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，发现新的抗动脉粥样硬化的治疗策略需要深入了解血管细胞用于消炎的内源性机制。使用基于细胞的模型和小鼠模型，我们发现巨噬细胞中L13a的遗传缺陷导致了不受控制的炎症和结果疾病，可能是通过取消L13a依赖的一组编码炎症蛋白的mRNAs的翻译沉默，例如趋化因子和趋化因子受体。巨噬细胞特异性L13a基因敲除(KO)小鼠在诱导实验性内毒素血症后不能消除炎症，并在apoE-/-背景下饲养这些小鼠，随后用高脂饮食挑战显示显著增加了动脉粥样硬化。我们还发现，在单核/巨噬细胞中，L13a从60S核糖体亚基释放并组装到干扰素-γ激活的翻译抑制物(GATT)复合体中，该复合体与位于靶ʼ3 MRNA非翻译区的步态元件结合，是翻译沉默所必需的。然而，一个靶mRNA的步态元件并不能有效地与其他靶mRNAs的步态元件竞争与步态复合体的结合。这表明，要么相同的步态复合体对这些元素有不同的亲和力，要么存在不同的含有L13a的步态复合体。综上所述，这些结果导致我们假设，适当的含有L13a的步态复合体的存在及其与靶mRNAs的同源步态元素的结合，通过防止血管斑块和循环白细胞的细胞景观的改变，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验证我们的假设：(1)测试L13a缺陷对血管斑块细胞景观以及对循环和组织白细胞的影响。为此，我们将利用L13a-KO动物对L13a耗尽的巨噬细胞进行全面的分析，并测试它们动员、代谢胆固醇、提呈抗原、参与吞噬活性、极化和Nlrp3炎性小体激活的能力。(2)检测顺式作用步态元件和RNA结合步态复合体的异质性。为此，我们将进行RNA结合分析，进行竞争研究和RNA亲和纯化，以确定步态复合体中新的辅助蛋白。(3)原代细胞中L13a核糖体掺入机制的研究。为此，我们将用野生型和核糖体掺入缺陷突变体补充来自胚胎的L13a/-成纤维细胞和巨噬细胞特异性的L13a-KO小鼠的L13a-/-巨噬细胞，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.