世界生命科學(xué)前沿動(dòng)態(tài)周報(bào)（六十七）

（11.21-11.27/2011）
美寶國(guó)際集團(tuán):陶國(guó)新 

　　主要內(nèi)容：指（趾）的形成顯示了“垃圾基因”的作用；糖尿病藥降低患癌風(fēng)險(xiǎn)；細(xì)胞移植受體的免疫系統(tǒng)控制移植干細(xì)胞的再生；線蟲研究揭示傷口愈合反應(yīng)的秘密；維持Sip2的乙?；娱L(zhǎng)酵母壽命；發(fā)現(xiàn)端粒酶延長(zhǎng)端粒的關(guān)鍵分子開關(guān)。

　　焦點(diǎn)動(dòng)態(tài)：發(fā)現(xiàn)端粒酶延長(zhǎng)端粒的關(guān)鍵分子開關(guān)。

1. 指（趾）的形成顯示了“垃圾基因”的作用
【動(dòng)態(tài)】
　　指（趾）頭的進(jìn)化是四足動(dòng)物成功的必要步驟，在指（趾）頭發(fā)育的關(guān)鍵因素中，Hoxd基因受到協(xié)調(diào)控制，幫助管理生長(zhǎng)和模式。瑞士、荷蘭和德國(guó)的科學(xué)家通過(guò)探查發(fā)育中的四肢的三位構(gòu)造確定了與這些基因有關(guān)的肢端調(diào)節(jié)位點(diǎn)。該方法結(jié)合體內(nèi)刪除特定的調(diào)節(jié)區(qū)，顯示基因簇的活性部位與數(shù)個(gè)增強(qiáng)子樣的序列接觸。這些序列分散在臨近的所謂垃圾基因序列中，各自對(duì)預(yù)期的指（趾）的Hox基因轉(zhuǎn)錄做出定量或定性的貢獻(xiàn)。他們提出名為“調(diào)節(jié)列島”的基因體系，提供一種遺傳靈活性可能部分支持四足動(dòng)物中指（趾）數(shù)目和形態(tài)的多樣性以及順應(yīng)劇烈變化的能力。

【點(diǎn)評(píng)】
　　該研究表明在曾經(jīng)錯(cuò)以為沒(méi)有任何作用的垃圾基因序列中的一些序列能夠相互結(jié)合，調(diào)節(jié)負(fù)責(zé)形成指（趾）頭的基因的作用。這樣的結(jié)果表明目前人類對(duì)基因的研究仍未成熟，尤其是對(duì)功能基因的作用機(jī)制還有很多工作要做。

【參考論文】
Cell, 2011; 147 (5): 1132 DOI: 10.1016/j.cell.2011.10.023 
A Regulatory Archipelago Controls Hox Genes Transcription in Digits
Thomas Montavon, Natalia Soshnikova, Bénédicte Mascrez, et al.
Highlights
Hox genes active in digits integrate the input of multiple regulatory elements
The global regulatory architecture of the HoxD locus involves flanking gene deserts
Alterations in this regulatory structure may fine tune digital morphology in tetrapods
Summary
The evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a regulatory archipelago, provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations.
 
2. 糖尿病藥降低患癌風(fēng)險(xiǎn)
【動(dòng)態(tài)】
　　一種廉價(jià)的2型糖尿病治療藥物，抑制糖異生基因轉(zhuǎn)錄的二甲雙胍證明能夠預(yù)防很多天然或人工合成的化合物刺激乳腺癌細(xì)胞生長(zhǎng)，降低糖尿病相關(guān)腫瘤的風(fēng)險(xiǎn)。最近，腫瘤干細(xì)胞被認(rèn)為是負(fù)責(zé)維持腫瘤生長(zhǎng)，抵抗治療。韓國(guó)和美國(guó)科學(xué)家為了驗(yàn)證二甲雙胍可能減少乳腺癌風(fēng)險(xiǎn)的假設(shè)，將生長(zhǎng)于代表人體乳腺癌干細(xì)胞群的三維乳球體中的MCF-7人體乳腺癌細(xì)胞系在有/無(wú)非細(xì)胞毒性濃度的二甲雙胍存在下用各種已知的和可能的乳腺癌誘發(fā)劑處理，OCT4表達(dá)作為腫瘤干細(xì)胞的標(biāo)志，測(cè)定這些細(xì)胞的數(shù)目和大小。結(jié)果表明100nM的TCDD和10µM的雙酚A像10µM的雌激素一樣增加了三維乳球體的數(shù)目和大小。通過(guò)檢測(cè)標(biāo)志物OCT4，這些致癌物的刺激與OCT4表達(dá)增多相關(guān)。另一方面，1mM和10mM的二甲雙胍能夠大大減少三維乳球體的數(shù)目和大小。結(jié)果還顯示二甲雙胍能夠減少雌激素和TCDD誘生的三維乳球體中OCT4的表達(dá)，但在雙酚A誘生的三維乳球體中則不能，表明雙酚A對(duì)人體乳腺癌細(xì)胞有不同的作用機(jī)制。另外，這些結(jié)果支持使用三維人體乳腺癌干細(xì)胞作為一種手段篩選潛在的人體乳腺癌致癌物以及化學(xué)預(yù)防和治療劑。

【點(diǎn)評(píng)】
　　該研究發(fā)現(xiàn)了廉價(jià)的糖尿病藥物二甲雙胍能夠通過(guò)抑制雌激素受體介導(dǎo)的癌基因OCT4的表達(dá)，減少人體乳腺癌干細(xì)胞的自我復(fù)制。同時(shí)為乳腺癌的防治和二甲雙胍的老藥新用提供了新的可能。

【參考論文】
PLoS ONE, 2011; 6 (11): e28068 DOI: 10.1371/journal.pone.0028068 
Metformin Represses Self-Renewal of the Human Breast Carcinoma Stem Cells via Inhibition of Estrogen Receptor-Mediated OCT4 Expression
Ji-Won Jung, Sang-Bum Park, Soo-Jin Lee, et al.
Metformin, a Type II diabetic treatment drug, which inhibits transcription of gluconeogenesis genes, has recently been shown to lower the risk of some diabetes-related tumors, including breast cancer. Recently, “cancer stem cells” have been demonstrated to sustain the growth of tumors and are resistant to therapy. To test the hypothesis that metformin might be reducing the risk to breast cancers, the human breast carcinoma cell line, MCF-7, grown in 3-dimensional mammospheres which represent human breast cancer stem cell population, were treated with various known and suspected breast cancer chemicals with and without non-cytotoxic concentrations of metformin. Using OCT4 expression as a marker for the cancer stem cells, the number and size were measured in these cells. Results demonstrated that TCDD (100 nM) and bisphenol A (10 µM) increased the number and size of the mammospheres, as did estrogen (10 nM E2). By monitoring a cancer stem cell marker, OCT4, the stimulation by these chemicals was correlated with the increased expression of OCT4. On the other hand, metformin at 1 and 10 mM concentration dramatically reduced the size and number of mammospheres. Results also demonstrated the metformin reduced the expression of OCT4 in E2 & TCDD mammospheres but not in the bisphenol A mammospheres, suggesting different mechanisms of action of the bisphenol A on human breast carcinoma cells. In addition, these results support the use of 3-dimensional human breast cancer stem cells as a means to screen for potential human breast tumor promoters and breast chemopreventive and chemotherapeutic agents.

3. 細(xì)胞移植受體的免疫系統(tǒng)控制移植干細(xì)胞的再生
【動(dòng)態(tài)】
　　受體的T淋巴細(xì)胞通過(guò)IFN-γ和TNF-α控制基于間充質(zhì)干細(xì)胞的組織再生。在組織重建中基于干細(xì)胞的再生醫(yī)學(xué)是一種很有希望的方法。南加大的美國(guó)科學(xué)家最近報(bào)道了促炎T細(xì)胞抑制外源骨髓間充質(zhì)干細(xì)胞（BMMSCs）引導(dǎo)骨頭修復(fù)的能力。這一抑制作用來(lái)自干擾素γ（IFN-γ）誘導(dǎo)的干細(xì)胞中runt相關(guān)轉(zhuǎn)錄因子2（Runx-2）途徑下調(diào)和腫瘤壞死因子α（TNF-α）信號(hào)的增強(qiáng)。他們還發(fā)現(xiàn)，通過(guò)抑制核因子κB (NF-κB)，TNF-α將BMMSCs中IFN-γ激活的，非凋亡形態(tài)的TNF受體超家族成員6（Fas）信號(hào)轉(zhuǎn)變?yōu)榧?xì)胞凋亡蛋白酶3和8相關(guān)的促凋亡級(jí)聯(lián)反應(yīng)信號(hào)，導(dǎo)致這些細(xì)胞凋亡。相反地，通過(guò)全身輸入Foxp3(+)調(diào)節(jié)性T細(xì)胞或局部給藥阿司匹林來(lái)減少IFN-γ 和 TNF-α的濃度，顯著地促進(jìn)了C57BL/6老鼠基于BMMSC的骨骼再生和顱骨缺損的修復(fù)。這些數(shù)據(jù)合起來(lái)表明前所未知的受體T細(xì)胞在基于BMMSC的組織工程中的作用。

【點(diǎn)評(píng)】
　　該研究表明基于干細(xì)胞的再生醫(yī)學(xué)研究需要關(guān)注機(jī)體自身的免疫系統(tǒng)對(duì)組織再生的調(diào)控，干細(xì)胞研究和再生醫(yī)學(xué)的突破離不開干細(xì)胞所處機(jī)體環(huán)境的研究。

【參考論文】
Nature Medicine, 2011 DOI: 10.1038/nm.254 
Mesenchymal stem cell-based tissue regeneration is governed by recipient T lymphocytes via IFN-γ and TNF-α
Yi Liu, Lei Wang, Takashi Kikuiri, et al.
Stem cell-based regenerative medicine is a promising approach in tissue reconstruction. Here we show that proinflammatory T cells inhibit the ability of exogenously added bone marrow mesenchymal stem cells (BMMSCs) to mediate bone repair. This inhibition is due to interferon γ (IFN-γ)-induced downregulation of the runt-related transcription factor 2 (Runx-2) pathway and enhancement of tumor necrosis factor α (TNF-α) signaling in the stem cells. We also found that, through inhibition of nuclear factor κB (NF-κB), TNF-α converts the signaling of the IFN-γ-activated, nonapoptotic form of TNF receptor superfamily member 6 (Fas) in BMMSCs to a caspase 3- and caspase 8-associated proapoptotic cascade, resulting in the apoptosis of these cells. Conversely, reduction of IFN-γ and TNF-α concentrations by systemic infusion of Foxp3(+) regulatory T cells, or by local administration of aspirin, markedly improved BMMSC-based bone regeneration and calvarial defect repair in C57BL/6 mice. These data collectively show a previously unrecognized role of recipient T cells in BMMSC-based tissue engineering.

4. 線蟲研究揭示傷口愈合反應(yīng)的秘密
【動(dòng)態(tài)】
　　在嚴(yán)酷的環(huán)境中生存，動(dòng)物必須能夠修復(fù)皮膚創(chuàng)傷，然而體內(nèi)啟動(dòng)創(chuàng)傷修復(fù)的信號(hào)途徑還不明了。在線蟲中，p38分裂素激活的蛋白激酶（MAPK）級(jí)聯(lián)反應(yīng)促進(jìn)應(yīng)對(duì)創(chuàng)傷的先天免疫反應(yīng)，但不是創(chuàng)傷愈合其他方面所需的。美國(guó)科學(xué)家為此在線蟲表皮中探查了其他的創(chuàng)傷反應(yīng)信號(hào)途徑。結(jié)果表明線蟲表皮創(chuàng)傷引發(fā)迅速而持續(xù)的表皮中鈣離子濃度的上升，這是創(chuàng)傷后存活的關(guān)鍵。創(chuàng)傷觸發(fā)的鈣離子增多需要表皮瞬時(shí)受體電壓通道、melastatin家族（TRPM）通道GTL-2和IP3R刺激的內(nèi)部?jī)?chǔ)存池的釋放。他們確立了一個(gè)表皮信號(hào)傳導(dǎo)途徑，包括Gαq EGL-30 及其效應(yīng)器 PLCβ EGL-8。該途徑失效的話會(huì)導(dǎo)致?lián)p害創(chuàng)傷后存活。Gαq-Ca2+途徑對(duì)已知的應(yīng)對(duì)創(chuàng)傷的先天免疫反應(yīng)不是必須的 ，但會(huì)促進(jìn)肌動(dòng)蛋白依賴的傷口閉合。傷口閉合需要Cdc42小GTP酶和Arp2/3-依賴的肌動(dòng)蛋白聚合，并被Rho 和非肌肉的肌球蛋白負(fù)向調(diào)節(jié)。他們還發(fā)現(xiàn)死亡相關(guān)蛋白激酶DAPK-1負(fù)向調(diào)節(jié)傷口閉合。

【點(diǎn)評(píng)】
　　該研究表明線蟲皮膚創(chuàng)傷會(huì)觸發(fā)鈣離子依賴的信號(hào)級(jí)聯(lián)反應(yīng)促進(jìn)傷口閉合，同時(shí)伴有損傷引起的先天免疫反應(yīng)。對(duì)于認(rèn)識(shí)創(chuàng)傷早期的生化事件和促進(jìn)創(chuàng)傷愈合研究有幫助。

【參考論文】
Current Biology, 2011; DOI: 10.1016/j.cub.2011.10.050 
A Gαq-Ca2 Signaling Pathway Promotes Actin-Mediated Epidermal Wound Closure in C. elegans
Suhong Xu, Andrew D. Chisholm.
Background
Repair of skin wounds is essential for animals to survive in a harsh environment, yet the signaling pathways initiating wound repair in vivo remain little understood. In Caenorhabditis elegans, a p38 mitogen-activated protein kinase (MAPK) cascade promotes innate immune responses to wounding but is not required for other aspects of wound healing. We therefore set out to identify additional wound response pathways in C. elegans epidermis.
Results
We show here that wounding the adult C. elegans skin triggers a rapid and sustained rise in epidermal Ca2+ that is critical for survival after wounding. The wound-triggered rise in Ca2+ requires the epidermal transient receptor potential channel, melastatin family (TRPM) channel GTL-2 and IP3R-stimulated release from internal stores. We identify an epidermal signal transduction pathway that includes the Gαq EGL-30 and its effector PLCβ EGL-8. Loss of function in this pathway impairs survival after wounding. The Gαq-Ca2+ pathway is not required for known innate immune responses to wounding but instead promotes actin-dependent wound closure. Wound closure requires the Cdc42 small GTPase and Arp2/3-dependent actin polymerization and is negatively regulated by Rho and nonmuscle myosin. Finally, we show that the death-associated protein kinase DAPK-1 acts as a negative regulator of wound closure.
Conclusions
Skin wounding in C. elegans triggers a Ca2+-dependent signaling cascade that promotes wound closure, in parallel to the innate immune response to damage. Wound closure requires actin polymerization and is negatively regulated by nonmuscle myosin.

5. 維持Sip2的乙?；娱L(zhǎng)酵母壽命
【動(dòng)態(tài)】
　　蛋白乙?；怯绊懞芏嗉?xì)胞生命過(guò)程的重要的翻譯后修飾。美國(guó)和臺(tái)灣的科學(xué)家最近發(fā)現(xiàn)隨著細(xì)胞變老，單細(xì)胞酵母中AMP激活的蛋白激酶Snf1復(fù)合物的β調(diào)節(jié)亞基Sip2的NuA4乙?；瘻p少。通過(guò)拮抗NuA4乙?；D(zhuǎn)移酶和Rpd3脫乙酰基酶控制的Sip2乙?；鰪?qiáng)了與Snf1復(fù)合物的催化亞基Snf1的相互作用。Sip2-Snf1相互作用抑制Snf1的活性，減少下游靶點(diǎn)Sch9（Akt/S6K同源物）的磷酸化，最終導(dǎo)致生長(zhǎng)變慢但是延長(zhǎng)了細(xì)胞復(fù)制的壽命（復(fù)制次數(shù)）。Sip2乙?；M物更能抵抗氧化壓力。他們還發(fā)現(xiàn)Sip2乙?；目顾ダ献饔貌灰蕾囃鈦?lái)營(yíng)養(yǎng)和TORC1活性。他們提出蛋白乙?；?磷酸化連鎖反應(yīng)調(diào)節(jié)Sch9活性，控制本身的衰老，延長(zhǎng)酵母繁殖的壽命（繁殖更多代）。天然酵母的正常繁殖壽命是25代，而通過(guò)基因工程改造恢復(fù)乙?；瘜W(xué)修飾的酵母繁殖壽命達(dá)到38代，延長(zhǎng)了50%。

【點(diǎn)評(píng)】
　　在酵母中維持Snf1復(fù)合物β調(diào)節(jié)亞基Sip2的乙?；哂锌顾ダ献饔?。文中實(shí)驗(yàn)了通過(guò)基因改造模擬這一乙酰化維持過(guò)程，達(dá)到了延長(zhǎng)酵母壽命達(dá)50%的效果。如果不用改造基因，通過(guò)其他更自然的方法也能維持Sip2的乙?；?，可能是抗衰老的重大突破。

【參考論文】
Cell, 2011; 146 (6): 969 DOI: 10.1016/j.cell.2011.07.044
Acetylation of Yeast AMPK Controls Intrinsic Aging Independently of Caloric Restriction
Jin-Ying Lu, Yu-Yi Lin, Jin-Chuan Sheu, et al. 
Acetylation of histone and nonhistone proteins is an important posttranslational modification affecting many cellular processes. Here, we report that NuA4 acetylation of Sip2, a regulatory β subunit of the Snf1 complex (yeast AMP-activated protein kinase), decreases as cells age. Sip2 acetylation, controlled by antagonizing NuA4 acetyltransferase and Rpd3 deacetylase, enhances interaction with Snf1, the catalytic subunit of Snf1 complex. Sip2-Snf1 interaction inhibits Snf1 activity, thus decreasing phosphorylation of a downstream target, Sch9 (homolog of Akt/S6K), and ultimately leading to slower growth but extended replicative life span. Sip2 acetylation mimetics are more resistant to oxidative stress. We further demonstrate that the anti-aging effect of Sip2 acetylation is independent of extrinsic nutrient availability and TORC1 activity. We propose a protein acetylation-phosphorylation cascade that regulates Sch9 activity, controls intrinsic aging, and extends replicative life span in yeast.

6. 發(fā)現(xiàn)端粒酶延長(zhǎng)端粒的關(guān)鍵分子開關(guān)
【動(dòng)態(tài)】
　　進(jìn)化上保守的端粒庇護(hù)蛋白復(fù)合物在哺乳動(dòng)物和裂殖酵母的端粒酶調(diào)節(jié)中既有正面作用也有負(fù)面作用。盡管哺乳動(dòng)物細(xì)胞的庇護(hù)蛋白防止檢查點(diǎn)激酶ATM和ATR在端粒處充分激活DNA損傷反應(yīng)，這些激酶也促進(jìn)端粒的維護(hù)。缺少Tel1（ATM直接同源物）和Rad3（ATR直接同源物）的裂殖酵母細(xì)胞無(wú)法招募端粒酶到端粒上，無(wú)法通過(guò)環(huán)形染色體存活。但是，還不知道Tel1ATM和Rad3ATR對(duì)應(yīng)的端粒底物。美國(guó)科學(xué)家最近的研究表明Tel1ATM和/或Rad3ATR調(diào)節(jié)的庇護(hù)蛋白亞基Ccq1在93號(hào)蘇氨酸上的磷酸化是端粒酶聯(lián)系端粒所必需的。另外，端粒酶亞基Est1直接與Ccq1的磷酸化的93號(hào)蘇氨酸相互作用以保證端粒的維護(hù)。庇護(hù)蛋白亞基Taz1, Rap1 和 Poz1 (以前已確認(rèn)的端粒酶抑制劑) 也負(fù)向調(diào)節(jié) Ccq1磷酸化 。所有這些發(fā)現(xiàn)證明Tel1ATM/Rad3ATR依賴的 Ccq1 的93號(hào)蘇氨酸磷酸化是裂殖酵母維護(hù)端粒的關(guān)鍵調(diào)節(jié)器。

【點(diǎn)評(píng)】
　　該研究進(jìn)一步闡釋了端粒酶途徑維護(hù)端粒長(zhǎng)度的機(jī)制，對(duì)于認(rèn)識(shí)各細(xì)胞組分如何協(xié)調(diào)一致維持端粒的正常功能和尋找預(yù)防癌癥的方法有重要意義。

【參考論文】
Nature Structural & Molecular Biology, 2011; DOI: 10.1038/nsmb.2187 
Tel1ATM and Rad3ATR kinases promote Ccq1-Est1 interaction to maintain telomeres in fission yeast
Bettina A Moser, Ya-Ting Chang, Jorgena Kosti, et al.
The evolutionarily conserved shelterin complex has been shown to play both positive and negative roles in telomerase regulation in mammals and fission yeast. Although shelterin prevents the checkpoint kinases ATM and ATR from fully activating DNA damage responses at telomeres in mammalian cells, those kinases also promote telomere maintenance. In fission yeast, cells lacking both Tel1 (ATM ortholog) and Rad3 (ATR ortholog) fail to recruit telomerase to telomeres and survive by circularizing chromosomes. However, the critical telomere substrate(s) of Tel1ATM and Rad3ATR was unknown. Here we show that phosphorylation of the shelterin subunit Ccq1 on Thr93, redundantly mediated by Tel1ATM and/or Rad3ATR, is essential for telomerase association with telomeres. In addition, we show that the telomerase subunit Est1 interacts directly with the phosphorylated Thr93 of Ccq1 to ensure telomere maintenance. The shelterin subunits Taz1, Rap1 and Poz1 (previously established inhibitors of telomerase) were also found to negatively regulate Ccq1 phosphorylation. These findings establish Tel1ATM/Rad3ATR-dependent Ccq1 Thr93 phosphorylation as a critical regulator of telomere maintenance in fission yeast.