ALS precursor finally shaken into fibrils.

T here is now little doubt that the prime cause of the motorneuron disease amyotrophic lateral sclerosis (ALS) is related to misfolding and aggregation of the metalloenzyme Cu/Zn superoxide dismutase (SOD1). Yet, SOD1 has turned out remarkably resistant to aggregation under physiological conditions in vitro. Pathologic mutant proteins that are destabilized to the extent that they fail to fold can still be stored at room temperature for long periods of time without notable signs of self-association. To produce SOD1 aggregates one has to resort to more radical measures, such as high temperature at low pH (1, 2), organic solvents (1), or oxidative cross-linking of the protein’s solventaccessible cysteines (3). Because these conditions are different from those in the cell the connection between SOD1 aggregation and ALS has remained elusive; after all, most if not all proteins contain sequences capable of nucleating aggregation (4) and can thus be forced to aggregate in vitro if the conditions are made sufficiently ‘‘persuasive.’’ In this issue of PNAS, Valentine and collaborators (5) present a new interesting piece to the puzzle by showing that SOD1 can be made to aggregate under conditions that are physiologically relevant. The trick seems to be simply that the solution requires appropriate agitation. After a few hours of this treatment in an orbital shaker at 250 rpm, immature molecules of wild-type SOD1 nucleate into ordered fibrillar structures similar to those observed in classical amyloidoses such as Alzheimer’s and Huntington’s diseases. For nucleation to occur, however, the protein also needs to be demetallated and without its stabilizing disulfide bridge. Once the seeds are formed the process can recruit more stable SOD1 species for the fibril elongation. Similar results were published independently by Furukawa et al. (6) just a few months ago. The question then arises, which part of the SOD1 structure promotes the fibrillation? For disordered proteins like the A peptide associated with Alzheimer’s disease, the aggregation propensity is simply determined by the local sequence signatures that are constantly accessible to the solvent (7). In the case of SOD1, such sequence signatures could be hidden inside the folded structure (F) and exposed for aggregation only in the unfolded state (U) (Fig. 1). The level of aggregation-competent material becomes then controlled by protein stability, i.e., G RT ln[F]/[U]. The less stable the protein is, the higher is the fraction of reactive molecules. Consistent with this model, the ALSprovoking SOD1 mutations are found to decrease protein stability and/or the protein’s net repulsive charge (8, 9). This seems to be a typical feature for the aggregation of proteins: the exposure of the sticky sequence through loss of stability promote aggregation nucleation and the repulsive charges help to reduce the rate of aggregation (10). Even so, it is not directly obvious which part of the protein that causes the problem because the SOD1 sequence seems completely devoid of the ‘‘sticky’’ hydrophobic patches that characterize most other globular proteins. A simple way to convince oneself about this peculiarity is to run SOD1 through one of the predictors for protein aggregation, e.g., TANGO (http://tango.crg.es/). It is conceivable that the overall hydrophilic nature of the SOD1 sequence is required for maintaining the immature states of the protein soluble inside the cell. Nevertheless, SOD1 displays two distinct sequence segments with intrinsic propensities for fibril formation as determined by the WALTZ algorithm (http:// switpc7.vub.ac.be/cgi-bin/submit.cgi), one in the center of the major -sheet ( 2) and one at its edge ( 6). This second level of aggregation signals emphasizes that protein aggregation is not a uniform phenomenon but encompasses considerable diversity at the level of both macroscopic morphology and mechanism of formation. At one extreme is the formation of amorphous precipitates that is typically driven by sequence hydrophobicity, and at the other end of the scale is the nucleated and highly ordered assembly of fibrils that can also involve more hydrophilic sequence signatures (11). It will be interesting to see whether these predictions at local sequence level capture also the fibrillation mechanism of the fulllength protein. We are then left with the big question, what is the coupling between the SOD1 fibrillation observed by Valentine and collaborators (5) and ALS? The identity of the SOD1 species that causes