Abstract
Human RNASET2 has been implicated in antitumorigenic and antiangiogenic activities, independent of its ribonuclease capacities. We constructed a truncated version of human RNASET2, starting at E50 (trT2-50) and devoid of ribonuclease activity. trT2-50 maintained its ability to bind actin and to inhibit angiogenesis and tumorigenesis. trT2-50 binds to cell surface actin and formed a complex with actin in vitro. The antiangiogenic effect of this protein was demonstrated in human umbilical vein endothelial cells (HUVECs) by its ability to arrest tube formation on Matrigel, induced by angiogenic factors. Immunofluorescence staining of HUVECs showed nuclear and cytosolic RNASET2 protein that was no longer detectable inside the cell following trT2-50 treatment. This effect was associated with disruption of the intracellular actin network. trT2-50 co-localized with angiogenin, suggesting that both molecules bind (or compete) for similar cellular epitopes. Moreover, trT2-50 led to a significant inhibition of tumor development. Histological analysis demonstrated abundant necrotic tissue and a substantial loss of endothelial structure in trT2-50-treated tumors. Collectively, the present results indicate that trT2-50, a molecule engineered to be deficient of its catalytic activity, still maintained its actin binding and anticancer-related biological activities. We therefore suggest that trT2-50 may serve as a potential cancer therapeutic agent.
Highlights
Ribonucleases (RNases) are classified into the RNase T1, RNase A and RNase T2 families, according to their base specificity, structure, function, optimal pH and origin [1]
Supernatant of heparantreated confluent starved Human umbilical vein endothelial cell (HUVEC) was incubated with trT2-50 and was analyzed by Western Blot with antitrT2-50 showing trT2-50 and with anti-actin showing a 42kDa protein which indicates that actin is present in the fraction of the supernatant of heparan-treated cells (Figure 2A, lane 2)
We examined the actin-binding capacities and the antitumorigenic and antiangiogenic effects of trT250, a novel RNase- inactive truncated form of hRNASET2, expressed in and purified from E. coli
Summary
Ribonucleases (RNases) are classified into the RNase T1, RNase A and RNase T2 families, according to their base specificity, structure, function, optimal pH and origin [1]. Therein], RNase T1 (e.g. Aspergillus orizae origin) [4, 5] and RNase T2 (e.g. ACTIBIND and RNASET2) [6, 7, 8, 9] family members have been implicated in inhibition of tumorigenesis. Members of the RNase T2 family resemble the Aspergillus orizae T2-RNase (EC 3.1.27.1) and are widely distributed in living organisms, from viruses to mammals [10]. Animal and plant T2-RNases contain a total of eight cysteine residues, four of which are common to all T2-RNases and likely of fundamental importance to protein stability and/or function [1, 11]. Human RNASET2 disulfide bridges (Fig. 1A) have been characterized by Thorn et al, 2012 [12]
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