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1.
Mol Cell Biol ; 34(22): 4177-85, 2014 Nov 15.
Article in English | MEDLINE | ID: mdl-25202122

ABSTRACT

In response to genotoxic stress, DNA damage checkpoints maintain the integrity of the genome by delaying cell cycle progression to allow for DNA repair. Here we show that the degradation of the basic helix-loop-helix (bHLH) transcription factor DEC1, a critical regulator of cell fate and circadian rhythms, controls the DNA damage response. During unperturbed cell cycles, DEC1 is a highly unstable protein that is targeted for proteasome-dependent degradation by the SCF(ßTrCP) ubiquitin ligase in cooperation with CK1. Upon DNA damage, DEC1 is rapidly induced in an ATM/ATR-dependent manner. DEC1 induction results from protein stabilization via a mechanism that requires the USP17 ubiquitin protease. USP17 binds and deubiquitylates DEC1, markedly extending its half-life. Subsequently, during checkpoint recovery, DEC1 proteolysis is reestablished through ßTrCP-dependent ubiquitylation. Expression of a degradation-resistant DEC1 mutant prevents checkpoint recovery by inhibiting the downregulation of p53. These results indicate that the regulated degradation of DEC1 is a key factor controlling the DNA damage response.


Subject(s)
DNA Damage , Endopeptidases/metabolism , Proteolysis , Tumor Suppressor Proteins/metabolism , beta-Transducin Repeat-Containing Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Cycle , Cell Line , Down-Regulation , Humans , Mutation , Proteasome Endopeptidase Complex/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/genetics , Ubiquitination
2.
J Biol Chem ; 289(11): 7730-7, 2014 Mar 14.
Article in English | MEDLINE | ID: mdl-24500709

ABSTRACT

TFAP4, a basic helix-loop-helix transcription factor that regulates the expression of a multitude of genes involved in the regulation of cellular proliferation, stemness, and epithelial-mesenchymal transition, is up-regulated in colorectal cancer and a number of other human malignancies. We have found that, during the G2 phase of the cell division cycle, TFAP4 is targeted for proteasome-dependent degradation by the SCF(ßTrCP) ubiquitin ligase. This event requires phosphorylation of TFAP4 on a conserved degron. Expression of a stable TFAP4 mutant unable to interact with ßTrCP results in a number of mitotic defects, including chromosome missegregation and multipolar spindles, which eventually lead to the activation of the DNA damage response. Our findings reveal that ßTrCP-dependent degradation of TFAP4 is required for the fidelity of mitotic division.


Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation , Mitosis , Proteasome Endopeptidase Complex/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , Transcription Factors/metabolism , Cell Line, Tumor , Cell Proliferation , DNA Damage , DNA-Binding Proteins/genetics , Epithelial-Mesenchymal Transition , G2 Phase , HEK293 Cells , HeLa Cells , Humans , Mass Spectrometry , Microscopy, Fluorescence , Mutation , Phosphorylation , Plasmids/metabolism , SKP Cullin F-Box Protein Ligases/genetics , Transcription Factors/genetics
3.
Biometals ; 24(2): 269-78, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21140194

ABSTRACT

The role of copper in the toxicity of mutant copper-dependent enzyme superoxide dismutase (SOD1) found in patients affected with the familial form of amyotrophic lateral sclerosis (fALS) is widely debated. Here we report that treatment of human neuroblastoma cells SH-SY5Y with a specific copper chelator, triethylene tetramine (Trien) induces the decrease of intracellular copper level, paralleled by decreased activity of SOD1. A comparable effect is observed in mouse NSC-34-derived cells, a motoneuronal model, transfected for the inducible expression of either wild-type or G93A mutant human SOD1, one of the mutations associated with fALS. In both cell types, the drop of SOD1 activity is not paralleled by the same extent of decrease in SOD1 protein content. This discrepancy can be explained by the occurrence of a fraction of copper-free SOD1 upon copper depletion, which is demonstrated by the partial recovery of the enzyme activity after the addition of copper sulphate to homogenates of SH-SY5Y cells. Furthermore, copper depletion produces the enrichment of the physiological mitochondrial fraction of SOD1 protein, in both cells models. However, increasing the fraction of mitochondrial, possibly copper-free, mutant human SOD1 does not further alter mitochondrial morphology in NSC-34-derived cells. Thus, copper deficiency is not a factor which may worsen mitochondrial damage, which is one of the earliest events in fALS associated with mutant SOD1.


Subject(s)
Copper/deficiency , Mitochondria/metabolism , Neurons/metabolism , Superoxide Dismutase/metabolism , Animals , Blotting, Western , Cell Line , Cell Line, Tumor , Humans , Immunohistochemistry , Mice , Mitochondria/drug effects , Neurons/drug effects , Superoxide Dismutase-1 , Trientine/pharmacology
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