TIFA介导DNA损伤诱发的NF-kappaB激活和衰老相关分泌表型的分子生物学机制

Challenges to the genome integrity would elicit DNA damage response (DDR). DNA damages and DDR events, including the special irreparable telomeric DNA damage could be detected in replicative and stress-induced cellular senescence, as well as in over-activated oncogene-associated senescence barrier models. Among the arrays of distinctive phenotypesand biomarkers in cellular senescence, there was a burst of nvestigations on secretory factors associated with inflammation and malignancy, which were designed as "senescence-associated secretory phenotype" (SASP) owing to their induction by replicative or stress-triggered senescence. These myriad factors, including panels of cytokines, chemokines and interferons, contribute to aging process in an autocrine, paracrine or endocrine fashion via their collaborations with DNA damage response. Nuclear factor-kappaB (NF-kappaB) is the master regulators responsible for transcriptional activation of inflammation machinery, and subunit of this family was identified as major transcription factor accumulated on chromatin of senescent cells. However, the molecular mechanisms linking nuclear DDR and cytoplasmic IKK (Inhibitor of Nuclear Factor kappa-B Kinase) activation in the context of cellular senescence remains not fully understood. In our pilot experiments, a FHA (Forkhead-associated) domain-containing protein TIFA (TRAF-interacting protein with an FHA domain) without any previously identified roles in DDR-elicited NF-kappaB activation, was observed to be translocated into nucleus and accumulated on damaged chromatin following genotoxic stress. Biochemical and molecular biological experiments further revealed that under DNA damage conditions TIFA promoted ubiquitination of NEMO (NF-kappaB Essential Modulator), which was a key transducer of DDR signals to cytoplasmic IKK complex assembly, and that TIFA could induce NF-kappaB activation and SASP subsequently. We believe the elucidation of mechanistic details for TIFA-mediated transduction of DDR signals to NF-kappaB activation and TIFA-elicited SASP would not only contribute to the molecular dissection of aging and senescence barrier in caners, but also provide novel pharmatheutical target for taking advantage of SASP in cancer development and metastasis.

基因组DNA的损伤会激活细胞内的损伤应答(DDR)通路。复制性衰老、应激诱导的衰老、以及原癌基因激活的衰老屏障等细胞衰老模型中都有明显的DNA损伤和DDR。衰老相关的分泌表型(SASP)是新近发现的细胞衰老关键分子特征之一。NF-kappaB作为调控诸多炎性分泌基因表达水平的核心转录因子，在SASP的发生和发展过程中起着重要作用。通过前期的功能筛选实验，我们找到一个具有FHA结构域的炎症相关蛋白TIFA，可以在DNA损伤条件下入核并明显富集于染色质上。初步实验发现TIFA可以通过促进DNA损伤时NEMO的泛素化修饰，诱导NF-kappaB的激活以及衰老相关分泌表型。我们相信深入探索以TIFA为核心节点的、能在DNA损伤条件下转导损伤信号到NF-kappaB的信号通路的分子机制和生物学功能，对于我们理解SASP的分子机理并通过SASP来调控衰老进程、肿瘤发生和转移有非常重要的意义。

衰老相关分泌表型（senescence-associated secretory phenotypes，SASP）是由老化细胞分泌的一组蛋白的总称，主要包括炎症及免疫调节相关的细胞因子、趋化因子、生长因子以及一些细胞表面分子。SASP是细胞衰老一个特征性表现，与衰老进展和老年疾病关系极为密切。转录因子NF-kappaB是调节SASP发生的枢纽，而DNA损伤是NF-kappaB通路激活的重要刺激信号之一。DNA损伤应激条件下，关键蛋白NEMO（NF-kappaB essential modulator）通过SUMO化，磷酸化和泛素化等一系列转录后修饰，完成从细胞浆到细胞核再到细胞浆的穿梭，将核内的损伤信号扩散到核外，最终引起NF-kappaB信号通路的活化。然而，这些分子过程的链接和偶联机制仍有很大的探索空间。通过本项目的实施，我们发现TIFA作为细胞浆中NF-kappaB的激活因子，在DNA损伤后转位到细胞核并在染色质上聚集。而过表达TIFA可以显著增强DNA损伤诱导的NEMO泛素化修饰，促进NF-kappaB下游靶基因的转录激活，引起SASP相关细胞因子如白介素-6和白介素-8的分泌增加。进一步，我们发现TIFA参与的NF-kappaB通路激活依赖于其第9位苏氨酸及FHA结构域；而当此苏氨酸或FHA结构域突变后，NF-kappaB通路的活化作用减弱，并伴随NEMO泛素化修饰水平的降低。通过蛋白质组学实验我们阐明：TRAF2作为TIFA的互作蛋白和E3泛素连接酶，可以在NF-kappaB激活过程中联合TIFA增加NEMO的泛素化水平；而TRAF2的敲低极大地阻碍了此修饰的建立。在多发性骨髓瘤和细胞衰老SASP模型中，TIFA同样可以促进DNA损伤诱导的NF-kappaB通路激活以及下游基因的转录和相关因子的分泌。这项研究既有助于我们深入理解SASP的分子机理，也为通过SASP干预衰老和肿瘤的发生提供了新的分子靶标。