Identification of novel proteins affected by rotenone in mitochondria of dopaminergic cells.
BACKGROUND
Many studies have shown that mitochondrial dysfunction, complex I inhibition in particular, is involved in the pathogenesis of Parkinson's disease (PD). Rotenone, a specific inhibitor of mitochondrial complex I, has been shown to produce neurodegeneration in rats as well as in many cellular models that closely resemble PD. However, the mechanisms through which complex I dysfunction might produce neurotoxicity are as yet unknown. A comprehensive analysis of the mitochondrial protein expression profile affected by rotenone can provide important insight into the role of mitochondrial dysfunction in PD.


RESULTS
Here, we present our findings using a recently developed proteomic technology called SILAC (stable isotope labeling by amino acids in cell culture) combined with polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry to compare the mitochondrial protein profiles of MES cells (a dopaminergic cell line) exposed to rotenone versus control. We identified 1722 proteins, 950 of which are already designated as mitochondrial proteins based on database search. Among these 950 mitochondrial proteins, 110 displayed significant changes in relative abundance after rotenone treatment. Five of these selected proteins were further validated for their cellular location and/or treatment effect of rotenone. Among them, two were confirmed by confocal microscopy for mitochondrial localization and three were confirmed by Western blotting (WB) for their regulation by rotenone.


CONCLUSIONS
Our findings represent the first report of these mitochondrial proteins affected by rotenone; further characterization of these proteins may shed more light on PD pathogenesis.
Jin
Jinghua
J
Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA. jhjin@zju.edu.cn
Davis
Jeanne
J
Zhu
David
D
Kashima
Daniel T
DT
Leroueil
Marc
M
Pan
Catherine
C
Montine
Kathleen S
KS
Zhang
Jing
J
D002460:Cell Line; D004298:Dopamine / Q000378:metabolism*; D042967:Electron Transport Complex I / Q000187:drug effects / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012402:Rotenone / Q000633:toxicity*; D058305:Sorting Nexins; D014475:Uncoupling Agents / Q000633:toxicity*; D051040:Vesicle-Associated Membrane Protein 3 / Q000187:drug effects / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000187:drug effects / Q000378:metabolism
D006360:Heat-Shock Proteins; D024101:Mitochondrial Proteins; C404403:SACS protein, human; C513978:SNX1 protein, human; D058305:Sorting Nexins; D014475:Uncoupling Agents; C494946:VAMP3 protein, human; D051040:Vesicle-Associated Membrane Protein 3; D033921:Vesicular Transport Proteins; D012402:Rotenone; D042967:Electron Transport Complex I; D004298:Dopamine
J Neurosci. 2002 Aug 15;22(16):7006-15
12177198
Int J Neurosci. 1998 Jul;95(1-2):33-42
9845014
Brain Res. 2002 Nov 22;956(1):156-65
12426058
J Neurosci. 2002 Dec 15;22(24):10690-8
12486162
Neuron. 2003 Feb 20;37(4):583-95
12597857
Nat Biotechnol. 2003 Mar;21(3):281-6
12592411
Ann N Y Acad Sci. 2003 Jun;991:120-31
12846981
J Neurochem. 2003 Nov;87(4):914-21
14622122
Anal Chem. 2003 Dec 1;75(23):6648-57
14640741
J Neuropathol Exp Neurol. 2004 Apr;63(4):302-13
15099020
J Biol Chem. 2004 Sep 10;279(37):39155-64
15234983
Neuroscience. 2004;128(2):281-91
15350641
J Neurochem. 2004 Nov;91(3):667-82
15485497
Biochem J. 1980 Nov 1;191(2):421-7
6263247
Genomics. 1999 Dec 1;62(2):156-64
10610707
Nat Genet. 2000 Feb;24(2):120-5
10655055
J Neurochem. 2000 Mar;74(3):970-8
10693927
Am J Pathol. 2000 Aug;157(2):401-10
10934145
Appl Microbiol Biotechnol. 2000 Jul;54(1):44-51
10952004
Nat Neurosci. 2000 Dec;3(12):1301-6
11100151
Nat Biotechnol. 2001 Mar;19(3):242-7
11231557
Am J Med Genet. 2001 Spring;106(1):27-36
11579422
J Biochem. 2001 Dec;130(6):765-71
11726276
Gene. 2002 Feb 20;285(1-2):229-37
12039050
Exp Lung Res. 2002 Jul-Aug;28(5):373-89
12097231
Mol Cell Proteomics. 2002 May;1(5):376-86
12118079
OMICS. 2002;6(2):207-12
12143966
Annu Rev Neurosci. 1999;22:123-44
10202534
J Biol Chem. 2004 Nov 26;279(48):50366-74
15448140
Nucleic Acids Res. 2005 Jan 1;33(Database issue):D514-7
15608251
J Neurochem. 2005 Mar;92(5):1265-76
15715675
Mol Cell Proteomics. 2005 Feb;4(2):169-81
15598749
Brain Res Mol Brain Res. 2005 Mar 24;134(1):119-38
15790536
Traffic. 2005 May;6(5):366-73
15813747
Hum Mol Genet. 2005 May 1;14(9):1231-41
15790595
J Biol Chem. 2005 Apr 22;280(16):15587-94
15710606
J Neurochem. 2005 Jun;93(5):1199-208
15934940
Hum Mol Genet. 2005 Sep 15;14(18):2749-55
16126732
Mol Cell Proteomics. 2005 Oct;4(10):1471-9
15975914
Can J Neurol Sci. 2005 Aug;32(3):277-86
16225167
NeuroRx. 2005 Jul;2(3):484-94
16389312
Proteomics. 2006 Jan;6(2):614-22
16342138
Mol Biol Cell. 2006 Mar;17(3):1228-38
16407403
Biochem Pharmacol. 2006 Jun 28;72(1):80-90
16678137
Mol Cell Proteomics. 2006 Jul;5(7):1193-204
16565515
Lancet Neurol. 2006 Aug;5(8):677-87
16857573
Neurochem Int. 2006 Sep;49(4):347-50
16600434
J Proteome Res. 2006 Aug;5(8):1948-58
16889417
Mol Cell Proteomics. 2007 May;6(5):845-59
16854843
Mol Cell Proteomics. 2004 May;3(5):441-55
14739257
Acta Neurol Scand Suppl. 1984;100:49-54
6333134
Adv Neurol. 1987;45:35-43
3030070
J Neurochem. 1990 Mar;54(3):823-7
2154550
J Neurosci. 1992 Sep;12(9):3392-8
1356145
Adv Neurol. 1993;60:288-91
8420145
Science. 1996 May 17;272(5264):1008-10
8638121
Biochem Biophys Res Commun. 1998 Jul 30;248(3):812-6
9704010
Anal Chem. 2002 Oct 15;74(20):5383-92
12403597