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

描述（由申请人提供）：基因表达在多个水平上受到调节，包括mRNA到蛋白质的帽依赖性翻译。真核翻译起始因子4 E（eIF 4 E）与所有真核细胞质mRNA上存在的5 '-m7 GTP结合。eIF 4 E是帽依赖性翻译起始复合物（eIF 4F）的中心组分。eIF 4F的组装受4 E结合蛋白（4 E-BP或BP）的负调控，4 E结合蛋白结合eIF 4 E并阻断eIF 4F组装以抑制帽依赖性翻译。三种BP已被鉴定为BP 1、BP 2和BP 3（BP 3）。我们发现，LV BP 1蛋白表达在晚期充血性心力衰竭（CHF）中显著增加。我们研究了BP 1和BP 2双基因敲除（DKO）对小鼠横主动脉缩窄（TAC）或心肌梗死诱导的CHF的影响，结果表明BP 1/2 DKO显著提高了这些小鼠的存活率和左室功能。我们还发现，BP 1/2 DKO使LV肌浆网Ca++ ATP酶（SERCA 2a）蛋白含量在蛋白水平上增加约2.5倍。基于这些初步数据，本提案的总体目标是通过实现以下研究目标来确定通过BP 1/2 DKO和BP 1 KO增强帽依赖性翻译起始如何在小鼠中发挥其针对CHF发展的深刻心脏保护作用：（i）确定通过DKO增强帽依赖性翻译起始对应激诱导的CHF的后果。我们的工作假设是通过DKO增强帽依赖性翻译起始将保护心脏免受TAC或梗塞诱导的CHF;（ii）鉴定在DKO小鼠中观察到的心脏保护作用是否主要是由于BP 1或BP 2的功能丧失。我们的工作假设是，心脏保护作用主要是由于BP 1的缺失，心肌细胞特异性BP 1过表达将导致或加剧LV功能障碍;（iii）确定潜在的分子机制，提高翻译启动BP 1/2 DKO的心脏保护作用。我们的工作假设是，BP 1/2 DKO将提高在5 '-非翻译区（5'-UTR）中具有过量二级结构的mRNA（如SERCA 2a）的翻译效率。该项目将利用具有BP 1、BP 2和BP 1/2靶向破坏的小鼠模型、心脏特异性BP 1转基因小鼠以及用于转录组和翻译组组合分析的技术。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CHF模型（Chen博士）、多核糖体微阵列（Bitterman博士）和心肌细胞中参与钙转运的膜蛋白复合物（如SERCA 2a）（大卫托马斯博士）。这些研究将提供新的见解帽依赖性翻译启动的作用，CHF的发展。我们的初步发现表明，通过靶向4 E结合蛋白调节帽依赖性翻译起始可能是治疗CHF的新方法。

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.