二硫代蘇糖醇對溶菌酶聚集行為的影響

目前已發現至少有二十種蛋白質有可能產生類澱粉沉積而造成類澱粉症。在發病的過程中，此類蛋白質會轉變為具有大量ß-sheet構形的纖維結構（fibril），進一步自我聚集成大量的糾結體（aggregate）。雖然已知這類糾結體會在細胞或組織附近沉積並導致細胞死亡，但是類澱粉蛋白誘發疾病的真正原因和機制尚未清楚。　　本研究以具有四對雙硫鍵的母雞蛋白溶菌酶為蛋白質模式系統，在含鹽類之酸性溶液環境下誘導生成類澱粉纖維，再配合二硫代蘇糖醇（DTT）破壞溶菌酶中的雙硫鍵，探討：（1）不同溫度下加入不同DTT濃度對溶菌酶類澱粉纖維形成之影響，（2）不同生長時期加入DTT對溶菌酶類澱粉纖維形成之影響，（3）不同比例之DTT和氧化態DTT濃度對溶菌酶類澱粉纖維形成之影響。我們研究結果發現，溫度會加速類澱粉纖維的生成，但不會增加生成的總量；而DTT不管在任何溫度下，破壞雙硫鍵後皆有抑制類澱粉纖維生長的效果，但如果在延遲期（lag phase）過後才加入DTT則沒有抑制效果。此外，加入氧化態DTT並不會對溶菌酶類澱粉纖維結構的生長或抑制產生任何效果。本研究不但針對溶菌酶類澱粉纖維形成機制進行探討，對於其形成過程中蛋白質結構變化也有深入的討論，相信本研究之成果應有助於近一步瞭解類澱粉症之分子機制。

關鍵字

溶菌酶二硫代蘇糖醇；類澱粉纖維；雙硫鍵；聚集體

並列摘要

At least twenty different human proteins can fold abnormally to form fibrillar protein deposits which lead to so-called amyloid disease or amyloidoses. During the progression of these disease, these amyloidogenic proteins can self-assemble into stable fibrils with extensive ß-sheet conformation. While it is widely recognized that the dysfunction and concomitant death of cells is caused by the nearby protein aggregates, the underlying mechanism of amyloid diseases remains far from clear. The research presented here is aimed at exploring the effect of DTT on the in vitro fibril formation of hen egg-white lysozymes with four disulfide bonds. In this work, we specifically addressed (a) the effect of DTT concentration under different incubation temperatures on the hen lysozyme structure and fibril formation, (b) the effect of addition of DTT at later time points on the amyloid fibrillization of hen lysozymes, and (c) the effect of concentration ratio between reduced and oxidized forms of DTT on the fibril formation of lysozymes. We first demonstrated that the rate of fibril formation of hen lysozyme increased with incubation temperature; however, the content of amyloid fibrils was not the function of incubation temperature. Next, we showed that the inhibition of lysozyme amyloid formation by DTT owing to the breakage of disulfide bonds. Also, no inhibitory effect was observed if DTT was added at 12 hours after the launch of aggregation on hen lysozyme fibrillogenesis. Finally, we found that the oxidized form of DTT did not exhibit any inhibitory potency against amyloid fibril formation. The current research not only investigates the mechanism of amyloid protein self-assembly, but also examines the structures of lysozyme species during the course of aggregation process. We believe the outcome from this work will certainly further our understanding of the molecular mechanism(s) of amyloid diseases.

並列關鍵字

lysozyme ； DTT ； amyloid fibril ； disulfide bonds ； aggregate

參考文獻

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二硫代蘇糖醇對溶菌酶聚集行為的影響

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