Purification and characterization of delta3Trx-1, a splicing variant of human thioredoxin-1 lacking exon 3.
Thioredoxins comprise a growing family of proteins that function as general protein-disulfide reductases and are maintained in their reduced active form by the flavoenzyme thioredoxin reductase. Human Trx-1 is mainly a cytosolic protein, although it has been shown to translocate into the nucleus upon certain stimuli and can also be secreted. We report here the expression and characterization of delta3Trx-1, a splicing variant of human Trx-1, lacking exon 3, which spans from residues 44 to 63 in the wild-type protein. Structure-based prediction of this splicing form indicates that delta3Trx-1 lacks helix alpha2 and strand beta3, which are implicated in substrate positioning and three-dimensional stabilization of the active site residues. Recombinant human delta3Trx-1 is recognized by polyclonal antibodies raised against full-length human Trx-1. However, delta3Trx-1 retains no enzymatic activity either with DTT or thioredoxin reductase and NADPH as reducing systems. Delta3Trx-1 competes with full-length Trx-1 for the interaction with thioredoxin reductase. The absence of helix alpha2 and strand beta3 in delta3Trx-1 is consistent with the lack of enzymatic activity and its potential dominant negative properties.
Jiménez
Alberto
A
Department of Biosciences at NOVUM, Center for Biotechnology, Karolinska Institutet, S-14157 Huddinge, Sweden.
Miranda-Vizuete
Antonio
A
D021581:Active Transport, Cell Nucleus; D017398:Alternative Splicing; D001665:Binding Sites; D015153:Blotting, Western; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D005799:Genes, Dominant; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D008957:Models, Genetic; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D013997:Time Factors; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D008565:Membrane Proteins; D011994:Recombinant Proteins; C491203:TXN protein, human; D013879:Thioredoxins; D004229:Dithiothreitol