Direct association of hepatopoietin with thioredoxin constitutes a redox signal transduction in activation of AP-1/NF-kappaB.
It has been demonstrated that growth factors quiescin Q6 family was created by the fusion of the sulfhydryl oxidase fragment of the yeast essential for respiration and vegetative growth (ERV)1 prototype [an orthologue of hepatopoietin (HPO)] and thioredoxin (TRX)/disulfide isomerase domain during evolution. In this paper, our results demonstrated that two components of this composite protein, i.e., HPO and TRX, were involved in the same signal transduction and interacted physically in eukaryocyte. When HPO and TRX were cotransfected into COS7 cells, the activity of activator protein-1 (AP-1) and NF-kappaB was evidently enhanced compared with the transfection with HPO or TRX alone, at the same time, the phosphorylation of c-Jun was increased. They were colocalized in the cells. By Co-IP and GST pull-down experiments, we found that HPO could physically interact with TRX, which was also confirmed by yeast two-hybrid assay. By further investigation, we found both HPO and TRX were sensitive to cellular oxidative state. HPO dimer is in its natural state and could be reduced by dithiothreitol (DTT) in vitro and in vivo. Under the treatment of oxidants such as H(2)O(2) and diamide, the amount of HPO monomer was decreased significantly and assembled into dimer, and the free thiol in TRX was oxidized. HPO could transfer oxidizing equivalents to TRX via direct thiol-disulfide exchange in vitro, the redox state of TRX was also affected by HPO in vivo. Taken together, it was implicated that the oxidizing equivalents might flow from HPO to TRX and then to substrate protein by the dimerization of HPO, and its interaction with TRX finally activates the redox-sensitive transcription factor, suggesting a new redox signal pathway conducted by thiol-disulfide transformation in eukaryocytic cytoplasm.
Li
Yingxian
Y
Laboratory of Systems Biology, Beijing Institute of Radiation Medicine, Chinese Human Genome Center at Beijing.
Liu
Wanying
W
Xing
Guichun
G
Tian
Chunyan
C
Zhu
Yunping
Y
He
Fuchu
F
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D008156:Luciferases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D015398:Signal Transduction; D013438:Sulfhydryl Compounds; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D004220:Disulfides; D016328:NF-kappa B; D013438:Sulfhydryl Compounds; D018808:Transcription Factor AP-1; D049452:Green Fluorescent Proteins; D013879:Thioredoxins; D017228:Hepatocyte Growth Factor; D004247:DNA; D006861:Hydrogen Peroxide; D008156:Luciferases; D005982:Glutathione Transferase; D010100:Oxygen