Effects of DJ-1 mutations and polymorphisms on protein stability and subcellular localization

Blackinton, Jeff; Ahmad, Rili; Miller, David W.; Brug, Marcel P. van der; Canet-Avilés, Rosa M.; Hague, Stephen; Kaleem, Mona; Cookson, Mark

doi:10.1016/j.molbrainres.2004.09.004

Cited by 83 publications

(103 citation statements)

References 22 publications

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“…Surprisingly, however, the Cookson group reported data showing the opposite trend-namely, that M26I has a greater propensity to relocalize to mitochondria in oxidatively stressed cells than the wild-type protein (61). Therefore, we infer that oxidation to the 2O form may not always be a key regulatory event that determines whether wild-type or mutant forms of DJ-1 localize to mitochondria (i.e.…”

Section: Discussionmentioning

confidence: 67%

Effect of Single Amino Acid Substitution on Oxidative Modifications of the Parkinson’s Disease-Related Protein, DJ-1

Madian

Hindupur

Hulleman

et al. 2012

Molecular & Cellular Proteomics

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Mutations in the gene encoding DJ-1 have been identified in patients with familial Parkinson's disease (PD) and are thought to inactivate a neuroprotective function. Oxidation of the sulfhydryl group to a sulfinic acid on cysteine residue C106 of DJ-1 yields the "2O " form, a variant of the protein with enhanced neuroprotective function. We hypothesized that some familial mutations disrupt DJ-1 activity by interfering with conversion of the protein to the 2O form. To address this hypothesis, we developed a novel quantitative mass spectrometry approach to measure relative changes in oxidation at specific sites in mutant DJ-1 as compared with the wild-type protein. Treatment of recombinant wild-type DJ-1 with a 10-fold molar excess of H 2 O 2 resulted in a robust oxidation of C106 to the sulfinic acid, whereas this modification was not detected in a sample of the familial PD mutant M26I exposed to identical conditions. Methionine oxidized isoforms of wild-type DJ-1 were depleted, presumably as a result of misfolding and aggregation, under conditions that normally promote conversion of the protein to the 2O form. These data suggest that the M26I familial substitution and methionine oxidation characteristic of sporadic PD may disrupt DJ-1 function by disfavoring a site-specific modification required for optimal neuroprotective activity. Our findings indicate that a single amino acid substitution can markedly alter a protein's ability to undergo oxidative modification, and they imply that stimulating the

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Section: Discussionmentioning

confidence: 67%

Effect of Single Amino Acid Substitution on Oxidative Modifications of the Parkinson’s Disease-Related Protein, DJ-1

Madian

Hindupur

Hulleman

et al. 2012

Molecular & Cellular Proteomics

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“…The M26I and D149A mutations appear milder than L166P, with M26I showing 50% reduced expression in cell culture and decreased dimerization and stability in vitro (Blackinton et al 2005;Malgieri and Eliezer 2008). The D149A mutant appears the mildest, showing decreased stability in vitro but normal dimerization and only slightly reduced levels in cultured cells (Blackinton et al 2005;Malgieri and Eliezer 2008). Yeast expressing wildtype DJ-1 inserted in DHFR (dhDJ1) grew similar to yeast expressing mutant forms at 30°, while at 37°, dhDJ1 M26I and dhDJ1 L166P showed a slight reduction in growth (61-69% of wild-type at 48 hr) ( Figure 5).…”

Section: Tunability Of Idesa With a Variety Of Protein Targetsmentioning

confidence: 96%

Section: Tunability Of Idesa With a Variety Of Protein Targetsmentioning

confidence: 99%

“…The L166P mutation disrupts dimer formation and greatly reduces stability in vitro and in vivo (Blackinton et al 2005;Malgieri and Eliezer 2008). The M26I and D149A mutations appear milder than L166P, with M26I showing 50% reduced expression in cell culture and decreased dimerization and stability in vitro (Blackinton et al 2005;Malgieri and Eliezer 2008). The D149A mutant appears the mildest, showing decreased stability in vitro but normal dimerization and only slightly reduced levels in cultured cells (Blackinton et al 2005;Malgieri and Eliezer 2008).…”

Section: Tunability Of Idesa With a Variety Of Protein Targetsmentioning

confidence: 99%

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Rapid Profiling of Disease Alleles Using a Tunable Reporter of Protein Misfolding

et al. 2012

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Many human diseases are caused by genetic mutations that decrease protein stability. Such mutations may not specifically affect an active site, but can alter protein folding, abundance, or localization. Here we describe a high-throughput cell-based stability assay, IDESA (intra-DHFR enzyme stability assay), where stability is coupled to cell proliferation in the model yeast, Saccharomyces cerevisiae. The assay requires no prior knowledge of a protein's structure or activity, allowing the assessment of stability of proteins that have unknown or difficult to characterize activities, and we demonstrate use with a range of disease-relevant targets, including human alanine:glyoxylate aminotransferase (AGT), superoxide dismutase (SOD-1), DJ-1, p53, and SMN1. The assay can be carried out on hundreds of disease alleles in parallel or used to identify stabilizing small molecules (pharmacological chaperones) for unstable alleles. As demonstration of the general utility of this assay, we analyze stability of disease alleles of AGT, deficiency of which results in the kidney stone disease, primary hyperoxaluria type I, identifying mutations that specifically affect the protein-active site chemistry.

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“…DJ-1 knockout mice lacking exon 2 have been described in detail (27). DJ-1 expression constructs have been described (6,34,35). Stable M17 human neuroblastoma cell lines were cultured as described (6).…”

Section: Dj-1 Knockoutmentioning

confidence: 99%

RNA binding activity of the recessive parkinsonism protein DJ-1 supports involvement in multiple cellular pathways

Brug

Blackinton

Chandran

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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201

166

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Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.gene expression ͉ oxidative stress ͉ Parkinson's disease ͉ translation M utations in any of three genes cause a recessively inherited early-onset movement disorder reminiscent of Parkinson's disease (PD). Parkin is an E3 protein-ubiquitin ligase and PINK1 is a mitochondrial kinase (1). Results from Drosophila models suggest that PINK1 and parkin define a single pathway that, when disrupted, leads to mitochondrial damage (2, 3). The third, and rarest, gene for recessive parkinsonism is DJ-1. The DJ-1 protein responds to oxidative stress evidenced by a pI shift in sporadic PD (4, 5) and in cell (6, 7) and animal (8) models. DJ-1 knockout models also show increased sensitivity to toxins that cause mitochondrial dysfunction or oxidative stress (9-13). Cys-106 of DJ-1, which is oxidized to form a cysteine-sulfinic acid, is critically required for DJ-1 to protect against these types of damage both in vitro (6) and in vivo (12). However, the molecular function of DJ-1 is unclear. DJ-1 is part of the ThiJ/PfPI superfamily but the proteins most similar to DJ-1 form a distinct clade away from other members with known function (14, 15), implying a novel activity. As well as effects on oxidation and mitochondrial function, DJ-1 enhances Ras-mediated oncogenesis (16), modulates the PTEN/Akt survival pathway (17,18), suppresses Ask1-mediated apoptosis (19), and increases glutathione (GSH) synthesis, Hsp70 (20) and tyrosine hydroxylase (21, 22) expression. DJ-1 is a small, dimeric, single-domain protein (23), so if all of these effects are true then either the protein has multiple functions or there is a single biochemical activity that explains all of them.One of the original descriptions of the cloning of DJ-1 was as RS, a regulatory component of an RNA binding complex (24). Therefore, we examined whether DJ-1 associates with R...

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Effects of DJ-1 mutations and polymorphisms on protein stability and subcellular localization

Cited by 83 publications

References 22 publications

Effect of Single Amino Acid Substitution on Oxidative Modifications of the Parkinson’s Disease-Related Protein, DJ-1

Effect of Single Amino Acid Substitution on Oxidative Modifications of the Parkinson’s Disease-Related Protein, DJ-1

Rapid Profiling of Disease Alleles Using a Tunable Reporter of Protein Misfolding

RNA binding activity of the recessive parkinsonism protein DJ-1 supports involvement in multiple cellular pathways

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