Translational Silencing in Monocytes: Role of L13a

单核细胞的翻译沉默：L13a 的作用

• 批准号: 8269844
• 负责人: BARSANJIT MAZUMDER
• 金额: $ 35.15万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8269844
• 关键词: 3' Untranslated Regions; Address; Alleles; Anti-Inflammatory Agents; Anti-inflammatory; Apolipoprotein E; Atherosclerosis; Binding; Binding Sites; Biological Assay; Breeding; Cardiovascular Diseases; Cells; Ceruloplasmin; Chronic; Code; Complex; Copper; Data; Defense Mechanisms; Dependence; Disease; Elements; Event; Fractionation; Funding; Generations; Genes; Genetic Translation; Human; Immunoblot Analysis; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Interferon Type II; Interferons; Interleukin-1; Internal Ribosome Entry Site; Investigation; Knock-out; Knockout Mice; Label; Laboratories; Low Density Lipoprotein oxidation; Mediating; Messenger RNA; Metabolic; Modeling; Mononuclear; Mus; Normal Cell; Operon; Pathway interactions; Phosphorylation; Physiological; Plasma; Polyribosomes; Protein Biosynthesis; Proteins; RNA Interference; Reaction; Research; Ribonucleoproteins; Ribosomal Proteins; Ribosomes; Role; Severities; Site; TNF gene; Testing; Therapeutic Agents; Translational Repression; Translations; atherogenesis; base; cellular targeting; chemokine; cis acting element; genome wide association study; genome-wide analysis; in vivo; inhibitor/antagonist; insight; macrophage; meetings; member; monocyte; novel; novel strategies; novel therapeutics; prevent; public health relevance; receptor; reconstitution; response; small molecule

DESCRIPTION (provided by applicant): Atherosclerosis is a pathogenic consequence of uncontrolled inflammation of the resident cells of vessel wall. Using cell-based model we showed that ribosomal protein L13a-dependent translational silencing pathway could terminate the expression of a group of inflammatory genes. In this proposal we will test whether this mechanism can resolve inflammation. We have demonstrated that IFN-3 induced synthesis of Ceruloplasmin (Cp) and a group of other inflammatory proteins in monocytic cells is under translational control. Further, we discovered a crucial role of ribosomal protein L13a and its release from 60S ribosome in the formation of IFN- Gamma-Activated Inhibitor of Translation (GAIT) complex. The translational silencing mechanism relies on the recognition of L13a-dependent GAIT complex by the GAIT element present in the 3< untranslated region (UTR) of the target mRNAs. This is based on our observation that shows the depletion of L13a by RNA silencing can overcome the translation inhibition of these target mRNAs. To our surprise, the depletion of L13a had no effect on overall protein synthesis, however several Internal Ribosome Entry Site (IRES) dependent translation were inhibited. This proposal wants to test the hypothesis that L13a-dependent translational silencing of these target mRNAs coding a cluster of inflammatory proteins may serve as an endogenous defense mechanism against uncontrolled inflammation and atherosclerosis. On the other hand, the role of L13a may be confined to specialized rather than overall function of the ribosome. We will test our hypothesis by pursuing the following three specific aims: (1) Analysis of the L13a regulated and inflammation responsive post-transcriptional operon in IFN-3 activated monocytes. In these aim we will undertake a comprehensive analysis of the new target mRNAs the member of the post-transcriptional operon by assessing the de novo translation, identifying the cis-active elements and the ribonucleoprotein complex recognized by these elements of the new target mRNAs. (2) Studies on the physiological role of the GAIT complex mediated translational silencing. We have generated mice harboring the conditional null allele (floxed allele) of L13a. Crossing LysMCre mouse with the floxed mouse will generate the macrophage specific knockout of L13a. Macrophages isolated from these mice will be tested ex-vivo for translational silencing. On the other hand these mice will be challenged with the inducer of inflammation and the response will be examined on multiple levels. (3) Role of L13a in ribosome function and IRES activities. In this aim using a combined approach involving ribosome fractionation, immunoblot analysis and in vitro ribosome binding we will determine the ribosome-binding site of L13a. We will use the translation competent extract of L13a depleted cell to determine the mechanism of L13a dependence of the IRES activities. We believe these studies will discover novel cellular targets for new generation of anti-inflammatory small molecules against atherosclerosis and other inflammatory diseases. PUBLIC HEALTH RELEVANCE: Uncontrolled inflammation is the cause of many diseases such as atherosclerosis or cardiovascular disease. This research will uncover the insight about the endogenous cellular mechanisms to control the expression of inflammatory molecules and help to generate novel therapeutic agents against inflammatory diseases.

描述（由申请人提供）：动脉粥样硬化是血管壁驻留细胞不受控制的炎症的致病后果。利用基于细胞的模型，我们发现核糖体蛋白L13 a依赖的翻译沉默途径可以终止一组炎症基因的表达。在这项提案中，我们将测试这种机制是否可以解决炎症。我们已经证明，IFN-3诱导的铜蓝蛋白（Cp）和一组其他炎症蛋白在单核细胞中的合成是在翻译控制下。此外，我们发现核糖体蛋白L13 a及其从60 S核糖体释放在IFN-γ激活的翻译抑制剂（GAIT）复合物的形成中的关键作用。翻译沉默机制依赖于存在于靶mRNA的3 '非翻译区（UTR）中的GAIT元件对L13 a依赖性GAIT复合物的识别。这是基于我们的观察，其显示通过RNA沉默消耗L13 a可以克服这些靶mRNA的翻译抑制。令我们惊讶的是，L13 a的缺失对整体蛋白质合成没有影响，然而几种内部核糖体进入位点（IRES）依赖性翻译被抑制。该提议想要测试以下假设：编码炎性蛋白簇的这些靶mRNA的L13 a依赖性翻译沉默可能作为对抗不受控制的炎症和动脉粥样硬化的内源性防御机制。另一方面，L13 a的作用可能局限于核糖体的专门功能而不是整体功能。我们将通过以下三个具体目标来验证我们的假设：（1）分析IFN-3激活的单核细胞中L13 a调节的和炎症反应的转录后操纵子。在这些目标中，我们将进行一个全面的分析，新的目标mRNA的转录后操纵子的成员，通过评估从头翻译，确定顺式活性元件和核糖核蛋白复合物识别的这些元件的新的目标mRNA。(2)GAIT复合体介导的翻译沉默的生理作用研究。我们已经产生了携带L13 a的条件无效等位基因（floxed等位基因）的小鼠。将LysMCre小鼠与floxed小鼠杂交将产生L13 a的巨噬细胞特异性敲除。将离体测试从这些小鼠分离的巨噬细胞的翻译沉默。另一方面，将用炎症诱导剂激发这些小鼠，并在多个水平上检查反应。(3)L13 a在核糖体功能和IRES活性中的作用。在这个目标中，我们将使用一种包括核糖体分离、免疫印迹分析和体外核糖体结合的组合方法来确定L13 a的核糖体结合位点。我们将利用L13 a缺失细胞的翻译感受态提取物来确定IRES活性的L13 a依赖性机制。我们相信这些研究将为新一代抗动脉粥样硬化和其他炎症性疾病的抗炎小分子发现新的细胞靶点。 公共卫生相关性：不受控制的炎症是许多疾病的原因，如动脉粥样硬化或心血管疾病。这项研究将揭示有关内源性细胞机制的见解，以控制炎症分子的表达，并有助于产生新的治疗药物对炎症性疾病。