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Characterization of the angiogenic activity of zebrafish ribonucleases.

ABSTRACT: Ribonucleases identified from zebrafish possess angiogenic and bactericidal activities. Zebrafish RNases have three intramolecular disulfide bonds, a characteristic structural feature of angiogenin, different from the typical four disulfide bonds of the other members of the RNase A superfamily. They also have a higher degree of sequence homology to angiogenin than to RNase A. It has been proposed that all RNases evolved from these angiogenin-like progenitors. In the present study, we characterize, in detail, the function of zebrafish RNases in various steps in the process of angiogenesis. We report that zebrafish RNase-1, -2 and -3 bind to the cell surface specifically and are able to compete with human angiogenin. Similar to human angiogenin, all three zebrafish RNases are able to induce phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase. They also undergo nuclear translocation, accumulate in the nucleolus and stimulate rRNA transcription. However, zebrafish RNase-3 is defective in cleaving rRNA precursor, even though it has been reported to have an open active site and has higher enzymatic activity toward more classic RNase substrates such as yeast tRNA and synthetic oligonucleotides. Taken together with the findings that zebrafish RNase-3 is less angiogenic than zebrafish RNase-1 and -2 as well as human angiogenin, these results suggest that zebrafish RNase-1 is the ortholog of human angiogenin and that the ribonucleolytic activity of zebrafish RNases toward the rRNA precursor substrate is functionally important for their angiogenic activity.

SUBMITTER: Monti DM

PROVIDER: S-EPMC2744113 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Characterization of the angiogenic activity of zebrafish ribonucleases.

Monti Daria M DM Yu Wenhao W Pizzo Elio E Shima Kaori K Hu Miaofen G MG Di Malta Chiara C Piccoli Renata R D'Alessio Giuseppe G Hu Guo-Fu GF

The FEBS journal 20090622 15

Ribonucleases identified from zebrafish possess angiogenic and bactericidal activities. Zebrafish RNases have three intramolecular disulfide bonds, a characteristic structural feature of angiogenin, different from the typical four disulfide bonds of the other members of the RNase A superfamily. They also have a higher degree of sequence homology to angiogenin than to RNase A. It has been proposed that all RNases evolved from these angiogenin-like progenitors. In the present study, we characteriz ...[more]

PMID: 19549190

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Project description:BACKGROUND: Anti-angiogenic therapy in certain cancers has been associated with improved control of tumor growth and metastasis. Development of anti-angiogenic agents has, however, been saddled with higher attrition rate due to suboptimal efficacy, narrow therapeutic windows, or development of organ-specific toxicities. The aim of this study was to evaluate the translational ability of the zebrafish efficacy-toxicity model to stratify anti-angiogenic agents based on efficacy, therapeutic windows, and off-target effects to streamline the compound selection process in anti-angiogenic discovery. METHODS: The embryonic model of zebrafish was employed for studying angiogenesis and toxicity. The zebrafish were treated with anti-angiogenic compounds to evaluate their effects on angiogenesis and zebrafish-toxicity parameters. Angiogenesis was measured by scoring the development of subintestinal vessels. Toxicity was evaluated by calculating the median lethal concentration, the lowest observed effect concentration, and gross morphological changes. Results of efficacy and toxicity were used to predict the therapeutic window. RESULTS: In alignment with the clinical outcomes, the zebrafish assays demonstrated that vascular endothelial growth factor receptor (VEGFR) inhibitors are the most potent anti-angiogenic agents, followed by multikinase inhibitors and inhibitors of endothelial cell proliferation. The toxicity assays reported cardiac phenotype in zebrafish treated with VEGFR inhibitors and multikinase inhibitors with VEGFR activity suggestive of cardiotoxic potential of these compounds. Several other pathological features were reported for multikinase inhibitors suggestive of off-target effects. The predicted therapeutic window was translational with the clinical trial outcomes of the anti-angiogenic agents. The zebrafish efficacy-toxicity approach could stratify anti-angiogenic agents based on the mechanism of action and delineate chemical structure-driven biological activity of anti-angiogenic compounds. CONCLUSION: The zebrafish efficacy-toxicity approach can be used as a predictive model for translational anti-angiogenic drug discovery to streamline compound selection, resulting in safer and efficacious anti-angiogenic agents entering the clinics.

Project description:Four ribonucleases (RNAases I-IV) have been purified to homogeneity from human seminal plasma by precipitation with 40-75%-satd. (NH4)2SO4, followed by chromatographies on concanavalin A-Sepharose 4B, DEAE-cellulose phosphocellulose, agarose-5'-(4-aminophenylphospho)uridine 2'(3')-phosphate (RNAase affinity column) and Sephadex G-75 or G-100. The homogeneity of these RNAases was confirmed by polyacrylamide-gel electrophoresis. Mr values for these purified RNAases were 78 000, 16 000, 13 300 and 5000 as estimated by gel filtration. Enzyme activities of RNAases I, III and IV were inhibited by Mn2+, Zn2+ and Cu2+ and activated by Na+, K+, Ba2+, Mg2+, Fe2+ and EDTA, whereas that of RNAase II was inhibited by Ba2+, Mg2+, Fe2+, Mn2+, Zn2+ and Cu2+ and activated by Na+, K+ and EDTA. RNAases I, II and IV demonstrated a higher affinity for poly(C) and poly(U) or yeast RNA, whereas RNAase III preferentially hydrolysed poly(U) over poly(C) and yeast RNA. In the presence of 5 mM-spermine, RNAase I was dissociated to a low-Mr (5000) enzyme with an increase in total RNAase enzymic activity. Xenoantiserum to each RNAase was raised and evaluated by immunoprecipitation and immunohistochemical methods. Anti-(seminal RNAase III) antiserum showed no immunological cross-reaction with RNAases of other human origin, whereas anti-(seminal RNAase I), -(RNAase II) and -(RNAase IV) antisera exhibited indistinguishable immunological reactions with serum RNAase and other human RNAases, except that anti-(seminal RNAase I) and -(RNAase antisera IV) did not react with pancreatic RNAases. Seminal RNAases I and IV were identical immunologically as shown by anti-(RNAase I) and anti-(RNAase IV) in immunodiffusion. Immunohistochemical study revealed that, among human tissues examined, only prostate expressed seminal RNAase III. These results suggested that human seminal RNAase I may be an aggregated molecule of RNAase IV and that seminal RNAases II and IV are similar to serum RNAases, whereas seminal RNAase III is a prostate-specific enzyme.

Dataset Information

Characterization of the angiogenic activity of zebrafish ribonucleases.

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Characterization of the angiogenic activity of zebrafish ribonucleases.

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