RESUMO
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
A genomic DNA clone encompassing the gene (cy beta) encoding rat cystatin beta (Cy beta) was isolated by screening with a rat cy beta cDNA as a probe. The gene spans about 2.6 kb and comprises three exons. The first intron is located between Lys22 and Val23 and the second between Lys56 and Val57 in the deduced amino acid sequence of Cy beta. The second exon contains the highly conserved QVVAG sequence which, unlike the sequence of other cystatin family members, is not split by an intron. In the 5'-upstream region, three SP-1-binding sites exist, but no typical TATA-box or CAAT-box sequences are found. The difference in the organization of the rat cy beta gene, encoding a family-1 cystatin, from that encoding members of the other cystatin families, suggests that cy beta diverged from a common ancestral gene earlier than the separation of genes encoding family-2 and family-3 cystatins.
Assuntos
Cistatinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Cistatina M , DNA/isolamento & purificação , Sondas de DNA , Éxons , Íntrons , Dados de Sequência Molecular , Família Multigênica , Ratos , Mapeamento por Restrição , Alinhamento de SequênciaRESUMO
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 structure of rat cathepsin L gene has been determined. The gene spans 8.5 kilobase pairs comprising 8 exons, and has an intron located near the active site cysteine residue. The gene structure does not correspond well to the functional units of the proteinase. These characteristics are found to be in common with the cysteine proteinase gene family. In the 5'-upstream region, one CAAT-box and four SP-1 binding sites, together with two AP-2 binding sites and CRE, but no typical TATA-box are found. Further, SP-1 and AP-2 binding sites and an octamer motif are also found in the 1st intron, suggesting a complex regulatory mechanism for the expression of the cathepsin L gene.
Assuntos
Catepsinas/genética , Endopeptidases , Genes , Sequências Reguladoras de Ácido Nucleico , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina L , Clonagem Molecular , Cisteína Endopeptidases , Regulação Enzimológica da Expressão Gênica , Íntrons , Dados de Sequência Molecular , Ratos , Mapeamento por Restrição , Transcrição GênicaRESUMO
The gene structure of rat cathepsin H was determined. It comprises at least 12 exons of various lengths (32-433 bp) spanning in total more than 17.5 kbp. The gene structure does not correspond well to the functional unit of the proteinase. The region around the active site Cys residue, the most conserved region among cysteine proteinases, is split by an intron. This is a common characteristic among the gene structures of cysteine proteinases.
Assuntos
Catepsinas/genética , Cisteína Endopeptidases , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina H , DNA/genética , Éxons , Genes , Dados de Sequência Molecular , Ratos , Mapeamento por RestriçãoRESUMO
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.
Assuntos
Anticorpos/imunologia , Catepsinas/imunologia , Endopeptidases , Precursores Enzimáticos/imunologia , Células 3T3 , Animais , Especificidade de Anticorpos , Western Blotting , Catepsina L , Catepsinas/isolamento & purificação , Cromatografia em Gel , Cisteína Endopeptidases , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Camundongos , RatosRESUMO
A near full-length cDNA for rat cathepsin L was isolated. The deduced protein comprises 334 amino acid residues (Mr 37,685) containing a typical signal sequence (N-terminal 17 residues), pro-peptide (96 residues), and the sequence for mature cathepsin L (221 residues). Rat cathepsin L shows 94% amino acid identity with mouse cysteine proteinase. Amino acid sequence homologies of rat cathepsin L with rat cathepsins H and B are 45 and 25%, respectively. These facts indicate that mouse cysteine proteinase is probably mouse cathepsin L and that cathepsin L is more closely related to cathepsin H than cathepsin B.
Assuntos
Catepsinas/genética , Endopeptidases , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina L , Clonagem Molecular , Cisteína Endopeptidases , DNA/genética , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
A cDNA for rat cathepsin H was isolated and sequenced. The deduced protein comprising 333 amino acid residues is composed of a typical signal sequence (21 residues), a pro-peptide region (92 residues) and a mature enzyme region (220 residues). The amino acid sequence in the pro-peptide region, in particular, residues Phe-(-41) to Ser-(-29) of cathepsin H, is highly homologous to the pro-peptide regions of other cysteine proteinases. This homologous region may play a role in the processing of cysteine proteinases.
Assuntos
Catepsinas/genética , Clonagem Molecular , Cisteína Endopeptidases/genética , DNA/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina H , Precursores Enzimáticos/genética , Dados de Sequência Molecular , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
Peptidylarginine deiminase (PAD) is the enzyme responsible for converting protein-bound arginine residues to citrulline. It has recently been shown that a number of epidermal proteins, including filaggrin, trichohyalin, and keratins, are deiminated by the action of PAD, suggesting a possible role for protein deimination during the final stages of epidermal differentiation. We report here a novel PAD substrate found during the course of identifying deiminated proteins in cultured rat epidermal keratinocytes. We found that a 70-kD protein localized to the periphery of the nucleus was preferentially deiminated after ionomycin treatment in the presence of 2 mM calcium and was associated with apoptotic events in these cells. Furthermore, we discovered that the deimination of nuclear protein could be induced by transfection of a PAD cDNA into rat epidermal keratinocytes. These data suggest that PAD may act on the 70-kD nuclear protein to induce disassembly of the nuclear lamina and promote apoptosis during terminal epidermal differentiation.
Assuntos
Apoptose , Hidrolases/metabolismo , Queratinócitos/enzimologia , Proteínas Nucleares/metabolismo , Animais , Western Blotting , Diferenciação Celular , Linhagem Celular , Núcleo Celular/metabolismo , Fragmentação do DNA , Técnica Indireta de Fluorescência para Anticorpo , Hidrolases/genética , Ionomicina/farmacologia , Queratinócitos/efeitos dos fármacos , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Ratos , TransfecçãoRESUMO
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
Decreased lysosomal proteolysis in regenerating liver after 70% hepatectomy was analyzed. The activities of cathepsins B and L increased transiently 4 h after hepatectomy, began to decrease gradually reaching about 30% of the control level at 24 h, then returned to near control level after 7 days. Immunoblot and RNA blot analyses confirmed that the changes in cathepsin activities coincided with changes in protein levels and mRNA levels. In parallel with the changes in cathepsins, we found that the amounts of LGP120, LGP110, and LGP85, three integral lysosomal membrane proteins, declined significantly after hepatectomy, suggesting that the lysosomal levels are also diminished in regenerating liver. We isolated dextran-loaded lysosomes and found that the protein content and marker enzyme activities of dextran-loaded lysosomes from partially hepatectomized livers are lower by 50 and 40%, respectively, compared with control livers. This indicates that there is a significant reduction in the cellular lysosomal level in regenerating liver. In addition, we used a sensitive biochemical assay to quantify leupeptin-induced autolysosomes and found that the autophagic activity is markedly suppressed in regenerating liver as compared with normal liver. Thus, the suppression of lysosomal proteolysis in regenerating liver is attained through three steps, i. e., decreased biosynthesis of cathepsins, decreased lysosomal biogenesis, and decreased cellular autophagy.
Assuntos
Catepsinas/metabolismo , Regeneração Hepática , Fígado/enzimologia , Lisossomos/metabolismo , Animais , Sondas de DNA , DNA Complementar , Hepatectomia , Hidrólise , Fígado/metabolismo , Fígado/fisiologia , Masculino , Proteínas de Membrana/metabolismo , Hibridização de Ácido Nucleico , Ratos , Ratos WistarRESUMO
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
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
In order to determine the functional role of the procathepsin L propeptide region for the preparation of active recombinant rat cathepsin L (CL), cDNAs encoding two short-length propeptides (C-terminal 2 and 27 residues) and the full-length (96 residues) one plus the entire CL were expressed as two soluble fusion proteins with a fragment of maltose-binding protein and an insoluble fusion protein with glutathione-S-transferase in Escherichia coli, respectively. After refolding of the insoluble fusion protein, each gene product was purified to homogeneity by amylose or glutathione-Sepharose-4B affinity column, and digestion with factor Xa and alpha-thrombin under alkaline conditions (pH approximately 8.0) led to the elution of two pure short-length procathepsin Ls (PCLs) and a full-length one, respectively. The enzymatic activity, estimated by hydrolytic assaying of benzoxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide under acidic conditions (pH 5.5), indicated that the two short-length PCLs exhibited in a great loss of the activity, as compared with the full-length PCL. The CD spectra of the short-length PCLs were different from that of the full-length one. The present results clearly show that the full-length propeptide is essential for construction of the active tertiary structure of CL at the stage of recombinant protein expression, although the expression of CL itself in E. coli does not require the propeptide. Based on the tertiary structure of PCL, the propeptide region necessary for the construction of the CL active structure has been discussed.
Assuntos
Catepsinas/genética , Catepsinas/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Escherichia coli/genética , Animais , Domínio Catalítico , Catepsina L , Catepsinas/química , Dicroísmo Circular , Precursores Enzimáticos/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Choroideremia (CHM), an X-linked recessive hereditary disease, is an intractable chorioretinal dystrophy. The rate of disease progression of CHM reportedly shows considerable variability. A number of mutations involving the gene that codes for Rab escort protein-1 (REP-1) have been detected in CHM patients. We have analyzed REP-1 gene mutations of Japanese CHM patients. The present study was designed to investigate the clinical variability and the genotype to phenotype relationship in 15 Japanese CHM patients referred to the Department of Ophthalmology of Juntendo University Hospital. The clinical investigation of visual acuity, visual field, color vision and refraction revealed inter-individual variability. Mutation analyses of the REP-1 gene revealed 10 types of mutations in 13 patients from 11 families, including an insertion, small deletions, nonsense mutations and an A to CC mutation. In 13 CHM patients with detectable REP-1 gene mutations, no relationship of genotype to phenotype was detected. At present, we consider the REP-1 genotype to be an unreliable prognostic factor for counseling of CHM patients. In two patients from one family, no mutations were detected in coding regions of the REP-1 gene. These patients may have intron mutations of the REP-1 gene, not detectable by the techniques employed in this study, or other causative genes. Both were observed to have somewhat slower disease progression than the other 13 patients. More advanced analyses are necessary to answer questions regarding the genotype-phenotype relationship in CHM patients.
Assuntos
Alquil e Aril Transferases , Proteínas de Transporte/genética , Coroideremia/genética , Mutação/genética , Transtornos da Visão/genética , Proteínas rab de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Criança , Coroideremia/diagnóstico , Defeitos da Visão Cromática/genética , Angiofluoresceinografia , Fundo de Olho , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Miopia/genética , Transtornos da Visão/fisiopatologia , Acuidade Visual , Campos VisuaisRESUMO
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
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
BACKGROUND: CDK5 is a member of proline-directed serine/threonine kinases. Although its cDNA was originally cloned as a homologue to those for the other members of the cyclin-dependent kinase (CDK) family, CDK5 has been shown to function differently from other CDKs. CDK5 is activated by non-cyclin partners, p35 and p39, and important during brain development by influencing adhesion, migration and differentiation of neurones. OBJECTIVES: We sought to investigate the expression and functions of CDK5 in human keratinocytes. METHODS: Expression of CDK5/p35, interaction of CDK5/p35 with adhesion molecules, and its roles in cell-cell and cell-matrix adhesion were studied by reverse transcriptase-polymerase chain reaction, immunoblotting and aggregation/adhesion assays in primary cultured normal human keratinocytes from infant foreskins and a human keratinocyte HaCaT cell line. Localization of CDK5 and p35 in normal human epidermis and psoriatic epidermis was studied by immunohistochemistry. RESULTS: Both CDK5 and p35 were expressed in primary cultured keratinocytes, HaCaT cells and normal human epidermis. Roscovitine, an inhibitor of CDK5, enhanced Ca2+-dependent (cadherin-dependent) aggregation of HaCaT cells whereas it inhibited adhesion of HaCaT cells to fibronectin associated with reduced active states of beta1 integrin. Interestingly, psoriatic skin showed reduced CDK5 and p35 expression in the lower half of the epidermis, which might be associated with decreased amount of activated beta1 integrin in the epidermis of psoriatic skin. CONCLUSIONS: CDK5/p35 may be involved in cell-cell and cell-matrix adhesion in human keratinocytes by differently regulating cadherins and integrins. Furthermore, reduced expression of CDK5/p35 in the epidermis might be involved in the pathogenesis of psoriasis.
Assuntos
Junções Célula-Matriz/fisiologia , Quinases Ciclina-Dependentes/fisiologia , Queratinócitos/enzimologia , Psoríase/enzimologia , Cálcio/fisiologia , Adesão Celular/fisiologia , Agregação Celular/efeitos dos fármacos , Agregação Celular/fisiologia , Células Cultivadas , Quinase 5 Dependente de Ciclina , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Epiderme/enzimologia , Fibronectinas/metabolismo , Expressão Gênica , Humanos , Lactente , Integrina beta1/metabolismo , Queratinócitos/citologia , Proteínas do Tecido Nervoso/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , RoscovitinaRESUMO
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.