RESUMO
Proteases are an important class of enzymes, whose activity is central to many physiologic and pathologic processes. Detailed knowledge of protease specificity is key to understanding their function. Although many methods have been developed to profile specificities of proteases, few have the diversity and quantitative grasp necessary to fully define specificity of a protease, both in terms of substrate numbers and their catalytic efficiencies. We have developed a concept of "selectome"; the set of substrate amino acid sequences that uniquely represent the specificity of a protease. We applied it to two closely related members of the Matrixin family-MMP-2 and MMP-9 by using substrate phage display coupled with Next Generation Sequencing and information theory-based data analysis. We have also derived a quantitative measure of substrate specificity, which accounts for both the number of substrates and their relative catalytic efficiencies. Using these advances greatly facilitates elucidation of substrate selectivity between closely related members of a protease family. The study also provides insight into the degree to which the catalytic cleft defines substrate recognition, thus providing basis for overcoming two of the major challenges in the field of proteolysis: 1) development of highly selective activity probes for studying proteases with overlapping specificities, and 2) distinguishing targeted proteolysis from bystander proteolytic events.
Assuntos
Modelos Biológicos , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico/genética , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Teoria da Informação , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Modelos Moleculares , Peptídeo Hidrolases/classificação , Biblioteca de Peptídeos , Dobramento de Proteína , Proteólise , Proteômica/métodos , Proteômica/estatística & dados numéricos , Especificidade por Substrato/genética , Especificidade por Substrato/fisiologiaRESUMO
Genomic sequencing and structural genomics produced a vast amount of sequence and structural data, creating an opportunity for structure-function analysis in silico [Radivojac P, et al. (2013) Nat Methods 10(3):221-227]. Unfortunately, only a few large experimental datasets exist to serve as benchmarks for function-related predictions. Furthermore, currently there are no reliable means to predict the extent of functional similarity among proteins. Here, we quantify structure-function relationships among three phylogenetic branches of the matrix metalloproteinase (MMP) family by comparing their cleavage efficiencies toward an extended set of phage peptide substrates that were selected from â¼ 64 million peptide sequences (i.e., a large unbiased representation of substrate space). The observed second-order rate constants [k(obs)] across the substrate space provide a distance measure of functional similarity among the MMPs. These functional distances directly correlate with MMP phylogenetic distance. There is also a remarkable and near-perfect correlation between the MMP substrate preference and sequence identity of 50-57 discontinuous residues surrounding the catalytic groove. We conclude that these residues represent the specificity-determining positions (SDPs) that allowed for the expansion of MMP proteolytic function during evolution. A transmutation of only a few selected SDPs proximal to the bound substrate peptide, and contributing the most to selectivity among the MMPs, is sufficient to enact a global change in the substrate preference of one MMP to that of another, indicating the potential for the rational and focused redesign of cleavage specificity in MMPs.
Assuntos
Domínio Catalítico , Metaloproteinases da Matriz/química , Metaloproteinases da Matriz/metabolismo , Peptídeos/metabolismo , Algoritmos , Sequência de Aminoácidos , Sítios de Ligação/genética , Biocatálise , Humanos , Cinética , Metaloproteinases da Matriz/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Filogenia , Proteólise , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Understanding the active site preferences of an enzyme is critical to the design of effective inhibitors and to gaining insights into its mechanisms of action on substrates. While the subsite specificity of thrombin is understood, it is not clear whether the enzyme prefers individual amino acids at each subsite in isolation or prefers to cleave combinations of amino acids as a motif. To investigate whether preferred peptide motifs for cleavage could be identified for thrombin, we exposed a phage-displayed peptide library to thrombin. The resulting preferentially cleaved substrates were analyzed using the technique of association rule discovery. The results revealed that thrombin selected for amino acid motifs in cleavage sites. The contribution of these hypothetical motifs to substrate cleavage efficiency was further investigated using the B1 IgG-binding domain of streptococcal protein G as a model substrate. Introduction of a P(2)-P(1)' LRS thrombin cleavage sequence within a major loop of the protein led to cleavage of the protein by thrombin, with the cleavage efficiency increasing with the length of the loop. Introduction of further P(3)-P(1) and P(1)-P(1)'-P(3)' amino acid motifs into the loop region yielded greater cleavage efficiencies, suggesting that the susceptibility of a protein substrate to cleavage by thrombin is influenced by these motifs, perhaps because of cooperative effects between subsites closest to the scissile peptide bond.
Assuntos
Modelos Químicos , Trombina/química , Trombina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófago M13/química , Bacteriófago M13/genética , Hidrólise , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Engenharia de Proteínas/métodos , Distribuição Aleatória , Reprodutibilidade dos Testes , Streptococcus , Especificidade por Substrato/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD/L(50) site initiates the MT1-MMP activation, whereas the (108)RRKR(111)/Y(112) cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP.
Assuntos
Furina/metabolismo , Metaloproteinase 14 da Matriz/química , Metaloproteinase 14 da Matriz/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Humanos , Metaloproteinase 14 da Matriz/genética , Inibidores de Metaloproteinases de Matriz , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Estrutura Terciária de ProteínaRESUMO
There is a need to develop inhibitors of mosquito-borne flaviviruses, including WNV (West Nile virus). In the present paper, we describe a novel and efficient recombinant-antibody technology that led us to the isolation of inhibitory high-affinity human antibodies to the active-site region of a viral proteinase. As a proof-of-principal, we have successfully used this technology and the synthetic naive human combinatorial antibody library HuCAL GOLD(R) to isolate selective and potent function-blocking active-site-targeting antibodies to the two-component WNV NS (non-structural protein) 2B-NS3 serine proteinase, the only proteinase encoded by the flaviviral genome. First, we used the wild-type enzyme in antibody screens. Next, the positive antibody clones were counter-screened using an NS2B-NS3 mutant with a single mutation of the catalytically essential active-site histidine residue. The specificity of the antibodies to the active site was confirmed by substrate-cleavage reactions and also by using proteinase mutants with additional single amino-acid substitutions in the active-site region. The selected WNV antibodies did not recognize the structurally similar viral proteinases from Dengue virus type 2 and hepatitis C virus, and human serine proteinases. Because of their high selectivity and affinity, the identified human antibodies are attractive reagents for both further mutagenesis and structure-based optimization and, in addition, for studies of NS2B-NS3 activity. Conceptually, it is likely that the generic technology reported in the present paper will be useful for the generation of active-site-specific antibody probes for multiple enzymes.
Assuntos
Anticorpos Antivirais/isolamento & purificação , Serina Proteases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Vírus do Nilo Ocidental/enzimologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Western Blotting , Domínio Catalítico/genética , Domínio Catalítico/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína , Serina Proteases/química , Serina Proteases/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Vírus do Nilo Ocidental/genéticaRESUMO
Multiple sclerosis (MS) is a disease of the central nervous system with autoimmune etiology. Susceptibility to MS is linked to viral and bacterial infections. Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination. The splice variants of the single MBP gene are expressed in the oligodendrocytes of the central nervous system (classic MBP) and in the immune cells (Golli-MBPs). Our data suggest that persistent inflammation caused by environmental risk factors is a step to MS. We have discovered biochemical evidence suggesting the presence of the inflammatory proteolytic pathway leading to MS. The pathway involves the self-activated furin and PC2 proprotein convertases and membrane type-6 MMP (MT6-MMP/MMP-25) that is activated by furin/PC2. These events are followed by MMP-25 proteolysis of the Golli-MBP isoforms in the immune system cells and stimulation of the specific autoimmune T cell clones. It is likely that the passage of these autoimmune T cell clones through the disrupted blood-brain barrier to the brain and the recognition of neuronal, classic MBP causes inflammation leading to the further up-regulation of the activity of the multiple individual MMPs, the massive cleavage of MBP in the brain, demyelination, and MS. In addition to the cleavage of Golli-MBPs, MMP-25 proteolysis readily inactivates crystallin alphaB that is a suppressor of MS. These data suggest that MMP-25 plays an important role in MS pathology and that MMP-25, especially because of its restricted cell/tissue expression pattern and cell surface/lipid raft localization, is a promising drug target in MS.
Assuntos
Células Apresentadoras de Antígenos/patologia , Encéfalo/imunologia , Inflamação/enzimologia , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Esclerose Múltipla/etiologia , Pró-Proteína Convertases/metabolismo , Células Apresentadoras de Antígenos/metabolismo , Encéfalo/enzimologia , Encéfalo/patologia , Proteínas Ligadas por GPI , Humanos , Inflamação/etiologia , Metaloproteinases da Matriz Associadas à Membrana/genética , Microdomínios da Membrana , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Proteína Básica da Mielina/metabolismo , Transdução de Sinais , Distribuição Tecidual , Regulação para CimaRESUMO
West Nile Virus (WNV) is a potentially deadly mosquito-borne flavivirus which has spread rapidly throughout the world. Currently there is no effective vaccine against flaviviral infections. We previously reported the identification of pyrazole ester derivatives as allosteric inhibitors of WNV NS2B-NS3 proteinase. These compounds degrade rapidly in pH 8 buffer with a half life of 1-2h. We now report the design, synthesis and in vitro evaluation of pyrazole derivatives that are inhibitors of WNV NS2B-NS3 proteinase with greatly improved stability in the assay medium.
Assuntos
Antivirais/química , Pirazóis/química , Inibidores de Serina Proteinase/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Vírus do Nilo Ocidental/efeitos dos fármacos , Regulação Alostérica , Antivirais/síntese química , Antivirais/farmacologia , Desenho de Fármacos , Meia-Vida , Concentração de Íons de Hidrogênio , Hidrólise , Pirazóis/síntese química , Pirazóis/farmacologia , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismo , Febre do Nilo Ocidental/tratamento farmacológicoRESUMO
Bioinformatics-based prediction of protease substrates can help to elucidate regulatory proteolytic pathways that control a broad range of biological processes such as apoptosis and blood coagulation. The majority of published predictive models are position weight matrices (PWM) reflecting specificity of proteases toward target sequence. These models are typically derived from experimental data on positions of hydrolyzed peptide bonds and show a reasonable predictive power. New emerging techniques that not only register the cleavage position but also measure catalytic efficiency of proteolysis are expected to improve the quality of predictions or at least substantially reduce the number of tested substrates required for confident predictions. The main goal of this study was to develop new prediction models based on such data and to estimate the performance of the constructed models. We used data on catalytic efficiency of proteolysis measured for eight major human matrix metalloproteinases to construct predictive models of protease specificity using a variety of regression analysis techniques. The obtained results suggest that efficiency-based (quantitative) models show a comparable performance with conventional PWM-based algorithms, while less training data are required. The derived list of candidate cleavage sites in human secreted proteins may serve as a starting point for experimental analysis.
Assuntos
Algoritmos , Biologia Computacional , Peptídeo Hidrolases , Proteólise , HumanosRESUMO
Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.
Assuntos
Vírus da Dengue/enzimologia , Proteínas não Estruturais Virais/metabolismo , Vírus do Nilo Ocidental/enzimologia , Sítios de Ligação , Mutação , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
West Nile virus (WNV), a member of the Flavividae family, is a mosquito-borne, emerging pathogen. In addition to WNV, the family includes dengue, yellow fever, and Japanese encephalitis viruses, which affect millions of individuals worldwide. Because countermeasures are currently unavailable, flaviviral therapy is urgently required. The flaviviral two-component nonstructural NS2B-NS3 proteinase (protease [pro]) is essential for viral life cycle and, consequently, is a promising drug target. We report here the results of the miniaturization of an NS2B-NS3pro activity assay, followed by high-throughput screening of the National Institutes of Health's 65,000 compound library and identification of novel, uncompetitive inhibitors of WNV NS2B-NS3pro that appear to interfere with the productive interactions of the NS2B cofactor with the NS3pro domain. We anticipate that following structure optimization, the identified probes could form the foundation for the design of novel and specific therapeutics for WNV infection. We also provide the structural basis for additional species-selective allosteric inhibitors of flaviviruses.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Vírus do Nilo Ocidental/enzimologia , Cromatografia Líquida , Interpretação Estatística de Dados , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Indicadores e Reagentes , Ligantes , Espectrometria de Massas , Modelos Moleculares , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/isolamento & purificação , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/efeitos dos fármacosRESUMO
Mosquito-borne WNV (West Nile virus) is an emerging global threat. The NS3 proteinase, which is essential for the proteolytic processing of the viral polyprotein precursor, is a promising drug target. We have isolated and biochemically characterized the recombinant, highly active NS3 proteinase. We have determined that the NS3 proteinase functions in a manner that is distantly similar to furin in cleaving the peptide and protein substrates. We determined that aprotinin and D-arginine-based 9-12-mer peptides are potent inhibitors of WNV NS3 with K(i) values of 26 nM and 1 nM respectively. Consistent with the essential role of NS3 activity in the life cycle of WNV and with the sensitivity of NS3 activity to the D-arginine-based peptides, we showed that nona-D-Arg-NH2 reduced WNV infection in primary neurons. We have also shown that myelin basic protein, a deficiency of which is linked to neurological abnormalities of the brain, is sensitive to NS3 proteolysis in vitro and therefore this protein represents a convenient test substrate for the studies of NS3. A three-dimensional model of WNV NS3 that we created may provide a structural guidance and a rationale for the subsequent design of fine-tuned inhibitors. Overall, our findings represent a foundation for in-depth mechanistic and structural studies as well as for the design of novel and efficient inhibitors of WNV NS3.
Assuntos
Arginina/análise , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Vírus do Nilo Ocidental/enzimologia , Sequência de Aminoácidos , Animais , Arginina/química , Células Cultivadas , Sequência Conservada , Furina/química , Furina/metabolismo , Camundongos , Dados de Sequência Molecular , Bainha de Mielina/metabolismo , Neurônios/metabolismo , Peptídeos/química , Peptídeos/farmacologia , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , RNA Helicases/isolamento & purificação , RNA Helicases/metabolismo , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/genética , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Serpinas/farmacologia , Especificidade por Substrato , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação , Vírus do Nilo Ocidental/genéticaRESUMO
CleavPredict (http://cleavpredict.sanfordburnham.org) is a Web server for substrate cleavage prediction for matrix metalloproteinases (MMPs). It is intended as a computational platform aiding the scientific community in reasoning about proteolytic events. CleavPredict offers in silico prediction of cleavage sites specific for 11 human MMPs. The prediction method employs the MMP specific position weight matrices (PWMs) derived from statistical analysis of high-throughput phage display experimental results. To augment the substrate cleavage prediction process, CleavPredict provides information about the structural features of potential cleavage sites that influence proteolysis. These include: secondary structure, disordered regions, transmembrane domains, and solvent accessibility. The server also provides information about subcellular location, co-localization, and co-expression of proteinase and potential substrates, along with experimentally determined positions of single nucleotide polymorphism (SNP), and posttranslational modification (PTM) sites in substrates. All this information will provide the user with perspectives in reasoning about proteolytic events. CleavPredict is freely accessible, and there is no login required.
Assuntos
Metaloproteinases da Matriz/metabolismo , Software , Navegador , Algoritmos , Bases de Dados Genéticas , Proteólise , Curva ROC , Reprodutibilidade dos TestesRESUMO
Viruses of the genus Flavivirus are responsible for significant human disease and mortality. The N-terminal domain of the flaviviral nonstructural (NS)3 protein codes for the serine, chymotrypsin-fold proteinase (NS3pro). The presence of the nonstructural (NS)2B cofactor, which is encoded by the upstream gene in the flaviviral genome, is necessary for NS3pro to exhibit its proteolytic activity. The two-component NS2B-NS3pro functional activity is essential for the viral polyprotein processing and replication. Both the structure and the function of NS2B-NS3pro are conserved in the Flavivirus family. Because of its essential function in the posttranslational processing of the viral polyprotein precursor, NS2B-NS3pro is a promising target for anti-flavivirus drugs. To identify selective inhibitors with the reduced cross-reactivity and off-target effects, we focused our strategy on the allosteric inhibitors capable of targeting the NS2B-NS3pro interface rather than the NS3pro active site. Using virtual ligand screening of the diverse, â¼275,000-compound library and the catalytic domain of the two-component West Nile virus (WNV) NS2B-NS3pro as a receptor, we identified a limited subset of the novel inhibitory scaffolds. Several of the discovered compounds performed as allosteric inhibitors and exhibited a nanomolar range potency in the in vitro cleavage assays. The inhibitors were also potent in cell-based assays employing the sub-genomic, luciferase-tagged WNV and Dengue viral replicons. The selectivity of the inhibitors was confirmed using the in vitro cleavage assays with furin, a human serine proteinase, the substrate preferences of which are similar to those of WNV NS2B-NS3pro. Conceptually, the similar in silico drug discovery strategy may be readily employed for the identification of inhibitors of other flaviviruses.
Assuntos
Bases de Dados de Proteínas , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Modelos Químicos , Mapeamento de Interação de Proteínas/métodos , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Sítios de Ligação , Simulação por Computador , Ligantes , Ligação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/química , Serina Endopeptidases/químicaRESUMO
BACKGROUND: Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination leading to autoimmune multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The classic MBP isoforms are predominantly expressed in the oligodendrocytes of the CNS. The splice variants of the single MBP gene (Golli-MBP BG21 and J37) are widely expressed in the neurons and also in the immune cells. The relative contribution of the individual MMPs to the MBP cleavage is not known. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate which MMP plays the primary role in cleaving MBP, we determined the efficiency of MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP and MT6-MMP in the cleavage of the MBP, BG21 and J37 isoforms in the in vitro cleavage reactions followed by mass-spectroscopy analysis of the cleavage fragments. As a result, we identified the MMP cleavage sites and the sequence of the resulting fragments. We determined that MBP, BG21 and J37 are highly sensitive to redundant MMP proteolysis. MT6-MMP (initially called leukolysin), however, was superior over all of the other MMPs in cleaving the MBP isoforms. Using the mixed lymphocyte culture assay, we demonstrated that MT6-MMP proteolysis of the MBP isoforms readily generated, with a near quantitative yield, the immunogenic N-terminal 1-15 MBP peptide. This peptide selectively stimulated the proliferation of the PGPR7.5 T cell clone isolated from mice with EAE and specific for the 1-15 MBP fragment presented in the MHC H-2(U) context. CONCLUSIONS/SIGNIFICANCE: In sum, our biochemical observations led us to hypothesize that MT6-MMP, which is activated by furin and associated with the lipid rafts, plays an important role in MS pathology and that MT6-MMP is a novel and promising drug target in MS especially when compared with other individual MMPs.
Assuntos
Metaloproteinases da Matriz/metabolismo , Esclerose Múltipla/imunologia , Proteína Básica da Mielina/metabolismo , Peptídeos/imunologia , Isoformas de Proteínas/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Humanos , Ativação Linfocitária , Metaloproteinases da Matriz/genética , Metalotioneína 3 , Camundongos , Dados de Sequência Molecular , Esclerose Múltipla/genética , Proteína Básica da Mielina/genética , Peptídeos/genética , Isoformas de Proteínas/genética , Alinhamento de Sequência , Linfócitos T/imunologiaRESUMO
Similar to many flavivirus types including Dengue and yellow fever viruses, the nonstructural NS3 multifunctional protein of West Nile virus (WNV) with an N-terminal serine proteinase domain and an RNA triphosphatase, an NTPase domain, and an RNA helicase in the C-terminal domain is implicated in both polyprotein processing and RNA replication and is therefore a promising drug target. To exhibit its proteolytic activity, NS3 proteinase requires the presence of the cofactor encoded by the upstream NS2B sequence. During our detailed investigation of the biology of the WNV helicase, we characterized the ATPase and RNA/DNA unwinding activities of the full-length NS2B-NS3 proteinase-helicase protein as well as the individual NS3 helicase domain lacking both the NS2B cofactor and the NS3 proteinase sequence and the individual NS3 proteinase-helicase lacking only the NS2B cofactor. We determined that both the NS3 helicase and NS3 proteinase-helicase constructs are capable of unwinding both the DNA and the RNA templates. In contrast, the full-length NS2B-NS3 proteinase-helicase unwinds only the RNA templates, whereas its DNA unwinding activity is severely repressed. Our data suggest that the productive, catalytically competent fold of the NS2B-NS3 proteinase moiety represents an essential component of the RNA-DNA substrate selectivity mechanism in WNV and, possibly, in other flaviviruses. Based on our data, we hypothesize that the mechanism we have identified plays a role yet to be determined in WNV replication occurring both within the virus-induced membrane-bound replication complexes in the host cytoplasm and in the nuclei of infected cells.
Assuntos
DNA/química , Proteínas não Estruturais Virais/química , Vírus do Nilo Ocidental/enzimologia , Sequência de Aminoácidos , Membrana Celular/virologia , Clonagem Molecular , Citoplasma/metabolismo , Cinética , Conformação Molecular , Dados de Sequência Molecular , Ligação Proteica , Desnaturação Proteica , RNA/química , RNA Helicases/química , Proteínas Recombinantes/química , Serina Endopeptidases/químicaRESUMO
West Nile virus (WNV) is an emerging mosquito-borne flavivirus that causes neuronal damage in the absence of treatment. In many flaviviruses, including WNV, the NS2B cofactor promotes the productive folding and the functional activity of the two-component NS3 (pro)teinase. Based on an analysis of the NS2B-NS3pro structure, we hypothesized that the G(22) residue and the negatively charged patch D(32)DD(34) of NS2B were part of an important configuration required for NS2B-NS3pro activity. Our experimental data confirmed that G(22) and D(32)DD(34) substitution for S and AAA, respectively, inactivated NS2B-NS3pro. An additional D42G mutant, which we designed as a control, had no dramatic effect on either the catalytic activity or self-proteolysis of NS2B-NS3pro. Because of the significant level of homology in flaviviral NS2B-NS3pro, our results will be useful for the development of specific allosteric inhibitors designed to interfere with the productive interactions of NS2B with NS3pro.
Assuntos
Regulação Viral da Expressão Gênica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Vírus do Nilo Ocidental/enzimologia , Sequência de Aminoácidos , Dicroísmo Circular , Cristalização , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Alinhamento de Sequência , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Relação Estrutura-Atividade , Vírus do Nilo Ocidental/genéticaRESUMO
We present the data and the technology, a combination of which allows us to determine the identity of proprotein convertases (PCs) related to the processing of specific protein targets including viral and bacterial pathogens. Our results, which support and extend the data of other laboratories, are required for the design of effective inhibitors of PCs because, in general, an inhibitor design starts with a specific substrate. Seven proteinases of the human PC family cleave the multibasic motifs R-X-(R/K/X)-R downward arrow and, as a result, transform proproteins, including those from pathogens, into biologically active proteins and peptides. The precise cleavage preferences of PCs have not been known in sufficient detail; hence we were unable to determine the relative importance of the individual PCs in infectious diseases, thus making the design of specific inhibitors exceedingly difficult. To determine the cleavage preferences of PCs in more detail, we evaluated the relative efficiency of furin, PC2, PC4, PC5/6, PC7, and PACE4 in cleaving over 100 decapeptide sequences representing the R-X-(R/K/X)-R downward arrow motifs of human, bacterial, and viral proteins. Our computer analysis of the data and the follow-on cleavage analysis of the selected full-length proteins corroborated our initial results thus allowing us to determine the cleavage preferences of the PCs and to suggest which PCs are promising drug targets in infectious diseases. Our results also suggest that pathogens, including anthrax PA83 and the avian influenza A H5N1 (bird flu) hemagglutinin precursor, evolved to be as sensitive to PC proteolysis as the most sensitive normal human proteins.
Assuntos
Furina/química , Pró-Proteína Convertases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Biologia Computacional/métodos , Humanos , Espectrometria de Massas/métodos , Modelos Biológicos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Peptídeos/química , Pró-Proteína Convertases/fisiologia , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Especificidade por SubstratoRESUMO
Regulated proteolysis of the polyprotein precursor of West Nile virus (WNV) by the essential NS2B-NS3(pro)tease, a promising drug target for WNV inhibitors, is required for the propagation of infectious virions. Structural and drug design studies, however, require pilot-scale quantities of a pure and catalytically active WNV protease that is resistant to self-proteolysis. Autolytic cleavage at the NS2B-NS3 boundary leads to individual, non-covalently associated, NS2B and NS3 domains, together with residual amounts of the intact NS2B-NS3, in the NS2B-NS3pro samples. We modified the cleavage site sequence of the NS2B-NS3 junction region and then developed expression and purification procedures to prepare a covalently linked, single-chain, NS2B-NS3pro K48A mutant construct. This construct exhibits high stability and functional activity and is thus well suited for the follow-up purification and structural and drug design studies.
Assuntos
Endopeptidases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Vírus do Nilo Ocidental/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Expressão Gênica , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação , Vírus do Nilo Ocidental/genéticaRESUMO
Membrane type-1 matrix metalloproteinase (MT1-MMP) exerts its enhanced activity in multiple cancer types. Understanding the activation process of MT1-MMP is essential for designing novel and effective cancer therapies. Like all of the other MMPs, MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its inhibitory prodomain. Proteolytic processing of the prodomain transforms the zymogen into a catalytically active enzyme. A sequential, two-step activation process is normally required for MMPs. Our in silico modeling suggests that the prodomain of MT1-MMP exhibits a conserved three helix-bundled structure and a "bait" loop region linking helixes 1 and 2. We hypothesized and then confirmed that in addition to furin cleavage there is also a cleavage at the bait region in the activation process of MT1-MMP. A two-step sequential activation of MT1-MMP is likely to include the MMP-dependent cleavage at either P47GD downward arrowL50 or P58QS downward arrowL61 or at both sites of the bait region. This event results in the activation intermediate. The activation process is then completed by a proprotein convertase cleaving the inhibitory prodomain at the R108RKR111 downward arrowY112 site, where Tyr112 is the N-terminal residue of the mature MT1-MMP enzyme. Our findings suggest that the most efficient activation results from a two-step mechanism that eventually is required for the degradation of the inhibitory prodomain and the release of the activated, mature MT1-MMP enzyme. These findings shed more light on the functional role of the inhibitory prodomain and on the proteolytic control of MT1-MMP activation, a crucial process that may be differentially regulated in normal and cancer cells.