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: 9797039
• 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 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9797039
• 关键词: Acetylation; Affect; Affinity; Alkynes; Area; Azides; Biological; Biomedical Research; Biotin; Carbon; Cells; Chemicals; Chemistry; Chronic; Communities; Disease; Dissection; Enzymes; Fluorine; Goals; Human; Hydrogen Bonding; Image; Immune response; Immunosuppressive Agents; In Situ; Inflammatory; Label; Length; Malignant Neoplasms; 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; base; cancer cell; cancer survival; cancer type; chemical function; 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

主要研究者/项目负责人（最后，第一，中间）：王，罗斯，E。 无空间标记策略研究疾病相关的非组蛋白底物后， 翻译修饰 拟议的研究将询问基于氟-硫醇的生物正交反应，用于无空间标记 翻译后修饰（PTMs）。尽管最近在生物医学研究方面取得了进展，但疾病仍然是 困扰着人类许多癌症类型仍然是致命的，对传统疗法有抵抗力，而大多数癌症类型仍然是致命的。 炎症性疾病导致慢性或长期负担，并且缺乏选择性免疫抑制剂。 市场上因此，需要新的治疗方法。PTM最近已经成为一类 重要的生物学途径，可以解开潜在的新的治疗靶点。PTM修改现有 具有额外化学功能的蛋白质，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， 如癌症和炎症性疾病。然而，这些非组蛋白蛋白的身份尚未完全确定。 阐明。目前在这一领域的研究严重依赖于化学蛋白质组学，它标记的目标蛋白质与 炔或叠氮化物修饰的PTM辅因子或前体。PTM位点上的这些化学标签可以稍后被 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原位衍生 用于下拉和基于蛋白质组学的目标识别。然而，这些基于炔/叠氮化物的标记物体积庞大， 长度和大小，以及对于许多情况，炔或叠氮化物标记的PTM前体/辅因子（例如用于乙酰化， 甲基化）几乎不掺入底物上，从而限制了PTM相关的靶标鉴定。 为了解决这个问题，我们将开发一种创新的化学标记方法，它可以是无空间位阻的， 并可广泛用于与重要的各种类型的PTM相关的蛋白质的全局谱分析 人类疾病的发作或复发。我们假设，与叠氮化物和炔不同，氟标记可以 最好地模拟固有的碳-氢键，因此是无空间位阻的，通常适用于标记PTM 辅因子/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，以标记底物蛋白与氟， 最后用巯基衍生的探针修饰氟化底物蛋白， 识别.第二个研究重点涉及这种氟-巯基标记策略的应用， 询问PTM的底物蛋白，其对癌细胞的存活具有潜在的重要性，但不 普通细胞最后的重点将研究介导人类T细胞活化的PTM的底物， 对自身反应性免疫应答的调节具有潜在的关键作用。 这些研究目标的完成将导致发明一种化学标记方法， 准确识别疾病相关的亚细胞成分。这种策略会导致系统性解剖 与PTM相关的疾病信号。所取得的成果将大大加快疾病的诊断和治疗， 并且还将为科学界带来一个无空间位阻PTM探针的工具箱。 第1页