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1.
Molecules ; 26(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34443484

RESUMO

The COVID-19 outbreak has rapidly spread on a global scale, affecting the economy and public health systems throughout the world. In recent years, peptide-based therapeutics have been widely studied and developed to treat infectious diseases, including viral infections. Herein, the antiviral effects of the lysine linked dimer des-Cys11, Lys12,Lys13-(pBthTX-I)2K ((pBthTX-I)2K)) and derivatives against SARS-CoV-2 are reported. The lead peptide (pBthTX-I)2K and derivatives showed attractive inhibitory activities against SARS-CoV-2 (EC50 = 28-65 µM) and mostly low cytotoxic effect (CC50 > 100 µM). To shed light on the mechanism of action underlying the peptides' antiviral activity, the Main Protease (Mpro) and Papain-Like protease (PLpro) inhibitory activities of the peptides were assessed. The synthetic peptides showed PLpro inhibition potencies (IC50s = 1.0-3.5 µM) and binding affinities (Kd = 0.9-7 µM) at the low micromolar range but poor inhibitory activity against Mpro (IC50 > 10 µM). The modeled binding mode of a representative peptide of the series indicated that the compound blocked the entry of the PLpro substrate toward the protease catalytic cleft. Our findings indicated that non-toxic dimeric peptides derived from the Bothropstoxin-I have attractive cellular and enzymatic inhibitory activities, thereby suggesting that they are promising prototypes for the discovery and development of new drugs against SARS-CoV-2 infection.


Assuntos
Venenos de Crotalídeos/química , Dimerização , Papaína/antagonistas & inibidores , Peptídeos/química , Peptídeos/farmacologia , SARS-CoV-2/enzimologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Simulação de Acoplamento Molecular , Papaína/química , Papaína/metabolismo , Peptídeos/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Conformação Proteica , SARS-CoV-2/efeitos dos fármacos
2.
J. venom. anim. toxins incl. trop. dis ; 27: e20200098, 2021. graf, ilus
Artigo em Inglês | ID: biblio-1154770

RESUMO

Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect and the main vector of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae). In the present study, the authors investigated whether a serine protease activity from the saliva of T. infestans has a role in vasomotor modulation, and in the insect-blood feeding by cleaving and activating protease-activated receptors (PARs). Methods T. infestans saliva was chromatographed as previously reported for purification of triapsin, a serine protease. The cleavage activity of triapsin on PAR peptides was investigated based on FRET technology. Mass spectrometry was used to analyze the sites of PAR-2 peptide cleaved by triapsin. NO measurements were performed using the DAN assay (2,3-diaminonapthalene). The vasorelaxant activity of triapsin was measured in vessels with or without functional endothelium pre-contracted with phenylephrine (3 µM). Intravital microscopy was used to assess the effect of triapsin on mouse skin microcirculation. Results Triapsin was able to induce hydrolysis of PAR peptides and showed a higher preference for cleavage of the PAR-2 peptide. Analysis by mass spectrometry confirmed a single cleavage site, which corresponds to the activation site of the PAR-2 receptor. Triapsin induced dose-dependent NO release in cultured human umbilical vein endothelial cells (HUVECs), reaching a maximum effect at 17.58 nM. Triapsin purified by gel-filtration chromatography (10-16 to 10-9 M) was applied cumulatively to mouse mesenteric artery rings and showed a potent endothelium-dependent vasodilator effect (EC30 = 10-12 M). Nitric oxide seems to be partially responsible for this vasodilator effect because L-NAME (L-NG-nitroarginine methyl ester 300 µM), a nitric oxide synthetase inhibitor, did not abrogate the vasodilation activated by triapsin. Anti-PAR-2 antibody completely inhibited vasodilation observed in the presence of triapsin activity. Triapsin activity also induced an increase in the mouse ear venular diameter. Conclusion Data from this study suggest a plausible association between triapsin activity mediated PAR-2 activation and vasodilation caused by T. infestans saliva.(AU)


Assuntos
Animais , Peptídeos , Triatoma , Trypanosoma cruzi , Vasodilatação , Cromatografia , Receptor PAR-2 , Óxido Nítrico
3.
Sci Rep ; 10(1): 18513, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33116174

RESUMO

Nuclear distribution element-like 1 (NDEL1) enzyme activity is important for neuritogenesis, neuronal migration, and neurodevelopment. We reported previously lower NDEL1 enzyme activity in blood of treated first episode psychosis and chronic schizophrenia (SCZ) compared to healthy control subjects, with even lower activity in treatment resistant chronic SCZ patients, implicating NDEL1 activity in SCZ. Herein, higher NDEL1 activity was observed in the blood and several brain regions of a validated animal model for SCZ at baseline. In addition, long-term treatment with typical or atypical antipsychotics, under conditions in which SCZ-like phenotypes were reported to be reversed in this animal model for SCZ, showed a significant NDEL1 activity reduction in blood and brain regions which is in line with clinical data. Importantly, these results support measuring NDEL1 enzyme activity in the peripheral blood to predict changes in NDEL1 activity in the CNS. Also, acute administration of psychostimulants, at levels reported to induce SCZ-like phenotype in normal rat strains, increased NDEL1 enzyme activity in blood. Therefore, alterations in NDEL1 activity after treatment with antipsychotics or psychostimulants may suggest a possible modulation of NDEL1 activity secondary to neurotransmission homeostasis and provide new insights into the role of NDEL1 in SCZ pathophysiology.


Assuntos
Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/fisiologia , Esquizofrenia/metabolismo , Animais , Antipsicóticos/farmacologia , Encéfalo/metabolismo , Estimulantes do Sistema Nervoso Central/uso terapêutico , Clozapina/farmacologia , Cisteína Endopeptidases/sangue , Haloperidol/farmacologia , Hipocampo/metabolismo , Masculino , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/metabolismo , Transtornos Psicóticos/tratamento farmacológico , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Esquizofrenia/fisiopatologia
4.
FEBS J ; 287(18): 4068-4081, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-31995266

RESUMO

Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which contribute to the regulation of infection and of inflammatory processes. In physiological conditions, endogenous inhibitors including α2-macroglobulin (α2-M), serpins [α1-proteinase inhibitor (α1-PI)], monocyte neutrophil elastase inhibitor (MNEI), α1-antichymotrypsin, and locally produced chelonianins (elafin, SLPI) control excessive proteolytic activity of neutrophilic serine proteinases. In contrast to human NE (hNE), hPR3 is weakly inhibited by α1-PI and MNEI but not by SLPI. α2-M is a large spectrum inhibitor that traps a variety of proteinases in response to cleavage(s) in its bait region. We report here that α2-M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between α2-M and hPR3 is governed by a kass in the ≤ 105  m-1 ·s-1 range. Since α2-M-trapped proteinases retain peptidase activity, we first predicted the putative cleavage sites within the α2-M bait region (residues 690-728) using kinetic and molecular modeling approaches. We then identified by mass spectrum analysis the cleavage sites of hPR3 in a synthetic peptide spanning the 39-residue bait region of α2-M (39pep-α2-M). Since the 39pep-α2-M peptide and the corresponding bait area in the whole protein do not contain sequences with a high probability of specific cleavage by hPR3 and were indeed only slowly cleaved by hPR3, it can be concluded that α2-M is a poor inhibitor of hPR3. The resistance of hPR3 to inhibition by endogenous inhibitors explains at least in part its role in tissue injury during chronic inflammatory diseases and its well-recognized function of major target autoantigen in granulomatosis with polyangiitis.


Assuntos
Simulação de Acoplamento Molecular , Mieloblastina/química , alfa 2-Macroglobulinas Associadas à Gravidez/química , Proteínas Recombinantes/química , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia Líquida/métodos , Humanos , Cinética , Espectrometria de Massas/métodos , Mieloblastina/genética , Mieloblastina/metabolismo , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , alfa 2-Macroglobulinas Associadas à Gravidez/genética , alfa 2-Macroglobulinas Associadas à Gravidez/metabolismo , Ligação Proteica , Domínios Proteicos , Proteólise , Proteínas Recombinantes/metabolismo
5.
Biochem Biophys Res Commun ; 522(2): 368-373, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31761323

RESUMO

Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (NEL, EC 3.4.24.16) are closely related zinc-dependent metalo-oligopeptidases, which take part in the metabolism of oligopeptides (from 5 to 17 amino acid residues) inside and outside cells. Both peptidases are ubiquitously distributed in tissues. TOP is one of the main intracellular peptide-processing enzymes being important for the antigen selection in the MHC Class I presentation route, while NEL function has been more associated with the extracellular degradation of neurotensin. Despite efforts being made to develop specific inhibitors for these peptidases, the most used are: CPP-Ala-Ala-Tyr-PABA, described by Orlowski et al. in 1988, and CPP-Ala-Aib-Tyr-PABA (JA-2) that is an analog more resistant to proteolysis, which development was made by Shrimpton et al. in 2000. In the present work, we describe other analogs of these compounds but, with better discriminatory capacity to inhibit specifically NEL or TOP. The modifications introduced in these new analogs were based on a key difference existent in the extended binding sites of NEL and TOP: the negatively charged Glu469 residue of TOP corresponds to the positively charged Arg470 residue of NEL. These residues are in position to interact with the residue at the P1' and/or P2' of their substrates (mimicked by the Ala-Ala/P1'-P2' residues of the CPP-Ala-Ala-Tyr-PABA). Therefore, exploring this single difference, the following compounds were synthesized: CPP-Asp-Ala-Tyr-PABA, CPP-Arg-Ala-Tyr-PABA, CPP-Ala-Asp-Tyr-PABA, CPP-Ala-Arg-Tyr-PABA. Confirming the predictions, the replacement of each non-charged residue of the internal portion Ala-Ala by a charged residue Asp or Arg resulted in compounds with higher selectivity for NEL or TOP, especially due to the electrostatic attraction or repulsion by the NEL Arg470 or TOP Glu469 residue. The CPP-Asp-Ala-Tyr-PABA and CPP-Ala-Asp-Tyr-PABA presented higher affinities for NEL, and, the CFP-Ala-Arg-Tyr-PABA showed higher affinity for TOP.


Assuntos
Metaloendopeptidases/metabolismo , Oligopeptídeos/farmacologia , Cinética , Metaloendopeptidases/antagonistas & inibidores , Mutação/genética , Oligopeptídeos/síntese química , Oligopeptídeos/química , Especificidade por Substrato/efeitos dos fármacos
6.
Sci Rep, v. 10, 18513, out. 2020
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-3297

RESUMO

Nuclear distribution element-like 1 (NDEL1) enzyme activity is important for neuritogenesis, neuronal migration, and neurodevelopment. We reported previously lower NDEL1 enzyme activity in blood of treated first episode psychosis and chronic schizophrenia (SCZ) compared to healthy control subjects, with even lower activity in treatment resistant chronic SCZ patients, implicating NDEL1 activity in SCZ. Herein, higher NDEL1 activity was observed in the blood and several brain regions of a validated animal model for SCZ at baseline. In addition, long-term treatment with typical or atypical antipsychotics, under conditions in which SCZ-like phenotypes were reported to be reversed in this animal model for SCZ, showed a significant NDEL1 activity reduction in blood and brain regions which is in line with clinical data. Importantly, these results support measuring NDEL1 enzyme activity in the peripheral blood to predict changes in NDEL1 activity in the CNS. Also, acute administration of psychostimulants, at levels reported to induce SCZ-like phenotype in normal rat strains, increased NDEL1 enzyme activity in blood. Therefore, alterations in NDEL1 activity after treatment with antipsychotics or psychostimulants may suggest a possible modulation of NDEL1 activity secondary to neurotransmission homeostasis and provide new insights into the role of NDEL1 in SCZ pathophysiology.

7.
Appl Biochem Biotechnol ; 187(1): 282-297, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29936594

RESUMO

Composting operation systems are valuable sources of microorganisms and enzymes. This work reports the assessment of proteolytic enzymes from cultivable bacteria isolated from a composting facility of the São Paulo Zoo Park (SPZPF), São Paulo, Brazil. Three hundred bacterial isolates were obtained and identified based on 16S rRNA gene as belonging to 13 different genera. The most common genus among the isolates was Bacillus (67%); some of which show high proteolytic activity in their culture media. Biochemical assays of hydrolytic activities using FRET peptides as substrates allowed the characterization of a repertoire of serine proteases and metalloproteases with different molecular weights secreted by Bacillus strains isolated from composting. Furthermore, thermostable serine and metalloproteases were detected in the composting leachate, which might be of interest for industrial applications.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/biossíntese , Compostagem , Peptídeo Hidrolases/biossíntese , Bacillus/classificação , Bacillus/genética , Bacillus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Brasil , Peptídeo Hidrolases/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo
8.
Braz. j. microbiol ; 49(4): 900-908, Oct.-Dec. 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-974290

RESUMO

ABSTRACT Matrix Assisted Laser Desorption/Ionization and Time of Flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the identification of bacteria through the detection and analysis of their proteins or fragments derived from ribosomes. Slight sequence variations in conserved ribosomal proteins distinguish microorganisms at the subspecies and strain levels. Characterization of Leptospira spp. by 16S RNA sequencing is costly and time-consuming, and recent studies have shown that closely related species (e.g., Leptospira interrogans and Leptospira kirschneri) may not be discriminated using this technology. Herein, we report an in-house Leptospira reference spectra database using Leptospira reference strains that were validated with a collection of well-identified Brazilian isolates kept in the Bacterial Zoonosis Laboratory at the Veterinary Preventive Medicine and Animal Health Department at Sao Paulo University. In addition, L. interrogans and L. kirschneri were differentiated using an in-depth mass spectrometry analysis with ClinProTools™ software. In conclusion, our in-house reference spectra database has the necessary accuracy to differentiate pathogenic and non-pathogenic species and to distinguish L. interrogans and L. kirschneri.


Assuntos
Humanos , Técnicas de Tipagem Bacteriana/métodos , Espectrometria de Massas em Tandem/métodos , Leptospira/isolamento & purificação , Leptospirose/microbiologia , Brasil , DNA Bacteriano/genética , RNA Ribossômico 16S/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Leptospira/classificação , Leptospira/genética , Leptospira/química
9.
Braz J Microbiol ; 49(4): 900-908, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29691192

RESUMO

Matrix Assisted Laser Desorption/Ionization and Time of Flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the identification of bacteria through the detection and analysis of their proteins or fragments derived from ribosomes. Slight sequence variations in conserved ribosomal proteins distinguish microorganisms at the subspecies and strain levels. Characterization of Leptospira spp. by 16S RNA sequencing is costly and time-consuming, and recent studies have shown that closely related species (e.g., Leptospira interrogans and Leptospira kirschneri) may not be discriminated using this technology. Herein, we report an in-house Leptospira reference spectra database using Leptospira reference strains that were validated with a collection of well-identified Brazilian isolates kept in the Bacterial Zoonosis Laboratory at the Veterinary Preventive Medicine and Animal Health Department at Sao Paulo University. In addition, L. interrogans and L. kirschneri were differentiated using an in-depth mass spectrometry analysis with ClinProTools™ software. In conclusion, our in-house reference spectra database has the necessary accuracy to differentiate pathogenic and non-pathogenic species and to distinguish L. interrogans and L. kirschneri.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Leptospira/isolamento & purificação , Leptospirose/microbiologia , Espectrometria de Massas em Tandem/métodos , Brasil , DNA Bacteriano/genética , Humanos , Leptospira/química , Leptospira/classificação , Leptospira/genética , RNA Ribossômico 16S/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
10.
Int J Biol Macromol ; 113: 1134-1141, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29524492

RESUMO

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon Natrialba magadii. Many extracellular proteases have been characterized from archaea to bacteria as adapted to hypersaline environments retaining function and stability until 4.0M NaCl. As observed in other secreted halolysins, this stability can be related to the presence of a C-terminal extension (CTE) sequence. In the present work, we compared the biochemical properties of recombinant Nep protease with the truncated form at the 134 amino acids CTE (Nep∆CTE), that was more active in 4M NaCl than the non-truncated wild type enzyme. Comparable to the wild type, Nep∆CTE protease is irreversibly inactivated at low salt solutions. The substrate specificity of the truncated Nep∆CTE was similar to that of wild type form as demonstrated by a combinatorial library of FRET substrates. The enzyme stability, the effect of different salts and the thermodynamics assays using different lengths of substrates demonstrated similarities between the two forms. Altogether, these data provide further information on the stability and structural determinants of halolysins under different salinities, especially concerning the enzymatic behavior.


Assuntos
Espaço Extracelular/enzimologia , Halobacteriaceae/citologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Sais/farmacologia , Relação Dose-Resposta a Droga , Halobacteriaceae/enzimologia , Cinética , Solventes/química , Relação Estrutura-Atividade , Especificidade por Substrato
11.
Insect Biochem Mol Biol ; 89: 17-30, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28838758

RESUMO

Cathepsins L are the major digestive peptidases in the beetle Tenebrio molitor. Two digestive cathepsins L (TmCAL2 and TmCAL3) from it had their 3D structures solved. The aim of this paper was to study in details TmCAL3 specificity and properties and relate them to its 3D structure. Recombinant TmCAL3 was assayed with 64 oligopeptides with different amino acid replacements in positions P2, P1, P1' and P2'. Results showed that TmCAL3 S2 specificity differs from the human enzyme and that its specificities also explain why on autoactivation two propeptide residues remain in the enzyme. Data on free energy of binding and of activation showed that S1 and S2' are mainly involved in substrate binding, S1' acts in substrate binding and catalysis, whereas S2 is implied mainly in catalysis. Enzyme subsite residues were identified by docking with the same oligopeptide used for kinetics. The subsite hydrophobicities were calculated from the efficiency of hydrolysis of different amino acid replacements in the peptide and from docking data. The results were closer for S1 and S2' than for S1' and S2, indicating that the residue subsites that were more involved in transition state binding are different from those binding the substrate seen in docking. Besides TmCAL1-3, there are nine other cathepsins L, most of them more expressed at midgut. They are supposed to be directed to lysosomes by a Drosophila-like Lerp receptor and/or motifs in their prodomains. The mannose 6-phosphate lysosomal sorting machinery is absent from T. molitor transcriptome. Cathepsin L direction to midgut contents seems to depend on overexpression.


Assuntos
Catepsina L/metabolismo , Tenebrio/enzimologia , Sequência de Aminoácidos , Animais , Catálise , Catepsina L/química , Trato Gastrointestinal/enzimologia , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Lisossomos/enzimologia , Simulação de Acoplamento Molecular , Sinais Direcionadores de Proteínas , Especificidade por Substrato
12.
Biochim Biophys Acta Proteins Proteom ; 1865(5): 558-564, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28254587

RESUMO

Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R↓R-ACC and Z-R↓R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates.


Assuntos
Calicreínas/metabolismo , Cinética , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Aminoácidos Básicos/química , Aminoácidos Básicos/genética , Encefalinas/química , Encefalinas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Furina/química , Furina/metabolismo , Humanos , Hidrólise , Calicreínas/química , Calicreínas/genética , Metaloproteinase 14 da Matriz/química , Metaloproteinase 14 da Matriz/metabolismo , Modelos Moleculares , Fator de Crescimento Neural/química , Fator de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Neurotrofina 3 , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Peptídeos/metabolismo , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Proteólise , Especificidade por Substrato
13.
Proc Natl Acad Sci U S A ; 114(14): E2826-E2835, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28325868

RESUMO

Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, including insulin, amylin, and the amyloid ß peptides. Polyanions activate IDE toward some substrates, yet an endogenous polyanion activator has not yet been identified. Here we report that inositol phosphates (InsPs) and phosphatdidylinositol phosphates (PtdInsPs) serve as activators of IDE. InsPs and PtdInsPs interact with the polyanion-binding site located on an inner chamber wall of the enzyme. InsPs activate IDE by up to ∼95-fold, affecting primarily Vmax The extent of activation and binding affinity correlate with the number of phosphate groups on the inositol ring, with phosphate positional effects observed. IDE binds PtdInsPs from solution, immobilized on membranes, or presented in liposomes. Interaction with PtdInsPs, likely PtdIns(3)P, plays a role in localizing IDE to endosomes, where the enzyme reportedly encounters physiological substrates. Thus, InsPs and PtdInsPs can serve as endogenous modulators of IDE activity, as well as regulators of its intracellular spatial distribution.


Assuntos
Endossomos/metabolismo , Fosfatos de Inositol/metabolismo , Insulisina/metabolismo , Fosfatidilinositóis/metabolismo , Androstadienos/farmacologia , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Endossomos/efeitos dos fármacos , Ativação Enzimática , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Insulisina/química , Insulisina/genética , Lipossomos/química , Lipossomos/metabolismo , Mutação , Wortmanina
14.
Biochim Biophys Acta Proteins Proteom ; 1865(4): 388-394, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28089596

RESUMO

Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P1. In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed metacaspase-2 of Trypanosoma brucei (TbMCA2) is active; however, it has been shown that cleavages at Lys55 and Lys268 increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys55 and Lys268 with Gly via site-directed mutagenesis results in non-processed but enzymatically active mutant, TbMCA2 K55/268G. To investigate the importance of this processing for the activity and specificity of TbMCA2, we performed activity assays comparing the non-processed mutant (TbMCA2 K55/268G) with the processed TbMCA2 form. Significant differences between TbMCA2 WT (processed form) and TbMCA2 K55/268G (non-processed form) were observed. Specifically, we verified that although non-processed TbMCA2 is active when assayed with small synthetic substrates, the TbMCA2 form does not exhibit hydrolytic activity on large substrates such as azocasein, while processed TbMCA2 is able to readily digest this protein. Such differences can be relevant for understanding the physiological regulation and function of TbMCA2.


Assuntos
Caspases/química , Proteínas de Protozoários/química , Trypanosoma brucei brucei/enzimologia , Substituição de Aminoácidos , Caspases/genética , Caspases/metabolismo , Ativação Enzimática , Mutação de Sentido Incorreto , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Especificidade por Substrato , Trypanosoma brucei brucei/genética
15.
Biochimie ; 135: 72-81, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28115185

RESUMO

Human plasma kallikrein (huPK) potentiates platelet responses to subthreshold doses of ADP, although huPK itself, does not induce platelet aggregation. In the present investigation, we observe that huPK pretreatment of platelets potentiates ADP-induced platelet activation by prior proteolysis of the G-protein-coupled receptor PAR-1. The potentiation of ADP-induced platelet activation by huPK is mediated by the integrin αIIbß3 through interactions with the KGD/KGE sequence motif in huPK. Integrin αIIbß3 is a cofactor for huPK binding to platelets to support PAR-1 hydrolysis that contributes to activation of the ADP signaling pathway. This activation pathway leads to phosphorylation of Src, AktS473, ERK1/2, and p38 MAPK, and to Ca2+ release. The effect of huPK is blocked by specific antagonists of PAR-1 (SCH 19197) and αIIbß3 (abciximab) and by synthetic peptides comprising the KGD and KGE sequence motifs of huPK. Further, recombinant plasma kallikrein inhibitor, rBbKI, also blocks this entire mechanism. These results suggest a new function for huPK. Formation of plasma kallikrein lowers the threshold for ADP-induced platelet activation. The present observations are consistent with the notion that plasma kallikrein promotes vascular disease and thrombosis in the intravascular compartment and its inhibition may ameliorate cardiovascular disease and thrombosis.


Assuntos
Difosfato de Adenosina/farmacologia , Calicreína Plasmática/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Humanos , Fosforilação/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
Angiogenesis ; 20(1): 125-137, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27921229

RESUMO

Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.


Assuntos
Colágeno Tipo XVIII/metabolismo , Endostatinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Colágeno Tipo XVIII/química , Espaço Extracelular/enzimologia , Transferência Ressonante de Energia de Fluorescência , Hemangioendotelioma/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Peptídeos/metabolismo , Subunidades Proteicas/metabolismo , Proteólise
17.
Planta ; 245(2): 343-353, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27778107

RESUMO

MAIN CONCLUSION: A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3'RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M-1 cm-1. MpBBI inhibits strongly trypsin with K i in the 10-10 M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants.


Assuntos
Maclura/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sementes/química , Inibidores da Tripsina/metabolismo , Clonagem Molecular , Modelos Moleculares , Mapeamento de Peptídeos , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Conformação Proteica , Homologia de Sequência de Aminoácidos , Tripsina/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificação
18.
Bioorg Med Chem Lett ; 26(5): 1485-9, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26848109

RESUMO

The human tissue kallikreins (KLK1-KLK15) comprise a family of 15 serine peptidases detected in almost every tissue of the human body and that actively participate in many physiological and pathological events. Some kallikreins are involved in diseases for which no effective therapy is available, as for example, epithelial disorders, bacterial infections and in certain cancers metastatic processes. In recent years our group have made efforts to find inhibitors for all kallikreins, based on natural products and synthetic molecules, and all the inhibitors developed by our group presented a competitive mechanism of inhibition. Here we describe fukugetin, a natural product that presents a mixed-type mechanism of inhibition against KLK1 and KLK2. This type of inhibitor is gaining importance today, especially for the development of exosite-type inhibitors, which present potential to selectively inhibit the enzyme activity only against specific substrate.


Assuntos
Biflavonoides/farmacologia , Produtos Biológicos/farmacologia , Inibidores de Serino Proteinase/farmacologia , Calicreínas Teciduais/antagonistas & inibidores , Biflavonoides/química , Biflavonoides/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Relação Dose-Resposta a Droga , Garcinia/química , Humanos , Modelos Moleculares , Conformação Molecular , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/isolamento & purificação , Relação Estrutura-Atividade , Calicreínas Teciduais/metabolismo
19.
Peptides ; 76: 80-6, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26775801

RESUMO

Tripeptidyl peptidase I (TPP-I), also named ceroid lipofuscinosis 2 protease (CLN2p), is a serine carboxyl lysosomal protease involved in neurodegenerative diseases, and has both tripeptidyl amino- and endo- peptidase activities under different pH conditions. We developed fluorescence resonance energy transfer (FRET) peptides using tryptophan (W) as the fluorophore to study TPP-I hydrolytic properties based on previous detailed substrate specificity study (Tian Y. et al., J. Biol. Chem. 2006, 281:6559-72). Tripeptidyl amino peptidase activity is enhanced by the presence of amino acids in the prime side and the peptide NH2-RWFFIQ-EDDnp is so far the best substrate described for TPP-I. The hydrolytic parameters of this peptide and its analogues indicated that the S4 subsite of TPP-I is occluded and there is an electrostatic interaction of the positively charged substrate N-terminus amino group and a negative locus in the region of the enzyme active site. KCl activated TPP-I in contrast to the inhibition by Ca(2+) and NaCl. Solvent kinetic isotope effects (SKIEs) show the importance of the free N-terminus amino group of the substrates, whose absence results in a more complex solvent-dependent enzyme: substrate interaction and catalytic process. Like pure TPP-I, rat spleen and kidney homogenates cleaved NH2-RWFFIQ-EDDnp only at F-F bond and is not inhibited by pepstatin, E-64, EDTA or PMSF. The selectivity of NH2-RWFFIQ-EDDnp to TPP-I was also demonstrated by the 400 times higher k(cat)/K(M) compared to generally used substrate, NH2-AAF-MCA and by its resistance to hydrolysis by cathepsin D that is present in high levels in kidneys.


Assuntos
Aminopeptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Serina Proteases/química , Sequência de Aminoácidos , Animais , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Masculino , Proteólise , Ratos , Extratos de Tecidos/química
20.
Front Microbiol ; 7: 2141, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28119672

RESUMO

Enzymes have important features that may facilitate their application in industrial processes and have been used as alternatives to chemical catalysts. In particular, proteases can be isolated from microorganisms, which provide important sources of advantageous enzymes for industrial processes. For example, Eupenicillium javanicum is a filamentous fungus that has been shown to express industrially applicable enzymes and chemical components, such as antifungal compounds. The biotechnological potential of E. javanicum and proteases made us search a novel protease from this microorganism. The macromolecule was isolated, the main biochemical properties was evaluated, and the specificity of the protease subsites was determined. The protease was produced under solid-state bioprocess with wheat bran and isolated by two chromatography steps with yield of 27.5% and 12.4-fold purification. The molecular mass was estimated at 30 kDa. The N-terminal sequence of the first 20 amino acid residues was AVGAGYNASVALALEKALNN. The enzyme presented higher proteolytic activity at pH 6.0 and 60°C. The protease is stable at wide range of pH values and temperatures and in the presence of surfactants. The "primed" side of the catalytic site showed the highest catalytic efficiency of the enzyme isolated from E. javanicum. The S'1 subsite is responsible for catalyzing the protease reaction with substrates with tyrosine in P'1. These findings provide important insights into the biochemical characterization of a highly active protease from E. javanicum and may facilitate the development of industrial processes involving this protease.

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