Dataset Information

Affinity-Based Interactome Mapping of Inositol Pyrophosphates Reveals 4/6-PP-InsP5-–Binding Proteins in Plants

ABSTRACT: Inositol pyrophosphates (PP-InsPs) are central regulators of eukaryotic signaling events. While certain PP-InsP isomers have been conclusively linked to the regulation of phosphate homeostasis through interaction with SPX domain containing proteins in plants, the functions of the recently discovered isomer 4/6-PP-InsP5 remain largely unknown. Here, we employed two complementary affinity-based strategies – a matrix approach and a photoaffinity probe – to systematically identify 4/6-PP-InsP5-binding proteins in Arabidopsis thaliana. The two methods yielded partially overlapping protein sets, with photoaffinity enrichment likely capturing additional transient and/or weak interactions. Moreover, competition experiments with different isomers were applied to obtain information about potential isomer-specific interactions. As a proof-of-concept, one candidate interactor (FHA domain-containing protein AtFHA2) was shown to bind 4-PP-InsP5 in vitro with markedly higher affinity than InsP6, while no reliable binding parameters could be obtained for its the enantiomeric isomer 6-PP-InsP5. Thus, besides the SPX domain, FHA domain containing proteins, of which 18 exist in Arabidopsis, are potentially regulated by inositol pyrophosphates. More generally, our findings reveal a diverse protein network associated with 4/6-PP-InsP5 and establish a versatile platform for dissecting its biological roles in plants and other organisms.

INSTRUMENT(S):

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell

SUBMITTER: Sara Christina Stolze

LAB HEAD: Hirofumi Nakagami

PROVIDER: PXD073760 | Pride | 2026-06-08

REPOSITORIES: Pride

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Affinity-Based Interactome Mapping of Inositol Pyrophosphates Reveals 4/6-PP-InsP<sub>5</sub>-Binding Proteins in Plants.

Ritter Kevin K Gaugler Verena V Stolze Sara Christina SC Ghosh Riya R Jayamon Akhila A Yadav Ranjana R Wollensack Felix F Laha Debabrata D Nakagami Hirofumi H Schaaf Gabriel G Jessen Henning Jacob HJ

Advanced science (Weinheim, Baden-Wurttemberg, Germany) 20260302 26

Inositol pyrophosphates (PP-InsPs) are central regulators of eukaryotic signaling events. While certain PP-InsP isomers have been conclusively linked to the regulation of phosphate homeostasis through interaction with SPX domain-containing proteins in plants, the functions of the recently discovered isomer 4/6-PP-InsP<sub>5</sub> remain largely unknown. Here, we employ two complementary affinity-based strategies-a matrix approach and a photoaffinity probe-to systematically identify 4/6-PP-InsP<s ...[more]

PMID: 41766624

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Project description:Comparison of gene expression from subjects who resolved or formed pustules to H.ducreyi. In human inoculation experiments, the cutaneous immune response to Haemophilus ducreyi consists of serum, PMN, macrophages, T cells and myeloid DC. In reinfection experiments, some subjects form pustules twice (PP group) or resolve infection twice (RR group). Although pustule formation is associated with serum resistance and phagocytic failure, there are no differences in the ability of isolated phagocytes or serum obtained from PP and RR subjects to ingest or kill H. ducreyi. To identify the basis for differential host susceptibility to H. ducreyi, we used microarrays to profile gene expression in infected and uninfected tissue and monocyte-derived DC obtained from PP and RR subjects. In infected tissue, both groups had a core response to H. ducreyi. Many additional transcripts that signify active immune function were upregulated exclusively in RR tissue, while PP tissue exclusively contained differentially regulated transcripts consistent with immune dysregulation. The core response of infected DC from both groups was typical of a DC1 response. RR DC exclusively expressed many additional transcripts indicative of DC1 function, while PP DC uniquely expressed differentially regulated transcripts characteristic of both DC1 and DCreg. The data suggest that DC from PP and RR subjects are prewired to respond differentially to H. ducreyi. Experiment Overall Design: Six healthy adult volunteers (5 females, 1 male, 36 Â± 13 yrs, mean age Â± SD) who had been infected with H. ducreyi twice were inoculated a third time. Each volunteer was inoculated at 3 sites on the upper arm with live H. ducreyi 35000HP (a human passaged isolate of strain 35000) and at 1 site with sterile PBS. Forty-eight hours after inoculation, lesion size was measured and RNA was isolated from the infected site that had the largest diameter. RNA was also obtained from the PBS control site (uninfected site). We obtained peripheral blood 6 to 12 months after inoculation of the 6th subject and derived myeloid DC in vitro. In brief, CD14+ peripheral blood monocytes were isolated from PBMC by positive selection magnetic beads (Miltenyi Biotec, Auburn, CA) and grown in the presence of recombinant human (rh) IL-4 (1 ng/ml) and rhGM-CSF (0.2 ng/ml) (R&D Systems, Minneapolis, MN). The cells were HLA-DR+, CD86+ CD40+, CD3-, CD14-, and CD19- by flow cytometry. The experiment was done in pairs so that DC from 1 RR subject were exposed to the same inoculum as DC from 1 PP subject. DC were incubated with nonopsonized H. ducreyi for 90 minutes at an MOI of 30:1. The DC were washed to remove non-associated bacteria, and incubated an additional 22.5 hours. Cells were collected and used for microarray analysis. Supernatants were collected and analyzed for cytokines using the Human Th1/Th2 II Cytometric Bead Array Kit per manufacturerâs instructions (BD Biosciences).

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Dataset Information

Affinity-Based Interactome Mapping of Inositol Pyrophosphates Reveals 4/6-PP-InsP5-–Binding Proteins in Plants

Dataset's files

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Affinity-Based Interactome Mapping of Inositol Pyrophosphates Reveals 4/6-PP-InsP<sub>5</sub>-Binding Proteins in Plants.

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Dataset Information

Affinity-Based Interactome Mapping of Inositol Pyrophosphates Reveals 4/6-PP-InsP5-–Binding Proteins in Plants

Dataset's files

Publications

Affinity-Based Interactome Mapping of Inositol Pyrophosphates Reveals 4/6-PP-InsP&lt;sub&gt;5&lt;/sub&gt;-Binding Proteins in Plants.

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OmicsDI is part of the ELIXIR infrastructure

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Affinity-Based Interactome Mapping of Inositol Pyrophosphates Reveals 4/6-PP-InsP<sub>5</sub>-Binding Proteins in Plants.