ABSTRACT
Cytoplasmic type I DnaJ/Hsp40 chaperones contain a Cys-rich domain consisting of four CXXCXG motifs that are in a reduced state and coordinate zinc, stabilizing the intervening sequence in a loop structure. However, the Cys-rich region of the endoplasmic reticulum localized HEDJ (ERdj3/ERj3p), is considerably different in sequence and arrangement. Unlike the typical type I molecule, the HEDJ CXC, and CXXC motifs were demonstrated in this study to be predominantly oxidized in intramolecular disulfide bonds. In the native state, HEDJ bound to immobilized, denatured thyroglobulin. Unlike its binding partner GRP78, redox conditions affected the interaction of HEDJ with substrate. Substitution of the Cys-rich domain cysteine residues with serine diminished or abolished HEDJ binding in the in vitro assay. These findings suggest that the Cys-rich region of HEDJ and its oxidation state are important in maintaining the substrate interaction domain in a binding-competent conformation.
Subject(s)
Cysteine/chemistry , Cysteine/metabolism , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/metabolism , Microsomes/chemistry , Microsomes/metabolism , Binding Sites , Endoplasmic Reticulum Chaperone BiP , Oxidation-Reduction , Protein Binding , Protein Structure, Tertiary , Substrate SpecificityABSTRACT
Alpha-1-antitrypsin deficiency is a genetic disorder resulting in the expression of misfolded mutant protein that can polymerize and accumulate in hepatocytes, leading to liver disease in some individuals. Transgenic PiZ mice are a well-characterized model, which express human alpha-1-antitrypsin mutant Z protein (ATZ protein) and faithfully recapitulate the human liver disease. Liver tissue expressing alpha-1-antitrypsin mutant Z protein exhibits inflammation, injury and replacement of damaged cells. Fibrosis and hepatocellular carcinoma (HCC) develop in aging PiZ mice. In this study, microarray analysis was performed comparing young PiZ (ZY) mice to wild-type (WY), and indicated that there were alterations in gene expression levels that could influence a number of pathways leading to liver disease. Redox-regulating genes were up-regulated in ZY tissue, including carbonyl reductase 3 (CBR3), glutathione S-transferase alpha 1 + 2 (GSTA(1 + 2)) and glutathione S-transferase mu 3 (GSTM3). We hypothesized that oxidative stress could develop in Z mouse liver, contributing to tissue damage and disease progression with age. The results of biochemical analysis of PiZ mouse liver revealed that higher levels of reactive oxygen species (ROS) and a more oxidized, cellular redox state occurred in liver tissue from ZY mice than WY. ZY mice showed little evidence of oxidative cellular damage as assessed by protein carbonylation levels, malondialdehyde levels and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodG) staining. Aging liver tissue from PiZ older mice (ZO) had elevated ROS, generally lower levels of antioxidant enzymes than younger mice and evidence of cellular damage. These data indicate that oxidative stress is a contributing factor in the development of liver disease in this model of alpha-1-antitrypsin deficiency.
Subject(s)
Liver/pathology , Oxidative Stress , alpha 1-Antitrypsin Deficiency/metabolism , alpha 1-Antitrypsin/genetics , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Animals , DNA Damage , Disease Models, Animal , Glutathione/metabolism , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Liver/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phenotype , Reactive Oxygen Species/metabolism , alpha 1-Antitrypsin/metabolism , alpha 1-Antitrypsin Deficiency/pathologyABSTRACT
AIM: Individuals with homozygous (ZZ) alpha-1-antitrypsin (alpha1AT) deficiency are at an increased risk for liver damage, cirrhosis and hepatocellular carcinoma (HCC). The transgenic PiZ mouse, expressing the human alpha1AT mutant Z gene, is a valuable model for this disease. We studied PiZ mice in order to identify and characterize mechanisms involved in the development of HCC. METHODS: Tumor incidence and histology were studied, gene expression levels were surveyed with microarrays, RNA quantified with quantitative real time polymerase chain reaction and protein levels determined with immunoblots and immunohistochemistry. RESULTS: By 16-19 months of age, approximately 69% of the PiZ mice had developed tumors. HCC was present with no evidence of benign adenomas as pre-cancerous lesions. Tumors showed abnormal mitochondria, variable levels of steatosis, globular inclusions of alpha1AT mutant Z protein and metastases. PiZ mice that subsequently developed liver tumors had higher serum levels of alpha1AT mutant Z protein than those that did not develop tumors. Cyclin D1, a cell cycle protein, was upregulated in PiZ livers without tumors compared to Wt. cFOS, a component of AP-1 that may be involved in transforming cells and MCAM, an adhesion molecule likely involved in tumorigenesis and metastases, were elevated in tumors compared with livers without tumors. CONCLUSION: In the PiZ model, many of the histological characteristics of HCC recapitulated features seen in human HCC, whether from individuals with homozygous ZZ liver disease or from unrelated causes in individuals that were not homozygous ZZ. The accumulation of mutant Z protein altered the regulation of several genes driving proliferation and tumorigenesis.