Exploiting Trans-Suppression to Arrest hSOD Aggregation in ALS

利用反式抑制来阻止 ALS 中的 hSOD 聚集

• 批准号: 7876353
• 负责人: Inchan Kwon
• 金额: $ 21.58万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7876353
• 关键词: Affinity; Alanine; Amyloidosis; Amyotrophic Lateral Sclerosis; Behavior; Binding; Candidate Disease Gene; Cells; Cytosol; Dependovirus; Development; Disease; Fluorescence; Future; Gene Delivery; Genes; Goals; Human; Induced Mutation; Lead; Libraries; Mammalian Cell; Methods; Molecular; Monitor; Mutation; Neurons; Parkinson Disease; Point Mutation; Prealbumin; Process; Property; Proteins; Reporting; Research; Screening procedure; Skeletal Muscle; Superoxide Dismutase; Surface; Techniques; Technology; Testing; Therapeutic; Therapeutic Agents; Valine; Variant; base; copper zinc superoxide dismutase; design; dimer; directed evolution; high throughput screening; monomer; mutant; nervous system disorder; neurotoxic; programs; protein aggregation; public health relevance; self assembly; success; therapeutic gene; vector

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.

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS），也称为Lou Gehrig's病，是一种快速发展且总是致命的神经系统疾病，攻击负责控制随意肌的神经细胞。20%的家族性ALS （FALS）病例是由人铜/锌超氧化物歧化酶（hSOD）点突变引起的。许多报道表明，FALS不是由hSOD活性丧失引起的，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。我们假设通过重新设计的互补hSOD变体结合突变hSOD单体将减少突变hSOD的聚集。这项研究的目的是用最常见的fals相关的hSOD变异来验证这一假设，该变异在第四个残基（hSOD_A4V）中含有丙氨酸到缬氨酸的突变。将采用两种方法开发hSOD_A4V的互补结合伙伴。首先，计算蛋白设计将用于鉴定野生型hSOD的突变，这些突变赋予了与hSOD_A4V结合的能力。我们将开发一种合适的筛选方法来鉴定能够稳定哺乳动物细胞中单体hSOD_A4V表达的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的候选结合伙伴池。考虑到潜在的基因治疗应用，基于腺相关病毒的基因传递载体将被用于将大量hSOD突变基因传递到哺乳动物细胞中，并将应用所开发的筛选方法。最有希望的候选者将受到更详细的生物物理和聚集特性的表征。这些研究将确定潜在的候选基因治疗方法来治疗与hSOD_A4V相关的FALS。更广泛地说，这种方法的成功将为治疗其他FALS突变体以及包括帕金森氏症在内的其他蛋白质聚集性疾病带来希望。