PROGRESSIVE AGGREGATION DESPITE CHAPERONE ASSOCIATION

尽管有陪伴协会，但仍不断聚合

• 批准号: 8171476
• 负责人: ARTHUR L HORWICH
• 金额: $ 0.24万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171476
• 关键词: Age; Age-Months; Amyotrophic Lateral Sclerosis; Animals; Biochemical; Computer Retrieval of Information on Scientific Projects Database; Enzymes; Funding; Gel Chromatography; Grant; Guanine Nucleotide Exchange Factors; Human; In Situ; Institution; Intermediate Filament Proteins; Link; Mass Spectrum Analysis; Molecular Chaperones; Motor; Motor Neurons; Mus; Nucleotides; Paralysed; Process; RNA; Research; Research Personnel; Resources; Source; Spinal Cord; Spinal cord grey matter structure; Symptoms; Time; Transgenic Animals; Transgenic Organisms; United States National Institutes of Health; monomer; mutant; neuronal cell body; protein misfolding; superoxide dismutase 1

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially "compensated" to maintain the misfolded protein in a soluble state, become progressively unable to do so.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 最近的研究表明，超氧化物歧化酶1(SOD1)与肌萎缩侧索硬化相关 硬化症是由于这种丰富的突变形式的不稳定和错误折叠造成的 胞浆酶。在这里，我们追踪了一个容易出错的人的表情和命运 在转基因G85R SOD1-YFP小鼠中，将人SOD1、G85R与YFP融合。这些老鼠， 但不是野生型人类SOD1-YFP转基因人，发展出致命的瘫痪运动 9个月时出现症状。脊髓的原位RNA杂交显示优势 所有转基因动物脊髓灰质运动神经元的表达。 一致地，G85R SOD-YFP在动物的运动神经元中呈弥漫性荧光。 6个月龄，但在出现症状时，细胞内可见点状聚集体 机构和流程。脊髓可溶性提取物的生化分析表明 G85R SOD-YFP在所有年龄段都表现为错误折叠的单体。它逐渐变得 在6个月和9个月龄时不溶，与可观察到的可溶低聚物的存在有关 通过凝胶过滤。免疫亲和捕获和质谱分析揭示了 G85R SOD-YFP，而不是WT SOD-YFP，与胞浆伴侣Hsc70在所有年龄段均存在。在……里面 此外，在6号和9号位置捕获了3个Hsp110‘S，它们是Hsp70的核苷酸交换因子 月份。尽管存在这种伴侣相互作用，G85R SOD-YFP在 晚期，主要含有中间丝蛋白。我们的结论是 运动神经元最初被“补偿”以维持错误折叠的蛋白质处于可溶状态， 逐渐变得无法做到这一点。