A Study of Drosophila melanogaster Cytotoxic T-Lymphocyte Antigen-2-like Protein, Crammer

果蠅類第二型細胞毒性T淋巴抗原(Crammer)之特性分析

陳典君(Dian-Jiun Chen)

指導教授 ： 呂平江

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2009年)

https://doi.org/10.6843/NTHU.2009.00339

摘要

Drosophila melanogaster CTLA-2-like protein, crammer, is a small (9.5 kDa) protein with 79 amino acids. The protein sequence is homologous to the proregions of certain cysteine proteases, such as cathepsins, suggesting that it might act as a propeptide-like cysteine protease inhibitor. In addition, it has been reported to be a key factor in establishing D. melanogaster long-term memory (LTM). However, details of regulatory mechanism on crammer toward LTM remain unclear. Recent studies indicated that crammer may regulate the activity of cathepsins to control LTM formation. In this study, biochemical and biophysical methods are applied to clarify the relationship between crammer and cathepsins. Our data showed that crammer has monomeric and dimeric forms depend on distinct pH environment. Under neutral pH condition, crammer exists predominantly dimeric state, while at acidic pH, monomer is prevalent. Furthermore, our circular dichroism and NMR results revealed that the crammer at neutral pH can form a stable and well-packed structure but not for that at acidic pH. Besides, we also found that a disulfide bridge is critical for the dimerization of crammer. In vivo, crammer probably exists in monomeric form predominantly. According to our inhibitory assay, only monomer can inhibit D. melanogaster cathepsins, whereas dimer and mutant (C72S) cannot. This result indicated that the cysteine residue of crammer may play an important role in regulating cathepsins activities. Moreover, crammer can act as a strong competitive inhibitor against cathepsins, which the inhibitor constant (Ki) is 0.78 nM. Our NMR data also suggested that crammer at acidic pH can form a well-packed monomeric structure to associate with cathepsins. Finally, we proposed that crammer probably blocks the active site of cathepsin by using the staphostatin-like inhibitory mechanism.

關鍵字

半胱胺酸蛋白酶 ； 半胱胺酸蛋白酶抑制劑 ； 長期記憶

並列摘要

果蠅的類第二型細胞毒性T淋巴抗原(CTLA-2-like)，或稱Crammer，為一具有79個胺基酸的小分子蛋白質(分子量約9.5 kDa)。其蛋白質序列與某些半胱胺酸蛋白酶(Cathepsins)的前段區域(proregions)具有高度的相似性，這也意謂著Crammer可能與半胱胺酸蛋白酶的前段區域具有相似的功能，即可用來抑制此蛋白酶的活性。此外，文獻報導指出它也是建立果蠅長期記憶的重要因子之一。然而，對於Crammer調控長期記憶的詳細機制目前仍然未知。最近的研究指出，Crammer或許是藉由調控Cathepsins的活性來控制長期記憶的形成。在本研究中，我們應用生物化學以及生物物理的方法來闡明Crammer和Cathepsins之間的關係。實驗數據顯示，Crammer在不同的pH環境下具有單體和二聚體的狀態。在中性pH條件下，Crammer主要以二聚體的狀態存在，而在酸性環境下，則是以單體佔多數。另外，圓二色光譜和核磁共振光譜的結果顯示Crammer在中性pH之下可以形成穩定且摺疊好的結構，而在酸性pH之下則無法。我們也發現雙硫鍵是形成二聚體的主因。在活的生物體內，Crammer可能主要以單體的形式存在。根據活性抑制分析的結果顯示，只有單體可以抑制果蠅的Cathepsins，而二聚體和突變種(C72S)則沒有抑制的活性。此結果指出Crammer的半胱胺酸在調控Cathepsins的活性可能扮演著重要的角色。此外，Crammer對於Cathepsins是一種很強的競爭型抑制劑，其抑制常數(Ki)為0.78 nM。同時，核磁共振光譜的結果也指出在酸性pH之下，Crammer與Cathepsins結合後可形成一個折疊良好的單體結構。最後，我們提出Crammer可能藉由類似Staphostatin的抑制機制阻擋了Cathepsins的活性區域。

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