Stable isotope labeling and label-free proteomics of Drosophila parkin null mutants.
Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra and formation of intracytoplasmic Lewy bodies (LBs). Loss-of-function mutations in parkin which encodes an E3 ubiquitin protein ligase contribute to a predominant cause of a familial form of PD termed autosomal recessive juvenile Parkinsonism (AR-JP). Drosophila parkin null mutants display muscle degeneration and mitochondrial dysfunction, providing an animal model to study Parkin-associated molecular pathways in PD. To define protein alterations involved in Parkin pathogenesis, we performed quantitative proteomic analyses of Drosophila parkin null mutants and age-matched controls utilizing both global internal standard technology (GIST) and extracted ion chromatogram peak area (XICPA) label-free approaches. A total of 375 proteins were quantified with a minimum of two peptide identifications from the combination of the XICPA and GIST measurements applied to two independent biological replicates. Sixteen proteins exhibited significant alteration. Seven of the dysregulated proteins are involved in energy metabolism, of which six were down-regulated. All five proteins involved in transporter activity exhibited higher levels, of which larval serum protein 1alpha, larval serum protein 1beta, larval serum protein 1gamma, and fat body protein 1 showed >10-fold up-regulation and substantially higher level of fat body protein 1 was confirmed by Western blot analysis. These findings suggest that abnormalities in energy metabolism and protein transporter activity pathways may be associated with the pathogenesis of Parkin-associated AR-JP.
Xun
Zhiyin
Z
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
Kaufman
Thomas C
TC
Clemmer
David E
DE
D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D004330:Drosophila / Q000378:metabolism; D029721:Drosophila Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004734:Energy Metabolism; D005260:Female; D006454:Hemoglobins / Q000032:analysis; D007553:Isotope Labeling / Q000379:methods*; D008297:Male; D010446:Peptide Fragments / Q000032:analysis; D021381:Protein Transport; D020543:Proteome / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012107:Research Design; D044767:Ubiquitin-Protein Ligases; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism
D029721:Drosophila Proteins; D006454:Hemoglobins; D010446:Peptide Fragments; D020543:Proteome; D051844:alpha-Synuclein; D044767:Ubiquitin-Protein Ligases; C487430:park protein, Drosophila
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