Table_2_An Expanded Polyproline Domain Maintains Mutant Huntingtin Soluble in vivo and During Aging.XLSX

Huntington’s disease is a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat, encoding for the amino acid glutamine (Q), present in the first exon of the protein huntingtin. Over the threshold of Q39 HTT exon 1 (HTTEx1) tends to misfold and aggregate into large intracellular structures, but whether these end-stage aggregates or their on-pathway intermediates are responsible for cytotoxicity is still debated. HTTEx1 can be separated into three domains: an N-terminal 17 amino acid region, the polyglutamine (polyQ) expansion and a C-terminal proline rich domain (PRD). Alongside the expanded polyQ, these flanking domains influence the aggregation propensity of HTTEx1: with the N17 initiating and promoting aggregation, and the PRD modulating it. In this study we focus on the first 11 amino acids of the PRD, a stretch of pure prolines, which are an evolutionary recent addition to the expanding polyQ region. We hypothesize that this proline region is expanding alongside the polyQ to counteract its ability to misfold and cause toxicity, and that expanding this proline region would be overall beneficial. We generated HTTEx1 mutants lacking both flanking domains singularly, missing the first 11 prolines of the PRD, or with this stretch of prolines expanded. We then followed their aggregation landscape in vitro with a battery of biochemical assays, and in vivo in novel models of C. elegans expressing the HTTEx1 mutants pan-neuronally. Employing fluorescence lifetime imaging we could observe the aggregation propensity of all HTTEx1 mutants during aging and correlate this with toxicity via various phenotypic assays. We found that the presence of an expanded proline stretch is beneficial in maintaining HTTEx1 soluble over time, regardless of polyQ length. However, the expanded prolines were only advantageous in promoting the survival and fitness of an organism carrying a pathogenic stretch of Q48 but were extremely deleterious to the nematode expressing a physiological stretch of Q23. Our results reveal the unique importance of the prolines which have and still are evolving alongside expanding glutamines to promote the function of HTTEx1 and avoid pathology.

亨廷顿病（Huntington’s disease）是一种显性遗传性神经退行性疾病，由亨廷顿蛋白（huntingtin）首个外显子中编码谷氨酰胺（Q）的CAG重复序列（CAG repeat）扩增所引发。当谷氨酰胺残基数超过39（Q39）时，HTT外显子1（HTTEx1）易于错误折叠并聚集形成大型细胞内结构，但目前仍存在争议的是，究竟是这种终末期聚集体，还是其聚集通路上的中间产物介导了细胞毒性（cytotoxicity）。HTTEx1可分为三个结构域：N端17氨基酸区域、多聚谷氨酰胺（polyglutamine, polyQ）扩增区域，以及羧基端富脯氨酸结构域（proline rich domain, PRD）。除扩增的polyQ之外，这些侧翼结构域同样会影响HTTEx1的聚集倾向：其中N17结构域可启动并促进聚集，而PRD则对聚集过程起到调控作用。本研究聚焦于PRD的前11个氨基酸——一段纯脯氨酸序列，该序列是伴随polyQ扩增在进化中新近获得的结构。我们提出假说：这段脯氨酸区域与polyQ一同扩增，以对抗其错误折叠并引发毒性的能力，且扩增该脯氨酸区域总体上是有益的。我们构建了一系列HTTEx1突变体：分别缺失两个侧翼结构域、缺失PRD的前11个脯氨酸残基，或是扩增该脯氨酸序列。随后我们通过一系列生化实验体外追踪其聚集动态，并在全神经元表达HTTEx1突变体的新型秀丽隐杆线虫（C. elegans）模型中开展体内研究。借助荧光寿命成像技术，我们能够观测到所有HTTEx1突变体在衰老过程中的聚集倾向，并通过多种表型检测实验将其与毒性相关联。研究发现，无论polyQ长度如何，扩增的脯氨酸序列均有助于长期维持HTTEx1的可溶性状态。然而，扩增的脯氨酸序列仅在携带致病性Q48序列的生物体中，能够促进其存活与进化适合度；但对于表达生理性Q23序列的秀丽隐杆线虫而言，该扩增的脯氨酸序列却具有极强的有害性。本研究结果揭示了与谷氨酰胺扩增共同演化的脯氨酸残基的独特重要性：它们可促进HTTEx1的正常功能，避免病理发生。