RESUMO
Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.
Assuntos
Endopeptidases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Análise Serial de Proteínas , Proteômica , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Humanos , Especificidade por Substrato , UbiquitinaçãoRESUMO
As the importance of ubiquitylation in certain disease states becomes increasingly apparent, the enzymes responsible for removal of ubiquitin (Ub) from target proteins, deubiquitylases (DUBs), are becoming attractive targets for drug discovery. For rapid identification of compounds that alter DUB function, in vitro assays must be able to provide statistically robust data over a wide dynamic range of both substrate and enzyme concentrations during high throughput screening (HTS). The most established reagents for HTS are Ubs with a quenched fluorophore conjugated to the C-terminus; however, a luciferase-based strategy for detecting DUB activity (DUB-Glo™, Promega) provides a wider dynamic range than traditional fluorogenic reagents. Unfortunately, this assay requires high enzyme concentrations and lacks specificity for DUBs over other isopeptidases (e.g. desumoylases), as it is based on an aminoluciferin (AML) derivative of a peptide derived from the C-terminus of Ub (Z-RLRGG-). Conjugation of aminoluciferin to a full-length Ub (Ub-AML) yields a substrate that has a wide dynamic range, yet displays detection limits for DUBs 100- to 1000-fold lower than observed with DUB-Glo™. Ub-AML was even a sensitive substrate for DUBs (e.g. JosD1 and USP14) that do not show appreciable activity with DUB-Glo™. Aminoluciferin derivatives of hSUMO2 and NEDD8 were also shown to be sensitive substrates for desumoylases and deneddylases, respectively. Ub/Ubl-AML substrates are amenable to HTS (Z'=0.67) yielding robust signal, and providing an alternative drug discovery platform for Ub/Ubl isopeptidases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.
Assuntos
Endopeptidases/metabolismo , Ensaios de Triagem em Larga Escala , Medições Luminescentes , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Humanos , Proteína NEDD8 , Sensibilidade e Especificidade , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMO
Microarray-based proteomics expanded the information potential of DNA arrays to the level of protein translation and interaction, but so far, not much beyond. Although enzymatic activity from immobilized proteins has been reliably studied using surface plasmon resonance, a microarray of catalytically competent enzymes would facilitate high throughput, parallel study of their function. The ability to localize activity from soluble substrates has frustrated development of such an array. Here, we report the novel use of previously developed, highly specific suicide substrates for three families of enzymes: deubiquitylases, deSUMOylases, and deISGylases. We show specificity of each family to its cognate substrate, and demonstrate utility of the array in a secondary screen of small molecule inhibitors.
Assuntos
Endopeptidases/metabolismo , Ésteres/metabolismo , Análise Serial de Proteínas/métodos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Enzimas Imobilizadas/metabolismo , Humanos , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/antagonistas & inibidores , Coloração e Rotulagem , Especificidade por Substrato/efeitos dos fármacosRESUMO
Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusion of SUMO (small ubiquitin-related modifier) to several test proteins leads to enhanced expression levels in Escherichia coli. In eukaryotic expression systems, however, the SUMO tag could be cleaved by endogenous desumoylase. In order to adapt SUMO-fusion technology to these systems, we have developed an alternative SUMO-derived tag, designated SUMOstar, which is not processed by native SUMO proteases. In the present study, we tested the SUMOstar tag in a baculovirus/insect cell system with several proteins, i.e. mouse UBP43, human tryptase beta II, USP4, USP15, and GFP. Our results demonstrate that fusion to SUMOstar enhanced protein expression levels at least 4-fold compared to either the native or His(6)-tagged proteins. We isolated active SUMOstar tagged UBP43, USP4, USP15, and GFP. Tryptase was active following cleavage with a SUMOstar specific protease. The SUMOstar system will make significant impact in difficult-to-express proteins and especially to those proteins that require the native N-terminal residue for function.
Assuntos
Baculoviridae/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína SUMO-1/genética , Animais , Baculoviridae/metabolismo , Clonagem Molecular , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Camundongos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteína SUMO-1/metabolismo , Spodoptera/citologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/isolamento & purificação , Ubiquitina Tiolesterase/metabolismo , Proteases Específicas de UbiquitinaRESUMO
Human coagulation factor XIa (FXIa), a serine protease activated by site-specific cleavage of factor XI by thrombin, FXIIa, or autoactivation, is a critical enzyme in the amplification phase of the coagulation cascade. To investigate the potential of FXIa inhibitors as safe anticoagulants, a series of potent, selective peptidomimetic inhibitors of FXIa were designed and synthesized. Some of these inhibitors showed low nanomolar FXIa inhibitory activity with >1000-fold FXa selectivity and >100-fold thrombin selectivity. The X-ray structure of one of these inhibitors, 36, demonstrates its unique binding interactions with FXIa. Compound 32 caused a doubling of the activated partial thromboplastin time in human plasma at 2.4 microM and was efficacious in a rat model of venous thrombosis. These data suggest that factor XIa plays a significant role in venous thrombosis and may be a suitable target for the development of antithrombotic therapy.
Assuntos
Anticoagulantes/farmacologia , Desenho de Fármacos , Fator XIa/antagonistas & inibidores , Inibidores do Fator Xa , Fragmentos de Peptídeos/síntese química , Inibidores de Serina Proteinase/farmacologia , Animais , Anticoagulantes/síntese química , Anticoagulantes/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Masculino , Estrutura Molecular , Tempo de Tromboplastina Parcial , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Conformação Proteica , Ratos , Ratos Sprague-Dawley , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/química , Relação Estrutura-Atividade , Trombina/antagonistas & inibidores , Trombose Venosa/tratamento farmacológicoRESUMO
Since the dawn of time, or at least the dawn of recombinant DNA technology (which for many of today's scientists is the same thing), investigators have been cloning and expressing heterologous proteins in a variety of different cells for a variety of different reasons. These range from cell biological studies looking at protein-protein interactions, post-translational modifications, and regulation, to laboratory-scale production in support of biochemical, biophysical, and structural studies, to large scale production of potential biotherapeutics. In parallel, fusion-tag technology has grown-up to facilitate microscale purification (pull-downs), protein visualization (epitope tags), enhanced expression and solubility (protein partners, e.g., GST, MBP, TRX, and SUMO), and generic purification (e.g., His-tags, streptag, and FLAG™-tag). Frequently, these latter two goals are combined in a single fusion partner. In this review, we examine the most commonly used fusion methodologies from the perspective of the ultimate use of the tagged protein. That is, what are the most commonly used fusion partners for pull-downs, for structural studies, for production of active proteins, or for large-scale purification? What are the advantages and limitations of each? This review is not meant to be exhaustive and the approach undoubtedly reflects the experiences and interests of the authors. For the sake of brevity, we have largely ignored epitope tags although they receive wide use in cell biology for immunopreciptation.
Assuntos
Clonagem Molecular/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Animais , Humanos , Imunoprecipitação , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/isolamento & purificação , SolubilidadeRESUMO
Although they are the primary determinants of substrate specificity, few E3-substrate pairs have been positively identified, and few E3's profiled in a proteomic fashion. Praja1 is an E3 implicated in bone development and highly expressed in brain. Although it has been well studied relative to the majority of E3's, little is known concerning the repertoire of proteins it ubiquitylates. We sought to identify high confidence substrates for Praja1 from an unbiased proteomic profile of thousands of human proteins using protein microarrays. We first profiled Praja1 activity against a panel of E2's to identify its optimal partner in vitro. We then ubiquitylated multiple, identical protein arrays and detected putative substrates with reagents that vary in ubiquitin recognition according to the extent of chain formation. Gene ontology clustering identified putative substrates consistent with information previously known about Praja1 function, and provides clues into novel aspects of this enzyme's function.
Assuntos
Análise Serial de Proteínas/métodos , Proteínas/metabolismo , Proteômica/métodos , Ubiquitina-Proteína Ligases/metabolismo , Análise por Conglomerados , Proteínas/classificação , Especificidade por Substrato , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
The preparation of sufficient amounts of high-quality protein samples is the major bottleneck for structural proteomics. The use of recombinant proteins has increased significantly during the past decades. The most commonly used host, Escherichia coli, presents many challenges including protein misfolding, protein degradation, and low solubility. A novel SUMO fusion technology appears to enhance protein expression and solubility ( http://www.lifesensors.com ). Efficient removal of the SUMO tag by SUMO protease in vitro facilitates the generation of target protein with a native N-terminus. In addition to its physiological relevance in eukaryotes, SUMO can be used as a powerful biotechnology tool for enhanced functional protein expression in prokaryotes and eukaryotes.
Assuntos
Escherichia coli/genética , Dobramento de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteína SUMO-1/biossíntese , Animais , Células Eucarióticas/metabolismo , Exopeptidases/genética , Exopeptidases/metabolismo , Humanos , Proteínas Recombinantes de Fusão/genética , Proteína SUMO-1/genéticaRESUMO
Using an alpha-ketothiazole arginine moiety as a key recognition element, a series of small peptidomimetic molecules was designed and synthesized, and their co-crystal structures with factor XIa were studied in an effort to develop smaller, less peptidic inhibitors as antithrombotic agents.
Assuntos
Arginina/química , Arginina/farmacologia , Fator XIa/antagonistas & inibidores , Tiazóis/química , Tiazóis/farmacologia , Amidas/química , Arginina/análogos & derivados , Arginina/síntese química , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Tiazóis/síntese químicaRESUMO
Factor XIa (FXIa) is a serine protease important for initiating the intrinsic pathway of blood coagulation. Protease nexin 2 (PN2) is a Kunitz-type protease inhibitor secreted by activated platelets and a physiologically important inhibitor of FXIa. Inhibition of FXIa by PN2 requires interactions between the two proteins that are confined to the catalytic domain of the enzyme and the Kunitz protease inhibitor (KPI) domain of PN2. Recombinant PN2KPI and a mutant form of the FXI catalytic domain (FXIac) were expressed in yeast, purified to homogeneity, co-crystallized, and the structure of the complex was solved at 2.6 angstroms (Protein Data Bank code 1ZJD). In this complex, PN2KPI has a characteristic, disulfide-stabilized double loop structure that fits into the FXIac active site. To determine the contributions of residues within PN2KPI to its inhibitory activity, selected point mutations in PN2KPI loop 1 11TGPCRAMISR20 and loop 2 34FYGGC38 were tested for their ability to inhibit FXIa. The P1 site mutation R15A completely abolished its ability to inhibit FXIa. IC50 values for the wild type protein and the remaining mutants were as follows: PN2KPI WT, 1.28 nM; P13A, 5.92 nM; M17A, 1.62 nM; S19A, 1.86 nM; R20A, 5.67 nM; F34A, 9.85 nM. The IC50 values for the M17A and S19A mutants were not significantly different from those obtained with wild type PN2KPI. These functional studies and activated partial thromboplastin time analysis validate predictions made from the PN2KPI-FXIac co-crystal structure and implicate PN2KPI residues, in descending order of importance, Arg15, Phe34, Pro13, and Arg20 in FXIa inhibition by PN2KPI.
Assuntos
Proteínas de Transporte/química , Fator XIa/química , Inibidores de Serina Proteinase/química , Proteínas de Transporte Vesicular/química , Alanina/química , Doença de Alzheimer/patologia , Arginina/química , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Análise Mutacional de DNA , Dissulfetos/química , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Cinética , Lisina/química , Modelos Moleculares , Modelos Estatísticos , Mutação , Tempo de Tromboplastina Parcial , Fenilalanina/química , Plasmídeos/metabolismo , Mutação Puntual , Prolina/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Serina/química , Relação Estrutura-Atividade , Tromboplastina/química , Fatores de TempoRESUMO
Activated factor XI (FXIa) is a key enzyme in the amplification phase of the blood-coagulation cascade. Thus, a selective FXIa inhibitor may have lesser bleeding liabilities and provide a safe alternative for antithrombosis therapy to available drugs on the market. In a previous report, the crystal structures of the catalytic domain of FXIa (rhFXI(370-607)) in complex with various ecotin mutants have been described. However, ecotin forms a matrix-like interaction with rhFXI(370-607) and is impossible to displace with small-molecule inhibitors; ecotin crystals are therefore not suitable for iterative structure-based ligand design. In addition, rhFXI(370-607) did not crystallize in the presence of small-molecule ligands. In order to obtain the crystal structure of rhFXI(370-607) with a weak small-molecule ligand, namely benzamidine, several rounds of surface-residue mutation were implemented to promote crystal formation of rhFXI(370-607). A quadruple mutant of rhFXI(370-607) (rhFXI(370-607)-S434A,T475A,C482S,K437A) readily crystallized in the presence of benzamidine. The benzamidine in the preformed crystals was easily exchanged with other FXIa small-molecule inhibitors. These crystals have facilitated the structure-based design of small-molecule FXIa inhibitors.
Assuntos
Benzamidinas/química , Fator XI/antagonistas & inibidores , Fator XIa/química , Fator XIa/genética , Mutação , Sítios de Ligação , Fatores de Coagulação Sanguínea/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , DNA Complementar/metabolismo , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Ligantes , Substâncias Macromoleculares/química , Modelos Moleculares , Mutagênese , Mutagênese Sítio-Dirigida , Peptídeos/química , Pichia/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Serina/químicaRESUMO
Thrombosis can lead to life-threatening conditions such as acute myocardial infarction, pulmonary embolism, and stroke. Although commonly used anti-coagulant drugs, such as low molecular weight heparin and warfarin, are effective, they carry a significant risk of inducing severe bleeding complications, and there is a need for safer drugs. Activated Factor XI (FXIa) is a key enzyme in the amplification phase of the coagulation cascade. Anti-human FXI antibody significantly reduces thrombus growth in a baboon thrombosis model without bleeding problems (Gruber, A., and Hanson, S. R. (2003) Blood 102, 953-955). Therefore, FXIa is a potential target for anti-thrombosis therapy. To determine the structure of FXIa, we derived a recombinant catalytic domain of FXI, consisting of residues 370-607 (rhFXI370-607). Here we report the first crystal structure of rhFXI370-607 in complex with a substitution mutant of ecotin, a panserine protease protein inhibitor secreted by Escherichia coli, to 2.2 A resolution. The presence of ecotin not only assisted in the crystallization of the enzyme but also revealed unique structural features in the active site of FXIa. Subsequently, the sequence from P5 to P2' in ecotin was mutated to the FXIa substrate sequence, and the structures of the rhFXI370-607-ecotin mutant complexes were determined. These structures provide us with an understanding of substrate binding interactions of FXIa, the structural information essential for the structure-based design of FXIa-selective inhibitors.
Assuntos
Cristalografia por Raios X/métodos , Proteínas de Escherichia coli/química , Fator XIa/química , Proteínas Periplásmicas/química , Anticoagulantes/química , Sítios de Ligação , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Dimerização , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Risco , Serina/química , Estereoisomerismo , TromboseRESUMO
The ZAP-70 tyrosine kinase plays a critical role in T cell activation and the immune response and therefore is a logical target for immunomodulatory therapies. Although the crystal structure of the tandem Src homology-2 domains of human ZAP-70 in complex with a peptide derived from the zeta subunit of the T cell receptor has been reported (Hatada, M. H., Lu, X., Laird, E. R., Green, J., Morgenstern, J. P., Lou, M., Marr, C. S., Phillips, T. B., Ram, M. K., Theriault, K., Zoller, M. J., and Karas, J. L. (1995) Nature 377, 32-38), the structure of the kinase domain has been elusive to date. We crystallized and determined the three-dimensional structure of the catalytic subunit of ZAP-70 as a complex with staurosporine to 2.3 A resolution, utilizing an active kinase domain containing residues 327-606 identified by systematic N- and C-terminal truncations. The crystal structure shows that this ZAP-70 kinase domain is in an active-like conformation despite the lack of tyrosine phosphorylation in the activation loop. The unique features of the ATP-binding site, identified by structural and sequence comparison with other kinases, will be useful in the design of ZAP-70-selective inhibitors.