Optically Gated Discovery of Protein-Biomolecule Interactions

蛋白质-生物分子相互作用的光门发现

• 批准号: 10501385
• 负责人: Jacob Geri
• 金额: $ 42.38万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-24 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10501385
• 关键词: Affinity; Automobile Driving; Biological Process; Cells; Chemicals; Communities; Data; Dependence; Development; Dimensions; Disease; Exocytosis; Goals; Health; Human; Immunology; Label; Lead; Light; Location; Lymphocyte Function; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Membrane; Methods; Microscopy; Molecular Biology; Motivation; Neurology; Neurons; Operating System; Optics; Orphan; Pattern; Peptides; Proteins; Proteomics; Research; Resolution; Specificity; System; Technology; Thymus Gland; Time; Tissue Sample; Tonsil; Work; catalyst; crosslink; design; diagnostic strategy; drug development; human tissue; improved; interest; ion mobility; millisecond; new technology; new therapeutic target; novel diagnostics; novel therapeutics; optogenetics; receptor; spatiotemporal; temporal measurement; tool; transcriptomics

The overall goal of research in the Geri lab is to map protein interactomes using discovery technologies that provide orders of magnitude improvements in spatiotemporal resolution over the current state-of-the-art. The motivation for this work is that advancing the resolution of protein interactome discovery technology beyond key milestones, such as single cell and single protein thresholds, will have a field-wide impact analogous to similar advances in transcriptomics and microscopy. The first three years of work in the lab will be focused on creating new technologies by combining photocatalytic proximity labeling, in which light-powered catalysts attached to an affinity handle drive the crosslinking of synthetic affinity probes with nearby proteins, with patterned light and interaction-gated activation to simultaneously enforce multiple dimensions of specificity. The fourth and fifth years of work will focus on applying the mature technologies. The overall strategy is divided along two thrusts, in which labeling specificity is obtained through extrinsic optical control or intrinsic chemical control, and has been designed to be programmatically robust by minimizing project interdependency. Intrinsically selective systems will exploit the high spatial resolution of photocatalytic labeling (5 nm) and use “split” systems that operate when defined protein targets are in proximity. Initial work will use natively expressed orphan peptides as proximity labeling loci to discover their currently unknown receptors. The effort will cover thousands of peptides by using label free ion mobility mass spectrometry for proteomics, maximally leveraged by using optimized labeling probe designs. Split systems will combine multiple photocatalysts targeted to different proteins of interest to make colocalization a dimension of specificity, and will be initially applied to map proteins present at membrane junctions. Extrinsically controlled systems will enable subcellular resolution labeling in human tissue sections and ms-resolution temporal control for the study of transient protein interactions. Both approaches are enabled by combining optical tools for spatiotemporal control of light itself with the total and instantaneously responsive (<1µs) dependence between photocatalytic efficiency and the local supply of light, and each allow for a three order of magnitude increase in resolution vs current tools. Spatially selective labeling will focus on identifying protein interactions unique to cell subpopulations in human tissues, with initial studies focusing on discovering location-conditional interactions driving lymphocyte function in human tonsil and thymus. Later studies will focus on discovering interactome differences between translationally relevant tissue samples. Temporally resolved labeling will combine optogenetic tools and photocatalytic proximity labeling to synchronize and interrogate transient protein interactions. The power of this approach will be fully exploited by studying exocytosis in neurons with millisecond resolution, one of the fastest dynamic biological processes known. Successful development and deployment of these systems for protein interaction discovery will enable the study of large interactome spaces for the first time, and is expected to have a broad impact on the molecular biology community.

GERI实验室研究的总体目标是使用发现技术来绘制蛋白质相互作用， 提供比当前最新临时分辨率的空间临时分辨率的数量级改进。 这项工作的动机是，将蛋白质Interactome互动发现技术的分辨率超越关键 里程碑，例如单细胞和单蛋白阈值，将具有类似于类似的野外影响 转录组学和显微镜的进展。实验室工作的前三年将集中于创建 通过结合光催化接近性标记，新技术，其中附着在该标签上 亲和力手柄驱动与附近蛋白质，图案光和 相互作用门控激活，以简单地执行特异性的多个维度。第四和第五 多年的工作将集中于应用成熟的技术。总体策略沿两个推力分配， 其中通过外部光学控制或内在化学控制获得标记特异性，并且具有 通过最大程度地减少项目相互依存的方式，可以在编程上具有稳健性。本质上选择性 系统将利用光催化标签的高空间分辨率（5 nm），并使用“拆分”系统 当定义的蛋白质靶标处于接近度时。最初的工作将使用本地表达的孤儿肽作为 接近本地标签以发现其当前未知的受体。这项努力将涵盖成千上万的宠儿 通过使用蛋白质组学的标签游离离子迁移率质谱法，通过使用优化的 标签探针设计。拆分系统将结合针对不同感兴趣蛋白的多个光催化剂 使共定位成为特异性的维度，并将最初应用于膜上存在的蛋白质 连接。外部控制的系统将在人体组织中启用亚细胞分辨率标记 MS分辨率临时控制，用于研究瞬态蛋白质相互作用。两种方法均已启用 通过将光的光学工具结合起来，以使光本身的时空控制与总和瞬时响应 （<1µs）光催化效率与局部光的供应之间的依赖性，每种都允许三个 分辨率与当前工具的数量级增加。空间选择性标签将集中于识别 人类组织中细胞亚群特有的蛋白质相互作用，最初的研究重点是发现 促进人扁桃体和胸腺中淋巴细胞功能的位置条件相互作用。后来的研究将集中 发现翻译相关的组织样品之间的相互作用差异。暂时解决 标签将结合光遗传学工具和光催化接近标记，以同步和询问 瞬态蛋白质相互作用。通过研究神经元的胞吐作用，将完全利用这种方法的力量 毫秒分辨率是已知的最快动态生物过程之一。成功发展和 这些系统用于蛋白质相互作用发现的部署将使大型相互作用的空间进行研究 这是第一次，预计将对分子生物学界产生广泛的影响。