Signaling via proteolysis of the interferon receptor

描述（由申请人提供）：I型干扰素（IFN）调节对病毒感染的先天性免疫应答，并具有抗增殖和促凋亡作用，已用于治疗癌症。此外，I型IFN信号传导是螺旋细胞因子大家族的模型，其调节增殖、分化和免疫应答的许多方面。因此，研究I型IFN信号有望影响我们对癌症和其他疾病的理解，并促进治疗方式的发展。I型IFN可以通过JAK-STAT途径发出信号。简而言之，受体（IFNaR 1和IFNaR 2）依赖于JAK酪氨酸激酶（Tyk 2和Jak 1）来启动信号传导。IFN结合触发JAK活化和两种STAT（Statl和Stat 2）的磷酸化。STAT然后异源二聚化，与干扰素调节因子9（Irf 9）复合，易位到细胞核并结合IFN调节基因上游的应答元件。以前，这项资助支持了我们对这种典型信号级联的多个方面的研究。最近，在研究Stat 2和IFNaR 2之间的相互作用的过程中，我们发现I型IFN以类似于Notch和阿尔茨海默氏症前体蛋白所采用的机制的方式诱导IFNaR 2亚基的两步蛋白水解。裂解也自发发生，并响应于诱导PKC活化的各种刺激。由金属蛋白酶TACE介导的初始切割释放大部分胞外域，由膜内早老蛋白酶进行的第二次切割释放IFNaR 2的胞内结构域（ICD）。初步数据表明ICD能够进行核转位，并且IFNaR 2的该片段可以调节基因转录并抑制细胞增殖，这表明I型IFN可能通过受调节的膜内蛋白水解（RIP）机制发出信号。因此，本更新申请的总体目标是确定I型IFN是否可以在生理相关的情况下通过RIP发出信号。此外，RIP是否代替或补充经典的JAK-STAT信号通路？提出了四个具体目标来检验这一假设。目的1确定I型IFN是否诱导内源性IFNaR 2 ICD的裂解和核转位，并表征启动ICD产生的机制。接下来，该提案中的关键实验将通过鉴定IFNaR 2上的蛋白酶切割位点（目的2）并确定阻止切割的突变是否扰乱I型IFN的生理效应（目的3）来检验IFN的生理效应需要切割的假设。最后，目的4通过测试ICD与Stat 2和Irf 9复合物调节基因转录的假设来解决ICD介导的基因调控机制。

DESCRIPTION (provided by applicant): The type I interferons (IFNs) regulate the innate immune response to viral infection and have anti-proliferative and pro-apoptotic action which has been exploited in the treatment of cancer. Furthermore, type I IFN signaling is a model for the large family of helical cytokines, which regulate many aspects of proliferation, differentiation and the immune response. Thus, studying type I IFN signaling is expected to impact our understanding of cancer and other diseases and facilitate the development of therapeutic modalities. Type I IFNs can signal through the JAK-STAT pathway. In brief, receptors (IFNaRI and IFNaR2) rely on JAK tyrosine kinases (Tyk2 and Jak1) to initiate signaling. IFN binding triggers JAK activation and phosphorylation of two STATs (Statl and Stat2). The STATs then heterodimerize, complex with the interferon regulatory factor 9 (Irf9), translocate to the nucleus and bind a response element upstream of IFN-regulated genes. Previously, this grant has supported our investigation of multiple aspects of this canonical signaling cascade. Recently, in the course of investigating the interaction between Stat2 and IFNaR2, we found that type I IFNs induce a two-step proteolysis of the IFNaR2 subunit in a manner that resembles the mechanism employed by Notch and the Alzheimer's precursor protein. Cleavage also occurs spontaneously and in response to various stimuli that induce PKC activation. An initial cleavage, mediated by the metalloprotease TACE, releases most of the ectodomain and a second cleavage by the intramembrane presenilin proteases releases the intracellular domain (ICD) of IFNaR2. Preliminary data indicates the ICD is capable of nuclear translocation and that this fragment of IFNaR2 can modulate gene transcription and inhibit cell proliferation, suggesting that type I IFNs might signal via a regulated intramembranous proteolysis (RIP) mechanism. Thus, the overall goal of this renewal application is to determine if type I IFNs can signal, in a physiologically relevant context, via RIP. Moreover, does RIP act in lieu of, or in addition to, the canonical JAK-STAT signaling pathway? Four specific aims are proposed to test this hypothesis. Aim 1 determines if type I IFNs induce cleavage and nuclear translocation of the endogenous IFNaR2 ICD and characterizes the mechanisms(s) initiating production of the ICD. Next, the key experiments in this proposal will test the hypothesis that cleavage is required for physiological effects of IFN by identifying the protease cleavage sites on IFNaR2 (aim 2) and determining if mutations which prevent cleavage perturb the physiological effects of the type I IFNs (aim 3). Finally, aim 4 addresses the mechanism of ICD mediated gene regulation by testing the hypothesis that the ICD functions in a complex with Stat2 and Irf9 to regulate gene transcription.