The use of Fluorescence Resonance Energy Transfer (FRET) peptidesfor measurement of clinically important proteolytic enzymes

Proteolytic enzymes have a fundamental role in many biological processes and are associated with multiple pathological conditions. Therefore, targeting these enzymes may be important for a better understanding of their function and development of therapeutic inhibitors. Fluorescence Resonance Energy Transfer (FRET) peptides are convenient tools for the study of peptidases specificity as they allow monitoring of the reaction on a continuous basis, providing a rapid method for the determination of enzymatic activity. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that permits the measurement of the activity of nanomolar concentrations of the enzyme. The assays can be performed directly in a cuvette of the fluorimeter or adapted for determinations in a 96-well fluorescence plate reader. The synthesis of FRET peptides containing ortho-aminobenzoic acid (Abz) as fluorescent group and 2, 4-dinitrophenyl (Dnp) or N-(2, 4-dinitrophenyl)ethylenediamine (EDDnp) as quencher was optimized by our group and became an important line of research at the Department of Biophysics of the Federal University of São Paulo. Recently, Abz/Dnp FRET peptide libraries were developed allowing high-throughput screening of peptidases substrate specificity. This review presents the consolidation of our research activities undertaken between 1993 and 2008 on the synthesis of peptides and study of peptidases specificities.As enzimas proteolíticas têm um papel fundamental em muitos processos biológicos e estão associadas a vários estados patológicos. Por isso, o estudo da especificidade das peptidases pode ser importante para uma melhor compreensão da função destas enzimas e para o desenvolvimento de inibidores. Os substratos com supressão intramolecular de fluorescência constituem uma excelente ferramenta, pois permitem o monitoramento da reação de forma contínua, proporcionando um método prático e rápido para a determinação da atividade enzimática. A hidrólise de qualquer ligação da cadeia peptídica entre o grupo doador e o grupo supressor gera fluorescência que permite detectar concentração nanomolar de enzima. Os ensaios podem ser acompanhados diretamente na cubeta ou adaptados para determinações de fluorescência em leitoras de placa. A síntese dos peptídeos com supressão intramolecular de fluorescência contendo o grupo fluorescente Abz (orto-aminobenzóico) e o grupo supressor EDDnp (N-[2, 4-dinitrofenil]-etilenodiamino ou Dnp (2, 4-dinitrophenyl) foi otimizada pelo nosso grupo e tornou-se uma importante linha de pesquisa no Departamento de Biofísica da Universidade Federal de São Paulo (UNIFESP). Recentemente, foram desenvolvidas bibliotecas de peptídeos fluorogênico contendo Abz/Dnp como grupo doador/supressor trazendo um grande avanço no estudo de especificidade das peptidases. Esta revisão apresenta o trabalho desenvolvido pelo nosso grupo entre 1993 e 2008 sobre a síntese de peptídeos e o estudo da especificidade de peptidases.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BiofísicaUNIFESP, EPM, Depto. de BiofísicaSciEL

Extracellular ATP triggers proteolysis and cytosolic Ca²⁺ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites.

BACKGROUND: Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host. METHODS: Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca²⁺ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated. RESULTS: The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 μM) and PPADS (50 μM) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 μM), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 μM) to isolated parasites previously loaded with Fluo4/AM in a Ca²⁺-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 μM), TNP-ATP (50 μM) or the purinergic blockers KN-62 (10 μM) and Ip5I (10 μM). Incubating P. berghei infected cells with KN-62 (200 μM) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 μM) led to an increase in rings forms (82% ± 4, n = 11) and a decrease in trophozoite forms (18% ± 4, n = 11). CONCLUSIONS: The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway

Effects of 'casoparan', a peptide isolated from casein hydrolysates with mastoparan-like properties.

Casein, a protein found in milk of several species, is divided into different chains from 19 to 25 kDa. Casein is also considered as a source of amino acids and generating peptides with biological activities such as opiate, immunostimulating, antibacterial, peptidase inhibitors, among others. In this work, Sephadex G-10 chromatography followed by high-performance liquid chromatography isolation purified NZCase TT, an industrial culture media for tetanus toxin production. In the first step, four pools were isolated and tested in different assays: isolated smooth muscle assay (guinea pig ileum, rat uterus), phagocytosis in vitro of opsonized sheep red blood cells, and hydrogen peroxide (H2O2) release from mouse peritoneal macrophages. Pool III was the main active pool being able to potentiate bradykinin action in guinea pig ileum, stimulating phagocitic activity by resident macrophages and increasing H2O2 release from macrophages previously activated with bacille Calmette Guérin. Using mass spectra the primary structure of the main peptide from pool III was obtained--INKKI, which corresponds to beta-casein fragment 26-30. The immunostimulating action is probably related to a direct action in macrophage cells

Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.A01Universidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Foot-and-mouth disease virus leader proteinase: Structural insights into the mechanism of intermolecular cleavage

Translation of foot-and-mouth disease virus RNA initiates at one of two start codons leading to the synthesis of two forms of leader proteinase L(pr)o (Lab(pro) and Lb(pro)). These forms free themselves from the viral polyprotein by intra- and intermolecular self-processing and subsequently cleave the cellular eukaryotic initiation factor (eIF) 4G. During infection, Lb(pro) removes six residues from its own C-terminus, generating sLb(pro). We present the structure of sLb(pro) bound to the inhibitor E64-R-P-NH2, illustrating how sLb(pro) can cleave between Lys/Gly and Gly/Arg pairs. in intermolecular cleavage on polyprotein substrates, Lb(pro) was unaffected by P1 or P1' substitutions and processed a substrate containing nine eIF4GI cleavage site residues whereas sLb(pro) failed to cleave the eIF4GI containing substrate and cleaved appreciably more slowly on mutated substrates. Introduction of 70 eIF4GI residues bearing the Lb(pro) binding site restored cleavage. These data imply that Lb(pro) and sLb(pro) may have different functions in infected cells. (C) 2014 the Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).Austrian Science FoundationFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Med Univ Vienna, Max F Perutz Labs, A-1030 Vienna, AustriaUniv Vienna, Dept Struct & Computat Biol, Max F Perutz Labs, A-1030 Vienna, AustriaUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-0404420 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-0404420 São Paulo, BrazilAustrian Science Foundation: P20889Austrian Science Foundation: P24038FAPESP: 12/50191-4RCNPq: 471340/2011-1CNPq: 470388/2010-2Web of Scienc

Interaction of divalent cations with protein PARK9

Metals have been shown to play a role in the genesis and development of many neurodegenerative diseases. Park9 encoded protein can protect cells from manganese poisoning, an environmental risk factor for a Parkinson’s disease- like syndrome. Park9 belongs to a family of ATP-ases involved in metal coordination and transportation; familial mutations of this gene may result in early development of PD. We tested two peptide sequences from Park9, -P1D2E3K4H5E6L7- (1) and -F1C2G3D4G5A6N7D8C9G10- (2), for Mn(II), Zn(II) and Cu(II) binding. These fragments are located from 1165 to 1171 and from 1184 to 1193 residues in Park9 sequence, and are highly conserved in a number of organisms, from yeasts to humans. Experiments have been carried out at different pH values and ligand/metal molar ratios with both potentiometric and spectroscopic (NMR, UV-vis) techniques, showing that the three metals are able to effectively bind the examined peptides. Mn(II) and Zn(II) coordination with peptide (1) involves imidazol of His5 and carboxyl γ-O of Asp2, Glu3 and Glu6 residues, in a distorted octahedral geometry, possibly involving bidentate interaction of carboxyl groups; four donor atoms participate in Zn(II) binding, resulting in a tetracoordinated geometry. Mn(II) and Zn(II) coordination involves the two cysteines in peptide (2); Mn(II) accepts additional ligand bonds from D4 and D8 to complete the coordination sphere, together with some water molecules. Details of Cu(II) coordination are under study

Effect of pre-weaning solid feed and milk intake on caecal content characteristics and performance of rabbits around weaning

The aim of this study is to know the effect of different solid feed and milk intake during suckling on performance around weaning and on caecal content characteristics at weaning. In order to obtain different intakes of milk and solid feed, 13 litters of pregnant females (PF) inseminated the day after delivery and 14 litters of non-pregnant females (NPF) were compared. At birth the litters were equalized at eight pups and during lactation dead pups were replaced by pups of the same age from nursing does. Compared to the PF group, rabbits in the NPF group had a higher milk intake (26.0 versus 21.4 g/day; P < 0.01) and lower solid feed intake (9.1 versus 11.5 g/day; P < 0.01) between 20 and 28 days of age. No significant difference was observed between the two groups in weight gain before and post-weaning (28-49 days). At weaning, the rabbits in group PF showed higher values in caecal content (g 26.3 versus 22.6; P < 0.05) and volatile fatty acids (mmol/l 52.2 versus 43.6; P < 0.01) and lower values in empty caecal weight (g 7.18 versus 7.78; P < 0.05), C3 (6.4 versus 9.3%; P < 0.01) and C3/C4 ratio (0.39 versus 0.63; P < 0.01) than the group NPF. On the basis of the above results, it may be concluded that the quantity of solid feed and milk intake before weaning influenced the charac- teristics of the caecal content, but not the performance of rabbits around weaning

Lysosomal enzymes are decreased in the kidney of diabetic rats

The objective of the present study was to investigate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in diabetic rat kidney. Cathepsins, glycosidases and sulfatases were studied on the 10th (DM-10) and on the 30th (DM-30) day of streptozotocin-induced diabetes mellitus (DM). the activity of cathepsin B, the main kidney cysteine protease, was decreased both in DM-10 and DM-30. Gel filtration chromatography of urinary proteins has shown the prevalence of low molecular weight peptides in normal and DM-10 urine, in contrast to the prevalence of high molecular weight peptides and intact proteins in DM-30. These results show that the decrease in lysosomal proteases could explain, at least in part, the increased albuminuria detected by radial immunodiffusion (RID), due to the excretion of less degraded or intact albumin. Concerning sulfated polysaccharides, the activities of beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, and N-acetyl-beta-D-galactosaminidase were also decreased in DM-30, while aryl sulfatases did not vary. Increased toluidine blue metachromatic staining of the tissue suggests that the lower activities of glycosidases could lead to intracellular deposition of partially digested molecules, and this could explain the decreased urinary excretion and increased tissue buildup of these molecules. the main morphological changes observed in kidney were proximal convoluted tubules with thinner walls and thinner brush border. Immunohistochemistry revealed that most of cathepsin B was located in the brush border of proximal tubular cells, highlighting the involvement of proximal convoluted tubules in diabetic nephropathy. (C) 2012 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Disciplina Biol Mol, Dept Bioquim, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Morfol, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Disciplina Biol Mol, Dept Bioquim, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Morfol, Escola Paulista Med, BR-04044020 São Paulo, BrazilFAPESP: 2010/16022-5FAPESP: 2009/11817-2CNPq: 308642/2010-4Web of Scienc