Abstract
Angiogenin (hANG), a member of the Ribonuclease A superfamily has angiogenic, neurotrophic and neuroprotective activities. Mutations in hANG have been found in patients with Amyotrophic lateral sclerosis (ALS). The zebrafish (Danio rerio) rnasel-1, 2 and 3 are orthologues of hANG and of these only Rnasel-1 and Rnasel-2 have been shown to be angiogenic. Herein we show that NCI-65828, a potent and specific small molecule inhibitor of hANG inhibits Rnasel-1 to a similar extent. Treatment of early zebrafish embryos with NCI-65828, or with terrein, a fungal metabolite which prevents the secretion of hANG, resulted in spinal neuron aberrations as well defects in trunk vasculature. Our detailed expression analysis and inhibitor studies suggest that Rnasel-1 plays important roles in neuronal migration and pathfinding as well as in angiogenesis in zebrafish. Our studies suggest the usefulness of the zebrafish as a model to dissect the molecular consequences of the ANG ALS variants.
Highlights
Human angiogenin, a potent angiogenic factor is a single chain polypeptide of Mr ~ 14400 first isolated from human colon adenocarcinoma cell line (HT-29) conditioned media[1,2]
HANG is involved in many disease conditions and has been shown to be upregulated in various human cancers, diabetic retinopathy and arthritis17. Human angiogenin (hANG) has been implicated in neurodegenerative diseases as mutations in hANG have been identified in patients with Amyotrophic Lateral Sclerosis (ALS), Parkinson’s disease and Fronto-Temporal Dementia (FTD)[15,18,19,20,21,22]
Prior to studying the effects of NCI-65828 (8-amino-5-(4′-hydroxybiphenyl-4-azo) napathlene-2-sulphate) (Fig. 1D), a selective and potent cell permeable inhibitor of the catalytic activity of hANG, on zebrafish motor neurons and vasculature we sought to establish if NCI-65828 is able to inhibit the enzymatic activity of zebrafish Rnasels
Summary
Human angiogenin (hANG), a potent angiogenic factor is a single chain polypeptide of Mr ~ 14400 first isolated from human colon adenocarcinoma cell line (HT-29) conditioned media[1,2] It is a member of the Ribonuclease A (RNase A, known as RNase 5) superfamily and has weak ribonucleolytic (catalytic) activity[3]. We have shown (based on a detailed structure-function study) that Rnasel-1a cleaves tRNA with a specific activity similar to hANG. This is consistent with the finding that the active site in Rnasel-1a is blocked by its C-terminus as in hANG27. This is in contrast to Rnasel-3e in which the active site is open and which has 17–20 fold more RNase activity towards tRNA27
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