Angiostatin binds to endothelial cell (EC) surface F 1 -F 0 ATP synthase, leading to inhibition of EC migration and proliferation during tumor angiogenesis. This has led to a search for angiostatin mimetics specific for this enzyme. A naturally occurring protein that binds to the F1 subunit of ATP synthase and blocks ATP hydrolysis in mitochondria is inhibitor of F1 (IF1). The present study explores the effect of IF1 on cell surface ATP synthase. IF1 protein bound to purified F 1 ATP synthase and inhibited F 1 -dependent ATP hydrolysis consistent with its reported activity in studies of mitochondria. Although exogenous IF1 did not inhibit ATP production on the surface of EC, it did conserve ATP on the cell surface, particularly at low extracellular pH. IF1 inhibited ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. In cell-based assays used to model angiogenesis in vitro, IF1 did not inhibit EC differentiation to form tubes and only slightly inhibited cell proliferation compared with angiostatin. From these data, we conclude that inhibition of ATP synthesis is necessary for an anti-angiogenic outcome in cell-based assays. We propose that IF1 is not an angiostatin mimetic, but it can serve a protective role for EC in the tumor microenvironment. This protection may be overridden in a concentration-dependent manner by angiostatin. In support of this hypothesis, we demonstrate that angiostatin blocks IF1 binding to ATP synthase and abolishes its ability to conserve ATP. These data suggest that there is a relationship between the binding sites of IF1 and angiostatin on ATP synthase and that IF1 could be employed to modulate angiogenesis.The term angiogenesis refers to the development of new blood vessels from preexisting vessels. This process is essential for maintaining and promoting tumor growth. One of the first anti-angiogenic agents discovered with the aim of treating cancers was angiostatin (1). Our laboratory identified F 1 -F 0 ATP synthase as a receptor for angiostatin on the surface of human EC 1 (2). This non-mitochondrial ATP synthase catalyzes ATP synthesis and is inhibited by angiostatin at low, tumor-like extracellular pH. The pH dependence explains the selectivity of angiostatin for the tumor microenvironment, where it inhibits EC migration and proliferation (3-5). Angiostatin inhibited both ATP production and ATP hydrolysis in previous studies (6). It was also demonstrated that polyclonal antibodies against the catalytic ␤-subunit or the regulatory ␣-subunit of ATP synthase inhibit the enzyme bidirectionally and therefore act as angiostatin mimetics. However, it was unknown whether a specific inhibitor of ATP hydrolysis could also serve as an angiostatin mimetic. To address this question, we have studied the effects of IF1, a natural inhibitor protein of F 1 -F 0 ATP synthase, on EC surface ATP synthase.The IF1 protein is a 9.6-kDa basic protein that comprises of 84 amino acids (7) and is known to inhibit the hydrolytic activity of mitochondrial ATP synthase (7,8...

Angiostatin binds to endothelial cell (EC)-surface F 1 -F 0 ATP synthase, leading to inhibition of EC3 migration and proliferation during tumor angiogenesis. This has led to a search for angiostatinmimetics specific for this enzyme. A naturally occurring protein that binds to the F1 subunit of ATP synthase and blocks ATP hydrolysis in mitochondria is Inhibitor of F1 (IF1). The present study explores the effect of IF1 on cell surface ATP synthase. IF1 protein bound to purified F 1 ATP synthase and inhibited F 1 -dependent ATP hydrolysis consistent with its reported activity in studies of mitochondria. While exogenous IF1 did not inhibit ATP production on the surface of EC, it did conserve ATP on the cell surface, particularly at low extracellular pH. IF1 inhibited ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. In cell-based assays used to model angiogenesis in vitro, IF1 did not inhibit EC differentiation to form tubes and only slightly inhibited cell proliferation compared to angiostatin. From these data, we conclude that inhibition of ATP synthesis is necessary for an anti-angiogenic outcome in cell-based assays. We propose that IF1 is not an angiostatin-mimetic, but it can serve a protective role for EC in the tumor microenvironment. This protection may be overridden in a concentration-dependent manner by angiostatin. In support of this hypothesis, we demonstrate that angiostatin blocks IF1 binding to ATP synthase, and abolishes its ability to conserve ATP. These data suggest that there is a relationship between the binding sites of IF1 and angiostatin on ATP synthase and that IF1 could be employed to modulate angiogenesis.

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