Oligomerization of the cysteinyl-rich oligopeptidase EP24.15 is triggered by S-glutathionylation

Demasi, Marilene; Filho, Gilberto M. Piassa; Castro, Leandro M.; Ferreira, Juliana C.; Rioli, Vanessa; Ferro, Emer S.

doi:10.1016/j.freeradbiomed.2007.12.012

Cited by 30 publications

(41 citation statements)

References 31 publications

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“…It appeared that drug-induced glutathionylation can also generate intermolecular crosslinking of p53 (Fig. 3C) as has been observed for the oligopeptidase EP24.15 (42); such an event occurs through a thiol-disulfide exchange between thiolated and unthiolated protein molecules.…”

Section: Discussionmentioning

confidence: 61%

Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines

Yusuf

Chuang²,

Bhat

et al. 2010

Free Radical Biology and Medicine

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Previously, we reported that human p53 is functionally inactivated by S-glutathionylation at Cys-141 during oxidative and DNA-damaging treatments. Here, we describe the presence of thiolated p53 and dynamic nature of this modification in human tissues using unique and specific polyclonal antibodies raised against a 12-residue p53 peptide bearing a mixed disulfide at Cys-141. The affinity-purified antibodies (glut-p53) were sequence-specific in that they recognized the antigenic peptide but not the unthiolated peptide or a scrambled glutathionylated peptide in ELISAs. On immunoblots, the purified antibodies did not react with native p53 or recombinant p53 (rp53), but readily detected the glutathionylated or cysteinylated or ethanethioltreated rp53 only under non-reducing conditions. Untreated HCT116 cells showed low levels of glut-p53 which increased markedly after H 2 O 2 , diamide, cisplatin, and doxorubicin treatments. Glut-p53 levels decreased sharply after passing cells to oxidant-free media, suggesting efficient dethiolation. The mutant p53 present in HT29 and T47D human cancer cells was also recognized. In vitro, the glut-p53 was rapidly degraded by rabbit reticulocyte lysates. Human prostate and prostate cancer tissues showed abundant presence of glut-p53 in luminal epithelium, a site wellknown to generate ROS. Melanoma and colon cancer samples were also positive for glut-p53. Availability of the thiolation-specific antibodies should enhance our knowledge of p53 regulation in redox-perturbed states found in various diseases including cancer.

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Section: Discussionmentioning

confidence: 61%

Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines

Yusuf

Chuang²,

Bhat

et al. 2010

Free Radical Biology and Medicine

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“…It is therefore of interest that thimet oligopeptidase (EP24.15), a ubiquitously expressed thiol-rich metallopeptidase that is important in the degradation of peptides generated by the proteasome machinery, is constitutively glutathionylated intracellularly [96]. The findings suggested an unconventional property of protein glutathionylation, namely inducing oligomerization by interprotein thiol-disulfide exchange to a less active enzyme form [96]. These results may be relevant to the dual role of EP24.15 both in degrading antigenic peptides, which results in decreased antigen presentation, and in increasing production of antigenic peptides, for example, under conditions of oxidative stress.…”

Section: Biological Importance Of Protein Glutathionylationmentioning

confidence: 99%

“…These results may be relevant to the dual role of EP24.15 both in degrading antigenic peptides, which results in decreased antigen presentation, and in increasing production of antigenic peptides, for example, under conditions of oxidative stress. It is of further interest that the Rpn1 and Rpn2 subunits of purified human 26S proteasomes undergo glutathionylation, resulting in a less active complex when exposed to H 2 O 2 and GSH [96]. …”

Section: Biological Importance Of Protein Glutathionylationmentioning

confidence: 99%

Reversible and irreversible protein glutathionylation: biological and clinical aspects

Cooper

Pinto

Callery

2011

Expert Opinion on Drug Metabolism & Toxicology

133

103

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Introduction Depending in part on the glutathione to glutathione disulfide ratio, reversible protein glutathionylation to a mixed disulfide may occur. Reversible glutathionylation is important in protecting proteins against oxidative stress, guiding correct protein folding, regulating protein activity, and modulating proteins critical to redox signaling. The potential also exists for irreversible protein glutathionylation via Michael addition of an -SH group to a dehydroalanyl residue, resulting in formation of a stable, non-reducible thioether linkage. Areas covered This article reviews factors contributing to reversible and irreversible protein glutathionylation and their biomedical implications. It also examines the possibility that certain drugs such as busulfan may be toxic by promoting irreversible glutathionylation. The reader will gain an appreciation of the protective nature and control of function resulting from reversible protein glutathionylation. The reader is also introduced to the recently identified phenomenon of irreversible protein glutathionylation and its possible deleterious effects. Expert opinion The process of reversible protein glutathionylation is now well established but these findings need to be substantiated at the tissue and organ levels, and also with disease state. That being said, irreversible protein glutathionylation can also occur and this has implications in disease and aging. Toxicologists should consider this when evaluating the possible side effects of certain drugs such as busulfan that may generate a glutathionylating species in vivo.

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“…In addition, a higher-molecular-mass species was detected around 113 kDa (top panel, lane 3, arrow), albeit to a lesser degree, presumably representing an S-glutathionylation-triggered multimeric APE1 form. 37 Since S-glutathionylation can be reversed by deglutathionylation with reducing agents, 37 we tested whether the observed modification could be reversed by dithiothreitol (DTT) treatment. As would be expected for S-glutathionylation, the observed modification of APE1 was fully reversed by DTT exposure (top panel, lane 4).…”

Section: Resultsmentioning

confidence: 99%

S-Glutathionylation of Cysteine 99 in the APE1 Protein Impairs Abasic Endonuclease Activity

Kim

Illuzzi

et al. 2011

Journal of Molecular Biology

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Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a central participant in the base excision repair pathway, exhibiting AP endonuclease activity that incises the DNA backbone 5′ to an abasic site. Besides its prominent role as a DNA repair enzyme, APE1 was separately identified as a protein called redox effector factor 1, which is able to enhance the DNA binding activity of several transcription factors through a thiol-exchange-based reduction–oxidation mechanism. In the present study, we found that human APE1 is S-glutathionylated under conditions of oxidative stress both in the presence of glutathione in vitro and in cells. S-glutathionylated APE1 displayed significantly reduced AP endonuclease activity on abasic-site-containing oligonucleotide substrates, a result stemming from impaired DNA binding capacity. The combination of site-directed mutagenesis, biochemical assays, and mass spectrometric analysis identified Cys99 in human APE1 as the critical residue for the S-glutathionylation that leads to reduced AP endonuclease activity. This modification is reversible by reducing agents, which restore APE1 incision function. Our studies describe a novel posttranslational modification of APE1 that regulates the DNA repair function of the protein.

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Oligomerization of the cysteinyl-rich oligopeptidase EP24.15 is triggered by S-glutathionylation

Cited by 30 publications

References 31 publications

Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines

Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines

Reversible and irreversible protein glutathionylation: biological and clinical aspects

S-Glutathionylation of Cysteine 99 in the APE1 Protein Impairs Abasic Endonuclease Activity

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