Brd4 interactions with host and viral proteins via the extra-terminal domain

 描述（由申请人提供）：项目概述本R21探索/发展研究资助）提案的目标是确定通过蛋白质的溴结构域和末端外（BET）结构域家族（例如，Brd 2，3，4）。这一目标是重要的，因为ET结构域是蛋白质-蛋白质相互作用（PPI）的中心部分，PPI（i）将多种宿主因子募集到染色质上的表观遗传标记物，以及（ii）确定基于γ-逆转录病毒的基因治疗载体的DNA整合位点。我们期望这些开发研究能够导致（i）发现新的抗癌和抗病毒治疗方法，这些方法通过预防调节表观遗传基因表达的PPI发挥作用，以及（ii）改善消除其他有效的基于逆转录病毒的基因治疗方法的致癌副作用的策略。该建议的灵感来自于发现BET蛋白质（Brd 2，3，4）是主要的细胞内蛋白质。 MLV IN是Moloney鼠白血病病毒整合酶（MLV IN）的结合配偶体，并在靶向MLV整合至转录起始位点中发挥核心作用。此外，我们表明，来自极端的C-末端的MLV IN的片段是必要的和足够的特异性与Brd 4的ET结构域的相互作用。因此，IN-ET复合物的结构研究将阐明保守的ET结构域对配体识别的决定因素，从而揭示该结构域如何能够将蛋白质因子募集到染色质中。我们推测，通过定点突变和小分子抑制剂靶向这些相互作用将在癌症治疗和基因治疗中开辟新的研究方向。该提案的目的是（1）确定Brd 4 ET结构域和MLV IN ET结合基序（EBM）之间复合物的三维溶液结构，（2）通过热力学分析和定点诱变定义Brd 4 ET与病毒和细胞因子结合的特异性决定簇，以及（3）评估EBM启发的小分子抑制BET-宿主因子相互作用的效用。 我们在aim 1中的结构研究将揭示ET结构域如何识别其靶标。在目标2中，我们将补充热力学数据的结构见解，而我们测试潜在的肽配体从主机辅因子。最后，我们将利用这些知识来开发新的模型化合物，以抑制这些蛋白质-蛋白质相互作用，并测试其在体外和体内的效用。拟议的研究将揭示BET蛋白的ET结构域的配体结合的结构基础，并产生新的试剂（PPI抑制剂，突变体）用于探测转录调控途径。这些将有助于识别和验证天然细胞辅因子，并突出转录靶向治疗的潜在靶点。此外，γ-逆转录病毒如何识别BET蛋白的知识将阐明改变基于MLV的载体的染色体整合模式以避免原癌基因激活的策略。

 DESCRIPTION (provided by applicant): PROJECT SUMMARY The goal of this R21 Exploratory/Developmental Research Grant) proposal is to determine the structural basis for ligand recognition and binding by the extraterminal (ET) domain of the bromodomain and extraterminal (BET) domain family of proteins (e.g., Brd2, 3, 4). This objective is significant because the ET domain is the center piece for protein-protein interactions (PPIs) that (i) recruit a variety of host factors to epigenetic markers on chromatin, and (ii) determine DNA integration sites of γ-retrovirus-based gene therapy vectors. We expect these developmental studies to lead to (i) discovery of new anti-cancer and anti-viral therapeutics that function by preventing PPIs that modulate epigenetic gene expression, and (ii) improved strategies for eliminating the oncogenic side effects of otherwise effective retrovirus-based gene therapy approaches. The proposal is inspired by the discovery that the BET proteins (Brd2, 3, 4) are the principal cellular binding partners of Moloney murine leukemia virus integrase (MLV IN), and play a central role in targeting MLV integration to transcription start sites. Moreover, we showed that a fragment derived from the extreme C-terminus of MLV IN is necessary and sufficient for specific interaction with the ET domain of Brd4. Thus, structural studies of the IN-ET complex will illuminate determinants for ligand recognition by the conserved ET domain, thereby revealing how this domain is able to recruit protein factors to chromatin. We hypothesize that targeting these interactions via site-directed mutagenesis and small molecule inhibitors will open new research directions in cancer treatment and gene therapy. The aims of the proposal are to (1) determine the three-dimensional solution structure of the complex between the Brd4 ET domain and the MLV IN ET-binding motif (EBM), (2) define the specificity determinants for Brd4 ET binding to viral and cellular factors via thermodynamic analysis and site-directed mutagenesis, and (3) assess the utility of EBM-inspired small molecules to inhibit BET-host factor interactions. Our structural studies in aim 1 will reveal how the ET domain recognizes its targets. In aim 2 we will complement the structural insights with thermodynamic data, while we test potential peptide ligands from host cofactors. Finally, we will use that knowledge to develop new model compounds to inhibit those protein-protein interactions and test their utility in vitro and in vivo The proposed studies will reveal the structural basis for ligand binding by the ET domain of BET proteins, and yield new reagents (PPI inhibitors, mutants) for probing transcriptional regulatory pathways. These will be useful for both identifying and validating native cellular cofactors, and for highlighting potential targets for transcription-targeted therapies. Moreover, knowledge of how γ-retroviruses recognize BET proteins will illuminate strategies for altering the chromosomal integration patterns of MLV-based vectors in order to avoid activation of proto-oncogenes.