Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a rapidly progressing and invariably fatal neurological disease that attacks the nerve cells responsible for controlling voluntary muscles. 20% of familial form of ALS (FALS) cases are caused by point mutations in human copper/zinc superoxide dismutase (hSOD). Many reports indicate that FALS is not caused by loss of hSOD activity, but rather by mutation- induced destabilization and formation of neurotoxic aggregates of the normally dimeric hSOD. We hypothesize the binding of mutant hSOD monomer by a redesigned, complementary hSOD variant will reduce mutant hSOD aggregation. The goal of the proposed research is to test this hypothesis with the most common FALS-associated hSOD variant containing an alanine to valine mutation in the fourth residue (hSOD_A4V). Two approaches will be taken to develop complementary binding partners for hSOD_A4V. First, computational protein design will be used to identify mutations to wild type hSOD that confer the ability to bind to hSOD_A4V. An appropriate screening method will be developed to identify hSOD mutants that stabilize monomeric hSOD_A4V expressed in mammalian cells. Second, directed evolution techniques will be used in parallel with computational approaches to expand the pool of candidate binding partners for hSOD_A4V. Considering potential gene therapeutic application, adeno-associated virus-based gene delivery vectors will be used to deliver a large number of hSOD mutant genes into mammalian cells, where the screening method developed will be applied. The most promising candidates will be subjected to more detailed characterization of biophysical and aggregation properties. These studies will identify potential candidates for gene therapeutic approaches to treating FALS associated with hSOD_A4V. More broadly, success with this approach would establish promise for treating other FALS mutants, as well as other protein aggregation diseases, including Parkinson's.
Although the molecular mechanisms are still poorly understood, familial forms of amyotrophic lateral sclerosis (FALS) involves the formation of neurotoxic aggregates of human superoxide dismutase. The proposed research will lead to designed proteins that could be tested as gene therapeutic agents in future studies.
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