Demonstration of the interaction of thioredoxin with p40phox, a phagocyte oxidase component, using a yeast two-hybrid system.
Thioredoxin (TRX) has disulfide reducing activity and is reported to be involved in various cellular functions including the promotion of cell growth and apoptosis. To help understand the molecular mechanism through which TRX is involved in immunological systems, we screened a cDNA library derived from a B-cell population of Epstein-Barr virus-transformed human peripheral blood lymhocyte for TRX binding proteins by use of a yeast two-hybrid system. Among plasmids from positive clones, a plasmid contained an insert which has homology with human p40phox, a cytosolic component of phagocyte oxidase. This insert sequence extended from the base + 181 to the stop codon of p40phox. The entire coding region of p40phox was shown to interact with TRX both in assays of histidine prototrophy and beta-galactosidase activity; in contrast, no interaction was observed with substituted mutant TRX (C32S/C35S), which lacks reducing activity. These results showed that p40phox interacts with TRX and indicated the possibility of TRX-dependent regulation of phagocyte oxidase activity.
Nishiyama
A
A
Department of Biological Responses, Institute for Virus Research, Kyoto University, Sakyo, Japan.
Ohno
T
T
Iwata
S
S
Matsui
M
M
Hirota
K
K
Masutani
H
H
Nakamura
H
H
Yodoi
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015723:Gene Library; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D010586:Phagocytes / Q000737:chemistry / Q000201:enzymology*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D046911:Macromolecular Substances; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C085072:neutrophil cytosol factor 40K; D013879:Thioredoxins; D010088:Oxidoreductases