PDF(Pubmed)
Abstract：
Exposure to solar UV irradiation damages γ-crystallin, leading to cataract formation via aggregation. α-Crystallin, as a small heat shock protein, efficiently suppresses this irreversible aggregation by selectively binding the denatured γ-crystallin monomer. In this study, liquid chromatography tandem mass spectrometry was used to evaluate UV-325 nm irradiation-induced photodamage of human γD-crystallin in the presence of bovine α-crystallin, atomic force microscope (AFM) and dynamic light scattering (DLS) techniques were used to detect the quaternary structure changes of the α-crystallin oligomer, and Fourier transform infrared spectroscopy and temperature-jump nanosecond time-resolved IR absorbance difference spectroscopy were used to probe the secondary structure changes of bovine α-crystallin. We find that the thermal-induced subunit dissociation of the α-crystallin oligomer involves the breaking of hydrogen bonds at the dimeric interface, leading to three different spectral components at varied temperature regions as resolved from temperature-dependent IR spectra. Under UV-325 nm irradiation, unfolded γD-crystallin binds to the dissociated α-crystallin subunit to form an αγ-complex, then follows the reassociation of the αγ-complex to the partially dissociated α-crystallin oligomer. This prevents the aggregation of denatured γD-crystallin. The formation of the γD-bound α-crystallin oligomer is further confirmed by AFM and DLS analysis, which reveals an obvious size expansion in the reassociated αγ-oligomers. In addition, UV-325 nm irradiation causes a peptide bond cleavage of γD-crystallin at Ala158 in the presence of α-crystallin. Our results suggest a very effective protection mechanism for subunits dissociated from α-crystallin oligomers against UV irradiation-induced aggregation of γD-crystallin, at the expense of a loss of a short C-terminal peptide in γD-crystallin.
摘要：
暴露于太阳紫外线照射会损害γ-晶状体蛋白，通过聚集导致白内障形成。α-晶体蛋白,作为一种小的热休克蛋白，通过选择性结合变性的γ-晶状体蛋白单体有效地抑制这种不可逆的聚集。在这项研究中,液相色谱串联质谱法用于评估在牛α-晶状体蛋白存在下UV-325nm辐照诱导的人γD-晶状体蛋白的光损伤，原子力显微镜(AFM)和动态光散射(DLS)技术用于检测α-晶状体蛋白低聚物的四元结构变化,利用傅里叶变换红外光谱和温度跃迁纳秒时间分辨红外吸收差分光谱来探测牛α-晶状体蛋白的二级结构变化。我们发现，α-晶状蛋白低聚物的热诱导亚基解离涉及二聚体界面处氢键的断裂，导致三个不同的光谱成分在不同的温度区域，从温度相关的红外光谱解析。在UV-325nm照射下，未折叠的γD-晶状体蛋白与解离的α-晶状体蛋白亚基结合形成αγ复合物，然后将αγ-复合物重新结合到部分解离的α-晶状体蛋白低聚物上。这防止了变性的γD-晶状体蛋白的聚集。通过AFM和DLS分析进一步证实了γD结合的α-晶状体蛋白低聚物的形成。这揭示了重新缔合的αγ-低聚物的明显尺寸扩展。此外,在存在α-晶状体蛋白的情况下，UV-325nm照射会导致γD-晶状体蛋白在Ala158处的肽键断裂。我们的结果表明，从α-晶状蛋白低聚物中解离的亚基具有非常有效的保护机制，可以抵抗紫外线引起的γD-晶状蛋白聚集，以损失γD-晶状体蛋白中的短C末端肽为代价。
