Translational Silencing in Monocytes: Role of L13a

 描述（由申请人提供）：涉及血管壁细胞的未解决的炎症在动脉粥样硬化的发病机制中起着至关重要的作用。因此，发现抗动脉粥样硬化的新治疗策略需要深入了解血管细胞解决炎症的内源性机制。使用基于细胞的和鼠模型，我们表明巨噬细胞中L13 a的遗传缺陷导致不受控制的炎症和由此产生的疾病，这可能是通过消除编码炎性蛋白质的一组mRNA的L13 a依赖性翻译沉默，趋化因子和趋化因子受体。巨噬细胞特异性L13 a基因敲除（KO）小鼠在诱导实验性内毒素血症后未能解决炎症，并且在apoE-/-背景下饲养这些小鼠，随后用高脂饮食激发显示动脉粥样硬化显著增加。我们还表明，在单核细胞/巨噬细胞中，L13 a从60 S核糖体亚基释放并组装成IFN-γ激活的翻译抑制剂（GAIT）复合物，该复合物结合位于靶mRNA的3个非翻译区（UTR）中的GAIT元件，对于翻译沉默是必不可少的。然而，来自一种靶mRNA的GAIT元件不能有效地与其他靶mRNA的GAIT元件竞争结合GAIT复合物。这表明相同GAIT复合物对那些元件的不同亲和力或存在不同的含L13 a的GAIT复合物。总之，这些结果使我们假设适当的含L13 a的GAIT复合物的存在及其与靶mRNA的同源GAIT元件的结合通过防止血管斑块和循环白细胞的细胞景观的改变而导致炎症的消退和动脉粥样硬化的预防。我们将通过追求以下三个具体目标来测试我们的假设：（1）测试L13 a缺陷对血管斑块的细胞景观以及对循环和组织白细胞的影响。在这个目标中，使用L13 a-KO动物，我们将对L13 a耗尽的巨噬细胞进行全面分析，并测试它们动员、代谢胆固醇、呈递抗原、参与吞噬活性、极化和Nlrp 3炎性体活化的能力。(2)测试顺式作用GAIT元件和RNA结合GAIT复合物的异质性。在这个目标中，我们将进行RNA结合分析的竞争研究和RNA亲和纯化，以确定新的辅助蛋白的GAIT复合物（3）的核糖体掺入的机制的研究L13 a在原代细胞。在这个目标中，我们将补充来自胚胎的L13 a-/-成纤维细胞和来自巨噬细胞特异性L13 a-KO小鼠的L13 a-/-巨噬细胞与野生型和核糖体缺失突变体，以研究该蛋白的亚细胞定位及其与其他组装因子的相互作用。

 DESCRIPTION (provided by applicant): Unresolved inflammation involving cells of the blood vessel wall plays a crucial role in the pathogenesis of atherosclerosis. Therefore, discovery of novel therapeutic strategies against atherosclerosis requires an in- depth understanding of the endogenous mechanisms adopted by vascular cells to resolve inflammation. Using cell-based and murine models we showed that genetic deficiency of L13a in macrophages leads to uncontrolled inflammation and resultant disease, perhaps by abrogation of L13a-dependent translational silencing of a group of mRNAs encoding inflammatory proteins e.g., chemokines and chemokine receptors. Macrophage-specific L13a-knockout (KO) mice fail to resolve inflammation upon induction of experimental endotoxemia and breeding these mice on an apoE-/- background and subsequent challenge with a high-fat diet showed significantly increased atherosclerosis. We also showed that in monocytes/macrophages, the release of L13a from the 60S ribosomal subunit and its assembly into the IFN-γ-activated inhibitor of translation (GAIT) complex which binds to the GAIT element located in the 3ʼ untranslated region (UTR) of target mRNAs, is essential for translational silencing. However the GAIT element from one target mRNA does not efficiently compete with the GAIT elements of other target mRNAs for the binding to GAIT complexes. This suggests either different affinities of the same GAIT complex for those elements or the presence of distinct L13a- containing GAIT complexes. Together, these results lead us to hypothesize that the presence of appropriate L13a-containing GAIT complexes and their binding to cognate GAIT elements of the target mRNAs leads to resolution of inflammation and prevention of atherosclerosis by preventing alterations of the cellular landscape of vascular plaques and circulating leukocytes. We will test our hypothesis by pursuing the following three specific aims: (1) Testing the impact of L13a deficiencies on the cellular landscapes of vascular plaques as well as on circulating and tissue leukocytes. In this aim using L13a-KO animals we will undertake a comprehensive analysis of L13a-depleted macrophages and test their ability to mobilize, metabolize cholesterol, present antigen, engagement in phagocytic activity, polarization and Nlrp3 inflammasome activation. (2) Testing the heterogeneity of the cis-acting GAIT elements and RNA-binding GAIT complexes. In this aim we will perform RNA-binding analysis for competition studies and RNA-affinity purification to identify new accessory proteins in the GAIT complex (3) Studies of the mechanisms of ribosomal incorporation of L13a in primary cells. In this aim we will complement the L13a-/- fibroblasts from embryo and L13a-/- macrophages from macrophage-specific L13a-KO mice with wild type and ribosome incorporation-defective mutants to study the subcellular localization of this protein and its interactions with other assembly factors.