Verani Margherita, Bustamante Maria, Martufi Paola, Daldin Manuel, Cariulo Cristina, Azzollini Lucia, Fodale Valentina, Puglisi Francesca, Weiss Andreas, Macdonald Douglas, Petricca Lara, Caricasole Andrea
IRBM Promidis, Via Pontina km 30.600, 00071, Pomezia, Rome, Italy; IRBM Science Park, Via Pontina km 30.600, 00071, Pomezia, Rome, Italy.
IRBM Science Park, Via Pontina km 30.600, 00071, Pomezia, Rome, Italy.
Biochem Biophys Res Commun. 2016 Sep 16;478(2):949-55. doi: 10.1016/j.bbrc.2016.08.057. Epub 2016 Aug 9.
We have previously reported TR-FRET based immunoassays to detect a conformational change imparted on huntingtin protein by the polyglutamine expansion, which we confirmed using biophysical methodologies. Using these immunoassays, we now report that polyglutamine expansion influences the conformational properties of other polyglutamine disease proteins, exemplified by the androgen receptor (associated with spinal bulbar muscular atrophy) and TATA binding protein (associated with spinocerebellar ataxia 17). Using artificial constructs bearing short or long polyglutamine expansions or a multimerized, unrelated epitope (mimicking the increase in anti-polyglutamine antibody epitopes present in polyglutamine repeats of increasing length) we confirmed that the conformational TR-FRET based immunoassay detects an intrinsic conformational property of polyglutamine repeats. The TR-FRET based conformational immunoassay may represent a rapid, scalable tool to identify modulators of polyglutamine-mediated conformational change in different proteins associated with CAG triplet repeat disorders.
我们之前曾报道过基于时间分辨荧光共振能量转移（TR-FRET）的免疫测定法，用于检测聚谷氨酰胺扩增赋予亨廷顿蛋白的构象变化，我们使用生物物理方法对此进行了证实。现在，利用这些免疫测定法，我们报告聚谷氨酰胺扩增会影响其他聚谷氨酰胺疾病蛋白的构象特性，以雄激素受体（与延髓脊髓性肌萎缩相关）和TATA结合蛋白（与脊髓小脑共济失调17型相关）为例。通过使用带有短或长聚谷氨酰胺扩增序列的人工构建体或多聚化的无关表位（模拟长度增加的聚谷氨酰胺重复序列中抗聚谷氨酰胺抗体表位的增加），我们证实基于TR-FRET的构象免疫测定法检测到了聚谷氨酰胺重复序列的内在构象特性。基于TR-FRET的构象免疫测定法可能是一种快速、可扩展的工具，用于识别与CAG三联体重复序列紊乱相关的不同蛋白质中聚谷氨酰胺介导的构象变化的调节剂。
