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Endothelial apoptotic activity of angiocidin is dependent on its polyubiquitin binding activity.

Dimitrov S, Sabherwal Y, Raymond DD, L'Heureux DZ, Lu Q, Tuszynski GP - Br. J. Cancer (2005)

Bottom Line: We recently cloned the full-length cDNA of a tumour-associated protein.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.These data strongly argue that the apoptotic activity of angiocidin is dependent on its polyubiquitin binding activity.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
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.

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Effect of angiocidin, its mutants and antiubiquitin antibody on the viability of HUVE cells. Cells were plated on 96-well microtitre plates coated with either 1 μg of angiocidin or its mutants. Cells were cultured overnight and viability measured using the Alamar blue assay as previously described (Zhou et al, 2004). (A) Cells photographed using Hoffman interference microscopy at a magnification of × 100. (B) Viability measurements using the Alamar blue assay. The experiment is representative and shows the mean of three replicates and error bars represent the standard error of the mean. (C) Effect of antiubiquitin antibody on the viability of HUVE cells. HUVE cells were grown for 24 h on 96-well plates coated with 1 μg of angiocidin or M-1-2 or treated with 10 μl of control serum or rabbit antiubiquitin antibody. Cells were photographed using Hoffman interference microscopy at a magnification of × 200.
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fig3: Effect of angiocidin, its mutants and antiubiquitin antibody on the viability of HUVE cells. Cells were plated on 96-well microtitre plates coated with either 1 μg of angiocidin or its mutants. Cells were cultured overnight and viability measured using the Alamar blue assay as previously described (Zhou et al, 2004). (A) Cells photographed using Hoffman interference microscopy at a magnification of × 100. (B) Viability measurements using the Alamar blue assay. The experiment is representative and shows the mean of three replicates and error bars represent the standard error of the mean. (C) Effect of antiubiquitin antibody on the viability of HUVE cells. HUVE cells were grown for 24 h on 96-well plates coated with 1 μg of angiocidin or M-1-2 or treated with 10 μl of control serum or rabbit antiubiquitin antibody. Cells were photographed using Hoffman interference microscopy at a magnification of × 200.

Mentions: Angiocidin and its polyubiquitin-binding mutants were compared for their endothelial cell apoptotic activity using the Alamar blue viability assay. We found that mutants M-1, containing residues I290 AYAM294 substituted by alanines, and M-1-2, containing residues I290 AYAM294 and L216ALAL220 substituted by alanines, lost their apoptotic inducing activity as well as their polyubiquitin binding activity. However, M-2, containing L216ALAL220 substituted with alanines, still bound polyubiquitin with a favourable Kd of 1.52 nM, while displaying four-fold lower maximal binding than angiocidin. In contrast to the other mutants that completely lost their polyubiquitin binding activity and their apoptotic activity, M-2 had a two-fold diminished apoptotic activity as compared to angiocidin (Figure 3B). Many more cells although rounded were still viable in the M-2 treatment group (Figure 3A). Cells were treated overnight with angiocidin and its mutants. These results indicate that the extent to which angiocidin binds polyubiquitin correlates with its in vitro apoptotic activity.


Endothelial apoptotic activity of angiocidin is dependent on its polyubiquitin binding activity.

Dimitrov S, Sabherwal Y, Raymond DD, L'Heureux DZ, Lu Q, Tuszynski GP - Br. J. Cancer (2005)

Effect of angiocidin, its mutants and antiubiquitin antibody on the viability of HUVE cells. Cells were plated on 96-well microtitre plates coated with either 1 μg of angiocidin or its mutants. Cells were cultured overnight and viability measured using the Alamar blue assay as previously described (Zhou et al, 2004). (A) Cells photographed using Hoffman interference microscopy at a magnification of × 100. (B) Viability measurements using the Alamar blue assay. The experiment is representative and shows the mean of three replicates and error bars represent the standard error of the mean. (C) Effect of antiubiquitin antibody on the viability of HUVE cells. HUVE cells were grown for 24 h on 96-well plates coated with 1 μg of angiocidin or M-1-2 or treated with 10 μl of control serum or rabbit antiubiquitin antibody. Cells were photographed using Hoffman interference microscopy at a magnification of × 200.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2361611&req=5

fig3: Effect of angiocidin, its mutants and antiubiquitin antibody on the viability of HUVE cells. Cells were plated on 96-well microtitre plates coated with either 1 μg of angiocidin or its mutants. Cells were cultured overnight and viability measured using the Alamar blue assay as previously described (Zhou et al, 2004). (A) Cells photographed using Hoffman interference microscopy at a magnification of × 100. (B) Viability measurements using the Alamar blue assay. The experiment is representative and shows the mean of three replicates and error bars represent the standard error of the mean. (C) Effect of antiubiquitin antibody on the viability of HUVE cells. HUVE cells were grown for 24 h on 96-well plates coated with 1 μg of angiocidin or M-1-2 or treated with 10 μl of control serum or rabbit antiubiquitin antibody. Cells were photographed using Hoffman interference microscopy at a magnification of × 200.
Mentions: Angiocidin and its polyubiquitin-binding mutants were compared for their endothelial cell apoptotic activity using the Alamar blue viability assay. We found that mutants M-1, containing residues I290 AYAM294 substituted by alanines, and M-1-2, containing residues I290 AYAM294 and L216ALAL220 substituted by alanines, lost their apoptotic inducing activity as well as their polyubiquitin binding activity. However, M-2, containing L216ALAL220 substituted with alanines, still bound polyubiquitin with a favourable Kd of 1.52 nM, while displaying four-fold lower maximal binding than angiocidin. In contrast to the other mutants that completely lost their polyubiquitin binding activity and their apoptotic activity, M-2 had a two-fold diminished apoptotic activity as compared to angiocidin (Figure 3B). Many more cells although rounded were still viable in the M-2 treatment group (Figure 3A). Cells were treated overnight with angiocidin and its mutants. These results indicate that the extent to which angiocidin binds polyubiquitin correlates with its in vitro apoptotic activity.

Bottom Line: We recently cloned the full-length cDNA of a tumour-associated protein.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.These data strongly argue that the apoptotic activity of angiocidin is dependent on its polyubiquitin binding activity.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus