PROGRESSIVE AGGREGATION DESPITE CHAPERONE ASSOCIATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 最近的研究表明，超氧化物歧化酶1（SOD 1）连接的肌萎缩侧索硬化症， 硬化症是由这种丰富的蛋白质的突变形式的不稳定和错误折叠引起的。 胞质酶在这里，我们追踪了一个有错误折叠倾向的人的表达和命运。 在转基因G85 R SOD 1-YFP小鼠系中，人SOD 1，G85 R与YFP融合。这些老鼠 而不是野生型人SOD 1-YFP转基因，开发致命的麻痹电机 9个月的症状脊髓原位RNA杂交显示， 在所有转基因动物的脊髓灰质中的运动神经元中表达。 与此一致，G85 R SOD-YFP在1和10 ℃时在动物的运动神经元中呈弥漫性荧光。 6月龄，但在出现症状时，在细胞中观察到点状聚集物， 机构和进程。脊髓可溶性提取物的生化分析表明， G85 R SOD-YFP在所有年龄表现为错误折叠的单体。它逐渐变得 在6和9月龄时不溶，与可观察到的可溶性低聚物的存在相关 通过凝胶过滤。免疫亲和捕获和质谱分析显示， G85 R SOD-YFP，而不是WT SOD-YFP，在所有年龄具有细胞溶质伴侣Hsc 70。在 此外，在6和9处捕获了3个Hsp 110，Hsp 70的核苷酸交换因子 个月尽管有这样的伴侣相互作用，G85 R SOD-YFP在100 ℃时形成不溶性包涵体。 晚期，主要含有中间丝蛋白。我们的结论是 运动神经元，最初“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。

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.