Translational Silencing in Monocytes: Role of L13a

单核细胞的翻译沉默：L13a 的作用

• 批准号: 6859704
• 负责人: BARSANJIT MAZUMDER
• 金额: $ 27.36万
• 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 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6859704
• 关键词: ferroxidase; gene induction /repression; genetic translation; inflammation; interferon gamma; mass spectrometry; messenger RNA; microarray technology; monocyte; phosphorylation; protein structure function; ribosomal proteins; ribosomes; site directed mutagenesis; small interfering RNA

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关于促炎基因产物靶向沉默的内源性细胞策略的重要见解。该建议旨在阐明可能控制宿主对炎症反应的自然解决的翻译沉默机制。我们已经证明，干扰素γ (IFN-y)是一种炎症细胞因子，在小鼠动脉粥样硬化模型中促进病变进展，可诱导单核细胞合成铜蓝蛋白（Cp）。Cp是一种急性期炎症蛋白，可以氧化LDL，可能在单核细胞介导的血管壁氧化过程中起重要作用。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。在IFN-y处理约16小时后，即使存在丰富的Cp mRNA，单核细胞中Cp蛋白的合成也会终止。我们已经描述了一种新的翻译沉默机制来抑制Cp表达，这种机制依赖于通过t端与5'-翻译起始复合物的相互作用使Cp转录物端到端循环。经过16小时的IFN-y处理后，从单核细胞中提取的细胞质提取物含有一种蛋白质复合物，称为ifn - γ激活的翻译抑制剂（步态），它与存在于Cp mRNA 3'非翻译区的29-nt步态元件结合，并沉默其翻译。在该提议的初步研究中，我们已经确定了一种蛋白质，核糖体蛋白L13a，它特异性结合步态元件并阻断Cp的体外翻译。此外，我们的工作表明，用IFN-y长期治疗单核细胞会导致L13a从60S核糖体亚基释放，并使其磷酸化，这是沉默活性所必需的。本研究的目标是通过解决以下具体目标来进一步了解沉默机制：(1)L13a如何抑制Cp mRNA的翻译起始。我们将在网织细胞裂解液中使用重组L13a和不同核糖体亚基的离心分辨率的翻译沉默系统来解决这一问题。(2) L13a不同结构域磷酸化位点测定及功能分析。我们将使用磷氨基酸分析，质谱分析，位点定向和缺失突变分析，体内和体外重组L13a磷酸化，以解决这个问题。(3) L13a的其他靶mrna的鉴定。我们将通过从IFN-y激活的单核细胞以及表达L13a sirna的细胞中获得的多体中分离mrna来解决这一问题。多体mrna将进行RT-PCR和cDNA克隆和微阵列分析。由于炎症是包括动脉粥样硬化在内的许多疾病的发生和发展的重要和必要的组成部分，我们相信从所提出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，并可能有助于开发有效延缓炎症的新型治疗药物。