RESUMO
In this study, we investigated the dynamics of the molecular interactions of tetraspanin CD81 in T lymphocytes, and we show that CD81 controls the organization of the immune synapse (IS) and T cell activation. Using quantitative microscopy, including fluorescence recovery after photobleaching (FRAP), phasor fluorescence lifetime imaging microscopy-Föster resonance energy transfer (phasorFLIM-FRET), and total internal reflection fluorescence microscopy (TIRFM), we demonstrate that CD81 interacts with ICAM-1 and CD3 during conjugation between T cells and antigen-presenting cells (APCs). CD81 and ICAM-1 exhibit distinct mobilities in central and peripheral areas of early and late T cell-APC contacts. Moreover, CD81-ICAM-1 and CD81-CD3 dynamic interactions increase over the time course of IS formation, as these molecules redistribute throughout the contact area. Therefore, CD81 associations unexpectedly define novel sequential steps of IS maturation. Our results indicate that CD81 controls the temporal progression of the IS and the permanence of CD3 in the membrane contact area, contributing to sustained T cell receptor (TCR)-CD3-mediated signaling. Accordingly, we find that CD81 is required for proper T cell activation, regulating CD3ζ, ZAP-70, LAT, and extracellular signal-regulated kinase (ERK) phosphorylation; CD69 surface expression; and interleukin-2 (IL-2) secretion. Our data demonstrate the important role of CD81 in the molecular organization and dynamics of the IS architecture that sets the signaling threshold in T cell activation.
Assuntos
Sinapses Imunológicas/metabolismo , Linfócitos T/imunologia , Tetraspanina 28/metabolismo , Células Apresentadoras de Antígenos/imunologia , Complexo CD3/metabolismo , Diferenciação Celular , Células Cultivadas , Recuperação de Fluorescência Após Fotodegradação , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Células Jurkat , Ativação Linfocitária , Microscopia de Fluorescência , Transdução de Sinais , Linfócitos T/citologiaRESUMO
The formation of a complex between beta-catenin and members of the TCF/LEF family of high-mobility group proteins is a key regulatory event in the wnt-signaling pathway, essential for embryonal development as well as the growth of normal and malignant colon epithelium. We have characterized the binding of TCF4 to human beta-catenin by steady-state intrinsic fluorescence quenching experiments, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding studies in solution and in heterogeneous phase showed that TCF4 binds reversibly to beta-catenin with an affinity (KB) of 3(+/-1) 10(8) M(-1). Site-directed mutagenesis, together with calorimetric measurements, revealed that residue D16 in TCF4 plays a crucial role in high-affinity binding. Mutation of this residue to alanine resulted in a decrease of KB by two orders of magnitude as well as a significant reduction in binding enthalpy. Binding of TCF4 to beta-catenin gave rise to a large negative enthalpy change at 25 degrees C (-29.7 kcal/mol). Binding enthalpies were strongly temperature dependent, which resulted in the determination of a large heat capacity change upon binding of -1.5 kcal/(mol K). The molecular events that take place upon complex formation are discussed using the measured thermodynamic data together with the crystal structure of the beta-catenin arm repeat region/TCF complex.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Termodinâmica , Transativadores , Fatores de Transcrição/metabolismo , Sítios de Ligação , Dicroísmo Circular , Clonagem Molecular , Proteínas do Citoesqueleto/química , Primers do DNA/química , Fluorescência , Glutationa Transferase/metabolismo , Humanos , Modelos Moleculares , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Fatores de Transcrição TCF , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/química , Fatores de Transcrição/genética , beta CateninaRESUMO
The protein tyrosine kinase ZAP-70 is implicated in the early steps of the T-cell antigen receptor (TCR) signaling. Binding of ZAP-70 to the phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain through its two src-homology 2 (SH2) domains results in its activation coupled to phosphorylation on multiple tyrosine residues, mediated by Src kinases including Lck as well as by autophosphorylation. The mechanism of ZAP-70 activation following receptor binding is still not completely understood. Here we investigated the effect of intramolecular interactions and autophosphorylation by following the kinetics of recombinant ZAP-70 activation in a spectrophotometric substrate phosphorylation assay. Under these conditions, we observed a lag phase of several minutes before full ZAP-70 activation, which was not observed using a truncated form lacking the first 254 residues, suggesting that it might be due to an intramolecular interaction involving the interdomain A and SH2 region. Accordingly, the lag phase could be reproduced by testing the truncated form in the presence of recombinant SH2 domains and was abolished by the addition of diphosphorylated ITAM peptide. Preincubation with ATP or phosphorylation by Lck also abolished the lag phase and resulted in a more active enzyme. The same results were obtained using a ZAP-70 mutant lacking the interdomain B tyrosines. These findings are consistent with a mechanism in which ZAP-70 phosphorylation/autophosphorylation on tyrosine(s) other than 292, 315, and 319, as well as engagement of the SH2 domains by the phosphorylated TCR, can induce a conformational change leading to accelerated enzyme kinetics and higher catalytic efficiency.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Tirosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Dicroísmo Circular , Ativação Enzimática/genética , Humanos , Células Jurkat , Cinética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas de Membrana/genética , Dados de Sequência Molecular , Fosforilação , Ligação Proteica/genética , Estrutura Secundária de Proteína , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Proteína-Tirosina Quinase ZAP-70 , Domínios de Homologia de src/genéticaRESUMO
Camptothecin (CPT) is a potent, antitumour drug acting mainly through inhibition of topoisomerase I during the S-phase of the cell cycle. Despite its impressive antitumour activity, clinical development was halted for unpredictable toxic events. Two soluble N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers were synthesised to contain CPT (5 wt.% and 10 wt.%). CPT was covalently linked at its alpha-hydroxyl group to the polymers through a Gly-Phe-Leu-Gly- spacer. In-vitro, CPT-conjugates were fairly resistant to hydrolysis in plasma as in buffer at neutral pH (0.2-0. 4% free CPT/h), while elastase and cysteine-proteases were able to release the active drug. Plasma levels in mice after intravenous administration of CPT-conjugates confirmed the modest hydrolysis in plasma. Plasma levels were approximately 5-fold lower than those observed at the highest tolerated dose of CPT administered in classical vehicles. Biodistribution in HT29 human colon carcinoma bearing mice was carried out after i.v. injection of [3H]CPT-conjugate and free [3H]CPT. Radioactivity uptake in tumour was evident only after [3H]CPT-conjugate treatment. Repeated intravenous administration of CPT-conjugates to HT29-bearing mice gave more than 90% tumour inhibition, some complete tumour regressions and no toxic deaths. The improved pharmacological profile on HT29 human colon carcinoma xenografts of the first poly(HPMA)-CPT conjugates might be ascribed to their prolonged intra-tumour retention and sustained release of the active drug.
Assuntos
Antineoplásicos Fitogênicos/farmacocinética , Antineoplásicos Fitogênicos/uso terapêutico , Camptotecina/farmacocinética , Camptotecina/uso terapêutico , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Autorradiografia , Soluções Tampão , Camptotecina/administração & dosagem , Preparações de Ação Retardada , Células HT29 , Humanos , Hidrólise , Injeções Intravenosas , Cinética , Metacrilatos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Polímeros , Distribuição Tecidual , Transplante HeterólogoRESUMO
Binding of new chemical entities to serum proteins is an issue confronting pharmaceutical companies during development of potential therapeutic agents. Most drugs bind to the most abundant plasma protein, human serum albumin (HSA), at two major binding sites. Excepting fluorescence spectroscopy, existing methods for assaying drug binding to serum albumin are insensitive to higher-affinity compounds and can be labour-intensive, time-consuming, and usually require compound-specific assays. This led us to examine alternative ways to measure drug-albumin interaction. One method described here uses fluorescence quenching of the single tryptophan (Trp) residue in HSA excited at 295 nm to measure drug-binding affinity. Unfortunately, many compounds absorb, fluoresce, or both, in this UV wavelength region of the spectrum. Several types of binding phenomenon and spectral interference were identified by use of six structurally unrelated compounds and the equations necessary to make corrections mathematically were derived and applied to calculate binding constants accurately. The general cases were: direct quenching of Trp fluorescence by optically transparent ligands with low or high affinities; binding of optically transparent, non-fluorescent ligands to two specific sites where both sites or only one site result in Trp fluorescence quenching; and chromophores whose absorption either overlaps the Trp emission and quenches by energy transfer or absorbs light at the Trp fluorescence excitation wavelength producing absorptive screening as well as fluorescence quenching. Unless identification of the site specificity of drug binding to serum albumin is desired, quenching of the Trp fluorescence of albumin by titration with ligand is a rapid and facile method for determining the binding affinities of drugs for serum albumin.
Assuntos
Albumina Sérica/metabolismo , Espectrometria de Fluorescência/métodos , Triptofano/química , Sítios de Ligação , Fluorescência , Humanos , Técnicas In Vitro , Ligantes , Estatística como AssuntoRESUMO
PNU145156E (7,7-(carbonyl-bis[imino-N-methyl-4, 2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]) -bis-(1, 3-naphthalene disulfonate)) is a naphthalene sulfonic distamycin A derivative that interacts with heparin-binding growth factors. Because PNU145156E inhibits tumor angiogenesis, it was selected for clinical development. Picosecond time-resolved fluorescence emission and anisotropy were used to characterize the binding of PNU145156E to the basic fibroblast growth factor (a protein associated with tumor angiogenesis). A decrease in PNU145156E fluorescence lifetime was observed as a function of human basic fibroblast growth factor (bFGF) concentration. Nonlinear least-squares fitting of the binding isotherm yielded Kd = 145 nM for a single class of binding sites. Time-resolved anisotropy gave Kd = 174 nM. Kd = 150 nM was independently verified by quantitative high-performance affinity chromatography. The displaced volume of the complex, calculated from its rotational correlation time, fitted a sphere of 1:1 stoichiometry. These results account for the formation of a tight yet reversible PNU145156E:bFGF complex. An evaluation of PNU145156E fluorescence lifetimes in various solvents has highlighted the forces involved in stabilizing the complex. These are mostly electrostatic-hydrophobic in nature, with a relatively low contribution from hydrogen bonding. Both polar and nonpolar groups are involved on the protein-binding site within a largely hydrophobic cleft. A potential binding trajectory, based on a combination of these results with site-directed chemical modification and known bFGF x-ray structure, is suggested.
Assuntos
Distamicinas/química , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Distamicinas/farmacologia , Polarização de Fluorescência , Humanos , Cinética , Análise dos Mínimos Quadrados , Modelos Moleculares , Neovascularização Patológica/prevenção & controle , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The cytotoxic activities of 2 novel distamycin-A derivatives, FCE 24517 and FCE 25450A, alone and in combination with tumor-necrosis factor-alpha (TNF), were studied. Both drugs, especially FCE 25450A, analyzed extensively here, inhibited the growth of HL60 promyelocytic cells, and human SV80 and murine L929 transformed fibroblasts in a dose-dependent manner. The growth-inhibitory potential of sequential exposure to the distamycin-A analogs and TNF was determined. A 4-hr treatment of L929 fibroblasts with 100-1,000 ng/ml FCE 25450A, followed by 2 ng/ml TNF, resulted in a synergistic anti-proliferative effect. The synergism of FCE 24517 with TNF was less profound. Experiments to elucidate the mechanism underlying the cooperation revealed that FCE 25450A pre-treatment almost completely abolished the elevated tyrosine phosphorylation of a 137-kDa and other membranal proteins and prevented the de-phosphorylation of another protein band observed in L929 cells in the presence of TNF. FCE 25450A alone induced no changes in the phosphotyrosine profile of the cells. The effect of FCE 25450A was counteracted by the tyrosine-phosphatase inhibitor orthovanadate. In parallel, the inhibitor also diminished the antiproliferative action of the FCE 25450A/TNF combination. These findings suggest that, beyond their cytotoxic effects as single agents, the distamycin derivatives increase the sensitivity of cells to TNF. This effect is governed via the inhibition of TNF-induced tyrosine phosphorylation of specific proteins which are probably involved in the development of TNF resistance. Thus, protein de-phosphorylation might provide an additional mechanism of action of these novel distamycin-A-derived drugs.
Assuntos
Antineoplásicos/farmacologia , Distamicinas/agonistas , Distamicinas/farmacologia , Compostos de Mostarda Nitrogenada/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Camundongos , Proteínas Tirosina Fosfatases/metabolismo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/administração & dosagem , Vanadatos/farmacologiaRESUMO
The conversion of big endothelin to endothelin by alpha-chymotrypsin was determined by following its single Trp fluorescence polarization. This provides a novel, simple, fast, and sensitive identifying assay in the search for a native endothelin-converting enzyme.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Quimotripsina/metabolismo , Endotelinas/química , Endotelinas/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Endotelina-1 , Enzimas Conversoras de Endotelina , Polarização de Fluorescência/métodos , Humanos , Cinética , Metaloendopeptidases , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , TriptofanoRESUMO
Sequence-directed recognition peptides (SDRPs) were constructed on the basis of their hydropathic complementarity for big-endothelin (bigET). These peptides can inhibit in vitro the proteolytic cleavage that generates endothelin (ET) from its bigET precursor. Comparison of dissociation constants of the complexes SDRP:bigET with kinetic constants obtained for the cleavage of bigET by alpha-chymotrypsin (taken as a model proteinase) provides evidence of the potential of SDRPs. This is a novel application of SDRPs used as inhibitors of a proteolytic reaction.
Assuntos
Endotelinas/química , Oligopeptídeos/síntese química , Precursores de Proteínas/química , Sequência de Aminoácidos , Quimotripsina , Endotelinas/biossíntese , Humanos , Cinética , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Precursores de Proteínas/efeitos dos fármacosRESUMO
Endothelin-1 (ET-1), a 21-residue vasoconstrictor peptide, originates in human cells from a 212-amino acid precursor (preproET-1). Big ET-1, an intermediate form of 38 amino acids, is generated by cleavage at basic-pair residues of proET-1, while a specific "ET-converting enzyme" was proposed to process the unusual Trp-Val site at positions 21 and 22 of big ET-1. We have previously shown that expression of synthetic RNA encoding human preproET-1 in Xenopus oocytes results in secretion of putative ET-1 and big ET-1. Here, to further dissect the processing pathway of preproET-1, we designed and expressed in oocytes a set of preproET-1 mutants. Four mutants affecting the Trp-Val site always originated putative ET-1(s) at levels comparable to the wild type, suggesting that there is only a conformational requirement for cleavage at this site. An Arg-->Ile mutation at the basic-pair site after the C terminus of big ET-1 fully inhibited the formation of both big ET-1 and ET-1, indicating that processing at this site is an early event and that big ET-1 is an obligate intermediate for the synthesis of ET-1 in vivo. Also, a truncated mutant bearing a stop codon after the C terminus of the big ET-1 sequence was totally stable and further processed into mature big ET-1 and ET-1, indicating that the second part of the precursor is not necessary for maturation.
Assuntos
Endotelinas/genética , Mutagênese Sítio-Dirigida , Precursores de Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Códon/genética , Eletroforese em Gel de Poliacrilamida , Endotelina-1 , Endotelinas/biossíntese , Endotelinas/isolamento & purificação , Feminino , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Oócitos/metabolismo , Precursores de Proteínas/biossíntese , Precursores de Proteínas/isolamento & purificação , Processamento de Proteína Pós-Traducional , Radioimunoensaio , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Transcrição Gênica , XenopusRESUMO
To investigate biochemical and biological parameters involved in preproendothelin-1 (preproET-1) maturation we infected Spodoptera frugiperda (Sf21) cells with a suitable engineered baculovirus vector carrying the cDNA encoding the entire human 212 amino acids precursor. Culture supernatants were tested by RIA using an anti-ET-1 serum, ET-1-like immunoreactive material (IRM) was detected in the infected Sf21 cells but not in control, wild-type or mock-infected cells. Fractionation of the culture supernatant by RP-HPLC coupled to an ET-1 specific RIA yielded two main peaks corresponding to the retention times of human bigET-1 and ET-1. Furthermore, culture supernatant of preproET-1 expressing Sf21 cells elicited a characteristic dose-response vasoconstrictive activity on rabbit vena cava, consistent with the amount of ET-1 as estimated by RP-HPLC coupled to RIA. These results suggest that insect cells possess the enzymatic activities necessary for human preproET-1 full maturation even though no such peptide has ever been found in insect cells.
Assuntos
Baculoviridae/genética , Endotelinas/metabolismo , Vetores Genéticos , Precursores de Proteínas/metabolismo , Animais , Sequência de Bases , Bioensaio , Linhagem Celular , Endotelina-1 , Endotelinas/genética , Endotelinas/farmacologia , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Mariposas , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Oligodesoxirribonucleotídeos , Precursores de Proteínas/genética , Coelhos , Transfecção , Vasoconstrição/efeitos dos fármacos , Veias Cavas/efeitos dos fármacos , Veias Cavas/fisiologiaRESUMO
Endothelin (ET) is an extremely potent vasoconstrictor peptide of 21 amino acids, originally found in the supernatant of cultured vascular endothelial cells. To gain insights into its biosynthetic pathway, we expressed a synthetic RNA coding for the 212-amino acid precursor of human ET-1 (preproET-1) in Xenopus oocytes. Cell homogenates and oocyte incubation medium were tested by RIA using an anti-ET-1 serum. ET-1-like immunoreactivity was detected in oocytes injected with preproET-1 synthetic RNA but not in control oocytes and was much higher in medium than in cell homogenates. When preproET-1 was expressed in oocytes treated with monensin, a dramatic decrease in secretion of immunoreactive material was observed, indicating that secretion is mediated by the Golgi complex. ET-1-like immunoreactive material present in oocyte incubation medium was fractionated by reverse-phase HPLC into two main peaks, corresponding to the retention times of human big ET-1 and ET-1. Incubation medium of oocytes expressing the synthetic preproET-1 RNA elicited a characteristic vasoconstrictor response on rabbit vena cava, consistent with the biological activity that would be predicted from the amount of ET-1-like immunoreactivity measured. These results suggest that common pathways of ET maturation exist in widely different cells and that Xenopus oocytes may represent a useful tool in studying the cell biology of ET-1 synthesis.
Assuntos
Endotelinas/análise , Endotelinas/genética , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional , Animais , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Endotelina-1 , Endotelinas/farmacologia , Feminino , Biblioteca Gênica , Humanos , Técnicas In Vitro , Masculino , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Oligonucleotídeos , Oócitos/fisiologia , Mapeamento de Peptídeos , Placenta/fisiologia , Reação em Cadeia da Polimerase , Gravidez , RNA/genética , Coelhos , Radioimunoensaio , Veias Cavas/efeitos dos fármacos , Veias Cavas/fisiologia , XenopusRESUMO
A combination of HPLC elution patterns and peptide sequencing has been used to characterize two distinct activities present in subcellular fractions of bovine aortic endothelial cells (BAECs) capable of converting human big endothelin-1 (big ET-1) to mature (ET-1). A pepstatin-inhibitable activity with an acidic pH optimum present in a lysosome-enriched fraction cleaved big ET-1 at positions 18 and 21 at similar rates. A neutral pH activity present in a postlysosomal organelles subfraction was also able to convert big ET-1, and was inhibited by EDTA, but not by 1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), an inhibitor of chymotrypsin-like serine proteases.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Endotelinas/metabolismo , Endotélio Vascular/enzimologia , Precursores de Proteínas/metabolismo , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Bovinos , Cromatografia Líquida de Alta Pressão , Ácido Edético/farmacologia , Endotelina-1 , Enzimas Conversoras de Endotelina , Congelamento , Concentração de Íons de Hidrogênio , Metaloendopeptidases , Pepstatinas/farmacologia , Frações Subcelulares/enzimologia , Tosilfenilalanil Clorometil Cetona/farmacologiaRESUMO
We studied the interaction properties of synthetic antisense (AS) peptides encoded in the antisense strand of DNA corresponding to the N-terminal 20-residue sequence of the biosynthetic precursor of Arg8-vasopressin (AVP) and its binding protein bovine neurophysin II (BNPII). Binding affinities of sense polypeptides AVP and BNPII with AS peptides were measured by analytical affinity chromatography, in each case by the extent of chromatographic retardation of a soluble polypeptide interactor on an affinity matrix containing the other interactor as the immobilized species. Chromatographically calculated dissociation constants ranged from 10(-3) to 10(-6) M. Experiments were carried out to define the selectivity and underlying forces involved in the AS peptide interactions. For AS peptide elutions on sense peptide affinity supports, reduced binding affinity with increasing 1-propanol concentration and ionic strength suggested the presence of both ionic and hydrophobic contributions to AS peptide/immobilized sense peptide recognition. This same conclusion was reached with the antisense peptides as the immobilized species and measurement of elution of sequence-simplified, truncated, and charge-depleted forms of sense peptides. Immobilized AS 20-mer affinity matrix differentially retarded AVP versus oxytocin (OT) and BNPII versus BNPI (the neurophysin related biosynthetically to OT) and was used to separate these polypeptides from acid extracts of bovine posterior pituitaries. In addition, immobilized AS 12-mer corresponding to AVP-Gly-Lys-Arg could be used to separate AVP from OT. The results confirm that antisense peptides recognize sense peptides with significant selectivity in the AVP/BNPII precursor case.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Arginina Vasopressina/metabolismo , DNA , Neurofisinas/metabolismo , Ocitocina , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Arginina Vasopressina/genética , Bovinos , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , DNA/genética , Metilação , Dados de Sequência Molecular , Neurofisinas/genética , Precursores de Proteínas/genéticaRESUMO
On the basis of the homology with the Bacillus thermoproteolyticus zinc endopeptidase thermolysin, we hypothesized that Glu-143 and His-231 are the key residues for the catalytic activity of the Bacillus subtilis neutral protease. To test this possibility by site-directed mutagenesis, we substituted these two residues with Ala, Ser, Trp and Arg, and Leu, Val and Cys respectively. All these substitutions dramatically affected the amount of secreted mutant proteins, as determined by immunological methods, and their catalytic activities. No appreciable secretion was observed with the three Glu mutants Trp, Ser and Arg, whereas the Glu----Ala mutant enzyme was secreted at a level of a few hundred micrograms per litre of culture. The His mutants were all secreted at higher levels (in the order of a few milligrams per litre) and their residual catalytic activity could be determined using Z-Ala-Leu-Ala as substrate. Our results confirm the key role played by Glu-143 and His-231 in catalysis and moreover suggest the existence of a relationship between the catalytic activity of the enzyme and the extent of its secretion. In this context, we present data suggesting an autoproteolytic mechanism of cleavage of the precursor form of the enzyme, analogous to the one previously reported for the B. subtilis subtilisin.
Assuntos
Bacillus subtilis/enzimologia , Glutamatos , Histidina , Peptídeo Hidrolases/metabolismo , Engenharia de Proteínas , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Dados de Sequência MolecularRESUMO
A serine esterase called granzyme A, which is specifically expressed in cytolytic lymphocytes has been characterized. It is a disulfide-linked dimer and exhibits a trypsin-like specificity cleaving best after Arg. N-terminal sequence analysis revealed that granzyme A is identical to a protease recently predicted from a cloned CTL-specific gene.
Assuntos
Endopeptidases/sangue , Genes , Serina Endopeptidases , Linfócitos T Citotóxicos/enzimologia , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Endopeptidases/genética , GranzimasRESUMO
Limited proteolysis or autolysis of thermolysin under different experimental conditions leads to fission of a small number of peptide bonds located in exposed surface segments of the polypeptide chain characterized by highest mobility, as given by the temperature factors (B values) determined crystallographically [Holmes, M.A., & Matthews, B.W. (1982) J. Mol. Biol. 160, 623-639]. Considering also similar findings observed previously with other protein systems, it is proposed that this correlation between segmental mobility and sites of limited proteolysis in globular proteins is quite general. Thus, flexibility of the polypeptide chain of a globular protein at the site of proteolytic attack promotes optimal binding and proper interaction with the active site of the protease. These findings emphasize that apparent thermal motion seen in protein crystals is relevant to motion in solution and appear to be of general significance in protein-protein recognition processes.
Assuntos
Termolisina/metabolismo , Sequência de Aminoácidos , Bacillus/enzimologia , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Fragmentos de Peptídeos/análise , Especificidade por SubstratoRESUMO
Incubation of the neutral metalloendopeptidase thermolysin at pH 7.2 in the presence of EDTA and/or low concentrations of calcium ions produces fast enzyme inactivation as a result of autolysis. The 'nicked' protein is a folded species composed of three tightly associated protein fragments. Dissociation of this complex can be achieved under denaturing conditions, such as gel filtration on a column equilibrated with 5 M guanidine hydrochloride or reverse-phase high-performance liquid chromatography (HPLC) at acidic pH. The positions of the peptide bond cleavages were defined by isolation of the individual fragments by HPLC and their characterization by amino acid analysis after acid hydrolysis, end-group determination and partial amino acid sequencing. The results of these analyses indicated that the nicked protein is composed of fragments 1-196, 197-204 and 205-316 and thus that the corresponding sites of limited proteolysis occur at the polypeptide chain loop involved in the binding of Ca(4) in native thermolysin [Matthews, B. W., Weaver, L. H. and Kester, W. R. (1974) J. Biol. Chem. 249, 8030-8044]. The overall conformational properties of nicked thermolysin are quite similar to those of the intact protein, as judged by spectroscopic measurements and by the fact that rabbit antibodies against native thermolysin recognize and precipitate the nicked protein in immunodiffusion assays. The nicked protein was much less stable to heat and unfolding agents than intact thermolysin. These results contribute to a better knowledge of the molecular mechanism of stabilization of native thermolysin by the four bound calcium ions and demonstrate that the function of Ca(4) is to stabilize the loop 190-205 on the surface of the molecule against autolysis.