Abstract
The global effect of ubiquitin-proteasome (UP) inhibitors on leukemic cell proteome was analysed. A total of 39 protein spots, affected by UP inhibitors, were identified, including 11 new apoptosis-associated proteins. They are involved in different cellular functions and four were associated with caspase-3 activation. Eukaryotic initiation factor 5A (eIF-5A) was identified in two spots; however, the peptide mass-fingerprinting for the accumulated one included a peptide with lysine50, indicating that hypusine formation was suppressed during UP inhibitor-induced apoptosis. Hypusine modification ensues immediately following translation of eIF-5A precursor, unless cells are treated with the modification inhibitors diaminoheptane. However, UP inhibitors induced a much stronger accumulation of unmodified eIF-5A compared to the effect of diaminoheptane. We further showed the unmodified eIF-5A was regulated in a proteasome-dependent manner. Inhibition of hypusine formation by diaminoheptane triggered apoptosis, but of particular interest is the finding that eIF-5A expression inhibition by antisense oligodeoxynucleotides significantly enhanced the stimulating effect of GM-CSF on cell growth. Therefore, the eIF-5A accumulation played important roles in the apoptosis induced by UP inhibitors. Moreover, hypusine inhibition in apoptosis was further revealed to be associated with the subcellular localization of eIF-5A. Our data pave the way to a better understanding of the mechanisms by which UP system has been linked to apoptosis.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Acetylcysteine / analogs & derivatives*
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Acetylcysteine / pharmacology*
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Apoptosis / drug effects
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Apoptosis / genetics
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Caspase 3
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Caspases / metabolism
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Cysteine Endopeptidases / physiology*
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Diamines / pharmacology
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Electrophoresis, Gel, Two-Dimensional
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Enzyme Activation
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Eukaryotic Translation Initiation Factor 5A
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Gene Expression Profiling*
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Gene Expression Regulation / drug effects*
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Granulocyte-Macrophage Colony-Stimulating Factor / pharmacology
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Humans
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Leukemia, Megakaryoblastic, Acute / pathology
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Leupeptins / pharmacology*
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Lysine / analogs & derivatives*
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Lysine / metabolism
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Multienzyme Complexes / antagonists & inhibitors
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Multienzyme Complexes / physiology*
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Oligodeoxyribonucleotides, Antisense / pharmacology
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Peptide Initiation Factors / genetics
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Peptide Initiation Factors / physiology*
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Peptide Mapping
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Protease Inhibitors / pharmacology*
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Proteasome Endopeptidase Complex
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Proteomics
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RNA-Binding Proteins*
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Spectrometry, Mass, Electrospray Ionization
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Subcellular Fractions / chemistry
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Tumor Cells, Cultured / drug effects
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Tumor Cells, Cultured / metabolism
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Ubiquitin / physiology*
Substances
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Diamines
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Leupeptins
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Multienzyme Complexes
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Oligodeoxyribonucleotides, Antisense
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Peptide Initiation Factors
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Protease Inhibitors
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RNA-Binding Proteins
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Ubiquitin
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lactacystin
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hypusine
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1,7-diaminoheptane
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Granulocyte-Macrophage Colony-Stimulating Factor
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CASP3 protein, human
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Caspase 3
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Caspases
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Cysteine Endopeptidases
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Proteasome Endopeptidase Complex
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Lysine
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benzyloxycarbonylleucyl-leucyl-leucine aldehyde
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Acetylcysteine