Abstract
Combinatorial expression of the various isoforms of diphosphoinositol synthases and phosphohydrolases determines the rates of phosphorylation/dephosphorylation cycles that have been functionally linked to vesicle trafficking, stress responses, DNA repair, and apoptosis. We now describe two new 19-kDa diphosphoinositol polyphosphate phosphohydrolases (DIPPs), named types 3alpha and 3beta, which possess the canonical Nudix-type catalytic motif flanked on either side by short Gly-rich sequences. The two enzymes differ only in that Pro-89 in the alpha form is replaced by Arg-89 in the beta form, making the latter approximately 2-fold more active in vitro. Another Nudix substrate, diadenosine hexaphosphate, was hydrolyzed less efficiently (k(cat)/K(m) = 0.2 x 10(5) m(-1) s(-1)) compared with diphosphoinositol polyphosphates (k(cat)/K(m) = 2-40 x 10(5) m(-1) s(-1)). Catalytic activity in vivo was established by individual overexpression of the human (h) DIPP3 isoforms in HEK293 cells, which reduced cellular levels of diphosphoinositol polyphosphates by 40-50%. The hDIPP3 mRNA is preferentially expressed in testis, accompanied by relatively weak expression in the brain, contrasting with hDIPP1 and hDIPP2 which are widely expressed. The hDIPP3 genes (NUDT10 encodes hDIPP3alpha; NUDT11 encodes hDIPP3beta) are only 152 kbp apart at p11.22 on chromosome X and probably arose by duplication. Transcription of both genes is inactivated on one of the X chromosomes of human females to maintain appropriate gene dosage. The hDIPP3 pair add tissue-specific diversity to the molecular mechanisms regulating diphosphoinositol polyphosphate turnover.
Highlights
Hydrolases containing the GX5EX7REUXEEXGU1 “Nudixtype” motif comprise a protein superfamily whose members have been proposed to act as “surveillance enzymes” [2] that function both to eliminate potentially toxic metabolites from the cell as well as to regulate concentrations and availability of substrates, cofactors, and signaling molecules [3]
These compounds are formed when specific monoester phosphate groups on Ins[1,3,4,5,6]P5 and InsP6 are converted to diphosphates, by enzymes known as diphosphoinositol polyphosphate synthases (DINS)
In the current study we first searched for novel hDIPP isoforms by screening an arrayed human cDNA library with a 492-bp probe spanning the coding region of hDIPP2␣ (P1, see under “Experimental Procedures”)
Summary
Hydrolases containing the GX5EX7REUXEEXGU1 “Nudixtype” motif comprise a protein superfamily whose members have been proposed to act as “surveillance enzymes” [2] that function both to eliminate potentially toxic metabolites from the cell as well as to regulate concentrations and availability of substrates, cofactors, and signaling molecules [3]. A 100,000 ϫ g supernatant was prepared, 100-l aliquots of which were each incubated for 90 min at 37 °C with 0.5 Ci of [3H]InsP6 (specific activity ϳ20 Ci/mmol; PerkinElmer Life Sciences) in 500 l buffer containing 0.75 mM EGTA, 1.5 mM EDTA, 9 mM MgSO4, 7.5 mM ATP, 10 mM NaF, 20 mM phosphocreatine, 1 mM DTT, 20 mM HEPES (pH 6.8), 4 mM CHAPS, 20 Sigma units/ml creatine phosphokinase, and 4 g of human DINS type 1.
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