利用LABEL-seq实现细胞内蛋白丰度、活性、相互作用及药物靶向性的多重分析

Multiplexed profiling of intracellular protein abundance, activity, interactions and druggability with LABEL-seq

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NATURE METHODS

影响因子:32.1

分区:生物学1区 Top / 生化研究方法1区

发表日期:2024 Nov

作者: Jessica J Simon, Douglas M Fowler, Dustin J Maly

DOI: 10.1038/s41592-024-02456-7

摘要

本文介绍了LABEL-seq（带有条形码的标记与富集测序技术），一种用于高通量池化蛋白变异体分析的测序方法。通过利用RNA结合域（RBD）自组装形成稳定的变异体编码RNA条形码，LABEL-seq能够直接测量蛋白的性质和功能，采用简单的亲和富集技术对RBD融合蛋白进行富集，然后进行高通量测序以获得共同富集的条形码。对约1,600个BRaf变异体的2万余个变异效应的测定显示，在癌症中频繁突变的位点，变异对蛋白内在丰度影响有限，但对活性、蛋白质相互作用和药物靶向性可能产生巨大影响。整合分析揭示了具有相似生化作用的位点网络，并建立了变异体对细胞增殖和小分子促降解的模型。因此，LABEL-seq实现了在原生细胞环境中直接测量多种生物化学性质，为研究蛋白功能、疾病机制和药物靶向提供了有力工具。

Abstract

Here we describe labeling with barcodes and enrichment for biochemical analysis by sequencing (LABEL-seq), an assay for massively parallel profiling of pooled protein variants in human cells. By leveraging the intracellular self-assembly of an RNA-binding domain (RBD) with a stable, variant-encoding RNA barcode, LABEL-seq facilitates the direct measurement of protein properties and functions using simple affinity enrichments of RBD protein fusions, followed by high-throughput sequencing of co-enriched barcodes. Measurement of ~20,000 variant effects for ~1,600 BRaf variants revealed that variation at positions frequently mutated in cancer minimally impacted intracellular abundance but could dramatically alter activity, protein-protein interactions and druggability. Integrative analysis identified networks of positions with similar biochemical roles and enabled modeling of variant effects on cell proliferation and small molecule-promoted degradation. Thus, LABEL-seq enables direct measurement of multiple biochemical properties in a native cellular context, providing insights into protein function, disease mechanisms and druggability.