Ribonuclease Inhibitor Regulates Neovascularization by Human Angiogenin

Abstract

Human angiogenin (ANG) is a homologue of bovine pancreatic ribonuclease (RNase A) that induces neovascularization. ANG is the only human angiogenic factor that possesses ribonucleolytic activity.To stimulate blood-vessel growth, ANG must be transported to the nucleus and must retain its catalytic activity. Like other mammalian homologues of RNase A, ANG forms a femtomolar complex with the cytosolic ribonuclease inhibitor protein (RI). To determine whether RI affects ANG-induced angiogenesis, we created G85R/G86R ANG, which possesses 10 6 -fold lower affinity for RI but retains wild-type ribonucleolytic activity. The neovascularization of rabbit corneas by G85R/G86R ANG was more pronounced and more rapid than by wild-type ANG. These findings provide the first direct evidence that RI serves to regulate the biological activity of ANG in vivo.Angiogenin (ANG) is a potent inducer of blood vessel growth (1) and has been implicated in the establishment, growth, and metastasis of tumors (2,3). A homologue of bovine pancreatic ribonuclease (RNase A (4-6); EC 3.1.27.5), ANG is the only human angiogenic factor that displays ribonucleolytic activity. ANG was first isolated from the conditioned medium of human adenocarcinoma cells (1), and is present in normal human plasma (7) as well as numerous other tissues and organs (8). After receptor-mediated endocytosis (9), a nuclear localization sequence (NLS) directs ANG to the nucleus (10). The receptor-binding, nuclear localization, and ribonucleolytic activity of ANG are all required for angiogenic activity (9-11). In endothelial and smooth muscle cells, ANG induces a wide range of cellular responses, including transcriptional activation (12), differentiation (13), cell migration and invasion (14), and tube formation (13).The ribonuclease inhibitor (RI (15)), a cytosolic protein found in all mammalian tissues analyzed to date, binds to mammalian ribonucleases with extraordinary affinity. The RI·ANG complex ( Figure 1A) is among the tightest of known protein-protein interactions with K d = † This work was supported in part by Grants CA073808 (NIH) and M10749000231-08N4900-23110 (Korea Science and Engineering Foundation A known role for RI is to protect cellular RNA from invading ribonucleases (19,20). ANG, however, possesses <1% of the ribonucleolytic activity of its homologues with cytotoxic activity (21). Moreover, the IC 50 values for cytotoxic ribonucleases are ≥10 2 -fold greater than the concentration of ANG required to induce endothelial cell proliferation in vitro. Thus, a major role for RI as an antagonist of the cytotoxic activity of ANG is unlikely.Does RI play a role in ANG-induced angiogenesis? The exogenous addition of extracellular RI is known to antagonize angiogenesis (22,23). That experiment, however, puts RI in a nonnative location. We sought to determine whether endogenous, intracellular RI regulates ANG-induced neovascularization. We reasoned that we could do so by using a variant of ANG that evades RI.To disrupt its interaction with RI, we introduce...

Ribonuclease inhibitor 1 regulates erythropoiesis by controlling GATA1 translation

“…RNH1 affinity for ribonucleases is the key determinant factor for RNase cytotoxicity; only ribonucleases that evade RNH1 can kill a cell. RNH1 also binds to angiogenin (ANG), suggesting a possible role in neovascularization (11), but the extent to which RNH1 may regulate angiogenesis remains unclear. Further, RNH1 contains numerous cysteine residues (e.g., 32 in human RNH1), whose sulfhydryl groups might play key structural roles and protect from oxidative damage (7,12).…”

Engineered Versions of Granzyme B and Angiogenin Overcome Intrinsic Resistance to Apoptosis Mediated by Human Cytolytic Fusion Proteins

“…This would also allow the formation of tiRNA and would therefore shut down protein synthesis. The Ang G85R/G86R variant was shown to have a 10 6 -fold lower affinity for RNH1 than the wild-type enzyme [178]. Further analysis showed that this variant was 10-25 times more cytotoxic than the wild-type enzyme towards the human promyelocytic leukemia cell line HL-60 when fused to H22(scFv) [242].…”

“…It specifically targets smooth muscle cells, endothelial cells and motor neurons, stimulating proliferation, cell migration and tubular development in response to environmental conditions [176,177]. The expression of angiogenin is upregulated in several types of cancer and it promotes the establishment, growth and metastasis of tumors [178,179].…”

“…Some angiogenin mutants with a reduced affinity towards RNH1 have already been identified by in vitro protein interaction experiments and ex vivo within the scope of neovascularization studies [178,246,247]. We adapted this knowledge to our requirements and assumed that hyper-angiogenic variants with a lower inhibitor affinity would be more suitable as hCFP effector domains, given that neovascularization requires RNase activity even in the presence of the inhibitor.…”

Systematic analysis of molecular and physiological synergic effects of iron, manganese and copper on connective tissue

“…Ангиогенин стимулирует васкуляризацию тканей: связыва-ясь с актином на поверхнос ти эндотелиальных клеток, ангиогенин претерпевает эндоцитоз, перемещается в ядро, где активирует экспрессию соответствующих генов [34]. Проангиогенная ак-тивность белка зависит от концентрации ионов меди, которые связываются с доменом взаимо-действия с эндотелиальными клетками [35] и модулируют сродство ангиогенина к эндотели-альным клеткам.…”