Saleh Aliabbas Ahmedbhai, Bhadra Ankan Kumar, Roy Ipsita
Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER) , Sector 67, S.A.S. Nagar, Punjab 160 062, India.
ACS Chem Neurosci. 2014 Mar 19;5(3):205-15. doi: 10.1021/cn400171d. Epub 2014 Jan 8.
Conflicting reports exist in the literature regarding the role of wild-type huntingtin in determining the toxicity of the aggregated, mutant huntingtin in Huntington's disease (HD). Some studies report the amelioration of toxicity of the mutant protein in the presence of the wild-type protein, while others indicate sequestration of the wild-type protein by mutant huntingtin. Over the years, yeast has been established as a valid model organism to study molecular changes associated with HD, especially at the protein level. We have used an inducible system to express human huntingtin fragments harboring normal (25Q) and pathogenic (103Q) polyglutamine lengths under the control of a galactose promoter in a yeast model of HD. We show that the relative expression level of each allele (wild-type/mutant) decides the cellular phenotype. When the expression level of wild-type huntingtin is high, an increase in the solubility of the mutant protein is observed. Fluorescence-recovery-after-photobleaching (FRAP) studies show that solubility reaches ∼94% in these cells. This leads to reduction in oxidative stress and cytotoxicity, and increases cell viability. In-cell FRET studies show that interaction between these proteins does not require the presence of a mediator. When the expression of wild-type huntingtin is low, it is sequestered into aggregates by the mutant protein. Even under these conditions, cytotoxicity is attenuated. Our findings indicate that the presence of wild-type huntingtin has a beneficial role even when its relative expression level is lower than that of the mutant protein.
关于野生型亨廷顿蛋白在亨廷顿舞蹈病（HD）中决定聚集的突变型亨廷顿蛋白毒性方面所起的作用，文献中有相互矛盾的报道。一些研究报告称，在野生型蛋白存在的情况下，突变蛋白的毒性会减轻，而另一些研究则表明突变型亨廷顿蛋白会隔离野生型蛋白。多年来，酵母已被确立为研究与HD相关分子变化的有效模式生物，尤其是在蛋白质水平上。我们使用了一种诱导系统，在HD酵母模型中，在半乳糖启动子的控制下表达具有正常（25Q）和致病（103Q）多聚谷氨酰胺长度的人类亨廷顿蛋白片段。我们发现，每个等位基因（野生型/突变型）的相对表达水平决定了细胞表型。当野生型亨廷顿蛋白的表达水平较高时，会观察到突变蛋白的溶解度增加。光漂白后荧光恢复（FRAP）研究表明，在这些细胞中溶解度达到约94%。这导致氧化应激和细胞毒性降低，并提高细胞活力。细胞内荧光共振能量转移（FRET）研究表明，这些蛋白质之间的相互作用不需要介质的存在。当野生型亨廷顿蛋白的表达水平较低时，它会被突变蛋白隔离到聚集体中。即使在这些条件下，细胞毒性也会减弱。我们的研究结果表明，即使野生型亨廷顿蛋白的相对表达水平低于突变蛋白，其存在也具有有益作用。
