ABSTRACT
A growing body of evidence has indicated that cellular redox status modulates various aspects of cellular events including proliferation and apoptosis (1,7). TRX (1,8) is a smalli, ubiquitous protein with two re dox-active half-cystine residues in an active center, -Cys-Gly-Pro-Cys. It is also known as adult T-cell leukemia-derived factor (ADF), in volved in HTLV-1 leukemogenesis (9,10). TRX exists either in a re duced or oxidized form and participates in redox reactions through the reversible oxidation of this active center dithiol. TRX functions inside (2,3) and outside (11-13) the cell. One of its intracellular functions is the facilitation of certain protein-nucleotide interactions. In vitro and in vivo experiments showed that TRX augmented the DNA-binding and transcriptional activities of the p50 subunit of NF-kB by reducing Cys 62 (2,3) of p50. Recently, direct physical association of TRX and an oligopeptide from NF-kB p50 was revealed by NMR study in vitro (14), although their in vivo direct association needs to be confirmed. Redox regulation of Jun and Fos molecules has also been implicated. Various antioxidants strongly activate the DNA-binding and transactivation abilities of AP-1 complex (7,15). Interestingly, TRX enhances the DNA-
binding activity of Jun and Fos, and this process requires other mol ecules such as a novel protein called redox factor-1 (Ref-1). Ref-1 was identified in a cell-free system as one of the factors restoring the AP1-DNA binding (6,16) and found to be identical to an AP endonuclease (5,6). There has been no report describing whether TRX and Ref-1 di rectly associate or require any intervening factor(s) for their interaction. We studied their association by in vitro cross-linking and by mamma lian two-hybrid system using various mutants of TRX in order to in vestigate interaction on AP-1 mediated transcription. If an oxidoreductase directly interacts with its substrate, these molecules should form an intermediate through disulfide linkage. Therefore, we reasoned that it would be possible to trap their transient association using cross-link ing reagent(s) such as diamide, which converts free sulfhydryls to disulfides by cysteine oxidation (17,18). In order to determine which of the five cysteines in TRX is/are responsible for the intermolecular disulfide bond formation, three TRX mutants, which contained cysteines-to-serines or cysteine-to-alanine substitution(s), were purified as hexahistidine (6x His)-tagged form. A series of experiments was per formed with the 6x His-tagged Ref-1 and the various mutants of TRX (Fig. la). It is known that TRXC62S/C69S/C73S retains its reducing
Figure 1 TRX associates with Ref-1 through the redox-active cysteines in vitro. Various TRX mutants containing cysteine-to-serine or cysteine-to-alanine substitutions were cross-linked with Ref-1 by diamide. After incubation, the complexes were resolved by electrophoresis under reducing or oxidizing conditions and detected by antibodies, (a) Schematic representation of mu tant constructs of TRX used in this experiment, (b) Western blot analysis us ing anti-Ref-1 antibody. Methods: Mutagenesis of human TRX was performed by a PCR-based technique (29). Various TRX recombinant protiens (22,23) were expressed in E. coli fused with 6x His tag using the pQE30 expression plasmids (Qiagen, Chatsworth, CA). The crude lystate containing 6x His-TRX was loaded onto a Ni2+-NTA-agarose column. The column was washed, and the fusion protein was eluted by PBS containing 80 mM imidazol. The eluted protein was dialyzed against PBS containing 2mM DTT. Human Ref-1 was expressed based on the pDS56Ref-l 15 (a gift form S. Xanthoudakis) as a 6x His-tagged protein following the same protocol as the expression of TRX except that Ref-1 eluate was dialyzed against a storage buffer 15 [50 mM so dium phosphate, pH 7.3/50 mM NaCl/5 mM MgC12/l mM EDTA, 5%(vol/ voi) glycerol] containing 1 mM DTT. 6x His-TRX (200 ng) and 6x His-Ref-
1 (200 ng) were incubated for 30 min at room temperature (RT) with 1 mM DTT. Then, they were incubated together in the presence of 10 mM diamide for 30 min at RT. After incubation, the reaction mixture was denatured for 5 min at 90 °С in dissociation buffer with or without 2-ME (1%). Each com plex was applied to a 15 % SDS-PAGE and electrophoresed. After electroblotting, PVDF membranes were blocked and incubated with antibodies, fol lowed by peroxidase-conjugated antimouse IgG. The antigens on the mem brane were visualized with the ECL Western blot detection kit (Amersham). Antihuman TRX monoclonal antibody, 11 Ab, was established and provided by Fujirebio Inc., Tokyo, Japan. Rabbit polyclonal antibody raised against Cterminus (aa 299-318) of human Ref-1 was purchased from Santa Crutz, Palo Alto, CA. (From Ref. 31.)
(b)
activity (19,20), while substitution mutants (TRXC35A, TRXC32S/ C35) at either Cys32 or Cys35 lose their in vitro reducing activity (1). In spite of loss of reducing activity, TRXC35A, which retains Cys32, is able to involve the covalent mixed disulfide intermediate formation between Cys32 and protein substrate (1,14,21). Under reducing condi tions, the wild-type TRX and all the TRX mutants (TRXC32S/C35S, TRXC35A, and TRXC62S/C69S/C73S) migratred as a single 13-kDa band in SDS-PAGE. Treatment of TRX wild or TRXC32S/C35S with diamide generated multiple bands, suggesting oligomer formation. In the case of TRXC62S/C69S/C73S, however, we detected only a mo nomeric form (data not shown) after diamide treatment. Ref-1 has seven cysteines, and moved as a monomer under reducing conditions (Fig. lb; lanes 1, 3, 5, and 7). In oxidizing conditions, an additional band smaller than the reduced form was observed, representing probably a different monomeric form of Ref-1 with intramolecular disulfide bond (lanes 2, 4, 6, and 8).
