INTRODUCTION

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Thioredoxin, a small protein of approximately 100 amino acid residues, is ubiquitously present and is evolutionarily conserved from prokaryotes to higher eukaryotes, plants, and animals (1-3). It is characterized by an amino acid sequence of an active site, -Cys-Gly-Pro-Cys-, conserved throughout evolution. The active site of thioredoxin is localized in a protrusion of its three-dimensional structure (4), and the two cysteine residues provide the sulfhydryl groups involved in the thioredoxin-dependent reducing activity. Thioredoxin exists in either a reduced form or an oxidized form. Oxidized thioredoxin, Trx-S₂, where the two cysteine residues are linked by an intramolecular disulfide bond is reduced by flavoenzyme thioredoxin reductase and NADPH (2). Reduced thioredoxin, Trx-(SH)₂, contains two thiol groups and can efficiently catalyze the reduction of many exposed disulfides. Thus, thioredoxin participates in various redox reactions via the reversible oxidation and reduction of the two cysteine residues in the active center.

In Escherichia coli, thioredoxin was first identified as an electron donor for ribonucleotide reductase, the enzyme that reduces ribonucleotides to deoxynucleotides for DNA synthesis and repair (5). E. coli thioredoxin can also function as a hydrogen donor for 3'-phosphoadenosine 5'-phosphosulfate reductase in the sulfate assimilation pathway as well as methionine sulfoxide reductase (6, 7). Apart from these functions, E. coli thioredoxin functions as the essential subunit of T7 DNA polymerase and in the maturation of filamentous bacteriophages M13 and f1 (8-10). In eukaryotic cells, thioredoxin has been implicated in a wide variety of biochemical and biological functions. It can function as a hydrogen donor, similar to the prokaryotic thioredoxin (2). In addition, thioredoxin can facilitate refolding of disulfide-containing proteins (11) and modulate the activity of some transcription factors such as NF-B and AP-1 (12, 13). Thioredoxin is an efficient antioxidant, which can reduce hydrogen peroxide (14), scavenge free radicals (15), and protect cells against oxidative stress (16). Another role of thioredoxin is the growth stimulation of human T cells. Adult T cell leukemia-derived factor, which augments the expression of interleukin-2 receptor, was found to be identical to human thioredoxin (17). Furthermore, thioredoxin reportedly inhibits the expression of human immunodeficiency virus in macrophages (18).

A number of eukaryotic proteins are known to contain domains evolutionarily related to thioredoxin, and most of them appear to belong to the protein-disulfide isomerase (PDI)¹ family, endoplasmic reticulum (ER) enzymes that catalyze the rearrangement of disulfide bonds in various proteins (19-21). All members of the PDI family, which contain two or three Trx domains per one PDI molecule (22), are primarily retained within the ER lumen by the recognition system for their carboxyl-terminal tetrapeptide motif, (K/H)DEL. DsbA, the bacterial functional equivalent of PDI, also contains a Trx domain and acts as a thiol:disulfide interchange protein that allows disulfide bond formation in some periplasmic proteins (23).

Here we report on the biochemical purification and cloning of a novel protein, designated TRP32, which is a 32-kDa protein with an N-terminal Trx domain. TRP32 is ubiquitously expressed in human tissues and mammalian cell lines and localized in the cytoplasm.

	EXPERIMENTAL PROCEDURES

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Purification of a 32-kDa Protein-- Human thymoma-derived cell line, HPB-ALL was cultured to 5 × 10⁵ cells/ml in 20 liters of RPMI supplemented with 10% bovine fetal calf serum (FCS), 20 mM HEPES (pH 7.3), 50 µM -mercaptoethanol, 50 units/ml penicillin, and 50 µg/ml streptomycin. Cells were concentrated to a density of 2.5 × 10⁷ cells/ml and stimulated with 1 µg/ml anti-Fas monoclonal antibody (CH-11) (24) for 2 h. After washing once with cold PBS, cells were harvested and frozen in liquid nitrogen. Cell pellets were thawed on ice and suspended with 90 ml of 20 mM Tris·HCl (pH 7.5) containing 10% glycerol, 50 mM NaF, 10 mM -glycerol phosphate, 2 mM EDTA, 1 mM DTT, 1 mM vanadate, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml aprotinin (buffer IA), and cells were homogenized in a Dounce homogenizer with 10 strokes. After centrifugation for 1 h at 100 000 × g at 4 °C, the supernatant was loaded onto SP Sepharose FF (20 ml, Amersham Pharmacia Biotech). After adding NaCl to 0.18 M, the flow-through fraction was loaded onto HiPrep 16/10 Q Sepharose FF (Amersham Pharmacia Biotech). The column was eluted with a 200-ml linear gradient of 0.18-0.5 M NaCl, and the eluted fractions were assayed by an in-gel kinase assay using histone (0.3 mg/ml) as substrate as described previously (25). All subsequent kinase assays in this study were performed by this method. Fractions of the 34-kDa kinase were pooled and loaded onto a hydroxyapatite column (10 ml, Bio-Rad), which had been equilibrated with 10 mM potassium phosphate (pH 6.8) containing 1 mM vanadate, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml aprotinin (buffer HA). The column was eluted with a 250-ml gradient of 10-500 mM potassium phosphate. Fractions of the 34-kDa kinase were pooled and dialyzed against buffer QA (buffer IA containing 0.18 M NaCl) and loaded onto a Mono Q column (Amersham Pharmacia Biotech). The column was eluted with a 20-ml gradient of 0.18-0.5 M NaCl, and the fractions of the 34-kDa kinase were concentrated to 2.5 ml by vacuum evaporation and then resolved on Hiload 16/60 Superdex 75. An aliquot of the kinase fraction was subjected to two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by sliver staining and in-gel kinase assay.

Peptide Sequencing-- The purified fraction of the 34-kDa kinase was precipitated with an equal volume of cold acetone and dissolved in sample solution for isoelectric focusing (8 M urea, 65 mM DTT, 2% Pharmalyte 3-10, 0.5% Triton X-100). After being resolved by two-dimensional SDS-PAGE using the Multiphor II system (Amersham Pharmacia Biotech) and the Investigator two-dimensional electrophoresis system (Millipore Corp.), the protein spots that co-migrated with activity by the in-gel kinase assay were recovered, crushed, and digested with lysylendopeptidase (Wako, Japan) as described (26). After SDS was removed with a DEAE-Tosopearl 650S column (Toso, Japan), the resulting peptide solution was separated with a reverse phase Wakopak column (Wako, Japan) and subjected to amino acid sequence analysis (Procise 492; Perkin-Elmer).

cDNA Cloning and Sequencing-- Degenerate oligonucleotides were designed based on peptide sequences 1 and 2 (Fig. 1). Sense (5'-GAYCCNGAYTTYACNCCNGA-3') and antisense (5'-GCRTCNGCNCCYTGRTAYTG-3') primers were used to amplify cDNA fragments by reverse transcriptase PCR from the first strand cDNA of human HPB-ALL cells, which was synthesized using SuperScript II reverse transcriptase (Life Technologies, Inc.) with oligo(dT) primer. The resulting 290-nucleotide PCR products were subcloned into pCR2.1 vector (Invitrogen) and sequenced with a model 310 genetic analyzer (Perkin-Elmer). The 290-nucleotide cDNA fragment was used to screen an oligo(dT)-primed HPB-ALL cDNA library in the pME18S vector by colony hybridization. Nine positive clones were isolated at a rate of ~6 × 10⁵ colonies. The longest 1.3-kb clone was sequenced in both directions. A mouse homologue was obtained by colony hybridization from Ba/F3 cDNA library (27) using the human 1.3-kb cDNA as a probe. Nucleotide sequences were analyzed using GeneWorks (IntelliGenetics), and public data base searches were performed using BLAST.

Plasmids-- The 1.3-kb full-length TRP32 cDNA in pME18S vector (pME18S-G14) was used as a template for PCR to create in-frame constructs for further cloning. pME18S-HA vector in which a hemagglutinin (HA) tag sequence of 11-amino acid extension is inserted after the initial methionine of the pME18S vector, was used to create HA-tagged TRP32 for mammalian expression. pGEX-2T vector (Amersham Pharmacia Biotech) was used to create glutathione S-transferase (GST)-fused TRP32. pQE-30 vector (Qiagen) was used to create histidine-tagged TRP32 and human thioredoxin for bacterial expression. The two conserved cysteine residues of the Trx-like domain in TRP32 were replaced by serine using Quick change site-directed mutagenesis (Stratagene).

Purification of Bacterially Expressed Proteins-- The GST fusion proteins and His-tagged proteins were expressed in E. coli strain XL1blue. The expression of recombinant proteins was induced with 0.1 mM isopropyl-1--D-galactopyranoside for 5 h at 37 °C at a cell density of A₆₀₀ = 1.0. GST fusion proteins were bound to glutathione-Sepharose beads and eluted with glutathione as described previously (28). His-tagged proteins were loaded onto a His-Trap column (Amersham Pharmacia Biotech) and eluted with a 5-500 mM imidazole gradient.

Northern Blot Analysis-- Adult and fetal human multiple-tissue Northern blots (CLONTECH) were hybridized in accordance with manufacturer's instructions. A probe of 723 base pairs encoding the N-terminal 241 amino acid residues of TRP32 was generated by PCR, random-radiolabeled with [-³²P]dCTP, and spin column-purified (Amersham Pharmacia Biotech).

Preparation of Anti-TRP32 Monoclonal Antibody-- Mice were immunized with bacterially expressed GST-TRP32. Lymphocytes from the immunized mice were collected, fused with NS-1 myeloma cells, and grown in ASF104 medium (Ajinomoto, Japan) supplemented with 10% FCS and hypoxanthine/aminopterin/thymidine for the selection of fused cells. Two cloning procedures by serial dilution were carried out after the selection, and finally all of the wells containing single clones became positive for anti-TRP32 antibody production. Cloned hybridoma cells were grown in ASF104 medium with 10% FCS until confluent, and then the hybridoma cells were moved to ASF104 medium without FCS. Culture supernatant without FCS was loaded onto protein G-Sepharose column and eluted with 50 mM glycine·HCl (pH 2.3). The eluate was immediately neutralized with 1 M Tris·HCl (pH 9.0) and dialyzed against 20 mM Tris·HCl (pH 7.5).

Western Blot Analysis-- Cellular total proteins (30 µg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore). The membrane was blocked in TBST (20 mM Tris·HCl (pH 7.5) containing 150 mM NaCl and 0.1% Tween 20) with 5% skim milk at room temperature for 1 h. Antibodies were applied to TBST containing 5% skim milk at the appropriate dilutions for 1 h. The membranes were then washed with TBST and incubated for 1 h with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Pharmacia Biotech). The specific signals were detected on x-ray films using an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech).

Cell Culture and Transfections-- Jurkat, WR19L, HL-60, and HPB-ALL cells were cultured in RPMI 1640 with 10% FCS, 20 mM HEPES (pH 7.3), 50 µM -mercaptoethanol, 50 units/ml penicillin, and 50 µg/ml streptomycin. KB, NIH3T3, Balb/c 3T3, HT-29, COS-7, and 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS and 100 µg/ml kanamycin. NIH3T3 cells were seeded at 1 × 10⁵ cells/35-mm tissue culture dish 1 day before transfection and then transfected with lipofectAMINE (Life Technologies, Inc.) in accordance with the manufacturer's instructions.

Insulin Disulfide Reduction Assay-- E. coli thioredoxin (MBI Fermentas), His-tagged human thioredoxin, and His-tagged TRP32 were compared for the reducing activity of insulin disulfide bonds as described previously (29) with slight modifications. A reduced form of the proteins was prepared by incubating with 2 mM DTT, and oxidized proteins were prepared by incubation with 10 mM Na₂SeO₃ at 4 °C overnight. Then excess amounts of DTT or Na₂SeO₃ were removed by gel chromatography on a NAP-5 column (Amersham Pharmacia Biotech) with ice-cold nitrogen equilibrated 50 mM Tris·HCl (pH 7.5) buffer containing 1 mM EDTA. The 200-µl reaction mixture contained 50 mM Tris·HCl (pH 7.5), 1 mM EDTA, and 0.17 mM porcine insulin (Sigma). A reaction was initiated by adding 2 mM DTT, and the absorbance at 690 nm was immediately recorded at room temperature.

Subcellular Fractionation and Immunofluorescence-- For subcellular fractionation, HPB-ALL cells were homogenized with a Dounce homogenizer in lysis buffer (20 mM Tris·HCl (pH 7.5) containing 0.25 M sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml aprotinin). The lysates were centrifuged at 900 × g for 10 min to obtain the nuclear pellet, and the supernatant was centrifuged at 100,000 × g for 60 min to separate the cytoplasmic fraction from the membrane fraction. The nuclear pellet and membrane pellet were washed once with PBS and suspended in lysis buffer. To investigate the subcellular localization of TRP32, NIH3T3 cells were transfected with cDNA encoding HA-tagged TRP32 using lipofectAMINE (see above). After a 36-h cultivation of the cells on chamber slides (Nunc), the cells were washed with PBS, fixed in 3.7% formaldehyde in PBS containing 0.1% Triton X-100 for 10 min, blocked in 2% bovine serum albumin in PBS for 10 min, and stained with anti-HA monoclonal antibody (12CA5; Boehringer Mannheim) for 1 h. Slides were then washed in PBS. The secondary antibody, fluorescein isothiocyanate-conjugated anti-mouse antibody (Cappel), was used at a 1:500 dilution for 45 min. Cells were washed with PBS, incubated with Hoechst 33342 (5 µg/ml), and washed again. Slides were analyzed using a fluorescence microscope.

	RESULTS

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cDNA Cloning of TRP32-- Previously, we reported that a histone kinase with a molecular mass of 34 kDa is activated in Fas-mediated apoptosis, and this kinase was identified as catalytic fragment of MST, a STE20-related serine/threonine kinase (30). In the course of the purification of this kinase, we purified a protein of 32 kDa that was co-eluted with a catalytic fragment of MST to the final chromatographic purification step (see below). To clarify the molecular identity of this protein, we sequenced the polypeptides that were derived from proteolytic digestion with lysylendopeptidase. Eight peptide sequences were revealed (Fig. 1), and since a public data base search showed no homology to any known proteins, we tried cDNA cloning of this protein, designated TRP32. Degenerate oligonucleotides corresponding to peptides 1 and 2 (Fig. 1) were used for reverse transcriptase PCR using mRNA from HPB-ALL cells as a template. One cDNA fragment of 290 nucleotides was obtained and sequenced. The data base search showed that this cDNA fragment encoded a novel protein with a Trx-like domain. Using the amplified cDNA fragments as probes, we screened the human HPB-ALL cDNA library. A clone of 1.3 kb was isolated and contained a perfect Kozak consensus sequence (AGGATGG) and a stop codon upstream from the putative start codon at the 5' side (Fig. 1). The putative polyadenylation site and poly(A) tail were also identified at the 3' side. The 1.3-kb cDNA contained an open reading frame of 289 amino acid residues with a predicted molecular mass of 32 kDa that coincided well with the molecular weight of the purified TRP32 estimated by SDS-PAGE (see below). All eight peptide sequences obtained from the purified protein were confirmed in the open reading frame of TRP32. We also cloned a mouse homologue of human TRP32. Mouse TRP32 showed 99% homology to human TRP32, and only three amino acids were substituted.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Nucleotide and predicted amino acid sequence of human TRP32. The open reading frame (289 amino acids) is preceded by an in-frame stop codon indicated by the box, and the possible polyadenylation signals are indicated in boldface type with underlines. Amino acid sequences obtained by peptide sequencing are underlined and numbered. Three unconserved amino acid residues in mouse TRP32 are shown in boldface type, and the replaced residues are shown in parentheses. The numbering of the nucleotides and amino acids is indicated on the right.

Similarity of TRP32 to Other Thioredoxin Domain-containing Proteins-- Data base searches using the BLAST program showed that the predicted amino acid sequence of the TRP32 gene was a novel protein and that the N-terminal 100 amino acid residues shared significant similarities with thioredoxin, an evolutionarily conserved ubiquitous protein that participates in various redox reactions (Fig. 2A). The N-terminal domain of TRP32 has 43% identity and 56% similarity to human thioredoxin and has 26-29% identity and 40-43% similarity to the Trx domains of other Trx-related proteins (Fig. 2B). All thioredoxin molecules from prokaryotes to higher eukaryotes have the conserved active site sequence Cys-Gly-Pro-Cys, and in particular, two active site cysteines are preserved in all proteins containing the functional Trx domain. The Trx-like domain of TRP32 also has the active site sequence Cys-Gly-Pro-Cys. Taken together, the N-terminal 100 residues of TRP32 are closely related to thioredoxin in the primary structure.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Sequence comparison of Trx domain of TRP32 with other Trx domain-containing proteins. A, alignment of the Trx domain of TRP32 and human thioredoxin. Conserved residues are boxed, and the two cysteine residues in the active site are indicated by asterisks. Noncatalytic cysteine residues are indicated in boldface type. B, amino acid sequences surrounding the active site in the Trx domain-containing proteins are aligned. Conserved residues in at least three proteins are shown in boldface type.

Expression of mRNA and Protein-- Northern blot analysis using poly(A⁺) RNA from multiple human tissues demonstrated that the TRP32 transcript was ubiquitously expressed (Fig. 3). A specific 1.4-kb mRNA was expressed at the highest levels in the heart and skeletal muscle. We prepared anti-TRP32 monoclonal antibodies and performed immunoblot analysis using one clone, LE4, that recognized the C-terminal 190 amino acids. LE4 recognized a protein of 32 kDa in all human (HPB-ALL, Jurkat, 293, KB), mouse (NIH3T3, Balb/c 3T3), and monkey (COS-7) cells examined (Fig. 4). A band with the same molecular weight was also detected by LE4 in the highly purified TRP32 fraction. In addition, recombinant HA-tagged TRP32 (HA-TRP32), with an 11-amino acid extension at the N terminus, could be detected as a slower migrating band on Western blots with LE4.² These results show that LE4 specifically recognizes TRP32. Taken together, both the TRP32 transcript and protein are ubiquitously expressed in human tissues and various mammalian cell lines.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Expression of the TRP32 transcript. Human adult and fetal tissue poly(A+) RNA Northern blot (CLONTECH) was probed with the 723-base pair cDNA fragment encoding the N-terminal 241 amino acid residues of human TRP32. PBL, peripheral blood leukocytes.

View larger version (59K): [in this window] [in a new window]   Fig. 4.   Expression of the TRP32 proteins. TRP32 protein was detected by Western blotting using anti-TRP32 monoclonal antibody, LE4, in various cell lines as indicated. The experiment was performed as described under "Experimental Procedures." Highly purified TRP32 fraction from Mono-Q column chromatography was also detected by immunoblotting.

Analysis of Purified TRP32-- Final chromatographic fractions from the Superdex 75 gel filtration were analyzed by SDS-PAGE. A doublet with a molecular mass of 32 kDa, which was identified as TRP32 by peptide sequencing, was eluted at the expected elution volume from its molecular weight (Fig. 5A). The anti-TRP32 antibody, LE4, also specifically recognized doublet bands of 32 kDa, proving the LE4-reactive proteins to be TRP32 (Fig. 5B). The purified TRP32 might be modified or cleaved during chromatographic procedures because TRP32 was detected as a single band in the total cell lysates (Fig. 4). Only a single band of TRP32 was also observed in apoptosis-induced HPB-ALL cells.² The elution pattern of TRP32 by gel filtration chromatography was nearly identical to that of the kinase-active catalytic fragment of MST (Fig. 5, C and D), confirming the co-purification of these two proteins. TRP32 and the catalytic fragment of MST were superimposable even in the two-dimensional electrophoresis analysis (30) with a molecular mass of 32-34 kDa and isoelectric point (pI) of 5.2 (Fig. 5E). The pI of purified TRP32 was slightly higher than the theoretical value of 4.6. 

View larger version (50K): [in this window] [in a new window]   Fig. 5.   Analysis of purified TRP32. A-D, the eluted fractions from the Superdex 75 gel filtration column, resolved by SDS-PAGE (10% polyacrylamide gel), were silver-stained (A), immunoblotted with anti-TRP32 antibody (LE4) (B), immunoblotted with anti-MST antibody (C), or subjected to an in-gel phosphorylation assay (D) as described under "Experimental Procedures." Molecular masses determined from the elution volume of the standard proteins are indicated. E, aliquot of fraction 42 in A was subjected to two-dimensional electrophoresis and immunoblotted with LE4.

Reductase Activity of TRP32-- To investigate the Trx-like reducing activity of TRP32, we expressed recombinant TRP32 and human thioredoxin as His-tagged forms (His-TRP32 and His-Trx) in E. coli. A truncated TRP32 (His-TRP32N) consisting of N-terminal Trx-like 107 amino acid residues was also prepared. In addition, Cys  Ser mutants, His-TRP32(C34S,C37S) and His-TRP32N(C34S,C37S), in which the two cysteines (Cys³⁴ and Cys³⁷) homologous to those in the thioredoxin active site were mutated to serines, were generated from His-TRP32 and His-TRP32N, respectively. The expressed recombinant proteins were purified by His-Trap column chromatography. His-TRP32N showed reducing activity for the insulin disulfide bonds with kinetics slower than that of His-Trx (Fig. 6A) but faster than that of purified E. coli thioredoxin. His-TRP32N(C34S,C37S) completely failed to reduce insulin, indicating that the N-terminal Trx-like domain of TRP32 is functionally related to thioredoxin. However, full-length His-TRP32 failed to reduce insulin, suggesting that the C-terminal region may regulate the activity of the N-terminal Trx domain or that bacterially expressed His-TRP32 may not be folded to its native structure. We tested the effect of the C-terminal region on the N-terminal reducing activity by mixing His-TRP32C (residue 107-289). However, His-TRP32C did not inhibit the reducing activity of His-TRP32N even in the presence of a 5-fold molar excess. In addition, direct interaction between the N-terminal and the C-terminal domains of TRP32 could not be detected either in vitro or in vivo.² We studied the effect of oxidation on the reducing activity of the His-TRP32N (Fig. 6B). The reducing activity of oxidized His-TRP32N had not recovered after 2 h of incubation with DTT, although His-Trx rapidly recovered the reducing activity within 5 min in the presence of DTT, suggesting that reduction to reductase-active His-TRP32N by DTT is a time-consuming and rate-limiting process. These results imply that the Trx domain of TRP32 is functionally related to thioredoxin but not identical.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Reductase activity of Trx-like domain of TRP32. Thioredoxin and TRP32 were assayed for their ability to reduce the disulfide bonds of insulin. A, reducing activity was measured using reduced forms of recombinant His-tagged proteins. ×, Trx (E. coli); , His-Trx (human); , His-TRP32; , His-TRP32(C34S,C37S); , His-TRP32N; , His-TRP32N(C34S,C37S). B, reduced and oxidized recombinant proteins, prepared as described under "Experimental Procedures," were compared for their reducing activity. , oxidized His-Trx; , oxidized His-TRP32N; , oxidized His-TRP32N(C34S,C37S); , reduced His-Trx; , reduced TRP32N; , reduced TRP32N(C34S,C37S). Absorbance at 690 nm was measured at 5-min intervals with mixing. Similar results were obtained from two independent experiments.

Subcellular Distribution of TRP32-- To investigate the subcellular distribution of endogenous TRP32, we prepared subcellular fractions from HPB-ALL cells. The endogenous TRP32 was detected in the cytoplasmic fraction.² The cytoplasmic distribution of TRP32 did not change when HPB-ALL cells underwent apoptosis by treatment with the anti-Fas monoclonal antibody, CH-11.

	DISCUSSION

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We have purified a protein with a molecular mass of 32 kDa, which was co-purified with the catalytic fragment of protein kinase MST. cDNA cloning of the gene encoding this protein, TRP32 (thioredoxin-related protein of 32 kDa), shows that TRP32 is a novel protein containing one Trx domain. Human and mouse TRP32 proteins are 99% identical, and in particular, the Trx domains are completely homologous. We also identified a Drosophila expressed sequence tag (GenBank^TM accession number AA264961) whose predicted N-terminal 100 residues are similar to the Trx domain of TRP32 (48% identity). This clone may be a Drosophila homologue of TRP32 because the adjacent region to the Trx domain (100-150 residues) shows high homology to the C-terminal domain of TRP32, which has no similarity to any proteins in the public data bases. However, we could not identify any putative TRP32 homologue in E. coli or yeast genome sequences. Thus, TRP32 may be evolutionarily conserved in multicellular eukaryotes.

TRP32 seems to be closely related to thioredoxin. The Trx domain of TRP32 is most similar to thioredoxin in mammalian Trx-related proteins. TRP32 is widely expressed in human tissues, and expression of TRP32 protein has been confirmed in various mammalian cell lines (Figs. 3 and 4). The recombinant Trx domain of TRP32 (His-TRP32N) has reducing activity comparable with human thioredoxin (Fig. 6). TRP32 is localized in the cytoplasm. TRP32 might have acquired specialized functions in multicellular eukaryotes during evolution from thioredoxin, which is conserved from prokaryotes to animals.

Despite the high similarity of the two proteins, however, some functional and structural differences were observed. The Trx domain of TRP32 is more sensitive to oxidation than thioredoxin, and full-length TRP32 has no reducing activity (Fig. 6). Mammalian thioredoxin contains other conserved cysteine residues in addition to the two cysteine residues in the active site. The human thioredoxin contains three such extra cysteine residues (Fig. 2A). These noncatalytic cysteine residues, Cys⁶², Cys⁶⁹, and Cys⁷³, in human thioredoxin can reportedly undergo oxidation, which leads to inactivation and dimerization of thioredoxin (31). In particular, Cys⁷³, located on the surface close to the active site, is reportedly linked to the redox regulation of human thioredoxin (32, 33). In contrast, the Trx domain of TRP32 has only one extra cysteine residue, Cys⁶⁴, corresponding to the Cys⁶² of human thioredoxin. Cys⁷³ of human thioredoxin is replaced by Ala in TRP32, which is also changed to Gly⁷⁴ in E. coli thioredoxin. Thus, the reducing activity of TRP32 may be differently regulated from mammalian thioredoxin, and TRP32 is functionally different from thioredoxin, since the C-terminal region of TRP32 may regulate the reducing activity (Fig. 6). The C-terminal region of human TRP32 does not have homology to other proteins but is conserved in mouse and probably Drosophila, suggesting its functional importance. The role of the C-terminal region of TRP32 should be elucidated.

The function of TRP32 may be also different from that of the PDI family because TRP32 has no homology to PDI except in the Trx domain. Furthermore, the Trx domain of TRP32 is significantly different from PDI. TRP32 has one Trx domain, but two or three Trx domains exist in the PDI family. In addition, TRP32 and mammalian thioredoxins have the conserved active site sequence, Cys-Gly-Pro-Cys, in which the third Pro is replaced by His in the PDI family (Fig. 2B). Pro  His-replaced thioredoxin reportedly shows increased reduction potential and can complement the PDI null mutant of Saccharomyces cerevisae (34), implying that the Trx domain of TRP32 is biochemically and biologically different from that of PDI. Different localization of two proteins, PDI in ER and TRP32 in cytoplasm, also suggests different functions in the different environments.

Recently, two new mammalian Trx-related proteins have been reported (Table I). Trx2 (35), which consists of 166 amino acid residues with the active site sequences found in mammalian thioredoxin, has higher homology with the E. coli thioredoxin than with the known mammalian proteins. Trx2 has a mitochondrial translocation signal, and the mature protein is localized in mitochondria. Kurooka et al. (36) reported another Trx-related nuclear protein, nucleoredoxin. Nucleoredoxin has one Trx domain in the central region with a modified active site sequence, -Cys-Pro-Pro-Cys, but it can reduce insulin disulfide in vitro. The second Pro residue in the active site sequence is quite rare, and no other proteins with Pro in this residue have been reported. Nucleoredoxin is localized in the nucleus when overexpressed in NIH3T3 cells. TRP32 may be also functionally different from these two proteins. First, these proteins have no homologous regions except the Trx domain and show a low homology only at the sequences surrounding the active site of the Trx domain. Second, their subcellular localizations are completely different. Trx2 and nucleoredoxin are mitochondrial and nuclear protein, respectively, whereas TRP32 is a cytoplasmic protein. The restricted localization of these proteins including PDI may reflect their specific roles in different subcellular compartments.

One possible role of TRP32 may be the redox regulation of cytoplasmic proteins including transcription factors. The DNA binding activity of some transcription factors, including AP-1, NF-B, Myb, and Ets are known to be regulated by a thiol-redox control mechanism (37-40). Thioredoxin augments the DNA binding and transcription activities of the p50 subunit of NF-B by reducing Cys⁶² in its DNA-binding loop (41, 42). Thioredoxin enhances the DNA binding activity of Jun and Fos (12, 13). Furthermore, thioredoxin translocates from the cytoplasm into the nucleus by treatment with PMA and UV irradiation in a nucleus localization signal-independent manner (43, 44). It may be interesting to investigate whether TRP32 has such a redox activity on transcription factors and whether it signal-dependently translocates into the nucleus.

Another possible function of TRP32 is the regulation of apoptosis. Reactive oxygen intermediates are implicated in apoptosis (32). Some process of apoptosis might be regulated by the cellular redox state, and TRP32 may regulate apoptosis through the control of the redox state. TRP32 was co-purified with the catalytic fragment of MST, which is proteolytically activated by caspase in apoptosis. The possibility cannot be excluded that TRP32 controls apoptosis by regulating the activity of MST and/or vice versa, although our preliminary experiment does not show co-immunoprecipitation of TRP32 and the catalytic fragment of MST.² A study of TRP32 upon apoptosis is currently under way. Our cloning of a novel human thioredoxin-related protein may contribute to elucidating the regulation of the redox state in cells, and the function of TRP32 should be further clarified.