Triplexed Affinity Reagents to Sample the Mammalian Inositol Pyrophosphate Interactome. Issue 8 (20th August 2020)

• Record Type: Journal Article
• Authors: Furkert, David; Hostachy, Sarah; Nadler-Holly, Michal; Fiedler, Dorothea
• Is Part Of: Cell chemical biology. Volume 27:Issue 8(2020)
• Page Start: 1097
• Page End: 1108.e4
• Publication Date: 2020-08-20
• Subjects: inositol pyrophosphate -- affinity enrichment -- proteomics -- small-molecule messengers -- signal transduction -- inositol phosphatases; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2020.07.017
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: The inositol pyrophosphates (PP-InsPs) are a ubiquitous group of highly phosphorylated eukaryotic messengers. They have been linked to a panoply of central cellular processes, but a detailed understanding of the discrete signaling events is lacking in most cases. To create a more mechanistic picture of PP-InsP signaling, we sought to annotate the mammalian interactome of the most abundant inositol pyrophosphate 5PP-InsP5 . To do so, triplexed affinity reagents were developed, in which a metabolically stable PP-InsP analog was immobilized in three different ways. Application of these triplexed reagents to mammalian lysates identified between 300 and 400 putative interacting proteins. These interactomes revealed connections between 5PP-InsP5 and central cellular regulators, such as lipid phosphatases, protein kinases, and GTPases, and identified protein domains commonly targeted by 5PP-InsP5 . Both the triplexed affinity reagents, and the proteomic datasets, constitute powerful resources for the community, to launch future investigations into the multiple signaling modalities of inositol pyrophosphates. Graphical Abstract: Highlights: Analogs of Inositol pyrophosphate messengers were derivatized at different positions Multiplexed affinity matrices were generated and validated 300–400 putative interacting proteins were identified from mammalian lysates Label-free quantification uncovered binding preferences of closely related messengers Abstract : Furkert et al. generated triplexed inositol pyrophosphate affinity reagents and comprehensively identified protein binding partners from HEK293T and HCT116 cell lysates. The proteomic datasets revealed unprecedented connections between 5PP-InsP5 and central cellular regulators, showcased which proteins displayed high specificity toward 5PP-InsP5, and thus constitute useful resources for the community.