Ray Nicholas J, Hall Damien, Carver John A
Research School of Chemistry, The Australian National University, Acton, ACT 2601, Australia.
Research School of Chemistry, The Australian National University, Acton, ACT 2601, Australia.
Biochim Biophys Acta. 2016 Jan;1860(1 Pt B):315-24. doi: 10.1016/j.bbagen.2015.08.015. Epub 2015 Aug 28.
Cataract formation is often attributed to the build-up of post-translational modifications in the crystallin proteins of the eye lens. One such modification, the deamidation of N76 in human γS-crystallin to D76, is highly correlated with age-related cataract (Hooi et al. Invest. Ophthalmol. Vis. Sci. 53 (2012) 3554-3561). In the current work, this modification has been extensively characterised in vitro.
Biophysical characterisation was performed on wild type and N76D γS-crystallins using turbidity measurements to monitor aggregation, intrinsic fluorescence and circular dichroism spectroscopy to determine the folded state and NMR spectroscopy for identifying local changes in structure. Protein mass was determined using SEC-MALLS and analytical ultracentrifugation methods.
Relative to the wild type protein, deamidation at N76 in γS-crystallin causes an increase in the thermal stability and resistance to thermally induced aggregation alongside a decrease in stability to denaturants, a propensity to aggregate rapidly once destabilised and a tendency to form a dimer. We ascribe the apparent increase in thermal stability upon deamidation to the formation of dimer which prevents the unfolding of the inherently less stable monomer.
Deamidation causes a decrease in stability of γS-crystallin but this is offset by an increased tendency for dimer formation.
Deamidation at N76 in human γS-crystallin likely has a combinatorial effect with other post-translational crystallin modifications to induce age-related cataract. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
白内障的形成通常归因于眼晶状体中晶状体蛋白翻译后修饰的积累。其中一种修饰，即人γS-晶状体蛋白中N76脱酰胺化为D76，与年龄相关性白内障高度相关（Hooi等人，《Invest. Ophthalmol. Vis. Sci.》53（2012）3554 - 3561）。在当前的工作中，这种修饰已在体外进行了广泛的表征。
对野生型和N76D γS-晶状体蛋白进行生物物理表征，使用浊度测量来监测聚集情况，使用内源荧光和圆二色光谱来确定折叠状态，使用核磁共振光谱来识别结构的局部变化。使用尺寸排阻色谱-多角度激光光散射（SEC-MALLS）和分析超速离心方法测定蛋白质质量。
相对于野生型蛋白，γS-晶状体蛋白中N76的脱酰胺化导致热稳定性增加以及对热诱导聚集的抵抗力增强，同时对变性剂的稳定性降低，一旦不稳定就倾向于快速聚集并形成二聚体。我们将脱酰胺化后热稳定性的明显增加归因于二聚体的形成，这阻止了本质上较不稳定的单体的展开。
脱酰胺化导致γS-晶状体蛋白稳定性降低，但这被二聚体形成倾向的增加所抵消。
人γS-晶状体蛋白中N76的脱酰胺化可能与其他晶状体蛋白翻译后修饰具有协同作用，从而诱发年龄相关性白内障。本文是名为“健康与疾病中的晶状体生物化学”的特刊的一部分。
