Molecular mechanism of 4E-binding proteins on heart failure development

描述(申请人提供)：基因表达在多个水平上受到调控，包括依赖帽子的信使核糖核酸到蛋白质的翻译。真核细胞翻译起始因子4E(EIF4E)与所有真核细胞胞质mRNA5‘-m7GTP帽结合。EIF4E是帽依赖翻译起始复合体(EIF4F)的中心成分。EIF4F的组装受到4E结合蛋白(4E-Bps或Bps)的负调控，4E结合蛋白结合eIF4E并阻断eIF4F组装，以抑制帽依赖的翻译。已确定三个BP为BP1、BP2和BP3(BP3)。我们发现，在晚期充血性心力衰竭(CHF)中，LV BP1蛋白的表达显著增加。我们检测了BP1和BP2双基因敲除(DKO)对小鼠横断主动脉缩窄(TAC)或心肌梗死所致心力衰竭的影响，结果表明BP1/2 DKO显著提高了这些小鼠的存活率和心功能。我们还发现，BP1/2 DKO使心肌肌浆网钙ATPase(SERCA2a)的蛋白质含量在蛋白质水平上增加约2.5倍。基于这些初步数据，本建议的总体目标是确定BP1/2 DKO和BP1KO如何通过实现以下研究目标来增强BP1/2 DKO和BP1KO对小鼠CHF发展的心脏保护作用：(I)确定DKO增强帽子依赖翻译启动在应激性CHF中的后果。我们的工作假设是，加强DKO对帽子依赖的翻译启动将保护心脏免受TAC或梗死性心力衰竭的影响；(Ii)确定在DKO小鼠中观察到的心脏保护作用是否主要是由于BP1或BP2功能丧失所致。我们的工作假设是，BP1的心脏保护作用主要是由于BP1的缺失，心肌细胞特异性BP1的过度表达将导致或加剧左心功能障碍；(Iii)确定BP1/2 DKO增强翻译起始的心脏保护作用的潜在分子机制。我们的工作假设是，BP1/2 DKO将提高5‘-非翻译区(5’-UTRs)中具有过多二级结构的mRNAs(如SERCA2a)的翻译效率。该项目将利用靶向干扰BP1、BP2和BP1/2的小鼠模型，心脏特异的BP1转基因小鼠，以及转录组和翻译组的联合分析技术。我们的研究团队包括翻译控制(Bitterman博士)、分子心脏病学和实验性CHF模型(Chen博士)、多核糖体微阵列(Bitterman博士)以及参与心肌细胞钙转运的膜蛋白复合体(如SERCA2a)(David Thomas博士)方面的专家。这些研究将为研究依赖上限的翻译启动在慢性心力衰竭发生中的作用提供新的见解。我们的初步发现表明，通过靶向4E结合蛋白来调节帽依赖的翻译启动可能是治疗CHF的一种新的治疗方法。

DESCRIPTION (provided by applicant): Gene expression is regulated at multiple levels including the cap-dependent translation of mRNA into proteins. Eukaryotic translation initiation factor4E (eIF4E) binds to the 5'-m7GTP cappresent on all eukaryotic cytoplasmic mRNAs. eIF4E is the central component of the cap-dependent translation initiation complex (eIF4F). The assembly of eIF4F is negatively regulated by 4E-binding proteins (4E-BPs or BPs), which bind eIF4E and block eIF4F assembly to inhibit cap-dependent translation. Three BPs have been identified as BP1, BP2 and BP3 (BP3). We have found that LV BP1 protein expression is greatly increased in advanced congestive heart failure (CHF). We examined the effect of double gene knockout (DKO) of BP1 and BP2 on transverse aortic constriction (TAC) or myocardial infarct induced CHF in mice, and the results demonstrated that BP1/2 DKO profoundly improved the survival rate and LV function in these mice. We also found that BP1/2 DKO increased LV sarcoplasmic reticulum Ca++ ATPase (SERCA2a) protein content ~2.5 fold at the protein level. Based on these preliminary data, the overall goal of this proposal is to determine how enhancing cap-dependent translation initiation by BP1/2 DKO and BP1 KO exert their profound cardiac protective effect against the development of CHF in mice by achieving following research objectives: (i) Determine the consequences of enhancing cap-dependent translational initiation by DKO on stress-induced CHF. Our working hypothesis is that enhancing cap-dependent translation initiation by DKO will protect the heart from TAC or infarct induced CHF; (ii) Identify whether the cardioprotective effect observed in DKO mice is mainly due to loss of function of BP1 or BP2. Our working hypothesis is that the cardiac protective effect is mainly due to the deletion of BP1, and that cardiac myocyte specific BP1 overexpression will cause or exacerbates LV dysfunction; (iii) Identify underlying molecular mechanisms for the cardioprotective effect of enhancing translational initiation by BP1/2 DKO. Our working hypothesis is that BP1/2 DKO will improve the translational efficiency of mRNAs (such as SERCA2a) that have an excessive amount of secondary structure in the 5'-untranslatedregions (5'-UTRs). The project will take advantage of mouse models with targeted disruption of BP1, BP2 and BP1/2, cardiac specific BP1 transgenic mice and of technologies for combined analysis of the transcriptome and translatome. Our research team includes experts in translational control (Dr. Bitterman), molecular cardiology and experimental CHF models (Dr. Chen), polyribosome microarrays (Dr. Bitterman), and membrane protein complexes involved in calcium transport (such as SERCA2a) in cardiac myocytes (Dr. David Thomas). These studies will provide novel insights into the role of cap-dependent translation initiation on the development of CHF. Our preliminary finding indicates that regulation of cap-dependent translation initiation by targeting 4E-binding proteins may be a novel therapeutic approach to treat CHF.