RESUMEN
Keratin 8 (K8) and keratin 18 (K18) are the intermediate filament proteins whose phosphorylation/transamidation associate with their aggregation in Mallory-Denk bodies found in patients with various liver diseases. However, the functions of other post-translational modifications in keratins related to liver diseases have not been fully elucidated. Here, using a site-specific mutation assay combined with nano-liquid chromatography-tandem mass spectrometry, we identified K8-Lys108 and K18-Lys187/426 as acetylation sites, and K8-Arg47 and K18-Arg55 as methylation sites. Keratin mutation (Arg-to-Lys/Ala) at the methylation sites, but not the acetylation sites, led to decreased stability of the keratin protein. We compared keratin acetylation/methylation in liver disease-associated keratin variants. The acetylation of K8 variants increased or decreased to various extents, whereas the methylation of K18-del65-72 and K18-I150V variants increased. Notably, the highly acetylated/methylated K18-I150V variant was less soluble and exhibited unusually prolonged protein stability, which suggests that additional acetylation of highly methylated keratins has a synergistic effect on prolonged stability. Therefore, the different levels of acetylation/methylation of the liver disease-associated variants regulate keratin protein stability. These findings extend our understanding of how disease-associated mutations in keratins modulate keratin acetylation and methylation, which may contribute to disease pathogenesis.-Jang, K.-H., Yoon, H.-N., Lee, J., Yi, H., Park, S.-Y., Lee, S.-Y., Lim, Y., Lee, H.-J., Cho, J.-W., Paik, Y.-K., Hancock, W. S., Ku, N.-O. Liver disease-associated keratin 8 and 18 mutations modulate keratin acetylation and methylation.
Asunto(s)
Queratina-18/genética , Queratina-18/metabolismo , Queratina-8/genética , Queratina-8/metabolismo , Hepatopatías/genética , Hepatopatías/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Acetilación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular , Cricetinae , Células HT29 , Humanos , Queratina-18/química , Queratina-8/química , Cuerpos de Mallory/metabolismo , Metilación , Proteínas Mutantes/química , Mutación Missense , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Espectrometría de Masas en TándemRESUMEN
This paper is number 2 in a series developed through a partnership between ISIMM and NordiQC for the purpose of reporting research assessing the performance characteristics of immunoassays in an external proficiency testing program.
Asunto(s)
Inmunoensayo , Queratina-18/química , Queratina-8/química , Humanos , Ensayos de Aptitud de Laboratorios , Peso Molecular , Control de CalidadRESUMEN
The intermediate filament protein keratin 8 (K8) interacts with the nucleotide-binding domain 1 (NBD1) of the cystic fibrosis (CF) transmembrane regulator (CFTR) with phenylalanine 508 deletion (ΔF508), and this interaction hampers the biogenesis of functional ΔF508-CFTR and its insertion into the plasma membrane. Interruption of this interaction may constitute a new therapeutic target for CF patients bearing the ΔF508 mutation. Here, we aimed to determine the binding surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify the NBD1 fragments perturbed by the ΔF508 mutation, we used hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) on recombinant wild-type (wt) NBD1 and ΔF508-NBD1 of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head domain, and extended this investigation using bioinformatics procedures and surface plasmon resonance, which revealed regions affected by the peptide binding in both wt-NBD1 and ΔF508-NBD1. Finally, we performed HDX-MS analysis of the NBD1 molecules and full-length K8, revealing hydrogen-bonding network changes accompanying complex formation. In conclusion, we have localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also confirm the stronger binding of K8 to ΔF508-NBD1, which is supported by an additional binding site located in the vicinity of the ΔF508 mutation in NBD1.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Queratina-8/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Medición de Intercambio de Deuterio , Humanos , Queratina-8/genética , Queratina-8/metabolismo , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Dominios ProteicosRESUMEN
Most autotransporter passenger domains, regardless of their diversity in function, fold or are predicted to fold as right-handed ß-helices carrying various loops that are presumed to confer functionality. Our goal here was to identify the subdomain (loop) or amino acid sequence of the Pet passenger domain involved in the receptor binding site on the host cell for Pet endocytosis. Here, we show that d1 and d2 subdomains, as well as the amino acid sequence linking the subdomain d2 and the adjacent ß-helix (PDWET), are not required for Pet secretion through the autotransporter system and that none of our deletion mutants altered the predicted long right-handed ß-helical structure. Interestingly, Pet lacking the d2 domain (PetΔd2) was unable to bind on the epithelial cell surface, in contrast to Pet lacking d1 (PetΔd1) subdomain or PDWET sequences. Moreover, the purified d1 subdomain, the biggest subdomain (29.8 kDa) containing the serine protease domain, was also unable to bind the cell surface. Thus, d2 sequence (54 residues without the PDWET sequence) was required for Pet binding to eukaryotic cells. In addition, this d2 sequence was also needed for Pet internalization but not for inducing cell damage. In contrast, PetΔd1, which was able to bind and internalize inside the cell, was unable to cause cell damage. Furthermore, unlike Pet, PetΔd2 was unable to bind cytokeratin 8, a Pet receptor. These data indicate that the surface d2 subdomain is essential for the ligand-receptor (Pet-Ck8) interaction for Pet uptake and to start the epithelial cell damage by this toxin.
Asunto(s)
Enterotoxinas/metabolismo , Células Epiteliales/metabolismo , Queratina-8/metabolismo , Dominios y Motivos de Interacción de Proteínas , Sistemas de Secreción Tipo V/metabolismo , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Enterotoxinas/química , Enterotoxinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Queratina-8/química , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Sistemas de Secreción Tipo V/genéticaRESUMEN
UNLABELLED: Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated keratins 8/18 (K8/K18). MDBs are characteristic of alcoholic and nonalcoholic steatohepatitis (NASH) and discriminate between the relatively benign simple steatosis and the more aggressive NASH. Given the emerging evidence for a genetic predisposition to MDB formation and NASH development in general, we studied whether high-fat (HF) diet triggers MDB formation and liver injury in susceptible animals. Mice were fed a high-fat (HF) or low-fat (LF) diet plus a cofactor for MDB development, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Additionally, we fed nontransgenic and K8 overexpressing mice (K8tg) with the HF diet. The presence of MDB and extent of liver injury was evaluated using biochemical markers, histological staining, and immunofluorescence microscopy. In DDC-fed animals, an HF diet resulted in greater liver injury and up-regulation of inflammation-related genes. As a potential mechanism, K8/K18 accumulation and increased ecto-5'-nucleotidase (CD73) levels were noted. In the genetically susceptible K8tg mice, HF diet triggered hepatocellular injury, ballooning, apoptosis, inflammation, and MDB development by way of 1) decreased expression of the major stress-inducible chaperone Hsp72 with appearance of misfolded keratins; 2) elevated levels of the transglutaminase 2 (TG2); 3) increased K8 phosphorylation at S74 with subsequent TG2-mediated crosslinking of phosphorylated K8; and 4) higher production of the MDB-modifier gene CD73. CONCLUSION: Our data demonstrate that HF diet triggers aggregate formation and development of liver injury in susceptible individuals through misfolding and crosslinking of excess K8.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Hígado Graso/patología , Queratina-8/química , Cuerpos de Mallory/química , Cuerpos de Mallory/patología , Deficiencias en la Proteostasis/patología , Animales , Colestasis/metabolismo , Colestasis/patología , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/metabolismo , Dieta con Restricción de Grasas , Hígado Graso/metabolismo , Queratina-8/genética , Queratina-8/metabolismo , Masculino , Cuerpos de Mallory/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Enfermedad del Hígado Graso no Alcohólico , Pliegue de Proteína , Deficiencias en la Proteostasis/metabolismoRESUMEN
The deletion of Phe508 (ΔF508) in the first nucleotide binding domain (NBD1) of CFTR is the most common mutation associated with cystic fibrosis. The ΔF508-CFTR mutant is recognized as improperly folded and targeted for proteasomal degradation. Based on molecular dynamics simulation results, we hypothesized that interaction between ΔF508-NBD1 and housekeeping proteins prevents ΔF508-CFTR delivery to the plasma membrane. Based on this assumption we applied structure-based virtual screening to identify new low-molecular-weight compounds that should bind to ΔF508-NBD1 and act as protein-protein interaction inhibitors. Using different functional assays for CFTR activity, we demonstrated that in silico-selected compounds induced functional expression of ΔF508-CFTR in transfected HeLa cells, human bronchial CF cells in primary culture, and in the nasal epithelium of homozygous ΔF508-CFTR mice. The proposed compounds disrupt keratin8-ΔF508-CFTR interaction in ΔF508-CFTR HeLa cells. Structural analysis of ΔF508-NBD1 in the presence of these compounds suggests their binding to NBD1. We conclude that our strategy leads to the discovery of new compounds that are among the most potent correctors of ΔF508-CFTR trafficking defect known to date.
Asunto(s)
Bronquios/citología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Animales , Sitios de Unión , Bronquios/efectos de los fármacos , Bronquios/fisiología , Células Cultivadas , Canales de Cloruro/metabolismo , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Evaluación Preclínica de Medicamentos , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Células HeLa , Homocigoto , Humanos , Queratina-8/química , Queratina-8/metabolismo , Ratones , Técnicas de Placa-Clamp , Unión Proteica , Mapas de Interacción de Proteínas/efectos de los fármacos , Estructura Terciaria de Proteína , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Parkin was originally identified as a protein associated with Parkinson's disease. Recently, numerous research studies have suggested that parkin acts as a tumor suppressor. In accordance with these studies, we previously reported that overexpression of parkin in HeLa cells induced growth inhibition. To elucidate possible mechanisms by which parkin may inhibit cell growth, HeLa cells were infected with adenoviruses expressing either the parkin gene or adenovirus alone for 72 h and a total proteomic analysis was performed using 2-D gel electrophoresis followed by LC-MS/MS. We identified three proteins whose expression changed between the two groups: the 40S ribosomal protein SA (RPSA) was downregulated in parkin virus-infected cells, and cytokeratins 8 and 18 exhibited an acid shift in pI value without a change in molecular weight, suggesting that these proteins became phosphorylated in parkin virus-infected cells. The changes in these three proteins were first observed at 60 h postinfection and were most dramatic at 72 h postinfection. Because upregulation of RPSA and dephosphorylation of cytokeratins 8/18 have been linked with tumor progression, these data suggest that parkin may inhibit cell growth, at least in part, by decreasing RPSA expression and inducing phosphorylation of cytokeratin 8/18.
Asunto(s)
Queratina-18/metabolismo , Queratina-8/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptores de Laminina/metabolismo , Proteínas Ribosómicas/metabolismo , Ubiquitina-Proteína Ligasas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Neoplasias del Cuello Uterino/patología , Secuencia de Aminoácidos , Proliferación Celular/efectos de los fármacos , Femenino , Células HeLa , Humanos , Queratina-18/química , Queratina-8/química , Datos de Secuencia Molecular , Receptores de Laminina/química , Proteínas Ribosómicas/químicaRESUMEN
Serum PSA, Gleason score, pathological stage, and positive surgical margins are currently used as predictors for disease recurrence. However, these criteria are less than precise in predicting disease outcome, with only 10% specificity at the 90% sensitivity level. Keratins are intermediate filament proteins that are contained within normal epithelia. However, human prostate cancer tissue shows differential immunohistochemical staining of keratin 8 (K8) when compared to normal prostate tissue. Our immunofluorescence and flow cytometry data show that K8 is also present on the cell surface of transformed prostate cancer cell lines. K8 is expressed at high levels on the surfaces of DU-145 and PC-3 cells but is expressed at comparatively lower levels on the surfaces of LNCaP cells, BPH-1 cells, and RWPE-1 cells. We hypothesize that extracellular K8 (eK8) present on epithelial prostate cancer cells plays an integral role in migration and in vivo dissemination. We found that K8 increased the rate of activity of plasmin approximately fivefold over a 48-h period. Functionally, K8 also enhanced the plasmin-mediated proteolysis of vitronectin, an important component of the prostate extracellular matrix. Taken together, our data show that K8 enhances the proteolytic activity of the plasminogen activation system, indicating that eK8 may be an important distinguishing marker in prostate cancer and progression.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Fibrinolisina/metabolismo , Queratina-8/metabolismo , Neoplasias de la Próstata/metabolismo , Anticuerpos Monoclonales , Biomarcadores de Tumor/química , Ciclo Celular , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Humanos , Inmunohistoquímica , Queratina-8/química , Masculino , Microscopía Fluorescente , Neoplasias de la Próstata/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sensibilidad y Especificidad , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Vitronectina/metabolismoRESUMEN
We have generated human recombinant keratins K8 and K18 and describe conditions to quantitatively follow their assembly into filaments. When renatured individually from 8M urea into a low ionic strength/high pH-buffer, K8 was present in a dimeric to tetrameric form as revealed by analytical ultracentrifugation. In contrast, K18 sedimented as a monomer. When mixed in 8 M urea and renatured together, K8 and K18 exhibited s-value profiles compatible with homogeneous tetrameric complexes. This finding was confirmed by sedimentation equilibrium centrifugation. Subsequently, these tetrameric starter units were subjected to assembly experiments at various protein concentrations. At low values such as 0.0025 g/l, unit-length filaments were abundantly present after 2s of assembly. During the following 5 min, filaments grew rapidly and by measuring the length of individual filaments we were able to generate time-dependent length profiles. These data revealed that keratins K8/K18 assemble several times faster than vimentin and desmin. In addition, we determined the persistence length l(p) of K8/K18 filaments to be in the range of 300 nm. Addition of 1 mM MgCl(2) increases l(p) to 480 nm indicating that magnesium ions affect the interaction of keratin subunits within the filament during assembly to some extent.
Asunto(s)
Citoesqueleto/química , Queratina-18/química , Queratina-8/química , Desmina/química , Humanos , Queratina-18/genética , Queratina-8/genética , Cinética , Microscopía Electrónica/métodos , Proteínas Recombinantes/química , Vimentina/químicaRESUMEN
BACKGROUND & AIMS: Mallory-Denk bodies (MDBs) are cytoplasmic protein aggregates in hepatocytes in steatohepatitis and other liver diseases. We investigated the molecular structure of keratin 8 (K8) and 18 (K18), sequestosome 1/p62, and ubiquitin, which are the major constituents of MDBs, to investigate their formation and role in disease pathogenesis. METHODS: Luminescent conjugated oligothiophenes (LCOs), h-HTAA, and p-FTAA are fluorescent amyloid ligands that specifically bind proteins with cross ß-sheet conformation. We used LCOs to investigate conformational changes in MDBs in situ in human and murine livers as well as in transfection studies. RESULTS: LCO analysis showed cross ß-sheet conformation in human MDBs from patients with alcoholic and nonalcoholic steatohepatitis or hepatocellular carcinoma, but not in intracellular hyaline bodies, α1-antitrypsin deficiency, or ground-glass inclusions. LCOs bound to MDBs induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding of mice at all developmental stages. CHO-K1 cells transfected with various combinations of SQSTM1/p62, ubi, and Krt8/Krt18 showed that K8 was more likely to have cross ß-sheet conformation than K18, whereas p62 never had cross ß-sheet conformation. The different conformational properties of K8 and K18 were also shown by circular dichroism analysis. CONCLUSIONS: K8 can undergo conformational changes from predominantly α-helical to cross ß-sheet, which would allow it to form MDBs. These findings might account for the observation that krt8â»/â» mice do not form MDBs, whereas its excess facilitates MDB formation. LCOs might be used in diagnosis of liver disorders; they can be applied to formalin-fixed, paraffin-embedded tissues to characterize protein aggregates in liver cells.
Asunto(s)
Hepatocitos/metabolismo , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/ultraestructura , Queratina-8/química , Queratina-8/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células CHO , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Cricetinae , Cricetulus , Hígado Graso/metabolismo , Hígado Graso/patología , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Hepatocitos/patología , Hepatocitos/ultraestructura , Humanos , Queratina-18/química , Queratina-18/metabolismo , Queratina-8/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Noqueados , Estructura Secundaria de Proteína , Proteína Sequestosoma-1 , TransfecciónRESUMEN
AIM: To construct an eukaryotic expression vector containing the coding region of human full length cytokeratin 8 gene and to detect its expression in SMMC7721 cells. METHODS: CK8 cDNA was amplified by RT-PCR and cloned to pMD18-T simple vector. After confirming the sequence, the cDNA was inserted into pEGFP-C1 and the positive clone pEGFP-CK8 was obtained. The recombinant plasmid was transfected into SMMC7721 cells with Lipofectamine(TM);2000 and the expression was detected by fluorescence microscope, real time PCR and Western blot. The physical-chemical properties, signal peptide and functional motifs were predicted by the bioinformatics software. RESULTS: PCR, restriction enzyme digestion and DNA sequencing showed that the recombinant plasmid contained the coding region of full length CK8 gene. Observation under fluorescence microscope and the results of real time PCR and western blot indicated CK8 was over-expressed in SMMC7721 cells. CONCLUSION: The eukaryotic expression vector containing the CK8 gene was successfully constructed and expressed, which provides a basis for the study for biological function of CK8.
Asunto(s)
Biología Computacional , Regulación de la Expresión Génica , Queratina-8/genética , Queratina-8/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Línea Celular Tumoral , Clonación Molecular , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Queratina-8/química , TransfecciónRESUMEN
Keratins 8 and 18 (K8/18) are intermediate filament proteins expressed specifically in simple epithelial tissues. Dynamic equilibrium of these phosphoglycoproteins in the soluble and filament pool is an important determinant of their cellular functions, and it is known to be regulated by site-specific phosphorylation. However, little is known about the role of dynamic O-GlcNAcylation on this keratin pair. Here, by comparing immortalized (Chang) and transformed hepatocyte (HepG2) cell lines, we have demonstrated that O-GlcNAcylation of K8/18 exhibits a positive correlation with their solubility (Nonidet P-40 extractability). Heat stress, which increases K8/18 solubility, resulted in a simultaneous increase in O-GlcNAc on these proteins. Conversely, increasing O-GlcNAc levels were associated with a concurrent increase in their solubility. This was also associated with a notable decrease in total cellular levels of K8/18. Unaltered levels of transcripts and the reduced half-life of K8 and K18 indicated their decreased stability on increasing O-GlcNAcylation. On the contrary, the K18 glycosylation mutant (K18 S29A/S30A/S48A) was notably more stable than the wild type K18 in Chang cells. The K18-O-GlcNAc mutant accumulated as aggregates upon stable expression, which possibly altered endogenous filament architecture. These results strongly indicate the involvement of O-GlcNAc on K8/18 in regulating their solubility and stability, which may have a bearing on the functions of these keratins.
Asunto(s)
Acetilglucosamina/química , Queratina-18/química , Queratina-8/química , Línea Celular Transformada , Línea Celular Tumoral , Glicosilación , Hepatocitos/metabolismo , Humanos , Filamentos Intermedios/química , Mutación , Fosforilación , Complejo de la Endopetidasa Proteasomal/química , Conformación Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SolubilidadRESUMEN
Keratins 8 and 18 (K8/18) intermediate filament proteins are believed to play an essential role in the protection of hepatocytes against mechanical and toxic stress. This assertion is mainly based on increased hepatocyte fragility observed in transgenic mice deficient in K8/18, or carrying mutations on K8/18. The molecular mechanism by which keratins accomplish their protective functions has not been totally elucidated. Liver diseases such as alcoholic hepatitis and copper metabolism diseases are associated with modifications, in hepatocytes, of intermediate filament organisation and the formation of K8/18 containing aggregates named Mallory-Denk bodies. Treatment of mice with a diet containing griseofulvin induces the formation of Mallory-Denk bodies in hepatocytes. This provides a reliable animal model for assessing the molecular mechanism by which keratins accomplish their protective role in the response of hepatocytes to chemical injuries. In this study, we found that griseofulvin intoxication induced changes in keratin solubility and that there was a 5% to 25% increase in the relative amounts of soluble keratin. Keratin phosphorylation on specific sites (K8 pS79, K8 pS436 and K18 pS33) was increased and prominent in the insoluble protein fractions. Since at least six K8 phosphoepitopes were detected after GF treatment, phosphorylation sites other than the ones studied need to be accounted for. Immunofluorescence staining showed that K8 pS79 epitope was present in clusters of hepatocytes that surrounded apoptotic cells. Activated p38 MAPK was associated with, but not present in K8 pS79-positive cells. These results indicate that griseofulvin intoxication mediates changes in the physicochemical properties of keratin, which result in the remodelling of keratin intermediate filaments which in turn could modulate the signalling pathways in which they are involved by modifying their binding to signalling proteins.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Griseofulvina/envenenamiento , Queratina-18/metabolismo , Queratina-8/química , Hígado/metabolismo , Animales , Hepatocitos/metabolismo , Queratina-18/genética , Queratina-8/genética , Hígado/fisiología , Ratones , Ratones Endogámicos C3H , Fosforilación , SolubilidadRESUMEN
Using an optical biosensor Biacore 3000 the interaction of human recombinant cytokeratins (CK) with isatin analogues (5-aminocaproyl-isatin and 5-aminoisatin) immobilized on the CM-5 chip has been investigated. CK-14 effectively interacted with 5-aminocaproyl-isatin immobilized on the carboxymethyl dextran chip surface, but not with a "shorter" analogue (5-aminoisatin). In contrast to CK14 CK8 effectively interacted only with 5-aminoisatin. In both cases cytokeratin binding with the immobilized isatin analogues was characterized by rather high affinity (Kd of 0.7 microM for the pair CK14/immobilized 5-aminocaproylisatin and 1.7 microM for the pair CK8/immobilized 5-aminoisatin). CK20 did not interact with both immobilized isatin analogues. Taking into consideration non-specific binding of mouse CK14 and rat CK8 with 5-aminocaproyl-Sepharose we have performed comparative analysis of amino acid sequences of human, mouse, and rat CK8 and CK14. The data obtained suggest that in the case of human, mouse, and rat CK14 the N-terminal domain is the most variable among these species, whereas the major differences between amino acid sequences of human, mouse, and rat CK8 have been found both in N-terminal and C-terminal regions.
Asunto(s)
Isatina/análogos & derivados , Isatina/química , Queratina-14/química , Queratina-20/química , Queratina-8/química , Secuencia de Aminoácidos , Animales , Técnicas Biosensibles , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Ratas , Proteínas Recombinantes/químicaRESUMEN
BACKGROUND: Generation of plasmin is a characteristic of tumor cells, promoting the degradation of extracellular matrix, tumor progression and metastasis. The process is accelerated if plasminogen and plasminogen activator are bound to their cell surface receptors. RESULTS: In this study we show that the monoclonal antibody that recognizes an epitope on the cytokeratin 8 (CK8) ectoplasmic domain (anti-CK MAb) inhibits plasminogen activation mediated by urokinase-type plasminogen activator (uPA) in MCF-7 and MCF-10A neoT cells. The ectoplasmic domain of CK8 acts as a binding site for plasminogen, however, by using confocal microscopy, we demonstrated that it is also co-localized with uPA. CK8, therefore, function also as a receptor for uPA on the cell surface, and the presence of anti-CK MAb may prevent the binding of uPA to a designated CK8 motif. The consequent inhibition of plasmin generation resulted in changed cell morphology, enhanced cell adhesion to fibronectin, reduced invasion potential, and an enhanced G1/S transition. Moreover, surface plasmon resonance analysis showed that the synthetic dodecapeptide corresponding to the epitope sequence (VKIALEVEIATY), binds uPA in the nanomolar range. CONCLUSION: These novel findings suggest a model in which CK8, together with uPA, plasminogen and fibronectin, constitutes a signaling platform capable of modulating cell adhesion/growth-dependent signal transduction in breast tumor cells. Anti-CK MAb, which competes for the binding site for uPA, could be used as an agent to reduce the invasive potential of breast tumor cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Queratina-8/química , Queratina-8/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Femenino , Fibronectinas/farmacología , Fase G1/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Invasividad Neoplásica , Péptidos/química , Plasminógeno/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Fase S/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The application of a hypothesis-driven method for the sensitive determination of lysine acetylation sites on enzymatically digested proteins is described. Comparative sensitivity tests were carried out using serial dilution of an acetylated bovine serum albumin (AcBSA) digest to assess the performance of a multiple reaction monitoring (MRM)-based approach as compared to a more conventional precursor scanning (PS) method. Both methods were capable of selectively detecting an acetylated peptide at the low femtomole level when spiked into a background of 500 fmol six-protein tryptic digest. The MRM approach was roughly tenfold more sensitive than precursor scanning with one acetylated peptide detected and sequenced at the level of 2 fmol on-column. The technique was subsequently applied to a gel-derived sample of cytokeratin-8 (CK8) shown to contain acetylated lysine residues by Western blot analysis. The strategy applied herein, termed MRM-initiated detection and sequencing (MIDAS), resulted in the facile identification of novel sites of acetylation on this protein.