Human thioredoxin/adult T cell leukemia-derived factor activates the enhancer binding protein of human immunodeficiency virus type 1 by thiol redox control mechanism

Abstract

Transcription from the human immunodeficiency virus type 1 (HIV-1) provirus is activated by a cellular factor, NF kappa B, recognizing the tandemly repeated 10-base-pair sequences, termed the kappa B sequence, present in the enhancer region within the viral long terminal repeat (LTR). Using electrophoretic mobility shift assay (EMSA), which demonstrates specific DNA-protein interaction in vitro, we could demonstrate that reducto-oxidative modulation of NF kappa B dramatically changes its DNA binding activity and that a cellular physiological reducing catalyst, thioredoxin (TRX) also known as adult T cell leukemia derived factor (ADF), fully restored the DNA-binding activity of the oxidized NF kappa B. We also observed that purified TRX/ADF protein could augment gene expression from HIV LTR as demonstrated by transient chloramphenicol acetyltransferase (CAT) assay. These observations confirmed the previous notion that ADF might be an inducing factor of cellular interleukin-2 receptor alpha subunit (IL-2R alpha) through the kappa B sequence that is a common central cis-regulatory element in both IL-2R alpha and HIV gene expression. These observations indicate that reducto-oxidative regulation (or redox regulation) of a cysteine residue(s) on the NF kappa B molecule might play an important role in its specific DNA interaction and that it might provide a clue to the understanding of a pathway of cellular signal transduction to NF kappa B that is independent from the known pathways involving protein phosphorylation.

Inhibition of the DNA‐binding activity of NF‐κB by gold compounds in vitro

“…The redox regulation of NF-xB of its DNA binding activity has been demonstrated [7,20]. When the cysteineresidues of NF-xB were oxidized by diamide or modified by N-ethylmaleimide (NEM), the DNA binding activity of NF-xB was abrogated [7,20].…”

“…NF-xB was prepared from the nuclei of human primary lymphocytes as reported previously [7]. Briefly, the crude nuclear extract was fractionated on heparin-Sepharose and DEAE-Sepharose columns.…”

“…The fractions with the xB DNA binding activity were pooled and concentrated. The specificity of NF-xB was examined by immunoblotting using specific antiserum [12] and electrophoretic mobility shift assay (EMSA) using the tcB DNA probe [7,8].…”

“…The DNA binding activity was examined by EMSA using the specific 1¢B DNA probe as previously described [7,8]. The t¢B sequence was taken from the human immunodeficiency virus long terminal repeat (HIV-LTR) [8].…”

“…In recent years, we have demonstrated that the DNA binding activity of NF-xB is regulated by an oxido-reductive mechanism (redox regulation). We found that oxidation of NF-xB abolished the DNA binding activity and that the subsequent reduction completely restored the activity [7,8]. These observations have indicated that development of a novel therapeutic strategy might be feasible by using chemical reagents that could modulate the redox status of NF-xB.…”