Use of cysteine‐reactive cross‐linkers to probe conformational flexibility of human <scp>DJ</scp>‐1 demonstrates that Glu18 mutations are dimers

Prahlad, Janani; Hauser, David N.; Milkovic, Nicole M.; Cookson, Mark; Wilson, Mark A.

doi:10.1111/jnc.12763

Cited by 15 publications

(24 citation statements)

References 61 publications

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“…Cysteines 53 and 106 have been reported previously to be oxidatively modified [23, 30], but there is no evidence for glutathionylation of DJ-1 in a physiological setting. In order to determine if DJ-1 undergoes glutathionylation in vivo , DJ-1 was immunoprecipitated from whole mouse brain homogenates and subjected to non-reducing SDS-PAGE and western blot analysis.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2016

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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.

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Section: Resultsmentioning

confidence: 99%

“…Recombinant uniformly 15 N-labeled DJ-1 was generated as previously described [23]. Briefly, N-terminally 6x-His tagged DJ-1 was expressed from pET15b in BL21(DE3) E coli .…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2016

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“…The cells were lysed in the wells using RIPA buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) with protease and phosphatase inhibitor cocktails (Sigma P8340, P5726, P0044, 93,482, and T1952). For transfection experiments, DJ-1 knockout astrocytes were transfected using pDEST40 LacZ, wildtype DJ-1, or C106A DJ-1 plasmids [ 40 ]. The cells were serum starved for 3 h, stimulated with 10 nM IGF-1 for 5 min, then fixed and immunocytochemistry was performed as outlined above.…”

Section: Methodsmentioning

confidence: 99%

Hexokinases link DJ-1 to the PINK1/parkin pathway

Hauser

Mamais

Conti

et al. 2017

Mol Neurodegeneration

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BackgroundEarly onset Parkinson’s disease is caused by variants in PINK1, parkin, and DJ-1. PINK1 and parkin operate in pathways that preserve mitochondrial integrity, but the function of DJ-1 and how it relates to PINK1 and parkin is poorly understood.MethodsA series of unbiased high-content screens were used to analyze changes at the protein, RNA, and metabolite level in rodent brains lacking DJ-1. Results were validated using targeted approaches, and cellular assays were performed to probe the mechanisms involved.ResultsWe find that in both rat and mouse brains, DJ-1 knockout results in an age-dependent accumulation of hexokinase 1 in the cytosol, away from its usual location at the mitochondria, with subsequent activation of the polyol pathway of glucose metabolism in vivo. Both in the brain and in cultured cells, DJ-1 deficiency is associated with accumulation of the phosphatase PTEN that antagonizes the kinase AKT. In cells, addition of an inhibitor of AKT (MK2206) or addition of a peptide to dissociate association of hexokinases from mitochondria both inhibit the PINK1/parkin pathway, which works to maintain mitochondrial integrity.ConclusionHexokinases are an important link between three major genetic causes of early onset Parkinson’s disease. Because aging is associated with deregulated nutrient sensing, these results help explain why DJ-1 is associated with age-dependent disease.Electronic supplementary materialThe online version of this article (10.1186/s13024-017-0212-x) contains supplementary material, which is available to authorized users.

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“…Spectra were measured in continuous scanning mode with a grating of 3400 lines cm -1 , 1 nm bandwidth, and a rate of 20 nm min −1 . Four scans were accumulated per spectrum with a data pitch of 0.1 nm and a data integration time of 1 s. The mean molar residue ellipticity (deg cm 2 dmol −1 residue −1 ) was calculated using Scopes’ method 42 for protein concentration determination as previously described 43 .…”

Section: Methodsmentioning

confidence: 99%

Short Carboxylic Acid–Carboxylate Hydrogen Bonds Can Have Fully Localized Protons

2016

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Short hydrogen bonds (H-bonds) have been proposed to play key functional roles in several proteins. The location of the proton in short H-bonds is of central importance, as proton delocalization is a defining feature of low barrier hydrogen bonds (LBHBs). Experimentally determining proton location in H-bonds is challenging. Here, bond length analysis of atomic (1.15–0.98 Å) resolution X-ray crystal structures of the human protein DJ-1 and its bacterial homolog YajL was used to determine the protonation states of H-bonded carboxylic acids. DJ-1 contains a buried, dimer-spanning 2.49 Å H-bond between Glu15 and Asp23 that satisfies standard donor-acceptor distance criteria for a LBHB. Bond length analysis indicates that the proton is localized on Asp24, excluding a LBHB at this location. However, similar analysis of the E. coli homolog YajL shows both residues may be protonated at the H-bonded oxygen atoms, potentially consistent with an LBHB. A PDB-wide screen identifies candidate carboxylic acid H-bonds in approximately 14% of proteins, which are typically short (=2.542(2) Å). Chemically similar H-bonds between hydroxylated residues (Ser/Thr/Tyr) and carboxylates show a trend of lengthening O-O distance with increasing H-bond donor pKa. This trend suggests that conventional electronic effects provide an adequate explanation for short, charge-assisted carboxylic acid-carboxylate H-bonds in proteins, without the need to invoke LBHBs in general. This study demonstrates that bond length analysis of atomic resolution X-ray crystal structures provides a useful experimental test of certain candidate LBHBs.

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Use of cysteine‐reactive cross‐linkers to probe conformational flexibility of human DJ‐1 demonstrates that Glu18 mutations are dimers

Cited by 15 publications

References 61 publications

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

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

Hexokinases link DJ-1 to the PINK1/parkin pathway

Short Carboxylic Acid–Carboxylate Hydrogen Bonds Can Have Fully Localized Protons

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