title: Molecular ageing of alpha- and Beta-synucleins: protein damage and repair mechanisms.
abstract: Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[(3)H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated (3)H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of (3)H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.
authors: School of Biomedical Sciences, The University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom.
authors: Vasanthy
authors: V
authors: Vigneswara
authors: Simon
authors: S
authors: Cass
authors: Declan
authors: D
authors: Wayne
authors: Edward L
authors: EL
authors: Bolt
authors: David E
authors: DE
authors: Ray
authors: Wayne G
authors: WG
authors: Carter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D026581:Isoaspartic Acid / Q000737:chemistry; D007526:Isoelectric Point; D008745:Methylation; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010300:Parkinson Disease / Q000235:genetics; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051846:beta-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D026581:Isoaspartic Acid; D051844:alpha-Synuclein; D051846:beta-Synuclein; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase
references: Science. 1997 Jun 27;276(5321):2045-7
references: 9197268
references: Biochemistry. 2003 Apr 8;42(13):3696-700
references: 12667059
references: Nat Genet. 1998 Feb;18(2):106-8
references: 9462735
references: J Biol Chem. 2001 Oct 5;276(40):37161-5
references: 11479322
references: Proc Natl Acad Sci U S A. 1997 Jun 10;94(12):6132-7
references: 9177182
references: Biochem Biophys Res Commun. 1995 Jul 17;212(2):442-8
references: 7626058
references: Biochem Biophys Res Commun. 2009 Apr 17;381(4):523-7
references: 19239903
references: Biochemistry. 1996 Oct 29;35(43):13709-15
references: 8901511
references: J Comp Neurol. 2005 Dec 26;493(4):524-37
references: 16304629
references: Gene Ther. 2004 Dec;11(23):1713-23
references: 15483670
references: FEBS J. 2007 Apr;274(7):1862-77
references: 17381514
references: Biochim Biophys Acta. 1998 Apr 28;1406(3):291-8
references: 9630681
references: Biochemistry. 1996 Apr 23;35(16):5183-90
references: 8611502
references: Science. 2003 Oct 31;302(5646):841
references: 14593171
references: Neurochem Res. 1993 Jan;18(1):87-94
references: 8464537
references: Hippocampus. 2001;11(3):287-98
references: 11769310
references: Biochemistry. 2008 Oct 7;47(40):10757-64
references: 18795804
references: J Nutr. 2002 Jun;132(6):1357-60
references: 12042458
references: Curr Med Chem. 2008;15(26):2748-59
references: 18991634
references: Biol Chem. 2008 Aug;389(8):1055-66
references: 18979630
references: Nat Rev Neurosci. 2001 Jul;2(7):492-501
references: 11433374
references: Neuron. 2001 Oct 25;32(2):213-23
references: 11683992
references: Science. 2000 Feb 18;287(5456):1265-9
references: 10678833
references: J Biol Chem. 2006 Mar 31;281(13):8389-98
references: 16443604
references: J Biol Chem. 1990 Jul 25;265(21):12717-21
references: 2373708
references: Protein Sci. 2009 Aug;18(8):1766-73
references: 19521992
references: J Biol Chem. 1993 Mar 25;268(9):6174-81
references: 8454593
references: Clin Chem. 2009 Oct;55(10):1852-60
references: 19679632
references: Biol Pharm Bull. 2005 Sep;28(9):1590-6
references: 16141521
references: Ann Neurol. 2004 Feb;55(2):164-73
references: 14755719
references: Proc Natl Acad Sci U S A. 1987 May;84(9):2595-9
references: 3472227
references: J Biol Chem. 2006 Nov 3;281(44):33802-13
references: 16959769
references: J Chromatogr B Analyt Technol Biomed Life Sci. 2010 May 15;878(17-18):1312-9
references: 19879817
references: J Biol Chem. 1994 Oct 7;269(40):24586-95
references: 7929130
references: J Hepatol. 2007 Jun;46(6):1119-25
references: 17336420
references: J Biol Chem. 1987 Apr 25;262(12):5622-9
references: 3571226
references: Genome Biol. 2002;3(1):REVIEWS3002
references: 11806835
references: J Neurosci. 1998 Mar 15;18(6):2063-74
references: 9482793
references: J Biol Chem. 2002 Apr 5;277(14):11970-8
references: 11812782
references: J Neurochem. 2009 Jan;108(2):465-74
references: 19012742
references: J Biol Chem. 1987 Jan 15;262(2):785-94
references: 3805008
references: Aging Cell. 2005 Feb;4(1):1-12
references: 15659208
references: Brain. 2010 Dec;133(Pt 12):3724-33
references: 20959308
references: J Neurosci Res. 2002 Nov 1;70(3):451-61
references: 12391606
references: J Neurosci Res. 2002 Aug 1;69(3):341-52
references: 12125075
references: J Biol Chem. 1987 Sep 5;262(25):12283-7
references: 3624258
references: FEBS Lett. 1994 May 23;345(1):27-32
references: 8194594
references: J Biol Chem. 2006 Oct 27;281(43):32619-29
references: 16923807
references: J Pept Res. 2004 May;63(5):426-36
references: 15140160
references: Neurology. 2004 Sep 14;63(5):805-11
references: 15365127
references: Prog Brain Res. 2010;183:115-45
references: 20696318
references: J Biol Chem. 1999 Jul 16;274(29):20671-8
references: 10400700
references: PLoS One. 2010 Nov 10;5(11):e13863
references: 21085664
references: Molecules. 2011 Oct 11;16(10):8535-51
references: 21989313
references: J Biol Chem. 2006 Oct 6;281(40):29739-52
references: 16847063
references: J Biol Chem. 2001 Jun 8;276(23):20695-702
references: 11279164