果蠅 Crammer 蛋白之結構功能與特定胺基酸註解分析

果蠅類第二型細胞毒性T淋巴抗原(CTLA-2-like)，又名Crammer，為一79個胺基酸蛋白質(分子量約9.5 kDa)。其蛋白質序列與某些半胱胺酸蛋白脢(Cathepsins)的前段序列(proregions)有高度相似性，意謂Crammer聚有與半胱胺酸蛋白脢的前段序列相似功能(抑制蛋白脢活性)。相關文獻指出Crammer是建立果蠅長期記憶的主要因子之一，可能是藉由其調控Cathepsins的活性來控制長期記憶的形成，但詳細的機制仍然未知。在本研究中，藉由利用生物化學以及生物物理的方法來探討Crammer和Cathepsins之間的相互關係，更利用特定點突變的方法研究結構上特定胺基酸對於Crammer抑制半胱胺酸蛋白脢的重要性、結構穩定性、以及對於整個Crammer蛋白質結構折疊上的影響。實驗結果顯示，Crammer在不同的pH環境下聚有單體與二聚體的狀態。在中性與偏鹼性pH下，Crammer以一對分子間雙硫鍵所構成的二聚體存在 然而，在酸性環境下，則是以單體的狀態存在。活性抑制分析的結果顯示，單體Crammer能抑制果蠅的Cathepsins，而二聚體則無抑制的活性。此外，Crammer在酸性的環境下是以熔球體(molten globule)的狀態存在，此環境類似於Crammer可能所處的溶脢體 (lysosome)的生理環境。然而，當與Cathepsin L結合後，Crammer的蛋白質構型會從熔球體轉變成一個穩定且摺疊完整的單體結構。在核磁共振光譜結果指出，突變種C72S於pH 6.0下的HSQC圖譜高度相似於與Cathepsin L形成複合體Crammer的HSQC圖譜。因此，我們解出C72S的結構，也是第一個類前段序列蛋白脢抑制劑(propeptide-like protease inhibitor)的蛋白質結構。另外，特定點突變研究結果顯示具，將疏水核心區(hydrophobic cores)的芳香族胺基酸(W9, Y12, F16, Y20, Y32, W53)以及構成支鍊鹽橋(salt-bridges)的帶電性胺基酸 (E8, R28, R29, E67)以丙胺酸取代後，會大幅降低Crammer抑制果蠅 Cathepsin B的活性。同時，圓二色光譜、自身螢光分析(intrinsic fluorescence)與核磁共振的結果指出，移除芳香環以及帶電胺基酸的側鏈，會明顯影響pH值依賴性螺旋的形成(pH-dependent helix formation)、降低熱穩定性並且破壞Crammer結構的摺疊(molten globule-to-ordered structure transition)。此外，我們也發現W53在Crammer與Cathepsin B結合的交互作用上扮演了重要的角色，及R28與E67所形成的鹽橋對於 螺旋確切地結合在Cathepsin B活化位有重要的貢獻。

關鍵字

果蠅類第二型細胞毒性T淋巴抗原；半胱胺酸蛋白脢；長期記憶；蛋白質結構折疊；特定點突變；二聚體；支鍊鹽橋

並列摘要

Drosophila melanogaster crammer is a novel cathepsin inhibitor that is involved in long-term memory (LTM) formation. The mechanism by which the inhibitory activity is regulated remains unclear. Here we have shown that at neutral pH, crammer is predominantly dimeric in vitro as a result of disulfide bond formation, and is monomeric at acidic pH. Our inhibition assay shows that monomeric crammer is a strong competitive inhibitor of cathepsin L. Crammer is a monomeric molten globule in acidic solution, upon binding to cathepsin L; however, crammer undergoes a molten globule-to-ordered structural transition. Using high-resolution NMR spectroscopy, we have shown that the C72S variant renders crammer monomeric at pH 6.0 and that the structure of the C72S variant highly resembles that of wild-type crammer in complex with cathepsin L at pH 4.0. We have determined the first solution structure of a propeptide-like protease inhibitor in its active form and examined in detail using a variety of spectroscopic methods the folding properties of crammer in order to delineate its biomolecular recognition of cathepsin. In addition, alanine substitution for the aromatic residues W9, Y12, F16, Y20, Y32, and W53 within the hydrophobic cores, and charged residues E8, R28, R29, and E67 in the salt bridges considerably decrease the ability of crammer to inhibit Drosophila cathepsin B (CTSB). Far-UV circular dichroism (CD), intrinsic fluorescence and nuclear magnetic resonance (NMR) spectroscopies show that the removal of most the aromatic and charged side-chains substantially reduce the thermostability, alter pH-dependent helix formation, and disrupt the molten globule-to-ordered structure transition. Molecular modeling indicates that W53 is essential for the interaction between crammer and CTSB; the salt bridge R28-E67 is critical for the appropriate alignment of the -helix 4 towards the CTSB active cleft. Alanine scanning provides detailed residue-specific dissection of folding transition and functional contributions of the hydrophobic cores and salt bridges of crammer, and these insights could serve as a template for further development of therapeutic inhibitors against cathepsins.

並列關鍵字

Crammer ； Cathepsin ； Long-term memory ； Propeptide-like protease inhibitor ； Molten globule ； Alanine scanning ； Hydrophobic core ； Human cathepsin ； Molten globule-to-ordered structure transition of crammer propeptide-like cysteine ； Protease inhibitor ； Prosegment binding loop (PBL)

參考文獻

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國際替代計量

果蠅 Crammer 蛋白之結構功能與特定胺基酸註解分析

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