Regulation of DJ-1 by glutaredoxin 1 \u3ci\u3ein vivo – implications for Parkinson’s disease\u3c/i\u3e

Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide, caused by the degeneration of the dopaminergic neurons in the substantia nigra. Mutations in PARK7 (DJ-1) result in early onset autosomal recessive PD, and oxidative modification of DJ-1 has been reported to regulate the protective activity of DJ-1 in vitro. Glutathionylation is a prevalent redox modification of proteins resulting from the disulfide adduction of the glutathione moiety to a reactive cysteine-SH; and glutathionylation of specific proteins has been implicated in regulation of cell viability. Glutaredoxin 1 (Grx1) is the principal deglutathionylating enzyme within cells, and it has been reported to mediate protection of dopaminergic neurons in C. elegans, however many of the functional downstream targets of Grx1 in vivo remain unknown. Previously, DJ-1 protein content was shown to decrease concomitantly with diminution of Grx1 protein content in cell culture of model neurons (SH-SY5Y and Neuro-2A lines). In the current study we aimed to investigate the regulation of DJ-1 by Grx1 in vivo and characterize its glutathionylation in vitro. Here, with Grx−/− mice we provide evidence that Grx1 regulates protein levels of DJ-1 in vivo. Furthermore, with model neuronal cells (SH-SY5Y) we observed decreased DJ-1 protein content in response to treatment with known glutathionylating agents; and with isolated DJ-1 we identified two distinct sites of glutathionylation. Finally, we found that overexpression of DJ-1 in the dopaminergic neurons partly compensates for the loss of the Grx1 homolog in a C. elegans in vivo model of PD. Therefore; our results reveal a novel redox modification of DJ-1 and suggest a novel regulatory mechanism for DJ-1 content in vivo

J02 inhibits Grx1 in BV2 murine microglia cells.

<p><b>A</b>, Grx1 specific activity in BV2 cell lysates. Cells were treated with 32 μM J02 or DMSO for 30 min, medium changed, and cells were allowed to recover for 1 hour before lysing and assaying activity. <b>B</b>, <i>glrx1</i> mRNA levels in BV2 cells treated as in A, or BV2 cells pre-incubated with 32 μM J02 or DMSO for 30 min and then treated with 1 μg/ml LPS for 1 hour. <b>C</b>, Immunoblot of BV2 murine microglial cells treated as in <b>A</b>. Densitometric quantification on right. <b>D</b>, Immunoblot of purified Grx1 incubated with 96 μM J02 or DMSO. Incubation was performed in phosphate buffer pH 7.4 for 30 min at room temperature. Densitometric quantification is shown on right. E, Grx1 activity in BV2 cells treated with 40 μM gliotoxin (sporidesmin analog) or DMSO as in A. n = 3 ± SEM. *p<0.05, **p<0.01, ***p<0.001. RQ—relative quantity.</p

J02 interactome for BV2 cells–proteins involved in inflammatory responses.

<p>BV2 cells were treated with 40 μM J02 or DMSO. Resulting cell pellets were lysed, linked to a biotin azide probe, and run over a streptavidin column. <b>A</b>, Pulled down proteins were identified using mass spectrometry (see SI Materials and Methods section for further details). A, Inflammatory proteins shown to be regulated <i>via</i> S-glutathionylation and identified in the mass spectrometry dataset (supplemental <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187991#pone.0187991.s001" target="_blank">S1 Table</a></b>), including glutaredoxin-1. <b>B</b>, Grx1 and p65 are detected in J02-adducted samples. BV2 cells were treated with 40 μM J02 or equivalent volume DMSO for 30 min. Medium was changed, and cells were allowed to recover for 60 min. Resulting cell pellets were lysed, adducted with azide fluorescent fluorophore, and run over streptavidin beads to precipitate J02-adducted proteins. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187991#sec015" target="_blank">Materials and Methods</a> for further details. Eluted proteins were separated on SDS-PAGE gel, transferred to PVDF membrane, and probed with antibodies against Grx1 (left) and p65 (right).</p

Novel chloroacetamide J02 inhibits Grx1 as isolated enzyme.

<p><b>A</b>, J02 chemical structure. <b>B</b>, % enzyme inhibition by J02 of Grx1 or GR as isolated enzymes. Grx1 or GR were pre-incubated with indicated concentrations of J02 in complete assay mix for 30 min. Enzyme activity was then measured using standard spectrophotometric assays. n≥3±SEM. <b>C</b>, Identification of J02 adducted to the active site cysteine of Grx1 by mass spectrometry. The tandem spectrum was collected for the m/z 654.3 [M+H]⁺³ ion that corresponds to the peptide </p><p>¹⁴VVVFIKPTCPYCR²⁶</p> modified by J02 adduction at Cys-22 and carbamidomethylation at Cys-25. Fragmentation of the parent ion revealed the presence of the cysteinyl-J02 moiety identified by a series of unique and subsequent “y” ions (y₄ –y₁₂). These fragments unambiguously confirm Cys-22 as the site of J02 adduction. The inset on the upper left of panel C of <b>Fig 1</b> indicates the observed fragment ions of the peptide containing modified Cys-22, labeled according to Biemann nomenclature. Of the five cysteine residues on Grx1, only cysteine-22 was found to be adducted under these conditions. <b>D</b>, J02 inhibition of Grx1 isolated enzyme activity in a concentration- and time-dependent manner. Grx1 (40 milliunits (nmol substrate/min)) was incubated with indicated concentrations of J02 in 0.33 M sodium potassium phosphate buffer pH 7.4 at 30°C for indicated time. The mixture was then diluted 20-fold into complete assay mix, and standard spectrophotometric assay was performed. <b>E</b>, modified Kitz-Wilson plot for Grx1 inactivation by J02. Grx1 (4 milliunits) was pre-incubated with various (10–90 μM) concentrations of J02 in 0.33 M sodium potassium phosphate buffer pH 7.4 for 5 min at 30°C. A separate experiment verified the log linear relationship between J02 concentration and % Grx1 inhibition for the range of experimental conditions (see <b>Fig 1D</b>). K_<i>I</i> and k_<i>inact</i> were determined according to the relationship ln(E₀/E_t)/t = k_i<i>nact</i>[I]/(K_<i>I</i> + [I]), where E₀ refers to Grx1 activity at time zero, and Et refers to Grx1 activity after 5 min pre-incubation, [I] refers to J02 concentration, K_<i>I</i> is the concentration of J02 that gives half the maximal rate of inactivation, and k_<i>inact</i> is the net rate constant for inactivation. The K_<i>I</i> for J02 is 40 μM and k_<i>inact</i> is 0.5 min^-1. n = 3 ± SEM.<p></p

J02 inhibits cytokine expression in BV2 cells, not due to induction of cytotoxicity.

<p><b>A</b>, Cytokine mRNA levels in lysates from BV2 cells pre-treated with indicated concentrations of J02 for 30 min and stimulated with 1μg/ml LPS for 60 min. n = 3 ± SEM. **p<0.01. RQ—relative quantity. <b>B</b>, ATP content in lysates from BV2 cells pre-treated with indicated concentrations of J02 for 30 min, allowed to recover for indicated amount of time. ATP levels were normalized to those from control (DMSO treated) cells. n = 3 ± SEM. Arrow indicates J02 concentration used in cell-based assays. <b>C</b>, mRNA levels in BV2 treated with non-targeting (NT) scrambled siRNA (control) or Grx1-targted (GLRX) siRNA for 24 hours, treated with J02 (32 μM) or DMSO for 30 min, and stimulated with 100 ng/ml LPS for 24 hours. n ≥ 3 ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. RQ—relative quantity.</p

Regulation of DJ-1 by Glutaredoxin 1 in Vivo: Implications for Parkinson's Disease

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide, caused by the degeneration of the dopaminergic neurons in the substantia nigra. Mutations in PARK7 (DJ-1) result in early onset autosomal recessive PD, and oxidative modification of DJ-1 has been reported to regulate the protective activity of DJ-1 in vitro. Glutathionylation is a prevalent redox modification of proteins resulting from the disulfide adduction of the glutathione moiety to a reactive cysteine-SH, and glutathionylation of specific proteins has been implicated in regulation of cell viability. Glutaredoxin 1 (Grxl) is the principal deglutathionylating enzyme within cells, and it has been reported to mediate protection of dopaminergic neurons in Caenorhabditis elegans; however many of the functional downstream targets of protection of dopaminergic Grxl in vivo remain unknown. Previously, DJ-1 protein content was shown to decrease concomitantly with diminution of Grxl protein content in cell culture of model neurons (SH-SYSY and Neuro-2A lines). In the current study we aimed to investigate the regulation of DJ-1 by Grxl in vivo and characterize its glutathionylation in vitro. Here, with Grx(-/-) mice we provide show that Grxl regulates protein levels of DJ-1 in vivo. Furthermore, with model neuronal cells (SH-SY5Y) we observed decreased DJ-1 protein content in response to treatment with known glutathionylating agents, and with isolated DJ-1 we identified two distinct sites of glutathionylation. Finally, we found that overexpression of DJ-1 in the dopaminergic neurons partly compensates for the loss of the Grxl homologue in a C. elegans in vivo model of PD. Therefore, our results reveal a novel redox modification of DJ-1 and suggest a novel regulatory mechanism for DJ-1 content in vivo