中科院上海藥物研究所蔣華良課題組與美國芝加哥大學(xué)化學(xué)系何川課題組合作,，針對胸腺嘧啶DNA糖苷化酶及細菌轉(zhuǎn)錄因子AgrA在表觀遺傳與轉(zhuǎn)錄調(diào)控中的作用開展研究，取得了良好進展,。研究論文分別于2012年2月及2012年5月在線發(fā)表在Nature Chemical Biology和PNAS上,。
在針對胸腺嘧啶DNA糖苷酶（Thymine-DNA glycosylase， TDG）底物選擇性機制的研究中,，兩個課題組研究人員綜合運用化學(xué)生物學(xué),、理論模擬等手段，闡明了TDG選擇性切除DNA分子中被修飾的嘧啶堿基的分子機制。TDG能選擇性剪切由TET蛋白催化5-甲基胞嘧啶（5mC）氧化產(chǎn)生的5-醛基胞嘧啶（5fC）及5-羧基胞嘧啶（5caC）,，繼而啟動下游DNA剪切修復(fù)（BER,，base-excision repair）通路，將5mC還原成未修飾的胞嘧啶,。然而,，TDG如何在眾多正常的胞嘧啶及5-甲基胞嘧啶中選擇性切除5caC和5fC的機制并不明確。
何川教授課題組通過生物實驗發(fā)現(xiàn),，TDG對于含有不同修飾胞嘧啶的DNA結(jié)合能力有顯著不同,。其與含有5caC和5fC的DNA結(jié)合能力最強，而與含5hmC,，5mC及正常胞嘧啶的DNA幾乎不結(jié)合,。蔣華良課題組成員從計算生物學(xué)的角度出發(fā)，根據(jù)何川課題組解析得到的TDG與5caC的復(fù)合物晶體結(jié)構(gòu),，開展了分子動力學(xué)模擬與結(jié)合自由能計算研究,。通過計算發(fā)現(xiàn)，TDG活性口袋的Ile139可以和5caC形成較強的氫鍵相互作用,，而同5mC和正常胞嘧啶不存在此相互作用,。另外，TDG活性口袋中的His151,、Tyr152同5caC的5位羧基存在很強的極性相互作用,，而與其它修飾的胞嘧啶如5mC和5hmC相互作用較弱。因此可以認為,，TDG的活性口袋能選擇性地結(jié)合5位具有負電性取代基的底物,，如5caC或5fC。研究結(jié)果為進一步闡明TDG在表觀遺傳調(diào)控中扮演的作用提供了重要線索,。
在轉(zhuǎn)錄因子AgrA調(diào)控機制的研究中,，兩個課題組進一步合作，闡明了細菌轉(zhuǎn)錄因子AgrA的DNA結(jié)合域中分子內(nèi)二硫鍵的開關(guān)在群體感應(yīng)agr信號系統(tǒng)中發(fā)揮的重要功能,。何川課題組前期研究發(fā)現(xiàn),，在氧化信號刺激下，AgrA結(jié)構(gòu)中C199和C228會形成分子內(nèi)二硫鍵,，導(dǎo)致AgrA與DNA的解離,。蔣華良課題組成員從計算生物學(xué)角度出發(fā)，運用分子動力學(xué)模擬等手段,，從分子水平揭示了二硫鍵形成引起的空間位阻效應(yīng)是破壞AgrA與DNA的結(jié)合的重要原因,。同時，C199S的突變不會影響蛋白結(jié)構(gòu)的穩(wěn)定性,，而C228S的突變破壞了DNA結(jié)合域的三維結(jié)構(gòu),，暗示C199主要負責氧化感應(yīng)，而C228對于維持蛋白結(jié)構(gòu)的穩(wěn)定性至關(guān)重要。模擬結(jié)果與相關(guān)突變實驗結(jié)果相吻合,，證實了氧化感應(yīng)是agr群體感應(yīng)信號系統(tǒng)中的一個重要組分,，細菌通過這種功能調(diào)節(jié)，可以有效緩解由其自身代謝或宿主免疫造成的氧化壓力,。研究結(jié)果為深入理解細菌轉(zhuǎn)錄調(diào)控機制及新型抗菌藥物的開發(fā)提供重要依據(jù),。
該合作團隊有機結(jié)合生物實驗與計算模擬，積極探索表觀遺傳與轉(zhuǎn)錄調(diào)控的相互聯(lián)系,，以及它們在胚胎發(fā)育與疾病產(chǎn)生中扮演的作用,。這為進一步靶向新的表觀遺傳與轉(zhuǎn)錄調(diào)控靶標，開發(fā)高效特異的小分子活性候選化合物提供了理論基礎(chǔ),。
上述合作研究得到了美國國立衛(wèi)生研究院（NIH）,、中科院“干細胞與再生醫(yī)學(xué)”戰(zhàn)略先導(dǎo)專項、973,、863以及上海市科委“非政府間國際合作”項目的資助,。
三年來，兩個課題組通力合作,，在表觀遺傳分子機制和相關(guān)藥物發(fā)現(xiàn)研究中取得一系列進展,。除上述兩項合作外，其他合作正在進行中,。（生物谷Bioon.com）
doi:10.1073/pnas.1200603109
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Quorum-sensing agr mediates bacterial oxidation response via an intramolecular disulfide redox switch in the response regulator AgrA
Fei Suna,1, Haihua Lianga,1, Xiangqian Kongb, Sherrie Xiea, Hoonsik Choc, Xin Denga, Quanjiang Jia, Haiyan Zhanga, Sophie Alvarezd, Leslie M. Hicksd, Taeok Baec, Cheng Luob, Hualiang Jiangb, and Chuan Hea,2
Oxidation sensing and quorum sensing significantly affect bacterial physiology and host–pathogen interactions. However, little attention has been paid to the cross-talk between these two seemingly orthogonal signaling pathways. Here we show that the quorum-sensing agr system has a built-in oxidation-sensing mechanism through an intramolecular disulfide switch possessed by the DNA-binding domain of the response regulator AgrA. Biochemical and mass spectrometric analysis revealed that oxidation induces the intracellular disulfide bond formation between Cys-199 and Cys-228, thus leading to dissociation of AgrA from DNA. Molecular dynamics (MD) simulations suggest that the disulfide bond formation generates a steric clash responsible for the abolished DNA binding of the oxidized AgrA. Mutagenesis studies further established that Cys-199 is crucial for oxidation sensing. The oxidation-sensing role of Cys-199 is further supported by the observation that the mutant Staphylococcus aureus strain expressing AgrAC199S is more susceptible to H2O2 owing to repression of the antioxidant bsaA gene under oxidative stress. Together, our results show that oxidation sensing is a component of the quorum-sensing agr signaling system, which serves as an intrinsic checkpoint to ameliorate the oxidation burden caused by intense metabolic activity and potential host immune response.
doi:10.1038/nchembio.914
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Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA
Liang Zhang, Xingyu Lu, Junyan Lu, Haihua Liang, Qing Dai, Guo-Liang Xu, Cheng Luo, Hualiang Jiang & Chuan He
Human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC), a key oxidation product of 5-methylcytosine in genomic DNA, in a recently discovered cytosine demethylation pathway. We present here the crystal structures of the hTDG catalytic domain in complex with duplex DNA containing either 5caC or a fluorinated analog. These structures, together with biochemical and computational analyses, reveal that 5caC is specifically recognized in the active site of hTDG, supporting the role of TDG in mammalian 5-methylcytosine demethylation.
