PMC:4608092 / 38458-43457
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4608092","sourcedb":"PMC","sourceid":"4608092","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4608092","text":"Loss of Function of the Walker B Motif in the First AAA+ of RNF213 Lowers Angiogenic Activity Whereas a Deletion Mutation of the First AAA+ Does Not Lower Angiogenic Activity\nMutation of glutamic acid of the Walker B motif (DExxbox) (WEQ) causes loss of function of ATPase hydrolysis.28 The Walker B motif in the first AAA+ of RNF213 is shown to stabilize hexamer formation, whereas deletion of the first AAA+, which results in loss of ATPase activity, does not initiate oligomerization.13 Therefore, we hypothesized that RNF213 WEQ mutation stabilizes oligomers and can cause deleterious effects in ECs by capture in the oligomeric state. We also hypothesized that RNF213 deletion of AAA+, which does not allow formation of oligomers, does not result in any deleterious effects as silencing RNF213 in vitro14 and in Rnf213 null mice.14,17 Based on these hypotheses, we investigated the effects of expression of the vectors RNF213 WT, RNF213 R4810K, RNF213 WEQ, and RNF213 deletion of AAA+ on tube formation and migration of HUVECs (Figure5A). RNF213 WEQ decreased tube formation and migration, similar to RNF213 R4810K (Figure5). In contrast, expression of RNF213 deletion of AAA+ did not lower tube formation or migration, which is in accord with a finding that silencing RNF213 did not impair angiogenesis.14 Expression of RNF213 WT did not decrease angiogenic activity. Therefore, we consider that RNF213 trapped in the oligomeric state may lead to low angiogenic activity.\nFigure 15 Angiogenic activity of RNF213 WT, and R4810K, WEQ, and ΔAAA mutants. A, RNF213 protein expression in HUVECs transiently expressing the RNF213 mutant. HUVECs transfected with the FLAG-RNF213 expression vector (RNF213 WT, RNF213 R4810K, RNF213 WEQ, and RNF213 ΔAAA) were immunoblotted using anti-FLAG antibodies. Empty vector (“vector” in the figure)-transfected HUVECs served as the control. Representative western blotting findings are shown. Similar results were obtained in 3 independent experiments. B, Tube formation assays for HUVECs transiently expressing RNF213 WT, RNF213 R4810K, RNF213 WEQ, and RNF213 ΔAAA. The vector served as the positive control (100%). Scale bars indicate 100 μm. Representative images are shown in left panel. Tube area was quantified by imaging analysis (right panel). A column with a bar represents mean±SD (n=3). *P\u003c0.05, according to Student t test compared with the vector. C, Migration assays for HUVECs transiently expressing RNF213 WT, RNF213 R4810K, RNF213 WEQ, and RNF213 ΔAAA. The vector served as the control. Scale bars indicate 100 μm. Representative images are shown in left panel. Re-endothelialized areas were quantified by imaging analysis (right panel). A column with a bar represents mean±SD (n=3). *P\u003c0.05 according to Student t test compared with the vector. HUVECs indicates human umbilical vein endothelial cells; LPF, low-pass filter; WEQ, Walker B motif; WT, wild-type. We then investigated the effects of RNF213 R4810K on ATPase activity (Figure6). Although we detected ATPase activity in Walker motif in a recombinant fragment containing amino acids from 2319 to 2613,8 we had never determined ATPase activity with an entire 5207 amino acids. In the current study, we determined ATPase activity with the entire RNF213 proteins of WT, R4810K, and deletion AAA+. Surprisingly, RNF213 R4810K resulted in a loss of ATPase activity. Under the present experimental conditions using excessive detergent washing, RNF213 protein may not be able to maintain an oligomeric form. Therefore, we postulate that the results may represent monomeric ATPase activity. RNF213 R4810K can form oligomers, similar to RNF213 WT,13 suggesting that R4810K may stabilize oligomeric states of RNF213 by inhibiting ATP hydrolysis, thereby inhibiting ATPase activity.\nFigure 16 ATPase activity of RNF213 WT, R4810K, and ΔAAA mutants. A, Lysates from EGFP-RNF213–transfected HEK293 cells were IP with anti-GFP agarose. A total volume of 15 μL was subjected to SDS-PAGE followed by GelCode staining. RNF213 proteins fused with EGFP were detected in EGFP-RNF213–transfected cells (arrow heads, “EGFP-RNF213”). Representative SDS-PAGE images are shown. Similar results were obtained in 3 independent experiments. B, ATPase activity of immunoprecipitated extracts was assayed for ATPase activity. Indicated volumes (μL) of IP products were combined with buffer to yield a total volume of 50 μL for ATPase reaction for 30 minutes at room temperature (see details in the text). Phosphate release was measured using the Phosphate Sensor as ATPase activity. Relative activity was calculated based on average activity of EGFP at 0 μL, which was equal to 1. Data with bars represent mean±SD (n=3). *P\u003c0.05 according to Student t test compared with WT at 2 volumes. EGFP indicates enhanced green fluorescent proteins detected in EGFP-transfected cells (arrow head); IgG HC, IgG heavy chain; IgG LC, IgG light chain; IP, immunoprecipitated; Well, sample wells of the gel; WT, wild type.\n\nE","divisions":[{"label":"Title","span":{"begin":0,"end":174}},{"label":"Figure caption","span":{"begin":1478,"end":2918}},{"label":"Figure caption","span":{"begin":3790,"end":4998}}],"tracks":[{"project":"2_test","denotations":[{"id":"26126547-23946885-55294637","span":{"begin":284,"end":286},"obj":"23946885"},{"id":"26126547-24658080-55294638","span":{"begin":487,"end":489},"obj":"24658080"},{"id":"26126547-23850618-55294639","span":{"begin":807,"end":809},"obj":"23850618"},{"id":"26126547-23850618-55294640","span":{"begin":834,"end":836},"obj":"23850618"},{"id":"26126547-23410753-55294641","span":{"begin":837,"end":839},"obj":"23410753"},{"id":"26126547-23850618-55294642","span":{"begin":1309,"end":1311},"obj":"23850618"},{"id":"26126547-21799892-55294643","span":{"begin":3119,"end":3120},"obj":"21799892"},{"id":"26126547-24658080-55294644","span":{"begin":3656,"end":3658},"obj":"24658080"}],"attributes":[{"subj":"26126547-23946885-55294637","pred":"source","obj":"2_test"},{"subj":"26126547-24658080-55294638","pred":"source","obj":"2_test"},{"subj":"26126547-23850618-55294639","pred":"source","obj":"2_test"},{"subj":"26126547-23850618-55294640","pred":"source","obj":"2_test"},{"subj":"26126547-23410753-55294641","pred":"source","obj":"2_test"},{"subj":"26126547-23850618-55294642","pred":"source","obj":"2_test"},{"subj":"26126547-21799892-55294643","pred":"source","obj":"2_test"},{"subj":"26126547-24658080-55294644","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93c3ec","default":true}]}]}}