Functional Research Of Thioredoxin-1

Thioredoxins are small ubiquitous multifuncational proteins which belong to a conserved redox-active protein family containing the conserved active catalytic domain (-Cys-Gly-Pro-Cys-).Trx-1 is the most studied member in thioredoxin family,which has been rediscovered under different names,including adult T cell leukemia-derived factor (ADF),interleukin-2(IL-2) receptor-inducing factor produced by human T-lymphotrophic virus type 1(HTLV-1)-infected T cells and early pregnancy factor.Trx-1 has been discovered to have a multiplicity of biological functions.Besides the well-known antioxidant effect with thioredoxin reductase(TrxR) through the NADPH-dependent process(Trx system),Trx-1 also contributes to many of the hallmarks of cancer including increased development,proliferation,resistance to cell death and increased angiogenesis.Trx-1 regulates the apoptotic signaling pathways and the activity of some transcription factors or functional enzyme in tumor cells.In some cancer cell lines,Trx-1 expression may confer resistance to chemotherapy.These findings suggest that the Trx-1 may represent a novel attractive target for development of new cancer therapeutics.Trx-1 can undergo different post-translational modifications such as S-nitrosylation/S-nitrosation,thiol-oxidation and glutathionylation.These modifications have been demonstrated to significantly affect the Trx-1 function.In contrast, phosphorylation of Trx-1 has not been investigated.In 2005,Trx-1 was first reported phosphorated on Thr-100 via Global phosphoproteome analysis on human HepG2 hepatoeytes using reversed-phase diagonal LC.In order to explore how the phosphorylation of Trx-1 affects its function,we carried out the following work:Trx-1 gene was obtained from human HepG2 hepatocytes by RT-PCR and the eukaryotic vector pET32a-Trx was constructed.Recombinant protein His-Trx-1 was produced by E.coli after induced by IPTG and then puried by Ni-NTA agarose.Three stable hybridoma cell lines were successfully established to secret anti-Trx MAbs.All these MAbs belong to the G1 subclass withκlight chains with high affinity and specificity.Two recombinant eukaryotic vectors,pcDNA-Trx and pcDNA-muTrx(A299C) were constructed.The two transfected HepG2 cell lines,which could stably express Trx-1 or muTrx-1(T 100N) were established,respectively.Stable transfection of HepG2 cell line with muTrx-1 cDNA showed higher sensitivity and greater apoptosis extent to the different anti-tumor drags.Meanwhile,the binding affinity of muTrx-1 and ASK-1 decreased.Phosphorylation of Trx-1 at Thr-100 had been demonstrated to be critical of its anti- apoptosis activity by conducing to Trx-1 binding to ASK-1.This finding suggested that the Phosphorylation of Trx-1 may represent an attractive target for development of new cancer therapeutics.With the novel hypothesis of "Inflammation-cancer link" was developed,Trx-1 began to be a research interest for its important effects on inflammatory disease and signal pathways.A new multifunctional inflammatory protein DT/AIF-1 was identified in autoimmune typeⅠdiabetes pancreas tissues and Rat Cardiac Allografts with Chronic Rejection.It contributed to proliferation,migration and secretion of varied inflammatory cells including macrophage,lymphocyte and monoeytic.To investigate the commutative regulation between Trx-1 and DT/AIF-1,we carried out the following work:When Human mononuclear leukemic cells(U937) were transfected by pcDNA-AIF-1,the intracellular Trx-1 level did not change by increasing expression of DT/AIF-1.Whereas,the intracellular Trx-1 level could be increased by adding additional exogenous of DT/AIF-1 protein.Two recombinant plasmids peDNA-Trx and pcDNA-NLS-Trx were transiently transfected into U937 cells,respectively.The intra-cellular DT/AIF-1 protein expression and secretion of both cell lines were increased.Interestingly,only nuclear Trx-1 but not cytoplasmic Trx-1 could increase DT/AIF-1 mRNA level.These results demonstrated Trx-1 with different subeellular localization had different modes to regulate DT/AIF-1. Cytoplasmic Trx-1 may up-regulate on translational level and Trx-1 can also translocate to nucleus to up-regulate transcriptional level.The results suggest the regulations beween Trx-1 and DT/AIF-1 form a negative feedback loop:cytoplasmic Trx-1 increases DT/AIF-1 protein expression and secretion; the excreted DT/AIF-1 will turn over to promote Trx-1 expression in cells.This conclusion may contribute to investigate the role of DT/AIF-1 in inflammation reactions occur in cardiovascular diseases and organ transplantation.More evidences need to be find out to reveal The intra-cellular signal pathway of Trx-1 regulate DT/AIF-1 and extra -cellular signal pathway of DT/AIF-1 regulate Trx-1.