RESUMO
We compared the membrane proteins of autolysosomes isolated from leupeptin-administered rat liver with those of lysosomes. In addition to many polypeptides common to the two membranes, the autolysosomal membranes were found to be more enriched in endoplasmic reticulum lumenal proteins (protein-disulfide isomerase, calreticulin, ER60, BiP) and endosome/Golgi markers (cation-independent mannose 6-phosphate receptor, transferrin receptor, Golgi 58-kDa protein) than lysosomal membranes. The autolysosomal membrane proteins include three polypeptides (44, 35, and 32 kDa) whose amino-terminal sequences have not yet been reported. Combining immunoblotting and reverse transcriptase-polymerase chain reaction analyses, we identified the 44-kDa peptide as the intact subunit of betaine homocysteine methyltransferase and the 35- and 32-kDa peptides as two proteolytic fragments. Pronase digestion of autolysosomes revealed that the 44-kDa and 32-kDa peptides are present in the lumen, whereas the 35-kDa peptide is not. In primary hepatocyte cultures, the starvation-induced accumulation of the 32-kDa peptide occurs in the presence of E64d, showing that the 32-kDa peptide is formed from the sequestered 44-kDa peptide during autophagy. The accumulation is induced by rapamycin but completely inhibited by wortmannin, 3-methyladenine, and bafilomycin. Thus, detection of the 32-kDa peptide by immunoblotting can be used as a streamlined assay for monitoring autophagy.
Assuntos
Autólise/enzimologia , Lisossomos/enzimologia , Metiltransferases/metabolismo , Animais , Sequência de Bases , Betaína-Homocisteína S-Metiltransferase , Eletroforese em Gel Bidimensional , Endossomos/enzimologia , Complexo de Golgi/enzimologia , Fígado/citologia , Masculino , Metiltransferases/isolamento & purificação , Dados de Sequência Molecular , Ratos , Ratos WistarRESUMO
Lysosomal proteinases are translated as preproforms, transported through the Golgi apparatus as proforms, and localized in lysosomes as mature forms. In this study, we analyzed which subclass of proteinases participates in the processing of lysosomal proteinases using Bafilomycin A1, a vacuolar ATPase inhibitor. Bafilomycin A1 raises lysosomal pH resulting in the degradation of lysosomal proteinases such as cathepsins B, D, and L. Twenty-four hours after the withdrawal of Bafilomycin A1, NIH3T3 cells possess these proteinases in amounts and activities similar to those in cells cultured in DMEM and 5% BCS. In the presence of various proteinase inhibitors, procathepsin processing is disturbed by E-64-d, resulting in abnormal processing of cathepsins D and L, but not by APMSF, Pepstatin A, or CA-074. In the presence of Helicobacter pylori Vac A toxin, which prevents vesicular transport from late endosomes to lysosomes, the processing of procathepsins B and D occurs, while that of procathepsin L does not. Thus, procathepsins B and D are converted to their mature forms in late endosomes, while procathepsin L is processed to the mature form after its arrival in lysosomes by some cysteine proteinase other than cathepsin B.
Assuntos
Endopeptidases/metabolismo , Lisossomos/enzimologia , Macrolídeos , ATPases Vacuolares Próton-Translocadoras , Células 3T3 , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/farmacologia , Catepsina D/química , Catepsina D/metabolismo , Catepsinas/química , Catepsinas/metabolismo , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Helicobacter pylori , Lisossomos/efeitos dos fármacos , Camundongos , Inibidores de Proteases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , ATPases Translocadoras de Prótons/antagonistas & inibidores , Vacúolos/efeitos dos fármacos , Vacúolos/enzimologia , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Three amino-terminal-specific peptidic antibodies against cathepsin L were generated. These antibodies recognize in vitro processing products of procathepsin L in time-course-dependent fashion. Immunoblot analyses with these antibodies followed by immunoprecipitation with anti-cathepsin L antibody showed that the amino terminal amino acid sequences of intracellular cathepsin L are heterogeneous: the single chain form of cathepsin L starts with either EPLML, LKIPK or IPKSV, and the heavy chain of the two chain form with IPKSV. Percoll density gradient and fluorescence immunohistochemistry suggested that these three species of cathepsin L localize in the lysosomes where procathepsin L processing occurs.
Assuntos
Catepsinas/biossíntese , Catepsinas/metabolismo , Endopeptidases , Precursores Enzimáticos/metabolismo , Processamento de Proteína Pós-Traducional , Células 3T3 , Sequência de Aminoácidos , Animais , Catepsina L , Catepsinas/química , Catepsinas/isolamento & purificação , Transformação Celular Neoplásica , Centrifugação com Gradiente de Concentração , Cisteína Endopeptidases , Precursores Enzimáticos/química , Genes ras , Rim/enzimologia , Cinética , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Pele/enzimologiaRESUMO
Cathepsin L, a lysosomal cysteine proteinase, belongs to the papain family. This proteinase is different from the other members of the mammalian papain family cysteine proteinase in the following ways: (i) The cathepsin L gene is activated by a variety of growth factors and activated oncogenes. (ii) Procathepsin L, a precursor form of cathepsin L is secreted from various cells. (iii) The mRNA level of cathepsin L is related to the in vivo metastatic potential of the transformed cells. Thus, the regulation of the cathepsin L gene and the extracellular functions of secreted procathepsin L are tightly coupled. In this review, we describe these two points, which have recently been addressed in our laboratory.
Assuntos
Catepsinas/genética , Precursores Enzimáticos/genética , Regulação da Expressão Gênica , Animais , Catepsina L , Catepsinas/fisiologia , Precursores Enzimáticos/fisiologia , Matriz Extracelular/metabolismo , HumanosRESUMO
Matrix expansion in the glomerular mesangial area is observed in diabetic nephropathy. Intracellular breakdown of long-lived proteins was lower in mesangial cells in the high glucose medium than that in the control medium. Enzymatic activity of cathepsin L increased 1.4-fold after 6 h of treatment with the high glucose, and then declined gradually to 72% of control cells after treatment for 36 h. Change in the enzyme activity of cathepsin B showed a similar time course but less magnitude than that of cathepsin L. Immunoblot analysis with anti-cathepsin L antibody showed that change in the enzyme activity of cathepsin L was due to the change in the amount of cathepsin L, and that with anti-cathepsin B antibody showed no change in the amount of cathepsin B in the mesangial cells treated with high glucose. Intracellular cathepsin activities were controlled not only by the amounts but also by the inhibitor cystatin beta. Immunoblot analysis with anti-cystatin beta antibody showed that intracellular levels of cystatin beta increased slightly after 24 h of treatment with high glucose. These changes were derived from changes in mRNA level. These results, therefore, demonstrated that the decrease of intracellular protein breakdown in mesangial cells treated with high glucose medium was due to both suppression of cathepsins and increase of cystatin beta.
Assuntos
Catepsina B/genética , Catepsinas/genética , Cistatinas/genética , Cisteína Endopeptidases/genética , Endopeptidases , Regulação da Expressão Gênica , Mesângio Glomerular/metabolismo , Animais , Catepsina B/metabolismo , Catepsina L , Catepsinas/metabolismo , Células Cultivadas , Cistatina M , Cistatinas/metabolismo , Cisteína Endopeptidases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mesângio Glomerular/citologia , Mesângio Glomerular/efeitos dos fármacos , Glucose/farmacologia , Concentração Osmolar , Proteínas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Both the protein and mRNA levels of cathepsin L in SR-3Y1-2, v-src-transformed 3Y1 cell lines were higher than in 3Y1 cells. Results of CAT assays suggested that the v-src responsible region in the cathepsin L gene localizes to 300 bp in the 5'-upstream region and 60 bp in the first exon. DNaseI footprinting analyses showed that transcription factors bind to the region from 29bp to 55bp from the transcription start site. This region contains a CAAT-box and a 5'-GGCGGGGGCGG-3' sequence containing two repeat copies of consensus Sp-1 binding sites, overlapping a consensus Egr family binding site. DNA band shift assays showed that Sp-1 and NF-1 binding proteins bind to this region in 3Y1 cells while Egr family protein binds in SR-3Y1-2 cells. These results suggest that Egr family proteins are involved in the activation of the cathepsin L gene in SR-3Y1-2 cells.
Assuntos
Catepsinas/genética , Transformação Celular Neoplásica/genética , Endopeptidases , Regulação Neoplásica da Expressão Gênica , Genes src , Fatores de Transcrição/genética , Animais , Catepsina L , Linhagem Celular Transformada , Cisteína Endopeptidases , RatosRESUMO
The differentiation of keratinocytes involves numerous steps including the formation of the cornified envelope and the aggregation of keratin filaments by filaggrin monomer molecules. In this study, we investigated whether mu-calpain is involved in the processing of profilaggrin to filaggrin monomers by using both an active mu-calpain specific antibody and a 27-mer synthetic calpastatin peptide, a cell-permeable calpain-specific inhibitor. Upon incubation of cultured keratinocytes with Ca2+ for 96 hours, active mu-calpain with a molecular mass of 76 kDa appeared preceded by an increase in mu-calpain mRNA. In synchrony with the appearance of active mu-calpain, the processing of profilaggrin occurred. Furthermore, the Ca2+-induced activation of mu-calpain and the processing of profilaggrin were affected by the addition of the synthetic calpastatin inhibitor. These results indicate that the activation of mu-calpain plays a major role in the profilaggrin processing.
Assuntos
Calpaína/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Queratinócitos/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Pele/metabolismo , Sequência de Aminoácidos , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/farmacologia , Diferenciação Celular , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Citosol/metabolismo , Proteínas Filagrinas , Humanos , Recém-Nascido , Proteínas de Filamentos Intermediários/biossíntese , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Cinética , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Pele/citologiaRESUMO
Cystatin A is a cysteine proteinase inhibitor with a molecular mass of 11 kDa, and is located mainly in the keratohyaline granules of the stratum granulosum and the cornified envelope of the stratum corneum in the epidermis. In this study, we demonstrated the genomic structure of this proteinase inhibitor in which there were three exons of 111 bp, 102 bp and 226 bp in length, while the lengths of the 1st and 2nd intron were approximately 14 Kbp and 4 Kbp, respectively. The conserved sequence of QVVAG was encoded in the 2nd exon and was not inserted by any introns. There were binding sites for SP-1 and AP-2 in the promoter region and an AP-1 binding site in the 1st intron. The successful amplification of each exon of cystatin A may possibly contribute to the detection of the genomic abnormality of some skin disorders e.g. keratinization disorder, chronic bacterial infection or photophobia.
Assuntos
Cistatinas/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Southern Blotting , Clonagem Molecular , Sequência Conservada , Cistatinas/metabolismo , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Éxons , Humanos , Íntrons , Dados de Sequência Molecular , Podofilina/análogos & derivados , Podofilina/metabolismo , Podofilotoxina/análogos & derivados , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Fator de Transcrição AP-2 , Fatores de Transcrição/metabolismoRESUMO
To develop a general method for analysis of the mutation and prenatal diagnosis of X-linked hyper-IgM syndrome (XHM), the human CD40 ligand (hCD40L) gene was cloned and sequenced with special reference to the 5' and 3' flanking regions and exon/intron boundaries. The hCD40L gene consists of five exons and four introns, as already reported by others. Two major transcription initiation sites were identified at 67 bp and 64 bp upstream from the ATG initiation codon. The hCD40L mRNA transcripts terminated at 321 bp, 327 bp and 987 bp downstream from the TGA stop codon. Based on the intronic sequences, oligonucleotide primers were designed for amplifying the coding region of each exon separately. Polymerase chain reaction--single-strand conformational polymorphism (PCR-SSCP) analysis was successfully applied to screening for the defective hCD40L gene in a family with XHM. The nonsense mutation, Trp140 (TGG)-->stop (TAG) in exon 5, was found in the mother and an affected child. We also performed prenatal diagnosis by PCR-SSCP during the first trimester of pregnancy in this family.
Assuntos
Amostra da Vilosidade Coriônica/métodos , Hipergamaglobulinemia/diagnóstico , Imunoglobulina M , Glicoproteínas de Membrana/genética , Polimorfismo Conformacional de Fita Simples , Sequência de Aminoácidos , Sequência de Bases , Ligante de CD40 , Clonagem Molecular , Éxons/genética , Feminino , Genes/genética , Triagem de Portadores Genéticos , Ligação Genética , Testes Genéticos/métodos , Humanos , Hipergamaglobulinemia/genética , Masculino , Dados de Sequência Molecular , Mutação Puntual , Reação em Cadeia da Polimerase/métodos , Gravidez , Primeiro Trimestre da Gravidez , Análise de Sequência de DNA , Síndrome , Transcrição Gênica/genética , Cromossomo XRESUMO
The processing of procathepsin L on the surfaces of physiological glycosaminoglycans, heparan sulfate, chondroitin sulfate, etc., as well as dextran derivatives were studied. All glycosaminoglycans and dextran derivatives including dextran T-500 and DEAE dextran examined in this study accelerated the conversion of procathepsin L to processed cathepsin L in vitro with different time courses. Further, we examined whether procathepsin L digests protein substrates in the presence or absence of the surface materials. Laminin was degraded by both procathepsin L itself and the 31-kDa processed form in the presence of surface materials with the same profiles. In contrast, fibronectin was digested by procathepsin L without processing in the presence of surface materials. The proteolytic profiles of fibronectin by the processed form differed from those by procathepsin L. This is the first evidence that the proform of a cysteine proteinase proteolyzes protein substrates.
Assuntos
Catepsinas/metabolismo , Precursores Enzimáticos/metabolismo , Fibronectinas/metabolismo , Glicosaminoglicanos/metabolismo , Laminina/metabolismo , Células 3T3 , Animais , Catepsina L , Camundongos , Peso Molecular , Processamento de Proteína Pós-TraducionalRESUMO
Secretory granules of GH4C1 cells, a rat pituitary tumor cell line, are known to be induced by the treatment of estradiol (E2), insulin, and epidermal growth factor (EGF). We examined changes in the localization of cathepsins B, H, and L, lysosomal cysteine proteinases, in GH4C1 cells before and after hormonal treatment. Northern blotting and immunofluorescence microscopy showed that both mRNAs and intracellular protein concentrations of these enzymes were increased in the hormone-induced cells. By immunoelectron microscopy, immunogold particles indicating cathepsins B, H, and L were localized not only in lysosomes but also in some secretory granules. To further examine the molecular forms of these proteinases in secretory granules, radiolabeling and immunoprecipitation methods were applied to the media of the cells incubated with or without secretagogues (100 nM 12-O-tetradecanoylphorbol-13-acetate and 50 microM forskolin); the proforms of cathepsins B, H, and L were secreted from the cells by the constitutive pathway, whereas the mature forms of cathepsins B and H, and the proform and mature form of cathepsin L were secreted by the regulated pathway. These results suggest that in hormone-induced GH4C1 cells, cathepsins B, H, and L are sorted from the Golgi complex not only into lysosomes but also into secretory granules, in which proforms of cathepsins B and H, and a part of procathepsin L are processed into mature forms.
Assuntos
Cisteína Endopeptidases/metabolismo , Grânulos Citoplasmáticos/metabolismo , Endopeptidases , Adeno-Hipófise/metabolismo , Animais , Catepsina B/química , Catepsina B/metabolismo , Catepsina H , Catepsina L , Catepsinas/química , Catepsinas/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Estradiol/farmacologia , Insulina/farmacologia , Lisossomos , Masculino , Adeno-Hipófise/citologia , Ratos , Ratos Wistar , Receptor IGF Tipo 2/metabolismo , Células Tumorais CultivadasRESUMO
Subunit c is normally present as an inner mitochondrial membrane component of the F0 sector of the ATP synthase complex, but in the late infantile form of neuronal ceroid-lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. The rate of degradation of subunit c as measured by pulse-chase and immunoprecipitation showed a marked delay of degradation in patients' fibroblasts with late infantile form of NCL. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed that the accumulation of labeled subunit c in the mitochondrial fraction can be detected before lysosomal appearance of radioactive subunit c, suggesting that subunit c accumulated as a consequence of abnormal catabolism in the mitochondrion and is transferred to lysosomes through an autophagic process. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. These findings suggest that a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes.
Assuntos
Mitocôndrias/enzimologia , Lipofuscinoses Ceroides Neuronais/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Endopeptidases/metabolismo , Expressão Gênica , Humanos , Lactente , Membranas Intracelulares/enzimologia , Cinética , Lisossomos/enzimologia , Substâncias Macromoleculares , Lipofuscinoses Ceroides Neuronais/genética , ATPases Translocadoras de Prótons/biossíntese , Valores de ReferênciaRESUMO
C2C12 myoblasts fuse to form multinucleated myotubes and express muscle specific proteins during differentiation. To elucidate developmental regulation of intracellular proteolytic systems, enzymatic activities, protein and mRNA levels of proteasomes (20S and 26S) and lysosomal cathepsins (B, L, and H) were examined. Myoblasts were differentiated fully to myotubes 6 days after starting differentiation. In this developmental process, the 26S proteasome activity decreased, while the 20S proteasome activity increased. Expression of proteasome subunits of 20S (RC2, RC8) and regulatory components of 26S (S4, S7) was down-regulated, though total protein levels of proteasomes showed no remarkable changes. On the other hand, enzymatic activities of cathepsins B and B + L increased in association with an increase of their transcriptional and translational levels. Expression of their specific endogenous inhibitor, cystatin beta, also increased. Maturation of the lysosomal proteolytic system was tightly linked to the differentiation process. These results suggested that signals for differentiation of myoblasts mediate a change of intracellular proteolytic systems, involving up-regulation of lysosomal cathepsins and down-regulation of proteasomes.
Assuntos
Catepsinas/fisiologia , Cisteína Endopeptidases/fisiologia , Endopeptidases , Complexos Multienzimáticos/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Animais , Catepsina B/genética , Catepsina B/metabolismo , Catepsina H , Catepsina L , Catepsinas/genética , Catepsinas/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Cisteína Endopeptidases/genética , Expressão Gênica , Camundongos , Complexos Multienzimáticos/genética , Complexo de Endopeptidases do Proteassoma , Biossíntese de ProteínasRESUMO
Protein breakdown was monitored in C2C12 myotubes as the rate of release of radioactivity after prelabeling cell protein with [3H] tyrosine. IGF-1 (13 nM) and insulin (100 nM) prolonged the half-life of long-lived proteins. Enzymatic activities of cathepsins B and B+L were inhibited by the addition of IGF-1 or insulin. Immunoblotting of cathepsins B and L revealed extensive degradation of heavy chain forms by IGF-1. However, neither expression of cathepsins B and L genes nor expression of cystatin beta, an intrinsic inhibitor for cathepsins, was influenced. The addition of E-64, L-3-carboxy-trans-2,3-epoxypropionyl-leucylamide-(4-guanidin o) butane, a inhibitor of cathepsins B and L, increased protein contents of heavy chains of cathepsins B and L in the IGF-1 treated cells. Inhibition of protein breakdown by IGF-1 is mediated by autocatalytic inactivation of lysosomal cathepsins B and L.
Assuntos
Catepsinas/antagonistas & inibidores , Endopeptidases , Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Lisossomos/enzimologia , Proteínas/metabolismo , Análise de Variância , Animais , Northern Blotting , Catepsina B/biossíntese , Catepsina L , Catepsinas/biossíntese , Linhagem Celular , Cistatinas/biossíntese , Cisteína Endopeptidases , Inibidores de Cisteína Proteinase/farmacologia , Meia-Vida , Insulina/farmacologia , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos , Músculo Esquelético , Biossíntese de Proteínas , RNA/biossíntese , RNA/metabolismo , RatosRESUMO
Subunit c is normally present as an inner mitochondrial membrane component of the F0 section of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. To explore the mechanism of storage of subunit c, the rates of degradation and synthesis of subunit c were measured in fibroblast cell types from controls and patients with the late infantile form of NCL. The radiolabel from subunit c decreased with time in control cells, whereas no apparent loss of radioactivity of subunit c was found in patients' cells. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. A combination of pulse-chase and subcellular fractionation analysis showed that a delay of intramitochondrial loss from prelabeled subunit c was seen in all diseased cells tested. Lysosomal appearance of labeled subunit c could be detected after chase for more than 1 week and its radioactivities were variable among diseased cell types. The biosynthetic rate of subunit c was almost the same in both control and patient cells. Northern blotting analyses showed that mRNAs for P1 and P2 genes had no significant difference in lengths and amounts between control and patient cells. Results suggest a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes. This is the first direct evidence to show a delay of subunit c degradation in the cells from the late infantile form of NCL.
Assuntos
Mitocôndrias/enzimologia , Lipofuscinoses Ceroides Neuronais/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Fibroblastos/enzimologia , Expressão Gênica , Humanos , Lactente , Lisossomos/enzimologia , ATPases Translocadoras de Prótons/genética , Pele/enzimologia , Fatores de TempoRESUMO
The proteolytic processes involved in the conversion of procathepsin L to cathepsin L on a negatively charged surface, dextran sulfate, were studied. Upon incubation for 30 min at 37 degrees C, pH 5.5 with dextran-sulfate and dithiothreitol, purified procathepsin L showed maximal activation and, correspondingly, the complete conversion to the 30 kDa, single chain mature form of enzyme was observed. In contrast, incubation under the same conditions on ice rather than at 37 degrees C for 30 or 60 min resulted in partial proteolysis to produce a 31 kDa form without a significant increase in activity. Amino terminal amino acid sequence analyses showed that the 30 kDa form obtained by incubation at 37 degrees C corresponds to the purified form of mature cathepsin L with a 2 amino acid extension at the amino terminal, and that the 31 kDa form generated by incubation on ice possesses a 6 amino acid amino terminal extension, suggesting that the activation and processing of procathepsin L are different processes, and that 4 amino acid residues (Glu-Pro-Leu-Met) at the carboxyterminal in the propeptide function to prevent the activation of processed cathepsin L.
Assuntos
Catepsinas/metabolismo , Endopeptidases , Precursores Enzimáticos/metabolismo , Processamento de Proteína Pós-Traducional , Células 3T3 , Sequência de Aminoácidos , Animais , Catepsina L , Catepsinas/isolamento & purificação , Linhagem Celular Transformada , Cisteína Endopeptidases , Sulfato de Dextrana , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Precursores Enzimáticos/isolamento & purificação , Genes ras , Cinética , Camundongos , Dados de Sequência Molecular , Peso MolecularRESUMO
The biosynthesis and processing of a lysosomal cysteine proteinase, cathepsin C (dipeptidylaminopeptidase I), was investigated by pulse-chase experiments in cultured rat macrophages. Cathepsin C is first synthesized as procathepsin C with a molecular mass of 55 kDa. Procathepsin C is then cleaved and modified within 1 h into mature cathepsin C with two chains of 25 and 7.8 kDa. A combination of pulse-chase experiments and the subcellular fractionation analysis showed that procathepsin C and cathepsin C are located in low-buoyant-density organelles and lysosomes, respectively. The reactivity of endoglycosidase H and N-glycanase and analysis of phosphorylation indicated that both precursor and mature cathepsin C are phosphorylated and N-glycosylated to give a high-mannose-type. The addition of 300-kDa mannose 6-phosphate receptor antiserum to the chase medium caused extensive release of procathepsin C into the medium, whereas the addition of control serum did not. The membrane association of procathepsin C was tested by successive extraction of cells pulse labeled for 75 min with hypotonic buffer, alkaline solution, and Triton X-100. Procathepsin C was totally extracted by hypotonic solution, whereas procathepsin D was a membrane-associated form requiring Triton X-100 for its extraction. Gel-filtration chromatography analysis of the pulse-labeled products revealed that the precursor product exists as an oligomeric form. It is suggested that the oligomerization of cathepsin C occurs before its entry into lysosomes.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/biossíntese , Precursores Enzimáticos/metabolismo , Lisossomos/metabolismo , Macrófagos Peritoneais/enzimologia , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Anticorpos , Autorradiografia , Transporte Biológico , Catepsina C , Catepsina D/biossíntese , Catepsina D/isolamento & purificação , Células Cultivadas , Centrifugação com Gradiente de Concentração , Dipeptidil Peptidases e Tripeptidil Peptidases/isolamento & purificação , Precursores Enzimáticos/isolamento & purificação , Cinética , Metionina/metabolismo , Dados de Sequência Molecular , Peso Molecular , Ratos , Ratos Wistar , Radioisótopos de EnxofreRESUMO
Elastinolytic enzymes derived from alveolar macrophages (AM) are considered to play an important role in the development of emphysema associated with cigarette smoking. In this study, the enzyme activity and mRNA expression of cathepsin L were quantitated in AM and bronchoalveolar lavage (BAL) fluid obtained from current smokers and compared with those from nonsmokers. Activity was measured with the synthetic substrate Z-Phe-Arg-MCA combined with a novel cathepsin B inhibitor, CA-074. We found that the specific activity of cathepsin L was significantly elevated in BAL cells from smokers (7.1 +/- 0.7 mumol/mg protein/h, mean +/- SEM) compared with cells from nonsmokers (2.9 +/- 0.3) (p < 0.01). The expression of cathepsin L mRNA in BAL cells as determined by dot-blot analysis was also higher in BAL cells from smokers, which was comparable to the increase in the enzyme activity. About 5 to 6% of the specific activity of cathepsin L in BAL cell lysates was detected in unconcentrated BAL fluid; specific activity was also significantly higher in samples from smokers (0.38 +/- 0.04 mumol/mg protein/h) than from nonsmokers (0.14 +/- 0.02). In addition, procathepsin L (42 kD) and the mature form of cathepsin L (33 kD) were demonstrated in BAL fluid by immunoblot analyses. These data suggest that cigarette smoking induces mRNA expression and the synthesis of cathepsin L in AM and the release of procathepsin from AM into extracellular milieu. Furthermore, increased activity levels of cathepsin L in extracellular compartments may contribute to the proteolysis of elastin in the process of lung destruction associated with cigarette smoking.
Assuntos
Líquido da Lavagem Broncoalveolar/enzimologia , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Endopeptidases , Macrófagos Alveolares/enzimologia , Fumar/metabolismo , Adulto , Western Blotting , Catepsina B/análise , Catepsina B/metabolismo , Catepsina L , Catepsinas/análise , Catepsinas/genética , Cisteína Endopeptidases/análise , Cisteína Endopeptidases/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Macrófagos Alveolares/química , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fumar/genética , Especificidade por SubstratoRESUMO
Procathepsin L was purified to apparent homogeneity from the culture medium of v-Ha-ras transformed NIH3T3 (Ras-NIH) cells in three steps; anion-exchange chromatography, gel filtration, and re-gel filtration. SDS-PAGE analyses revealed that the purified samples contained only the precursor form, procathepsin L, but not the mature enzyme, cathepsin L. Antibodies against purified procathepsin L were raised. These recognized both rat cathepsin L and the purified procathepsin L. To isolate procathepsin L-specific antibodies that did not recognize cathepsin L, sequential affinity chromatography procedures were carried out. Immunoblot analyses showed that the procathepsin L-specific antibodies recognized only procathepsin L, but not cathepsin L.