Much as the enzyme superoxide dismutase 1 protects cells from dangerous free radicals, in amyotrophic lateral sclerosis it may succumb to peer pressure, where malformed enzymes convert others to aggregation-prone forms in a twisted game of molecular tag. In the September 19 Proceedings of the National Academy of Science USA online, Neil Cashman and colleagues at the University of British Columbia in Vancouver report on their use of a truncated enzyme and conformation-specific antibodies to analyze the folding and misfolding of superoxide dismutase 1 (SOD1). The protein’s transmogrification, the researchers say, depends on a hydrophobic tryptophan residue that pokes out of the soluble protein’s structure and acts as a sticky patch where bad SOD1 can attach and reconfigure the good molecule. Blocking the unhealthy protein-protein interaction and conversion could stop disease progression in its tracks, Cashman suggested.

If misfolded SOD1 can re-jigger the conformation of native SOD1 with a touch, then it would be acting somewhat like prions, those infectious particles that convert normal proteins to toxic ones. The aggregating proteins in many neurodegenerative conditions may also have prion-esque properties: for example, studies hint that ALS-linked TAR DNA binding protein 43 (see ARF related news story on Fuentealba et al., 2010), amyloid-β (see ARF related news story on Morales et al., 2010), and tau (see ARF related news story), too, corrupt their normal counterparts (see ARF Webinar and Aguzzi, 2009). To be truly prion-like, the misfolded proteins would have to travel from cell to cell, and earlier this year, scientists from the UK showed that mutant SOD1 does just that (Münch et al., 2011). Toxicity spreading from protein to protein and cell to cell means that pathology ought to move from one body part to the next. That phenomenon, in ALS, is what pointed Cashman and others to examine SOD1 for prion-like transmission (Ravits and La Spada, 2009).

In the current work, Cashman, along with joint first authors Leslie Grad and Will Guest, compared the full-length, wild-type enzyme to an ALS-linked frameshift mutant, G127X, that is truncated. They initially studied human versions, and developed tools to label, individually, each form. When they transfected human embryonic kidney cells with the G127X construct, they observed that the cells’ endogenous, wild-type enzyme lit up with a misfold-specific antibody, indicating endogenous SOD1 adopted a non-native conformation in the presence of the mutant. Transfection with an empty vector did not cause any SOD1 misfolding.

The researchers used immunoprecipitation to confirm that mutant and misfolded wild-type SOD1 associated and to obtain protein for biochemical studies. SOD1’s amino acid chain is normally tightly wound, like the wool strands within a baseball, Cashman said. That makes it resistant to proteinase K degradation. However, the proteinase easily chews up mutant versions of the enzyme, likely because the conformation is looser, like a ball of yarn. Wild-type SOD1 was sensitive to the proteinase in the presence of the G127X mutant, indicating that the mutant somehow loosened the structure of wild-type SOD1.

This kind of transfer of non-native conformation is not surprising in light of well-known, similar activity among misfolded polyglutamine proteins, noted Marc Diamond of Washington University in St. Louis, Missouri (Ren et al., 2009). However, he suggested there is another possible interpretation for the results: The presence of any malformed protein, in general, could stress cells and lead to SOD1 misfolding. For example, researchers have shown that the presence of polyglutamine proteins can destabilize unrelated proteins under some circumstances (see ARF related news story on Gidalevitz et al., 2006). Thus, Diamond said, it is not clear if G127X restructures the wild-type SOD1 by touching it, or simply by altering the cellular milieu the two proteins share.

Curiously, scientists have noticed that no such native-to-misfolded metamorphosis occurs in transgenic SOD1 mouse models of ALS. Despite an overabundance of misfolded mutant human SOD1, the native mouse protein remains tightly wound (see ARF related news story on Bruijn et al., 1998). Cashman and colleagues explain that discrepancy with a difference between the mouse and human SOD1 amino acid sequence. Human SOD1 contains a tryptophan at position 32, whereas the mouse has a serine. Other studies have already indicated that this tryptophan could participate in SOD1 aggregation (Taylor et al., 2007). When the Vancouver team swapped the human G127X tryptophan for serine, it was no longer effective at converting wild-type SOD1. “That handful of atoms [the tryptophan] is really dominating the ability of misfolded SOD1 to trigger the misfolding of wild-type SOD1,” Cashman concluded.

While misfolded SOD1 has long been associated with inherited ALS, recent data indicate that the enzyme is ill-formed in sporadic disease, too (see ARF related news story on Bosco et al., 2010; Forsberg et al., 2010). In fact, it is in sporadic ALS where Cashman thinks that the touch of misfolded SOD1 could be the kiss of death to a natively folded version. In familial cases, a person with a SOD1 mutation is likely to suffer SOD1 misfolding in any cell—but it would be a rare event for a wild-type sequence to misfold in someone with no such genotype. If one misfolded SOD1 could transmit its pathology to another, and another, that might explain how the disease progresses in people with no SOD1 mutations, Cashman believes.

The theory suggests a therapeutic to Cashman. An antibody that neutralizes the transmission of the SOD1 misfold could prevent the disease from spreading, isolating it where it began and, he imagines, turning ALS from a progressively fatal disease to one that disables only a limited part of the body. In July 2010, drug maker Biogen-Idec licensed misfolded SOD1 antibodies from a company Cashman founded—Amorfix Life Sciences of Mississauga, Canada (see company press release).—Amber Dance

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References

News Citations

  1. Toxic TDP-43 Too Tough to Degrade, Plays Prion?
  2. Amyloid and Prions—Co-Conspirators in Disease?
  3. Keystone: Tau, Huntingtin—Do Prion-like Properties Play a Role in Disease?
  4. Protein Aggregation In Disease—A New Theory Joins the Fold
  5. Casting Doubt on Role of Oxidative Damage in ALS
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  1. Seeded Aggregation and Transmissible Proteopathy—Creepy Stuff Not Just for Prions Anymore?

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External Citations

  1. company press release

Further Reading

Papers

  1. . Prion-like acceleration of a synucleinopathy in a transgenic mouse model. Neurobiol Aging. 2011 Aug 1; PubMed.
  2. . RNA-binding proteins with prion-like domains in ALS and FTLD-U. Prion. 2011 Jul 1;5(3) PubMed.
  3. . Self-propagation and transmission of misfolded mutant SOD1: prion or prion-like phenomenon?. Cell Cycle. 2011 Jun 1;10(11):1711. PubMed.
  4. . Prion protein interacts with BACE1 protein and differentially regulates its activity toward wild type and Swedish mutant amyloid precursor protein. J Biol Chem. 2011 Sep 23;286(38):33489-500. PubMed.
  5. . The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A. 2004 Oct 19;101(42):15094-9. PubMed.
  6. . Therapeutic effects of immunization with mutant superoxide dismutase in mice models of amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2495-500. PubMed.

Primary Papers

  1. . Intermolecular transmission of superoxide dismutase 1 misfolding in living cells. Proc Natl Acad Sci U S A. 2011 Sep 27;108(39):16398-403. PubMed.