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

Brd4 通过末端外结构域与宿主和病毒蛋白相互作用

• 批准号: 9207412
• 负责人: MARK P. FOSTER
• 金额: $ 18.82万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-01 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9207412
• 关键词: Adverse effects; Antiviral Agents; Applications Grants; Binding; Biomedical Research; Bromodomain; Chemicals; Chromatin; Complement; Complex; DNA Integration; Data; Development; Dimensions; Disease; Epigenetic Process; Exploratory/Developmental Grant; Gammaretrovirus; Gene Expression; Genetic Transcription; Goals; Histone H3; Histone H4; In Vitro; Inflammation; Integrase; Integration Host Factors; Knowledge; Lead; Ligand Binding; Ligands; Lysine; Malignant Neoplasms; Mediating; Modeling; Molecular Target; Moloney Leukemia Virus; Murine leukemia virus; N-terminal; NMR Spectroscopy; Obesity; Oncogenes; Oncogenic; Pathway interactions; Pattern; Peptides; Pharmaceutical Chemistry; Play; Protein Family; Proteins; Proto-Oncogenes; Reagent; Recruitment Activity; Regulatory Pathway; Research; Research Project Grants; Resolution; Retroviridae; Role; Site-Directed Mutagenesis; Specificity; Structure; Tail; Testing; Therapeutic; Thermodynamics; Transcription Initiation Site; Transcriptional Activation; Transcriptional Regulation; Viral; Viral Proteins; Virus Latency; anticancer research; base; cancer therapy; cellular targeting; cofactor; epigenetic marker; experimental study; gene therapy; high reward; improved; in vivo; inhibitor/antagonist; insight; integration site; mutant; novel; pleiotropism; prevent; protein E; protein protein interaction; public health relevance; small molecule; small molecule inhibitor; targeted treatment; tumor progression; vector

 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.

 描述（应用程序提供）：项目摘要此R21探索/发展研究赠款的目标）建议是确定bromodomain和外部（ET）结构域的配体识别和结合的结构基础，以及蛋白质的外部（BET）结构域（例如，BRD2、3、4）。该目标很重要，因为ET结构域是蛋白质 - 蛋白质相互作用（PPI）的中心部分，（i）（i）将各种宿主因子募集到染色质上的表观遗传标记，（ii）确定基于γ-逆转录病毒基因疗法的DNA整合位点。我们预计这些发展研究将导致（i）通过防止调节表观遗传基因表达的PPI来发现新的抗癌和抗病毒疗法，以及（ii）消除原本有效基于逆转录病毒的基因疗法的致癌副作用的改进策略。该提案的灵感来自于发现BET蛋白（BRD2，3，4）是主要细胞 结合Moloney鼠白血病病毒（MLV IN）的结合伴侣，并在将MLV整合靶向转录起始位点中起着核心作用。此外，我们表明，从MLV的极端C端得出的片段对于与BRD4的ET域的特定相互作用是必要的，并且足够足够。这是IN-ET复合物的结构研究将通过配置的ET域来照亮确定剂的配体识别，从而揭示该域如何能够募集蛋白质因子为染色质。我们假设通过定位的诱变和小分子抑制剂靶向这些相互作用将打开癌症治疗和基因治疗方面的新​​研究方向。该提案的目的是（1）确定brd4 et域与ET结合基序（EBM）中络合物之间复合物的三维溶液结构，（2）定义了BRD4 ET的特异性确定剂的特异性确定剂，通过热力学分析和（3）型号的摩尔（3）统一性，并在3号中与病毒和细胞因子结合。互动。 我们在AIM 1中的结构研究将揭示ET领域如何识别其目标。在AIM 2中，我们将使用热力学数据来完成结构性见解，同时测试宿主辅助因子的潜在肽配体。最后，我们将使用这些知识来开发新的模型化合物来抑制这些蛋白质蛋白质相互作用，并在体外和体内测试其效用。拟议的研究将揭示BET蛋白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，突变体），以探测转录转录调节途径。这些对于识别和验证天然细胞辅助因子以及突出转录靶向疗法的潜在靶标很有用。此外，了解γ-逆转录病毒如何识别BET蛋白将阐明基于MLV的载体的染色体整合模式的策略，以避免激活原始癌基因。