细胞内蛋白质丰度，活性，相互作用和吸毒性与标签seq的多重分析
Multiplexed profiling of intracellular protein abundance, activity, interactions and druggability with LABEL-seq
影响因子:32.10000
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
摘要
在这里，我们通过测序（Label-Seq）描述了具有条形码和富集生化分析的标记，这是人类细胞中合并蛋白质变体的大规模平行分析的测定。通过利用稳定的，可变化的RNA条形码的RNA结合结构域（RBD）的细胞内自组装，标记-Seq促进了直接测量蛋白质的性能和功能，并使用RBD蛋白融合的简单亲和力富集，然后对Co-Enroced barcoded barcoded barcoded recencected co-norpoded barcoded barcoded。 〜20,000个变体效应对约1,600个BRAF变体的测量表明，在癌症中经常突变的位置的变异微细胞内丰度很小，但可能会大大改变活性，蛋白质 - 蛋白质相互作用和可药用。综合分析确定了具有相似生化作用的位置网络，并启用了对细胞增殖和小分子促进降解的变异作用的建模。因此，Label-seq可以在天然细胞环境中直接测量多种生化特性，从而提供对蛋白质功能，疾病机制和可药物的见解。
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.