RESUMO
Cruzain is a member of the papain/cathepsin L family of cysteine proteases, and the major cysteine protease of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We report an autoinduction methodology that provides soluble cruzain in high yields (>30 mg/L in minimal medium). These increased yields provide sufficient quantities of active enzyme for use in nuclear magnetic resonance (NMR)-based ligand mapping. Using circular dichroism and NMR spectroscopy, we also examined the solution-state structural dynamics of the enzyme in complex with a covalently bound vinyl sulfone inhibitor (K777). We report the backbone amide and side chain carbon chemical shift assignments of cruzain in complex with K777. These resonance assignments were used to identify and map residues located in the substrate binding pocket, including the catalytic Cys25 and His162. Selective [(15)N]Cys, [(15)N]His, and [(13)C]Met labeling was performed to quickly assess cruzain-ligand interactions for a set of eight low-molecular weight compounds exhibiting micromolar binding or inhibition. Chemical shift perturbation mapping verified that six of the eight compounds bind to cruzain at the active site. Three different binding modes were delineated for the compounds, namely, covalent, noncovalent, and noninteracting. These results provide examples of how NMR spectroscopy can be used to screen compounds for fast evaluation of enzyme-inhibitor interactions to facilitate lead compound identification and subsequent structural studies.
Assuntos
Cisteína Endopeptidases/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , Domínio Catalítico , Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Ressonância Magnética Nuclear Biomolecular , Fenilalanina/análogos & derivados , Piperazinas , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Compostos de Tosil , Trypanosoma cruzi/enzimologia , Compostos de Vinila/farmacologiaRESUMO
The type II transmembrane serine protease family consists of 18 closely related serine proteases that are implicated in multiple functions. To identify selective, inhibitory antibodies against one particular type II transmembrane serine protease, matriptase [MT-SP1 (membrane-type serine protease 1)], a phage display library was created with a natural repertoire of Fabs [fragment antigen binding (Fab)] from human naïve B cells. Fab A11 was identified with a 720 pM inhibition constant and high specificity for matriptase over other trypsin-fold serine proteases. A Trichoderma reesei system expressed A11 with a yield of â¼200 mg/L. The crystal structure of A11 in complex with matriptase has been determined and compared to the crystal structure of another antibody inhibitor (S4) in complex with matriptase. Previously discovered from a synthetic single-chain variable fragment library, S4 is also a highly selective and potent matriptase inhibitor. The crystal structures of the A11/matriptase and S4/matriptase complexes were solved to 2.1 Å and 1.5 Å, respectively. Although these antibodies, discovered from separate libraries, interact differently with the protease surface loops for their specificity, the structures reveal a similar novel mechanism of protease inhibition. Through the insertion of the H3 variable loop in a reverse orientation at the substrate-binding pocket, these antibodies bury a large surface area for potent inhibition and avoid proteolytic inactivation. This discovery highlights the critical role that the antibody scaffold plays in positioning loops to bind and inhibit protease function in a highly selective manner. Additionally, Fab A11 is a fully human antibody that specifically inhibits matriptase over other closely related proteases, suggesting that this approach could be useful for clinical applications.
Assuntos
Anticorpos/química , Anticorpos/farmacologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/imunologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Sequência de Aminoácidos , Anticorpos/isolamento & purificação , Anticorpos/metabolismo , Catálise/efeitos dos fármacos , Domínio Catalítico/genética , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Biblioteca de Peptídeos , Inibidores de Proteases/química , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Serina Endopeptidases/metabolismoRESUMO
Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, dual envelope pseudovirion (DEP) assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes.
Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/normas , Ensaios de Triagem em Larga Escala/normas , Humanos , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/normas , Cultura de Vírus/métodos , Cultura de Vírus/normasRESUMO
Intracellular iron homeostasis is critical for survival and proliferation. Lipocalin 24p3 is an iron-trafficking protein that binds iron through association with a bacterial siderophore, such as enterobactin, or a postulated mammalian siderophore. Here, we show that the iron-binding moiety of the 24p3-associated mammalian siderophore is 2,5-dihydroxybenzoic acid (2,5-DHBA), which is similar to 2,3-DHBA, the iron-binding component of enterobactin. We find that the murine enzyme responsible for 2,5-DHBA synthesis, BDH2, is the homolog of bacterial EntA, which catalyzes 2,3-DHBA production during enterobactin biosynthesis. RNA interference-mediated knockdown of BDH2 results in siderophore depletion. Mammalian cells lacking the siderophore accumulate abnormally high amounts of cytoplasmic iron, resulting in elevated levels of reactive oxygen species, whereas the mitochondria are iron deficient. Siderophore-depleted mammalian cells and zebrafish embryos fail to synthesize heme, an iron-dependent mitochondrial process. Our results reveal features of intracellular iron homeostasis that are conserved from bacteria through humans.
Assuntos
Enterobactina/metabolismo , Gentisatos/metabolismo , Sideróforos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Escherichia coli/metabolismo , Gentisatos/química , Humanos , Hidroxibutirato Desidrogenase/química , Hidroxibutirato Desidrogenase/metabolismo , Ferro/metabolismo , Camundongos , Dados de Sequência Molecular , Estresse Oxidativo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Alinhamento de Sequência , Peixe-ZebraRESUMO
Interactions between urokinase plasminogen activator receptor (uPAR) and its various ligands regulate tumor growth, invasion, and metastasis. Antibodies that bind specific uPAR epitopes may disrupt these interactions, thereby inhibiting these processes. Using a highly diverse and naïve human fragment of the antigen binding (Fab) phage display library, we identified 12 unique human Fabs that bind uPAR. Two of these antibodies compete against urokinase plasminogen activator (uPA) for uPAR binding, whereas a third competes with beta1 integrins for uPAR binding. These competitive antibodies inhibit uPAR-dependent cell signaling and invasion in the non-small cell lung cancer cell line, H1299. Additionally, the integrin-blocking antibody abrogates uPAR/beta1 integrin-mediated H1299 cell adhesion to fibronectin and vitronectin. This antibody and one of the uPAR/uPA antagonist antibodies shows a significant combined effect in inhibiting cell invasion through Matrigel/Collagen I or Collagen I matrices. Our results indicate that these antagonistic antibodies have potential for the detection and treatment of uPAR-expressing tumors.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Movimento Celular/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais/imunologia , Spodoptera , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Mycobacterium tuberculosis uses the ESX-1 secretion system to deliver virulence proteins during infection of host cells. Here we report a mechanism of posttranscriptional control of ESX-1 mediated by MycP1, a M. tuberculosis serine protease. We show that MycP1 is required for ESX-1 secretion but that, unexpectedly, genetic inactivation of MycP1 protease activity increases secretion of ESX-1 substrates. We demonstrate that EspB, an ESX-1 substrate required for secretion, is a target of MycP1 in vitro and in vivo. During macrophage infection, an inactive MycP1 protease mutant causes hyperactivation of ESX-1-stimulated innate signaling pathways. MycP1 is required for growth in mice during acute infection, while loss of its protease activity leads to attenuated virulence during chronic infection. As the key ESX-1 substrates ESAT-6 and CFP-10 are highly immunogenic, fine-tuning of their secretion by MycP1 may balance virulence and immune detection and be essential for successful maintenance of long-term M. tuberculosis infection.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Subtilisinas/fisiologia , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Contagem de Colônia Microbiana , Técnicas de Inativação de Genes , Fígado/microbiologia , Pulmão/patologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Processamento de Proteína Pós-Traducional , Baço/microbiologia , Subtilisinas/genética , Análise de SobrevidaRESUMO
Fungi are capable of degrading proteins in their environment by secreting peptidases. However, the link between extracellular digestion and intracellular proteolysis has scarcely been investigated. Mycelial lysates of the filamentous fungus Talaromyces emersonii were screened for intracellular peptidase production. Five distinct proteolytic activities with specificity for the p-nitroanilide (pNA) peptides Suc-AAPF-pNA, Suc-AAA-pNA, K-pNA, F-pNA and P-pNA were identified. The native enzyme responsible for the removal of N-terminal proline residues was purified to homogeneity by ammonium sulfate fractionation followed by five successive chromatographic steps. The enzyme, termed Talaromyces emersonii prolyl aminopeptidase (TePAP), displayed a 50-fold specificity for cleaving N-terminal Pro-X (k(cat)/K(m)=2.1 x 10(6) M(-1) s(-1)) compared with Ala-X or Val-X bonds. This intracellular aminopeptidase was optimally active at pH 7.4 and 50 degrees C. Peptide sequencing facilitated the design of degenerate oligonucleotides from homologous sequences encoding putative fungal proline aminopeptidases, enabling subsequent cloning of the gene. TePAP was shown to be relatively uninhibited by classical serine peptidase inhibitors and to be sensitive to selected cysteine- and histidine-modifying reagents, yet gene sequence analysis identified the protein as a serine peptidase with an alpha/beta hydrolase fold. Northern analysis indicated that Tepap mRNA levels were regulated by the composition of the growth medium. Highest Tepap transcript levels were observed when the fungus was grown in medium containing glucose and the protein hydrolysate casitone. Interestingly, both the induction profile and substrate preference of this enzyme suggest potential co-operativity between extracellular and intracellular proteolysis in this organism. Gel filtration chromatography suggested that the enzyme exists as a 270 kDa homo-hexamer, whereas most bacterial prolyl aminopeptidases (PAPs) are monomers. Phylogenetic analysis of known PAPs revealed two diverse subfamilies that are distinguishable on the basis of primary and secondary structure and appear to correlate with the subunit composition of the native enzymes. Sequence comparisons revealed that PAPs with key conserved topological features are widespread in bacterial and fungal kingdoms, and this study identified many putative PAP candidates within sequenced genomes. This work represents, to our knowledge, the first detailed biochemical and molecular analysis of an inducible PAP from a eukaryote and the first intracellular peptidase isolated from the thermophilic fungus T. emersonii.
Assuntos
Aminopeptidases/metabolismo , Proteínas Fúngicas/metabolismo , Talaromyces/enzimologia , Sequência de Aminoácidos , Aminopeptidases/genética , Aminopeptidases/isolamento & purificação , Clonagem Molecular , DNA Fúngico/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Temperatura Alta , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Filogenia , Estrutura Secundária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por Substrato , Talaromyces/genéticaRESUMO
The thermophilic filamentous fungus Talaromyces emersonii secretes a variety of hydrolytic enzymes that are of interest for processing of biomass into fuel. Many carbohydrases have been isolated and characterized from this fungus, but no studies had been performed on peptidases. In this study, two acid-acting endopeptidases were isolated and characterized from the culture filtrate of T. emersonii. One of these enzymes was identified as a member of the recently classified glutamic peptidase family and was subsequently named T. emersonii glutamic peptidase 1 (TGP1). The second enzyme was identified as an aspartyl peptidase (PEP1). TGP1 was cloned and sequenced and shown to exhibit 64 and 47% protein identity to peptidases from Aspergillus niger and Scytalidium lignocolum, respectively. Substrate profiling of 16 peptides determined that TGP1 has broad specificity with a preference for large residues in the P1 site, particularly Met, Gln, Phe, Lys, Glu, and small amino acids at P1' such as Ala, Gly, Ser, or Thr. This enzyme efficiently cleaves an internally quenched fluorescent substrate containing the zymogen activation sequence (k(cat)/K(m)=2 x 10(5) m(-1) s(-1)). Maximum hydrolysis occurs at pH 3.4 and 50 degrees C. The reaction is strongly inhibited by a transition state peptide analog, TA1 (K(i)=1.5 nM), as well as a portion of the propeptide sequence, PT1 (K(i)=32 nM). Ex vivo studies show that hyphal extension of T. emersonii in complex media is unaffected by the aspartyl peptidase inhibitor pepstatin but is inhibited by TA1 and PT1. This study provides insight into the functional role of the glutamic peptidase TGP1 for growth of T. emersonii.
Assuntos
Endopeptidases/metabolismo , Endopeptidases/fisiologia , Regulação Fúngica da Expressão Gênica , Glutamina/química , Talaromyces/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Endopeptidases/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Biológicos , Dados de Sequência Molecular , Pepstatinas/química , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TemperaturaRESUMO
The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 A resolution structure of Kaposi's sarcoma-associated herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 A2 of surface are buried in the newly formed S4 pocket when tyrosine binds at this site. The peptide inhibitor also induces a rearrangement of residues that stabilizes the oxyanion hole and the dimer interface. Concomitant with the structural changes, an increase in catalytic efficiency of the enzyme results upon extended substrate binding. A nearly 20-fold increase in kcat/KM results upon extending the peptide substrate from a tetrapeptide to a hexapeptide exclusively due to a KM effect. This suggests that the mechanism by which herpesvirus proteases achieve their high specificity is by using extended substrates to modulate both the structure and activity of the enzyme.
Assuntos
Herpesviridae/enzimologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Sítios de Ligação , Catálise , Cristalografia por Raios X/métodos , Ativação Enzimática , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
The cell death-inducing serine protease granzyme A (GzmA) has a unique disulfide-linked quaternary structure. The structure of human GzmA bound to a tripeptide CMK inhibitor, determined at a resolution of 2.4 A, reveals that the oligomeric state contributes to substrate selection by limiting access to the active site for potential macromolecular substrates and inhibitors. Unlike other serine proteases, tetrapeptide substrate preferences do not correlate well with natural substrate cleavage sequences. This suggests that the context of the cleavage sequence within a macromolecular substrate imposes another level of selection not observed with the peptide substrates. Modeling of inhibitors bound to the GzmA active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft. The crystal structure, along with substrate library profiling and mutagenesis, has allowed us to identify and rationally manipulate key components involved in GzmA substrate specificity.
Assuntos
Biopolímeros/química , Serina Endopeptidases/química , Sequência de Aminoácidos , Biopolímeros/metabolismo , Dimerização , Granzimas , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/metabolismo , Especificidade por SubstratoRESUMO
First identified as a neutrophil granule component, neutrophil gelatinase-associated lipocalin (NGAL; also called human neutrophil lipocalin, 24p3, uterocalin, or neu-related lipocalin) is a member of the lipocalin family of binding proteins. Putative NGAL ligands, including neutrophil chemotactic agents such as N-formylated tripeptides, have all been refuted by recent biochemical and structural results. NGAL has subsequently been implicated in diverse cellular processes, but without a characterized ligand, the molecular basis of these functions remained mysterious. Here we report that NGAL tightly binds bacterial catecholate-type ferric siderophores through a cyclically permuted, hybrid electrostatic/cation-pi interaction and is a potent bacteriostatic agent in iron-limiting conditions. We therefore propose that NGAL participates in the antibacterial iron depletion strategy of the innate immune system.