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Steric-free labeling strategies to study disease-related non-histone substrates of post-translational modifications

研究翻译后修饰的疾病相关非组蛋白底物的无空间标记策略

基本信息

批准号：
10829202
负责人：
Rongsheng Wang
金额：
$ 6.59万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10829202
关键词：
Acceleration Acetylation Affect Affinity Alkynes Area Azides Biological Biomedical Research Biotin Carbon Cells Chemicals Chemistry Chronic Communities Derivation procedure Disease Dissection Enzymes Fluorine Goals Human Hydrogen Bonding Image Immune response In Situ Inflammatory Label Length Malignant Neoplasms Marketing Mediating Methods Methylation Pathway interactions Post-Translational Modification Site Post-Translational Protein Processing Principal Investigator Proteins Proteomics Reaction Relapse Reporting Research Resistance Signal Transduction Sulfhydryl Compounds T-Cell Activation Writing autoreactivity cancer cell cancer survival cancer type chemical synthesis cofactor disease diagnosis fluorophore human disease imaging probe innovation insight interest invention new therapeutic target non-histone protein novel novel therapeutic intervention programs protein function protein profiling tool traditional therapy

项目摘要

Principal Investigator/Program Director (Last, first, middle): Wang, Ross, E. Steric-free labeling strategies to study disease-related non-histone substrates of post- translational modifications The proposed research will interrogate fluorine-thiol based bioorthogonal reactions for steric-free labeling of post-translational modifications (PTMs). Despite recent advances in biomedical research, diseases are still haunting human beings. Many cancer types remain lethal and are resistant to traditional therapy, while most inflammatory diseases result in chronic or long-term burdens, and there is a lack of selective immunosuppressive on the market. Thus, new therapeutic approaches are needed. PTMs have recently emerged as a class of important biological pathways that could unravel potentially novel therapeutic targets. PTMs modify existing proteins with additional chemical functionalities to modulate protein function, and thereby mediate various cellular activities. Dysregulation of PTM-related proteins has been reported to be key to certain human diseases such as cancer and inflammatory disorders. Yet, the identity of these non-histone proteins has not been fully elucidated. Current research in this area heavily relies on chemical proteomics, which tags target proteins with alkyne or azide- modified PTM cofactors or precursors. These chemical tags on PTM sites can later be derivatized in situ through bio-orthogonal `click chemistry' with a fluorophore for imaging or a biotin affinity probe for pull down and proteomics-based target identification. However, these alkyne/azide- based tags are bulky in length and size, and for many cases alkyne or azide- tagged PTM precursors/cofactors (e.g. for acetylation, methylation) were barely incorporated onto substrates, thereby limiting PTM-related target identification. To solve this issue, an innovative chemical tagging approach will be developed here, which could be steric free, and can be broadly used for the global profiling of proteins related to various types of PTMs that are important to the onset or relapse of human diseases. We hypothesize that unlike azides and alkynes, fluorine labeling can best mimic the intrinsic carbon-hydrogen bond, and is thereby steric free and generally applicable to tag PTM cofactors/precursors. The first research thrust seeks to utilize chemical synthesis to derivatize common PTM cofactor/precursors with fluorine, then to hijack the PTM biological pathway to tag substrate proteins with fluorine, and finally to modify the fluorinated substrate proteins with thiol-derivatized probes for imaging or protein identification. The second research thrust involves the application of this fluorine-thiol tagging strategy to interrogate the substrate proteins of PTMs, which are potentially important to the survival of cancer cells, but not regular cells. The final thrust will investigate the substrates of PTMs which mediate human T cell activation, and are potentially pivotal to the modulation of auto-reactive immune responses. Completion of these research goals will result in the invention of a chemical labeling method that efficiently and accurately identifies disease-associated subcellular components. This strategy will lead to a systemic dissection of PTM-related disease signaling. The achieved results will greatly accelerate disease diagnosis and treatment, and will also result in a tool box of steric-free PTM probes for the benefit of the scientific community. Page 1

首席研究员/项目主任（后、一、中）：Wang, Ross, E. 研究疾病相关非组蛋白底物的无空间标记策略翻译修饰拟议的研究将探讨基于氟-硫醇的生物正交反应，以实现无空间标记翻译后修饰 (PTM)。尽管生物医学研究最近取得了进展，但疾病仍然存在困扰着人类。许多癌症类型仍然是致命的并且对传统疗法有抵抗力，而大多数炎症性疾病会造成慢性或长期负担，且缺乏选择性免疫抑制药物在市场上。因此，需要新的治疗方法。 PTM 最近作为一类出现可能揭示潜在新治疗靶点的重要生物学途径。 PTM 修改现有的具有额外化学功能的蛋白质可以调节蛋白质功能，从而介导各种细胞活动。据报道，PTM 相关蛋白的失调是某些人类疾病的关键，例如如癌症和炎症性疾病。然而，这些非组蛋白的身份尚未完全确定。阐明了。目前该领域的研究很大程度上依赖于化学蛋白质组学，它用炔烃或叠氮修饰的 PTM 辅因子或前体。 PTM 网站上的这些化学标签稍后可以通过生物正交“点击化学”与用于成像的荧光团或生物素亲和探针进行原位衍生化用于下拉和基于蛋白质组学的目标识别。然而，这些基于炔烃/叠氮化物的标签体积庞大长度和大小，并且在许多情况下，炔或叠氮标记的 PTM 前体/辅因子（例如乙酰化、甲基化）几乎没有整合到底物上，从而限制了 PTM 相关靶标的识别。为了解决这个问题，这里将开发一种创新的化学标记方法，该方法可以是无空间的，并可广泛用于与重要的各类 PTM 相关的蛋白质的整体分析人类疾病的发生或复发。我们假设与叠氮化物和炔烃不同，氟标记可以最好地模拟内在的碳-氢键，因此无空间限制，通常适用于标签 PTM 辅助因子/前体。第一个研究重点是利用化学合成来衍生常见的 PTM 辅因子/前体与氟，然后劫持 PTM 生物途径，用氟标记底物蛋白，最后用硫醇衍生的探针修饰氟化底物蛋白以用于成像或蛋白质鉴别。第二个研究重点涉及这种氟硫醇标记策略的应用询问 PTM 的底物蛋白，这对癌细胞的生存可能很重要，但不是常规细胞。最后的重点将研究介导人类 T 细胞激活的 PTM 底物，以及可能对调节自身反应性免疫反应至关重要。完成这些研究目标将导致发明一种化学标记方法，该方法可以有效且高效地进行标记。准确识别与疾病相关的亚细胞成分。这一策略将导致系统性剖析 PTM 相关疾病信号传导。所取得的成果将大大加速疾病的诊断和治疗，并且还将产生一个无空间 PTM 探针工具箱，以造福科学界。第1页