PROGRESSIVE AGGREGATION DESPITE CHAPERONE ASSOCIATION

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 最近的研究表明超氧化物歧化酶 1 (SOD1) 相关的肌萎缩侧索硬化 硬化是由于这种丰富的突变形式的不稳定和错误折叠造成的 胞质酶。在这里，我们追踪了一个容易折叠错误的人的表情和命运 人类 SOD1，G85R，与 YFP 融合，存在于转基因 G85R SOD1-YFP 小鼠系中。这些老鼠， 但不是野生型人类 SOD1-YFP 转基因，开发出致命的麻痹运动 9个月时出现症状。脊髓原位 RNA 杂交显示主要 在所有转基因动物的脊髓灰质运动神经元中表达。 一致地，G85R SOD-YFP 在 1 和 1 时在动物运动神经元中发出弥漫性荧光。 6个月大，但在出现症状时，细胞内观察到点状聚集物 机构和过程。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。逐渐变成了 在 6 个月和 9 个月大时不溶，与可观察到的可溶性低聚物的存在有关 通过凝胶过滤。免疫亲和捕获和质谱分析揭示了 G85R SOD-YFP，但不是 WT SOD-YFP，在所有年龄段均具有胞质伴侣 Hsc70。在 此外，在 6 和 9 处捕获了 3 个 Hsp110（Hsp70 的核苷酸交换因子） 几个月。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“补偿”以维持错误折叠的蛋白质处于可溶状态， 逐渐无法做到这一点。

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.