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1.
ChemMedChem ; 19(11): e202400145, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38445366

RESUMO

The binding process of insulin to its transmembrane receptor entails a sophisticated interplay between two proteins, each possessing two binding sites. Given the difficulties associated with the use of insulin in the treatment of diabetes, despite its remarkable efficacy, there is interest in smaller and more stable compounds than the native hormone that would effectively activate the receptor. Our study adopts a strategy focused on synthesizing extensive combinatorial libraries of bipodal compounds consisting of two distinct peptides linked to a molecular scaffold. These constructs, evaluated in a resin bead-bound format, were designed to assess their binding to the insulin receptor. Despite notable nonspecific binding, our approach successfully generated and tested millions of compounds. Rigorous evaluations via flow cytometry and specific antibodies revealed peptide sequences with specific interactions at either receptor binding Site 1 or 2. Notably, these sequences bear similarity to peptides discovered through phage display by other researchers. This convergence of chemical and biological methods underscores nature's beauty, revealing general principles in peptide binding to the insulin receptor. Overall, our study deepens the understanding of molecular interactions in ligand binding to the insulin receptor, highlighting the challenges of targeting large proteins with small synthetic peptides.


Assuntos
Técnicas de Química Combinatória , Receptor de Insulina , Receptor de Insulina/metabolismo , Receptor de Insulina/química , Humanos , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/síntese química , Sítios de Ligação , Biblioteca de Peptídeos , Ligantes , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Estrutura Molecular , Ligação Proteica , Insulina/metabolismo , Insulina/química
2.
Chempluschem ; : e202300567, 2023 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-37942669

RESUMO

Galectins, a class of carbohydrate-binding proteins, play a crucial role in various physiological and disease processes. Therefore, the identification of ligands that efficiently bind these proteins could potentially lead to the development of new therapeutic compounds. In this study, we present a method that involves screening synthetic click glycopeptide libraries to identify lectin-binding ligands with low micromolar affinity. Our methodology, initially optimized using Concanavalin A, was subsequently applied to identify binders for the therapeutically relevant galectin 1. Binding affinities were assessed using various methods and showed that the selected glycopeptides exhibited enhanced binding potency to the target lectins compared to the starting sugar moieties. This approach offers an alternative means of discovering galectin-binding ligands as well as other carbohydrate-binding proteins, which are considered important therapeutic targets.

3.
RSC Med Chem ; 14(1): 144-153, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36760748

RESUMO

The development of highly active and selective enzyme inhibitors is one of the priorities of medicinal chemistry. Typically, various high-throughput screening methods are used to find lead compounds from a large pool of synthetic compounds, and these are further elaborated and structurally refined to achieve the desired properties. In an effort to streamline this complex and laborious process, new selection strategies based on different principles have recently emerged as an alternative. Herein, we compare three such selection strategies with the aim of identifying potent and selective inhibitors of human carbonic anhydrase II. All three approaches, in situ click chemistry, phage-display libraries and synthetic peptide libraries, led to the identification of more potent inhibitors when compared to the parent compounds. In addition, one of the inhibitor-peptide conjugates identified from the phage libraries showed greater than 100-fold selectivity for the enzyme isoform used for the compound selection. In an effort to rationalize the binding properties of the conjugates, we performed detailed crystallographic and NMR structural analysis, which revealed the structural basis of the compound affinity towards the enzyme and led to the identification of a novel exosite that could be utilized in the development of isoform specific inhibitors.

4.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 5): 300-306, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29717998

RESUMO

Human aldo-keto reductase 1C3 (AKR1C3) stereospecifically reduces steroids and prostaglandins and is involved in the biotransformation of xenobiotics. Its role in various cancers makes it a potential therapeutic target for the development of inhibitors. Recombinant AKR1C3 with a thrombin-cleavable N-terminal His6 tag was expressed from a pET-28(+) vector for structural studies of enzyme-inhibitor complexes. A modified in situ proteolysis approach was applied to specifically remove the His tag by thrombin cleavage during crystallization screening trials. This improved the morphology and diffraction quality of the crystals and allowed the acquisition of high-resolution diffraction data and structure solution. This approach may be generally applicable to other proteins expressed using the pET-28(+) vector.


Assuntos
Membro C3 da Família 1 de alfa-Ceto Redutase/química , Membro C3 da Família 1 de alfa-Ceto Redutase/metabolismo , Histidina , Trombina/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase/genética , Sequência de Aminoácidos , Cristalização/métodos , Cristalografia por Raios X/métodos , Histidina/genética , Humanos , Proteólise , Difração de Raios X/métodos
5.
Amino Acids ; 45(1): 143-57, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23483218

RESUMO

Three novel antimicrobial peptides (AMPs), named panurgines (PNGs), were isolated from the venom of the wild bee Panurgus calcaratus. The dodecapeptide of the sequence LNWGAILKHIIK-NH2 (PNG-1) belongs to the category of α-helical amphipathic AMPs. The other two cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges of the pattern Cys8-Cys23 and Cys11-Cys19 have almost identical sequence established as LDVKKIICVACKIXPNPACKKICPK-OH (X=K, PNG-K and X=R, PNG-R). All three peptides exhibited antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, antifungal activity, and low hemolytic activity against human erythrocytes. We prepared a series of PNG-1 analogs to study the effects of cationicity, amphipathicity, and hydrophobicity on the biological activity. Several of them exhibited improved antimicrobial potency, particularly those with increased net positive charge. The linear analogs of PNG-K and PNG-R having all Cys residues substituted by α-amino butyric acid were inactive, thus indicating the importance of disulfide bridges for the antimicrobial activity. However, the linear PNG-K with all four cysteine residues unpaired, exhibited antimicrobial activity. PNG-1 and its analogs induced a significant leakage of fluorescent dye entrapped in bacterial membrane-mimicking large unilamellar vesicles as well as in vesicles mimicking eukaryotic cell membrane. On the other hand, PNG-K and PNG-R exhibited dye-leakage activity only from vesicles mimicking bacterial cell membrane.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Venenos de Abelha/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antifúngicos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Venenos de Abelha/química , Venenos de Abelha/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Himenópteros/metabolismo , Testes de Sensibilidade Microbiana , Análise de Sequência de Proteína , Relação Estrutura-Atividade , Tensoativos , Lipossomas Unilamelares/metabolismo
6.
Amino Acids ; 43(2): 751-61, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22038181

RESUMO

In the venom of eusocial bee Lasioglossum laticeps, we identified a novel unique antimicrobial peptide named lasiocepsin consisting of 27 amino acid residues and two disulfide bridges. After identifying its primary structure, we synthesized lasiocepsin by solid-phase peptide synthesis using two different approaches for oxidative folding. The oxidative folding of fully deprotected linear peptide resulted in a mixture of three products differing in the pattern of disulfide bridges. Regioselective disulfide bond formation significantly improved the yield of desired product. The synthetic lasiocepsin possessed antimicrobial activity against both Gram-positive and -negative bacteria, antifungal activity against Candida albicans, and no hemolytic activity against human erythrocytes. We synthesized two lasiocepsin analogs cyclized through one native disulfide bridge in different positions and having the remaining two cysteines substituted by alanines. The analog cyclized through a Cys8-Cys25 disulfide bridge showed reduced antimicrobial activity compared to the native peptide while the second one (Cys17-Cys27) was almost inactive. Linear lasiocepsin having all four cysteine residues substituted by alanines or alkylated was also inactive. That was in contrast to the linear lasiocepsin with all four cysteine residues non-paired, which exhibited remarkable antimicrobial activity. The shortening of lasiocepsin by several amino acid residues either from the N- or C-terminal resulted in significant loss of antimicrobial activity. Study of Bacillus subtilis cells treated by lasiocepsin using transmission electron microscopy showed leakage of bacterial content mainly from the holes localized at the ends of the bacterial cells.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Venenos de Abelha/química , Abelhas/química , Peptídeos Cíclicos/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antifúngicos/síntese química , Antifúngicos/química , Venenos de Abelha/síntese química , Venenos de Abelha/farmacologia , Candida albicans/efeitos dos fármacos , Cistina/síntese química , Cistina/química , Eritrócitos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/ultraestrutura , Hemólise , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Estrutura Secundária de Proteína , Análise de Sequência de Proteína
7.
Amino Acids ; 39(3): 763-75, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20198492

RESUMO

Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH2 (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys-His-Ile-Leu-Lys-NH2 (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic alpha-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, alpha-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Venenos de Abelha/química , Abelhas/química , Sequência de Aminoácidos , Animais , Antibacterianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Bactérias/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Proteínas Hemolisinas/química , Proteínas Hemolisinas/isolamento & purificação , Proteínas Hemolisinas/farmacologia , Hemólise/efeitos dos fármacos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Ratos
8.
Cell Mol Life Sci ; 67(3): 455-66, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19921400

RESUMO

A novel homologue of insect defensin designated lucifensin (Lucilia defensin) was purified from the extracts of various tissues (gut, salivary glands, fat body, haemolymph) of green bottle fly (Lucilia sericata) larvae and from their excretions/secretions. The primary sequence of this peptide of 40 residues and three intramolecular disulfide bridges was determined by ESI-QTOF mass spectrometry and Edman degradation and is very similar to that of sapecin and other dipteran defensins. We assume that lucifensin is the key antimicrobial component that protects the maggots when they are exposed to the highly infectious environment of a wound during the medicinal process known as maggot therapy. We also believe that lucifensin is that long-sought larger molecular weight antimicrobial factor of the Lucilia sericata excretions/secretions believed to be effective against pathogenic elements of the wound microbial flora.


Assuntos
Anti-Infecciosos/química , Defensinas/química , Dípteros/metabolismo , Sequência de Aminoácidos , Animais , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Cromatografia Líquida de Alta Pressão , Defensinas/isolamento & purificação , Defensinas/farmacologia , Larva/metabolismo , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Alinhamento de Sequência , Cicatrização/efeitos dos fármacos
9.
J Biol Chem ; 284(39): 26557-68, 2009 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-19620709

RESUMO

The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP(6)). In this study, we demonstrated that InsP(6) is not simply an allosteric cofactor, but rather binding of InsP(6) stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-A crystal structure of this InsP(6)-bound unprocessed form of CPD was determined and revealed the scissile bond Leu(3428)-Ala(3429) captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP(6), but was reactivated for high affinity binding of InsP(6) by cooperative binding of both a new substrate and InsP(6). Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.


Assuntos
Toxina da Cólera/metabolismo , Ácido Fítico/metabolismo , Estrutura Terciária de Proteína , Vibrio cholerae/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Arginina/química , Arginina/genética , Arginina/metabolismo , Sítios de Ligação/genética , Domínio Catalítico , Toxina da Cólera/química , Toxina da Cólera/genética , Cristalização , Eletroforese em Gel de Poliacrilamida , Interações Hidrofóbicas e Hidrofílicas , Leucina/química , Leucina/genética , Leucina/metabolismo , Lisina/química , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ácido Fítico/química , Ligação Proteica , Dobramento de Proteína , Eletricidade Estática , Termodinâmica , Tripsina/metabolismo , Vibrio cholerae/genética
10.
Chembiochem ; 10(12): 2089-99, 2009 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-19591185

RESUMO

Three novel structurally related pentadecapeptides, named lasioglossins, were isolated from the venom of the eusocial bee Lasioglossum laticeps. Their primary sequences were established as H-Val-Asn-Trp-Lys-Lys-Val-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-I), H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-II) and H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Lys-NH(2) (LL-III). These lasioglossins exhibited potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, low haemolytic and mast cell degranulation activity, and a potency to kill various cancer cells in vitro. The lasioglossin CD spectra were measured in the presence of trifluoroethanol and sodium dodecyl sulfate solution and indicated a high degree of alpha-helical conformation. NMR spectroscopy, which was carried out in trifluoroethanol/water confirmed a curved alpha-helical conformation with a concave hydrophobic and convex hydrophilic side. To understand the role of this bend on biological activity, we studied lasioglossin analogues in which the Gly in the centre of the molecule was replaced by other amino acid residues (Ala, Lys, Pro). The importance of the N-terminal part of the molecule to the antimicrobial activity was revealed through truncation of five residues from both the N and C termini of the LL-III peptide. C-terminal deamidation of LL-III resulted in a drop in antimicrobial activity, but esterification of the C terminus had no effect. Molecular modelling of LL-III and the observed NOE contacts indicated the possible formation of a bifurcated H-bond between hydrogen from the Lys15 CONH peptide bond and one H of the C-terminal CONH(2) to the Ile11 oxygen atom. Such interactions cannot form with C-terminal esterification.


Assuntos
Anti-Infecciosos/química , Venenos de Abelha/química , Abelhas/química , Peptídeos/química , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Testes de Sensibilidade Microbiana , Peptídeos/síntese química , Peptídeos/farmacologia
11.
Chembiochem ; 9(17): 2815-21, 2008 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-18942691

RESUMO

A novel antimicrobial peptide designated melectin was isolated from the venom of the cleptoparasitic bee Melecta albifrons. Its primary sequence was established as H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Ala-His-Met-Lys-NH(2) by Edman degradation and ESI-QTOF mass spectrometry. Synthetic melectin exhibited antimicrobial activity against both gram-positive and -negative bacteria and it degranulated rat peritoneal mast cells, but its hemolytic activity was low. The CD spectra of melectin measured in the presence of trifluoroethanol and sodium dodecyl sulfate showed a high content alpha-helices, which indicates that melectin can adopt an amphipathic alpha-helical secondary structure in an anisotropic environment such as the bacterial cell membrane. To envisage the role of the proline residue located in the middle of the peptide chain on biological activity and secondary structure, we prepared several melectin analogues in which the Pro11 residue was either replaced by other amino acid residues or was omitted. The results of biological testing suggest that a Pro kink in the alpha-helical structure of melectin plays an important role in selectivity for bacterial cells. In addition, a series of N- and C-terminal-shortened analogues was synthesized to examine which region of the peptide is related to antimicrobial activity.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Venenos de Abelha/química , Abelhas , Sequência de Aminoácidos , Animais , Antibacterianos/síntese química , Antibacterianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Degranulação Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Eritrócitos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Mastócitos/efeitos dos fármacos , Mastócitos/fisiologia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
12.
J Pept Sci ; 14(6): 670-82, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18044819

RESUMO

We chose the larvae of fleshfly Sarcophaga bullata to map the peptide and protein immune response. The hemolymph of the third-instar larvae of S. bullata was used for isolation. The larvae were injected with bacterial suspension to induce an antimicrobial response. The hemolymph was separated into crude fractions, which were subdivided by RP-HPLC, gel electrophoresis, and free-flow electrophoresis. In several fractions, we determined significant antimicrobial activities against the pathogenic bacteria Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa. Among antimicrobially active compounds we identified dipeptide beta-alanyl-L-tyrosine, protein transferrin, and two variants of peptide sapecin. We also partially characterized two novel antimicrobially active polypeptides; odorant-binding protein 99b, and a peptide which remains unidentified.


Assuntos
Dípteros/imunologia , Proteínas de Insetos/imunologia , Larva/imunologia , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Dípteros/crescimento & desenvolvimento , Eletroforese em Gel de Poliacrilamida , Proteínas de Insetos/química , Dados de Sequência Molecular , Peso Molecular , Mapeamento de Peptídeos , Espectrometria de Massas por Ionização por Electrospray
13.
Z Naturforsch C J Biosci ; 62(5-6): 382-8, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17708444

RESUMO

The presence of various enzyme forms with terminal action pattern on pectate was evaluated in a protein mixture obtained from parsley roots. Enzymes found in the soluble fraction of roots (juice) were purified to homogeneity according to SDS-PAGE, partially separated by preparative isoelectric focusing and characterized. Three forms with pH optima 3.6, 4.2 and 4.6 clearly preferred substrates with a lower degree of polymerization (oligogalacturonates) while the form with pH optimum 5.2 was a typical exopolygalacturonase [EC 3. 2.1.67] with relatively fast cleavage of polymeric substrate. The forms with pH optima 3.6, 4.2 and 5.2 were released from the pulp, too. The form from the pulp with pH optimum 4.6 preferred higher oligogalacturonates and was not described in plants previously. The production of individual forms in roots was compared with that produced by root cells cultivated on solid medium and in liquid one.


Assuntos
Petroselinum/enzimologia , Polissacarídeo-Liases/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Cinética , Camundongos , Oxigenases de Função Mista/isolamento & purificação , Oxigenases de Função Mista/metabolismo , Raízes de Plantas/enzimologia , Polissacarídeo-Liases/isolamento & purificação
14.
Phytochemistry ; 66(1): 31-9, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15649508

RESUMO

The primary structure and proteolytic processing of the alpha-amylase isoinhibitor alpha AI-1 from common bean (Phaseolus vulgaris cv. Magna) was determined by protein chemistry techniques. The inhibitory specificity of alphaAI-1 was screened with a panel of the digestive alpha-amylases from 30 species of insects, mites, gastropod, annelid worm, nematode and fungal phytopathogens with a focus on agricultural pests and important model species. This in vitro analysis showed a selective inhibition of alpha-amylases from three orders of insect (Coleoptera, Hymenoptera and Diptera) and an inhibition of alpha-amylases of the annelid worm. The inhibitory potential of alphaAI-1 against several alpha-amylases was found to be modulated by pH. To understand how alphaAI-1 discriminates among closely related alpha-amylases, the sequences of the alpha-amylases sensitive, respectively, insensitive to alphaAI-1 were compared, and the critical determinants were localized on the spatial alpha-amylase model. Based on the in vitro analysis of the inhibitory specificity of alphaAI-1, the in vivo activity of the ingested alphaAI-1 was demonstrated by suppression of the development of the insect larvae that expressed the sensitive digestive alpha-amylases. The first comprehensive mapping of alphaAI-1 specificity significantly broadens the spectrum of targets that can be regulated by alpha-amylase inhibitors of plant origin, and points to potential application of these protein insecticides in plant biotechnologies.


Assuntos
Inseticidas/química , Inseticidas/farmacologia , Phaseolus/química , Lectinas de Plantas/farmacologia , alfa-Amilases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caenorhabditis elegans/enzimologia , Fungos/enzimologia , Caracois Helix/enzimologia , Insetos/enzimologia , Ácaros/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Oligoquetos/enzimologia , Lectinas de Plantas/química , Ligação Proteica , Especificidade por Substrato
15.
Clin Chim Acta ; 328(1-2): 59-69, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12559599

RESUMO

BACKGROUND: Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP 5) is one of the most promising serologic markers with regard to an ability to prognose development of osteoarthritis (OA). Our aim was to map the epitopes of three monoclonal antibodies (mAb) to COMP and to develop and characterize a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring COMP levels in human body fluids. METHODS: COMP was digested with trypsin and the NH(2)-terminal sequence of the fragments recognized by each of the mAbs was determined. Steric competition among the mAbs was tested with an antibody capture assay. A sandwich ELISA was developed using unlabeled mAb 16-F12 as a capture antibody, and mAb 17-C10 labeled with biotin as the second antibody. RESULTS: Epitopes of the three mAbs were mapped to three different domains within the COMP subunit (16-F12, NH(2)-terminal domain; 17-C10, EGF-like domain; 12-C4, COOH-terminal domain). These epitopes did not overlap. mAbs 17-C10 and 12-C4 yielded similar serum COMP results when used as the secondary antibodies. Serum COMP levels measured with the new sandwich ELISA using mAbs 16-F12 and 17-C10 correlated strongly with results based on an inhibition ELISA with mAb 17-C10 alone (r(2) = 0.836; P < 0.0001). We characterized the new sandwich ELISA with regards to inter- and intra-assay variability, the range of COMP levels that can be expected in human synovial fluids (SF) and sera (controls and OA and rheumatoid arthritis (RA) patients), and the day-to-day and diurnal variability of COMP levels in sera. CONCLUSIONS: We have developed and characterized a sandwich ELISA for COMP that is sensitive and yields highly reproducible COMP results upon analysis of human sera and synovial fluids.


Assuntos
Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas da Matriz Extracelular/imunologia , Glicoproteínas/imunologia , Proteína de Matriz Oligomérica de Cartilagem , Mapeamento de Epitopos , Proteínas da Matriz Extracelular/análise , Glicoproteínas/análise , Humanos , Proteínas Matrilinas , Líquido Sinovial/química
16.
Anticancer Res ; 22(2A): 913-9, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12014671

RESUMO

Procathepsin D is over secreted by certain human cancer cells. This enzymatically inactive precursor has been established as playing an important role in the development of several types of cancer. Due to their pleiotropical effects, numerous cytokines have also been recognized as important immunotherapeutic agents. In this study, we focused on the role of IL-4, IL-10 and IL-13 on procathepsin D-stimulated proliferation of breast cancer cells. Our results clearly showed that only ER+ breast cancer cells responded to the presence of cytokines by proliferation; ER- cells were resistant to the addition of cytokines. Since addition of anti-procathepsin D antibodies blocked the growth potentiation, we concluded that addition of these cytokines resulted in stimulation of synthesis and/or release of procathepsin D. This conclusion was further supported by findings of procathepsin D in culture supematants of cells incubated with cytokines.


Assuntos
Neoplasias da Mama/patologia , Catepsina D/fisiologia , Precursores Enzimáticos/fisiologia , Interleucinas/farmacologia , Neoplasias da Mama/metabolismo , Catepsina D/biossíntese , Catepsina D/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/metabolismo , Humanos , Interleucina-10/farmacologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Receptores de Estrogênio/fisiologia , Células Tumorais Cultivadas
17.
Protein Sci ; 11(4): 933-43, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11910036

RESUMO

The mature bovine cathepsin C (CC) molecule is composed of four identical monomers, each proteolytically processed into three chains. Five intrachain disulfides and three nonpaired cysteine residues per monomer were identified. Beside catalytic Cys234 in the active site, free-thiol Cys331 and Cys424 were characterized. Cys424 can be classified as inaccessible buried residue. Selective modification of Cys331 results in dissociation of native CC tetramer into dimers. The 3D homology-based model of the CC catalytic core suggests that Cys331 becomes exposed as the activation peptide is removed during procathepsin C activation. The model further shows that exposed Cys331 is surrounded by a surface hydrophobic cluster, unique to CC, forming a dimer-dimer interaction interface. Substrate/inhibitor recognition of the active site in the CC dimer differs significantly from that in the native tetramer. Taken together, a mechanism is proposed that assumes that the CC tetramer formation results in a site-specific occlusion of endopeptidase-like active site cleft of each CC monomeric unit. Thus, tetramerization provides for the structural basis of the dipeptidyl peptidase activity of CC through a substrate access-limiting mechanism different from those found in homologous monomeric exopeptidases cathepsin H and B. In conclusion, the mechanism of tetramer formation as well as specific posttranslational processing segregates CC in the family of papain proteases.


Assuntos
Catepsina C/química , Catepsina C/metabolismo , Cisteína/química , Baço/enzimologia , Sequência de Aminoácidos , Animais , Catepsina C/isolamento & purificação , Bovinos , Cromatografia Líquida de Alta Pressão , Dissulfetos/química , Ativação Enzimática , Lisina/química , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos , Conformação Proteica , Dobramento de Proteína , Processamento de Proteína Pós-Traducional
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