RESUMEN
The aim of this study was to assess the validity of categorization of chronic hepatitis B viral infection into stages or phases based upon measures of disease activity and viral load, assuming these phenotypes will be useful for prognostication and determining the need for antiviral therapy. We assessed the phenotype of hepatitis B of 1,390 adult participants enrolled in the Hepatitis B Research Network Cohort Study, using a computer algorithm. Only 4% were immune tolerant, while 35% had chronic hepatitis B (18% e antigen positive and 17% e antigen negative) while 23% were inactive carriers. Strikingly, 38% of participants did not fit clearly into any one of these groups and were considered indeterminant. The largest subset of indeterminants had elevated serum aminotransferases with low levels of HBV DNA (less than 10,000 iu/mL). Subsequent determination of hepatitis B phenotype on the next available laboratory tests showed that 64% remained indeterminant. These findings call into question the validity of conventional staging of hepatitis B, in large part because of the substantial proportion of patients who do not fit readily into one of the usual stages or phases. Further studies are needed of the indeterminant category of chronic hepatitis B viral infection, including assessments of whether patients in this group are perhaps in transition to another phase or if they are a distinct phenotype with a need to assess liver disease severity and need for antiviral therapy. (ClinicalTrials.gov identifier NCT01263587).
Asunto(s)
Biomarcadores , Hepatitis B Crónica/clasificación , Hepatitis B Crónica/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , ADN Viral/sangre , Femenino , Hepatitis B Crónica/virología , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Transaminasas/sangre , Carga Viral , Adulto JovenRESUMEN
Because retention of mutant alpha(1)-antitrypsin (alpha(1)-AT) Z in the endoplasmic reticulum (ER) is associated with liver disease in alpha(1)-AT-deficient individuals, the mechanism by which this aggregated glycoprotein is degraded has received considerable attention. In previous studies using stable transfected human fibroblast cell lines and a cell-free microsomal translocation system, we found evidence for involvement of the proteasome in degradation of alpha(1)-ATZ (Qu, D., Teckman, J. H., Omura, S., and Perlmutter, D. H. (1996) J. Biol. Chem. 271, 22791-22795). In more recent studies, Cabral et al. (Cabral, C. M., Choudhury, P., Liu, Y., and Sifers, R. N. (2000) J. Biol. Chem. 275, 25015-25022) found that degradation of alpha(1)-ATZ in a stable transfected murine hepatoma cell line was inhibited by tyrosine phosphatase inhibitors, but not by the proteasomal inhibitor lactacystin and concluded that the proteasome was only involved in ER degradation of alpha(1)-ATZ in nonhepatocytic cell types or in cell types with levels of alpha(1)-AT expression that are substantial lower than that which occurs in hepatocytes. To examine this important issue in further detail, in this study we established rat and murine hepatoma cell lines with constitutive and inducible expression of alpha(1)-ATZ. In each of these cell lines degradation of alpha(1)-ATZ was inhibited by lactacystin, MG132, epoxomicin, and clasto-lactacystin beta-lactone. Using the inducible expression system to regulate the relative level of alpha(1)-ATZ expression, we found that lactacystin had a similar inhibitory effect on degradation of alpha(1)-ATZ at high and low levels of alpha(1)-AT expression. Although there is substantial evidence that other mechanisms contribute to ER degradation of alpha(1)-ATZ, the data reported here indicate that the proteasome plays an important role in many cell types including hepatocytes.
Asunto(s)
Acetilcisteína/análogos & derivados , Carcinoma Hepatocelular/metabolismo , Cisteína Endopeptidasas/fisiología , Retículo Endoplásmico/metabolismo , Hepatocitos/metabolismo , Complejos Multienzimáticos/fisiología , Mutación , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Acetilcisteína/farmacología , Animales , Antibióticos Antineoplásicos/farmacología , Línea Celular , Células Cultivadas , Cisteína Endopeptidasas/metabolismo , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Células HeLa , Humanos , Lactonas/metabolismo , Leupeptinas/farmacología , Hígado/citología , Ratones , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Oligopéptidos/farmacología , Pruebas de Precipitina , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Ratas , Factores de Tiempo , Transfección , Células Tumorales CultivadasRESUMEN
The classical form of alpha 1-antitrypsin (alpha 1-AT) deficiency is associated with a mutant alpha 1-ATZ molecule that polymerizes in the endoplasmic reticulum (ER) of liver cells. A subgroup of individuals homozygous for the protease inhibitor (PI) Z allele develop chronic liver injury and are predisposed to hepatocellular carcinoma. In this study we evaluated the primary structure of alpha 1-AT in a family in which three affected members had severe liver disease associated with alpha 1-AT deficiency. We discovered that one sibling was a compound heterozygote with one PI Z allele and a second allele, the PI Z + saar allele, bearing the mutation that characterizes alpha 1-ATZ as well as the mutation that characterizes alpha 1-AT Saarbrucken (alpha 1-AT saar). The mutation in PI saar introduces a premature termination codon resulting in an alpha 1-AT protein truncated for 19 amino acids at its carboxyl terminus. Studies of a second sib with severe liver disease and other living family members did not reveal the presence of the alpha 1-AT saar mutation and therefore do not substantiate a role for this mutation in the liver disease phenotype of this family. However, studies of alpha 1-AT saar and alpha 1-ATZ + saar expressed in heterologous cells show that there is prolonged intracellular retention of these mutants even though they do not have polymerogenic properties. These results therefore have important implications for further understanding the fate of mutant alpha 1-AT molecules, the mechanism of ER retention, and the pathogenesis of liver injury in alpha 1-AT deficiency.
Asunto(s)
Retículo Endoplásmico/metabolismo , Mutación , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Alelos , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Secuencia de Bases , Células CHO , Carcinoma Hepatocelular/genética , Codón de Terminación , Cricetinae , Análisis Mutacional de ADN , Exones , Salud de la Familia , Femenino , Predisposición Genética a la Enfermedad , Heterocigoto , Homocigoto , Humanos , Hepatopatías/genética , Masculino , Microscopía Fluorescente , Modelos Genéticos , Datos de Secuencia Molecular , Linaje , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , TransfecciónRESUMEN
Although there is evidence for specific subcellular morphological alterations in response to accumulation of misfolded proteins in the endoplasmic reticulum (ER), it is not clear whether these morphological changes are stereotypical or if they depend on the specific misfolded protein retained. This issue may be particularly important for mutant secretory protein alpha(1)-antitrypsin (alpha(1)AT) Z because retention of this mutant protein in the ER can cause severe target organ injury, the chronic hepatitis/hepatocellular carcinoma associated with alpha(1)AT deficiency. Here we examined the morphological changes that occur in human fibroblasts engineered for expression and ER retention of mutant alpha(1)ATZ and in human liver from three alpha(1)AT-deficient patients. In addition to marked expansion and dilatation of ER, there was an intense autophagic response. Mutant alpha(1)ATZ molecules were detected in autophagosomes by immune electron microscopy, and intracellular degradation of alpha(1)ATZ was partially reduced by chemical inhibitors of autophagy. In contrast to mutant CFTRDeltaF508, expression of mutant alpha(1)ATZ in heterologous cells did not result in the formation of aggresomes. These results show that ER retention of mutant alpha(1)ATZ is associated with a marked autophagic response and raise the possibility that autophagy represents a mechanism by which liver of alpha(1)AT-deficient patients attempts to protect itself from injury and carcinogenesis.
Asunto(s)
Adenina/análogos & derivados , Autofagia/fisiología , Retículo Endoplásmico/fisiología , alfa 1-Antitripsina/genética , Adenina/farmacología , Animales , Autofagia/efectos de los fármacos , Células CHO , Cricetinae , Retículo Endoplásmico/química , Retículo Endoplásmico/ultraestructura , Fibroblastos/química , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica/fisiología , Humanos , Hígado/citología , Hígado/fisiología , Hepatopatías/metabolismo , Hepatopatías/fisiopatología , Ratones , Ratones Transgénicos , Microscopía Inmunoelectrónica , Microsomas/química , Microsomas/fisiología , Microsomas/ultraestructura , Mutagénesis/fisiología , Vacuolas/química , Vacuolas/fisiología , Vacuolas/ultraestructura , Vimentina/análisis , alfa 1-Antitripsina/análisisRESUMEN
A delay in intracellular degradation of the mutant alpha(1)-antitrypsin (alpha(1)AT)Z molecule is associated with greater retention within the endoplasmic reticulum (ER) and susceptibility to liver disease in a subgroup of patients with alpha(1)AT deficiency. Recent studies have shown that alpha(1)ATZ is ordinarily degraded in the ER by a mechanism that involves the proteasome, as demonstrated in intact cells using human fibroblast cell lines engineered for expression of alpha(1)ATZ and in a cell-free microsomal translocation assay system programmed with purified alpha(1)ATZ mRNA. To determine whether the ubiquitin system is required for proteasomal degradation of alpha(1)ATZ and whether specific components of the ubiquitin system can be implicated, we have now used two approaches. First, we overexpressed a dominant-negative ubiquitin mutant (UbK48R-G76A) by transient transfection in the human fibroblast cell lines expressing alpha(1)ATZ. The results showed that there was marked, specific, and selective inhibition of alpha(1)ATZ degradation mediated by UbK48R-G76A, indicating that the ubiquitin system is at least in part involved in ER degradation of alpha(1)ATZ. Second, we subjected reticulocyte lysate to DE52 chromatography and tested the resulting well-characterized fractions in the cell-free system. The results showed that there were both ubiquitin-dependent and -independent proteasomal mechanisms for degradation of alpha(1)ATZ and that the ubiquitin-conjugating enzyme E2-F1 may play a role in the ubiquitin-dependent proteasomal mechanism.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , Complejos Multienzimáticos/metabolismo , Mutación , Ubiquitinas/fisiología , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Animales , Línea Celular , Perros , Humanos , Complejo de la Endopetidasa Proteasomal , Conejos , Ubiquitinas/farmacología , alfa 1-Antitripsina/efectos de los fármacosRESUMEN
alpha 1-Antitrypsin (alpha 1-AT) deficiency is the most common genetic cause of liver disease in children and genetic disease for which children undergo liver transplantation. It also causes cirrhosis and hepatocellular carcinoma in adults. Studies by Sveger in Sweden have shown that only a subgroup of the population with homozygous PiZZ alpha 1-AT deficiency develop clinically significant liver injury. Other studies have shown that the mutant alpha 1-AT Z molecule undergoes polymerization in the endoplasmic reticulum and that a subpopulation of alpha 1-AT-deficient individuals may be susceptible to liver injury because they also have a trait that reduces the efficiency by which the mutant alpha 1-AT Z molecule is degraded in the endoplasmic reticulum.
Asunto(s)
Hepatopatías/enzimología , Deficiencia de alfa 1-Antitripsina , Adulto , Animales , Niño , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Homocigoto , Humanos , Hepatopatías/patología , Modelos Biológicos , Mutación Puntual , Factores de Riesgo , alfa 1-Antitripsina/genéticaAsunto(s)
Hepatopatías/fisiopatología , Hígado/fisiopatología , Deficiencia de alfa 1-Antitripsina , alfa 1-Antitripsina/genética , Adolescente , Animales , Retículo Endoplásmico/metabolismo , Homocigoto , Humanos , Lactante , Recién Nacido , Hígado/patología , Hepatopatías/etiología , Hepatopatías/genética , Modelos Biológicos , Estructura Secundaria de Proteína , alfa 1-Antitripsina/químicaRESUMEN
Degradation of proteins that are retained in the quality control apparatus of the endoplasmic reticulum (ER) has been attributed to a third proteolytic system, distinct from the lysosomal and the cytoplasmic ubiquitin-dependent proteosomal proteolytic pathways. However, several recent studies have shown that ER degradation of a mutant membrane protein, CFTRdeltaF508, is at least in part mediated from the cytoplasmic side by the 26 S proteasome. In this study, we examined the possibility that ER degradation of mutant secretory protein alpha1-antitrypsin (alpha1-AT) Z, the mutant protein associated with infantile liver disease and adult-onset emphysema of alpha1-AT deficiency, is mediated by the proteasome. The results show that a specific proteasome inhibitor, lactacystin, inhibits ER degradation of alpha1-ATZ in transfected human fibroblast cell lines and in a cell-free microsomal translocation system. Although it is relatively easy to conceptualize how a transmembrane protein like CFTRDeltaF508 might be accessible on the cytoplasmic aspect of the ER membrane for ubiquitination and degradation by the proteasome, it is more difficult to conceptualize how this might occur for a luminal polypeptide. The results show that, once within the lumen of the ER, alpha1-ATZ interacts with the transmembrane molecular chaperone calnexin and specifically induces the polyubiquitination of calnexin. The results, therefore, provide evidence that the proteasome, from its cytoplasmic localization, induces the degradation of the luminal alpha1-ATZ molecule by first attacking the cytoplasmic tail of calnexin molecules that are associated with alpha1-ATZ.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , Complejos Multienzimáticos/metabolismo , alfa 1-Antitripsina/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Animales , Proteínas de Unión al Calcio/metabolismo , Calnexina , Sistema Libre de Células , Inhibidores de Cisteína Proteinasa/farmacología , Perros , Electroforesis en Gel de Poliacrilamida , Endopeptidasa K/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Hexosaminidasas/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal , ConejosRESUMEN
We have theorized that a subset of PiZZ alpha1-antitrypsin (alpha1-AT)-deficient individuals is more susceptible to liver injury by virtue of second inherited trait(s) or environmental factor(s), which exaggerate the accumulation of mutant alpha1-AT Z within the endoplasmic reticulum (ER) of liver cells. Using a complementation approach in which cell lines from PiZZ individuals with liver disease ("susceptible" hosts) and from PiZZ individuals without liver disease ("protected" hosts) are transduced with the mutant alpha1-AT Z gene, we have recently shown that there is a delay in ER degradation of mutant alpha1-AT Z protein that is only present in cell lines from susceptible hosts and correlates with the liver disease phenotype. In the present study we examined the specificity of this ER degradation pathway to determine if it is responsible for degrading other misfolded mutants of alpha1-AT and/or for unassembled membrane proteins. The S mutant of alpha1-AT and H2a subunit of the asialoglycoprotein receptor (ASGPR H2a) were expressed in skin fibroblast cell lines from susceptible and protected hosts. The results showed in both susceptible and protected hosts that alpha1-AT S was associated with a delay in secretion as compared with wild type alpha1-AT. The alpha1-AT S mutant was retained in ER, albeit to a lesser extent than the alpha1-AT Z mutant. There was, however, a significant increase in retention of alpha1-AT S in the ER of susceptible as compared with protected host cells. The same host cell lines were transduced to express an unassembled membrane protein, ASGPR H2a. There was no difference in the kinetics of ER degradation of ASGPR H2a in susceptible as compared with protected hosts. Taken together, the results show that alpha1-AT S is associated with a defect in biogenesis, intracellular retention, which is similar to but milder than alpha1-AT Z. Like alpha1-AT Z, alpha1-AT S is degraded by a pathway in the ER, which is relatively inefficient in PiZZ individuals with the liver disease phenotype. However, this pathway appears to be different from that previously described for a model unassembled membrane protein.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , alfa 1-Antitripsina/metabolismo , Línea Celular , ADN Complementario , Humanos , Inmunohistoquímica , Mutagénesis Sitio-Dirigida , alfa 1-Antitripsina/genética , Deficiencia de alfa 1-AntitripsinaAsunto(s)
Hepatopatías/etiología , Hepatopatías/terapia , Errores Innatos del Metabolismo/complicaciones , Niño , Enfermedad de Gaucher/etiología , Enfermedad de Gaucher/terapia , Degeneración Hepatolenticular/etiología , Degeneración Hepatolenticular/terapia , Humanos , Errores Innatos del Metabolismo/terapia , Tirosina/sangre , Deficiencia de alfa 1-AntitripsinaRESUMEN
Membrane cofactor protein (MCP or gp45-70) is a recently described regulatory glycoprotein of the complement system which binds iC3 or C3b and is present on human platelets, T cells, B cells, monocytes, and mononuclear-derived cell lines. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, MCP migrates as a doublet with an Mr of the upper band of 63,000 and the lower band of 58,000. The same pattern was found on all cell populations in a given individual and was stable over time. In order to further characterize the two band pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, MCP was isolated by affinity chromatography or immunoprecipitation from 72 healthy unrelated donors. All individuals expressed both bands and, based on the densitometric scanning of gels, three patterns were noted: upper band predominant in 65%, approximately equal distribution of upper and lower bands in 29%, and lower band predominant in 6%. These observed phenotypic frequencies fit with expectations based on Hardy-Weinberg equilibrium for a two-allele system. Family studies also support this model as none of the 26 offspring had a phenotype that deviated from the expected, assuming an autosomal codominant model of inheritance. These results are consistent with a simple two-allele system that controls the expression of the two bands of MCP.