Secretion of metalloendopeptidase 24.15 (EC 3.4.24.15)

The metalloendopeptidase EP24.15 (EC3.4.24.15) is a neuropeptide-metabolizing enzyme present in neural and endocrine tissues, presumably functioning extracellularly, Because the majority of the EP24.15 activity is identified in the soluble fraction of cellular homogenates, suggesting that the enzyme is primarily an intracellular protein, we addressed the issue of how EP24.15 arrives in the extracellular environment, We utilized a model system of neuroendocrine secretion, the AtT20 cell, According to both enzymatic activity and immunologic assays, EP24.15 was synthesized in and released from AtT20 cells. Under basal conditions and after stimulation by corticotropin-releasing hormone or the calcium ionophore A23187, EP24.15 activity accumulated in the culture medium. This secretion was not attributable to cell damage, as judged by the absence of release of cytosolic enzyme markers and the ability to exclude trypan blue dye. Pulse-chase analysis and subcellular fractionation of AtT20 cell extracts suggested that the mechanism of EP24.15 secretion is not solely via classical secretory pathways, Additionally, drugs which disrupt the classical secretory pathway, such as Brefeldin A and nocodazole, blocked A23187-stimulated EP24.15 release yet had no effect on basal EP24.15 release, suggesting differences in the basal and stimulated pathways of secretion for EP24.15. In summary, EP24.15 appears to be secreted from AtT20 pituitary cells into the extracellular milieu, where the enzyme can participate in the physiologic metabolism of neuropeptides.

Circadian-rhythm of the endopeptidase 22.19 (ec 3.4.22.19) in the rat-brain

The endopeptidase 22.19 (EC 3.4.22.19) has been associated with the metabolism of neuropeptides by its ability to convert small enkephalin-containing peptides (8 to 13 amino acids) into enkephalins. In addition, this enzyme cleaves the Arg8-Arg9 bond of neurotensin and the Phe5-Ser6 bond of bradykinin. We analyzed the circadian variation of endopeptidase 22.19 in the whole and individual areas of the rat brain. Endopeptidase 22.19 activity was analyzed by high-performance liquid chromatography (HPLC) using bradykinin as an operative substrate. Enzymatic specific activities were analyzed by rhythmometric methods and indicate a circadian fluctuation of endopeptidase 22.19 specific activity (mU of enzyme/mg of protein) in the whole brain [p < 0.001, mesor (M) = 7.62, amplitude (A) = 2.89, and acrophase (phi) = 23:08 h], striatum (p < 0.001, M = 2.92, A = 0.62, phi-23:03 h), hypothalamus (p < 0.001, M = 3.15, A = 0.86, phi-01:12 h), periaqueductal gray matter (p < 0.005, M = 2.62, A = 0.34, phi = 22:35 h), and cerebellum (p < 0.0 14, M = 4.27, A = 0.88, phi = 17:12 h). The circadian rhythmicity in endopeptidase 22.19 specific activity suggests that light may have an effect on the peptidase activity in whole brain and in areas of the central nervous system and may be essential for the mechanisms of circadian fluctuations of neuropeptides in the brain.

O Sistema tributário nacional na constituição de 1988 : estudo comparativo com a constituição anterior ; ec n. 1/69 e Ec N. 2 a 27 / Iduna E. Weinert. --

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The quantitation of lipoprotein lipase mRNA in biopsies of human adipose tissue, using the polymerase chain reaction, and the effect of increased consumption of n-3 polyunsaturated fatty acids

Objective: To examine the effects of the consumption of fish oils on the gene expression of lipoprotein lipase (LPL, EC 3.1.1.34) in human adipose tissue. In order to measure LPL mRNA in adipose tissue samples obtained by needle biopsy from human volunteers a competitive, reverse transcriptase PCR (RT-PCR) protocol was developed. Design: A randomised controlled, single blind cross over dietary study which compared the effects of a low level n-3 polyunsaturated fatty acids (PUFA) using normal foods enriched with eicosapentaenoic (EPA) and docosahexaenoic (DHA) (test diet), with non-enriched but otherwise identical foods (control). The diets were consumed for a period of 22 d with a wash out period of 5 months between the diets. Setting: Free-living individuals associated with the University of Surrey. Subjects: Six male subjects with a mean (±sd) age of 51.2±3.6 y were recruited. Major Outcome Measures: Pre-and postprandial blood samples were taken for the measurement of triacylglycerol (TAG), postheparin LPL activity and adipose tissue samples for the measurement of LPL mRNA levels. Results: Mean LPL expression values were 4.12´105 molecules of LPL mRNA per ng total RNA on the control diet and 4.60´105 molecules of LPL mRNA per ng total RNA on the n-3 PUFA enriched (test) diet. There was no significant difference between the levels of LPL expression following each diet, consistent with the lack of change in TAG levels in response to increased dietary n-3 PUFA intake. However, the change in LPL expression (Test-Control diet) correlated significantly with the change in fasting TAG levels (P=0.03, R=-0.87 and R2=0.75) and with the total area under the TAG-time response curve (P=0.003, R=-0.96 and R2=0.92) in individuals. Conclusions: These findings, although based on a small number of subjects, suggest that LPL expression may be a determinant of plasma TAG levels. The development of this methodology should allow further elucidation of the effects of dietary manipulation and disease processes on lipid clearance and regulation in human subjects.

Caracterização estrutural e biofísica de duas proteínas relacionadas: β-1,3-1,4-glicanase de Bacillus subtilis 168 e α-L-arabinofuranosidase termoestável de Thermotoga petrophila RKU-1

Pós-graduação em Biofísica Molecular - IBILCE

Effects of pH, light and temperature on (1→3,1→4)-β-glucanase stability in wheat leaves

Changes in (1→3,1→4)-β-D-glucan endohydrolase (EC 3.2.1.73) protein levels were investigated in segments from second leaves of wheat (Triticum aestivum L.). The abundance of the enzyme protein markedly increased when leaf segments were incubated in the dark whereas the enzyme rapidly disappeared when dark-incubated segments were illuminated or fed with sucrose. Addition of cycloheximide (CHI) to the incubation medium led to the disappearance of previously synthesized (1→3,1→4)-β-glucanase and suppressed the dark-induced accumulation indicating that the enzyme was rather unstable. The degradation of (1→3,1→4)-β-glucanase was analyzed without the interference of de-novo synthesis in intercellular washing fluid (IWF). The loss of the enzyme protein during incubation of IWF (containing naturally present peptide hydrolases) indicated that the stability increased from pH 4 to pH 7 and that an increase in the temperature from 25 to 35 °C considerably decreased the stability. Chelating divalent cations in the IWF with o-phenanthroline also resulted in a lowered stability of the enzyme. A strong temperature effect in the range from 25 to 35 °C was also observed in wheat leaf segments. Diurnal changes in (1→3,1→4)-β-glucanase activity were followed in intact second leaves from young wheat plants. At the end of the dark period, the activity was high but constantly decreased during the light phase and remained low if the light period was extended. Activity returned to the initial level during a 10-h dark phase. During a diurnal cycle, changes in (1→3,1→4)-β-glucanase activity were associated with reciprocal changes in soluble carbohydrates. The results suggest that the synthesis and the proteolytic degradation of an apoplastic enzyme may rapidly respond to changing environmental conditions.

Reversible accumulation of (1→3,1→4)‐β‐glucan endohydrolase in wheat leaves under sugar depletion

A (1→3,1→4)‐β‐D‐glucan endohydrolase [(1→3,1→4)‐β‐glucanase, EC 3.2.1.73] was detected in wheat (Triticum aestivum L.) leaves by Western analyses and activity measurements. This enzyme is able to degrade the (1→3,1→4)‐β‐glucans present in the cell walls of cereals and other grass species. In wheat, enzyme levels clearly increased during leaf development, reaching maximum values at full expansion and then decreasing upon leaf ageing. To test whether the abundance of (1→3,1→4)‐β‐glucanase might be controlled by the carbohydrate status, environmental and nutritional conditions capable of altering the leaf soluble sugar contents were used. Both the activity and enzyme protein levels rapidly and markedly increased when mature leaves were depleted of sugars (e.g. during extended dark periods), whereas elevated carbohydrate contents (e.g. following continuous illumination, glucose supply in the dark or nitrogen deficiency during a light/dark cycle) caused a rapid decrease in (1→3,1→4)‐β‐glucanase abundance or prevented its accumulation in the leaves. The physiological significance of (1→3,1→4)‐β‐glucanase accumulation under sugar depletion remains to be elucidated.

A new brain metalloendopeptidase which degrades the Alzheimer ß-amyloid 1-40 peptide producing soluble fragments without neurotoxic effects

A new metalloendopeptidase was purified to apparent homogeneity from a homogenate of normal human brain using successive steps of chromatography on DEAE-Trisacryl, hydroxylapatite and Sephacryl S-200. The purified enzyme cleaved the Gly33-Leu34 bond of the 25-35 neurotoxic sequence of the Alzheimer ß-amyloid 1-40 peptide producing soluble fragments without neurotoxic effects. This enzyme activity was only inhibited by divalent cation chelators such as EDTA, EGTA and o-phenanthroline (1 mM) and was insensitive to phosphoramidon and captopril (1 µM concentration), specific inhibitors of neutral endopeptidase (EC 3.4.24.11) and angiotensin-converting enzyme (EC 3.4.15.1), respectively. The high affinity of this human brain endopeptidase for ß-amyloid 1-40 peptide (Km = 5 µM) suggests that it may play a physiological role in the degradation of this substance produced by normal cellular metabolism. It may also be hypothesized that the abnormal accumulation of the amyloid ß-protein in Alzheimer's disease may be initiated by a defect or an inactivation of this enzyme.

Étude structure-fonction des fructose-1,6-bisphosphate aldolases métallo-dépendantes : mécanisme catalytique et développement d’antimicrobiens

Les fructose-1,6-bisphosphate aldolases (FBPA) sont des enzymes glycolytiques (EC 4.1.2.13) qui catalysent la transformation réversible du fructose-1,6-bisphosphate (FBP) en deux trioses-phosphates, le glycéraldéhyde-3-phosphate (G3P) et le dihydroxyacétone phosphate (DHAP). Il existe deux classes de FBPA qui diffèrent au niveau de leur mécanisme catalytique. Les classes I passent par la formation d’un intermédiaire covalent de type iminium alors que les classes II, métallodépendantes, utilisent généralement un zinc catalytique. Contrairement au mécanisme des classes I qui a été très étudié, de nombreuses interrogations subsistent au sujet de celui des classes II. Nous avons donc entrepris une analyse détaillée de leur mécanisme réactionnel en nous basant principalement sur la résolution de structures cristallographiques. De nombreux complexes à haute résolution furent obtenus et ont permis de détailler le rôle de plusieurs résidus du site actif de l’enzyme. Nous avons ainsi corrigé l’identification du résidu responsable de l’abstraction du proton de l’O4 du FBP, une étape cruciale du mécanisme. Ce rôle, faussement attribué à l’Asp82 (chez Helicobacter pylori), est en fait rempli par l’His180, un des résidus coordonant le zinc. L’Asp82 n’en demeure pas moins essentiel car il oriente, active et stabilise les substrats. Enfin, notre étude met en évidence le caractère dynamique de notre enzyme dont la catalyse nécessite la relocalisation du zinc et de nombreux résidus. La dynamique de la protéine ne permet pas d’étudier tous les aspects du mécanisme uniquement par l’approche cristallographique. En particulier, le résidu effectuant le transfert stéréospécifique du proton pro(S) sur le carbone 3 (C3) du DHAP est situé sur une boucle qui n’est visible dans aucune de nos structures. Nous avons donc développé un protocole de dynamique moléculaire afin d’étudier sa dynamique. Validé par l’étude d’inhibiteurs de la classe I, l’application de notre protocole aux FBPA de classe II a confirmé l’identification du résidu responsable de cette abstraction chez Escherichia coli (Glu182) mais pointe vers un résidu diffèrent chez H. pylori (Glu149 au lieu de Glu142). Nos validations expérimentales confirment ces observations et seront consolidées dans le futur. Les FBPA de classe II sont absentes du protéome humain mais sont retrouvées chez de nombreux pathogènes, pouvant même s'y révéler essentielles. Elles apparaissent donc comme étant une cible idéale pour le développement de nouveaux agents anti-microbiens. L’obtention de nouveaux analogues des substrats pour ces enzymes a donc un double intérêt, obtenir de nouveaux outils d’étude du mécanisme mais aussi développer des molécules à visée pharmacologique. En collaboration avec un groupe de chimistes, nous avons optimisé le seul inhibiteur connu des FBPA de classe II. Les composés obtenus, à la fois plus spécifiques et plus puissants, permettent d’envisager une utilisation pharmacologique. En somme, c’est par l’utilisation de techniques complémentaires que de nouveaux détails moléculaires de la catalyse des FBPA de classe II ont pu être étudiés. Ces techniques permettront d’approfondir la compréhension fine du mécanisme catalytique de l’enzyme et offrent aussi de nouvelles perspectives thérapeutiques.

Deciphering the synergism of endogenous glycoside hydrolase families 1 and 9 from Coptotermes gestroi

Termites can degrade up to 90% of the lignocellulose they ingest using a repertoire of endogenous and symbiotic degrading enzymes. Termites have been shown to secrete two main glycoside hydrolases, which are GH1 (EC 3.2.1.21) and GH9 (EC 3.2.1.4) members. However, the molecular mechanism for lignocellulose degradation by these enzymes remains poorly understood. The present study was conducted to understand the synergistic relationship between GH9 (CgEG1) and GH1 (CgBG1) from Coptotermes gestroi, which is considered the major urban pest of São Paulo State in Brazil. The goal of this work was to decipher the mode of operation of CgEG1 and CgBG1 through a comprehensive biochemical analysis and molecular docking studies. There was outstanding degree of synergy in degrading glucose polymers for the production of glucose as a result of the endo-β-1,4-glucosidase and exo-β-1,4-glucosidase degradation capability of CgEG1 in concert with the high catalytic performance of CgBG1, which rapidly converts the oligomers into glucose. Our data not only provide an increased comprehension regarding the synergistic mechanism of these two enzymes for cellulose saccharification but also give insight about the role of these two enzymes in termite biology, which can provide the foundation for the development of a number of important applied research topics, such as the control of termites as pests as well as the development of technologies for lignocellulose-to-bioproduct applications. © 2013 Elsevier Ltd.

Produção de lipase por Fusarium oxysporum em fermentação em estado sólido e sua aplicação em reações de síntese de ésteres de biodiesel

Pós-graduação em Ciências Biológicas (Microbiologia Aplicada) - IBRC