Overexpression of mutant SOD1 causes accumulation of tubulin in insoluble fractionsGiven that mutant SOD1 physically interacts with tubulin, and that mutant SOD1 forms insoluble
saha hdac when overexpressed in cells, we hypothesized that mutant SOD1 causes aggregation of tubulin. To examine the effects of mutant SOD1 on the aggregation of tubulin, we prepared 1% Triton X-100-soluble and insoluble fractions from cells overexpressing wild-type or mutant SOD1, and the amount of tubulin in each fraction was analyzed. An increased level of SOD1 was detected in the insoluble fraction of cells overexpressing mutant SOD1 compared with that of cells overexpressing wild-type SOD1 (Fig. 3D) We found that overexpression of mutant SOD1 resulted in increased levels of α- and β-tubulin in the insoluble fraction, but had no effect on the protein levels in the soluble fraction (Fig. 3D and E). By contrast, overexpression of wild-type SOD1 did not alter the levels of α- and β-tubulin in either fraction (Fig. 3D and E). In both fractions, the level of β-actin, which does not interact with mutant SOD1 (Fig. 1C), was not affected by the overexpression of wild-type or mutant SOD1 (Fig. 3D). These results indicate that the accumulation of tubulin in insoluble fractions is mediated by the interaction between mutant SOD1 and tubulin. Since the insoluble aggregates associated with neurodegenerative diseases exhibit protease resistance [14], it is possible that aggregated tubulin exhibits resistance to constitutive degradation, resulting in the accumulation of insoluble tubulin in cells overexpressing mutant SOD1.Mutant SOD1 directly modulates tubulin polymerizationTo elucidate the direct effect of mutant SOD1 on microtubule dynamics, we used a cell-free tubulin polymerization assay. We confirmed that paclitaxel, a microtubule-stabilizing agent, promotes tubulin polymerization, and that nocodazole, a microtubule-destabilizing agent inhibits tubulin polymerization in a cell-free system (Fig. 3F). We prepared wild-type and mutant SOD1 proteins without a tag (Supplemental Fig. 1C and Supplemental Table 1), and performed tubulin polymerization assays. Because apo wild-type SOD1 interacts with tubulin, we used holo wild-type SOD1 as a control native SOD1. Wild-type SOD1 had almost no effect on tubulin polymerization (Fig. 3G). Interestingly, mutant SOD1 inhibited tubulin polymerization in tubulin polymerization assays (Fig. 3H–J). These results are consistent with the results showing that tubulin interacts with mutant SOD1, but not with wild-type SOD1, and indicate that mutant SOD1 directly affects the stability of microtubules.DiscussionThe microtubule-destabilizing agent colchicine has been reported to induce neuronal cell death in mice [15]. Microtubule-stabilizing agents such as paclitaxel are effective chemotherapeutic agents for the treatment of many cancers. However, neuropathy is a major adverse effect of microtubule-stabilizing agent-based chemotherapy [16]. Paclitaxel is also reported to induce cell death in cortical neurons [17]. Thus tightly regulated microtubule dynamics, namely controlled tubulin polymerization/depolymerization, are required for neurons to remain viable, and our results may indicate that the physical interaction of mutant SOD1 with α/β-tubulin at least partly underlies the neurodegeneration mediated by mutant SOD1.A recent report has shown that microtubule dynamics are increased in the motor neurons of mutant G93A SOD1 transgenic mice, and that this increase in microtubule dynamics in motor neurons is associated with altered axonal transport and motor neuron degeneration [18]. We have shown that mutant SOD1 directly interacts with tubulin (Fig. 2A), and that mutant SOD1 has a similar effect on microtubules to a microtubule-destabilizing agent, but to a lesser extent (Fig. 3F–J). Since microtubules in neurons are relatively stable due to their interactions with microtubule-associated proteins, destabilization of microtubules may cause increased microtubule dynamics in neurons. Taken together, our results suggest that mutant SOD1 can directly increase microtubule dynamics through its destabilizing effect on microtubules in neurons.Using tubulin-binding agents, we have shown that the mutant SOD1-binding site within tubulin in part overlaps with the sites bound by colchicine and nocodazole (Fig. 2A–C). Considering that colchicine destabilizes microtubules through the interaction with tubulin at microtubule ends [19], mutant SOD1 may also modulate microtubule dynamics through the interaction with tubulin at microtubule ends. Structural analysis of the interaction of SOD1 with tubulin, such as X-ray crystallography, is required for further understanding of the mechanism that underlies the modulation of microtubule dynamics by mutant SOD1. Native SOD1 forms a stable homodimer, while mutant SOD1 partly exists as a monomer [20]. We showed that amino
acid residues 1–23 and 116–153 in SOD1 interact with tubulin (Fig. 2D–L). These amino acid residues contain residues 4–8 and 143–151, which are sequestered in the SOD1 dimer interface and are inaccessible in native SOD1 [20]. We also showed that tubulin interacts with apo wild-type SOD1 (Supplemental Fig. 2). Apo SOD1 has been reported to exist substantially as a monomer [8]. Together, our results suggest that tubulin interacts with monomeric SOD1. The inhibitory effects of holo mutant SOD1 on tubulin polymerization (Fig. 3J) may be due to the misfolded or remaining apo SOD1 in the recombinant SOD1 sample, considering that the enzymatic activities of holo mutant SOD1 were reduced compared with that of wild-type SOD1 (Supplemental Table 1).In addition to tubulin, we observed that SOD1 mutants specifically interact with multiple proteins (Fig. 1 and Fig. 3C). The C111S substitution in mutant SOD1 decreases the degree of interaction between mutant SOD1 and various proteins (Fig. 3C), suggesting that most mutant SOD1-interacting proteins bind to SOD1 in a similar way. These other interactors may also be involved in the mechanism underlying SOD1-mediated neurodegeneration. We have identified some of these interactors, and these proteins are currently under investigation.Our present study demonstrates that mutant SOD1 can directly modulates tubulin polymerization by physically interacting with tubulin. The findings in the present study provide novel insights into the mechanisms underlying the pathology of ALS.AcknowledgmentsWe thank Dr. Ryosuke Takahashi (Kyoto University) and Dr. Makoto Urushitani (Shiga University of Medical Science) for the gift of pcDNA3-hSOD1-FLAG plasmids. We also thank Dr. H. Akiko Popiel (National Institute of Neuroscience) for support with English. This work was supported by Grants-in-Aid for Scientific Research of Japan Society for the Promotion of Science; Research Grant in Priority Area Research of the Ministry of Education, Culture, Sports, Science and Technology, Japan; Grants-in-Aid for Scientific Research of the Ministry of Health, Labour and Welfare, Japan and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, Japan.Appendix A. Supplementary dataSupplementary Fig. S1. The 55-kDa protein (A) was analyzed by LC-MS/MS analysis. Matched peptides are shown in bold letters. (B) Recombinant SOD1 proteins with a His tag (apo SOD1, left panel) and purified tubulin (right panel) were subjected to SDS-PAGE and CBB staining. (C) Purification of recombinant SOD1. Recombinant SOD1 proteins without a tag (apo SOD1) were purified and subjected to SDS-PAGE and CBB staining.Figure optionsDownload full-size imageDownload as PowerPoint slideSupplementary Fig. S2. A pull-down assay was performed using the indicated purified proteins. Precipitants were analyzed by immunoblotting. Tubulin was precipitated with apo wild-type SOD1. Holo wild-type SOD1 did not interact with tubulin (data not shown).Figure optionsDownload full-size imageDownload as PowerPoint slideSupplemental materials and methods. Supplemental materials and methodsHelp with PDF filesOptionsDownload file (59 K)
