Exploiting Trans-Suppression to Arrest hSOD Aggregation in ALS

描述（由申请人提供）：肌萎缩侧索硬化症（ALS），也称为Lou Gehrig病，是一种进展迅速且总是致命的神经系统疾病，其攻击负责控制随意肌的神经细胞。20%的家族性ALS（familial form of ALS，FALS）病例是由人类铜/锌超氧化物歧化酶（human copper/zinc superoxide dismutase，hSOD）的点突变引起的。许多报道表明，FALS不是由hSOD活性的丧失引起的，而是由突变诱导的不稳定和正常二聚体hSOD的神经毒性聚集体的形成引起的。我们假设突变hSOD单体的重新设计，互补的hSOD变体的结合将减少突变hSOD聚集。所提出的研究的目标是用最常见的在第四个残基中含有丙氨酸到缬氨酸突变的与HMDS相关的hSOD变体（hSOD_A4V）来检验这一假设。将采用两种方法来开发hSOD_A4V的互补结合配偶体。首先，将使用计算蛋白质设计来鉴定赋予与hSOD_A4V结合的能力的野生型hSOD的突变。将开发适当的筛选方法以鉴定稳定在哺乳动物细胞中表达的单体hSOD_A4V的hSOD突变体。其次，定向进化技术将与计算方法并行使用，以扩大hSOD_A4V的候选结合配偶体库。考虑到潜在的基因治疗应用，基于腺相关病毒的基因递送载体将被用于将大量hSOD突变基因递送到哺乳动物细胞中，在哺乳动物细胞中将应用所开发的筛选方法。最有希望的候选人将受到更详细的生物物理和聚集特性的表征。这些研究将鉴定用于治疗与hSOD_A4V相关的FALS的基因治疗方法的潜在候选者。更广泛地说，这种方法的成功将为治疗其他FALS突变体以及其他蛋白质聚集疾病（包括帕金森氏症）奠定基础。 公共卫生相关性：虽然分子机制仍然知之甚少，但家族性肌萎缩侧索硬化症（FALS）涉及人类超氧化物歧化酶的神经毒性聚集体的形成。拟议的研究将导致设计的蛋白质，可以在未来的研究中作为基因治疗剂进行测试。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.