Translational Silencing in Monocytes: Role of L13a

描述(由申请人提供)：我们的长期目标是深入了解定向沉默促炎基因产物的内源性细胞策略。这一提议试图阐明翻译沉默的机制，该机制可能支配宿主对炎症的自然消解。我们已经证明，干扰素-γ是一种炎性细胞因子，也是动脉粥样硬化小鼠模型病变进展的贡献者，它诱导单核细胞合成铜蓝蛋白(CP)。CP是一种急性时相炎性蛋白，可氧化低密度脂蛋白，在单核细胞介导的血管壁氧化过程中可能起重要作用。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。单核细胞中CP蛋白的合成在干扰素-γ处理约16小时后被终止，即使在大量CP mRNA存在的情况下也是如此。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转录本通过T末端与5‘-翻译起始复合体的相互作用而端到端的环化。经干扰素-γ处理16小时后的单核细胞胞浆提取物含有一个蛋白质复合体(S)，命名为干扰素-γ激活的翻译抑制因子(GATT)，它与存在于CP基因3‘非翻译区的29个核苷酸步态元件结合并抑制其翻译。在对这一提议的初步研究中，我们已经鉴定出一种蛋白质，核糖体蛋白L13a，它与步态元件特异性结合，并阻断CP的体外翻译。此外，我们的工作表明，用干扰素-γ长期处理单核细胞会导致60S核糖体亚基释放L13a及其磷酸化，这是沉默活动所必需的。这项研究的目的是通过解决以下具体目标来进一步深入了解沉默机制：(1)L13a如何抑制CP mRNA的翻译起始。我们将在网织红细胞裂解液中使用重组的翻译沉默系统，通过重组L13a和离心拆分不同的核糖体亚基来解决这一目标。(2)L13a不同结构域的磷酸化位点测定和功能分析。我们将利用磷酸氨基酸分析、质谱学、定点分析和缺失突变分析、体内和体外重组重组L13a磷酸化来解决这一目标。(3)L13a其他靶基因的鉴定。我们将通过从干扰素-γ激活的单核细胞获得的多聚体以及表达L13a siRNA的细胞中分离mRNAs来实现这一目标。多倍体mRNAs将接受RT-PCR、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发生和发展的重要和必需的组成部分，我们相信这项拟议的研究结果将对促炎基因产物的翻译沉默产生重要的洞察，并可能有助于开发能够有效延缓炎症的新型治疗药物。

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.