發展脂雙層膜陣列為統計工具以研究如何調控磷脂酶─磷脂質複合物之生成時間

蛋白質的功能可以藉由蛋白質與脂質之結合形成複合物而產生重大的變化。於本論文中我們研究一種外周膜酵素，神經磷脂酶，可與其脂質受質，鞘磷脂，及其脂質產物，神經醯胺形成一功能性複合物。神經磷脂酶水解活性在形成此功能性複合物後大幅提升。我們更進一步調控此功能性複合物之生成時間並發現其生成時間之延遲受到脂質膜成分的影響。我們假設此一生成時間之延遲源自於複合物自平面脂質膜中成核聚集時所需跨過的成核能障。為了研究具有隨機性質的複合物成核現象，我們建構了擁有大量各自獨立的分隔脂質膜的微流道平台以收集複合物生成時間的資料。利用適當的脂質膜組成以避免神經磷脂酶水解作用後產生複雜的膜相分離現象及利用分隔脂質膜微流道平台收集大量資料以進行統計分析，我們發現神經磷脂酶─磷脂質複合物的生成時間可以藉由在流動相脂質膜中的酵素、受質及產物的過飽和度來調控；並且此一調控關係可藉由古典成核理論來描述。藉由比對實驗結果及理論公式，我們提出於複合物中可能的受質：產物比為9：1；更進一步指出產物微量存在於系統中具有調控酵素活性的重要性。本研究的重點在於提出成核現象可以作為調控酵素表現活性的時間延遲的機制之ㄧ。藉由控制成核能障的大小我們可以進而控制影響酵素活性的成核現象發生的速度。我們的實驗設計及分析方法也可以延伸應用到其他具有功能性聚集的蛋白質，例如澱粉樣蛋白纖維聚集。

關鍵字

鞘磷脂；神經磷脂酶磷脂膜；微流體

並列摘要

Aggregation or assembly of lipids and proteins to form complexes could significantly change the proteins’ function. Here we report that a peripheral membrane enzyme, sphingomyelinase (SMase), can form a functional complex with its lipid substrate, sphingomyelin (SM), and its lipid product, ceramide (Cer), and has a tunable formation time. The enzyme’s hydrolysis rate is found to significantly increase after the complex is formed. The peculiar behavior is that the complex formation has a time lag depending on the membrane composition. We hypothesized that the time lag is due to the significant nucleation energy barrier when the complex phase forms in its metastable parent phase in the 2-D lipid membrane. To study the stochastic nucleation of the complex, we built a corralled lipid membrane platform with numerous isolated membrane systems in parallel to capture the nucleation statistics. Using the high-throughput approach and the appropriate experimental design to circumvent the interplay of the complicated phase segregation in membranes induced by SMase, we found that the nucleation rate of the complex can be tuned by the supersaturation of the enzyme, the lipid substrate and the lipid product, in the fluid phase of the membrane. The correlation between the supersaturation and the nucleation rate can be well described by the classical nucleation theory. Comparing the experimental result with the theory further suggests an approximate 9: 1 molar ratio of SM and Cer in the SMase-lipid complex, indicating that small amount of Cer is important for regulating the enzyme’s behavior. Our finding suggests that nucleation could serve as a time lag control mechanism in this enzymatic system, while many of the time lags in biology are controlled by diffusion time or reaction kinetic delay. Ways to reduce nucleation energy barrier could be used to shorten the aggregation time lag and vice versa. Our methodology and insights could also extend to studies on other membrane associated functional protein aggregation, such as amyloid fiber formation.

並列關鍵字

Sphingomyelin ； sphingomyelinase ； supported lipid bilayers ； microfluidics

參考文獻

1. Simons, K. and E. Ikonen, Functional rafts in cell membranes. Nature, 1997. 387(6633): p. 569-572.

2. Jacobson, K., O.G. Mouritsen, and R.G. Anderson, Lipid rafts: at a crossroad between cell biology and physics. Nature cell biology, 2007. 9(1): p. 7-14.

3. Simons, K. and D. Toomre, Lipid rafts and signal transduction. Nature reviews Molecular cell biology, 2000. 1(1): p. 31-39.

4. Contreras, F.-X., et al., Specificity of intramembrane protein–lipid interactions. Cold Spring Harbor perspectives in biology, 2011. 3(6).

5. van der Linden, E. and P. Venema, Self-assembly and aggregation of proteins. Current Opinion in Colloid & Interface Science, 2007. 12(4): p. 158-165.

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發展脂雙層膜陣列為統計工具以研究如何調控磷脂酶─磷脂質複合物之生成時間

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