RESUMO
The serine peptidase neurotrypsin is stored in presynaptic nerve endings and secreted in an inactive zymogenic form by synaptic activity. After activation, which requires activity of postsynaptic NMDA (N-methyl-D-aspartate) receptors, neurotrypsin cleaves the heparan sulfate proteoglycan agrin at active synapses. The resulting C-terminal 22-kDa fragment of agrin induces dendritic filopodia, which are considered to be precursors of new synapses. In the present study, we investigated the role of GAGs (glycosaminoglycans) in the activation of neurotrypsin and neurotrypsin-dependent agrin cleavage. We found binding of neurotrypsin to the GAG side chains of agrin, which in turn enhanced the activation of neurotrypsin by proprotein convertases and resulted in enhanced agrin cleavage. A similar enhancement of neurotrypsin binding to agrin, neurotrypsin activation and agrin cleavage was induced by the four-amino-acid insert at the y splice site of agrin, which is crucial for the formation of a heparin-binding site. Non-agrin GAGs also contributed to binding and activation of neurotrypsin and, thereby, to agrin cleavage, albeit to a lesser extent. Binding of neurotrypsin to cell-surface glycans locally restricts its conversion from zymogen into active peptidase. This provides the molecular foundation for the local action of neurotrypsin at or in the vicinity of its site of synaptic secretion. By its local action at synapses with correlated pre- and post-synaptic activity, the neurotrypsin-agrin system fulfils the requirements for a mechanism serving experience-dependent modification of activated synapses, which is essential for adaptive structural reorganizations of neuronal circuits in the developing and/or adult brain.
Assuntos
Agrina/metabolismo , Glicosaminoglicanos/farmacologia , Terminações Pré-Sinápticas/metabolismo , Serina Endopeptidases/metabolismo , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Ativação Enzimática , Glicosaminoglicanos/metabolismo , Células HEK293 , Humanos , Pró-Proteína Convertases/metabolismo , Sindecana-2/farmacologiaRESUMO
Zinc metallopeptidases of bacterial pathogens are widely distributed virulence factors and represent promising pharmacological targets. In this work, we have characterized Zmp1, a zinc metallopeptidase identified as a virulence factor of Mycobacterium tuberculosis and belonging to the neprilysin (NEP; M13) family, whose X-ray structure has been recently solved. Interestingly, this enzyme shows an optimum activity toward a fluorogenic substrate at moderately acidic pH values (i.e., 6.3), which corresponds to those reported for the Mtb phagosome where this enzyme should exert its pathological activity. Substrate specificity of Zmp1 was investigated by screening a peptide library. Several sequences derived from biologically relevant proteins were identified as possible substrates, including the neuropeptides bradykinin, neurotensin, and neuropeptide FF. Further, subsequences of other small bioactive peptides were found among most frequently cleaved sites, e.g., apelin-13 and substance P. We determined the specific cleavage site within neuropeptides by mass spectrometry, observing that hydrophobic amino acids, mainly phenylalanine and isoleucine, are overrepresented at position P1'. In addition, the enzymatic mechanism of Zmp1 toward these neuropeptides has been characterized, displaying some differences with respect to the synthetic fluorogenic substrate and indicating that the enzyme adapts its enzymatic action to different substrates.
Assuntos
Proteínas de Bactérias/metabolismo , Metaloproteases/metabolismo , Mycobacterium tuberculosis/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Concentração de Íons de Hidrogênio , Hidrólise , Ácidos Hidroxâmicos , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cinética , Metaloproteases/antagonistas & inibidores , Metaloproteases/química , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Inibidores de Proteases/farmacologia , Ligação Proteica , Proteólise , Substância P/metabolismo , Especificidade por Substrato , Zinco/metabolismoRESUMO
A number of strategies and protocols for the expression, purification and kinetic characterization of human caspases are described in the literature. We have systematically revised these protocols and present comprehensive optimized expression and purification protocols for caspase-1 to -9 as well as improved assay conditions for their reproducible kinetic characterization. Our studies on active site titration revealed that the reproducibility is strongly affected by the presence of DTT in the assay buffer. Furthermore, we observed that not all caspases show a linear relationship between enzymatic activity and protein concentration, which explains the discrepancy between published values of specific activities from different laboratories. Our broad kinetic analysis allows the conclusion that the dependency of caspase activities on protein concentration is an effect of concentration-dependent dimerization, which can also be influenced by kosmotropic salts. The protocol recommendations as an outcome of this work will yield higher reproducibility regarding expression and purification of human caspases and contribute to standardization of enzyme kinetic data.
Assuntos
Caspases/genética , Caspases/metabolismo , Clonagem Molecular/métodos , Caspases/química , Caspases/isolamento & purificação , Domínio Catalítico , Cromatografia em Gel/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/genética , Expressão Gênica , Humanos , Cinética , Redobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The human cysteine peptidase cathepsin K is a key enzyme in bone homoeostasis and other physiological functions. In the present study we investigate the mechanism of cathepsin K action at physiological plasma pH and its regulation by modifiers that bind outside of the active site. We show that at physiological plasma pH the enzyme fluctuates between multiple conformations that are differently susceptible to macromolecular inhibitors and can be manipulated by varying the ionic strength of the medium. The behaviour of the enzyme in vitro can be described by the presence of two discrete conformations with distinctive kinetic properties and different susceptibility to inhibition by the substrate benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin. We identify and characterize sulfated glycosaminoglycans as natural allosteric modifiers of cathepsin K that exploit the conformational flexibility of the enzyme to regulate its activity and stability against autoproteolysis. All sulfated glycosaminoglycans act as non-essential activators in assays using low-molecular-mass substrates. Chondroitin sulfate and dermatan sulfate bind at one site on the enzyme, whereas heparin binds at an additional site and has a strongly stabilizing effect that is unique among human glycosaminoglycans. All glycosaminoglycans stimulate the elastinolytic activity of cathepsin K at physiological plasma pH, but only heparin also increases the collagenolytic activity of the enzyme under these conditions. Altogether these results provide novel insight into the mechanism of cathepsin K function at the molecular level and its regulation in the extracellular space.
Assuntos
Catepsina K/química , Catepsina K/metabolismo , Regulação Alostérica , Animais , Catepsina K/antagonistas & inibidores , Catepsina K/sangue , Bovinos , Colágeno/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/fisiologia , Elastina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Especificidade por Substrato , Triptofano/químicaRESUMO
Enzymatic assays are widely employed to characterize important allosteric and enzyme modulation effects. The high sensitivity of these assays can represent a serious problem if the occurrence of experimental errors surreptitiously affects the reliability of enzyme kinetics results. We have addressed this problem and found that hidden assay interferences can be unveiled by the graphical representation of progress curves in modified reaction coordinates. To render this analysis accessible to users across all levels of expertise, we have developed a webserver, interferENZY, that allows (i) an unprecedented tight quality control of experimental data, (ii) the automated identification of small and major assay interferences, and (iii) the estimation of bias-free kinetic parameters. By eliminating the subjectivity factor in kinetic data reporting, interferENZY will contribute to solving the "reproducibility crisis" that currently challenges experimental molecular biology. The interferENZY webserver is freely available (no login required) at https://interferenzy.i3s.up.pt.
Assuntos
Ensaios Enzimáticos/métodos , Ensaios Enzimáticos/normas , Internet , Software , Animais , Automação , Biocatálise , Galinhas , Cinética , Muramidase/metabolismo , Controle de Qualidade , Padrões de Referência , Especificidade por Substrato , Fatores de TempoRESUMO
Ammodytoxins are presynaptically neurotoxic snake venom group IIA secreted phospholipase A(2) enzymes that interact specifically with calmodulin in the cytosol of nerve cells. We show that calmodulin behaves as an activator of ammodytoxin under both nonreducing and reducing (cytosol-like) conditions by stimulating its enzymatic activity up to 21-fold. Kinetic analysis, using a general modifier mechanism, and surface plasmon resonance measurements reveal that calmodulin influences both the catalytic and the vesicle binding properties of the enzyme without affecting its calcium binding properties. The equilibrium dissociation constant of the ammodytoxin-calmodulin complex under cytosol-like conditions is in the low nanomolar range (3 nM), while under nonreducing conditions, the binding affinity is in the subnanomolar range (0.07-0.18 nM). Upon exposure to cytosol-like conditions, ammodytoxin undergoes a slow hysteretic transition to a less active state. Calmodulin stabilizes the conformation of ammodytoxin and thereby restores its activity. These results provide insights into the neurotoxic action of ammodytoxins and the mechanisms involved in the regulation of secreted phospholipase A(2) activity within the cytosol.
Assuntos
Calmodulina/metabolismo , Citosol/metabolismo , Fosfolipases A2/metabolismo , Venenos de Serpentes/enzimologia , Animais , Sítios de Ligação , Cálcio/química , Cálcio/metabolismo , Calmodulina/química , Calmodulina/toxicidade , Cátions Bivalentes , Citosol/química , Ativação Enzimática , Cinética , Modelos Moleculares , Neurônios/química , Neurônios/metabolismo , Fosfolipases A2/química , SuínosRESUMO
Enzyme activity can be modulated by the concurrent action of two modifiers, either activators or inhibitors. The kinetic mechanisms for the interaction of the individual modifiers with the target enzyme can change considerably when two modifiers bind simultaneously. We illustrate a general equation for this kind of interactions, which can unambiguously describe the behavior of activators and inhibitors acting by any combination of classical kinetic mechanisms. The flexibility of this model is exemplified by combinations of activators and/or inhibitors, which can be competitive, uncompetitive or mixed-type, bind the target enzyme in either compulsory or random order, and are able to drive or not enzyme activity to zero at saturation. The model shows that the effects of zero-interaction and synergy between simultaneously acting enzyme modifiers are common events. Yet, in disagreement with previous theories, this model shows that antagonism between enzyme modifiers is a rare effect, which can be predicted only under very particular circumstances.
Assuntos
Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Modelos Químicos , Animais , Interações Medicamentosas , Sinergismo Farmacológico , Ativação EnzimáticaRESUMO
Specific and potent caspase inhibitors are indispensable for the dissection of the intricate pathways leading to apoptosis. We selected a designed ankyrin repeat protein (DARPin) from a combinatorial library that inhibits caspase-2 in vitro with a subnanomolar inhibition constant and, in contrast to the peptidic caspase inhibitors, with very high specificity for this particular caspase. The crystal structure of this inhibitor (AR_F8) in complex with caspase-2 reveals the molecular basis for the specificity and, together with kinetic analyses, the allosteric mechanism of inhibition. The structure also shows a conformation of the active site that can be exploited for the design of inhibitory compounds. AR_F8 is a specific inhibitor of an initiator caspase and has the potential to help identify the function of caspase-2 in the complex biological apoptotic signaling network.
Assuntos
Repetição de Anquirina/fisiologia , Caspase 2/química , Inibidores de Caspase , Cisteína Endopeptidases/química , Engenharia de Proteínas , Sequência de Aminoácidos , Humanos , Dados de Sequência MolecularRESUMO
A systematic survey of the acetylcholine-mimetic 2,4-dioxa-3-phosphadecalins as irreversible inhibitors of acetylcholinesterase revealed hitherto overlooked properties as far as the kinetic mechanisms of interaction are concerned. As a support to past and future work in this field, we describe the kinetics of eight reaction schemes that may be found in irreversible enzyme modification and compare them with two mechanism of reversible, slow-binding inhibition. The relevant kinetic equations and their associated graphical representations are given for all mechanisms, and concrete examples illustrate their practical use. Since irreversible inhibition is a time-dependent phenomenon, kinetic analysis is greatly facilitated by fitting the appropriate integrated rate equations to reaction-progress curves by nonlinear regression. This primary scrutiny provides kinetic parameters that are indispensable tools for diagnosing the kinetic mechanism and for calculating inhibition constants. Numerical integration of sets of differential equations is an additional useful investigation tool in critical situations, e.g., when inhibitors are unstable and/or act as irreversible modifiers only temporarily.
Assuntos
Acetilcolinesterase/efeitos dos fármacos , Inibidores da Colinesterase/farmacologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Naftalenos/farmacologia , Acetilcolinesterase/metabolismo , Isoflurofato/farmacologia , Cinética , Modelos Químicos , Relação Estrutura-AtividadeRESUMO
The synaptic serine protease neurotrypsin is thought to be important for adaptive synaptic processes required for cognitive functions, because humans deficient in neurotrypsin suffer from severe mental retardation. In the present study, we describe the biochemical characterization of neurotrypsin and its so far unique substrate agrin. In cell culture experiment as well as in neurotrypsin-deficient mice, we showed that agrin cleavage depends on neurotrypsin and occurs at two conserved sites. Neurotrypsin and agrin were expressed recombinantly, purified, and assayed in vitro. A catalytic efficiency of 1.3 x 10(4) M(-1) x s(-1) was determined. Neurotrypsin activity was shown to depend on calcium with an optimal activity in the pH range of 7-8.5. Mutagenesis analysis of the amino acids flanking the scissile bonds showed that cleavage is highly specific due to the unique substrate recognition pocket of neurotrypsin at the active site. The C-terminal agrin fragment released after cleavage has recently been identified as an inactivating ligand of the Na+/K+-ATPase at CNS synapses, and its binding has been demonstrated to regulate presynaptic excitability. Therefore, dysregulation of agrin processing is a good candidate for a pathogenetic mechanism underlying mental retardation. In turn, these results may also shed light on mechanisms involved in cognitive functions.
Assuntos
Agrina/metabolismo , Deficiência Intelectual/enzimologia , Serina Endopeptidases/metabolismo , Sinapses/enzimologia , Agrina/química , Sequência de Aminoácidos , Animais , Catálise , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/genética , Serina Endopeptidases/isolamento & purificaçãoRESUMO
An increasing number of studies indicate that serine proteases play an important role in structural plasticity associated with learning and memory formation. Neurotrypsin is a multidomain serine protease located at the presynaptic terminal of neurons. It is thought to be crucial for cognitive brain functions. A deletion in the neurotrypsin gene causes severe mental retardation in humans. For a biochemical characterization, we produced murine neurotrypsin recombinantly in a eukaryotic expression system using myeloma cells. From the culture medium we purified neurotrypsin using heparin-, hydrophobic interaction- and immobilized metal affinity chromatography. For an enzymological characterization two fragments of agrin containing the natural cleavages sites of neurotrypsin were used as substrates. The highest catalytic activity of neurotrypsin was observed in the pH range between 7.0 and 8.5. Calcium ions were required for neurotrypsin activity and an ionic strength exceeding 500 mM decreased substrate cleavage. Site-specific mutations of the amino acids flanking the scissile bonds showed that cleavage is highly specific and requires a basic amino acid preceded by a glutamate residue on the N-terminal side of the scissile bond. This sequence requirement argues for a unique substrate binding pocket of neurotrypsin. This observation was further substantiated by the fact that almost all tested serine protease inhibitors except dichloroisocoumarin and PMSF did not affect neurotrypsin activity.
Assuntos
Serina Endopeptidases/isolamento & purificação , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Especificidade por SubstratoRESUMO
Standards for reporting enzymology data (STRENDA) DB is a validation and storage system for enzyme function data that incorporates the STRENDA Guidelines. It provides authors who are preparing a manuscript with a user-friendly, web-based service that checks automatically enzymology data sets entered in the submission form that they are complete and valid before they are submitted as part of a publication to a journal.
Assuntos
Bases de Dados de Proteínas/normas , Ensaios Enzimáticos/normas , Enzimas/metabolismo , Interface Usuário-Computador , Animais , Bactérias/metabolismo , Ensaios Enzimáticos/métodos , Enzimas/química , Enzimas/classificação , Fungos/metabolismo , Guias como Assunto , Humanos , Disseminação de Informação/métodos , Cinética , Publicações Periódicas como Assunto , Plantas/metabolismo , Estudos de Validação como AssuntoRESUMO
Development of disease-modifying therapeutics is urgently needed for treating Alzheimer disease (AD). AD is characterized by toxic ß-amyloid (Aß) peptides produced by ß- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP). ß-secretase inhibitors reduce Aß levels, but mechanism-based side effects arise because they also inhibit ß-cleavage of non-amyloid substrates like Neuregulin. We report that ß-secretase has a higher affinity for Neuregulin than it does for APP. Kinetic studies demonstrate that the affinities and catalytic efficiencies of ß-secretase are higher toward non-amyloid substrates than toward APP. We show that non-amyloid substrates are processed by ß-secretase in an endocytosis-independent manner. Exploiting this compartmentalization of substrates, we specifically target the endosomal ß-secretase by an endosomally targeted ß-secretase inhibitor, which blocked cleavage of APP but not non-amyloid substrates in many cell systems, including induced pluripotent stem cell (iPSC)-derived neurons. ß-secretase inhibitors can be designed to specifically inhibit the Alzheimer process, enhancing their potential as AD therapeutics without undesired side effects.
Assuntos
Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/metabolismo , Oligopeptídeos/farmacologia , Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Animais , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Células Cultivadas , Endocitose , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas , Cinética , Camundongos , Simulação de Dinâmica Molecular , Neuregulina-1/química , Neuregulina-1/metabolismo , Oligopeptídeos/química , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteólise , Especificidade por SubstratoRESUMO
Complex formation of the Hsp70 chaperone DnaK with the fluorescence-labeled peptide ALLLSAPRR shows a very rapid first phase that has as yet not been observed with other peptides. This first phase is completed within the dead time (1-2 ms) of the stopped-flow instrument and corresponds to two thirds of the total increase in fluorescence. It occurs both in the presence and in the absence of ATP and is followed by a fast, a slow and a very slow step. These binding kinetics that are vastly different from those observed with other peptides might indicate the existence of a second substrate-binding site of DnaK.
Assuntos
Proteínas de Escherichia coli , Proteínas de Choque Térmico HSP70/química , Oligopeptídeos/química , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Fluorescência , Proteínas de Choque Térmico HSP70/metabolismo , Cinética , Oligopeptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Espectrometria de Fluorescência , Fatores de TempoRESUMO
Targeting allosteric sites is gaining increasing recognition as a strategy for modulating the activity of enzymes, especially in drug design. Here we investigate the mechanisms of allosteric regulation of cathepsin K as a representative of cysteine cathepsins and a promising drug target for the treatment of osteoporosis. Eight novel modifiers are identified by computational targeting of predicted allosteric sites on the surface of the enzyme. All act via hyperbolic kinetic mechanisms in presence of low molecular mass substrates, as expected for allosteric effectors. Two compounds have sizable effects on enzyme activity using interstitial collagen as a natural substrate of cathepsin K and four compounds show a significantly stabilizing effect on cathepsin K. The concept of activity modification space is introduced to obtain a global perspective of the effects elicited by the modifiers. Analysis of the activity modification space reveals that the activity of cathepsin K is regulated via multiple, different allosteric mechanisms.
Assuntos
Catepsina K/química , Regulação Alostérica , Catepsina K/metabolismo , Estabilidade Enzimática , Humanos , Osteoporose/enzimologiaRESUMO
Allosteric modifiers have the potential to fine-tune enzyme activity. Therefore, targeting allosteric sites is gaining increasing recognition as a strategy in drug design. Here we report the use of computational methods for the discovery of the first small-molecule allosteric inhibitor of the collagenolytic cysteine peptidase cathepsin K, a major target for the treatment of osteoporosis. The molecule NSC13345 is identified by high-throughput docking of compound libraries to surface sites on the peptidase that are connected to the active site by an evolutionarily conserved network of residues (protein sector). The crystal structure of the complex shows that NSC13345 binds to a novel allosteric site on cathepsin K. The compound acts as a hyperbolic mixed modifier in the presence of a synthetic substrate, it completely inhibits collagen degradation and has good selectivity for cathepsin K over related enzymes. Altogether, these properties qualify our methodology and NSC13345 as promising candidates for allosteric drug design.
Assuntos
Sítio Alostérico , Catepsina K/metabolismo , Simulação de Acoplamento Molecular , Regulação Alostérica , Benzoatos/metabolismo , Desenho de Fármacos , Humanos , Terapia de Alvo Molecular , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Estrutura Terciária de ProteínaRESUMO
The cysteine peptidase cathepsin K is a major player in extracellular proteolysis. Here we describe the identification of the multifunctional extracellular chaperone clusterin as a cathepsin K-binding protein. Clusterin increases the stability of cathepsin K in dilute solution and in the presence of high protein concentration. It does not alter the activity of the enzyme but acts as a liberator by preventing substrate inhibition. Kinetic measurements show that clusterin binds cathepsin K with high affinity (K(d) = 0.5-0.6 nM). Altogether these results provide novel insights into the mechanisms involved in the fine-tuning of cysteine cathepsin activity in the extracellular space.
Assuntos
Catepsina K/metabolismo , Clusterina/metabolismo , Catepsina K/antagonistas & inibidores , Catepsina K/química , Catepsina K/genética , Cromatografia de Afinidade , Cumarínicos/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Espaço Extracelular , Humanos , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Modelos Biológicos , Concentração Osmolar , Proteólise , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoAssuntos
Catepsina B/metabolismo , Condrócitos/metabolismo , Osteoartrite/enzimologia , Processamento Alternativo , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Catepsina B/genética , Condrócitos/enzimologia , Condrócitos/patologia , Humanos , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Osteoartrite/patologia , Fenótipo , RNA Mensageiro/metabolismoRESUMO
The serine endopeptidase elastase-2 from human polymorphonuclear leukocytes is associated with physiological remodeling and pathological degradation of the extracellular matrix. Glycosaminoglycans bound to the matrix or released after proteolytic processing of the core proteins of proteoglycans are potential ligands of elastase-2. In vitro, this interaction results in enzyme inhibition at low concentrations of glycosaminoglycans. However, inhibition is reversed and even abolished at high concentrations of the ligands. This behavior, which can be interpreted by a mechanism involving at least two molecules of glycosaminoglycan binding the enzyme at different sites, may cause interference with the natural protein inhibitors of elastase-2, particularly the alpha-1 peptidase inhibitor. Depending on their concentration, glycosaminoglycans can either stimulate or antagonize the formation of the enzyme-inhibitor complex and thus affect proteolytic activity. This interference with elastase-2 inhibition in the extracellular space may be part of a finely-tuned control mechanism in the microenvironment of the enzyme during remodeling and degradation of the extracellular matrix.
Assuntos
Serina Endopeptidases/sangue , Animais , Sulfatos de Condroitina/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/enzimologia , Espaço Extracelular/enzimologia , Glicosaminoglicanos/farmacologia , Humanos , Cinética , Modelos Moleculares , Neutrófilos/enzimologia , Polissacarídeos/farmacologia , Proteínas/farmacologia , Serina Endopeptidases/química , Sulfatos/farmacologiaRESUMO
Most synthetic inhibitors of peptidases have been targeted to the active site for inhibiting catalysis through reversible competition with the substrate or by covalent modification of catalytic groups. Cathepsin B is unique among the cysteine peptidase for the presence of a flexible segment, known as the occluding loop, which can block the primed subsites of the substrate binding cleft. With the occluding loop in the open conformation cathepsin B acts as an endopeptidase, and it acts as an exopeptidase when the loop is closed. We have targeted the occluding loop of human cathepsin B at its surface, outside the catalytic center, using a high-throughput docking procedure. The aim was to identify inhibitors that would interact with the occluding loop thereby modulating enzyme activity without the help of chemical warheads against catalytic residues. From a large library of compounds, the in silico approach identified [2-[2-(2,4-dioxo-1,3-thiazolidin-3-yl)ethylamino]-2-oxoethyl] 2-(furan-2-carbonylamino) acetate, which fulfills the working hypothesis. This molecule possesses two distinct binding moieties and behaves as a reversible, double-headed competitive inhibitor of cathepsin B by excluding synthetic and protein substrates from the active center. The kinetic mechanism of inhibition suggests that the occluding loop is stabilized in its closed conformation, mainly by hydrogen bonds with the inhibitor, thus decreasing endoproteolytic activity of the enzyme. Furthermore, the dioxothiazolidine head of the compound sterically hinders binding of the C-terminal residue of substrates resulting in inhibition of the exopeptidase activity of cathepsin B in a physiopathologically relevant pH range.