Endothelial apoptotic activity of angiocidin is dependent on its polyubiquitin binding activity.
We recently cloned the full-length cDNA of a tumour-associated protein. The recombinant protein expressed in bacteria and referred to as angiocidin has potent antitumour activity in vivo and in vitro. Angiocidin inhibits tumour growth and angiogenesis by inducing apoptosis in endothelial cells. Based on the sequence similarity of angiocidin to S5a, one of the major polyubiquitin recognition proteins in eukaryotic cells, we postulated that the antiendothelial activity of angiocidin could be due in part to its polyubiquitin binding activity. In support of this hypothesis, we show that angiocidin binds polyubiquitin in vivo with high affinity and colocalises with ubiquitinated proteins on the surface of endothelial cells. Binding is blocked with an antiubiquitin antibody. Angiocidin treatment of endothelial cells transfected with a proteasome fluorescent reporter protein showed a dose-dependent inhibition of proteasome activity and accumulation of polyubiquitinated proteins. Full-length angiocidin bound polyubiquitin while three angiocidin recombinant proteins whose putative polyubiquitin binding sites were mutated either failed to bind polyubiquitin or had significantly diminished binding activity. The in vitro apoptotic activity of these mutants correlated with their polyubiquitin binding activity. These data strongly argue that the apoptotic activity of angiocidin is dependent on its polyubiquitin binding activity.
Dimitrov
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S
Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, 354 Biology Life Sciences Building (015-96), 1900 North 12th Street, Philadelphia, PA 19122, USA.
Sabherwal
Y
Y
Raymond
D D
DD
L'Heureux
D Z
DZ
Lu
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Q
Tuszynski
G P
GP
D000954:Antigens, Surface / Q000378:metabolism; D017209:Apoptosis; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004730:Endothelium, Vascular / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D006801:Humans; D025821:Polyubiquitin / Q000276:immunology / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000378:metabolism*; D014471:Umbilical Veins
D000954:Antigens, Surface; D002352:Carrier Proteins; C498506:PSMD4 protein, human; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D016650:Fluorescein-5-isothiocyanate