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Genetic wiring maps of single-cell protein states reveal an off-switch for GPCR signalling

机译：单细胞蛋白质状态的遗传接线图揭示了GPCR信号的开关

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摘要

As key executers of biological functions, the activity and abundance of proteins are subjected to extensive regulation. Deciphering the genetic architecture underlying this regulation is critical for understanding cellular signalling events and responses to environmental cues. Using random mutagenesis in haploid human cells, we apply a sensitive approach to directly couple genomic mutations to protein measurements in individual cells. Here we use this to examine a suite of cellular processes, such as transcriptional induction, regulation of protein abundance and splicing, signalling cascades (mitogen-activated protein kinase (MAPK), G-protein-coupled receptor (GPCR), protein kinase B (AKT), interferon, and Wingless and Int-related protein (WNT) pathways) and epigenetic modifications (histone crotonylation and methylation). This scalable, sequencing-based procedure elucidates the genetic landscapes that control protein states, identifying genes that cause very narrow phenotypic effects and genes that lead to broad phenotypic consequences. The resulting genetic wiring map identifies the E3-ligase substrate adaptor KCTD5 (ref. 1) as a negative regulator of the AKT pathway, a key signalling cascade frequently deregulated in cancer. KCTD5-deficient cells show elevated levels of phospho-AKT at S473 that could not be attributed to effects on canonical pathway components. To reveal the genetic requirements for this phenotype, we iteratively analysed the regulatory network linked to AKT activity in the knockout background. This genetic modifier screen exposes suppressors of the KCTD5 phenotype and mechanistically demonstrates that KCTD5 acts as an off-switch for GPCR signalling by triggering proteolysis of G beta gamma heterodimers dissociated from the G alpha subunit. Although biological networks have previously been constructed on the basis of gene expression(2,3), protein-protein associations(4-6), or genetic interaction profiles(7,8), we foresee that the approach described here will enable the generation of a comprehensive genetic wiring map for human cells on the basis of quantitative protein states.
机译：作为生物学功能的关键执行者，蛋白质的活性和丰度受到广泛的调节。破译此调控基础的遗传结构对于理解细胞信号事件和对环境提示的反应至关重要。使用单倍体人类细胞中的随机诱变，我们应用了一种灵敏的方法将基因组突变直接耦合到单个细胞中的蛋白质测量中。在这里，我们使用它来检查一系列细胞过程，例如转录诱导，蛋白丰度和剪接的调控，信号级联（促分裂原活化蛋白激酶（MAPK），G蛋白偶联受体（GPCR），蛋白激酶B（ AKT），干扰素，无翅和Int相关蛋白（WNT）途径）和表观遗传修饰（组蛋白巴豆酰化和甲基化）。这种可扩展的，基于测序的程序阐明了控制蛋白质状态的遗传格局，确定了导致非常窄的表型效应的基因和导致广泛的表型后果的基因。最终的遗传学图谱表明，E3-连接酶底物适配器KCTD5（参考文献1）是AKT途径的负调节剂，而AKT途径是癌症中经常失控的关键信号传导级联。缺乏KCTD5的细胞在S473处显示磷酸AKT水平升高，这不能归因于对经典途径成分的影响。为了揭示该表型的遗传要求，我们反复分析了与敲除背景中AKT活性相关的调控网络。此遗传修饰剂筛选暴露了KCTD5表型的抑制子，并通过机制证明KCTD5通过触发从Gα亚基解离的Gβγ异二聚体的蛋白水解作用，充当GPCR信号的开关。尽管以前已经根据基因表达（2,3），蛋白质-蛋白质关联（4-6）或遗传相互作用图谱（7,8）构建了生物网络，但我们可以预见，此处描述的方法将使产生定量蛋白质状态的人类细胞综合遗传图谱的建立。

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  • 来源
    《Nature》 |2017年第7657期|307-311|共5页
  • 作者

    Brockmann Markus; Blomen Vincent A.; Nieuwenhuis Joppe; Stickel Elmer; Raaben Matthijs; Bleijerveld Onno B.; Altelaar A. F. Maarten; Jae Lucas T.; Brummelkamp Thijn R.;

  • 作者单位

    Netherlands Canc Inst, Plesmanlaan 121, NL-1066 CX Amsterdam, Netherlands;

    Netherlands Canc Inst, Plesmanlaan 121, NL-1066 CX Amsterdam, Netherlands;

    Netherlands Canc Inst, Plesmanlaan 121, NL-1066 CX Amsterdam, Netherlands;

    Netherlands Canc Inst, Plesmanlaan 121, NL-1066 CX Amsterdam, Netherlands;

    Netherlands Canc Inst, Plesmanlaan 121, NL-1066 CX Amsterdam, Netherlands;

    Netherlands Canc Inst, Plesmanlaan 121, NL-1066 CX Amsterdam, Netherlands;

    Netherlands Canc Inst, Plesmanlaan 121, NL-1066 CX Amsterdam, Netherlands|Univ Utrecht, Utrecht Inst Pharmaceut Sci, Biomol Mass Spectrometry & Prote, Padualaan 8, NL-3584 CH Utrecht, Netherlands;

    Netherlands Canc Inst, Plesmanlaan 121, NL-1066 CX Amsterdam, Netherlands|Ludwig Maximilians Univ Munchen, Gene Ctr, Feodor Lynen Str 25, D-81377 Munich, Germany|Ludwig Maximilians Univ Munchen, Dept Biochem, Feodor Lynen Str 25, D-81377 Munich, Germany;

    Netherlands Canc Inst, Plesmanlaan 121, NL-1066 CX Amsterdam, Netherlands|Austrian Acad Sci, CeMM Res Ctr Mol Med, A-1090 Vienna, Austria|Cancergenom Nl, Plesmanlaan 121, NL-1066 CX Amsterdam, Netherlands;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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