase, show that IRAP is expressed in neurons. IRAP immunoreactive cells were observed in the hippocampal formation, cerebral cortex, thalamus, amygdala and, abundantly, hypothalamus. Double labeling experiments (IRAP and oxytocin/neurophysin 1, IRAP with vasopressin/neurophysin 2) revealed that IRAP is present in oxytocinergic and in vasopressinergic neurons. In schizophrenia patients, the numerical density of IRAP-expressing neurons in the paraventricular and the suprachiasmatic nuclei is significantly reduced, which might be associated with the reduction in neurophysin-containing neurons in these nuclei in schizophrenia. The pathophysiological role of lowered hypothalamic IRAP expression in schizophrenia remains to be established. [ABSTRACT FROM AUTHOR]

Athetis lepigone was a new lepidopteran pest and caused severe damage to maize crops in China. We have detected that Cry1Ac protoxin and toxin were highly active against the larvae of A. lepigone. However, there is no report about the mode of action of Bt Cry1Ac toxin against this pest until now. A 110 kDa APN5 protein from BBMV of A. lepigone was identified as the binding receptor of Cry1Ac toxin using Ligand blotting. The Cry1Ac receptor APN5 was cloned from A. lepigone larval midgut mRNA and named as AlAPN5 (GenBank accession no.: KU950745). AlAPN5 had a GATEN motif and been classified to Class 5 APNs. 79.2% reduction in mortality was observed when A. lepigone larvae were injected with siRNA of the AlAPN5 gene and treated with Cry1Ac toxin. These data demonstrate that AlAPN5 is a putative functional receptor and maybe the only receptor of Cry1Ac in A. lepigone. [ABSTRACT FROM AUTHOR]

The combined effect of high pressure processing and temperature on aminopeptidases activity of lactic acid bacteria used as starter cultures in brined cheese manufacturing, in order to find the optimum process conditions for acceleration of the significant long-duration cheese ripening step, was investigated. The effect of high hydrostatic pressure (HP) (100-450 MPa) combined with temperature (20-40 °C) on the activity of five aminopeptidases (PepN, PepX, PepY, PepC, and PepA) of Streptococcus thermophilus ACA-DC 0022 and Lactococcus lactis ACA-DC 0049, used as the starter culture for white Greek brine cheese ( feta) production, was studied. S. thermophilus aminopeptidases PepN, PepX, PepA, and PepC were activated at pressures up to 200 MPa, and all studied temperatures (20-40 °C), while for L. lactis, PepN, X, and Y were activated at pressures up to 300 MPa and temperatures up to 30 °C and PepA at the same temperature range but milder pressures (up to 200 MPa). For L. lactis, PepC an increase in activity was observed at all studied pressures but only at 20 °C. A multi-parameter equation was used for predicting the activation of all aminopeptidases in the pressure and temperature domain. Overall, processing at 200 MPa and 20 °C may be selected as the optimum conditions for maximum activation of all aminopeptidases of both S. thermophilus ACA-DC 0022 and L. lactis ACA-DC 0049. A 20-min treatment at these conditions leads to an average threefold increase in activity which could lead to better and faster maturation of white cheese. [ABSTRACT FROM AUTHOR]

A bioactive compound was purified from the culture medium of a new strain of Lactococcus BSN307 by solvent extraction followed by chromatographic techniques. This bioactive compound was identified to belong to phenazine class of compounds by MS, NMR and FTIR. The phenazine compound showed antifungal activity against Aspergillus niger, Penicillium chrysogenum as well as Fusarium oxysporum by disc diffusion assay in addition to antioxidant potential as demonstrated by DPPH scavenging assay. The compound demonstrated selective cytotoxicity against cancer cell lines HeLa and MCF-7 where IC was achieved with 20 and 24 µg/mL respectively. At the same time no cytotoxicity was occurred in normal H9c2 cells. The bioactive found to be inhibitory to both leucine and proline aminopeptidases and thus revealed its potential as metalloenzyme inhibitor. This study, for the first time reports the production of phenazine class of compounds by lactic acid bacteria. [ABSTRACT FROM AUTHOR]

Biopores are pores or voids in soil produced by roots, by earthworms, or by the occupation of earthworms in root pores, which are considered important microbial hotspots, especially in subsoil. We hypothesized that earthworms ( Lumbricus terrestris L.) exert stronger effects on microbial activities than decaying plant roots (of Cichorium intybus L.) in the subsoil because of the addition of pre-digested organic material. We tested this hypothesis by analyzing microbial biomass (C), total organic C (C), and activities of eight enzymes (cellobiohydrolase, β-glucosidase, xylanase, acid phosphomonoesterase, leucine aminopeptidase, tyrosine aminopeptidase, chitotriosidase, and n-acetylglucosaminidase) down to 105-cm depth. The C increase was associated with a two- to threefold increase of C content in biopores as compared to bulk soil. The highest percentage of C-to-C (3.7 to 7.3 %) in the drilosphere demonstrated the enhancement of microbial efficiency for organic matter decomposition by earthworms. The availability of organic matter in biopores increased the activities of C- and N-targeting enzymes by 1.2-11.3 times, but reduced acid phosphomonoesterase activity by 10-40 % in biopores versus bulk soil. Introducing earthworms in root biopores caused 1.5-1.8 times higher microbial biomass and 1.2-1.9 times increased enzyme activities compared to the sole effect of earthworms. Soil depth showed a strong effect on the drilosphere, but only slight effects on the biochemical properties of root biopores and bulk soil. In conclusion, biopores are important microbial hotspots of C, N, and P transformations in subsoil. Earthworms exerted stronger effects on biochemical properties of biopores than decaying roots. [ABSTRACT FROM AUTHOR]

Prolyl aminopeptidases are specific exopeptidases that catalyze the hydrolysis of the N-terminus proline residue of peptides and proteins. In the present study, the prolyl aminopeptidase gene ( pap) from Aspergillus oryzae JN-412 was optimized through the codon usage of Pichia pastoris. Both the native and optimized pap genes were inserted into the expression vector pPIC9 K and were successfully expressed in P. pastoris. Additionally, the activity of the intracellular enzyme expressed by the recombinant optimized pap gene reached 61.26 U mL, an activity that is 2.1-fold higher than that of the native gene. The recombinant enzyme was purified by one-step elution through Ni-affinity chromatography. The optimal temperature and pH of the purified PAP were 60 °C and 7.5, respectively. Additionally, the recombinant PAP was recovered at a yield greater than 65 % at an extremely broad range of pH values from 6 to 10 after treatment at 50 °C for 6 h. The molecular weight of the recombinant PAP decreased from 50 kDa to 48 kDa after treatment with a deglycosylation enzyme, indicating that the recombinant PAP was completely glycosylated. The glycosylated PAP exhibited high thermo-stability. Half of the activity remained after incubation at 50 °C for 50 h, whereas the remaining activity of PAP expressed in E. coli was only 10 % after incubation at 50 °C for 1 h. PAP could be activated by the appropriate salt concentration and exhibited salt tolerance against NaCl at a concentration up to 5 mol L. [ABSTRACT FROM AUTHOR]

We have found that isolated from mammalian brain (rat, bovine) axonal endings (synaptosomes) degrade peptides of different composition. With the use of low concentration of non ionic detergent Triton X-100 (0.05-0.1 %) four low specific metallopeptidases were detached from synaptosomes. These peptidases were named Neuronal EctoMetalloPeptidases (NEMPs). Using specially designed test-peptides they were characterized as: carboxypeptidase (NEMP1), aminopeptidase (NEMP2) and endopeptidases NEMP3 and NEMP4. NEMPs are true peptidases (oligopeptidases), because they are able efficiently degrade peptides containing less than 40 amino acid residues. Specific properties of some NEMPs were revealed. NEMP1 is a small protein (molecular mass of about 10 kDa), which tends to dynamic oligomerization. NEMP3 needs activation. Some amino acids activate this enzyme. As far as we know, these properties were not ascribed to the known similarly localized peptidases. A possible physiological function of low specific NEMPs is participation in control of wide range of neuropeptides secreted in the synaptic cleft. However, NEMPs also due to their low specificity can destroy introduced in brain therapeutic peptides. The data obtained in this study open new opportunities for the protection of synthetic therapeutic peptides in brain and, possibly, in other tissues. [ABSTRACT FROM AUTHOR]

Nowadays, general and specific aminopeptidases are of great interest, especially for protein hydrolysis in the food industry. As shown previously, it is confirmed that the general aminopeptidase N (PepN; EC 3.4.11.2) and the proline-specific peptidase PepX (EC 3.4.14.11) from Lactobacillus helveticus ATCC 12046 show a synergistic effect during protein hydrolysis which results in high degrees of hydrolysis and reduced bitterness. To combine both activities, the enzymes were linked and a fusion protein called PepN-L1-PepX (FUS-PepN-PepX) was created. After production and purification, the fusion protein was characterized. Some of its biochemical characteristics were altered in favor for an application compared to the single enzymes. As an example, the optimum temperature for the PepN activity increased from 30 °C for the single enzyme to 35 °C for FUS-PepN. In addition, the temperature stability of PepX was higher for FUS-PepX than for the single enzyme (50 % compared to 40 % residual activity at 50 °C after 14 days, respectively). In addition, the disulfide bridge-reducing reagent β-mercaptoethanol did not longer inactivate the FUS-PepN activity. Furthermore, the K values decreased for both enzyme activities in the fusion protein. Finally, it was found that the synergistic hydrolysis performance in a casein hydrolysis was not reduced for the fusion protein. The increase of the relative degree of hydrolysis of a prehydrolyzed casein solution was the same as it was for the single enzymes. As a benefit, the resulting hydrolysate showed a strong antioxidative capacity (ABTS-IC value: 5.81 μg mL). [ABSTRACT FROM AUTHOR]

The methionine aminopeptidase 1b from Plasmodium falciparum ( PfMetAP 1b) was cloned, expressed in Escherichia coli and characterized. Surprisingly, and in contrast to other methionine aminopeptidases (MetAPs) that require heavy-metal cofactors such as cobalt, the enzyme is reliably activated by zinc ions. Immobilization of the enzyme is possible by His-tag metal chelation to iminodiacetic acid-agarose and by covalent binding to chloroacetamido-hexyl-agarose. The covalently immobilized enzyme shows long-term stability, allowing a continuous, heterogenous processing of N-terminal methionines, for example, in recombinant proteins. Activation by zinc, instead of cobalt as for other MetAPs, avoids the introduction of heavy metals with toxicological liabilities and oxidative potential into biotechnological processes. The PfMetAP 1b therefore represents a useful tool for the enzymatic, posttranslational processing of recombinant proteins. [ABSTRACT FROM AUTHOR]

The production of hydrolytic enzymes is considered a key virulence determinant of Candida albicans. Aminopeptidase 2 (Ape2) facilitates the penetration of C. albicans into the host tissue, by providing free amino acids to support fungal growth and proliferation. The objective of this study was to estimate the APE2 expression profile in C. albicans cells during invasion of the human epithelium. Sixty-one clinical fungal isolates and five reference strains were included in this study. The wild-type APE2 sequence was analyzed by polymerase chain reaction (PCR) amplification using genomic DNA from pathogenic isolates. Amplicons were verified in 1 % agarose gel and visualized by illumination with ultraviolet (UV) light. The APE2 expression levels were analyzed with reverse transcription quantitative real-time PCR (RT-qPCR) and APE2 quantification was normalized against the reference gene in C. albicans cells grown in YEPD and during Caco-2 invasion. The APE2-specific PCR product band was found in all C. albicans and C. dubliniensis strains, but not in other common pathogenic fungi. APE2 transcript abundance was elevated in the clinical isolates growing on the Caco-2 cell line in comparison to their counterparts grown in YEPD. Our data indicate a potential role for Ape2 in the invasion of epithelial cells. APE2 expression is also strain-specific, and it is not related to isolation site or disease entity. [ABSTRACT FROM AUTHOR]

Mosquito-based malaria transmission-blocking vaccines (mTBVs) target midgut-surface antigens of the Plasmodium parasite's obligate vector, the Anopheles mosquito. The alanyl aminopeptidase N (AnAPN1) is the leading mTBV immunogen; however, AnAPN1's role in Plasmodium infection of the mosquito and how anti-AnAPN1 antibodies functionally block parasite transmission have remained elusive. Here we present the 2.65-Å crystal structure of AnAPN1 and the immunoreactivity and transmission-blocking profiles of three monoclonal antibodies (mAbs) to AnAPN1, including mAb 4H5B7, which effectively blocks transmission of natural strains of Plasmodium falciparum. Using the AnAPN1 structure, we map the conformation-dependent 4H5B7 neoepitope to a previously uncharacterized region on domain 1 and further demonstrate that nonhuman-primate neoepitope-specific IgG also blocks parasite transmission. We discuss the prospect of a new biological function of AnAPN1 as a receptor for Plasmodium in the mosquito midgut and the implications for redesigning the AnAPN1 mTBV. [ABSTRACT FROM AUTHOR]

Purpose: Peptides containing the asparagine-glycine-arginine (NGR) motif bind to aminopeptidase N (CD13), which is expressed on inflammatory cells, endothelial cells, and fibroblasts. It is unclear whether radiolabeled NGR-containing tracers could be used for in vivo imaging of the early wound-healing phase after myocardial infarction (MI) using positron emission tomography (PET). Procedures: Uptake of novel tracer [Ga]NGR was assessed together with [Ga]arginine-glycine-aspartic acid ([Ga]RGD) and 2-deoxy-2-[ F]fluoro- d-glucose after myocardial ischemia/reperfusion (MI/R) injury using μ-PET and autoradiography, and relative expressions of CD13 and integrin β were assessed in fibroblasts, inflammatory cells, and endothelial cells by immunohistochemistry. Results: In the infarcted myocardium, uptake of [Ga]NGR was maximal from days 3 to 7 after MI/R, and correlated with fibroblast and inflammatory cell infiltration as well as [Ga]RGD uptake. Conclusions: [Ga]NGR allows noninvasive and sequential determination of CD13 expression in fibroblasts and inflammatory cells by PET. This will facilitate monitoring of CD13 in the individual wound healing processes, allowing patient-specific therapies to improve outcome after MI. [ABSTRACT FROM AUTHOR]

The mechanism by which human leukocyte antigen B27 (HLA-B27) contributes to ankylosing spondylitis (AS) remains unclear. Genetic studies demonstrate that association with and interaction between polymorphisms of endoplasmic reticulum aminopeptidase 1 (ERAP1) and HLA-B27 influence the risk of AS. It has been hypothesised that ERAP1-mediated HLA-B27 misfolding increases endoplasmic reticulum (ER) stress, driving an interleukin (IL) 23-dependent, pro-inflammatory immune response. We tested the hypothesis that AS-risk ERAP1 variants increase ER-stress and concomitant pro-inflammatory cytokine production in HLA-B27+ but not HLA-B27− AS patients or controls. Forty-nine AS cases and 22 healthy controls were grouped according to HLA-B27 status and AS-associated ERAP1 rs30187 genotypes: HLA-B27+ERAP1risk, HLA-B27+ERAP1protective, HLA-B27−ERAP1risk and HLA-B27−ERAP1protective. Expression levels of ER-stress markers GRP78 (8 kDa glucose-regulated protein), CHOP (C/EBP-homologous protein) and inflammatory cytokines were determined in peripheral blood mononuclear cell and ileal biopsies. We found no differences in ER-stress gene expression between HLA-B27+ and HLA-B27− cases or healthy controls, or between cases or controls stratified by carriage of ERAP1 risk or protective alleles in the presence or absence of HLA-B27. No differences were observed between expression of IL17A or TNF (tumour necrosis factor) in HLA-B27+ERAP1risk, HLA-B27+ERAP1protective and HLA-B27−ERAP1protective cases. These data demonstrate that aberrant ERAP1 activity and HLA-B27 carriage does not alter ER-stress levels in AS, suggesting that ERAP1 and HLA-B27 may influence disease susceptibility through other mechanisms. [ABSTRACT FROM AUTHOR]

Endosplasmic reticulum aminopeptidase 1 (ERAP1), endoplasmic reticulum aminopeptidase 2 (ERAP2) and puromycin-sensitive aminopeptidase (NPEPPS) are key zinc metallopeptidases that belong to the oxytocinase subfamily of M1 aminopeptidase family. NPEPPS catalyzes the processing of proteosome-derived peptide repertoire followed by trimming of antigenic peptides by ERAP1 and ERAP2 for presentation on major histocompatibility complex (MHC) Class I molecules. A series of genome-wide association studies have demonstrated associations of these aminopeptidases with a range of immune-mediated diseases such as ankylosing spondylitis, psoriasis, Behçet's disease, inflammatory bowel disease and type I diabetes, and significantly, genetic interaction between some aminopeptidases and HLA Class I loci with which these diseases are strongly associated. In this review, we highlight the current state of understanding of the genetic associations of this class of genes, their functional role in disease, and potential as therapeutic targets. [ABSTRACT FROM AUTHOR]

Infertility is a devastating medical condition that adversely affects emotional health and well-being of couples who desire pregnancy and parenthood. The overall demographic data suggest that the indication for more than one-third of assisted reproductive technology cycles performed in the United States includes male factor infertility. There is increasing recognition of the role that peptides present in seminal plasma have in determining sperm motility. Several recent studies suggest that peptidases, such as neutral endopeptidase (NEP) and aminopeptidase N (APN), impose significant adverse effects on sperm motility. Interestingly, several recent studies demonstrate that there is an endogenous NEP/APN inhibitor peptide called opiorphin in human seminal plasma. Our pilot studies suggest opiorphin promotes sperm motility and may positively influence sperm motility parameters in some cases of males infertility characterized by asthenozoospermia. [ABSTRACT FROM PUBLISHER]

The assays of muscle arginyl (RAP) and alanyl (AAP) aminopeptidases and dipeptidyl peptidase IV (DPPIV) have been optimised using colorimetric (aminoacyl-p-nitroanilide) substrates in order to design an appropriate test in their assay in fresh pork meat. Kinetic parameters, Km and Vmax, were calculated for the three enzymes using pNa substrates. The rapid colorimetric analysis was developed and applied directly on the muscle extracts from 16 samples previously classified in different pork meat qualities. The exudative meat quality class (PSE) showed significant lower values for these enzyme activities than the ideal meat quality (RFN) and the dark, firm and dry (DFD) quality classes. The protocols can be easily transferred to food industry because the use of colorimetric substrates and a microplate colour reader are feasible to use in large-scale production systems. [ABSTRACT FROM AUTHOR]

Amyloid beta (Aβ) peptides play a central role in the pathogenesis of Alzheimer's disease. The accumulation of Aβ peptides in AD brain was caused due to overproduction or insufficient clearance and defects in the proteolytic degradation of Aβ peptides. Hence, Aβ peptide degradation could be a promising therapeutic approach in AD treatment. Recent experimental report suggests that aminopeptidase from Streptomyces griseus KK565 (SGAK) can degrade Aβ peptides but the interactive residues are yet to be known in detail at the atomic level. Hence, we developed the three-dimensional model of aminopeptidase (SGAK) using SWISS-MODEL, Geno3D and MODELLER. Model built by MODELLER was used for further studies. Molecular docking was performed between aminopeptidase (SGAK) with wild-type and mutated Aβ peptides. The docked complex of aminopeptidase (SGAK) and wild-type Aβ peptide (1IYT.pdb) shows more stability than the other complexes. Molecular docking and MD simulation results revealed that the residues His93, Asp105, Glu139, Glu140, Asp168 and His255 are involved in the hydrogen bonding with Aβ peptide and zinc ions. The interactions between carboxyl oxygen atoms of Glu139 of aminopeptidase (SGAK) with water molecule suggest that the Glu139 may be involved in the nucleophilic attack on Ala2-Glu3 peptide bond of Aβ peptide. Hence, amino acid Glu139 of aminopeptidase (SGAK) might play an important role to degrade Aβ peptides, a causative agent of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

This study evaluated the effects of the pro-oxidant buthionine sulfoximine (BSO) and of the interaction between BSO and TETRAC, an antagonist of αvß3 integrin, on tumor development and aminopeptidase (AP) activity in a murine model of implanted Lewis's carcinoma. Male CBA-C57 mice were untreated (controls) or treated with BSO (222 mg/100 mL in drinking water), TETRAC (10 mg/kg/day, i.p.), or BSO + TETRAC. BSO for 28 days and TETRAC were given for the last 20 days. Mice were subcutaneously inoculated with 1 × 10 Lewis carcinoma 3LL cells into the dorsum. Study variables were tumor weight (TW); Hb, as index of tumor-mediated angiogenesis; vascular endothelial growth factor (VEGF) protein abundance; protein carbonyl content; α-tubulin abundance; and GluAp, AlaAp, and AspAp activities. BSO produced a major decrease in TW (203 ± 18 mg) with respect to controls (365 ± 26) and a reduction in Hb content. The TETRAC group also showed marked reductions in TW (129 ± 15) and Hb concentration associated with a reduced VEGF content. The BSO + TETRAC group showed a major TW reduction (125 ± 13); although, the difference with the TETRAC group was not significant. BSO treatment increased protein carbonyl and tubulin abundance in comparison to controls. The activity of all APs was increased in the three experimental groups and was strongly and negatively correlated with TW. In conclusion, administration of BSO reduced the TW, which inversely correlated with protein carbonyl content, suggesting a loss of microtubule polymerization. The finding of a negative correlation between TW and AP activity opens up new perspectives for the study of APs as tumor growth modulators. [ABSTRACT FROM AUTHOR]

The changes in protease activities in embryonic axes during the first days of bean ( Phaseolus vulgaris L.) seed germination were investigated in response to copper stress. Synthetic substrates and specific protease inhibitors have been used to define qualitatively and quantitatively different catalytic classes, particularly endoproteases (EP), carboxypeptidases (CP) and aminopeptidases (AP), then identify which ones were affected in the presence of copper. In fact, a failure in storage proteins mobilization and a disorder of nitrogen supply at enzymatic level occurred in Cu. In fact, Cu inhibited azocaseinolytic activity (ACA) and cysteine-, aspartic-, serine-, and metallo-endopeptidases activities (Cys-EP, Asp-EP, Ser-Ep, and Met-EP, respectively). Besides, Cu affected leucine- and proline-aminopeptidases (LAP and PAP, respectively) and glycine-carboxypeptidases (Gly-CP). The proteolytic responses might also be associated with the decrease in defense capacity in the Cu-treated embryos. [ABSTRACT FROM AUTHOR]

Mutations of the puromycin-sensitive aminopeptidase ( Psa) orthologs of flies, mice, and plants result in meiotic errors and reduced embryonic viability. Genetic lesions of the Caenorhabditis elegans ortholog of Psa, pam- 1, similarly result in dramatic reductions of worm fecundity. The gonads of animals harboring mutant pam- 1 alleles display expanded populations of pachytene germinal nuclei and delayed nucleolar disassembly in the developing oocytes, phenotypes that ultimately hinder embryonic viability and overall brood sizes. PAM-1 is a member of the M1 aminopeptidase family and shares a high amount of homology with its M1 paralogs. Comparative analysis of the M1 aminopeptidase family reveals that only nine (including PAM-1) of the 17 annotated M1 aminopeptidases are predicted to be catalytically active. Interestingly, we demonstrate that three of these active M1 paralogs have roles independent of PAM-1 in promoting gametogenesis and fecundity. Simultaneous inhibition of pam- 1 and M1 paralogs produces synergistic decreases in overall brood sizes and embryonic viability, exacerbates the germinal phenotypes of pachytene extension and delayed nucleolar disassembly, and unmasks previously hidden phenotypes. Our data suggests that the interdependent functions of multiple M1 aminopeptidases are necessary for reproductive success in C. elegans and lend further credence to the redundant composition of an evolutionarily conserved enzyme family. [ABSTRACT FROM AUTHOR]

This study evaluated the effects of thyroid hormone-NO interaction on tumor development, vascularization, vascular endothelial growth factor (VEGF), and aminopeptidase (AP) activity in a murine model of implanted Lewis's carcinoma. Experiments were performed in male CBA-C57 mice. Animals were untreated (controls) or treated with: T, the antithyroid drug methimazole, the NO inhibitor L-NAME, T + L-NAME, methimazole + NAME, the αvß3 integrin antagonist tetrac, T + tetrac, the iNOS inhibitor aminoguanidine (AG), and T + AG; all treatments were for 6 weeks except for tetrac, administered for the last 11 days. Mice were subcutaneously inoculated with 1 × 10 exponentially growing Lewis carcinoma 3LL cells into the dorsum. Study variables 9 days later were tumor weight (TW), Hb content, an index of tumor vascularization, VEGF, and AP activity. T produced parallel increases in TW and angiogenesis. L-NAME reduced TW and angiogenesis in control, hyperthyroid, and hypothyroid mice, whereas AG had no effect on these variables. Tetrac arrested TW in normal and T-treated mice but did not decrease angiogenesis in T-treated animals. Negative correlations were found between TW and AP activity in tumors from control hyper- and hypothyroid groups and an inverse relationship was observed between TW and AP activities in tetrac-treated mice. T enhances TW and angiogenesis, in which NO participates, but requires activation of integrin αvß3 to promote carcinogenesis. NO blockade reduces TW, regardless of the thyroid status. Thyroid hormone negatively modulates AP activity in the tumor. Accordingly, blockade of the membrane TH receptor αvß3 integrin reduces TW associated with an increase in AP activity. [ABSTRACT FROM AUTHOR]

Leukotriene A hydrolase (LTA4H--EC 3.3.2.6) is a bifunctional zinc metalloenzyme, which processes LTA through an epoxide hydrolase activity and is also able to trim one amino acid at a time from N-terminal peptidic substrates via its aminopeptidase activity. In this report, we have utilized a library of 130 individual proteinogenic and unnatural amino acid fluorogenic substrates to determine the aminopeptidase specificity of this enzyme. We have found that the best proteinogenic amino acid recognized by LTA4H is arginine. However, we have also observed several unnatural amino acids, which were significantly better in terms of cleavage rate ( k/ K values). Among them, the benzyl ester of aspartic acid exhibited a k/ K value that was more than two orders of magnitude higher (1.75 × 10 M s) as compared to l-Arg (1.5 × 10 M s). This information can be used for design of potent inhibitors of this enzyme, but may also suggest yet undiscovered functions or specificities of LTA4H. [ABSTRACT FROM AUTHOR]

The variations and associations of abiotic and biotic variables in Baiyangdian Lake, China, were analyzed in situ. Abiotic variables included eleven water quality parameters, and were characterized by water quality index (WQI). Biotic variables included periphyton function and diversity metrics. WQI differed in different seasons at sampling sites and the highest value of WQI was observed in October 2009. Periphyton function metrics, expressed by extracellular enzyme activities of alkaline phosphatase, β-glucosidase and leucine aminopeptidase, gross primary productivity and daily respiration rate, and diversity indices, in terms of Shannon diversity index and Berger-Parker abundance index, showed significantly temporal and spatial variations. Regression linear analysis illustrated a fairly good correlation of WQI with periphyton function and diversity indices, Shannon diversity index was the best correlated with WQI (r = 0.904, P < 0.01), followed by leucine aminopeptidase (r = −0.847, P < 0.01) and Berger-Parker abundance index (r = −0.840, P < 0.01), alkaline phosphatase, β-glucosidase and gross primary productivity also showed a good inverse correlation with WQI. Redundancy analysis suggested that eleven environmental variables explained a significant amount of the variation in the periphyton community data. The study was helpful for us to understand chemical and ecological status of water quality, and give us messages for monitoring water quality accurately. [ABSTRACT FROM AUTHOR]

Aminopeptidases represent a class of (zinc) metalloenzymes that catalyze the cleavage of amino acids nearby the N-terminus of polypeptides, resulting in hydrolysis of peptide bonds. Aminopeptidases operate downstream of the ubiquitin-proteasome pathway and are implicated in the final step of intracellular protein degradation either by trimming proteasome-generated peptides for antigen presentation or full hydrolysis into free amino acids for recycling in renewed protein synthesis. This review focuses on the function and subcellular location of five key aminopeptidases (aminopeptidase N, leucine aminopeptidase, puromycin-sensitive aminopeptidase, leukotriene A hydrolase and endoplasmic reticulum aminopeptidase 1/2) and their association with different diseases, in particular cancer and their current position as target for therapeutic intervention by aminopeptidase inhibitors. Historically, bestatin was the first prototypical aminopeptidase inhibitor that entered the clinic 35 years ago and is still used for the treatment of lung cancer. More recently, new generation aminopeptidase inhibitors became available, including the aminopeptidase inhibitor prodrug tosedostat, which is currently tested in phase II clinical trials for acute myeloid leukemia. Beyond bestatin and tosedostat, medicinal chemistry has emerged with additional series of potential aminopeptidases inhibitors which are still in an early phase of (pre)clinical investigations. The expanded knowledge of the unique mechanism of action of aminopeptidases has revived interest in aminopeptidase inhibitors for drug combination regimens in anti-cancer treatment. In this context, this review will discuss relevant features and mechanisms of action of aminopeptidases and will also elaborate on factors contributing to aminopeptidase inhibitor efficacy and/or loss of efficacy due to drug resistance-related phenomena. Together, a growing body of data point to aminopeptidase inhibitors as attractive tools for combination chemotherapy, hence their implementation may be a step forward in a new era of personalized treatment of cancer patients. [ABSTRACT FROM AUTHOR]

Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes. [ABSTRACT FROM AUTHOR]