Inositol polyphosphates intersect with signaling and metabolic networks via two distinct mechanisms

Abstract

Inositol-based signaling molecules are central eukaryotic messengers and include the highly phosphorylated, diffusible inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs). Despite the essential cellular regulatory functions of InsPs and PP-InsPs (including telomere maintenance, phosphate sensing, cell migration, and insulin secretion), the majority of their protein targets remain unknown. Here, the development of InsP and PP-InsP affinity reagents is described to comprehensively annotate the interactome of these messenger molecules. By using the reagents as bait, >150 putative protein targets were discovered from a eukaryotic cell lysate (Saccharomyces cerevisiae). Gene Ontology analysis of the binding partners revealed a significant overrepresentation of proteins involved in nucleotide metabolism, glucose metabolism, ribosome biogenesis, and phosphorylation-based signal transduction pathways. Notably, we isolated and characterized additional substrates of protein pyrophosphorylation, a unique posttranslational modification mediated by the PP-InsPs. Our findings not only demonstrate that the PP-InsPs provide a central line of communication between signaling and metabolic networks, but also highlight the unusual ability of these molecules to access two distinct modes of action.

Keywords: affinity reagents; inositol pyrophosphates; metabolism; protein pyrophosphorylation; signal transduction.

Fig. 1.

Biosynthesis and signaling mechanisms of PP-InsPs. (A) Abbreviated biosynthetic pathway for PP-InsPs, involving the action of IP6K and PPIP5K in mammals (green). The corresponding enzymes in S. cerevisiae are shown in black. The pyrophosphate moiety can be cleaved by the mammalian phosphohydrolase hDIPP and the yeast proteins Ddp1 and Siw14. (B) PP-InsPs transmit information via two proposed mechanisms, protein binding and protein pyrophosphorylation, a covalent posttranslational modification. Specific examples for either mechanism are listed. hs, Homo sapiens; sc, S. cerevisiae.

Scheme 1.

Synthesis of affinity reagents. (A) Synthetic procedure to obtain InsP₆ affinity reagent 1. (B) Synthetic procedure to obtain 5PCP-InsP₅ affinity reagent 2. (C) Control reagent C. See also SI Appendix, Scheme S1.

Fig. 2.

Validation of affinity reagents and application to cell lysates. (A) hDIPP1 fusion proteins were retained by reagents 1 and 2 in the presence of cell lysate. A total of 5 μg of GST-hDIPP1 and 5 μg of His-hDIPP1 were applied to reagents 1, 2, or C, either in pure form or premixed with a cell lysate from S. cerevisiae at two different concentrations (0.1 and 1 mg/mL). After incubation at 4 °C for 2 h and removal of supernatants, the bound fraction was eluted with InsP₆ (10 mM). Samples were separated by SDS/PAGE and visualized by using silver staining. The results were replicated in two independent experiments. (B) General workflow for enrichment procedure from cell lysates. The head groups of the affinity reagents are drawn, and phosphate groups (OPO₃²⁻) are depicted as P; the linker and resin are shown as a cartoon.

Fig. 3.

The affinity reagents isolate a specific set of proteins in the presence of magnesium ions. GO analysis of proteins enriched with magnesium ions present, using GO terms related to cellular compartment, molecular function, and biological process, is shown (see also Dataset S2).

Fig. 4.

The affinity reagents isolate substrates of protein pyrophosphorylation. (A) In vitro pyrophosphorylation assays reveal targets of pyrophosphorylation. GST-fusion proteins purified from S. cerevisiae were treated with 1 μM β[³²P]5PP-InsP₅ (15 μCi) in 25 mM Tris (pH 7.4, 50 mM NaCl, 6 mM MgCl₂, and 1 mM DTT) at 37 °C for 40 min, while still on beads. Reactions were quenched, heated, resolved by SDS/PAGE, transferred to PVDF membranes, and visualized by autoradiography. Protein loading was analyzed by Western blot of the PVDF membrane with an anti-GST antibody (see also SI Appendix, Fig. S4). The predicted molecular masses are as follows: GST-Nsr1, 71 kDa; GST, 26 kDa; GST-Aad4, 63 kDa; GST-Ypi1, 44 kDa; GST-Nop15, 51 kDa; GST-Has1, 83 kDa; GST-Svf1, 80 kDa; GST-Sso1, 59 kDa; and GST-Kgd2, 76 kDa. All pyrophosphorylation reactions were confirmed in at least one additional independent experiment. (B) GST-Puf6 is a pyrophosphorylation substrate, confirmed by the experimental procedure outlined in A.

Fig. 5.

Enrichment in the absence of divalent ions uncovers a distinct set of protein targets of InsP₆ and 5PP-InsP₅. (A) Reagents 1 and 2 isolate different proteins, depending on the presence or absence of magnesium ions during affinity enrichment. (B) GO analysis of proteins enriched under conditions restricting metal ion availability, using GO terms related to cellular compartment, molecular function, and biological process (see also SI Appendix, Table S2).

Fig. 6.

Inositol polyphosphates interact with the VTC domain of Vtc4. (A) The interaction between a truncated version of Vtc4, His-Vtc4*, and reagents 1 and 2 was confirmed. Reagents 1, 2, and C were exposed to 20 μg of His-Vtc4*, incubated for 2 h at 4 °C, and eluted with either 10 mM InsP₆ or 10 mM 5PCP-InsP₅ solution (PCP). His-Vtc4* input (lane 1), supernatants (lanes 2–4), and eluents (elution with InsP₆: lanes 5–7; elution with 5PCP-InsP₅, lanes 8–10) were separated by SDS/PAGE and visualized by using silver staining. (B) Vtc4 is not pyrophosphorylated. Recombinant GST-Vtc4 was expressed and purified from S. cerevisiae and treated with 1 μM β[³²P]5PP-InsP₅ (15 μCi) in 25 mM Tris (pH 7.4, 50 mM NaCl, 6 mM MgCl₂, and 1 mM DTT) at 37 °C for 40 min, while still on beads. GST-Nsr1 and GST-Puf6 were included as positive control experiments.