Rachel L. Redler

Over 100 mutations in Cu/Zn-superoxide dismutase (SOD1) result in familial amyotrophic lateral sclerosis. Dimer dissociation is the first step in SOD1 aggregation, and studies suggest nearly every amino acid residue in SOD1 is dynamically connected to the dimer interface. Post-translational modifications of SOD1 residues might be expected to have similar effects to mutations, but few modifications have been identified. Here we show, using SOD1 isolated from human erythrocytes, that human SOD1 is phosphorylated at threonine 2 and glutathionylated at cysteine 111. A second SOD1 phosphorylation was observed and mapped to either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD1 monomer formation, a necessary initiating step in SOD1 aggregation, by causing a 2-fold increase in the Kd. This change in the dimer stability is expected to result in a 67% increase in monomer concentration, 315 nm rather than 212 nm at physiological SOD1 concentrations. Because protein glutathionylation is associated with redox regulation, our finding that glutathionylation promotes SOD1 monomer formation supports a model in which increased oxidative stress promotes SOD1 aggregation. Over 100 mutations in Cu/Zn-superoxide dismutase (SOD1) result in familial amyotrophic lateral sclerosis. Dimer dissociation is the first step in SOD1 aggregation, and studies suggest nearly every amino acid residue in SOD1 is dynamically connected to the dimer interface. Post-translational modifications of SOD1 residues might be expected to have similar effects to mutations, but few modifications have been identified. Here we show, using SOD1 isolated from human erythrocytes, that human SOD1 is phosphorylated at threonine 2 and glutathionylated at cysteine 111. A second SOD1 phosphorylation was observed and mapped to either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD1 monomer formation, a necessary initiating step in SOD1 aggregation, by causing a 2-fold increase in the Kd. This change in the dimer stability is expected to result in a 67% increase in monomer concentration, 315 nm rather than 212 nm at physiological SOD1 concentrations. Because protein glutathionylation is associated with redox regulation, our finding that glutathionylation promotes SOD1 monomer formation supports a model in which increased oxidative stress promotes SOD1 aggregation. Familial amyotrophic lateral sclerosis (FALS) 4The abbreviations used are: FALS, familial amyotrophic lateral sclerosis; SOD1, Cu/Zn-superoxide dismutase; μ-ESI-FT-ICR-MS, microcapillary electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry; ECD, electron-capture dissociation; CID, collision-induced dissociation; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; LC, liquid chromatography. 4The abbreviations used are: FALS, familial amyotrophic lateral sclerosis; SOD1, Cu/Zn-superoxide dismutase; μ-ESI-FT-ICR-MS, microcapillary electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry; ECD, electron-capture dissociation; CID, collision-induced dissociation; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; LC, liquid chromatography. is the hereditary form of amyotrophic lateral sclerosis, a fatal disease characterized by progressive motor neuron loss (1Cleveland D.W. Rothstein J.D. Nat. Rev. Neurosci. 2001; 2: 806-819Crossref PubMed Scopus (1160) Google Scholar). A subset of FALS is caused by mutations in the gene encoding homodimeric Cu/Zn-superoxide dismutase (SOD1), which forms intraneuronal aggregates (2Bruijn L.I. Houseweart M.K. Kato S. Anderson K.L. Anderson S.D. Ohama E. Reaume A.G. Scott R.W. Cleveland D.W. Science. 1998; 281: 1851-1854Crossref PubMed Scopus (973) Google Scholar). Although SOD1 aggregation is involved in SOD1-mediated FALS, it is generally believed that the functional properties of the enzyme are not related to the toxic gain of function imparted by mutations in SOD1 (3Reaume A.G. Elliott J.L. Hoffman E.K. Kowall N.W. Ferrante R.J. Siwek D.F. Wilcox H.M. Flood D.G. Beal M.F. Brown Jr., R.H. Scott R.W. Snider W.D. Nat. Genet. 1996; 13: 43-47Crossref PubMed Scopus (1030) Google Scholar). However, the discovery of roles for SOD1 in the regulation of the cellular phosphorylation balance (4Juarez J.C. Manuia M. Burnett M.E. Betancourt O. Boivin B. Shaw D.E. Tonks N.K. Mazar A.P. Donate F. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 7147-7152Crossref PubMed Scopus (193) Google Scholar) and redox state (5Harraz M.M. Marden J.J. Zhou W. Zhang Y. Williams A. Sharov V.S. Nelson K. Luo M. Paulson H. Schoneich C. Engelhardt J.F. J. Clin. Investig. 2008; 118: 659-670PubMed Google Scholar) provides additional avenues for connecting the cellular role of SOD1 to FALS. The classical studies of SOD1 were generally performed using bovine erythrocyte SOD1 or recombinant human SOD1. Although recombinant methods are widely used to produce SOD1 mutants, a disadvantage of studying recombinant SOD1 is the absence of potentially important post-translational modifications present in human tissues. The initial SOD1 crystal structure was solved using bovine erythrocyte SOD1 (6Richardson J. Thomas K.A. Rubin B.H. Richardson D.C. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 1349-1353Crossref PubMed Scopus (464) Google Scholar) and no structure of human erythrocyte SOD1 is available. Here we report results using human erythrocyte SOD1 rather than the recombinant enzyme and find that the native enzyme features a consistent pattern of post-translational modifications. Using a combination of “bottom-up” and “top-down” mass spectrometry (MS) approaches, we show that SOD1 isolated from human erythrocytes is post-translationally phosphorylated and glutathionylated. These modifications occur near the SOD1 dimer interface. Because monomer formation is thought to be the first intermediate leading to SOD1 aggregation (7Khare S.D. Caplow M. Dokholyan N.V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15094-15099Crossref PubMed Scopus (124) Google Scholar, 8Rakhit R. Crow J.P. Lepock J.R. Kondejewski L.H. Cashman N.R. Chakrabartty A. J. Biol. Chem. 2004; 279: 15499-15504Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), we tested the dimer stability of modified SOD1 found, as expected, that glutathionylation promotes the formation of SOD1 monomer. Isolation of hSOD1 from Erythrocytes—Expired human erythrocytes were obtained from the University of North Carolin-Chapel Hill Hospital blood bank. Human erythrocytes were preserved using one of several anticoagulants: AS-1, AS-3, or AS-5 (stored as long as 42 days before expiration). Bovine erythrocytes preserved with 0.38% sodium citrate were purchased from Pel-Freez Biologicals, Rogers, AR. SOD1 was isolated from human erythrocytes using a modification of the protocol originally used by McCord and Fridovich (9McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). Following acetone precipitation of SOD1, we dialyzed SOD1 against 20 mm Tris, pH 7.8, and performed hydrophobic interaction chromatography and anion exchange chromatography using an AKTA-FPLC to remove trace impurities. For hydrophobic interaction chromatography, dry (NH4)2SO4 was added to bring the final concentration to 55%. The protein was loaded onto a HiTrap phenyl-Sepharose column (GE Healthcare) and eluted with a gradient from 2 to 0 m (NH4)2SO4 in 20 mm Tris, pH 7.8. Fractions containing SOD1 were combined and dialyzed against salt-free 20 mm Tris, pH 7.8, and anion exchange chromatography was performed using a MonoQ column (GE Healthcare) with a gradient from 0 to 1 m NaCl in 20 mm Tris, pH 7.8. The fractions containing SOD1 were dialyzed against 20 mm Tris, 150 mm NaCl, pH 7.8, and concentrated. Isolation of hSOD1 from Saccharomyces cerevisiae—Human wild type SOD1 was expressed using plasmid yEP351-hwtSOD1 in the EG118 SOD1-knock out yeast strain (both kindly provided by J. S. Valentine). Growth was carried out at 30 °C for 72 h in YPD media. We uses a modified isolation protocol adapted from Goscin and Fridovich (10Goscin S.A. Fridovich I. Biochim. Biophys. Acta. 1972; 289: 276-283Crossref PubMed Scopus (68) Google Scholar) where we replaced all steps, after stirring the lysate at room temperature, with high speed centrifugation and dialysis of the supernatant against 20 mm Tris, pH 7.8, to remove the chloroform and ethanol. Trace impurities were removed by MonoQ and phenyl-Sepharose chromatography as described above. Remetallation of hSOD1 from yeast was performed by successive dialysis at 4 °C against: (a) 50 mm acetate, pH 3.5, 150 mm NaCl, 10 mm EDTA; (b) 50 mm acetate, pH 3.5, 150 mm NaCl; (c) 50 mm acetate, pH 3.5, 150 mm NaCl + CuSO4 (5-fold molar excess compared with [SOD1]); (d) 20 mm Tris, pH 7.8, 150 mm NaCl + ZnCl2 (5-fold molar excess compared with [SOD1]); and (e) 20 mm Tris, pH 7.8, 150 mm NaCl. Upon concentration with a YM-10 ultrafiltration membrane (Millipore, Bedford, MA), a blue-green coloration was observed, indicating incorporation of copper into the enzyme. Microcapillary (μ) ESI-FT-ICR-MS Analysis (Top-down Approach)—MS spectra are acquired using a hybrid Qe-Fourier Transform Ion Cyclotron Resonance (FT-ICR)-Mass Spectrometer, equipped with a 12.0-tesla actively shielded magnet (Apex Qe-FTICR-MS, 12.0 T AS, Bruker Daltonics, Billerica, MA), and an Apollo II microelectrospray source. The voltages on the μESI sprayer, interface plate, heated capillary exit, deflector, ion funnel, and skimmer were set at 4.3 kV, 3.9 kV, 300 V, 250 V, 175 V, and 80 V, respectively. The temperature of the μESI source was maintained at 180 °C. Desolvation was carried out using a nebulization gas flow (2.0 bar) and a countercurrent drying gas flow (4.0 liters/s). The electron capture dissociation (ECD) hollow dispenser cathode was heated to increase the temperature inside the ICR cell above 180 °C without application of ECD bias, eliminating non-covalent adducts. SOD1 sample solutions were directly infused using a syringe pump (Harvard Apparatus, Holliston, MA) and a 250-μl syringe (Hamilton, Reno, NV), and electrosprayed at an flow of ion are inside the cell for a of 50 were acquired in the ICR cell with a of at for spectra were obtained from collision-induced dissociation performed with dissociation by or from was used as the gas were isolated with and to dissociation using an isolation of 50 were acquired in the ICR cell with a of at ECD were isolated with and to ICR Isolation was 10 were by a heated hollow dispenser cathode with a of ECD was set at The by the hollow cathode were into the ICR cell with a of causing of the that were in the ICR the ion before the cell was with of ion from the cell of the isolated as R. J.P. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and ECD 50 were acquired in the ICR cell with a of at and Analysis protein were with at °C in an enzyme to of by with of the were directly on a matrix-assisted laser for using a mass an the were using a liquid chromatography MALDI, 10 of were and directly loaded on a capillary column 100 with a and using a gradient from A to and in 10 at a flow of The column are with a of acid in and mm on a at a of and by was in in the mass of by ion laser to with a than 30 were for which was performed with a and gas of an was laser from 50 laser were was by the at the M. O. H. M. R. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). and spectra were using in of SOD1 we The were using 20 mm Tris, 150 mm NaCl, pH 7.8, and added to a of SOD1 at a The was performed at °C. chromatography was carried out using a column and a The of the column was and a flow of was used at 4 °C. For of the dimer dissociation of SOD1 at pH were to nm to dimer dissociation using the column mm Tris, pH 7.8, 150 mm NaCl, and 10 bovine to SOD1 to the column and the are at °C for to h to 250-μl fractions were the chromatography for in Fractions were acetone by the of 4 of acetone to the sample and a at °C. The are for 30 at The SOD1 was in protein containing sodium and and by Following to a membrane and with bovine were with a and a The are in and using a of is performed using the (GE Using SOD1 tested for a in the of as described F. PubMed Scopus Google Scholar). The was to of SOD1 as as 100 The of glutathionylated and nm SOD1 was before and after a at °C. performed after and h at °C show no additional loss in indicating that the was at The dimer dissociation at was using the where is the initial of the is the initial of the containing SOD1, and is the initial of the of SOD1 from a a physiological SOD1 concentration of F. K. Kato K. Clin. Acta. PubMed Scopus Google Scholar), we the monomer concentration on the Kd. For a of we the monomer concentration to be 212 For a of we a monomer concentration of 315 a 67% SOD1 in Human a with mass and SOD1 from human erythrocytes was to have a mass of to the mass of for human SOD1. to the with of and to the of one or were observed, as as a to the modification of SOD1 by The protein was with which in the loss of the and are related to the non-covalent of or and a expected to result in the of we heated the ICR cell to above 180 °C. results in the of and to and but no on the The spectra of the which phosphorylation at threonine 2 are similar to of the indicating that the to phosphorylation one the spectra of the that it from phosphorylation one A to phosphorylation was observed in SOD1 from human erythrocytes However, the of the and were for in the second modification We that human erythrocyte SOD1 is phosphorylated at and but to in phosphorylated SOD1, after the of phosphorylated we report that a second phosphorylation is at either Thr-58 or and are in the in SOD1, which the at the and for several dimer interface The of glutathionylation in human SOD1 was by of human erythrocyte SOD1 with and The acquired and spectra were using in SOD1 we mass of from to to the spectra of the which that cysteine 111 is to containing and were not to be glutathionylated Human erythrocyte SOD1 was into by anion exchange chromatography modification in mass The SOD1 that at the and a at to the protein with The of phosphorylation in SOD1 1 and 2 that at and and A of SOD1 that is phosphorylated and glutathionylated was observed of in modifications described above were observed in SOD1 isolated from erythrocytes from and as modifications have to from a the of glutathionylation and phosphorylation we compared SOD1 from of human phosphorylation and glutathionylation sample but was of phosphorylation and glutathionylation of SOD1, we the of modifications to be the of the erythrocyte the of combined phosphorylation and glutathionylation is than the that phosphorylation and glutathionylation are in the of SOD1. However, the observed of combined modification in 4 and is than the expected and is the This from in or not for in of modifications in erythrocyte in a in Human phosphorylation and glutathionylation of human erythrocyte SOD1 not occur as a result of blood We isolated SOD1 from human erythrocytes from The results are in spectra from and related to SOD1 phosphorylation and we observed related to SOD1. These were and from the mass of SOD1 Because are no or to either phosphorylated or glutathionylated SOD1, we that is the of the as been observed in M. Kato S. H. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). residues in SOD1 might be SOD1 and the of residues in SOD1 been observed R. A. S. Crow J.P. Cashman N.R. Kondejewski L.H. Chakrabartty A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is to the of SOD1 in sample compared with sample A that the sample was from a sample A was from a However, a on is to a that the is related to of modifications in erythrocyte in a on S. a copper for SOD1, it is widely used for recombinant human SOD1, but phosphorylation and glutathionylation of human SOD1 occur in yeast or used to we and recombinant SOD1 from as as SOD1 from yeast and bovine spectra of human SOD1 isolated from S. an with the SOD1 in human erythrocytes We a sample of human SOD1 into of high and and similar mass in the mass spectra indicating SOD1 glutathionylation but not phosphorylation The mass of SOD1 isolated from bovine erythrocytes using the protocol used for human erythrocytes bovine is phosphorylated is yeast SOD1 and an expected result that a cysteine at 111 in SOD1. Cysteine is bovine and human SOD1 but we find no glutathionylation of residue in either We that SOD1 phosphorylation was not observed to the of bovine erythrocyte SOD1 or recombinant human SOD1. of on SOD1 Dimer to be at a as not to directly in the SOD1 dimer interface. However, phosphorylation change the structure and of J.M. Thomas J.D. Nat. Biol. PubMed Scopus Google Scholar) and the from the interface and the not phosphorylation from the stability or of SOD1. residues in SOD1 monomer are directly in a at the dimer interface. We that the of the to result in a in SOD1 dimer Because of the of phosphorylation and glutathionylation to the dimer interface and that dimer stability is important for SOD1 aggregation R.J. K. Brown Jr., R.H. Jr., 2004; PubMed Scopus Google Scholar, R.J. Brown Jr., R.H. Jr., Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), we the of glutathionylation and phosphorylation on the in stability of SOD1. Using chromatography, we compared SOD1 by anion exchange as described above 1 and in dimer stability of SOD1 in to 1 is expected to be the result of glutathionylation of phosphorylation are present in SOD1 to the high stability of the SOD1 dimer dissociation of human SOD1 isolated from yeast is than 10 nm B. J.C. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google it was necessary to the at concentrations. dissociation into we performed chromatography using nm SOD1. We compared SOD1 with and without glutathionylation and that glutathionylation a on the SOD1 SOD1 dimer dissociation was observed at °C for to We observed a increase in monomer formation in glutathionylated SOD1 to SOD1 by Using an for SOD1 SOD1 was to have on that mutations SOD1 show an loss M. W. I. S. J. Biol. PubMed Scopus Google Scholar). We that loss be used as an of dimer dissociation from high in the of SOD1. The is important that concentration of the SOD1 aggregation the of the monomer concentration, where is the of in the in the result in in the of as the of in the Using a to SOD1, we the in the in the of nm SOD1 at to the after We observed a loss in SOD1 with dimer as a in the initial to the We tested the of glutathionylation on dimer stability by SOD1 by anion exchange as described above 1 and in Although the initial was similar the glutathionylated SOD1 a at indicating SOD1 and dimer on the in we the to be nm for SOD1 and nm for modified SOD1. Post-translational of Human we report that SOD1 be phosphorylated at and at either Thr-58 or Ser-59. We of SOD1 glutathionylation and that modification to in our a of was carried out that that residues in every of SOD1 show high to the dimer interface S.D. Dokholyan N.V. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and that residues associated with stability on the of SOD1 S.D. F. Dokholyan N.V. J. Biol. PubMed Scopus Google Scholar). suggest that loss of copper and at the results in at the dimer interface F. Dokholyan N.V. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: PubMed Scopus Google Scholar), for in SOD1 aggregation dimer of we that post-translational mutations, have a similar on the structure and of SOD1. the of post-translational modifications on the we a sample of SOD1 into a that a of phosphorylation and a that was glutathionylated phosphorylated to a similar as the enzyme. We that forms of SOD1 in the glutathionylated SOD1 monomer finding that SOD1 is the first intermediate SOD1 aggregation that which a on the SOD1 the of SOD1 aggregation in FALS. been several in erythrocyte SOD1, erythrocytes from FALS M. A. A. Y. S. H. J. Neurosci. 1998; PubMed Scopus Google Scholar, A. J. PubMed Scopus Google Scholar), and of with aggregation in Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We show that glutathionylation is present in However, glutathionylation of SOD1 been performed in and to of the Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of SOD1 aggregation been I. A. S. M. M. J.P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). However, bovine SOD1 is of (7Khare S.D. Caplow M. Dokholyan N.V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15094-15099Crossref PubMed Scopus (124) Google Scholar). glutathionylation not with SOD1 aggregation by a in the of SOD1 in Human SOD1 in to phosphorylation and in human Although the sample is not to show a and the of SOD1 the show in are and have an of days in the SOD1 used in our was isolated from and Because all of the SOD1 in one of the was we that in SOD1 are not the result of a a of SOD1 is modified as erythrocyte but rather an in which the of SOD1 to in the that or not be as in erythrocytes or the in studying the effects of mutations on SOD1, it is important to have a for SOD1 as it inside human tissues. is from our of human SOD1 isolated from human erythrocytes and yeast that the model for native SOD1 at one of native SOD1, Because we wild type SOD1 isolated from human we that phosphorylation and glutathionylation are features of the SOD1 native state and not a result of a However, of from amyotrophic lateral sclerosis that are and in the phosphorylation of Zhang H. R. C. J. PubMed Scopus Google Scholar), that in SOD1 phosphorylation are in FALS. of and glutathionylation the function of SOD1 the of with SOD1 is involved in cellular redox regulation a interaction with the enzyme (5Harraz M.M. Marden J.J. Zhou W. Zhang Y. Williams A. Sharov V.S. Nelson K. Luo M. Paulson H. Schoneich C. Engelhardt J.F. J. Clin. Investig. 2008; 118: 659-670PubMed Google Scholar). This of a interaction SOD1 and a functional enzyme one which phosphorylation glutathionylation of SOD1 a role without as a a role in the redox state of the cell PubMed Scopus Google Scholar). The high of to is by which uses to to and the of from the I. U. M. J. 1996; PubMed Scopus Google Scholar). of oxidative the concentration of Biol. 2001; PubMed Scopus Google Scholar) and in is thought to be a against of by or PubMed Scopus Google Scholar). to several of functional roles for protein glutathionylation are B. Y. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, J. 2004; PubMed Scopus Google Scholar, E. S. J. 13: PubMed Scopus Google Scholar, J. W. E. H.M. S. J.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). that SOD1 be involved in the of which as a second in the of and the balance of phosphorylation in the cell (4Juarez J.C. Manuia M. Burnett M.E. Betancourt O. Boivin B. Shaw D.E. Tonks N.K. Mazar A.P. Donate F. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 7147-7152Crossref PubMed Scopus (193) Google Scholar). on we present a model for the of SOD1 modification on oxidative regulation SOD1 glutathionylation dimer aggregation in the of as oxidative or FALS a of glutathionylation on SOD1 dimer combined with the effects of mutations, aggregation a SOD1 to be to the wild type dimer M. W. I. S. J. Biol. PubMed Scopus Google Scholar), and we that by dimer SOD1 Dimer for SOD1 mutations, protein aggregation, and FALS is not but are that dimer dissociation is an SOD1 aggregation (7Khare S.D. Caplow M. Dokholyan N.V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15094-15099Crossref PubMed Scopus (124) Google Scholar, 8Rakhit R. Crow J.P. Lepock J.R. Kondejewski L.H. Cashman N.R. Chakrabartty A. J. Biol. Chem. 2004; 279: 15499-15504Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). finding that modifications SOD1 dimer dissociation a the of SOD1 and role in FALS. Although a 2-fold increase in from SOD1 modification to nearly a increase in SOD1 monomer Because of SOD1 aggregation is on at the of the monomer concentration, we a increase in monomer concentration to have a on the of SOD1 For the formation of a of glutathionylated SOD1 and a of to SOD1. We S. for the EG118 yeast strain and and for We the University of North for human with

Since the linking of mutations in the Cu,Zn superoxide dismutase gene (sod1) to amyotrophic lateral sclerosis (ALS) in 1993, researchers have sought the connection between SOD1 and motor neuron death. Disease-linked mutations tend to destabilize the native dimeric structure of SOD1, and plaques containing misfolded and aggregated SOD1 have been found in the motor neurons of patients with ALS. Despite advances in understanding of ALS disease progression and SOD1 folding and stability, cytotoxic species and mechanisms remain unknown, greatly impeding the search for and design of therapeutic interventions. Here, we definitively link cytotoxicity associated with SOD1 aggregation in ALS to a nonnative trimeric SOD1 species. We develop methodology for the incorporation of low-resolution experimental data into simulations toward the structural modeling of metastable, multidomain aggregation intermediates. We apply this methodology to derive the structure of a SOD1 trimer, which we validate in vitro and in hybridized motor neurons. We show that SOD1 mutants designed to promote trimerization increase cell death. Further, we demonstrate that the cytotoxicity of the designed mutants correlates with trimer stability, providing a direct link between the presence of misfolded oligomers and neuron death. Identification of cytotoxic species is the first and critical step in elucidating the molecular etiology of ALS, and the ability to manipulate formation of these species will provide an avenue for the development of future therapeutic strategies.

Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T. Glutathionylation results in profound destabilization of SOD1(WT) dimers, increasing the equilibrium dissociation constant K(d) to ~10-20 μM, comparable to that of the aggressive A4V mutant. SOD1(A4V) is further destabilized by glutathionylation, experiencing an ~30-fold increase in K(d). Dissociation kinetics of glutathionylated SOD1(WT) and SOD1(A4V) are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1(I112T) has a modestly increased dissociation rate but no change in K(d) when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS.

A systematic and robust approach to generating complex protein nanomaterials would have broad utility. We develop a hierarchical approach to designing multi-component protein assemblies from two classes of modular building blocks: designed helical repeat proteins (DHRs) and helical bundle oligomers (HBs). We first rigidly fuse DHRs to HBs to generate a large library of oligomeric building blocks. We then generate assemblies with cyclic, dihedral, and point group symmetries from these building blocks using architecture guided rigid helical fusion with new software named WORMS. X-ray crystallography and cryo-electron microscopy characterization show that the hierarchical design approach can accurately generate a wide range of assemblies, including a 43 nm diameter icosahedral nanocage. The computational methods and building block sets described here provide a very general route to de novo designed protein nanomaterials.