13 research outputs found
Regulation of DJ-1 by glutaredoxin 1 \u3ci\u3ein vivo ā implications for Parkinsonās disease\u3c/i\u3e
Parkinsonās 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 Grxā/ā 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
Upregulation of Glutaredoxin-1 Activates Microglia and Promotes Neurodegeneration: Implications for Parkinson's Disease
Novel chloroacetamido compound CWR-J02 is an anti-inflammatory glutaredoxin-1 inhibitor
<div><p>Glutaredoxin (Grx1) is a ubiquitously expressed thiol-disulfide oxidoreductase that specifically catalyzes reduction of S-glutathionylated substrates. Grx1 is known to be a key regulator of pro-inflammatory signaling, and Grx1 silencing inhibits inflammation in inflammatory disease models. Therefore, we anticipate that inhibition of Grx1 could be an anti-inflammatory therapeutic strategy. We used a rapid screening approach to test 504 novel electrophilic compounds for inhibition of Grx1, which has a highly reactive active-site cysteine residue (pKa 3.5). From this chemical library a chloroacetamido compound, CWR-J02, was identified as a potential lead compound to be characterized. CWR-J02 inhibited isolated Grx1 with an IC<sub>50</sub> value of 32 Ī¼M in the presence of 1 mM glutathione. Mass spectrometric analysis documented preferential adduction of CWR-J02 to the active site Cys-22 of Grx1, and molecular dynamics simulation identified a potential non-covalent binding site. Treatment of the BV2 microglial cell line with CWR-J02 led to inhibition of intracellular Grx1 activity with an IC<sub>50</sub> value (37 Ī¼M). CWR-J02 treatment decreased lipopolysaccharide-induced inflammatory gene transcription in the microglial cells in a parallel concentration-dependent manner, documenting the anti-inflammatory potential of CWR-J02. Exploiting the alkyne moiety of CWR-J02, we used click chemistry to link biotin azide to CWR-J02-adducted proteins, isolating them with streptavidin beads. Tandem mass spectrometric analysis identified many CWR-J02-reactive proteins, including Grx1 and several mediators of inflammatory activation. Taken together, these data identify CWR-J02 as an intracellularly effective Grx1 inhibitor that may elicit its anti-inflammatory action in a synergistic manner by also disabling other pro-inflammatory mediators. The CWR-J02 molecule provides a starting point for developing more selective Grx1 inhibitors and anti-inflammatory agents for therapeutic development.</p></div
J02 inhibits Grx1 in BV2 murine microglia cells.
<p><b>A</b>, Grx1 specific activity in BV2 cell lysates. Cells were treated with 32 Ī¼M 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 Ī¼M J02 or DMSO for 30 min and then treated with 1 Ī¼g/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 Ī¼M 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 Ī¼M gliotoxin (sporidesmin analog) or DMSO as in A. n = 3 Ā± SEM. *p<0.05, **p<0.01, ***p<0.001. RQārelative quantity.</p
J02 interactome for BV2 cellsāproteins involved in inflammatory responses.
<p>BV2 cells were treated with 40 Ī¼M 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 Ī¼M 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
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 1Ī¼g/ml LPS for 60 min. n = 3 Ā± SEM. **p<0.01. RQārelative 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 Ā± 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 Ī¼M) or DMSO for 30 min, and stimulated with 100 ng/ml LPS for 24 hours. n ā„ 3 Ā± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. RQārelative quantity.</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. nā„3Ā±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]<sup>+3</sup> ion that corresponds to the peptide </p><p><sup>14</sup>VVVFIKPTCPYCR<sup>26</sup></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 āyā ions (y<sub>4</sub> āy<sub>12</sub>). 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 30Ā°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 (10ā90 Ī¼M) concentrations of J02 in 0.33 M sodium potassium phosphate buffer pH 7.4 for 5 min at 30Ā°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<sub><i>I</i></sub> and k<sub><i>inact</i></sub> were determined according to the relationship ln(E<sub>0</sub>/E<sub>t</sub>)/t = k<sub>i<i>nact</i></sub>[I]/(K<sub><i>I</i></sub> + [I]), where E<sub>0</sub> refers to Grx1 activity at time zero, and Et refers to Grx1 activity after 5 min pre-incubation, [I] refers to J02 concentration, K<sub><i>I</i></sub> is the concentration of J02 that gives half the maximal rate of inactivation, and k<sub><i>inact</i></sub> is the net rate constant for inactivation. The K<sub><i>I</i></sub> for J02 is 40 Ī¼M and k<sub><i>inact</i></sub> is 0.5 min<sup>-1</sup>. n = 3 Ā± SEM.<p></p
Multistep Synthesis of CWR-J02.
<p>This diagram displays the starting compound and illustrates the step-by-step synthesis of J02 involving the preparation, purification, and documentation of purity of the intermediates and the final product. Details are provided under Materials and Methods.</p
MD simulations suggest that J02 forms an interaction with Grx1 in a distinct region of Grx1 compared to that of the glutathionyl moiety in the Grx1 glutathionyl mixed disulfide intermediate.
<p><b>A</b>, Grx1/J02 complex at the end of a 50 ns molecular dynamics simulation, using the NMR structure of Grx1 (PDB ID: 1JHB); the chlorine atom of J02 is shown in green. <b>B</b>, NMR structure of the glutathionyl mixed disulfide intermediate of the quadruple Cā>S mutant of Grx1 (QM-Grx1-Cys22-SSG) (PDB ID: 1B4Q). Approximately the same view of Grx1 is shown in panels A and B. <b>C</b>, Rotated view of the Grx1/J02 complex, highlighting the main interaction residues of Grx1 in orange (Glu-3, Phe-4, Thr-50, Asn-51, and His-52). The catalytic Cys-22 is labeled in all three panels.</p