Nucleolar Architecture Is Modulated by a Small Molecule, the Inositol Pyrophosphate 5-InsP7

Abstract

Inositol pyrophosphates (PP-InsPs); are a functionally diverse family of eukaryotic molecules that deploy a highly-specialized array of phosphate groups as a combinatorial cell-signaling code. One reductive strategy to derive a molecular-level understanding of the many actions of PP-InsPs is to individually characterize the proteins that bind them. Here, we describe an alternate approach that seeks a single, collective rationalization for PP-InsP binding to an entire group of proteins, i.e., the multiple nucleolar proteins previously reported to bind 5-InsP7 (5-diphospho-inositol-1,2,3,4,6-pentakisphosphate). Quantitative confocal imaging of the outer nucleolar granular region revealed its expansion when cellular 5-InsP7 levels were elevated by either (a) reducing the 5-InsP7 metabolism by a CRISPR-based knockout (KO) of either NUDT3 or PPIP5Ks; or (b), the heterologous expression of wild-type inositol hexakisphosphate kinase, i.e., IP6K2; separate expression of a kinase-dead IP6K2 mutant did not affect granular volume. Conversely, the nucleolar granular region in PPIP5K KO cells shrank back to the wild-type volume upon attenuating 5-InsP7 synthesis using either a pan-IP6K inhibitor or the siRNA-induced knockdown of IP6K1+IP6K2. Significantly, the inner fibrillar volume of the nucleolus was unaffected by 5-InsP7. We posit that 5-InsP7 acts as an ‘electrostatic glue’ that binds together positively charged surfaces on separate proteins, overcoming mutual protein–protein electrostatic repulsion the latter phenomenon is a known requirement for the assembly of a non-membranous biomolecular condensate.

Pools of independently cycling inositol phosphates revealed by pulse labeling with18O-water

“…or casein kinase 2 (Macbeth et al, 2005;Lee et al, 2013). Moreover, InsPs might be sequestered into biomolecular condensate type structures such as nucleoli (Sahu et al, 2023).…”

“…Examples include the capsid and Gag proteins of retroviruses (Obr et al , 2021; Renner et al , 2023) the RNA editing enzyme ADAR2 or casein kinase 2 (Macbeth et al , 2005; Lee et al , 2013). Moreover, InsPs might be sequestered into biomolecular condensate type structures such as nucleoli (Sahu et al , 2023).…”

Proteome-wide quantification of inositol pyrophosphate-protein interactions

“…In general, the nucleolus stands out as an interesting compartment where regulation by 5PP-InsP5 can take place 26,28,60,61 , and therefore we were curious if any of the recently identified pyrophosphoproteins were enriched by biotin-5PCP-InsP5 and/or biotin-1,5(PCP)2-InsP4 in the Mg 2+ containing nuclear sample. Indeed, 24 out of the 57 known pyrophosphoproteins (in HEK293T) were enriched by biotin-5PCP-InsP5 and/or biotin-1,5(PCP)2-InsP4 (Figure 5d).…”

Fluorination Influences the Bioisostery of Myo‐Inositol Pyrophosphate Analogs

“…The PP−IP diphosphate groups also contribute to ligand specificity through allosteric interactions with protein receptors; prominent mammalian examples include the SPX domain of mammalian XPR1, [8,9] and certain pleckstrin homology domains [10–12] . Separately, the high negative charge density of PP−IPs is also hypothesized to act as an intermolecular electrostatic ‘glue’ to promote formation of functionally significant macromolecular complexes [13,14] . These various mechanisms of action license PP−IPs to regulate an extremely wide range of biological activities [14–16] …”

“…[10][11][12] Separately, the high negative charge density of PPÀ IPs is also hypothesized to act as an intermolecular electrostatic 'glue' to promote formation of functionally significant macromolecular complexes. [13,14] These various mechanisms of action license PPÀ IPs to regulate an extremely wide range of biological activities. [14][15][16] Two classes of enzymes synthesize PPÀ IPs.…”