OsDer1在内质网相关蛋白降解途径中的功能及其作用机理

Regulatory mechanism of endoplasmic reticulum associated protein degradation (ERAD) is one of the basic biological questions and has been becoming one of the hot areas of modern biology. ERAD plays critical roles in removing misfolded proteins, regulating important biochemical/physiological processes, including abiotic stress tolerance and plant defense. To date, most of the ERAD studies were concentrated on yeast and mammalian systems. Recent studies revealed that the ERAD machinery is conserved in plants. Derlin 1 is involved in substrate recognition, ubiquitination, and extraction of ERAD substrates. However, less is known about the function of Derlins in plants. Previously, we identified a Derlin-like protein (OsDer1) through proteomic analysis of the ER isolated from ER-stressed developing rice seed. The expression of OsDer1 was dramatically upregulated under ER-stress conditions. We also obtained some candidate proteins that interact with OsDer1 through CoIP experiments. In this study, we will focus on the identification and characterization of OsDer1 via biochemical, molecular and cellular biological strategies. The expected results will help us to reveal the molecular mechanisms of ERAD in rice. We will also evaluate the potentials of OsDer1 in stress-tolerance rice breeding.

内质网相关蛋白降解（ERAD）调控机制是生物学的一个基本问题，已成为当代生物学的热点研究领域之一。ERAD在非正确折叠蛋白降解、缓解内质网胁迫、维持ER平衡过程中发挥重要功能，参与生物体的抗病及抗胁迫等多个重要生物学过程。ERAD是由许多因子参与的复杂调控过程，其中Derlin蛋白在错误折叠蛋白底物识别、泛素化和逆向运输中发挥重要作用。目前对Der1功能研究方面多限于哺乳动物和酵母中，对其在植物中的功能尚少见报道。在前期通过对ER胁迫水稻种子内质网膜蛋白进行蛋白质组学分析发现一个表达量极显著上调的Derlin-like（OsDer1）蛋白，并证明其表达受ER胁迫诱导。同时利用免疫共沉淀技术获得了一些可能与OsDer1相互作用的蛋白。在此基础上，本研究对其功能进行详尽研究，以期逐步揭示水稻ERAD的遗传调控机制，对高等植物的ERAD机理提出新的见解，具有重大的理论意义。

水稻种子是蛋白质和淀粉的储藏器官。在逆境环境下，灌浆期种子会积累大量错误折叠和非折叠蛋白，产生内质网胁迫，进而影响种子产量和品质。内质网相关蛋白降解（ERAD）途径在清除错误折叠蛋白以及内质网胁迫应答中发挥着重要作用。由于现有的ER胁迫诱导方法很难应用于种子系统中，因此，人们对植物种子中ERAD的机理仍知之甚少。我们在前期研究工作中发现，胚乳特异性抑制小G蛋白SAR1的表达，会导致分泌蛋白滞留在内质网中，导致水稻种子产生内质网胁迫。在此基础上，分离纯化了发生内质网胁迫的水稻种子的内质网，通过定量蛋白质组学分析，鉴定到一个内质网胁迫应答因子OsDER1。研究发现，OsDER1表达受ER胁迫诱导，过量表达或者抑制表达OsDER1会激活未折叠蛋白响应，使水稻对内质网胁迫更加敏感，泛素化蛋白的水平显著提高；抑制表达OsDER1转基因水稻种子呈现粉质、皱缩表型，淀粉粒和蛋白体I形态发生改变。进一步研究表明，OsDER1与经典的ERAD途径核心组分OsHRD1、OsHRD3以及OsCDC48均存在相互作用。研究还发现，OsDER1还与信号肽肽酶OsSPP相互作用。近期的一些研究表明，在酵母和哺乳动物系统中SPP和DER1能够形成一条新的ERAD途径，而OsDER1与OsSPP相互作用结果暗示这条ERAD途径在植物中也保守。该研究对于阐明植物ER胁迫应答机理具有重要意义。