近日來自美國德克薩斯大學(xué)加爾維斯頓醫(yī)學(xué)分部及加州大學(xué)圣地亞哥分校醫(yī)學(xué)院的研究人員,，在新研究中解析了一個(gè)在多種疾病例如糖尿病和癌癥的生理過程中起關(guān)鍵性作用的蛋白質(zhì),。這一研究成果將有助于推動(dòng)科學(xué)家們開發(fā)出治療這些疾病的新型藥物。相關(guān)研究論文被選為 “封面故事”發(fā)布在著名期刊《生物化學(xué)期刊》（Journal of Biological Chemistry）上,。
“在這項(xiàng)研究中我們應(yīng)用了一種功能強(qiáng)大的蛋白質(zhì)結(jié)構(gòu)分析方法,，研究了cAMP化學(xué)信號(hào)啟動(dòng)其蛋白質(zhì)開關(guān)Epac2的機(jī)制，”德克薩斯大學(xué)加爾維斯頓醫(yī)學(xué)分部Sealy結(jié)構(gòu)生物學(xué)和分子物理學(xué)中心成員,、藥理學(xué)與毒理學(xué)系教授程曉東說,。
cAMP分子在包括大腦學(xué)習(xí)和記憶,、心臟收縮與舒張以及胰腺胰島素分泌等多種生理過程中均起著重要的調(diào)控作用。在細(xì)胞內(nèi)cAMP主要通過結(jié)合,、激活并開啟特異性的受體蛋白質(zhì)的方式啟動(dòng)下游信號(hào)途徑,。
當(dāng)這一細(xì)胞信號(hào)通路出現(xiàn)異常時(shí)則會(huì)導(dǎo)致多種疾病例如糖尿病、癌癥及心力衰竭等發(fā)生,。深入了解cAMP介導(dǎo)的細(xì)胞信號(hào)通路對(duì)于開發(fā)出特異性靶向cAMP-Epac2信號(hào)元件的新型治療策略具有重要的意義,。
在這一研究項(xiàng)目中，程曉東領(lǐng)導(dǎo)的研究小組與加州大學(xué)圣地亞哥分校醫(yī)學(xué)系教授Virgil Woods Jr及同事展開合作,，開發(fā)和利用了一種氫/氘交換質(zhì)譜測(cè)定技術(shù)（DXMS）對(duì)cAMP信號(hào)通路進(jìn)行了研究,。相對(duì)于其他的蛋白質(zhì)分析技術(shù)，DXMS尤其適用于研究蛋白質(zhì)的結(jié)構(gòu)運(yùn)動(dòng),。
利用這一新型技術(shù),，研究人員詳細(xì)地解析了cAMP逐步與Epac2上的兩個(gè)已知位點(diǎn)結(jié)合產(chǎn)生相互作用，并以一種非常特異性方式改變Epac2蛋白形狀,，啟動(dòng)Epac2活性的過程,。研究結(jié)果表明cAMP誘導(dǎo)的Epac2激活受到cAMP第二結(jié)合域C末端鉸鏈運(yùn)動(dòng)的調(diào)控。這一構(gòu)象的改變促使Epac2調(diào)控元件重新排列遠(yuǎn)離催化核心,，以便于隨后的效應(yīng)器結(jié)合,。此外，研究人員發(fā)現(xiàn)cAMP第一結(jié)合域與第二結(jié)合域的接口處于高度動(dòng)態(tài),，這一特征揭示了cAMP能夠進(jìn)入晶體結(jié)構(gòu)中被其他cAMP結(jié)合域相互阻隔的配體結(jié)合位點(diǎn)之謎,。
“DXMS分析方法已被證實(shí)是一種功能極其強(qiáng)大的技術(shù)，它可以單獨(dú)運(yùn)用亦可與其他技術(shù)結(jié)合使用,，用于解析蛋白質(zhì)在正常情況下改變形狀發(fā)揮功能的機(jī)制,，”Woods說：“這一技術(shù)可在將來廣泛地運(yùn)用到鑒別及開發(fā)靶向這些蛋白質(zhì)運(yùn)動(dòng)的治療性藥物中去。”（生物谷Bioon.com）
生物谷推薦原文出處：
The Journal of Biological Chemistry DOI:10.1074/jbc.M111.224535
Mechanism of Intracellular cAMP Sensor Epac2 Activation cAMP-INDUCED CONFORMATIONAL CHANGES IDENTIFIED BY AMIDE HYDROGEN/DEUTERIUM EXCHANGE MASS SPECTROMETRY (DXMS)
Sheng Li, Tamara Tsalkova, Mark A. White, Fang C. Mei, Tong Liu, Daphne Wang, Virgil L. Woods Jr and Xiaodong Cheng
Epac2, a guanine nucleotide exchange factor, regulates a wide variety of intracellular processes in response to second messenger cAMP. In this study, we have used peptide amide hydrogen/deuterium exchange mass spectrometry to probe the solution structural and conformational dynamics of full-length Epac2 in the presence and absence of cAMP. The results support a mechanism in which cAMP-induced Epac2 activation is mediated by a major hinge motion centered on the C terminus of the second cAMP binding domain. This conformational change realigns the regulatory components of Epac2 away from the catalytic core, making the later available for effector binding. Furthermore, the interface between the first and second cAMP binding domains is highly dynamic, providing an explanation of how cAMP gains access to the ligand binding sites that, in the crystal structure, are seen to be mutually occluded by the other cAMP binding domain. Moreover, cAMP also induces conformational changes at the ionic latch/hairpin structure, which is directly involved in RAP1 binding. These results suggest that in addition to relieving the steric hindrance imposed upon the catalytic lobe by the regulatory lobe, cAMP may also be an allosteric modulator directly affecting the interaction between Epac2 and RAP1. Finally, cAMP binding also induces significant conformational changes in the dishevelled/Egl/pleckstrin (DEP) domain, a conserved structural motif that, although missing from the active Epac2 crystal structure, is important for Epac subcellular targeting and in vivo functions.
