Analysis of Radiomitigative Cell Signaling

描述（由申请人提供）：核事故或“脏弹”爆炸造成的意外辐射暴露可能造成毁灭性后果。我们开发了磷脂生长因子溶血磷脂酸（LPA）的一种新的类似物，十八烯基硫代磷酸盐（OTP），当在用致死剂量的γ射线照射的小鼠中照射后24小时应用时，其通过拯救细胞免于凋亡和挽救生命而显示出强的放射性激动作用。实验表明，OTP的分子靶点是LPA 2 G蛋白偶联受体。本申请的目的是阐明LPA 2受体特有的放射性激动作用机制。所提出的工作的中心假设是，在LPA 2受体的C-末端尾部形成的大分子蛋白质复合物是OTP的辐射防护作用所必需的。这与LPA 2受体偶联G蛋白介导的信号传导事件足以引发放射性激动的概念有显著的偏离。LPA 2与其它LPA受体亚型在其C-末端尾部明显不同，其中其与PDZ结构域结合蛋白和与LIM结构域蛋白形成蛋白质-蛋白质相互作用。我们假设这些LPA 2-蛋白质相互作用调节多种促生存信号通路（ERK 1/2和AKT），也阻止了Siva-1的作用，Siva-1是一种将DNA损伤与凋亡级联反应联系起来的促凋亡蛋白。这一假设超越了LPA 2 GPCR，因为负责这些蛋白质-蛋白质相互作用的序列基序存在于许多其他GPCR中。具体目标是：1.检验C-末端PDZ基序是辐射防护作用所需的假设。2.通过确定抗凋亡作用是否需要与LIM结构域蛋白TRIP 6和Siva-1的物理相互作用，表征LPA 2受体C末端55个氨基酸中与LIM结构域蛋白TRIP 6和Siva-1相互作用所需的基序。3.探讨LPA 2-TRIP 6- c-Src - ERK/AKT信号轴在抗凋亡作用中的作用。4.阐明LPA 2- Siva-1- BCL-XL信号轴在抗凋亡作用中的作用。深入了解OTP的作用机制对于科学，监管和治疗原因至关重要。不可忽视的是，OTP还可能在减轻放疗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。了解促存活GPCR和凋亡机制之间的信号相互作用将为鉴定新的治疗靶点以及GPCR配体铺平道路。核事故或“脏弹”爆炸造成的意外辐射暴露可能对公众造成毁灭性后果。我们开发了一种新的类似物的磷脂生长因子溶血磷脂酸（LPA），十八烯基硫代磷酸（OTP）。当在用致死剂量的γ-辐射照射的小鼠中照射后24小时应用时，OTP通过拯救细胞免于凋亡和挽救生命而显示出显著的辐射诱导作用。OTP具有理想的辐射防护剂/辐射抑制剂的许多特性：a）它在室温下稳定多年; B）它可以配制用于各种患者类型; c）它相对容易合成和制造，并且便宜;以及d）当在辐射损伤后长达12小时给予时，它是有效的。在体外和敲除（KO）小鼠中进行的实验表明，OTP的分子靶标是LPA 2 G蛋白偶联受体（GPCR）。该基金申请的目的是阐明LPA 2受体亚型独特的抗凋亡作用机制

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