Analysis of Radiomitigative Cell Signaling

描述(由申请人提供)：通过核事故意外暴露在辐射中，或爆炸“脏弹”，可能会造成毁灭性的后果。我们开发了一种新的磷脂生长因子溶血磷脂酸(LPA)的类似物，十八烯基硫代磷酸盐(OTP)，当它在受到致死剂量的伽马射线照射的小鼠照射后24小时内应用时，通过从细胞凋亡中拯救细胞和挽救生命，显示出强大的放射减肥作用。实验表明，OTP的分子靶点是LPA2 G蛋白偶联受体。本应用的目的是阐明LPA2受体特有的放射增敏作用机制。这项工作的中心假设是，在LPA2受体的C末端形成的大分子蛋白质复合体是OTP辐射防护作用所必需的。这与LPA2受体偶联的G蛋白介导的信号事件足以引发放射缓解的概念有很大不同。LPA2明显不同于其C末端的其他LPA受体亚型，它与PDZ结构域结合蛋白和LIM结构域蛋白形成蛋白质-蛋白质相互作用。我们假设，这些LPA2与蛋白质的相互作用调节多个促生存信号通路(ERK1/2和AKT)，并抑制SIVA-1的作用，SIVA-1是一种将DNA损伤与凋亡级联联系起来的促凋亡蛋白。这一假设超出了LPA2 GPCR，因为负责这些蛋白质-蛋白质相互作用的序列基序在许多其他GPCR中都存在。其具体目的是：1.验证C-末端PDZ基序是辐射防护所必需的假设。2.通过确定是否需要与LIM结构域蛋白TRIP6和SIVA-1的物理相互作用来表征LPA2受体C端55个氨基酸与LIM结构域蛋白TRIP6和SIVA-1相互作用所需的基序。3.剖析LPA2-TRIP6-c-Src-ERK/AKT信号轴在抗细胞凋亡中的作用。4.阐明LPA2-SIVA-1-BCL-XL信号轴在抗细胞凋亡中的作用。深入了解OTP的作用机制对于科学、调控和治疗具有极其重要的意义。不容忽视的是，OTP还可能在减轻放射和化疗的副作用以及与程序性细胞死亡增加相关的疾病方面提供潜在的治疗应用。了解促进生存的gpr和凋亡机制之间的信号相互作用将为识别新的治疗靶点和gpr配体铺平道路。通过核事故或“脏弹”爆炸意外暴露在辐射中，可能会给公众带来毁灭性的后果。我们开发了一种新的磷脂生长因子溶血磷脂酸(LPA)的类似物，十八烯基硫代磷酸(OTP)。在受到致死剂量的伽马射线照射的小鼠照射后24小时内，OTP通过将细胞从凋亡中解救出来并挽救生命而显示出显著的放射防护作用。OTP具有理想的辐射防护剂/放射增强剂的许多特性：a)它在室温下稳定多年；b)它可以为各种类型的患者配制；c)它相对容易合成和制造，并且价格低廉；d)它在辐射损伤后12小时内给予时有效。体外和基因敲除(KO)小鼠的实验表明，OTP的分子靶点是LPA2 G蛋白偶联受体(GPCR)。这项拨款申请的目的是阐明LPA2受体亚型特有的抗凋亡作用机制。

DESCRIPTION (provided by applicant): Unintended exposure to radiation via a nuclear accident, or explosion of a "dirty bomb," can have devastating consequences. We developed a novel analog of the phospholipid growth factor lysophosphatidic acid (LPA), octadecenyl thiophosphate (OTP), which when applied up to 24h post-irradiation in mice irradiated with lethal doses of gamma-irradiation shows strong radiomitigating action by rescuing cells from apoptosis and saving life. Experiments indicate that the molecular target of OTP is the LPA2 G protein-coupled receptor. The objective of this application is to elucidate the radiomitigating mechanism of action unique to the LPA2 receptor. The central hypothesis of the proposed work is that macromolecular protein complexes formed at the C-terminal tail of the LPA2 receptor are required for the radioprotective effect of OTP. This is a significant departure from the concept that the LPA2 receptor coupled G protein-mediated signaling events are sufficient to elicit radiomitigation. LPA2 is distinctly different from the other LPA receptor subtypes in its C- terminal tail, where it forms protein-protein interactions with PDZ domain-binding proteins and with LIM domain proteins. We hypothesize that these LPA2-protein interactions regulate multiple prosurvival signaling pathways (ERK1/2 and AKT) and also arrest the actions Siva-1, a proapoptotic protein that links DNA damage to the apoptotic cascade. This hypothesis goes beyond the LPA2 GPCR because the sequence motifs responsible for these protein-protein interactions are present in many other GPCR. The specific aims are: 1. Test the hypothesis that the C-terminal PDZ motif is required for the radioprotective effect. 2. Characterize the motif in the C terminal 55 amino acids of the LPA2 receptor required for the interactions with the LIM domain proteins TRIP6 and Siva-1 by determining whether physical interaction with them is required for the antiapoptotic effect. 3. Dissect the role of LPA2-TRIP6 - c-Src - ERK/AKT signaling axis in the antiapoptotic effect. 4. Elucidate the role of the LPA2 - Siva-1- BCL-XL signaling axis in the antiapoptotic effect. An in-depth understanding of OTP's mechanism of action is of paramount importance for scientific, regulatory, and therapeutic reasons. Not negligible is the fact that OTP might also provide potential therapeutic applications in the attenuation of the side effects of radiation- and chemotherapy, as well as diseases associated with increased program cell death. Understanding the signaling interactions between pro-survival GPCR and the apoptotic machinery will pave the way to the identification of new therapeutic targets as well as GPCR ligands. Unintended exposure to radiation via a nuclear accident, or explosion of a "dirty bomb," can have devastating consequences to the public. We developed a novel analog of the phospholipid growth factor lysophosphatidic acid (LPA), octadecenyl thiophosphate (OTP). When applied up to 24 h post-irradiation in mice irradiated with lethal doses of gamma-irradiation, OTP shows significant radiomitigating action by rescuing cells from apoptosis and saving life. OTP has many of the properties of an ideal radioprotectant/radiomitigator: a) it is stable at room temperature for years; b) it can be formulated for a wide variety of patient types; c) it is relatively easy to synthesize and manufacture, and inexpensive; and d) it is effective when given up to 12 h after radiation injury. Experiments conducted in vitro and with knockout (KO) mice indicate that the molecular target of OTP is the LPA2 G protein-coupled receptor (GPCR). The objective of this grant application is to elucidate the an- tiapoptotic mechanism of action unique to the LPA2 receptor subtype