IP6K1 upregulates the formation of processing bodies by influencing protein-protein interactions on the mRNA cap.

Inositol hexakisphosphate kinase 1 (IP6K1) is a small molecule kinase that catalyzes the conversion of the inositol phosphate IP6 to 5-IP7. We show that IP6K1 acts independently of its catalytic activity to upregulate the formation of processing bodies (P-bodies), which are cytoplasmic ribonucleoprotein granules that store translationally repressed mRNA. IP6K1 does not localise to P-bodies, but instead binds to ribosomes, where it interacts with the mRNA decapping complex - the scaffold protein EDC4, activator proteins DCP1A/B, decapping enzyme DCP2 and RNA helicase DDX6. Along with its partner 4E-T, DDX6 is known to nucleate protein-protein interactions on the 5' mRNA cap to facilitate P-body formation. IP6K1 binds the translation initiation complex eIF4F on the mRNA cap, augmenting the interaction of DDX6 with 4E-T (also known as EIF4ENIF1) and the cap-binding protein eIF4E. Cells with reduced IP6K1 show downregulated microRNA-mediated translational suppression and increased stability of DCP2-regulated transcripts. Our findings unveil IP6K1 as a novel facilitator of proteome remodelling on the mRNA cap, tipping the balance in favour of translational repression over initiation, thus leading to P-body assembly. This article has an associated First Person interview with the first author of the paper.

Inositol pyrophosphates promote MYC polyubiquitination by FBW7 to regulate cell survival.

The transcription factor MYC regulates cell survival and growth, and its level is tightly controlled in normal cells. We report that serine pyrophosphorylation - a posttranslational modification triggered by inositol pyrophosphate signaling molecules - controls MYC levels via regulated protein degradation. We find that endogenous MYC is stabilized and less polyubiquitinated in cells with reduced inositol pyrophosphates. We show that the inositol pyrophosphate 5-IP7 transfers its high-energy beta phosphate moiety to pre-phosphorylated serine residues in the central PEST domain of MYC. Loss of serine pyrophosphorylation in the PEST domain lowers the extent of MYC polyubiquitination and increases its stability. Fusion to the MYC PEST domain lowers the stability of GFP, but this effect is dependent on the extent of PEST domain pyrophosphorylation. The E3 ubiquitin ligase FBW7 can bind directly to the PEST domain of MYC, and this interaction is exclusively dependent on serine pyrophosphorylation. A stabilized, pyrophosphorylation-deficient form of MYC increases cell death during growth stress in untransformed cells. Splenocytes from mice lacking IP6K1, a kinase responsible for the synthesis of 5-IP7, have higher levels of MYC, and show increased cell proliferation in response to mitogens, compared with splenocytes from wild type mice. Thus, control of MYC stability through a novel pyro-phosphodegron provides unexpected insight into the regulation of cell survival in response to environmental cues.

A high energy phosphate jump - From pyrophospho-inositol to pyrophospho-serine.

Inositol pyrophosphates (PP-IPs) are a class of energy rich metabolites present in all eukaryotic cells. The hydroxyl groups on these water soluble derivatives of inositol are substituted with diphosphate and monophosphate moieties. Since the discovery of PP-IPs in the early 1990s, enormous progress has been made in uncovering pleiotropic roles for these small molecules in cellular physiology. PP-IPs exert their effect on proteins in two ways - allosteric regulation by direct binding, or post-translational regulation by serine pyrophosphorylation, a modification unique to PP-IPs. Serine pyrophosphorylation is achieved by Mg2+-dependent, but enzyme independent transfer of a ß-phosphate from a PP-IP to a pre-phosphorylated serine residue located in an acidic motif, within an intrinsically disordered protein sequence. This distinctive post-translational modification has been shown to regulate diverse cellular processes, including rRNA synthesis, glycolysis, and vesicle transport. However, our understanding of the molecular details of this phosphotransfer from pyrophospho-inositol to generate pyrophospho-serine, is still nascent. This review discusses our current knowledge of protein pyrophosphorylation, and recent advances in understanding the mechanism of this important yet overlooked post-translational modification.

Inositol Pyrophosphates: Energetic, Omnipresent and Versatile Signalling Molecules.

Inositol pyrophosphates (PP-IPs) are a class of energy-rich signalling molecules found in all eukaryotic cells. These are derivatives of inositol that contain one or more diphosphate (or pyrophosphate) groups in addition to monophosphates. The more abundant and best studied PP-IPs are diphosphoinositol pentakisphosphate (IP7) and bis-diphosphoinositol tetrakisphosphate (IP8). These molecules can influence protein function by two mechanisms: binding and pyrophosphorylation. The former involves the specific interaction of a particular inositol pyrophosphate with a binding site on a protein, while the latter is a unique attribute of inositol pyrophosphates, wherein the ß-phosphate moiety is transferred from a PP-IP to a pre-phosphorylated serine residue in a protein to generate pyrophosphoserine. Both these events can result in changes in the target protein's activity, localisation or its interaction with other partners. As a consequence of their ubiquitous presence in all eukaryotic organisms and all cell types examined till date, and their ability to modify protein function, PP-IPs have been found to participate in a wide range of metabolic, developmental, and signalling pathways. This review highlights many of the known functions of PP-IPs in the context of their temporal and spatial distribution in eukaryotic cells.

Independent amino acid residues in the S2 pocket of falcipain-3 determine its specificity for P2 residues in substrates.

Falcipain-3 (FP3) is an essential and drug target cysteine protease of the most lethal human malaria parasite Plasmodium falciparum. FP3 and its majority of homologs in malaria parasites prefer Leu at the P2 position in substrates and inhibitors, whereas its major host homolog cathepsin L prefers Phe. However, FP3 is much less active on peptide substrates and has negligible activity against a P2 Arg-containing substrate (Z-RR-AMC) compared to its paralog falcipain-2A (FP2A). To identify the specificity determinants, the S2/3 pocket residues of FP3 were substituted with the corresponding residues in FP2 or cathepsin L, and the wild type and mutant proteases were assessed for hydrolysis of peptide and protein substrates. Our results indicate that the S2 pocket residues I94 and P181 of FP3 are chiefly responsible for its P2 Leu preference and negligible activity for Z-RR-AMC, respectively. E243 in FP3 and the corresponding residue D234 in FP2 have a key role in Z-RR-AMC hydrolysing activity, possibly through stabilization of side chain interactions, as their substitution with Ala abolished the activity. Several FP3 mutants, which retained P2 Leu preference and showed similar or more activity than wild type FP3 on peptide substrates, degraded haemoglobin less efficiently than wild type FP3, suggesting that multiple residues contribute to haemoglobinase activity. Furthermore, P181 and E243 appear to contribute to the optimum activity of FP3 in the food vacuole milieu (≈pH 5.5). The identification of residues determining specificity of FP3 could aid in developing specific inhibitors of FP3 and its homologs in malaria parasites.