Translational Silencing in Monocytes: Role of L13a

单核细胞的翻译沉默：L13a 的作用

• 批准号: 7367833
• 负责人: BARSANJIT MAZUMDER
• 金额: $ 26.59万
• 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 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7367833
• 关键词: 3' Untranslated Regions; Acute; Address; Amino Acids; Atherosclerosis; Binding; Biochemical; Cardiovascular Diseases; Cells; Ceruloplasmin; Cloning; Complementary DNA; Complex; Constitution; Copper; Dimerization; Disease; Elements; Epidemiologic Studies; Event; Gene Expression; Genetic Translation; Goals; Hour; Human; Hybrids; Immune response; In Vitro; Inflammation; Inflammatory; Initiator Codon; Injury; Intercept; Interferon Type II; Interferons; Lead; Lesion; Leucine Zippers; Low Density Lipoprotein oxidation; Mass Spectrum Analysis; Mediating; Messenger RNA; Methods; Microarray Analysis; Modeling; Molecular; Mus; Myocardial Infarction; Peptide Initiation Factors; Phase; Phosphoamino Acids; Phosphorylation; Phosphorylation Site; Plasma; Poly(A) Tail; Poly(A)-Binding Proteins; Polyribosomes; Process; Production; Proteins; Recombinants; Reporting; Research; Research Personnel; Resolution; Reticulocytes; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Proteins; Ribosomes; Risk Factors; Role; Scanning; Series; Site; Small Interfering RNA; System; Testing; Therapeutic Agents; Transcript; Translating; Translation Initiation; Translations; U937 Cells; Work; Yeasts; atherogenesis; base; cytokine; deletion analysis; in vivo; inhibitor/antagonist; insight; macrophage; monocyte; mutant; novel; novel therapeutics; oxidized low density lipoprotein; peripheral blood; prevent; programs; reconstitution; research study

DESCRIPTION (provided by applicant): Our long-term goal is to gain significant insight about the endogenous cellular strategy of targeted silencing of pro-inflammatory gene products. This proposal seeks to elucidate the mechanisms of translational silencing that may govern the natural resolution of the host response to inflammation. We have shown that interferon gamma (IFN-y), an inflammatory cytokine and a contributor to lesion progression in murine model of atherosclerosis, induces ceruloplasmin (Cp) synthesis in monocytes. Cp is an acute phase inflammatory protein that can oxidize LDL and may have an important role in monocytic cell mediated oxidative process in the vessel wall. In addition epidemiological studies also described Cp as an independent risk factor for cardiovascular disease. The synthesis of Cp protein in monocytes is terminated after about 16 hours of IFN-y treatment even in the presence of abundant Cp mRNA. We have described a novel translational silencing mechanism for this inhibition of Cp expression that depends on end-to-end circularization of the Cp transcript via interaction of the T-terminus with the 5'-translation-initiation complex. Cytosolic extracts made from monocytes after 16 hours of IFN-y treatment contain a protein complex(s), denoted IFN-Gamma Activated Inhibitor of Translation (GAIT) that binds to the 29-nt GAIT element present in the 3' untranslated region of Cp mRNA and silences its translation. In preliminary studies of this proposal, we have identified a protein, ribosomal protein L13a that specifically binds to the GAIT element and blocks in vitro translation of Cp. In addition, our work shows that prolonged treatment of monocytes with IFN-y causes release of L13a from the 60S ribosomal subunit and its phosphorylation that is required for silencing activity. The goal of this proposed study is to gain further insight in to the silencing mechanism by addressing the following Specific Aims: (1) How L13a inhibits translation initiation of Cp mRNA. We will use the reconstituted translational silencing system in reticulocyte lysate with recombinant L13a and centrifugal resolution of the different ribosomal subunit, to address this aim. (2) Determination of phosphorylation site and functional analysis of different domains of L13a. We will use phosphoamino acid analysis, mass spectrometry, site-directed and deletion-mutational analysis, in vivo and in vitro re-constitution of recombinant L13a phosphorylation, to address this aim. (3) Identification of other target mRNAs for L13a. We will address this aim by isolating mRNAs from the polysome obtained from the IFN-y activated monocytes as well as cells expressing siRNAs of L13a. The polysomal mRNAs will be subjected to RT-PCR and cDNA cloning and microarray analysis. Since inflammation is an important and obligatory component of initiation and progression of many diseases including atherosclerosis, we believe results obtained from the proposed study will yield significant insight into the translational silencing of pro-inflammatory gene products and may help to develop novel therapeutic agents that could effectively retard inflammation.

描述（由申请人提供）：我们的长期目标是获得有关促炎基因产物靶向沉默的内源性细胞策略的重要见解。这一建议旨在阐明翻译沉默的机制，可能支配自然决议的主机响应炎症。我们已经表明，干扰素γ（IFN-γ），一种炎性细胞因子和动脉粥样硬化的小鼠模型中的病变进展的贡献者，诱导铜蓝蛋白（Cp）在单核细胞中的合成。Cp是一种急性期炎症蛋白，可氧化LDL，并可能在单核细胞介导的血管壁氧化过程中发挥重要作用。此外，流行病学研究也将Cp描述为心血管疾病的独立危险因素。即使存在丰富的Cp mRNA，单核细胞中Cp蛋白的合成在IFN-y处理约16小时后也会终止。我们已经描述了一种新的抑制Cp表达的翻译沉默机制，该机制依赖于Cp转录物通过T-末端与5 '-转录起始复合物的相互作用的端到端环化。IFN-γ处理16小时后由单核细胞制备的胞质提取物含有蛋白质复合物，称为IFN-γ激活的翻译抑制剂（GAIT），其结合Cp mRNA的3'非翻译区中存在的29-nt GAIT元件并使其翻译沉默。在这个建议的初步研究中，我们已经确定了一种蛋白质，核糖体蛋白L13 a，它特异性地结合到GAIT元件上，并阻止Cp的体外翻译。此外，我们的工作表明，用IFN-γ长时间处理单核细胞会导致L13 a从60 S核糖体亚基释放，并导致其磷酸化，这是沉默活性所需的。本研究的主要目的是通过以下几个方面来进一步了解基因沉默的机制：（1）L13 a如何抑制Cp mRNA的翻译起始。我们将使用重组L13 a和离心分离不同核糖体亚基的网织红细胞裂解物中的重建翻译沉默系统来解决这一目标。(2)L13 a不同结构域磷酸化位点的确定及功能分析。我们将使用磷酸化氨基酸分析，质谱，定点和缺失突变分析，在体内和体外重组L13 a磷酸化，以解决这一目标。(3)L13 a的其他靶mRNA的鉴定。我们将通过从获自IFN-γ活化的单核细胞以及表达L13 a的siRNA的细胞的多核糖体中分离mRNA来解决该目的。将对多聚体mRNA进行RT-PCR和cDNA克隆以及微阵列分析。由于炎症是包括动脉粥样硬化在内的许多疾病的启动和进展的重要和必要组成部分，我们相信从拟议的研究中获得的结果将对促炎基因产物的翻译沉默产生重要的见解，并可能有助于开发可有效延缓炎症的新型治疗药物。