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

项目摘要 了解和调控蛋白质组的功能是生物医学科学的主要目标。癌 特别地，生物学是目前极大地受益于高通量功能分析的领域， 基因和蛋白质。癌细胞对共同细胞通路的选择性依赖，这种现象被称为 非癌基因成瘾，经常通过高通量筛选方法进行研究，从而发现新的 潜在的治疗不幸的是，基于CRISPR和RNA干扰的现代功能筛选，尽管 信息，不能严重影响蛋白质组，因此不仅在技术上受到限制，而且特别是在 容易受到补偿机制的影响，使蛋白质的作用模糊不清。此外，在建立 基因敲除后，许多对生存力至关重要的蛋白质丢失，无法检测到感兴趣的表型 非活性筛选格式。为了促进对蛋白质组的更广泛和更全面的研究，我 建议开发一种新的筛选模式，基于快速和直接调节蛋白质， 全基因组规模。这个筛选平台最初将被开发，以揭示许多新的见解，非 癌细胞中的致癌成瘾。该技术是基于一个多路复用的细胞库，其中每个基因是 用配体结合的生物正交蛋白质标记，一次一个细胞。小分子配体将双向 调节标记蛋白质的稳定性。在第一个目标中，我将确定有效的大规模 蛋白质标记随后，在第二个目标中，我将构建癌症全基因组多重标签文库 细胞，并通过快速降解进行蛋白质必要性筛选，证明该平台能够 发现新的热门用传统的遗传扰动工具进行的遗传筛选，如CRISPR和 RNA干扰，被预测更容易受到细胞补偿机制的影响，所以我将 直接比较从该筛选中获得的结果与从其他可比较的重要性中获得的结果 卡位在第三个目标中，我将利用开发的平台，全面探讨蛋白质稳态的作用 非致癌基因成瘾具体来说，我将揭示蛋白毒性应激的急性反应的亚细胞图谱 在癌细胞和健康细胞中。这将通过使用多路复用标签细胞文库的子集来实现， 诱导隔室特异性蛋白质去稳定化，这将通过单细胞RNA测序进行分析。在 总之，我将开发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，这将大大有助于我们了解癌细胞中的非致癌成瘾， 从而导致新型癌症疗法的发展。该项目的成功将大大促进其 环境，它提供了从儿童医院的专家获得设施和见解， 费城和宾夕法尼亚大学。令人兴奋的，附近的合作可以很容易地建立一次， 这一平台一经开发，立即将其潜力用于最有利的领域。

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.