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

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.

CYLD-Mediated Signaling and Diseases

“…However, emerging evidence has implicated a potential role of CYLD. A report of polyubiquitin as a discriminatory marker for synthetic versus contractile VSMCs, along with a subsequent finding that angiocidin is regulated by polyubiquitination, has raised the possibility that CYLD is intimately involved in the vascular lesion process, thus revealing a potential therapeutic target [42,52]. Indeed, CYLD is upregulated in injured carotid arteries of rats, reduces cyclin D1 levels, and is capable of suppressing vascular lesion formation.…”

The interaction of angiocidin with tissue transglutaminase

“…We previously reported that tTgase present in HUVE cell extracts bound to recombinant angiocidin immobilized on Sepharose beads (Dimitrov et al, 2005). The peptide eluted from the angiocidin-Sepharose beads was identified by mass spectral analysis of its tryptic peptides.…”

“…While investigating the interaction of angiocidin with potential HUVE cell protein binding partners using angiocidin-affinity chromatography (Dimitrov et al, 2005), we discovered that one of the protein bands that was eluted from the column was a 150 kDa protein whose tryptic fragments matched those of tissue transglutaminase 2 (tTgase) as revealed by mass spectral fingerprinting. In this study we have attempted to investigate the significance of this finding as it relates to the anti-tumor activity of recombinant angiocidin.…”

“…While the anti-angiogenic mechanism of action of angiocidin has not been fully characterized, we have observed that the exogenous application of the recombinant protein to endothelial cell culture inhibits adhesion, migration, and proliferation and that these activities are related to the ability of angiocidin to inhibit cell proteasome activity and cell-extracellular matrix interactions (Dimitrov et al, 2005; Sabherwal et al, 2006). In a previous study, we made the interesting observation that angiocidin bound tissue transglutaminase-2 (tTgase) from endothelial cell lysates when passed over columns containing immobilized angiocidin (Dimitrov et al, 2005).…”

“…In a previous study, we made the interesting observation that angiocidin bound tissue transglutaminase-2 (tTgase) from endothelial cell lysates when passed over columns containing immobilized angiocidin (Dimitrov et al, 2005). Establishing the significance of this observation is the topic of the present paper.…”