Leukotriene A4 hydrolase: mapping of a henicosapeptide involved in mechanism-based inactivation.

Leukotriene A4 (LTA4) hydrolase [7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7,9 ,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme which converts LTA4 into the chemotactic agent leukotriene B4 (LTB4). Suicide inactivation, a typical feature of LTA4 hydrolase/aminopeptidase, occurs via an irreversible, apparently mechanism-based, covalent binding of LTA4 to the protein in a 1:1 stoichiometry. Differential lysine-specific peptide mapping of unmodified and suicide-inactivated LTA4 hydrolase has been used to identify a henicosapeptide, encompassing the amino acid residues 365-385 of human LTA4 hydrolase, which is involved in the binding of LTA4, LTA4 methyl ester, and LTA4 ethyl ester to the native enzyme. A modified form of this peptide, generated by lysine-specific digestion of LTA4 hydrolase inactivated by LTA4 ethyl ester, could be isolated for complete Edman degradation. The sequence analysis revealed a gap at position 14, which shows that binding of the leukotriene epoxide had occurred via Tyr-378 in LTA4 hydrolase. Inactivation of the epoxide hydrolase and the aminopeptidase activity was accompanied by a proportionate modification of the peptide. Furthermore, both enzyme inactivation and peptide modification could be prevented by preincubation of LTA4 hydrolase with the competitive inhibitor bestatin, which demonstrates that the henicosapeptide contains functional elements of the active site(s). It may now be possible to clarify the molecular mechanisms underlying suicide inactivation and epoxide hydrolysis by site-directed mutagenesis combined with structural analysis of the lipid molecule, covalently bound to the peptide.

A study of the growth and differentiation of the human adenocarcinoma of the colon cell line HT-29

The HT-29 human colon adenocarcinoma cell line, like many epithelial cells, displays an undifferentiated phenotype when cultured on plastic substrata. Biochemical markers of differentiation, such as brush border associated enzymes and carcinoembryonic antigen were expressed at very low levels. The differentiation-inducing effects of the culture of HT-29 cells on collagen type I gels were evaluated, and were assessed by morphological appearance, brush border associated enzyme activities and the secretion of CEA. The effect that this more physiological environment had on their chemosensitivity to a panel of chemotherapeutic agents was determined, so as to indicate whether this system could be used to improve the selectivity of screening for novel anticancer agents. Initial studies were performed on HT-29 cells derived from cells seeded directly from plastic substrata onto the collagen gels (designated Non-PPC gels). Their time of exposure to the collagen was limited to the time course of a single experiment and the results suggested that a longer, more permanent exposure might produce a more pronounced differentiation. HT-29 cells were then passaged continuously on collagen gels for a minimum of 10 passages prior to experimentation (designated PPC gels). The same parameters were measured, and compared to those for the cells grown on plastic and on the non-passaged collagen gels (Non-PPC) from the original studies. Permanently passaged cells displayed a similar degree of morphological differentiation as the non-passaged cells, with both culture conditions resulting in a more pronounced differentiation than that achieved by culture on plastic. It was noted that the morphological differentiation observed was very heterogeneous, a situation also seen in xenografted tumours in vivo. The activity of alkaline phosphatase and the production of CEA was higher in the cells passaged on collagen (PPC) than the cells cultured on non-passaged collagen gel (Non-PPC) and plastic. The biochemical determination of aminopeptidase activity showed that collagen gel culture enhanced the activity in both non-passaged and passaged HT-29 cells above that of the cells cultured on plastic. However, immunocytochemical localization of aminopeptidase and sucrase-isomaltase of samples of cells grown on the various substrata for 7, 14, 21 and 28 days showed a reduction in both enzymes in the cells grown on collagen gels when compared to cells grown on plastic. The reason for the discrepancy between the two assays for aminopeptidase is at this stage unexplained. Although, there was evidence to suggest that the culture of HT-29 cells on collagen gels was capable of inducing morphological and biochemical markers of enterocytic differentiation, there were no differences in the chemosensitivity of the different cell groups to a panel of anticancer agents. Preliminary studies suggested that the ability of the cells to polarize by their culture on porous filter chambers without any exogenous ECM was sufficient to enhance HT-29 differentiation and the onset of differentiation was probably correlated with the production of ECM by the cells themselves.

Biological barriers to the nasal delivery of peptide drugs

The nasal absorption of larger peptide and protein drugs is generally low. The importance of the mucus layer and enzymic degradation in reducing absorption were investigated. Reversed-phase high-performance liquid chromatographic (HPLC) methods were developed to assay a variety of compounds. Pig gastric mucus (PGM) was selected to investigate the importance of the mucus layer. A method of treating and storing PGM was developed and evaluated which was representative of the gel in vivo. The nature of the mucus barrier was evaluated in vitro with three-compartment diffusion cells and a series of compounds with differing physicochemical properties. Mucus retarded the diffusion of all the compounds with molecular weight and charge exerting a marked effect. Binding to mucus was investigated by a centrifugation method. All of the compounds tested were found to bind to mucus with the exception of the negatively charged molecule benzoic acid. The small peptides did not demonstrate greater binding to mucus than any of the other compounds evaluated. The effect of some absorption enhancers upon the rate of diffusion of tryptophan through mucus was determined in vi tro. At the concentrations employed the enhancers EDTA, N-acetylcysteine and taurodeoxycholic acid exerted no effect, whilst taurocholic acid and cholic acid, were found to slightly reduce the rate of diffusion. The intracellular and luminal proteolytic activity of the nose was investigated in the sheep animal model with a nasal mucosal homogenate and a nasal wash preparation respectively and a series of chemically similar peptides. Hydrolysis was also investigated with the proteolytic enzymes carboxypeptidase A, cytosolic leucine aminopeptidase and microsomal leucine aminopeptidase. Sheep nasal mucosa possesses significant peptide hydrolase activity capable of degrading all the substrates tested. Considerable variation in susceptibility was observed. Degradation occurred excl us i ve ly at the pept ide bond between the aromatic amino ac id and glycine, indicating some specificity for aromatic amino acids. Hydrolysis profiles indicated the presence of both aminopeptidase and carboxypeptidase enzymes. The specific activity of the microsomal fraction was found to be greater than the cytosolic fraction. Hydrolysis in the nasal wash indicated the presence of either luminal or loosely-bound proteases, which can degrade peptide substrates. The same specificity for aromatic amino acids was observed and aminopeptidase activity demonstrated. The specific activity of the nasal wash was smaller than that of the homogenate.

Bacteria growth, enzyme activities and TEP during KOSMOS 2011 mesocosm experiment in the Raunefjord

Marine bacteria are the main consumers of freshly produced organic matter. Many enzymatic processes involved in the bacterial digestion of organic compounds were shown to be pH sensitive in previous studies. Due to the continuous rise in atmospheric CO2 concentration, seawater pH is presently decreasing at a rate unprecedented during the last 300 million years but the consequences for microbial physiology, organic matter cycling and marine biogeochemistry are still unresolved. We studied the effects of elevated seawater pCO2 on a natural plankton community during a large-scale mesocosm study in a Norwegian fjord. Nine Kiel Off-Shore Mesocosms for Future Ocean Simulations (KOSMOS) were adjusted to different pCO2 levels ranging initially from ca. 280 to 3000 µatm and sampled every second day for 34 days. The first phytoplankton bloom developed around day 5. On day 14, inorganic nutrients were added to the enclosed, nutrient-poor waters to stimulate a second phytoplankton bloom, which occurred around day 20. Our results indicate that marine bacteria benefit directly and indirectly from decreasing seawater pH. During the first phytoplankton bloom, 5-10% more transparent exopolymer particles were formed in the high pCO2 mesocosms. Simultaneously, the efficiency of the protein-degrading enzyme leucine aminopeptidase increased with decreasing pH resulting in up to three times higher values in the highest pCO2/lowest pH mesocosm compared to the controls. In general, total and cell-specific aminopeptidase activities were elevated under low pH conditions. The combination of enhanced enzymatic hydrolysis of organic matter and increased availability of gel particles as substrate supported up to 28% higher bacterial abundance in the high pCO2 treatments. We conclude that ocean acidification has the potential to stimulate the bacterial community and facilitate the microbial recycling of freshly produced organic matter, thus strengthening the role of the microbial loop in the surface ocean.

Experiment: Response of Nodularia spumigena to pCO2

The filamentous and diazotrophic cyanobacterium Nodularia spumigena plays a major role in the productivity of the Baltic Sea as it forms extensive blooms regularly. Under phosphorus limiting conditions Nodularia spumigena has a high enzyme affinity for dissolved organic phosphorus (DOP) by production and release of alkaline phosphatase. Additionally, it is able to degrade proteinaceous compounds by expressing the extracellular enzyme leucine aminopeptidase. As atmospheric CO2 concentrations are increasing, we expect marine phytoplankton to experience changes in several environmental parameters including pH, temperature, and nutrient availability. The aim of this study was to investigate the combined effect of CO2-induced changes in seawater carbonate chemistry and of phosphate deficiency on the exudation of organic matter, and its subsequent recycling by extracellular enzymes in a Nodularia spumigena culture. Batch cultures of Nodularia spumigena were grown for 15 days aerated with three different pCO2 levels corresponding to values from glacial periods to future values projected for the year 2100. Extracellular enzyme activities as well as changes in organic and inorganic compound concentrations were monitored. CO2 treatment-related effects were identified for cyanobacterial growth, which in turn was influencing exudation and recycling of organic matter by extracellular enzymes. Biomass production was increased by 56.5% and 90.7% in the medium and high pCO2 treatment, respectively, compared to the low pCO2 treatment and simultaneously increasing exudation. During the growth phase significantly more mucinous substances accumulated in the high pCO2 treatment reaching 363 µg Gum Xanthan eq /l compared to 269 µg Gum Xanthan eq /l in the low pCO2 treatment. However, cell-specific rates did not change. After phosphate depletion, the acquisition of P from DOP by alkaline phosphatase was significantly enhanced. Alkaline phosphatase activities were increased by factor 1.64 and 2.25, respectively, in the medium and high compared to the low pCO2 treatment. In conclusion, our results suggest that Nodularia spumigena can grow faster under elevated pCO2 by enhancing the recycling of organic matter to acquire nutrients.

Metabolites of PPI-2458, a selective, irreversible inhibitor of methionine aminopeptidase-2: structure determination and In vivo activity

The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, (3R,4S,5S,6R)-5-methoxy-4-(2R,3R)-2-methyl-3-(3-methylbut-2-enyl) oxiran-2-yl]-1-oxaspiro2.5]octan-6-yl] N-(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only similar to 10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ((3R, 4S, 5S, 6R)-5-methoxy-4-(2R, 3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro2.5]octan-6 -yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases.

Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X)18-E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K 528R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.

Molecular modeling of Bt Cry1Ac (DI–DII)–ASAL (Allium sativum lectin)–fusion protein and its interaction with aminopeptidase N (APN) receptor of Manduca sexta

Genetic engineering of Bacillus thuringiensis (Bt) Cry proteins has resulted in the synthesis of various novel toxin proteins with enhanced insecticidal activity and specificity towards different insect pests. In this study, a fusion protein consisting of the DI–DII domains of Cry1Ac and garlic lectin (ASAL) has been designed in silico by replacing the DIII domain of Cry1Ac with ASAL. The binding interface between the DI–DII domains of Cry1Ac and lectin has been identified using protein–protein docking studies. Free energy of binding calculations and interaction profiles between the Cry1Ac and lectin domains confirmed the stability of fusion protein. A total of 18 hydrogen bonds was observed in the DI–DII–lectin fusion protein compared to 11 hydrogen bonds in the Cry1Ac (DI–DII–DIII) protein. Molecular mechanics/Poisson–Boltzmann (generalized-Born) surface area [MM/PB (GB) SA] methods were used for predicting free energy of interactions of the fusion proteins. Protein–protein docking studies based on the number of hydrogen bonds, hydrophobic interactions, aromatic–aromatic, aromatic–sulphur, cation–pi interactions and binding energy of Cry1Ac/fusion proteins with the aminopeptidase N (APN) of Manduca sexta rationalised the higher binding affinity of the fusion protein with the APN receptor compared to that of the Cry1Ac–APN complex, as predicted by ZDOCK, Rosetta and ClusPro analysis. The molecular binding interface between the fusion protein and the APN receptor is well packed, analogously to that of the Cry1Ac–APN complex. These findings offer scope for the design and development of customized fusion molecules for improved pest management in crop plants.

Dipeptidyl-Peptidase IV Activity Is Correlated with Colorectal Cancer Prognosis

Background Dipeptidyl-peptidase IV (EC 3.4.14.5) (DPPIV) is a serine peptidase involved in cell differentiation, adhesion, immune modulation and apoptosis, functions that control neoplastic transformation. Previous studies have demonstrated altered expression and activity of tissue and circulating DPPIV in several cancers and proposed its potential usefulness for early diagnosis in colorectal cancer (CRC). Methods and principal findings The activity and mRNA and protein expression of DPPIV was prospectively analyzed in adenocarcinomas, adenomas, uninvolved colorectal mucosa and plasma from 116 CRC patients by fluorimetric, quantitative RT-PCR and immunohistochemical methods. Results were correlated with the most important classic pathological data related to aggressiveness and with 5-year survival rates. Results showed that: 1) mRNA levels and activity of DPPIV increased in colorectal neoplasms (Kruskal-Wallis test, p<0.01); 2) Both adenomas and CRCs displayed positive cytoplasmic immunostaining with luminal membrane reinforcement; 3) Plasmatic DPPIV activity was lower in CRC patients than in healthy subjects (Mann-U test, p<0.01); 4) Plasmatic DPPIV activity was associated with worse overall and disease-free survivals (log-rank p<0.01, Cox analysis p<0.01). Conclusion/significance 1) Up-regulation of DPPIV in colorectal tumors suggests a role for this enzyme in the neoplastic transformation of colorectal tissues. This finding opens the possibility for new therapeutic targets in these patients. 2) Plasmatic DPPIV is an independent prognostic factor in survival of CRC patients. The determination of DPPIV activity levels in the plasma may be a safe, minimally invasive and inexpensive way to define the aggressiveness of CRC in daily practice.

Leucine Aminopeptidase (Aminopeptidase-I) N-Acetyl-Beta-Hexosaminidase And Lysosomes In The Mussel Mytilus-Edulis-L In Response To Salinity Changes

The intracellular distribution of aminopeptidase-I in the intestinal and digestive cells of Mytilus edulishas been shown to be the same as the lysosomal marker enzymes β-glucuronidase and N-acetyl-β-hexosaminidase. Activity for these enzymes was also associated with the intestinal apical cytoplasm and microvillous border where there was pronounced staining for aminopeptidase-I. Experimental alterations of salinity induced changes in both microdensitometrically and spectrophotometrically determined aminopeptidase-I activity, as an increase with raised salinity and a decrease with lowered salinity. Lysosomal hexosaminidase showed similar changes in activity with altered salinity. Cytochemically determined lysosomal stability was also responsive to salinity changes, indicative of alterations in lysosomal functional capability. The lysosomal distribution of aminopeptidase-I is discussed in terms of the function of lysosomes in intracellular protein turnover, their high concentrations of free amino acids, and the possible roles which these might play in intracellular osmoregulation in response to salinity change.

Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in > 2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH2] Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.