Mutational analysis of lysine-coordinated superoxide ionophores on the surface of SOD1 Aron S. Workman and David R. BorcheltDepartment of Neuroscience and the McKnight Brain Institute University of Florida, Gainesville, FL 32612 III. Inducible neural cells AmyotrophicLateralSclerosis(ALS) isacommonneuromuscular diseasewithouteffectivetreatment characterizedbytheselective degenerationofmotorneurons resultinginparalyticdeath.Ina familialsubsetofALS,mutant Cu/Znsuperoxidedismutase(SOD1 ) enzymesinitiateandsustainthis disorderdose-dependently.SOD1is ahighlyconserved153-residue barrelhomodimerthatservesasthe predominantintracellularscavenger ofneurotoxicsuperoxideanion radical.Determiningthegain-offunctionmechanismofSOD1 toxicityfortherapeuticintervention inALShasbeenthesubjectof continuingresearchforoverfifteen years. colored formazan NBT riboflavin O2 -+ H+TEMED (catalyst) superoxide antioxidant 24 h S1 5 g 24 h P2 20 g 48 h S1 5 g 48 h P2 20 gSUMMARYANDCONCLUSIONS Wehavediscoveredstructuralmotifs onthesurfaceofSOD1thatactasnodal ionophoresforsuperoxideanion substrate. Usingthisparadigmwehavecloned theenzymaticallyfastestspeciesthus farassayed(E40Q),fasterthanthe nucleophilicshiftmutantE133Qatthe electrostatictriad(Getzoff etal .) UnlikedozensoftoxicSOD1species thusfarexamined,E133Vretainsan oxidizedintradisulfideandtherefore doesnotaggregateat48h. Theseearlyandexcitingfindingsdepict asuperelectrophilicapo-SOD1that acquiresradicalchargeatfocallesions, nucleatingpolymerizationandtoxicity. Thisintermediateisclearedintheactive intraoxidized/metallatedstate. Fig 2. Cutaways showing ionophores (from referenced crystal structure PDB ID 2c9v). E21 E24 S25 K23 T2 E40 D90 D92 K91 T39 -barrel plug E78 E77 S68 D76 K70 K75loop IVE100 D101 S102 K30 E121 D124 D125 S142 K122loop VIIE133 E132 S134 K136 T135 T137electrostatic loop (loop VII)Fig 3. Mechanism of riboflavin -nitro blue tetrazolium su peroxide radical antioxidant activity assay and representative native-PAGE demonstrating variable mutant SOD1 scavenging activities. high exposureR Olow exposure ------------S1 ------------------------P2 ------------Fig 1. Detergent extracts in reducing (left) or alkylating (above) conditions. Mutant SOD1 samples are artificial (*) or confirmed toxic from clinical data (E133V, A4V). REFERENCESEDGetzoffetal."Fastersuperoxidedismutasemutants designedbyenhancingelectrostaticguidance.1992 Nature 358:347-351. BeauchampCandFridovichI.Superoxidedismutase:Improved assaysandanassayapplicabletoacrylamidegels.1971 Analytical Biochemistry 244:276-287. RWStrangeetal."Variablemetallationofhumansuperoxide dismutase:atomicresolutioncrystalstructuresofCu-Zn,Zn-Znand as-isolatedwild-typeenzymes."2006 JMolBio 356:1152-62. MATERIALSANDMETHODSCell culture and transfections. SOD1 cDNAs coded in the mammalian expression vector pEF-BOS were prepared by double CsCl/EtBr density gradient and confirmed by automated sequencing and agarose electrophoresis. 4 g cDNA transfectant was prepared with Lipofectamine 2000 (Invitrogen) and added to confluent 60mmdi shes of human embryonic kidney cells (HEK cells, line HEK293FT). Cells were passaged in high-glucose DMEM with 10% horse serum supplemented with L-glutamine. Fresh media was added 4h after tra nsfection, and cell pellets were harvested to -80C for storage after rinsing three times in phos phate-buffered saline (PBS). SOD1 aggregate extraction and immunoblotting Cell pellets were thawed on ice and resuspended in 100 L 1x TEN (10 mM Tris pH 7.4, 1 mM EDTA pH 8.0, 100 mM NaCl). 100 L1x TEN with 1% Nonidet P40 (NP-40) and 1% protease inhibitor cocktail (PI, Roche) was then added for a final concentration of 0.5% NP-40. This mixture was then sonicated and centrifuged for 5 min at >100,000 x g in a Beckman Airfuge. The soluble portion was saved as S1 and the pellet resuspended in 200 L of rinse buffer: 1x TEN with 0.5% NP-40 and 1% PI. The pellet was sonicated and centrifuge d again, this time discarding the soluble fraction. The pellet was finally resuspended and resonicated in 1x TEN with 0.5% NP-40, 0.25% SDS, 0.5% deoxycholate, and 1% PI and saved as P2 Protein concentrations were assayed by the BCA method (BSA standard). 5 g S1 or 20 g P2 were brought up to 20 L in 1x TEN with laemmli buffer (5% -mercaptoethanol). Samples were boiled at 95 C for 6 min and loaded on 18% tris-glycine acrylamide gels. Gels were transferred to nitrocellulose membrane for 2 h at 400 mA and then blocked for 15-40 mins in 5% lowfat dry milk in PBS-T (PBS with 0.1% Tween-20). Primary anti-m/hSOD1 antibody was added 1:5000 in milk from PBS-T for 116 h followed by 3x rinse in PBS-T alone and secondary goat ant i-rabbit HRP at 1:2500 in milk from PBS-T for 1 h. Blots were rinsed again and visualized and pixel-quantified with ECL chemiluminescence on a Fujifilm LAS-3000. Superoxide anion scavenging activity assay. < 100 g total cell lysate prepared from 10x pellet volume sonicated 0.1% NP-40 in 1x TN was run on 8% or 4-20% tri-glycine acrylamide gels in 1x TG with 20% methanol at 4 C and 100 constant V for 4-6 h, without denaturant or reductant added. The gel was retrieved and soaked in 50 mM potassium bicarbonate buffer containing 65 g/mL riboflavin and 280 g/mL nitro blue tetrazolium, pH 7.6 (5). After incubating 2-40 m, the solution was aspirated and 0.1% TEMED in 50 mM potassium bicarbonate buffer was added to the gel. T he gel was immediately exposed to white light from a bright box and imaged on a Agfa Duoscan, with contrast increased.