外膜蛋白是一類非常獨特但又非常重要的蛋白質(zhì),他們廣泛的存在于原核生物的細(xì)胞外膜和真核生物的細(xì)胞器外膜中。結(jié)構(gòu)上,它們都具有β-桶狀結(jié)構(gòu),不同的外膜蛋白的β-桶由不同偶數(shù)個β-折疊片組成,從8個到22個不等;功能上,它們具有為外膜提供通透性,維持外膜結(jié)構(gòu)穩(wěn)定的作用。折疊好的外膜蛋白具有極強的穩(wěn)定性,常溫下,在2%的強離子型表面活性劑SDS中,外膜蛋白仍然具有正常的折疊結(jié)構(gòu);而在此溫度下,其他可溶蛋白在0.2%的SDS中就可以迅速失去結(jié)構(gòu)。因此通常將加樣前是否對樣品進(jìn)行95C加熱去折疊的SDS-PAGE稱為denatured SDS-PAGE和semi-native SDS-PAGE,并被用來研究外膜蛋白的折疊狀況。 外膜蛋白的研究經(jīng)歷了漫長而又曲折的過程。起初人們并沒有意識到外膜蛋白的重要性,雖然有一些關(guān)于外膜脂多糖生成障礙與熱致死相關(guān)的文獻(xiàn)報道,但是人們還是把更多的注意力集中到膜間質(zhì)蛋白的生成上,或者模糊的認(rèn)為膜間質(zhì)蛋白的生成就是外膜的生成,或者認(rèn)為膜間質(zhì)蛋白的錯誤折疊是引起細(xì)胞在脅迫條件下死亡的主要原因。隨著認(rèn)識的深入,Ried, G.et al.發(fā)現(xiàn)與耐熱相關(guān)的脂多糖實際上幫助了外膜蛋白的正確生成,Mecsas, J.et al.通過遺傳篩選證明只有外膜蛋白的過表達(dá)才會對細(xì)胞產(chǎn)生來自胞外的蛋白質(zhì)生成壓力,激活主要膜間質(zhì)脅迫信號途徑σE通路。進(jìn)一步的證據(jù)表明,膜間質(zhì)的主要分子伴侶SurA和Skp都參與了外膜蛋白的正常生成,過表達(dá)膜間質(zhì)分子伴侶可以補救脂多糖合成缺陷的表型,挽救外膜蛋白的異常生成。此后,Rizzitello, A.E.et al.和Sklar, J.G.et al.分別發(fā)現(xiàn)還發(fā)現(xiàn)膜間質(zhì)的分子伴侶SurA與DegP和Skp存在平行結(jié)構(gòu)的協(xié)作關(guān)系,surA和skp,surA和degP的雙敲除都會表現(xiàn)出致死表型并影響外膜蛋白的正常生成。整個認(rèn)識過程中最重要的發(fā)現(xiàn)出現(xiàn)在2003年,Voulhoux, R.et al.發(fā)現(xiàn),一種特殊的外膜蛋白Omp85,能夠捕捉膜間質(zhì)中的自內(nèi)膜轉(zhuǎn)運而來的其他外膜蛋白中間體,并引導(dǎo)它們進(jìn)入外膜,它的敲除株會使外膜蛋白的生成出現(xiàn)嚴(yán)重的障礙并導(dǎo)致細(xì)菌的死亡,這一發(fā)現(xiàn)使得外膜蛋白生成這一鮮有問津的生物學(xué)領(lǐng)域真正引起了學(xué)術(shù)界的高度重視。這之后,Wu, T.et al.發(fā)現(xiàn)Omp85/YaeT/BamA這一因子是通過與YfgL (BamB), NlpB (BamC), YfiO (BamD)和SmpA (BamE)形成巨大的復(fù)合物(>230kDa)來發(fā)揮作用。最近,Krojer, T.et al.發(fā)現(xiàn)膜間質(zhì)中的另一個與耐熱相關(guān)的蛋白DegP/HtrA,能夠和外膜蛋白形成穩(wěn)定的復(fù)合物DegP12/24-OMP,雖然其形成的機制尚不清楚,但是這一發(fā)現(xiàn)仍然說明膜間質(zhì)中的脅迫更多是由于外膜蛋白的異常生成造成的。 最近我們實驗室通過生化和遺傳學(xué)的方法證明,主要外膜蛋白都存在穩(wěn)定的折疊中間態(tài),并且這些折疊中間態(tài)的存在是革蘭氏陰性菌能在脅迫條件下能正常存活的基礎(chǔ)條件。耐熱相關(guān)蛋白DegP/HtrA通過在Skp和SurA下游識別并分選這種折疊中間狀態(tài)來使細(xì)菌抵抗環(huán)境脅迫并為其提供致病性。DegP的分子伴侶活性和蛋白酶活性在分選外膜蛋白折疊中間狀態(tài)的過程中同時發(fā)揮著重要的作用,分子伴侶活性負(fù)責(zé)捕捉和穩(wěn)定不同折疊狀態(tài)的外膜蛋白,幫助部分折疊的外膜蛋白插膜,防止錯誤折疊的外膜蛋白吸附到外膜上;蛋白酶活性對于清除錯誤折疊的外膜蛋白至關(guān)重要。 Burgess, N.K., Dao, T.P., Stanley, A.M., & Fleming, K.G., Beta-barrel proteins that reside in theEscherichia coliouter membrane in vivo demonstrate varied folding behavior in vitro.J. Biol. Chem.283 (39), 26748-26758 (2008).
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