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A novel context for the 'MutT' module, a guardian of cell integrity, in a diphosphoinositol polyphosphate
S T Safrany1, J J Caffrey, X Yang
1Inositide Signaling Group, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, PO Box 12233, NC 27709, USA.
Abstract:
Diphosphoinositol pentakisphosphate (PP-InsP5 or 'InsP7') and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4 or 'InsP8') are the most highly phosphorylated members of the inositol-based cell signaling family. We have purified a rat hepatic diphosphoinositol polyphosphate phosphohydrolase (DIPP) that cleaves a beta-phosphate from the diphosphate groups in PP-InsP5 (Km = 340 nM) and [PP]2-InsP4 (Km = 34 nM). Inositol hexakisphophate (InsP6) was not a substrate, but it inhibited metabolism of both [PP]2-InsP4 and PP-InsP5 (IC50 = 0.2 and 3 microM, respectively). Microsequencing of DIPP revealed a 'MutT' domain, which in other contexts guards cellular integrity by dephosphorylating 8-oxo-dGTP, which causes AT to CG transversion mutations. The MutT domain also metabolizes some nucleoside phosphates that may play roles in signal transduction. The rat DIPP MutT domain is conserved in a novel recombinant human uterine DIPP. The nucleotide sequence of the human DIPP cDNA was aligned to chromosome 6; the candidate gene contains at least four exons. The dependence of DIPP's catalytic activity upon its MutT domain was confirmed by mutagenesis of a conserved glutamate residue. DIPP's low molecular size, Mg2+ dependency and catalytic preference for phosphoanhydride bonds are also features of other MutT-type proteins. Because overlapping substrate specificity is a feature of this class of proteins, our data provide new directions for future studies of higher inositol phosphates.
Insights
Researchers purified a rat enzyme, diphosphoinositol polyphosphate phosphohydrolase (DIPP), that breaks down highly phosphorylated inositol signaling molecules. This enzyme contains a MutT domain, suggesting a role in cellular integrity and signal transduction pathways.
Area of Science:
- Biochemistry
- Cell Signaling
- Enzymology
Background:
- Diphosphoinositol pentakisphosphate (PP-InsP5 or InsP7) and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4 or InsP8) are key highly phosphorylated inositol signaling molecules.
- Understanding the enzymes that regulate these molecules is crucial for deciphering cellular processes.
Purpose of the Study:
- To purify and characterize a diphosphoinositol polyphosphate phosphohydrolase (DIPP) from rat liver.
- To investigate the enzymatic activity and structural features of DIPP, particularly its conserved MutT domain.
- To explore the potential role of DIPP in regulating higher inositol phosphate metabolism and its conservation in humans.
Main Methods:
- Purification of rat hepatic diphosphoinositol polyphosphate phosphohydrolase (DIPP).
- Enzymatic assays to determine substrate specificity and kinetic parameters (Km, IC50) for PP-InsP5 and [PP]2-InsP4.
- Microsequencing and mutagenesis to identify the functional domains (MutT domain) and conserved residues.
- Bioinformatic analysis to identify homologous human genes and their genomic organization.
Main Results:
- A rat hepatic DIPP was purified, demonstrating efficient cleavage of beta-phosphate from PP-InsP5 (Km = 340 nM) and [PP]2-InsP4 (Km = 34 nM).
- Inositol hexakisphosphate (InsP6) acted as an inhibitor, not a substrate.
- DIPP possesses a conserved MutT domain, known for guarding cellular integrity by dephosphorylating 8-oxo-dGTP and metabolizing nucleoside phosphates.
- The rat DIPP MutT domain is conserved in a human uterine DIPP homolog, with the human gene located on chromosome 6 and containing at least four exons.
- Mutagenesis confirmed the dependence of DIPP's catalytic activity on its MutT domain, specifically a conserved glutamate residue.
Conclusions:
- Rat hepatic DIPP effectively metabolizes highly phosphorylated inositol phosphates, PP-InsP5 and [PP]2-InsP4.
- The presence of a conserved MutT domain in DIPP suggests a dual role in regulating inositol phosphate signaling and potentially guarding genomic integrity.
- The conservation of DIPP across species and its enzymatic properties provide new avenues for studying higher inositol phosphate functions.