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免疫反应，具有抗增殖和促凋亡作用，已被用于癌症的治疗。此外，I型IFN信号传导是螺旋细胞因子大家族的一个模型，它调节增殖、分化和免疫反应的许多方面。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通路发出信号。简而言之，受体（IFNaRI和IFNaR2）依赖于JAK酪氨酸激酶（Tyk2和Jak1）启动信号传导。IFN结合触发JAK激活和两种stat （Statl和Stat2）的磷酸化。然后STATs异二聚化，与干扰素调节因子9 （Irf9）复合，转移到细胞核并结合干扰素调节基因上游的应答元件。在此之前，这项拨款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Stat2和IFNaR2相互作用的过程中，我们发现I型ifn诱导IFNaR2亚基的两步蛋白水解，其机制类似于Notch和阿尔茨海默病前体蛋白所采用的机制。劈裂也会自发发生，并响应于诱导PKC激活的各种刺激。由金属蛋白酶TACE介导的初始裂解释放大部分外结构域，由膜内早老素蛋白酶介导的第二次裂解释放IFNaR2的胞内结构域（ICD）。初步数据表明，ICD具有核易位能力，IFNaR2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，这项更新应用的总体目标是确定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是否替代或补充了标准的JAK-STAT信号通路？为了验证这一假设，提出了四个具体目标。目的1确定I型ifn是否诱导内源性IFNaR2 ICD的裂解和核易位，并表征ICD产生的机制。接下来，本提案中的关键实验将通过鉴定IFNaR2上的蛋白酶裂解位点（目的2）和确定阻止裂解的突变是否会干扰I型IFN的生理作用（目的3）来验证IFN的生理作用需要裂解的假设。最后，目的4通过验证ICD与Stat2和Irf9复合物调控基因转录的假设，阐述了ICD介导基因调控的机制。

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.