Evidence for a second messenger function of dUTP during Bax mediated apoptosis of yeast and mammalian cells

Williams, Drew; Norman, Grant; Khoury, Chamel; Metcalfe, Naomi; Briard, Jennie G.; Laporte, Aimée N.; Sheibani, Sara; Portt, Liam; Mandato, Craig A.; Greenwood, Michael T.

doi:10.1016/j.bbamcr.2010.11.021

Cited by 24 publications

(21 citation statements)

References 56 publications

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“…Uncovering unexpected cell survival sequences such as the Metalloprotease 15 (MMP15) suggests a novel role for extracellular matrix or alternative functions to well-known proteins (Abraham et al, 2005). The ability to increase cell survival by increasing the expression of the gene encoding dUTPase supports the emerging consensus that nucleotides such as dUTP serve as stress-induced pro-apoptotic second messengers (Williams et al, 2011; Wilson et al, 2012). On the other hand, the cytoprotective effects of the ceramide utilizing enzyme sphingomyelin synthase is consistent with the long standing role of ceramide as a stress-induced pro-apoptotic second messenger (Yang et al, 2006; Separovic et al, 2007, 2008).…”

Section: Regulation Of Programmed Cell Deathmentioning

confidence: 57%

Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

Clapp¹,

Portt²,

Khoury³

et al. 2012

Front. Oncol.

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Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti-apoptosis, we screened a human heart cDNA expression library in yeast cells undergoing PCD due to the conditional expression of a mammalian pro-apoptotic Bax cDNA. Analysis of the multiple Bax suppressors identified revealed several previously known as well as a large number of clones representing potential novel anti-apoptotic sequences. The focus of this review is to report on recent achievements in the use of humanized yeast in genetic screens to identify novel stress-induced PCD suppressors, supporting the use of yeast as a unicellular model organism to elucidate anti-apoptotic and cell survival mechanisms.

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Section: Regulation Of Programmed Cell Deathmentioning

confidence: 57%

Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

Clapp¹,

Portt²,

Khoury³

et al. 2012

Front. Oncol.

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“…Previously, several studies indicated that the level of dUTPase protein decreases during apoptosis [36], [37], [38]. Moreover, the potentially significant role of dUTPase as an anti-apoptotic protein has been recently suggested [39]. This pathway of inducing apoptosis by calpain action may act in parallel to other described roles of calpain in apoptosis (e.g.…”

Section: Discussionmentioning

confidence: 92%

Calpain-Catalyzed Proteolysis of Human dUTPase Specifically Removes the Nuclear Localization Signal Peptide

et al. 2011

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BackgroundCalpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca2+-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.Methodology/Principal FindingsLimited 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 Ca2+-calpain-induced cleavage of human dUTPase. The catalytic rate constant of dUTPase was unaffected by calpain cleavage. Gel electrophoretic analysis showed that Ca2+-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 4SE5; 7TP8; and 31LS32). The cleavage between the 31LS32 peptide bond specifically removes the flexible N-terminal nuclear localization signal, indispensable for cognate localization.Conclusions/SignificanceResults argue for a mechanism where Ca2+-calpain may regulate nuclear availability and degradation of dUTPase.

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“…We speculate that if dUTPase activity could be impaired, under conditions that provoke dUTP accumulation and enhanced misincorporation into DNA, p53 exonuclease-proofreading activity might avoid mtDNA damage produced by the dUTP–DNA misincorporation pathway. Remarkably, the level of dUTP increases in response to certain stress signals [43]. Enhanced error-correction responses were observed following the IR stress-facilitated mitochondrial localization of p53, implying the increased DNA repair potential of p53 in stressed cells.…”

Section: Discussionmentioning

confidence: 99%

p53 in the mitochondria, as a trans-acting protein, provides error-correction activities during the incorporation of non-canonical dUTP into DNA

et al. 2016

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Mutations in mitochondrial DNA is an outcome of errors produced by DNA polymerase γ during replication and failure of the repair mechanism. Misincorporation of non-canonical dUTP leads to mutagenesis or apoptosis, and may contribute to the cytotoxic effects of 5′-fluorouracil chemotherapy. Tumor suppressor p53 protein in the mitochondria displays physical and functional interactions with mitochondrial DNA and polymerase γ, and by its intrinsic 3′→5′ exonuclease activity can diminish the polymerization errors. Here we demonstrate the impact of p53 on incorporation of uracil into DNA examined with mitochondrial fractions, as the source of polymerase γ. p53 in mitochondria facilitates DNA damage repair functions resulting from uracil–DNA misincorporation. Our biochemical studies revealed that the procession of U:A and mismatched U:G lesions enhances in the presence of recombinant or endogenous cytoplasmic p53. p53 in mitochondria can function as an exonuclease/proofreader for polymerase γ by either decreasing the incorporation of non-canonical dUTP into DNA or by promoting the excision of incorporated nucleotide from nascent DNA, thus expanding the spectrum of DNA damage sites exploited for proofreading as a trans-acting protein. The data suggest that p53 may contribute to defense of the cells from consequences of dUTP misincorporation in both normal and tumor cells.

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Evidence for a second messenger function of dUTP during Bax mediated apoptosis of yeast and mammalian cells

Cited by 24 publications

References 56 publications

Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

Calpain-Catalyzed Proteolysis of Human dUTPase Specifically Removes the Nuclear Localization Signal Peptide

p53 in the mitochondria, as a trans-acting protein, provides error-correction activities during the incorporation of non-canonical dUTP into DNA

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