Molecular mechanisms of nuclear translocation of TRAIL-R2

质膜表达的TRAIL-R1和TRAIL-R2可以诱导细胞死亡和多种非凋亡信号通路，作为对其配体TRAIL结合的反应。在肿瘤中，这些受体主要存在于细胞内、细胞质和细胞核中。有趣的是，TRAIL-R2在细胞内的高表达往往与患者的预后不良有关。我们发现，在肿瘤细胞中，TRAIL-R2主要位于细胞核内，在那里它与核心微处理器复合体及其附属蛋白相互作用。通过这些相互作用，核TRAIL-R2(nTRAIL-R2)抑制miRNA let-7的成熟，从而增加肿瘤细胞的恶性程度。此外，我们未发表的数据表明，核TRAIL-R2与P53肿瘤抑制蛋白相互作用，并对其功能产生负面影响。此外，我们观察到TRAIL-R2与染色质部分相关，这表明nTRAIL-R2可能在基因表达调控中发挥作用。TRAIL-R2参与调节let-7的成熟以及wt-p53的功能，对于理解这些受体在恶性转化中的作用是至关重要的，并为治疗策略开辟新的视角。然而，为了制定这样的策略，必须了解TRAIL-R2的核转位机制。在本项目中，我们将阐明：i)TRAIL受体和ii)其他TRAIL受体对TRAIL-R2核转位的影响。3)揭示TRAIL-R2核进出口机制。特别是，我们将确定依赖和非依赖细胞内吞作用的重要性，Sec61转运蛋白以及Importinβ和Cm-1/Exportin 1。iv)此外，我们还将确定TRAIL-R2负责其核转位的蛋白质结构域。V)使用染色质免疫沉淀技术结合质谱仪和芯片序列，我们将分别鉴定与TRAIL-R2相互作用的染色质相关蛋白和DNA序列。我们相信，我们的项目结果将有助于揭示nTRAIL-R2的进一步功能，从而揭示TRAIL受体生物学的新方面。了解TRAIL-R2在细胞内运输的机制，特别是其核转位，将有助于设计药物来操纵其在细胞内的分布，从而导致新的治疗策略的发展。

Plasma membrane expressed TRAIL-R1 and TRAIL-R2 can induce cell death and diverse non-apoptotic signaling pathways as a response to the binding of their ligand TRAIL. In tumors, these receptors are present mainly intracellularly, in the cytoplasm and in the nucleus. Interestingly, high intracellular expression of particularly TRAIL-R2 frequently correlates with bad patient’s prognosis. We found that in tumor cells TRAIL-R2 is located predominantly in the nucleus where it interacts with the core microprocessor complex and its accessory proteins. Via these interactions, nuclear TRAIL-R2 (nTRAIL-R2) inhibits the maturation of the miRNA let-7 thereby increasing tumor cell malignancy. In addition, our unpublished data show that nuclear TRAIL-R2 interacts with the p53 tumor suppressor protein and negatively influences its functions. Furthermore, our observation that TRAIL-R2 associates with the chromatin fraction indicates a possible role of nTRAIL-R2 in the regulation of the gene expression. The involvement of TRAIL-R2 in the regulation of let-7 maturation as well as the functions of wt p53 is of utmost importance for the understanding of the role of these receptors in the malignant transformation and opens new perspectives for therapeutic strategies. However, for the development of such strategies, it is mandatory to understand the mechanisms of the nuclear translocation of TRAIL-R2. In this project, we will clarify: i) the impact of TRAIL and ii) other TRAIL receptors on the nuclear translocation of TRAIL-R2. iii) We will uncover the mechanisms of nuclear import/export of TRAIL-R2. In particular, we will determine the importance of Clathrin-dependent and -independent endocytosis, Sec61-translocons as well as Importin β and CRM-1/Exportin 1. iv) In addition, we will identify the protein domains of TRAIL-R2 responsible for its nuclear translocation. v) Using chromatin immunoprecipitation techniques coupled to mass spectrometry and ChIP-Seq, we will identify TRAIL-R2-interacting chromatin-associated proteins and DNA-sequences, respectively. We are convinced that the results of our project will allow uncovering further functions of nTRAIL-R2 and thereby novel aspects of TRAIL-Receptor biology. Understanding the mechanisms of intracellular trafficking of TRAIL-R2, in particular its nuclear translocation will allow designing drugs for manipulating its intracellular distribution which subsequently will lead to the development of novel therapeutic strategies.