Dozens of mutations in the gene for superoxide dismutase 1 can cause amyotrophic lateral sclerosis, and they all do so by making the normally rigid enzyme floppy and aggregation-prone, according to a paper in the October 14 Proceedings of the National Academy of Sciences online. By examining six SOD1 mutants, all carrying substitutions at the same disease hotspot, first author Ashley Pratt of Lawrence Berkeley National Laboratory (LBNL) in Berkeley, California, determined that the mutations that cause the worst disease were those that most disrupted SOD1 structure and weakened its grip on copper, which is essential for enzyme activity. This information could help researchers predict how rapidly ALS progresses, and help them develop an enzyme-stabilizing treatment, suggested John Tainer of LBNL, co-senior author on the paper with Elizabeth Getzoff of The Scripps Research Institute in La Jolla, California.
SOD1 mutations are the source for about one-fifth of inherited ALS cases, and scientists have catalogued 178 different SOD1 mutations that cause disease (see ALSoD database). Though researchers still do not understand precisely how the myriad defects lead to neurodegeneration, it is clear that the altered enzymes aggregate to form large protein inclusions that disrupt motor neurons. To study how mutations affect protein structure, Pratt focused on mutations at position 93 of the 154 amino-acid protein, which is normally a glycine. Alanine, valine, arginine, serine, cysteine, or aspartic acid can take that place in people with ALS. Of these six possibilities, G93A causes the most aggressive disease, followed by G93V. G93R results in rather variable disease severity, and the serine, cysteine and aspartic acid replacements create slower-moving ALS.
Pratt used an X-ray-scattering technique to examine the shape taken by each of these different SOD1 proteins. While wild-type SOD1 assembles into an unusually stable dimer (Perry et al., 2010), the mutants assumed flexible, unstable structures (see image below). They also formed aggregates faster than wild-type SOD1, though all proteins formed snake-like fibrils if given sufficient time. “There is a striking agreement between severity of clinical outcome and the propensity to destabilize and aggregate,” Tainer said.
SOD1 dimers normally contain two copper ions, each held in place by four amino acids. To examine how the different disease mutations influenced that part of the structure, the authors used electron spin resonance spectroscopy to measure the distance between those two coppers in three of the SOD1 mutants: G93A, G93D, and G93R. One or zero coppers in a dimer would not produce a signal. They observed that the mutants contained copper when they were first purified, but tended to lose it over time. The protein that causes the most aggressive ALS, G93A, was quickest to drop the ion.
Tainer suggested that SOD1 mutants are different from the start because they are more flexible than the wild-type enzyme. Then, because the mutants are prone to losing copper, they destabilize even further and eventually aggregate. This theory could explain why other scientists have reported that missing copper ions underlie SOD1-based ALS, Tainer said (Goto et al., 2000). He believes structural instability likely explains how all ALS-linked SOD1 mutations cause disease. Moreover, because some studies have indicated that even wild-type SOD1 misfolds in sporadic cases of ALS, stabilizing SOD1 might treat many or all forms of the disease, suggested Tainer (see Oct 2010 news story). He said the researchers are working on some stabilizer ideas, but did not elaborate.
Elizabeth Meiering of the University of Waterloo in Canada, who was not involved in the study, noted that SOD1 adopts many forms as it matures from a metal-free monomer to a copper- and zinc-bound, disulfide-bonded dimer. A caveat to many SOD1 studies, she noted, is that the form studied—here, the mature dimer—might not be the version that aggregates in people with ALS. However, she thought that a SOD1-stabilizing therapeutic would be worth considering, noting a precedent for such a treatment. The medicine diflunisal stabilizes the enzyme transthyretin in its native tetramer conformation, preventing a mutant version from disassembling into monomers that form amyloids. In so doing, it stops progression of familial amyloid polyneuropathy, a fatal disease if left untreated (see Dec 2013 news story).—Amber Dance
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