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Aminopeptidases catalyze the hydrolysis of peptides into constituent amino acids and are therefore a key step in nitrogen (N) depolymerization and downstream mineralization. Differences in carbon (C):N as well as sulfur (S) content of amino acids present the possibility of amino acid-specific aminopeptidase activities revealing distinct information on N and non-N processes in soils. However, chromogenic assays of soil aminopeptidases using para-nitroaniline (pNA) substrates are underutilized and have largely focused on just two aminopeptidases to-date. We evaluated activities of eight amino acid-specific aminopeptidases using pNA substrates for arginine (ARG), glycine (GLY), alanine (ALA), lysine (LYS), glutamic acid (GLU), methionine (MET), proline (PRO) and leucine (LEU) aminopeptidases in a high-resolution sampling (30 samples per 0.01 ha−1 treatment plot) of nine 145-year treatment plots resulting from a factorial combination of three crop rotations and three fertility treatments on a Argiudoll in North Central United States. Across treatments, we identified non-detectable activities of ARG and PRO, and detectable activities of GLY, ALA, LYS, GLU, MET, and LEU ranging 0.2 to 400 nmol pNA h−1 g−1. Except for LYS and GLU activities, aminopeptidase activities were homogeneously distributed within treatments (n=30 locations per plot), and exhibited a mean coefficient of variation (CV) of 9 to 30 % and spatial range of 4.1 to 9.0 m. Activities of ALA and MET had the lowest CV and greatest contribution in explaining overall variability of aminopeptidase activities and were more sensitive to treatments compared to GLY and LEU activity. Multivariate analysis revealed a positive correlation among aminopeptidase activities, with major variation explained by β-glucosidase activity followed by pH and total N (TN). Absolute aminopeptidase activities generally reflected soil organic C (SOC) and TN concentrations, with greater activities under diverse rotation with organic fertilization (manure) and lower activities under continuous maize without fertilization. However, aminopeptidase activities normalized to SOC concentration were greater in soils without fertilization relative to organic fertilization. This study provides benchmark values of soil aminopeptidase activities assayed by pNA substrates using the world’s second oldest continuous agricultural experiment, including six aminopeptidases that have not been previously assayed with these substrates. Given their low magnitude of variability, high spatial correlation, and sensitivity to management, assays of aminopeptidase activities – particularly MET and ALA activities – could be fruitful tools for better understanding soil N cycling. Future work should evaluate assay optimization and expand to additional amino acid-specific substrates using pNA-based substrates.

A consortium of microbial community was used for the treatment of acid mine drainage wastewater laden with sulphate and heavy metals. The wastewater was treated in an anaerobic continuously stirred tank bioreactor. The microbial community activity increased the pH from 5.6 to 6.5, and improved sulphate removal up to 85% from an initial sulphate concentration of 8080 mg SO42−/L in a continuous mode, following enrichment for 21 d. The maximum heavy metal removal percentage was observed for Cd (98%), Al (97%), Mn (95%), Pb (94%), Sr (94%) and Cu (91%). The microbial community showed synergy between strictly anaerobic and facultative Firmicutes sp., which were responsible for the bioreactor performance. The biochemical reaction indicated the microbial community has a wider range of substrates dominated by metallo-aminopeptidases.

Products of microbial protein metabolism in the gut can influence the health of the host in many ways. Members of the Bacteriodales, major commensals of the human colon have been associated with long-term intake of high-protein diets. Undigested proteins or peptides that reach the colon can be hydrolyzed by extra-cellular proteases found in some Bacteroides species into amino acids and peptides which can be further catabolized. In this communication, we have characterized one such secreted aminopeptidase (BfAP) from Bacteroides fragilis belonging to the M28 family which is capable of degrading peptides released from soybean protein after predigestion in the small intestine. The BfAP enzyme was cloned, expressed in E. coli, and purified to homogeneity. It is a metallopeptidase requiring Co 2+ ion for optimum activity at 55 °C and pH 8 and preferentially cleaves neutral aliphatic (Met/Leu) and positively charged (Arg/Lys) amino acids from the N-terminus of peptides. It showed high specificity for long peptides as well as proteins like β-casein. Structural analysis of BfAP and its orthologues using AlphaFold2 reveal a shared highly conserved M28 domain, but vary with respect to their N-terminal region with some of them possessing an additional cap domain which may be important for regulation of substrate binding. Although BfAP lacks the typical cap domain, it shows small extensions that can form a loop adjacent to the proposed active site and may affect substrate binding. We suggest that this secreted enzyme may play an important role in protein metabolism in the colon where Bacteroides species are abundant., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023. Published by Elsevier B.V.)

The development of a healthy gut during prestarter and starter phases is crucial to drive chicken's productivity. This study aimed to evaluate the effects of a thermomechanical, enzyme-facilitated, coprocessed yeast and soybean meal (pYSM) on growth performance, organ weights, leg health, and gut development in broiler chickens. A total of 576 as-hatched broiler chicks were randomly allotted to 3 dietary treatments (8 replicates/treatment, 24 chickens/replicate): a control group (C) without the pYSM, a treatment group 1 (T1), in which the pSYM was included at 20, 10, 5, 0, and 0% levels in the prestarter, starter, grower, finisher I, and finisher II feeding phases, respectively, and a treatment group 2 (T2), in which the pSYM was included at 5, 5, 5, 0, and 0% levels in each feeding phase. On d 3 and 10, 16 broilers/treatment were euthanized. The T1 broilers tended to show higher live weight (d 3 and 7) and average daily gain (prestarter and starter phases) than the other groups (P ≤ 0.10). Differently, pYSM-based diets did not influence the growth performance of the other feeding phases and the whole experimental period (P > 0.05). Relative weights of pancreas and liver were also unaffected by pYSM utilization (P > 0.05). Litter quality tended to have higher average scores in C group (P = 0.079), but no differences were observed for leg health (P > 0.05). Histomorphometry of gut, liver, and bursa of Fabricius was not affected by diet (P > 0.05). Gut immunity was driven to an anti-inflammatory pattern, with the reduction of IL-2, INF-γ, and TNF-α in the duodenum of treated birds (d 3, P < 0.05). Also, MUC-2 was greater in the duodenum of C and T2 group when compared to T1 (d 3, P = 0.016). Finally, T1-fed chickens displayed greater aminopeptidase activity in the duodenum (d 3 and 10, P < 0.05) and jejunum (d 3, P < 0.05). Feeding high levels of pYSM (10-20%) to broilers in the first 10 d tended to improve growth performance in the prestarter and starter phases. It also positively downregulated proinflammatory cytokines during the first 3 d, as well as stimulated the aminopeptidase activity in the prestarter and starter periods., Competing Interests: DISCLOSURES Two of the authors, Mark Karimi and Mai Anh Ton Nu, are employees of the AB NEO company. This interest has been fully disclosed to the journal., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The short peptides, containing the amino acid sequence asparagine-glycine-arginine (NGR) and arginine-glycine-aspartic acid (RGD), possess the strong binding ability to N (APN/CD13) aminopeptidase receptor and integrin proteins involved in antitumor properties are overexpressed. A novel short N-terminal modified hexapeptides P1 and P2 was designed and synthesized using the Fmoc-chemistry solid phase peptide synthesis protocol. Notably, the cytotoxicity of the MTT assay demonstrated the viability of normal and cancer cells up to lower peptide concentrations. Interestingly, both peptides show good anticancer activities against the four cancer cells and normal cells namely, Hep-2, HepG2, MCF-7, A375, and Vero and compared with standard drugs, doxorubicin and paclitaxel. Additionally, in silico studies were applied to predict the binding sites and binding orientation of the peptides for potential anticancer targets. Steady-state fluorescence measurements showed that peptide P1 exhibits preferential interactions with POPC/POPG anionic bilayers rather than the zwitterionic POPC lipid bilayers and peptide P2, did not show any preferential interaction with lipids bilayers. But impressively, peptide P2 shows anticancer activity due to the NGR/RGD motif. Circular dichroism studies demonstrated that the peptide's secondary structure changes only minimally upon binding to the anionic lipid bilayers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Bufadienolides are steroids that inhibit Na + /K + -ATPase; recent evidence shows that bufalin inhibits the activity of porcine aminopeptidase N (pAPN). We evaluated the selectivity of some bufadienolides on metallo-aminopeptidases. Among the enzymes of the M1 and M17 families, pAPN and porcine aminopeptidase A (pAPA) were the only targets of some bufadienolides. ѱ-bufarenogin, telocinobufagin, marinobufagin, bufalin, cinobufagin, and bufogenin inhibited the activity of pAPN in a dose-dependent manner in the range of 10 -7 -10 -6  M. The inhibition mechanism was classical reversible noncompetitive for telocinobufagin, bufalin and cinobufagin. Bufogenin had the lowest K i value and a non-competitive behavior. pAPA activity was inhibited by ѱ-bufarenogin, cinobufagin, and bufogenin, with a classical competitive type of inhibition. The models of enzyme-inhibitor complexes agreed with the non-competitive type of inhibition of pAPN by telocinobufagin, bufalin, cinobufagin, and bufogenin. Since APN is a target in cancer therapy, we tested the effect of bufadienolides on the MeWo APN+ human melanoma cell line; they induced cell death, but we obtained scant evidence that inhibition of APN contributed to their effect. Thus, APN is a selective target of some bufadienolides, and we suggest that inhibition of APN activity by bufadienolides is not a major contributor to their antiproliferative properties in MeWo cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Trichinella spiralis is a major foodborne zoonotic parasitic nematode which has a serious threat to meat food safety. Development of anti-Trichinella vaccine is requisite for control and elimination of Trichinella infection in food animals to ensure meat safety. Aminopeptidase P (TsAPP) and cathepsin X (TsCX) are two novel proteins identified in T. spiralis intestinal infectious L1 larvae (IIL1). The objective of this study was to investigate the protective immunity elicited by immunization with TsAPP and TsCX alone and TsAPP-TsCX in combination in a mouse model. The results demonstrate that subcutaneous vaccination of mice with rTsAPP, rTsCX or rTsAPP + rTsCX elicited a systemic humoral response (high levels of serum IgG, IgG1/IgG2a and IgA) and significant local gut mucosal sIgA responses. The vaccination with rTsAPP, rTsCX or rTsAPP + rTsCX also induced a systemic and local mixed Th1/Th2 response, as demonstrated by clear elevation levels of IFN-γ and IL-4 in vaccinated mice. Vaccination of mice with rTsAPP+rTsCX exhibited a 63.99 % reduction of intestinal adult worms and 68.50% reduction of muscle larva burdens, alleviated inflammation of intestinal mucosal and muscle tissues, and provided a higher immune protection than that of vaccination with rTsAPP or rTsCX alone. The results demonstrated that TsAPP and TsCX might be considered novel candidate target molecules for anti-Trichinella vaccines., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Lipid oxidation and proteolysis are essential processes in Serrano dry-cured ham quality. The influence of high pressure processing (HPP) at 600 MPa for 6 min on lipid oxidation, aminopeptidase (AP) activities and free amino acids (FAA) in ripened Serrano hams of different chemical composition after 5 months at 4 °C were studied. HPP increased lipid peroxidation indexes. Composition influenced both indexes, with higher levels in hams of medium or high intramuscular fat (IMF) content and in hams of low or medium salt content or salt-in-lean ratio. HPP lowered AP activities by more than 50%. Composition also affected AP activities, with lower levels in hams of low a w , high IMF content, low salt content or low salt-in-lean ratio. At the end of refrigerated storage, HPP only affected Arg and Tyr levels. Many of the individual FAA reached higher levels in hams of low a w , medium or high IMF content, low or medium salt content, or low or medium salt-in-lean ratio., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The degradation of the immunomodulatory octapeptide, thymic humoral factor γ2 (THF-γ2, thymoctonan) has been studied in whole blood samples from human, rat and mouse. The peptide, Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu, was shown to be rapidly degraded by peptidases. The half-life of the intact peptide was less than 6 min at 37 °C in blood from the three species tested. The main fragments formed from THF-γ2 were found to be Glu-Asp-Gly-Pro-Lys-Phe-Leu (2-8), Asp-Gly-Pro-Lys-Phe-Leu (3-8) and Glu-Asp-Gly-Pro-Lys (2-6) in human and in rat blood and 2-8 and 2-6 in mouse blood. Analysis of the time course of degradation revealed a sequential removal of single amino acids from the N-terminus (aminopeptidase activities) in a process that was apparently unable to cleave the Gly-Pro bond (positions 4-5 in the peptide) together with an independent cleavage of the Lys-Phe bond (positions 6-7 in the peptide) to release the dipeptide Phe-Leu. This behaviour and the effects of inhibitors showed the involvement of metallo-exopeptidases in the N-terminal digestion and a phosphoramidon-sensitive metallo-endopeptidase in the cleavage of the Lys-Phe bond. The degradation patterns in human blood were modelled in terms of the competing pathways involved approximating to first-order kinetics, and an analytical solution obtained via the method of Laplace Transforms. The half-life of THF degradation in whole rat blood sample was found to be significantly lower than in human or mouse., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aims: Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) differ in their renin-angiotensin system function and sympathetic tone. The metabolism of angiotensins and vasopressin depends on the action of certain aminopeptidases whose activity may be influenced by the autonomic nervous system. Their regulation may differ between WKY and SHR in hypothalamus and plasma according to the sympathetic tone. We analyzed aminopeptidases responsible for the hydrolysis of certain angiotensins, vasopressin, cholecystokinin or enkephalins in hypothalamus and plasma of WKY and SHR in untreated controls rats and under beta-adrenoceptor blockade. Systolic blood pressure, food intake, water intake and diuresis were measured as parameters modulated by the autonomic nervous system and the above mentioned peptides., Main Methods: Glutamyl-, aspartyl-, cystinyl- and alanyl-aminopeptidase activities were analyzed fluorimetrically in plasma and hypothalamus of control and propranolol-treated (100mg/kg/day administered in drinking water for 1month) WKY and SHR, using arylamide derivatives as substrates., Key Findings: An opposite response of aminopeptidases to propranolol treatment between plasma and hypothalamus was observed in either WKY and SHR. Furthermore, the behavior of aminopeptidases was inversed between WKY and SHR either in hypothalamus and plasma: while the activity increased in hypothalamus and decreased in plasma of WKY, it decreased in hypothalamus and increased in plasma of SHR., Significance: These results revealed an inverse response of aminopeptidases between hypothalamus and plasma and also an opposite behavior of these enzymes between WKY and SHR in hypothalamus and plasma. These observations support the involvement of the sympathetic system in the modulation of aminopeptidase activities., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Schistosoma mansoni enzymes play important roles in host-parasite interactions and are potential targets for immunological and/or pharmacological attack. The aim of this study was to comparatively assess the presence of hydrolytic activities (phosphatases, glycosidases, aminopeptidases) in soluble (SF) and membrane (MF) fractions from different S. mansoni developmental stages (schistosomula 0 and 3h, juveniles, and adult worms of 28 and 45days-old, respectively), by using simple enzyme-substrate microassays. Our results show and confirm the prominent presence of alkaline phosphatase (AlP) activity in the MF of all the above parasite stages, highlighting also the relevant presence of MF-associated α-mannosidase (α-MAN) activity in juveniles. A soluble AlP activity, together with β-N-D-acetylglucosaminidase (β-NAG), and α-MAN activities, was detected in SF of schistosomulum 0h. Soluble β-NAG, α-MAN, acid phosphatase (AcP), leucin (LAP) and alanine (AAP) aminopeptidase activities were also seen in the SF of the other different developmental stages. This work shows different soluble and membrane-associated hydrolytic capacities in each S. mansoni developmental stage from schistosomula to adults that might be exploitable as potential new targets for immune and/or chemoprophylactic strategies., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

At least one-third of enzymes contain metal ions as cofactors necessary for a diverse range of catalytic activities. In the case of polymetallic enzymes (i.e., two or more metal ions involved in catalysis), the presence of two (or more) closely spaced metal ions gives an additional advantage in terms of (i) charge delocalisation, (ii) smaller activation barriers, (iii) the ability to bind larger substrates, (iv) enhanced electrostatic activation of substrates, and (v) decreased transition-state energies. Among this group of proteins, enzymes that catalyze the hydrolysis of ester and amide bonds form a very prominent family, the metallohydrolases. These enzymes are involved in a multitude of biological functions, and an increasing number of them gain attention for translational research in medicine and biotechnology. Their functional versatility and catalytic proficiency are largely due to the presence of metal ions in their active sites. In this chapter, we thus discuss and compare the reaction mechanisms of several closely related enzymes with a view to highlighting the functional diversity bestowed upon them by their metal ion cofactors., (© 2014 Elsevier Inc. All rights reserved.)

Activities of 19 hydrolases were estimated in cell suspensions of 40 Brochothrix thermosphacta strains, isolated from meat and meat products packaged under various conditions, by using API ZYM® test. These strains produced: acidic phosphatase, esterase C4, esterase/lipase C8, α-chymotrypsin, leucine arylamidase, β-glucosidase and α-glucosidase. The predominating biotype produced only the first four of the listed enzymes. The extracellular lipolytic activity for bromo-chloro-indolyl-caprylate was detected only in culture broth supernatants of 4 strains thereby indicating that esterase/lipase C8 is cell-bound. 13 of the strains displayed proteolytic activity for albumin at 4 °C (0.31-2.07 U) while 9 strains showed this activity at 25 °C (0.11-1.21 U). Only 4 strains digested albumin at both temperatures. Thus the meat spoilage potential of B. thermosphacta strains results not only from digestion of carbohydrates but also from their proteolytic activity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A new analytical method for the detection and the quantitative evaluation of the undecapeptide substance P by capillary electrophoresis coupled with ion trap mass spectrometry (CE-MS) by a co-axial sheath liquid interface has been developed. Conditions of analysis employed an acidic buffer and a 60 cm fused silica capillary installed by overcoming the UV window position, thus allowing to perform the analysis in a brief time. The method has been applied to the evaluation of substance P enzymatic hydrolysis during incubation with the human osteosarcoma SaOS-2 cell line. The analysis of amino acids derived from the cleavage of substance P has been also carried out simultaneously under the same electrophoretic conditions allowing the description of a kinetic of amino acid formation, parallel with substance P disappearance. The amounts of intact substance P and of free amino acids were monitored along 600 s of incubation time. A steady decrease of substance P as function of reaction time was observed. Peptide's half-life was found to be about 4.3s, indicating an extremely fast hydrolysis in the presence of the SaOS-2 cells. Proline, phenilalanine and methionine were the predominant free amino acids recorded. Obtained results lead to hypothesize the occurrence of endopeptidases activity, followed by aminopeptidases responsible for the release of free amino acids originated after primary bond cleavage., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to gamma-irradiation, and that nuclear expression of TPPII was present in most gamma-irradiated transformed cell lines. We used a panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after gamma-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following gamma-irradiation (at 1-4h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in gamma-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII-/- transformed fibroblasts showed no influences of TPPII expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.

The effective control of the infection of mice with the facultatively intracellular bacterium Listeria monocytogenes requires CD8 T cells which recognize bacterial antigenic peptides presented in the context of host MHC class I molecules. It is generally accepted that bacterial antigens are processed by the proteasome, a proteolytic cytoplasmic multiprotein complex. We observed that presentation of the L. monocytogenes-derived CD8 T cell epitope LLO 91-99 by infected cells can not be totally suppressed by inhibitors of the proteasome alone. Further analysis revealed that inhibitors of the cytoplasmic tripeptidyl peptidase II suppressed the presentation of the epitopes LLO 91-99 and p60 449-457. While significant suppression of the presentation of LLO 91-99 required the simultaneous inhibition of the proteasome and tripeptidyl peptidase II, presentation of p60 449-457 was suppressed by inhibitors of either the proteasome or TPPII alone. Thus, these data indicate that both, the proteasome and tripeptidyl protease II play a role in the processing of L. monocytogenes-derived antigenic peptides.

Tripeptidyl-peptidase II (TPP II) is a cytosolic peptidase that has been implicated in fat formation and cancer, apparently independent of the enzymatic activity. In search for alternative functional regions, conserved motifs were identified and eleven signatures were constructed. Seven of the signatures covered previously investigated residues, whereas the functional importance of the other motifs is unknown. This provides directions for future investigations of alternative activities of TPP II. The obtained signatures provide an efficient bioinformatic tool for the identification of TPP II homologues. Hence, a TPP II sequence homologue from fission yeast, Schizosaccharomyces pombe, was identified and demonstrated to encode the TPP II-like protein previously reported as multicorn. Furthermore, an homologous protein was found in the prokaryote Blastopirellula marina, albeit the TPP II function was apparently not conserved. This gene is probably the result of a rare gene transfer from eukaryote to prokaryote.

Overexpression of TPPII correlates with accelerated growth and the appearance of centrosome and chromosome aberrations, suggesting that the activity of this enzyme may be critical for the induction and/or maintenance of genetic instability in malignant cells. We now find that the length of mitosis and of the entire cell cycle is significantly reduced in TPPII overexpressing HEK293 cells compared to untransfected and control transfected cells. Functional TPPII knockdown by shRNA interference caused a significant slowdown in cell growth and the accumulation of cells that delayed or failed to complete mitosis. TPPII overexpressing cells evade mitotic arrest induced by spindle poisons and display high levels of polyploidy despite the constitutively high expression of major components of the spindle checkpoint. TPPII overexpression correlated with upregulation of IAPs and with resistance to mitochondria dependent apoptosis induced by p53 stabilization. Thus, TPPII appears to promote malignant cell growth by allowing exit from mitosis and the survival of cells with severe mitotic spindle damage.

The mutational analysis of potassium (KCNQ2, KCNQ3), sodium (SCN1A, SCN2A), and chloride (CLCN2) ion channels was performed in three children with typical features of the recently described syndrome of migrating partial seizures in infancy. Mutational analysis was performed by PCR and automatic sequencing. The coding regions, including the exon-intron boundaries, were amplified in the patients using appropriate primers sets. No mutations associated to migrating partial seizures have been found. Mutational screening of CLCN2 gene, revealed a homozygous mutation G2003C (exon 17), leading to a Ser/Thr substitution at the codon 668, in two of the three patients. The same variation has been found in 38 out of 100 control alleles. The identification of the genetic basis of this new epileptic encephalopathy requires further studies that might be enforced by familial cases.

Tripeptidyl-peptidase II is a high-molecular weight peptidase with a widespread distribution in eukaryotic cells. The enzyme sequentially removes tripeptides from a free N-terminus of longer peptides and also displays a low endopeptidase activity. A role for tripeptidyl-peptidase II in the formation of peptides for antigen presentation has recently become evident, and the enzyme also appears to be important for the degradation of some specific substrates, e.g. the neuropeptide cholecystokinin. However, it is likely that the main biological function of tripeptidyl-peptidase II is to participate in a general intracellular protein turnover. This peptidase may act on oligopeptides generated by the proteasome, or other endopeptidases, and the tripeptides formed would subsequently be good substrates for other exopeptidases. The fact that tripeptidyl-peptidase II activity is increased in sepsis-induced muscle wasting, a situation of enhanced protein turnover, corroborates this biological role.

Late-infantile neuronal ceroid lipofuscinosis (CLN2), previously known as the late-infantile form of Batten disease, is a lysosomal storage disease which results from mutations in the gene that codes for tripeptidyl peptidase-I (TPP-I). This disease is characterised by progressive neurodegeneration in young children although the molecular mechanisms responsible for neuronal cell death are unclear. TPP-I is an exopeptidase which removes N-terminal tripeptides from small peptides, including several peptide hormones. We report that the degradation of the neuropeptide, neuromedin B, by mouse brain cells is restricted to lysosomes and that the pattern of degradation products is consistent with a predominant role for TPP-I. Neuromedin B is degraded by a similar pathway in a mouse neuronal cell line and also in cultured human fibroblasts. A specific inhibitor of TPP-I is able to abolish neuromedin B degradation in a variety of cell types. Fibroblasts from CLN2 patients, which are deficient in TPP-I activity, are unable to degrade neuromedin B. These observations suggest that TPP-I is the predominant proteolytic enzyme responsible for the intracellular degradation of neuromedin B. The inability of cells from CLN2 patients to degrade neuromedin B and other neuropeptides may contribute to the pathogenesis of the disease.

Eukaryotic initiation factor 2 (eIF2)-associated glycoprotein p67 protects eIF2alpha phosphorylation from kinases. The N-terminal lysine-rich domains increase this activity and the acidic residue-rich domain inhibits it. Conserved amino acid residues D251, D262, E364, and E459 are involved in this inhibition. During heat shock, the overall protein synthesis rate decreases due to the increased levels of eIF2alpha phosphorylation. In this study, we examined whether the above inhibition is also found during heat shock. Indeed, the acidic residue-rich domain mutant (D6/2) showed a decreased level of eIF2alpha phosphorylation, and its second-site alanine substitutions at D251, D262, and E459 reversed this effect, whereas second-site alanine substitution at H331 and E364 residues further augmented it. A high-molecular-weight phosphoprotein and at least two faster-migrating phosphoproteins were detected by the monospecific polyclonal antibody against eIF2alpha(P) form in rat tumor hepatoma cells constitutively expressing the double mutant D6/2+D251A. Although the levels of p67 mutants were unaffected during heat shock, those of p67 and p67-deactivating enzyme varied. Furthermore, the overall rate of protein synthesis correlated with the level of eIF2alpha phosphorylation. Taken together, these results suggest that the lysine-rich domains and conserved amino acid residues of p67 are involved in the regulation of eIF2alpha phosphorylation during heat shock.

A spore-based biosensor for detecting low levels of bacteria in real-time has been recently developed. The system (termed LEXSAS, label-free exponential signal-amplification system) exploits spore's ability to produce fluorescence when sensing neighboring bacterial cells. We studied the LEXSAS as a possible approach for identifying bacterially contaminated platelet concentrates prior to transfusion because the system offers rapid analysis, high sensitivity, and low cost. If successful, this approach could reduce the risk of morbidity and mortality from transfusion-related bacteremia and sepsis. In this study, we used the LEXSAS for detecting bacteria in platelet concentrates spiked with Bacillus cereus, Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, or Streptococcus pyogenes. Bacteria were separated from platelets using a 2-min procedure based on bacterial resistance to detergents and osmotic shock. The results indicate that the LEXSAS could be used to design a practical biosensor for identifying bacterially contaminated platelets in real-time.

Ubiquitin-proteasome-dependent protein degradation plays a central role in sepsis-induced muscle wasting. Because the proteasome degrades proteins into small peptides rather than free amino acids, it is likely that additional mechanisms downstream of the proteasome are involved in sepsis-induced muscle proteolysis. Recent studies suggest that the extralysosomal peptidase tripeptidyl-peptidase II (TPP II) degrades peptides generated by the proteasome. We hypothesized that TPP II expression and activity are increased in skeletal muscle during sepsis. Sepsis was induced in rats by cecal ligation and puncture. Control rats were sham-operated. TPP II activity was determined by using the specific substrate Ala-Ala-Phe-7-amido-4-methylcoumarin (AAF-AMC). TPP II protein and gene expression were determined by Western blot and real-time PCR, respectively. Sepsis resulted in increased activity and protein and gene expression of TPP II in extensor digitorum longus muscles. This result was blunted by the glucocorticoid receptor antagonist RU 38486, indicating that glucocorticoids participate in the upregulation of TPP II in skeletal muscle during sepsis. The results suggest that proteolytic mechanisms downstream of the proteasome may be important for the complete degradation of muscle proteins during sepsis.

Muscle cachexia induced by sepsis, severe injury, cancer, and a number of other catabolic conditions is mainly caused by increased protein degradation, in particular breakdown of myofibrillar proteins. Ubiquitin-proteasome-dependent proteolysis is the predominant mechanism of muscle protein loss in these conditions, but there is evidence that several other regulatory mechanisms may be important as well. Some of those mechanisms are reviewed in this article and they include pre-, para-, and postproteasomal mechanisms. Among preproteasomal mechanisms, mediators, receptor binding, signaling pathways, activation of transcription factors, and modification of proteins are important. Several paraproteasomal mechanisms may influence the trafficking of ubiquitinated proteins and their interaction with the proteasome, including the expression and activity of the COP9 signalosome, the carboxy terminus of heat shock protein 70-interacting protein (CHIP) and valosin-containing protein (VCP). Finally, because the proteasome does not degrade proteins completely into free amino acids but into peptides, postproteasomal degradation of peptides by the giant protease tripeptidyl peptidase II (TPP II) and various aminopeptidases is important in muscle catabolism. Thus, multiple mechanisms and regulatory steps may influence the breakdown of ubiquitinated muscle proteins by the 26S proteasome., ((c)2002 Elsevier Science.)

Purpose: Late-infantile neuronal ceroid lipofuscinosis (LINCL), an autosomal recessively inherited lysosomal storage disorder characterized by autofluorescent inclusions and rapid progression of neurodegeneration, is due to CLN2 gene mutations. However, CLN2 mutation analysis has failed to identify some clinically diagnosed "late-infantile" NCL cases. This study was conducted to further characterize genetic heterogeneity in families affected by LINCL., Methods: DNA mutations in the CLN1, CLN2, and CLN3 genes that underlie INCL (infantile NCL), LINCL, and JNCL (juvenile NCL), respectively, were studied with molecular analyses., Results: A total of 252 families affected by childhood NCL were studied. Of 109 families clinically diagnosed as having LINCL, 3 were determined to have either INCL or JNCL by identification of mutation(s) in CLN1 or CLN3. Six families diagnosed initially as having JNCL were found to have LINCL based on the finding of mutations in the CLN2 gene. In addition, several novel mutations were identified., Conclusions: Clinical and genetic heterogeneity of LINCL was demonstrated in nine LINCL families studied.

Tripeptidyl-peptidase II is an unusually large exopeptidase. The subunits (M(r) = 138,000) form an active complex with an M(r) > 10(6). This paper demonstrates that the complex can spontaneously dissociate in vitro into dimers which retain 110th of the original specific activity. The dissociated enzyme can reassociate at elevated temperatures, provided the protein concentration is sufficiently high. This reassociation was accompanied by a reactivation. The rate of reactivation was increased by the presence of competitive peptide inhibitors. It is speculated that association/dissociation may be a way of regulating the enzyme activity in vivo., (Copyright 2000 Academic Press.)

Endopeptidase activities of the CLN2 gene product (Cln2p)/tripeptidyl peptidase I (TPP-I), purified from rat spleen, were studied using the synthetic fluorogenic substrates. We designed and constructed decapeptides, based on the known sequence cleavage specificities of bacterial pepstatin-insensitive carboxyl proteases (BPICP). MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH(2) is readily hydrolyzed by Cln2p/TPP-I (K(cat)/K(m) = 7.8 s(-1) mM(-1)). The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity. Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CH(2)Cl, but not inhibited by tyrostatin, an inhibitor of bacterial pepstatin-insensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases. Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin-insensitive endopeptidase activity. Cln2p/TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity., (Copyright 2000 Academic Press.)

A highly sensitive assay for mammalian lysosomal pepstatin-insensitive proteinase (LPIP) is described using a synthetic peptide substrate coupled to aminotrifluoromethyl coumarin (AFC). LPIP is an endocarboxyl proteinase which has specific sequence requirements of Phe-Phe around the carboxyl terminal. This HPLC based assay can detect patients suffering from late-infantile neuronal ceroid lipofuscinosis (LINCL) and also heterozygote carriers in cultured lymphoid cells and skin fibroblasts. None of the patients analyzed had detectable enzyme activity confirming the defective gene product, while carriers had about 50% activity when compared with the normal controls. Neurological controls comprised of patients with other neurodegenerative disorders have LPIP activities similar to normal controls. LPIP activity is also detectable in amniocytes and chorionic villi. Thus the assay reported can also be used for prenatal diagnosis of LINCL.

p67, a cellular glycoprotein, protects eIF2 alpha from phosphorylation by inhibitory kinases such as PKR and HCR. p67 promoter contains heat shock element (HSE). To investigate whether this HSE of p67 has any role during heat-shock, rat tumor hepatoma cells were transiently transfected with CAT reporters linked to p67 promoter with HSE and without HSE. Heat shock induced CAT activity when p67 promoter contained HSE and this induction was not observed when HSE was deleted from the p67 promoter. In response to heat-shock, the endogenous p67 mRNA was also induced to more than 36-fold, and much of it translated into protein which was modified by GlcNAc moieties. The time of induced glycosyl modification at the later stages of the heat-shock correlates with the reduced level of eIF2 alpha phosphorylation. During later stages of the heat shock of animal cells, there is a preferential translation of a small class of messages encoding heat shock proteins. Our results suggest that the expression and activity of p67 are induced at the later stages of the heat-shock, and may be involved in the preferential translation of the heat-shock messages.

From the neuritic plaques and vascular walls of the brains of patients with Alzheimer disease, we have purified and quantified an A beta peptide which starts at residue 3Glu in the form of pyroglutamyl (A beta3pE). The N-terminally truncated A beta3pE comprised 51% of the A beta in the neuritic plaques. This was followed by 30% starting at position 1Asp which included 20% in the isomerized form (IsoAsp). In contrast, the vascular amyloid only contained an average of 11% in the form of A beta3pE with the major component starting at residue 1Asp (69%), which included only 6% in the form of IsoAsp. The presence of A beta3pE has important structural consequences since it is more hydrophobic than other forms of A beta, thus increasing the insolubility of A beta. In addition, A beta3pE, with its blocked N-terminus to the action of common aminopeptidases, may result in the profuse accumulation of A beta in the neuritic plaques of Alzheimer disease.

Regulation of vaccinia viral infection was studied using three animal cell lines: KRC-7 (rat hepatoma), L929 (mouse fibroblast), and CV-1 (African green monkey kidney). KRC-7 is highly enriched in p67, a glycoprotein which protects eIF-2 alpha-subunit from phosphorylation by eIF-2 kinases. We report: (i) At 5 pfu per cell of the virus, KRC-7 is resistant to the virus. Other cells are sensitive. At 25 pfu per cell of the virus, KRC-7 is also sensitive to the virus. After productive viral infection, the cell extracts showed strong p67-DG activity and actively deglycosylated exogenous p67. After p67-deglycosylation, the cell extracts also phosphorylated eIF-2. (ii) The rate of synthesis of a major host protein (approximately 45 kDa) in infected L929 cells measured after 2 h of viral infection declined more than 50%. The rate declined thereafter. The rate of synthesis of host proteins in viral-resistant KRC-7 cells (infected with 5 pfu per cell of the virus) remained unchanged. The mechanism of resistance of KRC7 cells to vacinia virus at 5 pfu per cell of the virus was investigated. The p67 level in these cells was varied by growing the cells under different physiological conditions such as serum starvation and expression of p67-sense and p67-antisense DNA. At low p67 level in the cells, p67-DG is activated. This deglycosylates p67 and inactivates p67. This accompanies eIF-2 phosphorylation and shutoff of host protein synthesis. At high p67 level in the cells, activation of p67-DG is prevented. This prevents shut-off of host protein synthesis and viral growth.

Tripeptidyl peptidase II is an intracellular exopeptidase, which has been purified from rat liver and human erythrocytes. An efficient specific inhibitor was obtained through beta-elimination of phosphate from the phosphopeptide Arg-Ala-Ser(P)-Val-Ala. The dehydroalanine-containing peptide formed was a competitive inhibitor with a Ki of 0.02 +/- 0.01 microM. This study demonstrated that replacing a serine residue in a good inhibitor with a dehydroalanine residue reduced the Ki 45 times. It is proposed that dehydroalanine-containing peptides could be of interest in the development of inhibitors for other peptidases as well.

Tripeptidyl peptidase I (EC 3.4.14.9), which cleaves tripeptides from the N-terminus of synthetic substrates, has been purified from human osteoclastomas (a bone tumor containing large numbers of normal osteoclasts). The enzyme has an M(r) of 48 kDa but forms aggregates with an M(r) of about 700 kDa. The tripeptidyl peptidase has an acidic pH optimum (approximately pH 5.0), suggesting that it has a lysosomal localization and prefers substrates with a hydrophobic amino acid in the P1 position. There is an absolute requirement for a nonsubstituted N-terminus. The enzyme is inhibited by reagents which modify serine and histidine residues. Lysosomal tripeptidyl peptidase is known to be capable of cleaving Gly-Pro-X triplets from synthetic collagen-like polypeptides. Ala-Ala-Phe-CH2Cl, a potent inhibitor of osteoclastoma tripeptidyl peptidase, inhibits osteoclastic bone resorption in an in vitro test system. This suggests that tripeptidyl peptidase I, secreted by osteoclasts, is involved at some stage in the degradation of bone collagen.

Capillary electrophoresis (CE) was used to assay the activity of a tripeptidase from a crude extract of Lactococcus lactis subsp. lactis NCDO 712 against the substrate, Gly-Gly-Phe and a comparison with a standard ninhydrin assay was made. Standard curves of the substrates and products showed a significantly variable colorimetric reaction to ninhydrin making accurate quantification of the tripeptidase problematic. The CE assay further demonstrated that the presence of contaminating enzymes in crude cell-free extracts can cause secondary reactions that are not apparent from the ninhydrin assay data. The CE assay was also able to generate enzyme kinetics data and monitor, during purification, the presence of co-eluting contaminating activities. The speed and sensitivity with CE allows routine analysis of the tripeptidase activity without any derivatization normally required for this enzyme.

A micromethod for nondestructive amino acid analysis was developed. Peptides were digested with amino-peptidase M followed by prolidase. The whole procedure was performed in a microvial for the autosampler of the analyzer. The resulting method has a working range from 10 to 600 pmol, at least, of sample taken into work. The usefulness of the method was demonstrated by its ability to determine the sulfation of tyrosine preceding proline in chicken gastrin.

In an attempt to identify improved biochemical markers for the diagnosis of early kidney damage via urinary analysis of kidney derived enzymes, we have undertaken the systematic identification, quantification and characterization (following purification via anion exchange and gel filtration chromatography, and preparative electrophoresis) of the dipeptidyl and tripeptidyl aminopeptidases in normal human kidney soluble extract (with which the majority of the cellular activity of these enzymes is associated). Four chromatographically separable enzyme types were identified as follows (% relative activity in parentheses): dipeptidyl aminopeptidase I (EC 3.4.14.1; 24%); dipeptidyl aminopeptidase II (EC 3.4.14.2; 8%); dipeptidyl aminopeptidase IV (EC 3.4.14.5; 61%); tripeptidyl aminopeptidase (unclassified; 7%). Comparison of the levels of activity for the above enzyme types in normal and pathological urine may lead to an improvement upon existing procedures for the early detection of renal damage.

N alpha-acetyl-gamma-endorphin (Ac gamma E) was identified in the rat neurointermediate pituitary, based on its immunological properties, comigration with synthetic Ac gamma E on HPLC and resistance to aminopeptidase-M degradation. The peptide appeared to be the main form of gamma-endorphin (gamma E) in this tissue and in brain areas remote from the hypothalamus (hippocampus, septum, amygdala). The anterior pituitary, the hypothalamus and the thalamus contained almost exclusively the non-acetylated form of gamma E. In contrast to gamma E, Ac gamma E was completely devoid of specific affinity for brain opiate binding sites. Yet, the peptide mimicked gamma E in that it potently attenuated passive avoidance behaviour in rats, when injected topically into the nucleus accumbens. It is concluded that Ac gamma E is an endogenous neuropeptide with non-opioid biological activity. N alpha-acetylation may not merely represent a mechanism for the inactivation of opioid activities of endorphins, but rather allow the organism to select specific sets of biological activities that reside in the endorphin structure.

The stimulated alveolar macrophage is a potent source of neutrophil chemotactic activity. The release of this chemotactic activity can be inhibited by pretreating alveolar macrophages with anti-C5 antibody. We hypothesized that C5a, a fragment cleaved from C5 when C5 is activated, might activate the alveolar macrophage to release neutrophil chemotactic activity and that chemotactic factor inactivator, a serum inhibitor of C5a, could decrease this release. Activated complement components including C5a were found to stimulate guinea pig macrophages to release chemotactic activity into their culture supernatants at levels that were significantly higher than the chemotactic activity of C5a alone (P less than 0.001). Chemotactic factor inactivator was found to cause a marked reduction in the chemotactic activity released by macrophages stimulated with phagocytic and nonphagocytic stimuli (P less than 0.001, all comparisons). These data indicate that C5a can stimulate alveolar macrophages to release chemotactic activity in vitro, and that chemotactic factor inactivator may play a role in modulating this process.