title: Calpain-catalyzed proteolysis of human dUTPase specifically removes the nuclear localization signal peptide.
abstract: BACKGROUND
  Calpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca(2+)-induced activation. Recently, dUTPase, an enzyme essential to maintain genomic integrity, was identified as a physiological calpain substrate in Drosophila cells. Here we investigate the potential structural/functional significance of calpain-activated proteolysis of human dUTPase.
  
  
  RESULTS
  Limited proteolysis of human dUTPase by mammalian m-calpain was investigated in the presence and absence of cognate ligands of either calpain or dUTPase. Significant proteolysis was observed only in the presence of Ca(II) ions, inducing calpain action. The presence or absence of the dUTP-analogue α,β-imido-dUTP did not show any effect on Ca(2+)-calpain-induced cleavage of human dUTPase. The catalytic rate constant of dUTPase was unaffected by calpain cleavage. Gel electrophoretic analysis showed that Ca(2+)-calpain-induced cleavage of human dUTPase resulted in several distinctly observable dUTPase fragments. Mass spectrometric identification of the calpain-cleaved fragments identified three calpain cleavage sites (between residues (4)SE(5); (7)TP(8); and (31)LS(32)). The cleavage between the (31)LS(32) peptide bond specifically removes the flexible N-terminal nuclear localization signal, indispensable for cognate localization.
  
  
  CONCLUSIONS
  Results argue for a mechanism where Ca(2+)-calpain may regulate nuclear availability and degradation of dUTPase.
authors.affiliation: Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Bozóky
authors.forename: Gergely
authors.initials: G
authors.lastname: Róna
authors.forename: Éva
authors.initials: É
authors.lastname: Klement
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
authors.forename: Gábor
authors.initials: G
authors.lastname: Merényi
authors.forename: Beáta G
authors.initials: BG
authors.lastname: Vértessy
authors.forename: Peter
authors.initials: P
authors.lastname: Friedrich
mesh_terms: D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D002384:Catalysis; D002467:Cell Nucleus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019913:Nuclear Localization Signals / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011755:Pyrophosphatases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D019913:Nuclear Localization Signals; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; D002154:Calpain; C449000:m-calpain; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; D002118:Calcium