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Calpain-catalyzed proteolysis of human dUTPase specifically removes the nuclear localization signal peptide.

ABSTRACT: 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.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 ?,?-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.Results argue for a mechanism where Ca(2+)-calpain may regulate nuclear availability and degradation of dUTPase.

SUBMITTER: Bozoky Z

PROVIDER: S-EPMC3098232 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Calpain-catalyzed proteolysis of human dUTPase specifically removes the nuclear localization signal peptide.

Bozóky Zoltán Z Róna Gergely G Klement Éva É Medzihradszky Katalin F KF Merényi Gábor G Vértessy Beáta G BG Friedrich Peter P

PloS one 20110519 5

<h4>Background</h4>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.<h4>Methodology/principal findings</h4>Limited proteolysis of human dUTPase by ma ...[more]

PMID: 21625588

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Project description:Histone deacetylase 5 (HDAC5), a class IIa deacetylase, is a prominent regulator of cellular and epigenetic processes that underlie the progression of human disease, ranging from cardiac hypertrophy to cancer. Although it is established that phosphorylation mediates 14-3-3 protein binding and provides the essential link between HDAC5 nucleo-cytoplasmic shuttling and transcriptional repression, thus far only four phospho-acceptor sites have been functionally characterized. Here, using a combinatorial proteomics approach and phosphomutant screening, we present the first evidence that HDAC5 has at least 17 in vivo phosphorylation sites within functional domains, including Ser278 and Ser279 within the nuclear localization signal (NLS), Ser1108 within the nuclear export signal, and Ser755 in deacetylase domain. Global and targeted MS/MS analyses of NLS peptides demonstrated the presence of single (Ser278 and Ser279) and double (Ser278/Ser279) phosphorylations. The double S278/279A mutation showed reduced association with HDAC3, slightly decreased deacetylation activity, and significantly increased cytoplasmic localization compared with wild type HDAC5, whereas the S278A and S1108A phosphomutants were not altered. Live cell imaging revealed a deficiency in nuclear import of S278/279A HDAC5. Phosphomutant stable cell lines confirmed the cellular redistribution of NLS mutants and revealed a more pronounced cytoplasmic localization for the single S279A mutant. Proteomic analysis of immunoisolated S278/279A, S279A, and S259/498A mutants linked altered cellular localization to changes in protein interactions. S278/279A and S279A HDAC5 showed reduced association with the NCoR-HDAC3 nuclear corepressor complex as well as protein kinase D enzymes, which were potentiated in the S259/498A mutant. These results provide the first link between phosphorylation outside the known 14-3-3 sites and downstream changes in protein interactions. Together these studies identify Ser279 as a critical phosphorylation within the NLS involved in the nuclear import of HDAC5, providing a regulatory point in nucleo-cytoplasmic shuttling that may be conserved in other class IIa HDACs-HDAC4 and HDAC9.

Project description:BackgroundOncogenic roles of epidermal growth factor receptor pathway substrate no.8 (EPS8) have been widely reported in various tumors, making targeting of EPS8 an appealing prospect. Here, we describe the role of EPS8 in acute myeloid leukemia (AML) and consider the potential of EPS8 as an anti-AML target. Nuclear localization signal (NLS) residues of tumor-associated proteins are crucial for cell cycle progression, and specific inhibitors derived from the NLS have inhibitory effect on cancer cells. The NLS in EPS8 has potential as a specific anti-AML target.MethodsGene Expression Omnibus expression profiles of AML patients were used to test associations between EPS8 expression and AML patient outcome. The biological characteristics of AML cells after EPS8 knockdown were analyzed in vitro and in vivo. A specific peptide (CP-EPS8-NLS) derived from the NLS of EPS8 (amino acids 298-310) was synthesized, and the anti-AML effects of CP-EPS8-NLS were analyzed in cancer cells and in xenograft models. Mutated CP-EPS8-NLS and penetratin served as controls.ResultsWe observed that elevated EPS8 expression in AML patients is associated with poor outcome. Knockdown of EPS8 significantly suppressed the survival of AML cells in vitro and in vivo. CP-EPS8-NLS interfered with EPS8-associated signaling and consequently exerted anti-AML activity. Importantly, CP-EPS8-NLS displayed anti-AML activity in various AML cell types, with diminished activity in PBMCs. CP-ESP8-NLS suppressed U937 cell proliferation, and injection of CP-EPS8-NLS exerted potent antitumor activity in the xenograft tumor models. A synergistic effect of CP-EPS8-NLS and chemotherapeutic agents was also observed in vitro and in vivo. Mechanistically, treatment of various AML cells with CP-EPS8-NLS downregulated the expression of EPS8 and its downstream pathways.ConclusionsThe function of CP-EPS8-NLS is explained by the presence of a NLS in EPS8, which has been shown to induce nuclear translocation, consequently resulting in EPS8 overexpression. These results indicate that EPS8 is a potential target for AML treatment.

Dataset Information

Calpain-catalyzed proteolysis of human dUTPase specifically removes the nuclear localization signal peptide.

Publications

Calpain-catalyzed proteolysis of human dUTPase specifically removes the nuclear localization signal peptide.

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