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Proteome-wide quantification of inositol pyrophosphate-protein interactions

ABSTRACT: Inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs) constitute a group of highly phosphorylated molecules that are involved in many cellular signaling processes. To characterize discrete signaling events of these structurally closely related molecules, a mass spectrometry approach was developed to derive apparent binding constants for these ligands on a proteome-wide scale. The method employed a series of chemically synthesized, biotinylated affinity reagents for inositol hexakisphosphate (InsP6), and the inositol pyrophosphates 1PP-InsP5, 5PP-InsP5 and 1,5(PP)2-InsP4 (also termed InsP8). Application of these affinity reagents at different concentrations, in combination with tandem mass tag (TMT) labeling, provided binding data for thousands of proteins from a mammalian cell lysate. Investigation of different enrichment conditions, where Mg2+ ions were either available or not, showcased a strong influence of Mg2+ on the protein binding capacities of PP-InsPs. Gene ontology analysis closely linked PP-InsPs-interacting proteins to transcriptional processes in the nucleus. Subsequent data analysis enabled a targeted search for protein pyrophosphorylation among PP-InsP interactors, and identified four new targets . The data presented here constitute a valuable resource for the community, and application of the method reported here to other biological contexts will enable the exploration of PP-InsP dependent signaling pathways across species.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Dorothea Fiedler

PROVIDER: PXD052682 | JPOST Repository | Mon Apr 27 00:00:00 BST 2026

REPOSITORIES: jPOST

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			Action	DRS
	L1_20230825_AR_02_185_cytoMg_IP8_rep1.raw	Raw
	L1_20230825_AR_02_185_cytoMg_IP8_rep2.raw	Raw
	L1_20230825_AR_02_185_cytoMg_IP8_rep3.raw	Raw
	L1_20230901_AR_03_001_nuclMg_1IP7_rep1.raw	Raw
	L1_20230901_AR_03_001_nuclMg_1IP7_rep2.raw	Raw

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Project description:Inositol pyrophosphates (PP-InsPs) are important signaling molecules that regulate diverse cellular processes in eukaryotes, including energy homeostasis and phosphate (Pi) signaling. However, the enzymes responsible for their turnover in plants remain largely unknown. Using a non-hydrolysable PP-InsP analog in a pull-down approach, we identified a family of Arabidopsis Nudix hydrolases (NUDTs) that group into two closely related subclades. Through in vitro assays, heterologous expression systems, and higher-order gene-edited mutants, we explored the substrate specificities and physiological roles of these hydrolases. Using a combination of strong anion exchange (SAX)-HPLC, PAGE, and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that their PP-InsP pyrophosphatase activity is enantiomer-selective and Mg2+-dependent. Specifically, subclade I NUDTs preferentially hydrolyze 4-InsP7, while subclade II NUDTs target 3-InsP7, with minor activity against other PP-InsPs, including 5-InsP7. In higher-order mutants of subclade II NUDTs, we observed defects in both Pi and iron homeostasis, accompanied by increased levels of 1/3-InsP7 and 5-InsP7, with a markedly larger increase in 1/3-InsP7. Furthermore, ectopic expression of NUDTs from both subclades induced local Pi starvation responses (PSRs). However, RNA-seq analysis of WT and nudt12/13/16 plants suggests that these NUDTs have PSR-independent roles, potentially involving 1/3-InsP7 in the regulation of plant defense. Expanding beyond subclade II NUDTs, we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs, and found that such activities are conserved across plants and humans. Additionally, we found that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to subclade I NUDTs. Collectively, our study provides new insights into the roles of NUDTs in regulating PP-InsP signaling pathways in plants and highlights cross-kingdom conservation of enzymes involved in PP-InsP metabolism.

Project description:Cryptococcus neoformans is the most common cause of fungal meningitis, with high mortality and morbidity. The reason for the frequent occurrence of Cryptococcus infection in the central nervous system (CNS) is poorly understood. In this study, we find that inositol plays an important role in the transversal of Cryptococcus across the blood-brain barrier (BBB) both in an in vitro human BBB model and in vivo animal models. The inositol stimulation of BBB crossing is dependent upon fungal inositol transporters. The upregulation of genes involved in the inositol catabolism pathway is evident in a microarray analysis. The expression of CPS1, a gene encoding the hyaluronic acid synthase in Cryptococcus, is also upregulated by the inositol treatment. The production of hyaluronic acid increased in cells treated with inositol, which leads to the enhanced binding ability of Cryptococcus cells to the human brain microvascular endothelial cells (HBMECs) constituting the BBB. Overall, our studies provide a mechanism for inositol-dependent Cryptococcus transversal of the BBB, supporting our hypothesis that host inositol utilization by the fungus contributes to Cryptococcus CNS infection. To understand the effect of inositol on gene expression profiles during cell development, microarray experiments were performed to monitor the genes regulated by inositol. H99 overnight culture was washed with dH2O once, and cells were inoculated on SD medium with or without inositol. Cells were collected from SD plates 24 hr post-inoculation, washed with dH2O, and total RNA was purified. Total RNAs were extracted using Trizol Reagents (Invitrogen) and purified using the Qiagen RNeasy cleanup kit (Qiagen). Cy3 and Cy5-labeled cDNA were generated by incorporating amino-allyl-dUTP during reverse transcription of 5 Âµg of total RNA as described previously and competitively hybridized to a JEC21 whole-genome array generated previously at Washington University in Saint Louis. After hybridization, arrays were scanned with a GenePix 4000B scanner (Axon Instruments, http://www.axon.com) and analyzed by using GenePix Pro version 4.0 and BRB array tools (developed by Richard Simon and Amy Peng Lam at the National Cancer Institute; http://linus.nci.nih.gov/BRB-ArrayTools.html)

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Proteome-wide quantification of inositol pyrophosphate-protein interactions

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