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Nucleation and kinetics of SOD1 aggregation in human cells for ALS1

Abstract

Aberrant structural formations of Cu/Zn superoxide dismutase enzyme (SOD1) are the probable mechanism by which circumscribed mutations in the SOD1 gene cause familial amyotrophic lateral sclerosis (ALS1). SOD1 forms aberrant structures which can proceed by nucleation to insoluble aggregates. Here, the SOD1 aggregation reaction was investigated predominantly by time-course studies on ALS1 variants G85R, G37R, D101G, and D101N in human embryonic kidney cells (HEK293FT), with analysis by detergent ultracentrifugation extractions and high-resolution PAGE methodologies. Nucleation was found to be pseudo-zeroth order and dependent on time and concentration at constant 37.0 °C and pH 7.4. The predominant subsets of the total SOD1 expression set which comprised the nucleation phase were both soluble and insoluble inactive monomers, trimers, and hexamers with reduced intra-disulfide bonds. Superoxide exposure via paraquat initiated the formation of SOD1 trimers in untransfected SH-SY5Y cells and increased the aggregation propensity of G85R in HEK293FT. These data show the kinetic formation of aberrant SOD1 subsets implicated in ALS1 and indicate that superoxide substrate may initiate its radical polymerization. In an instance of the utility of methodological reductionism in molecular theory: though many ALS1 variants retain their global enzymatic activity, the SOD1 subsets most implicated in causing ALS1 do not retain their specific activity.

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References

  1. 1.

    Pardo CA, Xu Z, Borchelt DR, Price DL, Sisodia SS, Cleveland DW (1995) Superoxide dismutase is an abundant component in cell bodies, dendrites, and axons of motor neurons and in a subset of other neurons. Proc Natl Acad Sci USA 92:954–958

  2. 2.

    Dupeyrat F, Vidaud C, Lorphelin A, Berthomieu C (2004) Long distance charge redistribution upon Cu, Zn-superoxide dismutase reduction: significance for dismutase function. J Biol Chem 279:48091–48101

  3. 3.

    Rotilio G, Bray RC, Fielden EM (1972) A pulse radiolysis study of superoxide dismutase. Biochim Biophys Acta 268:605–609

  4. 4.

    Forman HJ, Fridovich I (1973) Superoxide dismutase: a comparison of rate constants. Arch Biochem Biophys 158:396–400

  5. 5.

    Reddi AR, Culotta VC (2013) SOD1 integrates signals from oxygen and glucose to repress respiration. Cell 152:224–235

  6. 6.

    Liochev SI, Fridovich I (2000) Copper- and zinc-containing superoxide dismutase can act as a superoxide reductase and a superoxide oxidase. J Biol Chem 275:38482–38485

  7. 7.

    Workman AS (2017) Real-time TD-DFT study on the dioxygen/superoxide radical charge transfer reaction. Comput Theor Chem 1117:207–214

  8. 8.

    Nedd S, Redler RL, Proctor EA, Dokholyan NV, Alexandrova AN (2014) Cu, Zn-superoxide dismutase without Zn is folded but catalytically inactive. J Mol Biol 426:4112–4124

  9. 9.

    Banci L, Bertini I, Cantini F, Kozyreva T, Massagni C, Palumaa P, Rubino JT, Zovo K (2012) Human superoxide dismutase 1 (hSOD1) maturation through interaction with human copper chaperone for SOD1 (hCCS). Proc Natl Acad Sci USA 109:13555–13560

  10. 10.

    Girotto S, Cendron L, Bisaglia M, Tessari I, Mammi S, Zanotti G, Bubacco L (2014) DJ-1 is a copper chaperone acting on SOD1 activation. J Biol Chem 289:10887–10899

  11. 11.

    Culotta VC, Klomp LW, Strain J, Casareno RLB, Krems B, Gitlin JD (1997) The copper chaperone for superoxide dismutase. J Biol Chem 272:23469–23472

  12. 12.

    Banci L, Cantini F, Kozyreva T, Rubino JT (2013) Mechanistic aspects of hSOD1 maturation from the solution structure of CuI-Loaded hCCS domain 1 and analysis of disulfide-free hSOD1 mutants. ChemBioChem 14:1839–1844

  13. 13.

    Leitch JM, Jensen LT, Bouldin SD, Outten CE, Hart PJ, Culotta VC (2009) Activation of Cu, Zn-superoxide dismutase in the absence of oxygen and the copper chaperone CCS. J Biol Chem 284:21863–21871

  14. 14.

    Brown NM, Torres AS, Doan PE, O'Halloran TV (2004) Oxygen and the copper chaperone CCS regulate posttranslational activation of Cu, Zn superoxide dismutase. Proc Natl Acad Sci USA 101:5518–5523

  15. 15.

    Seetharaman SV, Prudencio M, Karch C, Holloway SP, Borchelt DR, Hart PJ (2009) Immature copper–zinc superoxide dismutase and familial amyotrophic lateral sclerosis. Exp Biol Med 234:1140–1154

  16. 16.

    Hörnberg A, Logan DT, Marklund SL, Oliveberg M (2007) The coupling between disulphide status, metallation and dimer interface strength in Cu/Zn superoxide dismutase. J Mol Biol 365:333–342

  17. 17.

    Furukawa Y, Torres AS, O'Halloran TV (2004) Oxygen-induced maturation of SOD1: a key role for disulfide formation by the copper chaperone CCS. EMBO J 23:2872–2881

  18. 18.

    Saccon RA, Bunton-Stasyshyn RK, Fisher EM, Fratta P (2013) Is SOD1 loss of function involved in amyotrophic lateral sclerosis? Brain 136:2342–2358

  19. 19.

    Bunton-Stasyshyn RK, Saccon RA, Fratta P, Fisher EM (2015) SOD1 function and its implications for amyotrophic lateral sclerosis pathology: new and renascent themes. Neuroscientist 21:519–529

  20. 20.

    Grad LI, Rouleau GA, Ravits J, Cashman NR (2017) Clinical spectrum of amyotrophic lateral sclerosis (ALS). Cold Spring Harb Perspect Med

  21. 21.

    Black HA, Leighton DJ, Cleary EM, Rose E, Stephenson L, Colville S, Ross D, Warner J, Porteous M, Gorrie GH, Swingler R, Goldstein D, Harms MB, Connick P, Pal S, Aitman TJ, Chandran S (2016) Genetic epidemiology of motor neuron disease-associated variants in the Scottish population. Neurobiol Aging 51:178

  22. 22.

    Finsterer J, Burgunder J (2014) Recent progress in the genetics of motor neuron disease. Eur J Med Genet 57:103–112

  23. 23.

    Abel O, Powell JF, Andersen PM, Al-Chalabi A (2012) ALSoD: a user-friendly online bioinformatics tool for amyotrophic lateral sclerosis genetics. Hum Mutat 33:1345–1351

  24. 24.

    Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O'Regan JP, Deng H, Rahmani Z, Krizus A, McKenna-Yasek D, Cayabyab A, Gaston SM, Berger R, Tanzi RE, Halperin JJ, Herzfeldt B, Van dB, Hung W, Bird T, Deng G, Mulder DW, Smyth C, Laing NG, Soriano E, Pericak-Vance M, Haines J, Rouleau GA, Gusella JS, Horvitz HR, Brown RH, (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362(6415):59–62

  25. 25.

    Wang J, Xu G, Slunt HH, Gonzales V, Coonfield M, Fromholt D, Copeland NG, Jenkins NA, Borchelt DR (2005) Coincident thresholds of mutant protein for paralytic disease and protein aggregation caused by restrictively expressed superoxide dismutase cDNA. Neurobiol Dis 20:943–952

  26. 26.

    Andersen PM (2006) Amyotrophic lateral sclerosis associated with mutations in the CuZn superoxide dismutase gene. Curr Neurol Neurosci Rep 6:37–46

  27. 27.

    Borchelt DR, Lee MK, Slunt HS, Guarnieri M, Xu ZS, Wong PC, Brown RH Jr, Price DL, Sisodia SS, Cleveland DW (1994) Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activity. Proc Natl Acad Sci USA 91:8292–8296

  28. 28.

    Prudencio M, Hart PJ, Borchelt DR, Andersen PM (2009) Variation in aggregation propensities among ALS-associated variants of SOD1: correlation to human disease. Hum Mol Genet 18:3217–3226

  29. 29.

    Ayers J, Lelie H, Workman A, Prudencio M, Brown H, Fromholt S, Valentine J, Whitelegge J, Borchelt D (2013) Distinctive features of the D101N and D101G variants of superoxide dismutase 1; two mutations that produce rapidly progressing motor neuron disease. J Neurochem 128:305–314

  30. 30.

    Bystrom R, Andersen PM, Grobner G, Oliveberg M (2010) SOD1 mutations targeting surface hydrogen bonds promote amyotrophic lateral sclerosis without reducing apo-state stability. J Biol Chem 285:19544–19552

  31. 31.

    Bruijn L, Becher M, Lee M, Anderson K, Jenkins N, Copeland N, Sisodia S, Rothstein J, Borchelt D, Price D (1997) ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions. Neuron 18:327–338

  32. 32.

    Wang J, Caruano-Yzermans A, Rodriguez A, Scheurmann JP, Slunt HH, Cao X, Gitlin J, Hart PJ, Borchelt DR (2007) Disease-associated mutations at copper ligand histidine residues of superoxide dismutase 1 diminish the binding of copper and compromise dimer stability. J Biol Chem 282:345–352

  33. 33.

    Winkler DD, Schuermann JP, Cao X, Holloway SP, Borchelt DR, Carroll MC, Proescher JB, Culotta VC, Hart PJ (2009) Structural and biophysical properties of the pathogenic SOD1 variant H46R/H48Q. Biochemistry 48:3436–3447

  34. 34.

    Elam JS, Taylor AB, Strange R, Antonyuk S, Doucette PA, Rodriguez JA, Hasnain SS, Hayward LJ, Valentine JS, Yeates TO (2003) Amyloid-like filaments and water-filled nanotubes formed by SOD1 mutant proteins linked to familial ALS. Nat Struct Mol Biol 10:461–467

  35. 35.

    Ray SS, Nowak RJ, Strokovich K, Brown RH, Walz T, Lansbury PT (2004) An intersubunit disulfide bond prevents in vitro aggregation of a superoxide dismutase-1 mutant linked to familial amytrophic lateral sclerosis. Biochemistry 43:4899–4905

  36. 36.

    Rakhit R, Crow JP, Lepock JR, Kondejewski LH, Cashman NR, Chakrabartty A (2004) Monomeric Cu, Zn-superoxide dismutase is a common misfolding intermediate in the oxidation models of sporadic and familial amyotrophic lateral sclerosis. J Biol Chem 279:15499–15504

  37. 37.

    Svensson AE, Bilsel O, Kayatekin C, Adefusika JA, Zitzewitz JA, Matthews CR (2010) Metal-free ALS variants of dimeric human Cu, Zn-superoxide dismutase have enhanced populations of monomeric species. PLoS ONE 5:e10064

  38. 38.

    Banci L, Bertini I, Durazo A, Girotto S, Gralla EB, Martinelli M, Valentine JS, Vieru M, Whitelegge JP (2007) Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: a possible general mechanism for familial ALS. Proc Natl Acad Sci USA 104:11263–11267

  39. 39.

    Kayatekin C, Zitzewitz JA, Matthews CR (2010) Disulfide-reduced ALS variants of Cu, Zn superoxide dismutase exhibit increased populations of unfolded species. J Mol Biol 398:320–331

  40. 40.

    Abdolvahabi A, Shi Y, Chuprin A, Rasouli S, Shaw BF (2016) Stochastic formation of fibrillar and amorphous superoxide dismutase oligomers linked to amyotrophic lateral sclerosis. ACS Chem Neurosci 7:799–810

  41. 41.

    Karch CM, Prudencio M, Winkler DD, Hart PJ, Borchelt DR (2009) Role of mutant SOD1 disulfide oxidation and aggregation in the pathogenesis of familial ALS. Proc Natl Acad Sci USA 106:7774–7779

  42. 42.

    Shaw BF, Lelie HL, Durazo A, Nersissian AM, Xu G, Chan PK, Gralla EB, Tiwari A, Hayward LJ, Borchelt DR, Valentine JS, Whitelegge JP (2008) Detergent-insoluble aggregates associated with amyotrophic lateral sclerosis in transgenic mice contain primarily full-length, unmodified superoxide dismutase-1. J Biol Chem 283:8340–8350

  43. 43.

    Wang J, Xu G, Borchelt DR (2002) High molecular weight complexes of mutant superoxide dismutase 1: age-dependent and tissue-specific accumulation. Neurobiol Dis 9:139–148

  44. 44.

    Wang J, Farr GW, Zeiss CJ, Rodriguez-Gil DJ, Wilson JH, Furtak K, Rutkowski DT, Kaufman RJ, Ruse CI, Yates JR, Perrin S, Feany MB, Horwich AL (2009) Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS. Proc Natl Acad Sci USA 106(5):1392–1397

  45. 45.

    Wang J, Xu G, Gonzales V, Coonfield M, Fromholt D, Copeland NG, Jenkins NA, Borchelt DR (2002) Fibrillar inclusions and motor neuron degeneration in transgenic mice expressing superoxide dismutase 1 with a disrupted copper-binding site. Neurobiol Dis 10:128–138

  46. 46.

    Ratovitski T, Corson LB, Strain J, Wong P, Cleveland DW, Culotta VC, Borchelt DR (1999) Variation in the biochemical/biophysical properties of mutant superoxide dismutase 1 enzymes and the rate of disease progression in familial amyotrophic lateral sclerosis kindreds. Hum Mol Genet 8:1451–1460

  47. 47.

    Synofzik M, Ronchi D, Keskin I, Basak AN, Wilhelm C, Gobbi C, Birve A, Biskup S, Zecca C, Fernandez-Santiago R, Kaugesaar T, Schols L, Marklund SL, Andersen PM (2012) Mutant superoxide dismutase-1 indistinguishable from wild-type causes ALS. Hum Mol Genet 21:3568–3574

  48. 48.

    Wang Q, Johnson JL, Agar NY, Agar JN (2008) Protein aggregation and protein instability govern familial amyotrophic lateral sclerosis patient survival. PLoS Biol 6:e170

  49. 49.

    Parge HE, Hallewell RA, Tainer JA (1992) Atomic structures of wild-type and thermostable mutant recombinant human Cu, Zn superoxide dismutase. Proc Natl Acad Sci USA 89:6109–6113

  50. 50.

    Getzoff ED, Cabelli DE, Fisher CL, Parge HE, Viezzoli MS, Banci L, Hallewell RA (1992) Faster superoxide dismutase mutants designed by enhancing electrostatic guidance. Nature 358:347–351

  51. 51.

    Cleveland DW, Liu J (2000) Oxidation versus aggregation-how do SOD1 mutants cause ALS? Nat Med 6:1320–1321

  52. 52.

    Salo DC, Pacifici RE, Lin SW, Giulivi C, Davies KJ (1990) Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation. J Biol Chem 265:11919–11927

  53. 53.

    Cocheme HM, Murphy MP (2008) Complex I is the major site of mitochondrial superoxide production by paraquat. J Biol Chem 283:1786–1798

  54. 54.

    Turner BJ, Lopes EC, Cheema SS (2004) Inducible superoxide dismutase 1 aggregation in transgenic amyotrophic lateral sclerosis mouse fibroblasts. J Cell Biochem 91:1074–1084

  55. 55.

    Khare SD, Caplow M, Dokholyan NV (2004) The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis. Proc Natl Acad Sci USA 101:15094–15099

  56. 56.

    Hong S, Lee S, Choi I, Yang YI, Kang T, Yi J (2013) Real-time analysis and direct observations of different superoxide dismutase (SOD1) molecules bindings to aggregates in temporal evolution step. Colloids Surf B 101:266–271

  57. 57.

    McAlary L, Aquilina JA, Yerbury JJ (2016) Susceptibility of mutant SOD1 to form a destabilized monomer predicts cellular aggregation and toxicity but not in vitro aggregation propensity. Front Neurosci 10:499

  58. 58.

    Lang L, Zetterstrom P, Brannstrom T, Marklund SL, Danielsson J, Oliveberg M (2015) SOD1 aggregation in ALS mice shows simplistic test tube behavior. Proc Natl Acad Sci USA 112:9878–9883

  59. 59.

    Karch CM, Borchelt DR (2008) A limited role for disulfide cross-linking in the aggregation of mutant SOD1 linked to familial amyotrophic lateral sclerosis. J Biol Chem 283:13528–13537

  60. 60.

    Wang J, Slunt H, Gonzales V, Fromholt D, Coonfield M, Copeland NG, Jenkins NA, Borchelt DR (2003) Copper-binding-site-null SOD1 causes ALS in transgenic mice: aggregates of non-native SOD1 delineate a common feature. Hum Mol Genet 12:2753–2764

  61. 61.

    Mizushima S, Nagata S (1990) pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res 18:5322

  62. 62.

    Prudencio M, Lelie H, Brown HH, Whitelegge JP, Valentine JS, Borchelt DR (2012) A novel variant of human superoxide dismutase 1 harboring amyotrophic lateral sclerosis-associated and experimental mutations in metal-binding residues and free cysteines lacks toxicity in vivo. J Neurochem 121:475–485

  63. 63.

    Henderson LJ (1908) Concerning the relationship between the strength of acids and their capacity to preserve neutrality. Am J Physiol 21:173–179

  64. 64.

    Hasselbalch KA (1916) Die berechnung der wasserstoffzahl des blutes aus der freien und gebundenen Kohlensäure desselben, und die sauerstoffbindung des blutes als funktion der wasserstoffzahl. Springer, Berlin

  65. 65.

    Chauhan OP, Singh S (1992) Order of reaction for sigmoidal curves. Biochem Educ 20:230–230

  66. 66.

    Akaike H (1978) On the likelihood of a time series model. Stat 27(3–4):217–235

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Acknowledgements

The author thanks David Borchelt for advice and resources.

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The author designed, performed, and analyzed the experiments and prepared the manuscript.

Correspondence to Aron Workman.

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Workman, A. Nucleation and kinetics of SOD1 aggregation in human cells for ALS1. Mol Cell Biochem 466, 117–128 (2020). https://doi.org/10.1007/s11010-020-03693-y

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Keywords

  • Superoxide dismutase 1 (SOD1)
  • Polyacrylamide gel electrophoresis (PAGE)
  • High-resolution colorless native PAGE (hrcN-PAGE)
  • Familial amyotrophic lateral sclerosis (ALS)