ABSTRACT
The phenomenon that heme oxygenase-1 (HO-1) protects cell from injury yet its enzymatic product, iron, may facilitate generation of free radical has been long puzzling. Here we establish a functional connection between ferritin heavy chain (FHC) and HO-1. In human lupus nephritis HO-1 and FHC are colocalized within the glomeruli. In rodent anti-Thy1 (thymocyte antigen 1) induced glomerulonephritis, heme oxygenase blockade lowers the expression of FHC and accelerates mesangial cell death. Stimulation of heme oxygenase in cultured rat mesangial cell enhances its resistance to hydrogen peroxide, whereas FHC knockdown by RNA interference compromises this salutary effect. RNA interference of HO-1 makes the cell more susceptible to hydrogen peroxide, which can be rescued by forced expression of wild-type FHC but not mutants that lose the capacity of iron storage and ferroxidase activity. Phosphorylation of JunD was not sustained in these cells. Microarray analysis identifies four candidate transcriptional factors that may regulate the HO-1-induced transcription of FHC. Our results support the role of FHC in neutralizing the iron toxicity as well as mediating the protective effect of HO-1 in response to oxidative stress.
Subject(s)
Apoferritins/physiology , Heme Oxygenase-1/physiology , Oxidative Stress , Animals , Glomerular Mesangium/cytology , Glomerular Mesangium/metabolism , RatsABSTRACT
BACKGROUND: Dioscorins are the major storage proteins of yam tubers. However, the molecular nature of their heterogeneity in tubers has not been fully elucidated. In this study the authors isolated the dioscorin gene families of Dioscorea japonica and Dioscorea pseudojaponica, performed matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and elucidated which dioscorin isoforms are the major constituents in tubers. RESULTS: The dioscorin gene families of D. japonica (Dj-dioA1-Dj-dioA4, Dj-dioB1 and Dj-dioB2) and D. pseudojaponica (Dp-dioA1-Dp-dioA5 and Dp-dioB1) were cloned from cDNA libraries of yam tubers. The dioscorins isolated from Dioscorea alata (Da-dioscorins), D. japonica (Dj-dioscorins) and D. pseudojaponica (Dp-dioscorins) were mainly monomers, with a few dimers. The monomers contained one intramolecular disulfide bond (Cys(28)-Cys(187)) and belonged to Class A dioscorins with two cysteine residues. The dimers consisted of Class B dioscorins with one intermolecular disulfide bond (Cys(40)-Cys(40)). Results of MALDI-TOF-MS revealed that the Da-dioscorins were mainly encoded by Da-dioA2, Da-dioA3 and Da-dioA4. The majority of the Dj-dioscorins were encoded by Dj-dioA1, Dj-dioA2, Dj-dioA3 and Dj-dioB2. The Dp-dioscorins mainly comprised proteins encoded by Dp-dioA1, Dp-dioA3, Dp-dioA4, Dp-dioB1 and Dp-dioB2. CONCLUSION: Determination of the constituents of dioscorin isoforms in yam tubers provides a basis for future studies of their physiological and biomedical functions.
Subject(s)
Dietary Proteins/analysis , Dioscorea/chemistry , Genes, Plant , Plant Proteins/chemistry , Plant Tubers/chemistry , Cloning, Molecular , Diet , Dietary Proteins/isolation & purification , Dioscorea/genetics , Gene Library , Humans , Mass Spectrometry , Molecular Structure , Plant Proteins/isolation & purification , Protein Isoforms , Species SpecificityABSTRACT
Dioscorins, the major storage proteins in yam tubers, exhibit biochemical and immunomodulatroy activities. To investigate the potential application of dioscorins in biomedical research, we expressed the dioscorin genes Dj-dioA3 and Dp-dioA2 from Dioscorea japonica and Dioscorea pseudojaponica, respectively, in E. coli and routinely obtained approximately 15 mg proteins per liter Escherichia coli culture (mg/L) to 30 mg/L of rDj-dioscorinA3 and 4 to 8 mg/L of rDp-dioscorinA2. Western blot analyses revealed that both recombinant dioscorins contained epitopes with similar antigenicities to those of the native dioscorins. Results from dithiothreitol (DTT) treatment followed by monobromobimane (mBBr) staining showed that both recombinant dioscorins, like the native dioscorins, contain an intramolecular disulfide bond between Cys(28) and Cys(187) residues. Circular dichroism spectroscopy findings indicated that the secondary structural contents of the recombinant dioscorins showed high similarity to those of their corresponding native dioscorins. Both recombinant dioscorins, like the native dioscorins, exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and Toll-like receptor 4 signaling activities, and stimulated the phagocytosis of E. coli by macrophage. Overall, our results indicated that substantial amounts of recombinant dioscorins can be purified easily from E. coli and that these recombinant dioscorins are appropriate for application in future investigations of the biomedical functions of dioscorins.