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Proteomic identification of dopamine-conjugated proteins from isolated rat brain mitochondria and SH-SY5Y cells.

ABSTRACT: Dopamine oxidation has been previously demonstrated to cause dysfunction in mitochondrial respiration and membrane permeability, possibly related to covalent modification of critical proteins by the reactive dopamine quinone. However, specific mitochondrial protein targets have not been identified. In this study, we utilized proteomic techniques to identify proteins directly conjugated with (14)C-dopamine from isolated rat brain mitochondria exposed to radiolabeled dopamine quinone (150 microM) and differentiated SH-SY5Y cells treated with (14)C-dopamine (150 microM). We observed a subset of rat brain mitochondrial proteins that were covalently modified by (14)C-dopamine, including chaperonin, ubiquinol-cytochrome c reductase core protein 1, glucose regulated protein 75/mitochondrial HSP70/mortalin, mitofilin, and mitochondrial creatine kinase. We also found the Parkinson's disease associated proteins ubiquitin carboxy-terminal hydrolase L1 and DJ-1 to be covalently modified by dopamine in both brain mitochondrial preparations and SH-SY5Y cells. The susceptibility of the identified proteins to covalent modification by dopamine may carry implications for their role in the vulnerability of dopaminergic neurons in Parkinson's disease pathogenesis.

SUBMITTER: Van Laar VS

PROVIDER: S-EPMC2759724 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Proteomic identification of dopamine-conjugated proteins from isolated rat brain mitochondria and SH-SY5Y cells.

Van Laar Victor S VS Mishizen Amanda J AJ Cascio Michael M Hastings Teresa G TG

Neurobiology of disease 20090328 3

Dopamine oxidation has been previously demonstrated to cause dysfunction in mitochondrial respiration and membrane permeability, possibly related to covalent modification of critical proteins by the reactive dopamine quinone. However, specific mitochondrial protein targets have not been identified. In this study, we utilized proteomic techniques to identify proteins directly conjugated with (14)C-dopamine from isolated rat brain mitochondria exposed to radiolabeled dopamine quinone (150 microM) ...[more]

PMID: 19332121

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Project description:Parkinson's disease is the second most common age-related, neurodegenerative disease. A small collection of genes has been linked to Parkinson's disease including LRRK2, SAT1, and SNCA, the latter of which encodes the protein alpha-synuclein that aggregates in Lewy bodies as a hallmark of the disease. Overexpression of even wild-type versions of these genes can lead to pathogenesis, yet the regulatory mechanisms that control protein production of the genes are not fully understood. Pumilio proteins belong to the highly conserved PUF family of eukaryotic RNA-binding proteins that post-transcriptionally regulate gene expression through binding conserved motifs in the 3' untranslated region (UTR) of mRNA targets known as PUF Recognition Elements (PREs). The 3'UTRs of LRRK2, SNCA and SAT1 each contain multiple putative PREs. Knockdown (KD) of the two human Pumilio homologs (Pumilio 1 and Pumilio 2) in a neurodegenerative model cell line, SH-SY5Y, resulted in increased SNCA and LRRK2 mRNA, as well as alpha-synuclein levels, suggesting these genes are normally repressed by the Pumilio proteins. Some studies have indicated a relationship between Pumilio and microRNA activities on the same target, especially when their binding sites are close together. LRRK2, SNCA, and SAT1 each contain several putative microRNA-binding sites within the 3'UTR, some of which reside near PREs. Small RNA-seq and microRNA qPCR assays were performed in both wild type and Pumilio KD SH-SY5Y cells to analyze global and differential microRNA expression. One thousand four hundred and four microRNAs were detected across wild type and Pumilio KD cells. Twenty-one microRNAs were differentially expressed between treatments, six of which were previously established to be altered in Parkinson's disease patient samples or research models. Expression of ten miRs predicted to target LRRK2 and SNCA was verified by RT-qPCR. Collectively, our results demonstrate that Pumilios and microRNAs play a multi-faceted role in regulating Parkinson's disease-associated genes.

Project description:Background: Neurotoxicity induced by the amyloid beta (Aβ) peptide is one of the most important pathological mechanisms of Alzheimer's disease (AD). Activation of the adaptive IRE1α-XBP1 pathway contributes to the pathogenesis of AD, making it a potential target for AD therapeutics. However, the mechanism of IRE1α-XBP1 pathway involvement in AD is unclear. We, therefore, investigated the effect of the IRE1α-XBP1 axis in an in vitro AD model and explored its potential mechanism. Methods: The human neuroblastoma cell line, SH-SY5Y, was used. Cells were treated with Aβ25-35, with or without 4μ8c, an inhibitor of IRE1α. Cells were collected and analyzed by Western blotting, quantitative real-time PCR, electron microscopy, fluorescence microscopy, calcium imaging, and other biochemical assays. Results: Aβ-exposed SH-SY5Y cells showed an increased expression of XBP1s and p-IRE1α. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and calcium imaging analysis showed that the IRE1α inhibitor, 4μ8c, reduced Aβ-induced cytotoxicity. Increased levels of ATP, restoration of mitochondrial membrane potential, and decreased production of mitochondrial reactive oxygen species after Aβ treatment in the presence of 4μ8c showed that inhibiting the IRE1α-XBP1 axis effectively mitigated Aβ-induced mitochondrial dysfunction in SH-SY5Y cells. Furthermore, Aβ treatment increased the expression and interaction of IP3R, Grp75, and vdac1 and led to an increased endoplasmic reticulum (ER)-mitochondria association, malfunction of mitochondria-associated ER-membranes (MAMs), and mitochondrial dysfunction. These deficits were rescued by inhibiting the IRE1α-XBP1 axis. Conclusion: These findings demonstrate that Aβ peptide induces the activation of the IRE1α-XBP1 axis, which may aggravate cytotoxicity and mitochondrial impairment in SH-SY5Y cells by targeting MAMs. Inhibition of the IRE1α-XBP1 axis provides the protection against Aβ-induced injury in SH-SY5Y cells and may, therefore, be a new treatment strategy.

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Proteomic identification of dopamine-conjugated proteins from isolated rat brain mitochondria and SH-SY5Y cells.

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Proteomic identification of dopamine-conjugated proteins from isolated rat brain mitochondria and SH-SY5Y cells.

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