Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jul;67(3):203-8.
doi: 10.1016/j.neures.2010.04.002. Epub 2010 Apr 10.

DJ-1 protects against oxidative damage by regulating the thioredoxin/ASK1 complex

Joo-Young Im  1 , Kang-Woo Lee, Eunsung Junn, M Maral Mouradian
Affiliations

DJ-1 protects against oxidative damage by regulating the thioredoxin/ASK1 complex

Joo-Young Im et al. Neurosci Res. 2010 Jul.

Abstract

DJ-1 is a multifunctional protein linked to recessively inherited Parkinson's disease (PD) due to loss of function mutations. Among its activities is anti-oxidant property leading to cytoprotection under oxidative stress conditions. A key effector of oxidant-induced cell death is the MAP3 kinase apoptosis signal-regulating kinase 1 (ASK1) which is bound to and inhibited by thioredoxin 1 (Trx1) under basal conditions. Upon oxidative stimuli, however, ASK1 dissociates from this physiological inhibitor and is activated. In the present study, we investigated the role of DJ-1 in regulating Trx1/ASK1 interaction. Over-expression of DJ-1 suppressed ASK1 activation in response to H(2)O(2) in a time-dependent manner. Wild-type DJ-1, but not the PD-associated L166P mutant, prevented the dissociation of ASK1 from Trx1 in response to H(2)O(2). Among cysteine mutants of DJ-1, C46S, C53S, and C106S, only C106S failed to inhibit this dissociation implying that cysteine 106 is essential for Trx1/ASK1 regulation. Furthermore, compared to wild-type mice, DJ-1 null mouse brain homogenates and embryonic fibroblasts were more susceptible to oxidant-induced dissociation of ASK1 from Trx1, activation of the downstream kinase c-Jun N-terminal kinase, and to cell death. These findings point to yet another mechanism through which DJ-1 has anti-oxidant and cytoprotective properties by regulating the Trx1/ASK1 complex and controlling the availability of ASK1 to effect apoptosis.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. DJ-1 represses ASK1 activation
HEK293T cells were transfected with Myc-ASK1 along with Flag-DJ-1 or empty Flag vector. Thirty six h later, cells were incubated with 0.5 mM H2O2 for the indicated times. Cell lysates were subjected to Western blot analysis with anti-p-ASK1, anti-Myc and anti-Flag antibodies. For each sample, the relative intensity of p-ASK1 was normalized to total Myc-ASK1 using NIH ImageJ. Data represent means±S.E.M. of three independent experiments. ** p<0.01 compared to empty Flag vector-transfected cells by Student t-test.
Fig. 2
Fig. 2. DJ-1 inhibits the dissociation of Trx1 from ASK
HEK293T cells were transfected with the indicated expression plasmids and incubated with 0.5 mM H2O2 for 30 min. (A) Cell lysates were immunoprecipitated with anti-Myc and Western blot analysis was performed with anti-GST and anti-Myc. The relative intensity of GST-Trx1 was normalized to Myc-ASK1 (B) GST-pull down experiment. GST-Trx1 was pulled down with glutathione-4B-sepharose beads, and Western blots performed using anti-Myc and anti-GST. Data shown are representative from three independent experiments. The relative density of Myc-ASK1 was normalized to GST-Trx1. Data represent means±S.E.M. * p<0.01.
Fig. 3
Fig. 3. L166P mutant DJ-1 fails to regulate Trx1/ASK1 interaction
HEK293T cells were transfected for 36 h with GST-Trx1, Myc-ASK1, and 1 μg of WT Flag-DJ-1, or 3 μg of L166P mutant Flag-DJ-1. Cells were exposed to 0.5 mM H2O2 for 30 min, followed by immunoprecipitation with anti-Myc. Western blot analysis was performed with anti-GST and anti-Myc. The relative intensity of GST-Trx1 was normalized to Myc-ASK1. Data are shown as means±S.E.M. of three independent experiments. * p<0.01.
Fig. 4
Fig. 4. DJ-1 cysteine 106 mutant fails to regulate Trx1/ASK1 interaction
HEK293T cells were transfected for 36 h with GST-Trx1, Myc-ASK1, along with WT Flag-DJ-1, or cysteine mutants C46S, C53S, or C106S Flag-DJ-1. Cells were incubated with 0.5 mM H2O2 for 30 min, followed by immunoprecipitation with anti-Myc. Western blot analysis was performed with anti-GST and anti-Myc. As Flag Western of total lysates shows, C46S mutant has lower expression than the other constructs, as reported previously (Junn et al., 2009; Waak et al., 2009). The results shown are representative from three independent experiments. The relative intensity of GST-Trx1 was normalized to Myc-ASK1. Data are shown as means±S.E.M. * p<0.001.
Fig. 5
Fig. 5. DJ-1 deletion promotes the dissociation of ASK1/Trx1 and cell death
(A) Brain homogenates from WT and DJ-1 null mice were exposed to 0.1 mM H2O2 (H) or buffer (C) for 30 min, followed by immunoprecipitation with anti-Trx1 or normal rabbit IgG. Western blot analysis was performed with anti-ASK1, anti-Trx1, and anti-DJ-1 antibodies. The relative intensity of ASK1 was normalized to Trx1. Data represent means±S.E.M. of three independent experiments. * p<0.05, ** p<0.01. (B) MEFs isolated from WT and DJ-1 null mice were challenged with 0.5 mM H2O2 for the indicated times. Western blot analysis was performed with anti-pJNK, anti-JNK, or anti-DJ-1. The relative intensity of pJNK was normalized to JNK. Data shown are means±S.E.M. of three independent experiments. * p<0.05, ** p<0.01. (C) WT and DJ-1 null MEFs were incubated with 0.1 mM H2O2 for 24 h, and cell death was measured by LDH assay. Data represent means±S.E.M (n=6). * p<0.05 compared to WT H2O2-treated cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Andres-Mateos E, Perier C, Zhang L, Blanchard-Fillion B, Greco TM, Thomas B, Ko HS, Sasaki M, Ischiropoulos H, Przedborski S, Dawson TM, Dawson VL. DJ-1 gene deletion reveals that DJ-1 is an atypical peroxiredoxin-like peroxidase. Proc Natl Acad Sci U S A. 2007;104:14807–14812. - PMC - PubMed
    1. Bandopadhyay R, Kingsbury AE, Cookson MR, Reid AR, Evans IM, Hope AD, Pittman AM, Lashley T, Canet-Aviles R, Miller DW, McLendon C, Strand C, Leonard AJ, Abou-Sleiman PM, Healy DG, Ariga H, Wood NW, de Silva R, Revesz T, Hardy JA, Lees AJ. The expression of DJ-1 (PARK7) in normal human CNS and idiopathic Parkinson’s disease. Brain. 2004;127:420–430. - PubMed
    1. Bonifati V, Rizzu P, van Baren MJ, Schaap O, Breedveld GJ, Krieger E, Dekker MC, Squitieri F, Ibanez P, Joosse M, van Dongen JW, Vanacore N, van Swieten JC, Brice A, Meco G, van Duijn CM, Oostra BA, Heutink P. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science. 2003;299:256–259. - PubMed
    1. Canet-Aviles RM, Wilson MA, Miller DW, Ahmad R, McLendon C, Bandyopadhyay S, Baptista MJ, Ringe D, Petsko GA, Cookson MR. The Parkinson’s disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. Proc Natl Acad Sci U S A. 2004;101:9103–9108. - PMC - PubMed
    1. Chang HY, Nishitoh H, Yang X, Ichijo H, Baltimore D. Activation of apoptosis signal-regulating kinase 1 (ASK1) by the adapter protein Daxx. Science. 1998;281:1860–1863. - PubMed

Publication types

MeSH terms