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

• 批准号: 10608102
• 负责人: Rongsheng Wang
• 金额: $ 39.1万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 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/10608102
• 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

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/计划主任（最后，第一，中间）：王，罗斯，E。 无狭窄的标签策略，用于研究与疾病相关的非途径底物 翻译修改 拟议的研究将询问基于氟硫醇的生物正交反应，以进行无稳定的标记 翻译后修改（PTMS）。尽管生物医学研究最近取得了进步，但疾病仍然是 困扰人类。许多癌症类型仍然致命，并且对传统疗法具有抵抗力，而大多数 炎症性疾病会导致慢性或长期负担，并且缺乏选择性免疫抑制作用 在市场上。因此，需要新的治疗方法。 PTM最近出现了 重要的生物学途径可以揭示潜在的新型治疗靶标。 PTMS修改现有 具有其他化学功能的蛋白质调节蛋白质功能，从而介导了各种细胞 活动。据报道，与PTM相关蛋白的失调是某些人类疾病的关键 作为癌症和炎症性疾病。然而，这些非历史蛋白的身份尚未完全 阐明。目前在该领域的研究严重依赖化学蛋白质组学，该化学蛋白质组学将靶向蛋白质的标签 炔烃或叠氮化物修饰的PTM辅因子或前体。 PTM站点上的这些化学标签稍后可以 通过生物正交的“单击化学”与荧光团进行成像或生物素亲和探针的原位衍生化。 用于下拉和基于蛋白质组学的目标识别。但是，这些藻类/基于叠氮化物的标签在 长度和大小，在许多情况下，炔烃或叠氮化物标记的PTM前体/辅助因子（例如，对于乙酰化， 几乎没有将甲基化掺入底物上，从而限制了与PTM相关的靶标识别。 为了解决此问题，这里将开发一种创新的化学标记方法，该方法可能是无空间的， 并且可以广泛用于与重要的PTM相关的蛋白质的全球分析，这些蛋白质很重要 开始或复发人类疾病。我们假设与叠氮化物和炔烃不同，氟标记可以 最佳模仿固有的碳 - 氢键，因此不含空间，通常适用于标记PTM 辅助因子/前体。第一个研究力量试图利用化学合成来衍生普通PTM 与氟的辅助因子/前体，然后劫持PTM生物途径，用氟，将底物蛋白标记 最后，用硫醇衍生的探针修饰氟化的底物蛋白进行成像或蛋白 鉴别。第二项研究推力涉及将此氟硫醇标签策略应用于 询问PTM的底物蛋白，这些蛋白可能对癌细胞的存活至关重要，但却不重要 常规细胞。最终推力将研究介导人T细胞激活的PTM的底物，并 对于自身反应性免疫反应的调节可能是关键的。 完成这些研究目标将导致有效和有效的化学标签方法发明 准确地识别与疾病相关的亚细胞成分。该策略将导致系统性解剖 PTM相关疾病信号传导的。实现的结果将大大加速疾病的诊断和治疗， 并将导致一个无固定PTM探针的工具盒，以使科学界受益。 第1页