Rapid and direct control of the proteome through a multiplexed tag system

通过多重标签系统快速直接控制蛋白质组

• 批准号: 9760412
• 负责人: Yevgeniy Vladimirovich Serebrenik
• 金额: $ 6.12万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9760412
• 关键词: Acute; Adopted; Affect; Aftercare; Binding; Bioinformatics; Biological Assay; CRISPR interference; CRISPR/Cas technology; Cancer Biology; Cancerous; Cell Separation; Cell Survival; Cells; Cellular biology; Chemicals; Chimeric Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collaborations; Critical Pathways; DNA; DNA cassette; Data; Development; Environment; Essential Genes; Experimental Designs; Financial compensation; Gene Proteins; Generations; Genes; Genetic; Genetic Transcription; Genomics; Goals; Growth; Human; Individual; Introns; Knock-out; Knowledge; Libraries; Ligand Binding; Ligands; Link; Malignant Neoplasms; Maps; Masks; Measures; Mediating; Mentors; Mentorship; Messenger RNA; Methods; Modernization; Nonhomologous DNA End Joining; Pathway interactions; Pediatric Hospitals; Pennsylvania; Phenotype; Philadelphia; Proteins; Proteome; RNA Interference; RNA Splicing; Resources; Role; Science; Site; Societies; Stress; System; Techniques; Technology; Tertiary Protein Structure; Testing; Time; Toxic effect; Universities; addiction; anticancer research; base; biological adaptation to stress; cancer cell; cancer therapy; combat; driving force; experience; genome editing; genome-wide; high throughput screening; insertion/deletion mutation; insight; knock-down; non-oncogenic; novel; novel therapeutics; protein degradation; proteostasis; proteotoxicity; repaired; response; screening; single cell analysis; single-cell RNA sequencing; small molecule; success; targeted cancer therapy; technology development; tool

Project Summary Understanding and regulating the functions of the proteome is a primary goal in the biomedical sciences. Cancer biology, in a particular, is a field that is currently greatly benefiting from high-throughput functional analysis of genes and proteins. The selective reliance of cancer cells on common cell pathways, a phenomenon known as non-oncogene addiction, is often studied via high-throughput screening approaches, thus uncovering new potential therapies. Unfortunately, modern functional screens based on CRISPR and RNA interference, although informative, cannot affect the proteome acutely and are thus not only technically limiting, but also especially susceptible to compensation mechanisms that obscure protein roles. Additionally, in the time it takes to establish genetic knockdown, many proteins essential for viability are lost and cannot be tested for interesting phenotypes in non-viability screen formats. To facilitate a broader and more comprehensive study of the proteome, I propose to develop a new screening paradigm based on rapid and direct modulation of proteins at a genome-wide scale. This screening platform will initially be developed to uncover many new insights on non- oncogenic addiction in cancer cells. The technology is based on a multiplexed cell library where each gene is tagged with a ligand-binding, bioorthogonal protein, one cell at a time. Small-molecule ligands will bidirectionally regulate the stability of tagged proteins. In the first aim, I will determine the rules required for efficient large-scale protein tagging. Subsequently, in the second aim, I will construct a genome-wide multiplexed tag library in cancer cells and perform a protein essentiality screen by rapid degradation, demonstrating the ability of the platform to uncover novel hits. Essentiality screens performed with traditional genetic perturbation tools, like CRISPR and RNA interference, are predicted to be much more susceptible to cell compensation mechanisms, and so I will directly compare the results obtained from this screen to results obtained from other, comparable essentiality screens. In the third aim, I will utilize the developed platform to comprehensively explore the role of proteostasis in non-oncogene addiction. Specifically, I will uncover a subcellular map of acute responses to proteotoxic stress in both cancerous and healthy cells. This will be achieved by using a subset of the multiplexed tag cell library to induce compartment-specific protein destabilization, which will be analyzed by single-cell RNA sequencing. In summary, I will develop a strategy to rapidly and directly modulate the proteome in a high-throughput manner, facilitating studies that will greatly contribute to our understanding of non-oncogenic addiction in cancer cells and leading to the development of novel cancer therapies. The success of this project will be greatly facilitated by its environment, which provides access to facilities and insight from experts from both the Children’s Hospital of Philadelphia and the University of Pennsylvania. Exciting, nearby collaborations can be easily established once this platform is developed, immediately putting its potential to use where it will be most beneficial.

项目概要 了解和调节蛋白质组的功能是生物医学的首要目标。癌症 尤其是生物学，是目前从高通量功能分析中受益匪浅的领域。 基因和蛋白质。癌细胞对常见细胞途径的选择性依赖，这种现象称为 非癌基因成瘾，通常通过高通量筛选方法进行研究，从而发现新的 潜在的疗法。不幸的是，尽管现代功能筛选基于 CRISPR 和 RNA 干扰， 信息丰富，不能严重影响蛋白质组，因此不仅在技术上受到限制，而且特别 容易受到掩盖蛋白质作用的补偿机制的影响。此外，在建立过程中 基因敲低，许多生存必需的蛋白质都会丢失，无法测试有趣的表型 在非生存屏幕格式中。为了促进对蛋白质组进行更广泛和更全面的研究，我 提议开发一种基于蛋白质快速和直接调节的新筛选范例 全基因组规模。该筛选平台最初的开发目的是发现许多关于非 癌细胞的致癌成瘾。该技术基于多重细胞库，其中每个基因都是 一次用配体结合生物正交蛋白标记一个细胞。小分子配体将双向 调节标记蛋白的稳定性。在第一个目标中，我将确定高效大规模所需的规则 蛋白质标记。随后，在第二个目标中，我将构建癌症中的全基因组多重标签库 细胞并通过快速降解进行蛋白质必需性筛选，展示了该平台的能力 发现热门小说。使用传统的遗传扰动工具（例如 CRISPR 和 RNA干扰预计更容易受到细胞补偿机制的影响，所以我会 直接将从该屏幕获得的结果与从其他可比较的重要性获得的结果进行比较 屏幕。第三个目标，我将利用所开发的平台全面探讨蛋白质稳态的作用 在非癌基因成瘾中。具体来说，我将揭示对蛋白毒性应激的急性反应的亚细胞图 在癌细胞和健康细胞中。这将通过使用多路复用标签单元库的子集来实现 诱导隔室特异性蛋白质不稳定，这将通过单细胞 RNA 测序进行分析。在 总之，我将制定一种以高通量方式快速直接调节蛋白质组的策略， 促进研究，这将极大地有助于我们了解癌细胞的非致癌成瘾， 导致新型癌症疗法的开发。该项目的成功将得到极大的促进 环境，可以使用来自儿童医院的设施和专家的见解 费城和宾夕法尼亚大学。一旦建立，就可以轻松建立令人兴奋的、附近的合作 该平台的开发立即将其潜力发挥到最有利的地方。