内质网应激下IRE1alpha活性调控机制研究

The unfolded protein response (UPR) is an evolutionarily conserved intracellular signaling pathway triggered when unfolded proteins accumulate in the endoplasmic reticulum (ER) to cause “ER stress”. While the UPR initially serves to maintain ER homeostasis (“Adaptive UPR”), under irremediable ER stress it causes critical loss of cell function or even cell death (“Terminal UPR”). Irregular balance between Adaptive and Terminal UPR could lead to ER-stress-related diseases, such as neurodegenerative disease and diabetes. .The most ancient UPR component, Inositol-requiring enzyme-1alpha (IRE1alpha) plays a pivotal role in the UPR system. Upon sensing ER stress, active IRE1alpha as an RNase initiates splicing of XBP1 mRNA (which encodes a transcription factor). XBP1 up-regulates expression of chaperones and foldases, allowing the ER to adapt to ER stress. However, IRE1alpha could also degrade many RNAs that are spatially close to the ER, which largely contributes to the Terminal UPR. While these two IRE1alpha RNase activities have different influence on the cell fate, little is known about the regulatory mechanism of the RNase substrate specificity and the physiological significance of the RNA degradation by IRE1alpha. We propose to address these questions by performing deep sequencing, biochemical and cell biology assays. We anticipate to identify IRE1alpha’s RNase substrate genome-wide, and to understand how the RNase activity towards different substrates are regulated. The study will advance our understanding on the regulation of UPR balance, and may also facilitate development of new strategies against ER stress-related diseases.

内质网内蛋白质错误折叠和聚集（“内质网应激”）可启动 “去折叠蛋白响应”（UPR）以维持蛋白质内稳态（“适应性UPR”），但长期、不可逆的内质网应激下UPR也可造成细胞功能丧失和细胞凋亡（“终结性UPR”）。IRE1a是最保守的UPR启动分子。作为RNase，IRE1a能特异剪切转录因子XBP1 的mRNA，启动有活性XBP1的翻译及下游适应性UPR，也能降解多种靠近内质网膜的mRNA及非编码RNA，造成终结性UPR。虽然IRE1a的这两种RNA酶活性对细胞造成截然不同的影响，但目前人们对IRE1a的RNA酶底物选择性、哪些RNA可能被IRE1a降解及其生理意义知之甚少。本项目将针对这一问题，综合运用高通量测序、生化、细胞生物学实验手段，鉴定IRE1a的RNA底物，考察IRE1a底物特异性的调控机制及其生理意义。该工作将帮助我们更好的认识UPR的调节机制，为治疗UPR相关疾病提供新思路。

内质网腔中的蛋白质发生错误折叠和聚集会导致内质网应激，进而启动未折叠蛋白响应（UPR）。一方面，UPR能通过上调分子伴侣和折叠酶的表达、促进错误折叠蛋白降解以帮助细胞恢复稳态；另一方面，UPR也能导致细胞凋亡。IRE1alpha是最为保守的UPR通路，本研究关注IRE1alpha活性的调控机制，并试图发现IRE1的新功能，揭示IRE1alpha活性调控的生理意义。.我们发现Mn2+调控IRE1alpha的激酶活性和二聚化水平。在THP-1、B16.F10和MDA-MB-231等细胞上，Mn2+均能显著促进IRE1alpha-TRAF2互作，激活下游JNK和NFkB磷酸化，进而上调细胞因子IL6的表达。利用重组表达蛋白和体外生化实验，我们证明了Mn2+作用于IRE1alpha胞浆段。虽然Mn2+不影响xbp1 mRNA剪接，但XBP1S和JNK磷酸化均在IL6的表达调控中发挥重要作用。我们的研究揭示了IRE1alpha在天然免疫应答中的新功能，特别是鉴定到Mn2+能有效调控IRE1alpha活性，而这种调控能大大促进其在天然免疫应答中的功能发挥。这为认识UPR的功能提供了新思路，也有望为肿瘤免疫治疗提供新策略。.我们还观察到持续性内质网应激下xbp1 mRNA剪接水平发生升-降-升的波动，而这似乎与细胞增殖能力相关，也与细胞在内质网应激下抗凋亡能力有关。相关分子机制还在进一步研究中。该工作发现了持续性内质网应激下UPR调控的新现象。由于生理和生理病理条件下的内质网应激往往是长期的，该研究对于我们深入理解这些条件下UPR的动态调控机制和生理功能提供了新视角。.我们还与生物物理研究所王志珍、王磊研究员课题组合作，揭示了PDI在内质网应激早期结合并负调控IRE1alpha活性的分子机制。.综上，我们围绕IRE1alpha活性的调控机制，揭示了内质网腔和胞浆区IRE1alpha调控的新机制，发现了持续性内质网应激下IRE1alpha活性动态调控的新现象，推进了对UPR调控机制和生理功能的理解。