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Quantitative fragmentomics to map affinities of interactomes

ABSTRACT: ~500,000 protein interactions have been identified in human but most binding affinities remain unknown, limiting quantitative investigation of interactome-function relationships. This gap can be filled by systematically measuring the affinities of minimal interacting protein fragments, the building blocks of interactomes. As a proof of principle, we experimentally measured 65,000 affinities involving 266 human PDZ domains (practically the complete "PDZome"), 424 human PDZ-Binding Motifs (~10% of the "PBMome") and 24 viral motifs. We determined binding constants for ~18,000 interactions that passed the detection threshold. Independent AP-MS experiments and database surveys demonstrated the strong agreement of quantitative fragmentomics with full-length protein interactomics. We evidence hot spots for viral interference, which allow a viral PBM, such as that of HPV-E6 oncoprotein, to impact the cell proteome way beyond its immediate binders. We propose to distinguish interactomes and complexomes, viewed as intrinsic and extrinsic properties linked by the law of mass action.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Luc Negroni

LAB HEAD: Gergo Gogl

PROVIDER: PXD027743 | Pride | 2022-10-14

REPOSITORIES: Pride

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Quantitative fragmentomics allow affinity mapping of interactomes.

Gogl Gergo G Zambo Boglarka B Kostmann Camille C Cousido-Siah Alexandra A Morlet Bastien B Durbesson Fabien F Negroni Luc L Eberling Pascal P Jané Pau P Nominé Yves Y Zeke Andras A Østergaard Søren S Monsellier Élodie É Vincentelli Renaud R Travé Gilles G

Nature communications 20220917 1

Human protein networks have been widely explored but most binding affinities remain unknown, hindering quantitative interactome-function studies. Yet interactomes rely on minimal interacting fragments displaying quantifiable affinities. Here, we measure the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer. We calculate interactomic distances, identify hot spots ...[more]

PMID: 36115835

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Project description:The earliest stages of development in most metazoans are driven by maternally deposited proteins and mRNAs, with widespread transcriptional activation of the zygotic genome occurring hours after fertilization, at a period known as the maternal-to-zygotic transition (MZT). In Drosophila, the MZT is preceded by the transcription of a small number of genes that initiate sex determination, patterning and other essential developmental processes. The zinc-finger transcription factor Zelda (ZLD) plays a key role in the transcriptional activation of these earliest-expressed genes. To better understand the mechanisms of ZLD activation and the range of its targets, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to map regions bound by ZLD prior to (mitotic cycles 8 and 9), during (mitotic cycles 13 and early 14) and after (late mitotic cycle 14) the MZT. Although only a handful of genes are transcribed prior to mitotic cycle 10, we identified thousands of regions bound by ZLD in cycle 8-9 embryos, most of which remain bound through mitotic cycle 14. As expected, these ZLD-bound regions include the promoters and enhancers of the small subset of genes transcribed at this early stage. However we also observed ZLD bound at cycle 8-9 to the promoters of a large fraction of the several thousand genes whose first transcription does not occur until roughly an hour and four mitotic cycles later. These early ZLD-bound regions include virtually all of the thousands of known and presumed enhancers bound at cycle 14 by the transcription factors that regulate patterned gene activation during the MZT. The association between early ZLD binding and MZT activity is so strong that ZLD binding alone can be used to identify active promoters and regulatory sequences with high specificity and selectivity. This strong early association of ZLD with regions not active until the MZT suggests that ZLD is not only required for the earliest wave of transcription, but also plays a major role in activating the genome at the MZT. Genome-wide mapping of Zelda in wild-type Drosophila melanogaster embryos prior (mitotic cycles 8-9), during (cycles 13-14), and after (late cycle 14) maternal-to-zygotic transition

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Quantitative fragmentomics to map affinities of interactomes

Publications

Quantitative fragmentomics allow affinity mapping of interactomes.

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