Identification of Rotenone-Induced Modifications in α-Synuclein Using Affinity Pull-Down and Tandem Mass Spectrometry

Department of Medicinal Chemistry and Molecular Pharmacology (MCMP), Purdue University, ウェストラファイエット, Indiana, United States
Analytical Chemistry (Impact Factor: 5.64). 05/2006; 78(7):2422-31. DOI: 10.1021/ac051978n
Source: PubMed

ABSTRACT Parkinson's disease is a movement disorder that results from a loss of dopaminergic neurons in the substantia nigra. The disease is characterized by mitochondrial dysfunction, oxidative stress, and the presence of "Lewy body" inclusions enriched with aggregated forms of alpha-synuclein, a presynaptic protein. Although alpha-synuclein is modified at various sites in Lewy bodies, it is unclear how sequence-specific posttranslational modifications modulate the aggregation of the protein in oxidatively stressed neurons. To begin to address this problem, we developed an affinity pull-down/mass spectrometry method to characterize the primary structure of histidine-tagged alpha-synuclein isolated from catecholaminergic neurons. Using this method, we mapped posttranslational modifications of alpha-synuclein from untreated neurons and neurons exposed to rotenone, an inhibitor of mitochondrial complex I. Various posttranslational modifications suggestive of oxidative damage or repair were identified in a region comprising a 20-residue stretch in the C-terminal part of the protein. The results indicate that alpha-synuclein is subject to discrete posttranslational modifications in neurons with impaired mitochondrial function. Our affinity pull-down/mass spectrometry method is a useful tool to examine how specific modifications of alpha-synuclein contribute to neurologic disorders such as Parkinson's disease.

12 Reads
  • Source
    • "Treatment of neurons in cell cultures with the rotenone is known to induce several modifications in the a-synuclein protein (Mirzaei et al., 2006). These modifications include oxidation of methionine, introduction of nitrotyrosine , phosphotyrosine, and aminotyrosine (Mirzaei et al., 2006). Introduction of such modifications in the native polypeptide cause it to misfold and subsequently form aggregates. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein aggregation and dysfunction of ubiquitin proteasome system (UPS) have been implicated in Parkinson's disease (PD) pathology for a long time. Heat shock proteins (HSPs) have neuro-protective effects in PD as they assist in protein refolding and targeting of irreparable proteins to UPS. To realize their benefits in a chronically progressing disease like PD, it is imperative to maintain slightly up-regulated levels of HSPs consistently over a longer period of time. Here, we evaluate the possible beneficial effects of HSP inducer carbenoxolone (cbx) in a rotenone-based rat model of PD. Simultaneously with rotenone, a low dose of cbx (20 mg/kg body weight) was administered for five weeks to male SD rats. Weekly behavioral analysis along with end-point evaluation of HSPs, UPS activity, apoptosis, and oxidative stress were performed. The activation of heat shock factor-1 (HSF-1) and up-regulation of HSP70, HSP40, and HSP27 levels in mid-brain following cbx administration resulted in the reduction of α-synuclein and ubiquitin aggregation. This decrease seems to be mediated by reduction in protein carbonylation as well as up-regulation of UPS activity. In addition, the decrease in apoptosis and oxidative stress following HSP upregulation prevented the decline in tyrosine hydroxylase (TH) and dopamine levels in mid-brain region, which in turn resulted in improved motor functions. Thus, persistent HSP induction at low levels by cbx could improve the PD pathophysiology.
    Neuropharmacology 11/2013; 79. DOI:10.1016/j.neuropharm.2013.11.016 · 5.11 Impact Factor
  • Source
    • "Oxidative stress has been proposed as a possible mechanism that may link αsyn posttranslational modifications to subsequent aggregation (Bieschke et al., 2006; Ischiropoulos, 2003; Jenner, 1996; Maguire-Zeiss et al., 2005; Norris and Giasson, 2005; Schapira, 2008). A recent study (Mirzaei et al., 2006) examining the effects of rotenone, a mitochondrial complex I inhibitor, on a C-terminal 20-residue stretch of αsyn, revealed multiple modifications on five specific residues (M116, Y125, M127, Y133, and Y136), including phosphorylation, nitration, and amination. Interestingly, each residue demonstrated a number of possible modifications upon exposure to rotenone, but not all residues demonstrated the same propensity for different modifications; for instance, Y125 could be phosphorylated, nitrated, or aminated, whereas Y133 was shown to be nitrated or aminated but never phosphorylated. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Multiple system atrophy (MSA) is a progressive neurodegenerative disease characterized by autonomic failure, parkinsonism, cerebellar ataxia, and oligodendrocytic accumulation of alpha-synuclein (alphasyn). Oxidative stress has been linked to neuronal death in MSA and the mitochondrial toxin 3-nitropropionic acid (3NP) is known to enhance the motor deficits and neurodegeneration in transgenic mice models of MSA. However, the effect of 3NP administration on alphasyn itself has not been studied. In this context, we examined the neuropathological effects of 3NP administration in alphasyn transgenic mice expressing human alphasyn (halphasyn) under the control of the myelin basic protein (MBP) promoter and the effect of this administration on posttranslational modifications of alphasyn, on levels of total alphasyn, and on its solubility. We demonstrate that 3NP administration altered levels of nitrated and oxidized alphasyn in the MBP-halphasyn tg while not affecting global levels of phosphorylated or total alphasyn. 3NP administration also exaggerated neurological deficits in the MBP-halphasyn tg mice, resulting in widespread neuronal degeneration and behavioral impairment.
    Journal of Neuroscience Research 09/2009; 87(12):2728-39. DOI:10.1002/jnr.22089 · 2.59 Impact Factor
    • "α-Synuclein has been reported to be phosphorylated on serine residues, at Ser-87 and Ser-129 (10), although to date only the Ser-129 phosphorylation has been identified in the central nervous system (11, 12). Phosphorylation at tyrosine residues has been observed by some investigators (13, 14) but not by others (10–12). Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the majority of synuclein in Lewy bodies contains this modification (15). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of alpha-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to alpha-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates alpha-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited alpha-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in alpha-synuclein phosphorylation in central nervous system.
    Journal of Biological Chemistry 12/2008; 284(5):2598-602. DOI:10.1074/jbc.C800206200 · 4.57 Impact Factor
Show more