2011-08-012024-05-16https://scholars.lib.ntu.edu.tw/handle/123456789/670068摘要：腸病毒 71 型感染可以說是後小兒麻痺時代，最容易造成兒童中樞神經系統感染與傷害的病毒。然而，至今醫界對於腸病毒71 型的分子致病機轉依然了解有限。我們先前的實驗發現腸病毒 71 型的2B 蛋白可以與Mcl-1 直接的交互作用，並且調降Mcl-1 的代謝，穩定Mcl-1。Mcl-1 半衰期很短，在體內經由泛素-蛋白脢體(ubiquitin-proteasome)路徑進行快速的代謝，是一個受到非常嚴密調控的蛋白。我們證明腸病毒71 型的2B 蛋白可以佔據Mcl-1 的BH3 domain，因為Mcl-1 的BH3 domain 剛好就是Mcl-1 的泛素E3 ligase (MULE)作用的位置，腸病毒71 型的2B 蛋白使得MULE 無法作用於Mcl-1，Mcl-1 也就不會被泛素化與代謝。2B 這種對Mcl-1 泛素化形同競爭性抑制的作用穩定了細胞內Mcl-1 的量，也就抑制了細胞凋亡的進行。藉由在腸病毒Infectious DNA clone 的2B 蛋白上製造變異，我們也證實了腸病毒71 型2B 藉由這樣的機轉，確保其有足夠的時間完成病毒的複製，一旦2B 蛋白無法跟Mcl-1 交互作用，病毒將幾乎無法完成複製。我們這樣的研究結果是一種全新的發現，腸病毒 71 型2B 藉由與Mcl-1 的交互作用，增進了Mcl-1 在細胞內的穩定度，進一步抑制了細胞的凋亡，更進一步確保了病毒複製的完成。這些結果，大幅增進了我們對於腸病毒71 型如何調控細胞凋亡的了解。也開啟了更多值得進一步研究的領域。首先，腸病毒的2B 蛋白是否可以藉由同樣的機轉調控其他具有BH3 domain 的Bcl-2 家族蛋白，甚至調控其他非Bcl-2 家族的蛋白的泛素化。我們將研究腸病毒71 型2B 蛋白對Mcl-1 以外的Bcl-2 家族蛋白，包括Bcl-2、Bcl-2A1、Bik、Noxa 等等，作何種調控，以及其機制為何。這將是本計畫的第一部分。這部份的研究，除了近一步了解一種病毒如何調控宿主細胞的細胞凋亡以外，也可能有其它的應用，因為Bcl-2 家族蛋白在許多人類疾病包括腫瘤都扮演重要角色，透過對這些蛋白調控的了解，可以更了解這些疾病德致病機轉與尋找可能的治療方向。另一方面，我們利用 yeast two hybrid system 針對腸病毒71 型蛋白的交互作用對象做篩選時，同時也找到其他幾種可以與2B 蛋白交互作用的細胞蛋白，包括腫瘤壞死因子接受體家族的成員9 與18 (tumor necrosis factor receptor superfamily (TNFRSF)members: TNFRSF9 and TNFRSF18)以及甲型干擾素誘發蛋白27(interferonalpha-inducible protein 27, IFI27)。這些都與T 細胞活化的調控有關。本計畫的第二部分將探討腸病毒71 型2B 蛋白與這些分子交互作用的生物意義。這部分亦屬前所未有的開創性研究，也頗具臨床意義，因為腸病毒71 型感染重症病患常常出現嚴重的全身性發炎反應，其臨床表現並不能單純用病毒侵犯與破壞來解釋。腸病毒71 型感染是否會過度激化T 細胞，導致免疫系統失調是一個可能性。了解腸病毒71 型2B 蛋白與這些T細胞活化路徑相關的接受體的關係，有可能有重要的新發現，也可能提供治療嚴重腸病71 型感染新的新思維。<br> Abstract: In the post-polio era, enterovirus 71 (EV71) is the most important etiology of CNSinfections resulting in mortalities or severe neurological deficits in children. However, themolecular pathogenesis of EV71 remained not fully understood.Our earlier studies have shown 2B protein of EV71 regulates expression levels of Mcl-1and several other Bcl-1 family members by direct interactions. Normally, Mcl-1 is tightlyregulated by degradation through the ubiquitin-proteasome pathway. We have proved thatEV71 2B competitively inhibits ubiquitin E3 ligase of Mcl-1. In the presence of EV71 2B,ubiquitination of Mcl-1 is inhibited due to the blocking of E3 liagase (MULE) docking site(Bcl-2 homologue domain 3) by EV71 2B. As a result, Mcl-1 stability is increased andcellular apoptosis is suppressed. Using EV71 infectious DNA clones, we also confirmed thatEV71 whose ability to interact with Mcl-1 was lost by mutation is defective in replication.Mcl-1 regulation by a viral protein is a novel finding. Our earlier studies have not onlyimproved our understanding of the mechanism by which EV71 modulates host cell apoptosisbut also raised several important questions worthy of further studies. Whether EV71 uses thesame molecular mechanism to regulate other Bcl-2 members remains unknown and will bethe aims of the first part of this proposal. We will first work on several Bcl-2 family members,including Bcl-2, Bcl-2 A1, BiK, and Noxa. The study will also investigate if EV71 2B proteinregulates ubiquitination of proteins other than Bcl-2 family members. Bcl-2 family proteinsare involved in many human diseases including cancers. Our study will advance ourunderstating of pathogenesis and possibly shade new lights on the therapy of these diseases.On the other hand, in the yeast two-hybrid screening, we also identified several othercellular factors as EV71 2B-interacting proteins. These included tumor necrosis factorreceptor superfamily (TNFRSF) members: TNFRSF9 and TNFRSF18. In addition, interferonalpha-inducible protein 27 (IFI27) was also picked up by the screening. These molecules areinvolved in signaling of T cell activations. The second part of this project is to study thebiological consequences of these interactions. Whether EV71 2B protein is involved in theregulation of host T cell activation is of special interests because severe EV71 infectionstrigger severe systemic inflammatory responses clinically. The clinical presentation cannot befully explained by direct viral invasions only. Over activation or dysregulation of T cellactivities may also play important roles. EV71 possibly execute its modulation on hostcytokine signaling through 2B/TNFRSF interactions. Understanding of these molecularpathways will provide basis not only for basic virologic studies but also studies of therapeutic options in the future.