Liang Jack J-N
Center for Ophthalmic Research, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, MA 02115, USA.
Protein Sci. 2004 Sep;13(9):2476-82. doi: 10.1110/ps.04815104.
T5P gammaC-crystallin mutation is associated with Coppock-like cataract, one of the autosomal dominant congenital cataracts. It is not known why the abundant alpha-crystallin cannot prevent the mutation-related aggregation. Our previous studies indicate that the mutation changes conformation and reduces solubility and stability, but it is not known whether it is these events or the loss of interaction with other crystallins that causes the cataract. It is also not known whether the alpha-crystallin can protect T5P mutant as effectively from heat-induced aggregation as the wild-type (WT) gammaC-crystallin. To investigate the mechanism of interactions and chaperone function between alphaA- and gammaC-crystallin, human alphaA-crystallin and W9F mutant as well as WT gammaC-crystallin and T5P mutant were cloned. Interactions between alphaA- and gammaC-crystallin were studied with fluorescence resonance energy transfer (FRET), and chaperone activity was assessed by the suppression of heat-induced aggregation of substrate proteins. Conformational changes of substrate proteins were studied by spectroscopic measurements. The results indicate that the T5P mutant showed a slightly greater FRET than WT gammaC-crystallin with alphaA-crystallin, and alphaA-crystallin could effectively prevent both WT and T5P gammaC-crystallin from heat-induced aggregation. Spectroscopic measurements show that both alphaA-crystallin and gammaC-crystallin underwent only slight conformational change after chaperone binding. Together with previous results obtained with a two-hybrid system assay of interactions between alphaA- and gammaC-crystallin, the present FRET and chaperone results indicate that loss of interactions of T5P mutant with other crystallins may play a larger role than the protection afforded by chaperone-like activity in Coppock-like cataract.
T5P γC-晶体蛋白突变与常染色体显性先天性白内障之一的科波克样白内障相关。目前尚不清楚为何大量的α-晶体蛋白无法阻止与突变相关的聚集。我们之前的研究表明，该突变会改变构象，降低溶解度和稳定性，但尚不清楚是这些事件还是与其他晶体蛋白相互作用的丧失导致了白内障。也不清楚α-晶体蛋白是否能像野生型（WT）γC-晶体蛋白一样有效地保护T5P突变体免受热诱导的聚集。为了研究αA-和γC-晶体蛋白之间的相互作用机制和伴侣功能，克隆了人αA-晶体蛋白和W9F突变体以及WT γC-晶体蛋白和T5P突变体。利用荧光共振能量转移（FRET）研究了αA-和γC-晶体蛋白之间的相互作用，并通过抑制底物蛋白的热诱导聚集来评估伴侣活性。通过光谱测量研究了底物蛋白的构象变化。结果表明，T5P突变体与αA-晶体蛋白的FRET略高于WT γC-晶体蛋白，并且αA-晶体蛋白可以有效地阻止WT和T5P γC-晶体蛋白受热诱导的聚集。光谱测量表明，伴侣结合后，αA-晶体蛋白和γC-晶体蛋白仅发生轻微的构象变化。结合之前通过αA-和γC-晶体蛋白之间相互作用的双杂交系统分析获得的结果，目前的FRET和伴侣结果表明，T5P突变体与其他晶体蛋白相互作用的丧失在科波克样白内障中可能比伴侣样活性提供的保护起更大作用。
