RESUMO
The ubiquitin-proteasome system (UPS) plays an essential role in a wide variety of cell regulatory signaling pathways. The clinical effectiveness of the proteasome inhibitor Velcade in the treatment of several human cancers underscores the importance of the UPS as a novel target area for pharmaceutical intervention. E3 ubiquitin ligases are key enzyme complexes that regulate and determine the ubiquitination of specific substrates, whose abnormal regulation has been implicated in multiple disease phenotypes. Targeting a selective E3 ligase may allow specific manipulation of distinct pathways and eventually lead to a better therapeutic index with reduced nonspecific side effects. Here, we aim to discuss the challenges of interfering with small molecules in this target class, as well as current strategies and progress in E3 ligase drug discovery.
Assuntos
Descoberta de Drogas , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Domínios RING Finger , Especificidade por Substrato , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
As contemporary "genomics" steadily reveals an increasing number of novel gene sequences, the need for efficient methodologies to functionally characterize these genes in vivo increases significantly. Reliable coupling of target gene expression to a variety of surrogate reporter functions is critical to properly assay novel gene function in complex cell populations. Ideally, independent target and reporter proteins would be derived from a single open reading frame creating a stoichiometric relationship without obscuring subcellular localization. We report here effective strategies for assaying gene function through the stable production of chimeric polyproteins, processed intracellularly by inclusion of an intervening 19-amino-acid sequence from the 2A region of the Foot and Mouth Disease virus. Using drug-resistance and flow cytometry-based assay systems, we demonstrate that diverse protein functions are effectively delivered to various cell types by retroviral constructs as single 2A-cleaved polyproteins. For example, cells infected with a retrovirus encoding a nuclear cell cycle regulator, linked via the 2A-motif to a marker membrane protein, showed a direct correlation between cell cycle arrest and surface marker level. This demonstrates the utility of this methodology for stable and stoichiometric delivery of distinctly localized protein functionalities. In particular, the ability to exploit multiple cellular functions will serve to accelerate the functional characterization of gene products and facilitate novel gene therapy approaches.
Assuntos
Técnicas Genéticas , Terapia Genética/métodos , Retroviridae/genética , Motivos de Aminoácidos , Western Blotting , Núcleo Celular/metabolismo , Separação Celular , Farmacorresistência Viral , Citometria de Fluxo , Vírus da Febre Aftosa/genética , Genes Virais , Proteínas de Fluorescência Verde , Humanos , Células Jurkat , Proteínas Luminescentes/metabolismo , Fases de Leitura Aberta , Proteínas/genética , Frações Subcelulares , Proteínas Virais/genéticaRESUMO
We used a N-biotinylated peptide analog of the C-terminal domain of the tumor suppressor protein, p21cip1/waf1 to elucidate peptide/protein interacting partners. The C-terminal domain of p21cip1/waf1 protein spanning 141-160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell-cycle control. This C-terminal 20-mer efficiently extracts PCNA in the presence of a variety of N- or C-terminally attached affinity tags. Using difference silver stained 2D gels combined with in-gel tryptic digests, we identified the difference spots using MALDI-TOF mass spectrometry-based peptide mass fingerprinting followed by a database search using PROFOUND against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor-1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion-trap LC-MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using SEQUEST confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90alpha, HSP40 and T-complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21cip1/waf1 protein. The use of N-biotinylated peptide derived from the C-terminal domain of p21cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.
Assuntos
Ciclinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteômica , Sequência de Aminoácidos , Western Blotting , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/química , Eletroforese em Gel Bidimensional , Substâncias Macromoleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Biblioteca de Peptídeos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Nucleares , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Domínios de Homologia de src , Sequência de Aminoácidos , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Complementar , Proteínas de Ligação a DNA/genética , Humanos , Células Jurkat , Lectinas Tipo C , Dados de Sequência Molecular , Ácido Mirístico/metabolismo , Fatores de Transcrição NFATC , Fosforilação , Regiões Promotoras Genéticas , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/imunologia , Homologia de Sequência de Aminoácidos , Tetraciclina/farmacologia , Transativadores , Fatores de Transcrição/genética , Ativação Transcricional , Tirosina/metabolismoRESUMO
BACKGROUND: Mammalian cell screens of peptide libraries for changes in cellular phenotype may identify novel functional peptides and their cognate binding partners, and allow identification of signal transduction network members or proteins important in disease processes. RESULTS: Green fluorescent protein (GFP) peptide libraries with different structural biases were tested by retroviral expression in A549 carcinoma cells, HUVEC and other cell types. Three different loop replacement libraries, containing 12 or 18 random residues, were compatible with enhanced GFP (EGFP) folding, as was a C-terminally fused random 20-mer library. Library concentrations in A549 cells ranged from ca. 1 to 54 microM. Replacement of loop 3 with known nuclear localization sequence (NLS) peptides, but not with inactive mutants, directed EGFP to the nucleus. Microscopy-based screens of three different libraries for non-uniform localization revealed novel NLS peptides, novel variants of a peroxisomal localization motif, a variety of partial NLS peptides, peptides localized to the nucleolus, and nuclear-excluded peptides. CONCLUSIONS: Peptides can be presented by EGFP in conformations that can functionally interact with cellular constituents in mammalian cells. A phenotypic screen resulting in the discovery of novel localization peptides that were not cell type-specific suggests that this methodology may be applied to other screens in cells derived from diseased organisms, and illustrates the use of intracellular combinatorial peptide chemistry in mammalian cells.
Assuntos
Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Biblioteca de Peptídeos , Animais , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Mamíferos/metabolismo , Programas de Rastreamento/métodos , Mutagênese Insercional/genética , Sinais de Localização Nuclear/genética , Peroxissomos/química , Peroxissomos/metabolismo , Fenótipo , Dobramento de Proteína , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade , Células Tumorais Cultivadas/metabolismoRESUMO
Proliferating cell nuclear antigen (PCNA) is an essential protein in both DNA replication and DNA damage repair. A novel 15 kD protein, p15(PAF), was identified as a PCNA-associated factor in a yeast two-hybrid screen using PCNA as the bait. p15(PAF) is localized primarily in the nucleus. p15(PAF) shares the conserved PCNA binding motif with several other PCNA binding proteins including CDK inhibitor p21. Overexpression of p15(PAF) competes with p21-PCNA binding. Mutation of this motif in p15(PAF) abolished its PCNA-binding activity. Notably, p15(PAF) expression in several types of tumor tissues was significantly increased, especially in esophageal tumors. Like PCNA, p15(PAF) may possess prognostic significance in a broad array of human cancers.
Assuntos
Proteínas de Transporte/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , Proteínas de Transporte/isolamento & purificação , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Proteínas de Ligação a DNA , Feminino , Humanos , Dados de Sequência Molecular , Ligação Proteica , RNA Mensageiro/isolamento & purificação , RNA Neoplásico/isolamento & purificação , Distribuição Tecidual , Técnicas do Sistema de Duplo-HíbridoRESUMO
We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.
Assuntos
Genes MDR/genética , Paclitaxel/farmacologia , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Motivos de Aminoácidos , Sequência de Aminoácidos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Dominantes/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Subunidades Proteicas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
Retrovirally mediated functional genomics enables identification of physiologically relevant cellular therapeutic targets. Unique properties of retroviruses make them ideal tools for the introduction of large and diverse libraries of potential genetic effectors to a variety of cell types. The identification and recovery of intracellular library elements responsible for altered disease responses establishes a direct basis for pharmaceutical development. Recent innovations in retroviral infection efficiency and expression control have broadened application of the methodology to include libraries of mutagenized cDNAs, peptides and ribozyme genetic effectors.
Assuntos
Indústria Farmacêutica/métodos , Genoma , Retroviridae/genética , Animais , DNA Complementar/metabolismo , Biblioteca Gênica , Técnicas de Transferência de Genes , Genes Reporter , Humanos , Camundongos , Modelos Biológicos , Oligonucleotídeos Antissenso/farmacologia , RNA Catalítico/químicaRESUMO
Green fluorescent protein (GFP) is useful as an intracellular scaffold for the display of random peptide libraries in yeast. GFPs with a different sequence from Aequorea victoria have recently been identified from Renilla mulleri and Ptilosarcus gurneyi. To examine these proteins as intracellular scaffolds for peptide display in human cells, we have determined the expression level of retrovirally delivered human codon-optimized versions in Jurkat-E acute lymphoblastic leukemia cells using fluorescence activated cell sorting and Western blots. Each wild type protein is expressed at 40% higher levels than A. victoria mutants optimized for maximum fluorescence. We have compared the secondary structure and stability of these GFPs with A. victoria GFP using circular dichroism (CD). All three GFPs essentially showed a perfect beta-strand conformation and their melting temperatures (Tm) are very similar, giving an experimental evidence of a similar overall structure. Folded Renilla GFP allows display of an influenza hemagglutinin epitope tag in several internal insertion sites, including one which is not permissive for such display in Aequorea GFP, giving greater flexibility in peptide display options. To test display of a functional peptide, we show that the SV-40 derived nuclear localization sequence PPKKKRKV, when inserted into two different potential loops, results in the complete localization of Renilla GFP to the nucleus of human A549 cells.
Assuntos
Cnidários/química , Proteínas Luminescentes/genética , Peptídeos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Dicroísmo Circular , Clonagem Molecular , Primers do DNA , Mapeamento de Epitopos , Proteínas de Fluorescência Verde , Humanos , Dados de Sequência Molecular , Peptídeos/química , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.
Assuntos
Ciclinas/genética , Fase G1/fisiologia , Fase G2/fisiologia , Terapia Genética/métodos , Antígeno Nuclear de Célula em Proliferação/genética , Retroviridae/genética , Motivos de Aminoácidos , Animais , Western Blotting , Medula Óssea/fisiologia , Cromatografia Líquida de Alta Pressão , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Primers do DNA/química , Inibidores Enzimáticos/farmacologia , Exocitose/fisiologia , Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde , Células HeLa/metabolismo , Células HeLa/virologia , Humanos , Células Jurkat , Proteínas Luminescentes/biossíntese , Espectrometria de Massas , Mastócitos/efeitos dos fármacos , Mastócitos/fisiologia , Mastócitos/virologia , Microscopia de Fluorescência , Testes de Precipitina , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transdução GenéticaRESUMO
BACKGROUND: Few examples exist of peptides of < 35 residues that form a stable tertiary structure without disulfide bonds. A method for stabilization and noncovalent constraint of relatively short peptides may allow the construction and use of intracellular peptide libraries containing protein minidomains. RESULTS: We have examined a novel method for the noncovalent constraint of peptides by attaching the peptide EFLIVKS (single-letter amino acid code), which forms dimers, to the amino and carboxyl termini of different peptide inserts. An 18 residue random coil taken from the inhibitor loop of barley chymotrypsin inhibitor 2 was inserted between the peptides to produce a 32-mer minidomain that is attacked only slowly by elastase, has numerous slowly exchanging protons, contains a high beta-structure content and has a T(m) above 37 degrees C. A point mutation disrupting the hydrophobic interior in both dimerizing peptides causes a loss of all slowly exchanging protons and of secondary structure. Adding specific charged residues to each terminus substantially increased the T(m), as did point mutants designed to add interdimerizer ion pairs. Three flexible epitope tag inserts and a nonamer insert do not appear to be folded in a stable structure by EFLIVKS. The properties of two peptides selected for expression in HeLa cells suggest they do form a stable tertiary structure. CONCLUSIONS: Attaching short dimerizing peptides to both the amino and carboxyl termini of several 18-mer peptides appears to create stable monomeric tertiary structures. Mutations in the dimerizers can either destabilize or significantly stabilize a standard 18-mer insert. Dimerizing peptides flanking random insert sequences could be used as a strategy to generate heterogeneous peptide libraries with both extended and folded members.
Assuntos
Peptídeos/química , Peptídeos/metabolismo , Proteínas de Plantas/química , Engenharia de Proteínas , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Cromatografia em Gel , Dicroísmo Circular , Deutério/metabolismo , Dimerização , Inibidores Enzimáticos , Dados de Sequência Molecular , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/metabolismo , Biblioteca de Peptídeos , Peptídeos/síntese química , Peptídeos/isolamento & purificação , Proteínas de Plantas/metabolismo , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Retroviral vectors are ideally suited to the study of gene function, allowing efficient, stable expression. Many biological systems (e.g., cell cycle, apoptosis) require the use of regulated expression systems. We therefore developed a regulated retroviral vector system, TRA99, based on a tetracycline transactivator-dependent LTR, where the MMLV enhancer was replaced with a tetracycline-response element. Using fluorescence-activated flow cytometric analysis of a destabilized green fluorescent protein to monitor expression levels, we optimized the minimal promoter configuration with respect to both activated and repressed transcription. The TRA99 vectors demonstrate regulated expression with activated levels comparable to those of standard retroviral vectors and repressed levels indistinguishable from background. This was achieved without using an internal promoter cassette, thus retaining the cis-packaging elements requisite for helper-mediated transfer.
Assuntos
Regulação Viral da Expressão Gênica/genética , Vetores Genéticos/genética , Vírus da Leucemia Murina/genética , Regiões Promotoras Genéticas/genética , Sequências Repetidas Terminais/genética , Animais , Sequência de Bases , Linhagem Celular , Doxorrubicina/farmacologia , Elementos Facilitadores Genéticos/genética , Citometria de Fluxo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Genes Reporter/genética , Proteínas de Fluorescência Verde , Humanos , Cinética , Vírus da Leucemia Murina/fisiologia , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Mutagênese Insercional/genética , Elementos de Resposta/genética , Tetraciclina/farmacologia , Transdução Genética/genética , Células Tumorais Cultivadas , Montagem de VírusRESUMO
GTPases regulate a myriad of cellular functions including signal transduction, cytoskeletal organization and membrane trafficking. Rab GTPases act to coordinate the membrane dynamics of cells by organizing and regulating the activity of effector proteins important in vesicle trafficking. Rab37 is a novel Rab GTPase specifically expressed in the MC-9 mast cell line and bone marrow mast cells. Rab37 is 74% identical to Rab26 and 47% identical to Rab8, a GTPase important in Golgi to plasma membrane vesicle trafficking in mammalian cells. When green fluorescent protein tagged Rab37 is expressed in bone marrow mast cells, the secretory granules are labeled. These data suggest that Rab37 may play an important role in mast cell degranulation making this protein a potentially important target for therapeutic intervention in the treatment of allergy.
Assuntos
Grânulos Citoplasmáticos/enzimologia , GTP Fosfo-Hidrolases/metabolismo , Mastócitos/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Transformada , DNA Complementar , GTP Fosfo-Hidrolases/genética , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Germinal center kinases (GCKs) compose a subgroup of the Ste20 family of kinases. Here we describe the cloning and characterization of a novel GCK family kinase, Traf2- and Nck-interacting kinase (TNIK) that interacts with both Traf2 and Nck. TNIK encodes a polypeptide of 1360 amino acids with eight spliced isoforms. It has 90% amino acid identity to the Nck-interacting kinase in both the N-terminal kinase domain and the C-terminal germinal center kinase homology region. The homology drops to 53% in the intermediate region. TNIK specifically activates the c-Jun N-terminal kinase pathway when transfected into Phoenix-A cells (derivatives of 293 cells), similar to many GCKs. However, in contrast to other GCKs, this activation is mediated solely by the GCK homology region of TNIK. In addition, in Phoenix-A, NIH-3T3, and Hela cells, overexpression of wild type TNIK, but not the kinase mutant form of TNIK, results in the disruption of F-actin structure and the inhibition of cell spreading. Furthermore, TNIK can phosphorylate Gelsolin in vitro. This is the first time that a GCK family kinase is shown to be potentially involved in the regulation of cytoskeleton.
Assuntos
Citoesqueleto/fisiologia , Centro Germinativo/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células 3T3 , Proteínas Adaptadoras de Transdução de Sinal , Processamento Alternativo , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Linhagem Celular , Clonagem Molecular , Biblioteca Gênica , Quinases do Centro Germinativo , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Dados de Sequência Molecular , Proteínas Oncogênicas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fator 2 Associado a Receptor de TNF , Transfecção , Domínios de Homologia de srcRESUMO
BACKGROUND: Mast cells are primary mediators of allergic inflammation. Antigen-mediated crosslinking of their cell surface immunoglobulin E (IgE) receptors results in degranulation and the release of proinflammatory mediators including histamine, tumor necrosis factor-alpha, and leukotrienes. METHODS: Mast cells were stimulated to degranulate by using either IgE crosslinking or ionophore treatment. Exogenously added annexin-V was used to stain exocytosing granules, and the extent of binding was measured flow cytometrically. Release of the enzyme beta-hexosaminidase was used for population-based measurements of degranulation. Two known inhibitors of degranulation, the phosphatidylinositol 3 kinase inhibitor wortmannin and overexpression of a mutant rab3d protein, were used as controls to validate the annexin-V binding assay. RESULTS: Annexin-V specifically bound to mast cell granules exposed after stimulation in proportion to the extent of degranulation. Annexin-V binding was calcium dependent and was blocked by phosphatidylserine containing liposomes, consistent with specific binding to this membrane lipid. Visualization of annexin-V staining showed granular cell surface patches that colocalized with the exocytic granule marker VAMP-green fluorescent protein (GFP). Wortmannin inhibited both annexin-V binding and beta-hexosaminidase release in RBL-2H3 cells, as did the expression of a dominant negative rab3d mutant protein. CONCLUSIONS: The annexin-V binding assay represents a powerful new flow cytometric method to monitor mast cell degranulation for functional analysis.
Assuntos
Anexina A5 , Degranulação Celular , Citometria de Fluxo/métodos , Mastócitos/fisiologia , Androstadienos/farmacologia , Animais , Anexina A5/metabolismo , Cálcio/farmacologia , Degranulação Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Fluorescência Verde , Lipossomos/farmacologia , Proteínas Luminescentes , Camundongos , Microscopia de Fluorescência , Fosfatidilserinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Wortmanina , beta-N-Acetil-Hexosaminidases/análise , Proteínas rab3 de Ligação ao GTPRESUMO
The tumor necrosis factor receptor 1 (TNFR1) and the Fas receptor recruit complexes formed by the interactions between RIP kinase, TRADD, FADD and RAIDD - adaptor proteins that contain death domains - which in turn recruit other proteins to initiate signaling [1][2][3][4][5]. To identify proteins associated with the TNF signaling pathway, we performed a yeast two-hybrid interaction screen using RIP as bait. We isolated a kinase, RIP3, which shares homology with the kinase domain of RIP and RIP2 (also known as Rick or CARDIAK). RIP3 could be co-immunoprecipitated with RIP, TRAF2 and TNFR1 in mammalian cells. The carboxy-terminal domain of RIP3, like that of RIP, could activate the transcription factor NFkappaB and induce apoptosis when expressed in mammalian cells. Interestingly, this region shares no significant sequence homology to the death domain of RIP, the caspase-recruiting domain (CARD) of RIP2 [6][7][8] or any other apoptosis-inducing domain. As with RIP and RIP2, the kinase domain of RIP3 was not required for either NFkappaB activation or apoptosis induction. Overexpression of a dominant-negative mutant of RIP3 strongly inhibited the caspase activation but not the NFkappaB activation induced by TNFalpha. Therefore, RIP3 appears to function as an intermediary in TNFalpha-induced apoptosis.
Assuntos
Apoptose , NF-kappa B/metabolismo , Proteínas Quinases/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Proteína Ligante Fas , Glicoproteínas de Membrana/farmacologia , Ligação Proteica , Proteínas/metabolismo , Fator 2 Associado a Receptor de TNF , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Fluorescence energy transfer (FRET) can be generated when green fluorescent protein (GFP) and blue fluorescent protein (BFP) are covalently linked together by a short peptide. Cleavage of this linkage by protease completely eliminates FRET effect. Caspase-3 (CPP32) is an important cellular protease activated during programmed cell death. An 18 amino acid peptide containing CPP32 recognition sequence, DEVD, was used to link GFP and BFP together. CPP32 activation can be monitored by FRET assay during the apoptosis process.
Assuntos
Apoptose , Caspases , Cisteína Endopeptidases/biossíntese , Transfecção/métodos , Sequência de Aminoácidos , Caspase 3 , Transferência de Energia , Precursores Enzimáticos/biossíntese , Citometria de Fluxo/métodos , Biblioteca Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/biossíntese , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossínteseRESUMO
Proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin-like enzymes. PAR-2 is highly expressed by small intestinal enterocytes where it is activated by luminal trypsin. The location, mechanism of activation, and biological functions of PAR-2 in the colon, however, are unknown. We localized PAR-2 to the muscularis externa of the rat colon by immunofluorescence. Myocytes in primary culture also expressed PAR-2, assessed by immunofluorescence and RT-PCR. Trypsin, SLIGRL-NH2 (corresponding to the PAR-2 tethered ligand), mast cell tryptase, and a filtrate of degranulated mast cells stimulated a prompt increase in [Ca2+]i in myocytes. The response to tryptase and the mast cell filtrate was inhibited by the tryptase inhibitor BABIM, and abolished by desensitization of PAR-2 with trypsin. PAR-2 activation inhibited the amplitude of rhythmic contractions of strips of rat colon. This response was unaffected by indomethacin, l-NG-nitroarginine methyl ester, a bradykinin B2 receptor antagonist and tetrodotoxin. Thus, PAR-2 is highly expressed by colonic myocytes where it may be cleaved and activated by mast cell tryptase. This may contribute to motility disturbances of the colon during conditions associated with mast cell degranulation.
Assuntos
Mastócitos/enzimologia , Receptores de Superfície Celular/fisiologia , Serina Endopeptidases/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Animais , Benzimidazóis/farmacologia , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Cálcio/metabolismo , Células Cultivadas , Quimases , Colo/química , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Técnica Indireta de Fluorescência para Anticorpo , Motilidade Gastrointestinal/efeitos dos fármacos , Técnicas In Vitro , Indometacina/farmacologia , Mediadores da Inflamação/farmacologia , Leupeptinas/farmacologia , Músculo Liso/química , NG-Nitroarginina Metil Éster/farmacologia , Nitroprussiato/farmacologia , Ratos , Receptor PAR-2 , Receptores de Superfície Celular/análise , Serina Endopeptidases/farmacologia , Substância P/farmacologia , Tetrodotoxina/farmacologia , Fatores de Tempo , Tripsina/farmacologia , TriptasesRESUMO
Plasma extravasation from postcapillary venules is one of the earliest steps of inflammation. Substance P (SP) and bradykinin (BK) mediate extravasation and cause hypotension. The cell-surface enzyme neutral endopeptidase (NEP) inactivates both peptides. Thus, absence of NEP may predispose development of inflammation and hypotension. We examined these possibilities in mice in which the NEP gene was deleted by homologous recombination. There was widespread basal plasma extravasation in postcapillary venular endothelia in NEP-/- mice, which was reversed by recombinant NEP and antagonists of SP (NK1) and BK (B2) receptors. Mean arterial blood pressure was 20% lower in NEP-/- animals, but this was unaffected by reintroduction of recombinant NEP and the kinin receptor antagonists. The hypotension was also independent of nitric oxide (NO), because NEP-/- mice treated with a NO synthase inhibitor remained hypotensive relative to the wild type. Thus, NEP has important roles in regulating basal microvascular permeability by degrading SP and BK, and may regulate blood pressure set point through a mechanism that is independent of SP, BK and NO. The use of NEP antagonists as candidate drugs in cardiovascular disease is suggested by the blood pressure data reported herein.
Assuntos
Pressão Sanguínea/fisiologia , Capilares/fisiologia , Permeabilidade Capilar/fisiologia , Neprilisina/fisiologia , Vênulas/fisiologia , Animais , Bradicinina/fisiologia , Antagonistas dos Receptores da Bradicinina , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neprilisina/genética , Antagonistas dos Receptores de Neurocinina-1 , Substância P/fisiologiaRESUMO
Proteinase-activated receptor 2 (PAR-2) is a recently characterized G-protein coupled receptor that is cleaved and activated by pancreatic trypsin. Trypsin is usually considered a digestive enzyme in the intestinal lumen. We examined the hypothesis that trypsin, at concentrations normally present in the lumen of the small intestine, is also a signaling molecule that specifically regulates enterocytes by activating PAR-2. PAR-2 mRNA was highly expressed in the mucosa of the small intestine and in an enterocyte cell line. Immunoreactive PAR-2 was detected at the apical membrane of enterocytes, where it could be cleaved by luminal trypsin. Physiological concentrations of pancreatic trypsin and a peptide corresponding to the tethered ligand of PAR-2, which is exposed by trypsin cleavage, stimulated generation of inositol 1,4,5-trisphosphate, arachidonic acid release, and secretion of prostaglandin E2 and F1alpha from enterocytes and a transfected cell line. Application of trypsin to the apical membrane of enterocytes and to the mucosal surface of everted sacs of jejunum also stimulated prostaglandin E2 secretion. Thus, luminal trypsin activates PAR-2 at the apical membrane of enterocytes to stimulate secretion of eicosanoids, which regulate multiple cell types in a paracrine and autocrine manner. We conclude that trypsin is a signaling molecule that specifically regulates enterocytes by triggering PAR-2.