RESUMO
ErbB2 amplification and overexpression in breast cancer correlates with aggressive disease and poor prognosis. To find novel ErbB2-interacting proteins, we used stable isotope labeling of amino acids in cell culture followed by peptide affinity pull-downs and identified specific binders using relative quantification by mass spectrometry. Copine-III, a member of a Ca(2+)-dependent phospholipid-binding protein family, was identified as binding to phosphorylated Tyr1248 of ErbB2. In breast cancer cells, Copine-III requires Ca(2+) for binding to the plasma membrane, where it interacts with ErbB2 upon receptor stimulation, an interaction that is dependent on receptor activity. Copine-III also binds receptor of activated C kinase 1 and colocalizes with phosphorylated focal adhesion kinase at the leading edge of migrating cells. Importantly, knockdown of Copine-III in T47D breast cancer cells causes a decrease in Src kinase activation and ErbB2-dependent wound healing. Our data suggest that Copine-III is a novel player in the regulation of ErbB2-dependent cancer cell motility. In primary breast tumors, high CPNE3 RNA levels significantly correlate with ERBB2 amplification. Moreover, in an in situ tissue microarray analysis, we detected differential protein expression of Copine-III in normal versus breast, prostate and ovarian tumors, suggesting a more general role for Copine-III in carcinogenesis.
Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Fosfoproteínas/metabolismo , Receptor ErbB-2/metabolismo , Sítios de Ligação/genética , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Modelos Biológicos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Fosfoproteínas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Interferência de RNA , Receptor ErbB-2/genética , Tirosina/genética , Tirosina/metabolismoRESUMO
We report that caveolin-1, one of the major structural protein of caveolae, interacts with TCP-1, a hetero-oligomeric chaperone complex present in all eukaryotic cells that contributes mainly to the folding of actin and tubulin. The caveolin-TCP-1 interaction entails the first 32 amino acids of the N-terminal segment of caveolin. Our data show that caveolin-1 expression is needed for the induction of TCP-1 actin folding function in response to insulin stimulation. Caveolin-1 phosphorylation at tyrosine residue 14 induces the dissociation of caveolin-1 from TCP-1 and activates actin folding. We show that the mechanism by which caveolin-1 modulates TCP-1 activity is indirect and involves the cytoskeleton linker filamin. Filamin is known to bind caveolin-1 and to function as a negative regulator of insulin-mediated signaling. Our data support the notion that the caveolin-filamin interaction contributes to restore insulin-mediated phosphorylation of caveolin, thus allowing the release of active TCP-1.
Assuntos
Caveolina 1/metabolismo , Chaperoninas/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Linhagem Celular , Chaperonina com TCP-1 , Chaperoninas/efeitos dos fármacos , Células HT29 , Humanos , Insulina/farmacologia , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Fosforilação , Transdução de SinaisRESUMO
Endochitinases contribute to the defence response of plants against chitin-containing pathogens. The vacuolar class I chitinases consist of an N-terminal cysteine-rich domain (CRD) linked by a glycine-threonine-rich spacer with 4-hydroxylated prolyl residues to the catalytic domain. We examined the functional role of the CRD and spacer region in class I chitinases by comparing wild-type chitinase A (CHN A) of Nicotiana tabacum with informative recombinant forms. The chitinases were expressed in transgenic N. sylvestris plants, purified to near homogeneity, and their structures confirmed by mass spectrometry and partial sequencing. The enzymes were tested for their substrate preference towards chitin, lipo-chitooligosaccharide Nod factors of Rhizobium, and bacterial peptidoglycans (lysozyme activity) as well as for their capacity to inhibit hyphal growth of Trichoderma viride. Deletion of the CRD and spacer alone or in combination resulted in a modest <50% reduction of hydrolytic activity relative to CHN A using colloidal chitin or M. lysodeikticus walls as substrates; whereas, antifungal activity was reduced by up to 80%. Relative to CHN A, a variant with two spacers in tandem, which binds chitin, showed very low hydrolytic activity towards chitin and Nod factors, but comparable lysozyme activity and enhanced antifungal activity. Neither hydrolytic activity, substrate specificity nor antifungal activity were strictly correlated with the CRD-mediated capacity to bind chitin. This suggests that the presence of the chitin-binding domain does not have a major influence on the functions of CHN A examined. Moreover, the results with the tandem-spacer variant raise the possibility that substantial chitinolytic activity is not essential for inhibition of T. viride growth by CHN A.
Assuntos
Antifúngicos/metabolismo , Quitinases/genética , Quitinases/metabolismo , Nicotiana/enzimologia , Proteínas de Plantas/metabolismo , Plantas Tóxicas , Motivos de Aminoácidos , Sequência de Aminoácidos , Antifúngicos/farmacologia , Domínio Catalítico , Quitina/metabolismo , Quitinases/farmacologia , Immunoblotting , Lipopolissacarídeos/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptidoglicano/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/farmacologia , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Nicotiana/genética , Nicotiana/metabolismo , Trichoderma/efeitos dos fármacos , Trichoderma/crescimento & desenvolvimentoRESUMO
O-Fucosylation is an unusual posttranslational modification present in several proteins that play important roles in physiological processes such as coagulation, cell signaling and metastasis. Although the exact function of the modification is still unclear, the number of proteins found to be modified is increasing, and there is a need for further structural and functional analyses. Here we report on a rapid and straightforward approach in the analysis of glycosylation status and determination of glycosylation sites in O-fucosylated glycopeptides using nano-electrospray quadrupole time-of-flight (nano-ESI Q-TOF) mass spectrometry. In a single measurement of previously chemically untreated O-fucosylated peptides originating from the thrombospondin-1 repeats, we were able to determine the glycosylation status of the analyzed peptide, the glycosylation site, and the glycan structure. The abundance of glycosylated peptide fragment ions in MS(2) spectra suggests that nano-ESI Q-TOF mass spectrometry can be used as a general approach in structural studies of O-fucosylation in proteins.
Assuntos
Glicopeptídeos/química , Espectrometria de Massas/métodos , Processamento de Proteína Pós-Traducional , Animais , Glicosilação , Humanos , Microquímica/instrumentação , Microquímica/métodos , Proteínas Recombinantes de Fusão/química , Trombospondina 1/químicaRESUMO
The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-beta-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.
Assuntos
Cromossomos Humanos Par 17 , Dolicol Monofosfato Manose/metabolismo , Glicosiltransferases/metabolismo , Monoterpenos , Proteínas Repressoras/metabolismo , Monoterpenos Acíclicos , Animais , Proteínas de Bactérias , Células CHO/efeitos dos fármacos , Sequência de Carboidratos , Permeabilidade da Membrana Celular , Cricetinae , Éxons , Glicolipídeos/metabolismo , Glicosilação , Humanos , Metabolismo dos Lipídeos , Lipídeos/química , Mamíferos , Dados de Sequência Molecular , Mutação , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Monossacarídeos de Poli-Isoprenil Fosfato , Proteínas Repressoras/genética , Estreptolisinas/farmacologiaRESUMO
Ribonuclease inhibitor (RI) is a cytoplasmic protein (50 kDa) that inhibits a variety of pancreatic type RNases. The porcine inhibitor contains 30 cysteine residues, all of which occur in the reduced state. It is well known that in vitro modification of the thiol groups inactivates the protein and greatly increases its susceptibility to proteolysis. Here we show that oxidation of thiol groups in RI can also occur within the cell. Induction of an oxidative insult in cultured LLC-PK1 cells, either with a general oxidant, H2O2, or with a thiol-specific oxidant, diamide, led to the loss of RI activity. By using specific antibodies it was demonstrated that the decrease correlated with a decline in the amount of RI protein in the cells. Furthermore, analysis of RI mRNA levels and half-life of the protein excluded inhibition of the synthesis of RI as the cause of its depletion. The results indicate that oxidation of thiol groups in RI is sufficient to cause its rapid inactivation and disappearance from the cell. Most likely this results from intracellular degradation of the protein.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ribonucleases/antagonistas & inibidores , Animais , Bovinos , Diamida/farmacologia , Peróxido de Hidrogênio/farmacologia , Técnicas In Vitro , Cinética , L-Lactato Desidrogenase/metabolismo , Células LLC-PK1 , Oxidantes/farmacologia , Oxirredução , Proteínas/metabolismo , Compostos de Sulfidrila/química , SuínosRESUMO
RNase PL3 is a structurally highly conserved, pyrimidine-specific RNase, which strongly prefers to cleave at the 3'-side of uridine. Here, question of which residues are involved in determining substrate specificity is addressed. The difference in the rate of cleavage of UpA and CpA was found to result from a 375-fold larger kcat for the former substrate, whereas the values of Km were essentially the same. The pyrimidine specificity of this class of RNases is thought to result from hydrogen bonds between the base and a threonine residue in the B1 subsite. Mutation of this residue (Thr-44) in RNase PL3 resulted in strongly reduced activity with UpA and poly(U). However, the activity with CpA and poly(C) had increased. Comparison with the effect of the same mutation in RNase A [delCardayre, S. B., & Raines, R. T. (1994) Biochemistry 33, 6031-6037] and angiogenin [Curran et al. (1993) Biochemistry 32, 2307-2313] showed that the function of this threonine in substrate recognition is different in three RNase subfamilies. Previous studies have shown that the 36-42 region contains one or more residues that are involved in substrate recognition [Vicentini et al. (1994) Protein Sci. 3, 459-466]. Site-directed mutagenesis of amino acids in this region identified Phe-42 as the only single residue that affected the cytidine/uridine specificity ratio. The mutation F42V resulted in a 10-fold increase in kcat and a 1.9-fold decrease in Km for CpA. The properties of the double mutant F42V/T44A suggested that a suboptimal binding of cytidine is caused by Phe-42, partially through an effect on Thr-44.
Assuntos
Endorribonucleases/química , Endorribonucleases/metabolismo , Fígado/enzimologia , Uridina/metabolismo , Animais , Sítios de Ligação , Citidina/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Escherichia coli/genética , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação , Poli C/metabolismo , Poli U/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Suínos , Treonina/metabolismoRESUMO
AU-rich elements within the 3' untranslated region of transcripts of lymphokines and some protooncogenes serve as signal for rapid mRNA degradation. By using an AUUUA matrix, we have affinity-purified a 32-kDa protein, microsequenced it, and cloned the corresponding cDNA. In vitro, the recombinant protein bound specifically to AU-rich transcripts, including those for interleukin 3, granulocyte/macrophage colony-stimulating factor, c-fos, and c-myc. Sequence analysis revealed an unexpected homology to enoyl-CoA hydratase (EC 4.2.1.17), and the recombinant protein showed a low degree of the enzymatic activity. Thus, this gene, designated AUH, encodes an RNA binding protein with intrinsic enzymatic activity. Protein immobilized on an AUUUA matrix was enzymatically active, suggesting that hydratase and AU-binding functions are located on distinct domains within a single polypeptide.
Assuntos
Encéfalo/metabolismo , Enoil-CoA Hidratase/biossíntese , Enoil-CoA Hidratase/metabolismo , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Cromatografia de Afinidade , Clonagem Molecular , Primers do DNA , Enoil-CoA Hidratase/isolamento & purificação , Biblioteca Gênica , Genes fos , Genes myc , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Humanos , Immunoblotting , Interleucina-3/biossíntese , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Peptídeos/química , Peptídeos/imunologia , Reação em Cadeia da Polimerase , Proteínas de Ligação a RNA/isolamento & purificação , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Within the superfamily of homologous mammalian ribonucleases (RNases) 4 distinct families can be recognized. Previously, representative members of three of these have been cloned and studied in detail. Here we report on the cloning of a cDNA encoding a member of the fourth family, RNase PL3 from porcine liver. The deduced amino acid sequence showed the presence of a signal peptide, confirming the notion that RNase PL3 is a secreted RNase. Expression of the cDNA in Escherichia coli yielded 1.5 mg of purified protein/liter of culture. The recombinant enzyme was indistinguishable from the enzyme isolated from porcine liver based on the following criteria: amino acid analysis, N-terminal amino acid sequence, molecular weight, specific activity toward yeast RNA, and kinetic parameters for the hydrolysis of uridylyl(3',5')adenosine and cytidylyl(3',5')adenosine. Interestingly, the kinetic data showed that RNase PL3 has a very low activity toward yeast RNA, i.e., 2.5% compared to pancreatic RNase A. Moreover, using the dinucleotide substrates and homopolymers it was found that RNase PL3, in contrast to most members of the RNase superfamily, strongly prefers uridine over cytidine on the 5' side of the scissile bond. Replacement, by site-directed mutagenesis, of residues 36-42 of RNase PL3 by the corresponding ones from bovine pancreatic RNase A resulted in a large preferential increase in the catalytic efficiency for cytidine-containing substrates. This suggests that this region of the molecule contains some of the elements that determine substrate specificity.
Assuntos
Endorribonucleases/genética , Endorribonucleases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , DNA Complementar/genética , Endorribonucleases/química , Escherichia coli/genética , Fígado/enzimologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/genética , Ribonuclease Pancreático/genética , Especificidade por Substrato , Suínos , UridinaRESUMO
Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisin-catalyzed semisynthesis starting from synthetic RNase 1-20 peptides and S-protein (RNase 21-124). The lysyl residue at position 1 was replaced by alanine, whereas Lys-7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl-3',5'-adenosine and yeast RNA. When Lys-7 was replaced by S-methyl-cysteine or S-carboxamido-contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A-RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys-1, Cys-7-methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol-1. Modifications that introduced a positive charge in position 7 (S-aminoethyl- or S-ethylpyridyl-cysteine) led to much smaller losses. The replacement of Lys-1 resulted in a 4-kJ mol-1 loss in binding energy. S-protein bound to RI with Ki = 63.4 pM, 800-fold weaker than RNase A. This corresponded to a 16-kJ mol-1 difference in binding energy. The results show that the N-terminal portion of RNase A contributes significantly to binding of ribonuclease inhibitor and that ionic interactions of Lys-7 and to a smaller extent of Lys-1 provide most of the binding energy.
Assuntos
Proteínas/metabolismo , Ribonuclease Pancreático/síntese química , Ribonuclease Pancreático/metabolismo , Ribonucleases/antagonistas & inibidores , Animais , Catálise , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Ligação de Hidrogênio , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Fígado/enzimologia , Concentração Osmolar , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , RNA Fúngico/metabolismo , Proteínas Recombinantes/metabolismo , Ribonuclease Pancreático/química , Relação Estrutura-Atividade , Especificidade por Substrato , Subtilisinas/metabolismo , Suínos , TermodinâmicaRESUMO
The pyrimidine-specific ribonuclease superfamily constitutes a group of homologous proteins so far found only in higher vertebrates. Four separate families are found in mammals, which have resulted from gene duplications in mammalian ancestors. To learn more about the evolutionary history of this superfamily, the primary structure and other characteristics of the pancreatic enzyme from iguana (Iguana iguana), a herbivorous lizard species belonging to the reptiles, have been determined. The polypeptide chain consists of 119 amino acid residues. The positions of insertions and deletions in the sequence are identical to those in the enzyme from snapping turtle. However, the two enzymes differ at 54% of the amino acid positions. Iguana ribonuclease contains no carbohydrate, although the enzyme possesses three recognition sites for carbohydrate attachment, and has a high number of acidic residues in a localized part of the sequence.
Assuntos
Pâncreas/enzimologia , Conformação Proteica , Ribonuclease Pancreático/química , Sequência de Aminoácidos , Animais , Bovinos , Galinhas , Humanos , Iguanas , Cinética , Fígado/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Ribonuclease Pancreático/isolamento & purificação , Ribonuclease Pancreático/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , SuínosRESUMO
The trimeric form of protein phosphatase 2A consisting of 36-, 65-, and 72-kDa subunits (previously termed polycation-stimulated protein phosphatase M) was purified from rabbit skeletal muscle. Amino acid sequence data of the 72-kDa regulatory subunit (termed PR72) were used to isolate cDNAs from human heart and fetal brain libraries and libraries derived from WI-38 and MCF-7 cells. The clones isolated from the heart cDNA library revealed an open reading frame encoding a protein with a predicted molecular mass of 62 kDa. All the peptides sequenced from the protein matched with the sequence predicted from the cDNA. However, in vitro transcription and translation from this cDNA yielded a protein with an apparent molecular mass of 72 kDa on sodium dodecyl sulfate-polyacrylamide gels. From brain we isolated cDNA clones spanning an open reading frame encoding a 130-kDa protein (termed PR130). The apparent molecular mass of the protein produced by in vitro transcription and translation was 130 kDa. This protein has exactly the same deduced C-terminal protein sequence as the PR72 subunit from amino acids 45 to 527 but has an N-terminal extension of 665 amino acids. It is likely, therefore, that these two proteins arise from the same gene by alternative splicing. In human tissues several transcripts were detected by Northern analysis generated probably by the use of different polyadenylation signals and alternative splicing. High levels of the PR72 mRNAs were detected in heart and muscle, while lower levels of PR130 transcripts were found in heart, brain, placenta, lung, muscle, and kidney.
Assuntos
Processamento Alternativo , Fosfoproteínas Fosfatases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Humanos , Soros Imunes , Dados de Sequência Molecular , Músculos/enzimologia , Fases de Leitura Aberta , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/isolamento & purificação , Testes de Precipitina , Proteína Fosfatase 2 , CoelhosRESUMO
Single crystals of the RNase inhibitor from porcine liver have been obtained from 30 to 34% saturated ammonium sulphate solutions at pH 6.0 to 7.2, containing 20 mM dithiothreitol, at room temperature over a period of two to three weeks. Because the inhibitor contains 30 1/2-cystinyl residues, all of which occur in the free thiol form, crystallization experiments were carried out in a desiccator under a nitrogen atmosphere. The crystals belong to the tetragonal space group I4, with cell dimensions a = b = 134.76 A and c = 83.65 A. The asymmetric part of the unit cell contains two molecules with a molecular mass of 49,093 Da, as could be shown with a self-rotation function calculated in the resolution range 10.0 to 3.2 A. The crystals diffract to at least 3.2 A resolution and are suitable for an X-ray structure determination.
Assuntos
Ribonuclease Pancreático/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Cristalização , Leucina , Fígado/química , Dados de Sequência Molecular , Família Multigênica , Sequências Repetitivas de Ácido Nucleico , Suínos , Difração de Raios XRESUMO
The fungicidal class I chitinases (EC 3.2.1.14) are believed to be important in defending plants against microbial pathogens. The vacuolar isoforms of tobacco (Nicotiana tabacum), chitinases A and B, are the first examples of a new type of hydroxyproline-containing protein with intracellular location, enzymic activity, and a small number of hydroxyprolyl residues restricted to a single, short peptide sequence. We have investigated the posttranslational processing and intracellular transport of transgene-encoded chitinase A in callus cultures of Nicotiana tabacum L. cv Havana 425 and leaves of Nicotiana sylvestris Spegazzini and Comes. Pulse-chase experiments and cell fractionation show that chitinase A is processed in two distinct steps. In the first step, the nascent protein undergoes an increase in apparent M(r) of approximately 1500 detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with the inhibitor of prolyl hydroxylation, alpha,alpha'-dipyridyl, and pulse-chase labeling of cells expressing recombinant forms of chitinase A indicate that the anomalous increase in M(r) is due to hydroxylation of prolyl residues. This step occurs in the endomembrane system before sorting for secretion and vacuolar transport and does not appear to be required for correct targeting of chitinase A to the vacuole. The second step is a proteolytic cleavage. Sequencing of tryptic peptides of the mature proteins indicates that during processing essentially all molecules of chitinase A and B lose a C-terminal heptapeptide, which has been shown to be a vacuolar targeting signal. This appears to occur primarily in the endomembrane system late in intracellular transport. A model for the posttranslational modification of chitinase A is proposed.
Assuntos
Quitinases/metabolismo , Hidroxiprolina/metabolismo , Nicotiana/genética , Plantas Tóxicas , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Fracionamento Celular , Quitinases/química , Quitinases/genética , Clonagem Molecular , Técnicas de Cultura , DNA , Eletroforese em Gel de Poliacrilamida , Hidroxilação , Dados de Sequência Molecular , Nicotiana/enzimologia , Nicotiana/metabolismo , Vacúolos/metabolismoRESUMO
One of cAMP-regulatory sites in the porcine urokinase-type plasminogen activator (uPA) gene resides 3.4 kb upstream of the transcription initiation site and is composed of three protein binding domains, FPA, FPB and FPC. Whereas FPA and FPB contain a CRE-like sequence, the FPC sequence is not related to any known protein recognition sequences, yet all three domains are required to mediate cAMP action on a heterologous promoter. To study the functional cooperation among these three domains we purified and cloned a FPC-binding protein (FPCB) from porcine kidney derived LLC-PK1 cells. Sequence comparisons showed that FPCB is homologous to mouse LFB3 and rat vHNF1. LFB3/vHNF1 is related to a liver specific transcription factor HNF1, it recognizes the same sequence as HNF1 and is highly expressed in kidney cells. FPCB and HNF1 recognition sequences are dissimilar, nevertheless both sequences are recognized by in vitro-translated LFB3 and FPCB, indicating that binding to the two different sequences is an intrinsic character of FPCB/LFB3/vHNF1. In HeLa cells, this cAMP-responsive site was inactive whether FPCB was overexpressed or not, suggesting a requirement for an additional cell-specific factor. These results may suggest a mechanism by which hormonal control is integrated into cell-specific gene regulation.
Assuntos
AMP Cíclico/metabolismo , Proteínas de Ligação a DNA , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/isolamento & purificação , Ativador de Plasminogênio Tipo Uroquinase/genética , Fatores Ativadores da Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Proteínas Sanguíneas/metabolismo , Clonagem Molecular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Dados de Sequência Molecular , Suínos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Porcine ribonuclease inhibitor (RI) contains 30 1/2-cystinyl residues, all of which occur in the reduced form. Reaction of the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) resulted in the release of 30 mol of the product 5-mercapto-2-nitrobenzoate, and the loss of the RNase inhibitory activity. A linear relationship between the degree of modification and inactivation was observed. The rate of modification was greatly increased in the presence of 6 M guanidinium HCl. Reaction with substoichiometric amounts of 5,5'-dithiobis(2-nitrobenzoic acid) was found to yield a mixture of fully reduced active molecules, and fully oxidized inactive ones, but no partially oxidized forms were detected. This suggests that an "all-or-none" type of modification and inactivation took place. All 1/2-cystinyl residues in the inactive, monomeric inhibitor had formed disulfide bridges, judged by the absence of either free thiol groups or mixed disulfides with 5-mercapto-2-nitrobenzoate. This fully disulfide-cross-linked molecule had an open conformation compared to the native one, as shown by gel filtration and limited proteolysis. Reaction of phenylarsinoxide with vicinal dithiols yields products that are much more stable than those with monothiols. Titration of RI with this reagent yielded complete inactivation at a reagent/thiol ratio of 0.5. Taken together, these observations suggest that the thiol groups in RI have a diminished reactivity due to three-dimensional constraints. After the initial modification of a small number of thiol groups, a conformational change occurs which causes an increase in reactivity of the remaining thiols. The thiol groups are situated close enough together to permit the formation of 15 disulfide bridges in the inactive molecule.
Assuntos
Ácido Ditionitrobenzoico/farmacologia , Proteínas/antagonistas & inibidores , Animais , Arsenicais/farmacologia , Bovinos , Cromatografia de Afinidade , Reagentes de Ligações Cruzadas , Dissulfetos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Conformação Proteica , Proteínas/isolamento & purificação , Proteínas/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Ribonuclease Pancreático/metabolismo , Saccharomyces cerevisiae/genética , SuínosRESUMO
The fungicidal type I chitinases contribute to the defense response of plants against pathogens. Two tobacco chitinases represent a different class of hydroxyproline-containing proteins. Hydroxyproline-rich proteins are predominantly extracellular, structural glycoproteins proteins that lack enzymatic activity and contain many hydroxyproline residues. In contrast, type I chitinases are vacuolar enzymes. They are not glycosylated and contain a small number of hydroxyproline residues restricted to a single, short peptide sequence.
Assuntos
Quitinases/química , Hidroxiprolina/análise , Nicotiana/enzimologia , Plantas Tóxicas , Vacúolos/enzimologia , Sequência de Aminoácidos , Quitinases/metabolismo , Glicosilação , Hidroxilação , Dados de Sequência Molecular , Peso Molecular , Conformação Proteica , Nicotiana/ultraestruturaRESUMO
Peptidyldiazomethanes with proline in the P1 position were found to be competitive slow-binding inhibitors of prolyl endopeptidase. Progress-curve experiments monitoring the increase in the degree of inhibition with time indicated that the kinetic mechanism involved an initial complex that isomerized to form a tighter complex. Reversibility of the inhibited complex was demonstrated by monitoring the regain of enzyme activity after removal of free inhibitor and dilution into an assay containing competing substrate. The kinetics of the reversal of inhibition indicated a more complicated inhibitory mechanism involving more than one pathway for reversal of the tight complex. A slow-binding mechanism of inhibition has not been previously observed with peptidyldiazomethanes. Incorporation of [3H]Ac-Ala-Ala-Pro-diazomethane into prolyl endopeptidase was observed after denaturation of the inhibited complex. The peptide labelled with [3H]Ac-Ala-Ala-Pro-diazomethane was isolated and found to contain the active-site serine residue.
Assuntos
Diazometano/análogos & derivados , Endopeptidases/metabolismo , Serina Endopeptidases , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Diazometano/metabolismo , Diazometano/farmacologia , Rim/enzimologia , Cinética , Dados de Sequência Molecular , Músculos/enzimologia , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Prolina/química , Prolil Oligopeptidases , Inibidores de Proteases/farmacologia , Desnaturação Proteica , Serina , SuínosRESUMO
Soluble beta-fructofuranosidase with an intracellular location and an isoelectric point of 3.8 (isoenzyme I) was purified and characterized from dry seeds and seedlings of carrot (Daucus carota). The enzyme hydrolyzed sucrose with a Km of 5 mM and a broad pH optimum around 5.0. The purified protein, which was N-glycosylated with high-mannose-containing and high-xylose-containing complex glycans, eluted as a monomeric polypeptide with a molecular mass of 68,000 from a gel-filtration column. On SDS/PAGE, the protein separated in the presence of SDS and 2-mercaptoethanol into three polypeptides with molecular masses of 68, 43 and 25 kDa. The amount of the 68-kDa polypeptide was highest in dry seeds and decreased with increasing age of carrot seedlings. Amino acid sequence analysis and immunological studies showed that the 43-kDa and 25-kDa polypeptides were N-terminal and C-terminal proteolytic fragments of the 68-kDa polypeptide. A comparison of partial amino acid sequences of the soluble beta-fructofuranosidase with the complete sequence of carrot cell-wall beta-fructofuranosidase showed that their N-terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.
Assuntos
Glicosídeo Hidrolases/isolamento & purificação , Isoenzimas/isolamento & purificação , Verduras/enzimologia , Sequência de Aminoácidos , Western Blotting , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Isoenzimas/metabolismo , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Tripsina/metabolismo , beta-FrutofuranosidaseRESUMO
Ribonuclease inhibitor (RI) is a protein that forms a very tight complex with ribonucleases (RNases) of the pancreatic type. RI contains 30 thiol groups, some of which are important for the enzyme-inhibitor interaction. To examine which thiols are affected by the binding of RNase, differential labeling experiments were performed. Reaction of porcine RI with the cysteine-specific labeling reagent 4-N,N-dimethylaminoazobenzene-4'-iodoacetamido-2'-sulfonic acid resulted in labeling of an average of 7.4 of the 30 cysteinyl residues. Binding of bovine pancreatic RNase A caused a 3.2-fold reduction in the extent of modification. Peptide mapping showed that in free RI, Cys-57, -371, and -404 were labeled to the greatest extent (yield, 0.4-0.6 mol/mol). RNase A did not protect Cys-57 against modification, whereas the labeling of Cys-371 and -404 was reduced by more than 90%. A second group of residues was labeled to a lesser extent in free RI (yield, 0.04-0.2 mol/mol). Within this group 11 residues were protected by RNase A by more than 90%, 2 were not affected at all, and 7 were protected between 10 and 90%. Seven cysteinyl residues in RI that were protected in the RI.RNase A complex were no longer protected in the RI.S-protein complex. These residues were mainly present in the N-terminal region of RI. However, when the S-peptide was included to yield the RI.RNase S complex, the same pattern of labeling was obtained as with the RI.RNase A complex. Addition of the S-peptide alone had no effect on the labeling. The implications of these observations with respect to RNase binding areas of RI are discussed in relation to the results obtained from the analysis of active RI molecules that contain deletions.