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控，包括mRNA转化为蛋白质的帽依赖翻译。真核生物翻译起始因子4e （eIF4E）与所有真核细胞质mrna上的5'-m7GTP cappresent结合。eIF4E是帽依赖翻译起始复合体（eIF4F）的核心成分。eIF4F的组装受到4e结合蛋白（4e - bp或bp）的负调控，这些蛋白结合eIF4E并阻断eIF4F组装以抑制帽依赖翻译。三种bp被确定为BP1、BP2和BP3 （BP3）。我们发现LV BP1蛋白在晚期充血性心力衰竭（CHF）中表达显著增加。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）或心肌梗死诱导的CHF的影响，结果表明BP1/2 DKO可显著提高小鼠的存活率和左室功能。我们还发现BP1/2 DKO在蛋白水平上使左室肌浆网Ca++ atp酶（SERCA2a）蛋白含量增加了2.5倍。基于这些初步数据，本研究的总体目标是通过以下研究目标来确定BP1/2 DKO和BP1 KO如何增强帽依赖性翻译启动对小鼠CHF发展的深远心脏保护作用：(i)确定DKO增强帽依赖性翻译启动对应激性CHF的影响。我们的工作假设是，增强DKO的帽依赖性翻译启动将保护心脏免受TAC或梗死诱导的CHF；（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。我们的工作假设是心脏保护作用主要是由于BP1的缺失，心肌细胞特异性BP1过表达会导致或加重左室功能障碍；（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将提高在5'-非翻译区（5'- utr）中具有过量二级结构的mrna（如SERCA2a）的翻译效率。该项目将利用靶向破坏BP1， BP2和BP1/2的小鼠模型，心脏特异性BP1转基因小鼠以及转录组和翻译组联合分析技术。我们的研究团队包括翻译控制专家（Dr. Bitterman），分子心脏病学和实验CHF模型专家（Dr. Chen），多核糖体微阵列专家（Dr. Bitterman），以及心肌细胞中参与钙转运的膜蛋白复合物（如SERCA2a）专家（Dr. David Thomas）。这些研究将为帽依赖性翻译起始在CHF发展中的作用提供新的见解。我们的初步发现表明，通过靶向4e结合蛋白调控帽依赖性翻译起始可能是治疗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. PUBLIC HEALTH RELEVANCE: Cardiovascular disease ranks as America's No. 1 killer that accounts for nearly one million deaths each year. 4E binding proteins (4E-BPs) play an important role in regulating the translation of mRNA into proteins. We recently found that 4E-BP1 was significantly increased in the advance heart failure. The purpose of this project is to determine the effect 4E-BP gene deletion on the development of congestive heart failure and to understand how 4E-BPs play a critical role in regulating the development of congestive heart failure. The findings from this project are going to advance our understanding of the development of heart failure and to identify novel new therapeutic targets for treating congestive heart failure.