title: Identification of novel nuclear targets of human thioredoxin 1.
abstract: The dysregulation of protein oxidative post-translational modifications has been implicated in stress-related diseases. Trx1 is a key reductase that reduces specific disulfide bonds and other cysteine post-translational modifications. Although commonly in the cytoplasm, Trx1 can also modulate transcription in the nucleus. However, few Trx1 nuclear targets have been identified because of the low Trx1 abundance in the nucleus. Here, we report the large-scale proteomics identification of nuclear Trx1 targets in human neuroblastoma cells using an affinity capture strategy wherein a Trx1C35S mutant is expressed. The wild-type Trx1 contains a conserved C32XXC35 motif, and the C32 thiol initiates the reduction of a target disulfide bond by forming an intermolecular disulfide with one of the oxidized target cysteines, resulting in a transient Trx1-target protein complex. The reduction is rapidly consummated by the donation of a C35 proton to the target molecule, forming a Trx1 C32-C35 disulfide, and results in the concurrent release of the target protein containing reduced thiols. By introducing a point mutation (C35 to S35) in Trx1, we ablated the rapid dissociation of Trx1 from its reduction targets, thereby allowing the identification of 45 putative nuclear Trx1 targets. Unexpectedly, we found that PSIP1, also known as LEDGF, was sensitive to both oxidation and Trx1 reduction at Cys 204. LEDGF is a transcription activator that is vital for regulating cell survival during HIV-1 infection. Overall, this study suggests that Trx1 may play a broader role than previously believed that might include regulating transcription, RNA processing, and nuclear pore function in human cells.
authors.affiliation: From the ‡Center for Advanced Proteomics Research and Department of Microbiology, Biochemistry & Molecular Genetics, Rutgers University-New Jersey Medical School Cancer Center, 205 S. Orange Ave., Newark, New Jersey 07103;
authors.forename: Changgong
authors.initials: C
authors.lastname: Wu
authors.affiliation: From the ‡Center for Advanced Proteomics Research and Department of Microbiology, Biochemistry & Molecular Genetics, Rutgers University-New Jersey Medical School Cancer Center, 205 S. Orange Ave., Newark, New Jersey 07103;
authors.forename: Mohit Raja
authors.initials: MR
authors.lastname: Jain
authors.affiliation: From the ‡Center for Advanced Proteomics Research and Department of Microbiology, Biochemistry & Molecular Genetics, Rutgers University-New Jersey Medical School Cancer Center, 205 S. Orange Ave., Newark, New Jersey 07103;
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.affiliation: ¶Department of Cell Biology and Molecular Medicine, Rutgers University-New Jersey Medical School, 185 S. Orange Ave., Newark, New Jersey 07103;
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Oka
authors.affiliation: ‖Albert Einstein College of Medicine, 1301 Morris Park Avenue, Bronx, New York 10461;
authors.forename: Wenge
authors.initials: W
authors.lastname: Li
authors.affiliation: **Department of Pharmaceutics, Rutgers University-Ernest Mario School of Pharmacy, Piscataway, New Jersey 08854.
authors.forename: Ah-Ng Tony
authors.initials: AN
authors.lastname: Kong
authors.affiliation: ¶Department of Cell Biology and Molecular Medicine, Rutgers University-New Jersey Medical School, 185 S. Orange Ave., Newark, New Jersey 07103;
authors.forename: Narayani
authors.initials: N
authors.lastname: Nagarajan
authors.affiliation: ¶Department of Cell Biology and Molecular Medicine, Rutgers University-New Jersey Medical School, 185 S. Orange Ave., Newark, New Jersey 07103;
authors.forename: Junichi
authors.initials: J
authors.lastname: Sadoshima
authors.affiliation: From the ‡Center for Advanced Proteomics Research and Department of Microbiology, Biochemistry & Molecular Genetics, Rutgers University-New Jersey Medical School Cancer Center, 205 S. Orange Ave., Newark, New Jersey 07103;
authors.forename: William J
authors.initials: WJ
authors.lastname: Simmons
authors.affiliation: From the ‡Center for Advanced Proteomics Research and Department of Microbiology, Biochemistry & Molecular Genetics, Rutgers University-New Jersey Medical School Cancer Center, 205 S. Orange Ave., Newark, New Jersey 07103; liho2@rutgers.edu.
authors.forename: Hong
authors.initials: H
authors.lastname: Li
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D009154:Mutation; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010084:Oxidation-Reduction; D025941:Protein Interaction Mapping; D015398:Signal Transduction; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004220:Disulfides; C504649:PSIP1 protein, human; C491203:TXN protein, human; D014157:Transcription Factors; D013879:Thioredoxins; D003545:Cysteine