Human p53 Is Inhibited by Glutathionylation of Cysteines Present in the Proximal DNA-Binding Domain during Oxidative Stress^,

Abstract

The cellular mechanisms that modulate the redox state of p53 tumor suppressor remain unclear, although its DNA binding function is known to be strongly inhibited by oxidative and nitrosative stresses. We show that human p53 is subjected to a new and reversible posttranslational modification, namely, S-glutathionylation in stressed states, including DNA damage. First, a rapid and direct incorporation of biotinylated GSH or GSSG into the purified recombinant p53 protein was observed. The modified p53 had a significantly weakened ability to bind its consensus DNA sequence. Reciprocal immunoprecipitations and a GST overlay assay showed that p53 in tumor cells was marginally glutathionylated; however, the level of modification increased greatly after oxidant and DNA-damaging treatments. GSH modification coexisted with the serine phophorylations in activated p53, and the thiol-conjugated protein was present in nuclei. When tumor cells treated with camptothecin or cisplatin were subsequently exposed to glutathione-enhancing agents, p53 underwent dethiolation accompanied by detectable increases in the level of p21waf1 expression, relative to the DNA-damaging drugs alone. Mass spectrometry of GSH-modified p53 protein identified cysteines 124, 141, and 182, all present in the proximal DNA-binding domain, as the sites of glutathionylation. Biotinylated maleimide also reacted rapidly with Cys141, implying that this is the most reactive cysteine on the p53 surface. The glutathionylatable cysteines were found to exist in a negatively charged microenvironment in cellular p53. Molecular modeling studies located Cys124 and -141 at the dimer interface of p53 and showed glutathionylation of either residue would inhibit p53-DNA association and also interfere with protein dimerization. These results show for the first time that shielding of reactive cysteines contributes to a negative regulation for human p53 and imply that such an inactivation of the transcription factor may represent an acute defensive response with significant consequences for oncogenesis.

Effective elimination of fludarabine-resistant CLL cells by PEITC through a redox-mediated mechanism

“…Under mild oxidative stress, protein glutathionylation was shown to regulate the functions of multiple proteins. 23,31 Here, we showed that the antiapoptotic protein MCL1 is a novel substrate of glutathionylation in CLL cells, and that such glutathionylation regulates its stability. Removal of glutathionylation by the glutathione-depleting agent PEITC rendered MCL1 susceptible to rapid proteolytic cleavage.…”

“…This suggests that the depletion of glutathione might play a role in MCL1 cleavage. Because glutathionylation of certain proteins may affect their functions and stability, 23,31 we speculated that MCL1 may be a target of glutathionylation, and its glutathionylation status might affect susceptibility to caspase-cleavage. To test the possibility, we first examined whether in vitro glutathionylation of MCL1 could protect it from cleavage by caspase-3.…”

Identification of Two Reactive Cysteine Residues in the Tumor Suppressor Protein p53 Using Top-Down FTICR Mass Spectrometry

“…This is in conflict with both their X-ray crystallography data and the top-down FTICR MS results described here. Intriguingly, Velu et al [46] also concluded that Cys141 is the most reactive cysteine residue in p53, after using a bottom-up MS approach to investigate cysteine alkylation. Cys141 has a solvent accessibility value of zero, and the X-ray crystal structure of the p53 core domain shows that it is buried within two sides of the β-sheet sandwich (see Table 1 and Fig.…”

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal

“…Several signaling proteins have been shown to undergo glutathionylation, including PTPs and transcription factors (36,63,(77)(78)(79)(80). Although the mechanism by which glutathionylation occurs is not agreed upon, this process has been shown occur in vivo in relevant cell types (81)(82)(83)(84)(85)(86). Although glutathionylation of PTP1B can be achieved in vitro with the purified enzyme through oxidation by H 2 O 2 and addition of GSH (through the sulfenic acid and sulfenyl-amide as described above) (26) (27) or by addition of a very high concentration of GSSG (63), it is not known if glutathionylation occurs enzymatically or non-enzymatically in vivo and kinetic considerations clearly argue against the latter (87).…”