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January 14, 2004 The Mutant Proteins of a Neurodegenerative DisorderJ.S. Elam1, A.B. Taylor1, R. Strange2, S. Antonyuk2, P.A. Doucette3, J.A. Rodriguez3, S.S. Hasnain2, L.J. Hayward4, J.S. Valentine3, T.O. Yeates3 and P.J. Hart1 Mutations in copper-zinc superoxide dismutase (SOD1) proteins cause the neurodegenerative disorder familial amyotrophic lateral sclerosis (FALS). In human spinal cord neurons and in transgenic mice expressing these proteins, proteinaceous inclusions (aggregates) containing pathogenic SOD1 are observed. These aggregates are believed to interfere with a variety of cellular processes, eventually leading to motor neuron death. We discovered that several metal-deficient, pathogenic SOD1 proteins can undergo conformational changes that do not occur in non-pathogenic (native) SOD1. This promotes a “gain-of-interaction” between the molecules, causing them to “stick” to each other. In turn, this leads to the formation of extensive helical and linear, or “amyloid-like,” arrays of SOD1. Such non-native protein-protein interactions, leading to larger, or “higher order,” SOD1 assemblies, could thus represent a toxic property that is common to mutants of SOD1 linked to FALS.
There has been increasing support for the concept that this “toxic gain of function” of pathogenic SOD1 is due to the propensity of these proteins to misfold and aggregate. This is supported by the manifestation of aggregates containing pathogenic SOD1 in human spinal cord neurons and in transgenic mice expressing these proteins. These pathogenic SOD1 aggregates (or their soluble precursors) could play a role in pathogenesis, either by occupying the neurons’ heat shock proteins and preventing them from doing their jobs, which leaves the neuron vulnerable, and/or interfering with the way the neurons transport materials along their length (or along their axons) and get rid of unwanted proteins. However, the molecular basis underlying the formation of pathogenic SOD1 oligomers has remained undefined.
To partially address this issue, we determined the crystal structures of the pathogenic SOD1 mutants H46R and S134N, which are members of a larger class of “metal-binding region mutants.” As the name suggests, members of this class of pathogenic SOD1 cannot properly bind either copper or zinc, or both. In the absence of the proper complement of these metal ions, structural features normally involved in metal binding, called loop elements, undergo conformational changes that allow H46R and S134N to polymerize into two types of higher-order filamentous arrays. Figure 1 shows that both metal-free H46R and metal-deficient S134N (which crystallize in different crystal systems) form nearly identical linear, amyloid-like fibers. A variation on this theme can be seen in Figure 2(iv), which shows that copper-free, but zinc-loaded H46R engages in slightly different pathogenic SOD1-SOD1 interactions, leading to the formation of helical, rather than linear, filamentous arrays. From the perspective in Figure 2(iv), the interactions (which occur in the red patches) form a donut shape. Thus, propagating them leads to hollow, water-filled nanotubes. Importantly, when native SOD1 dimers are structurally aligned with the pathogenic SOD1 dimers in these linear and helical arrays, it is obvious that the native variety cannot participate in filamentous assembly. This is due to clashes and unfavorable electrostatic interactions between the loop elements described above.
In summary, the filamentous arrangement of mutant, metal-deficient SOD1 proteins provides justification for a specific and testable hypothesis that links various pathogenic SOD1 mutations to protein aggregation, thereby making it possible to draw a parallel between ALS and other established amyloid diseases. BEAMLINE FUNDING PUBLICATION FOR MORE INFORMATION |
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Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s
disease or motor neuron disease) is a neurodegenerative condition
characterized by the loss of motor neurons in the spinal cord and
brain. The disorder results in paralysis, leading to the death of
the afflicted individual within two to five years of diagnosis. A
subset of ALS cases are familial (FALS), and most of these are
associated with dominantly inherited mutations in copper-zinc
superoxide dismutase (SOD1). SOD1 is an antioxidant enzyme that is
32 kilo-daltons (kDa) in size (where one Da is equal to one atomic
mass unit), and is homodimeric, meaning it consists of two identical
molecules. Initial hypotheses suggested that SOD1-linked FALS comes
from extensive oxidative damage in the neurons, arising from
diminished SOD1 activity. However, mice without SOD1 appear normal
and live to adulthood without developing motor neuron disease. In
contrast, transgenic mice expressing human FALS SOD1 mutants become
paralyzed, even though they possess normal levels of SOD1 activity
from their own endogenous SOD1. Together, these observations
strongly suggest that pathogenic SOD1 molecules act by gaining the
ability to kill other cells (becoming “cytotoxic”) and not by losing
their enzymatic function.
