Decreased stability and increased formation of soluble aggregates by immature superoxide dismutase do not account for disease severity in ALS.
Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfide-reduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chemically and structurally diverse ALS-associated mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quantitative thermodynamic stability analyses. In the absence of ALS-associated mutations, reduced apo SOD1 is marginally stable but predominantly folded. Mutations generally result in slight decreases to substantial increases in the fraction of unfolded protein. Calorimetry, ultracentrifugation, and light scattering show that all mutations enhance aggregation propensity, with the effects varying widely, from subtle increases in most cases, to pronounced formation of 40-100 nm soluble aggregates by A4V, a mutation that is associated with particularly short disease duration. Interestingly, although there is a correlation between observed aggregation and stability, there is minimal to no correlation between observed aggregation, predicted aggregation propensity, and disease characteristics. These findings suggest that reduced apo SOD1 does not play a dominant role in modulating disease. Rather, additional and/or multiple forms of SOD1 and additional biophysical and biological factors are needed to account for the toxicity of mutant SOD1 in ALS.
Department of Chemistry, Guelph-Waterloo Centre for Graduate Studies in Chemistry and Biochemistry, Waterloo, ON, Canada N2L 3G1.
Kenrick A
KA
Vassall
Helen R
HR
Stubbs
Heather A
HA
Primmer
Ming Sze
MS
Tong
Sarah M
SM
Sullivan
Ryan
R
Sobering
Saipraveen
S
Srinivasan
Lee-Ann K
LA
Briere
Stanley D
SD
Dunn
Wilfredo
W
Colón
Elizabeth M
EM
Meiering
D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072105:Superoxide Dismutase-1
C000606290:SOD1 protein, human; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1
J Biol Chem. 2006 Mar 10;281(10):6184-93
16407238
J Biol Chem. 2004 Dec 24;279(52):54558-66
15485869
J Mol Biol. 2008 Feb 8;376(1):258-68
18155723
Adv Protein Chem. 1979;33:167-241
44431
Prog Neurobiol. 2008 May;85(1):94-134
18282652
Proc Natl Acad Sci U S A. 2009 May 12;106(19):7774-9
19416874
Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18663-8
19022905
PLoS One. 2009;4(3):e5004
19325915
Methods Enzymol. 1986;131:4-51
3773768
J Biol Chem. 1990 Dec 15;265(35):21612-8
2254318
J Pharm Sci. 2008 Oct;97(10):4347-66
18240293
J Biol Chem. 2004 Nov 12;279(46):47998-8003
15326189
J Biol Chem. 2008 Aug 29;283(35):24167-76
18552350
Proc Natl Acad Sci U S A. 2007 Aug 28;104(35):14157-62
17715066
J Biol Chem. 2005 Oct 28;280(43):35815-21
16105836
Proc Natl Acad Sci U S A. 2004 Nov 9;101(45):15893-8
15522970
Protein Sci. 1995 Oct;4(10):2138-48
8535251
Proc Natl Acad Sci U S A. 2009 Feb 3;106(5):1392-7
19171884
Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11263-7
17592131
J Biol Chem. 2008 May 16;283(20):13528-37
18316367
Amyotroph Lateral Scler Other Motor Neuron Disord. 2000 Jun;1(3):163-84
11464950
J Biol Chem. 2010 Jun 18;285(25):19544-52
20189984
Protein Sci. 2007 Jan;16(1):125-34
17123959
J Mol Biol. 2006 Dec 15;364(5):1084-102
17046019
Proc Natl Acad Sci U S A. 2005 Jul 12;102(28):9754-9
15987780
PLoS Biol. 2008 Jul 29;6(7):e170
18666828
Acta Neuropathol. 2010 Mar;119(3):335-44
20111867
Biochemistry. 2006 Jun 13;45(23):7366-79
16752926
Nat Struct Biol. 2003 Jun;10(6):461-7
12754496
Annu Rev Biochem. 2005;74:563-93
15952898
Neurosci Lett. 1991 Aug 19;129(2):233-6
1660578
J Biol Chem. 2002 May 3;277(18):15923-31
11854284
J Biol Chem. 2005 Apr 29;280(17):17266-74
15691826
J Biol Chem. 2007 Jul 20;282(29):21230-6
17513298
J Mol Biol. 2003 Sep 19;332(3):601-15
12963370
J Biol Chem. 2003 Feb 21;278(8):5984-92
12458194
Biochemistry. 1999 Dec 14;38(50):16424-31
10600103
Neuron. 2006 Oct 5;52(1):39-59
17015226
Proc Natl Acad Sci U S A. 2005 Jul 26;102(30):10516-21
16020530
Protein Sci. 2004 Nov;13(11):3017-27
15459333
J Biol Chem. 2010 Dec 31;285(53):41701-11
20974846
Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15463-8
14665689
Biochemistry. 2004 Dec 28;43(51):16525-31
15610047
Lancet Neurol. 2009 Jan;8(1):94-109
19081518
Arch Neurol. 2008 Feb;65(2):184-9
18268186
J Biol Chem. 2008 Jan 11;283(2):866-74
18006498
J Mol Biol. 2006 Jan 6;355(1):106-23
16307756
Nature. 2003 Aug 14;424(6950):805-8
12917692
Proc Natl Acad Sci U S A. 2004 Oct 19;101(42):15094-9
15475574
Methods Enzymol. 1999;309:429-59
10507039
Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7021-6
12773627