Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is usually associated with

Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is usually associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). further incubations we observed that addition of the mammalian molecular chaperone Hsc70 abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice exerted partial correction of the transport defect associated with diminished phosphorylation of p38. Most striking the addition of the molecular chaperone Hsp110 in a concentration substoichiometric to the mutant SOD1 protein completely rescued both the transport defect and the phosphorylation of p38. Hsp110 Rabbit Polyclonal to PPP1R16A. has been demonstrated to act as a nucleotide exchange factor for Hsc70 and more recently to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein abrogating toxicity. (27). We find that added G85R mutant human SOD1 fused with yellow fluorescent protein (G85R SOD1YFP) a protein we previously associated with development of ALS in transgenic mice (28) produces inhibition of anterograde kinesin-dependent fast axonal transport in the isolated axoplasm which is usually associated with activation of a MAPK cascade. By contrast WT SOD1 fused with YFP exerts only a minor effect. We observe that addition of the cytosolic molecular chaperone mammalian Hsc70 previously observed as the predominant protein associating with the G85R SOD1-YFP in spinal cord of transgenic mice (28) can partially reverse the transport defect. Strikingly the molecular chaperone Hsp110 also associated with the mutant SOD1 in spinal cord (28) and established as a nucleotide exchange UMI-77 factor for Hsc70 (29 30 that assists it in protein disaggregation (31 32 completely reverses the transport defect when added at levels substoichiometric to the mutant protein. This establishes a role for molecular chaperones in potentially providing to bind and prevent the toxicity of disease-producing misfolded SOD1 species. Results G85R SOD1-YFP but Not WT SOD1-YFP Inhibits Anterograde Fast Axonal Transport in Squid Axoplasm. Although deficiencies in axonal transport have been explained in mouse models of ALS (16-20) the relative inaccessibility of mouse axons to biochemical manipulation led us UMI-77 to use axoplasm isolated from UMI-77 squid giant axon a preparation free of the axonal membrane to which it is possible to directly add purified proteins and small molecules and observe their effects on transport in real-time (27). Additionally this system allows for recovery of the incubated axoplasm for biochemical and immunochemical analysis. To provide proteins for measuring effects on axoplasmic transport we overexpressed both WT and ALS-associated G85R mutant forms of human SOD1 fused to YFP bearing a C-terminal hexahistidine tag in and purified the soluble protein UMI-77 (WT SOD1YFP and G85R SOD1YFP respectively; with and and BL21/DE3 cells by overexpression from pET vectors. The former transformant was induced at low heat in 50 μM isopropyl β-D-1-thiogalactopyranoside to optimize the portion that remained soluble (~2%). Both mutant and WT fusion proteins were purified on Talon resin and the eluted material further purified by chromatography on MonoQ 10/10 eluting at 0.1-0.15 M NaCl. Bovine Hsc70 was overexpressed in and purified by anion exchange chromatography on Q Sepharose Fast Circulation followed by UMI-77 ATP agarose chromatography (Sigma/Fluka/02065). Human Hsp110 (HSPA4L) was produced as a 6His-SUMO-2G-HSPA4L fusion in and purified on Talon resin. The eluted protein was treated with purified ULP1-His to cleave the SUMO moiety and the HSPA4L was recovered free of both His-SUMO and ULP1-His by passage through Talon resin (32 44 Vesicle Motility Assays in Isolated Axoplasm. Intact axoplasms were extruded from giant axons of the squid (Marine Biological Laboratory) as explained previously (45). Recombinant proteins and pharmacological inhibitors were diluted into X/2 buffer (175 mM potassium aspartate 65 mM taurine 35 mM betaine 25 mM glycine 10 mM Hepes 6.5 mM MgCl2 5 mM EGTA 1.5 mM CaCl2 0.5 mM glucose pH 7.2) supplemented with 2-5 mM ATP and 25 μL were perfused into chambers holding membrane-free axoplasms. The final concentration of the SOD1-YFP was 4.6 μM with respect to the fusion monomer; the final concentration of Hsc70 was 10 μM and that of Hsp110 was 0.6 μM. Axoplasms were visualized on a Zeiss Axiomat microscope with a 100× 1.3 n.a. objective and DIC optics used. Images were acquired with a Hamamatsu.