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Gonadotropin regulation of ankyrin-repeat and SOCS-box protein 9 (ASB9) in ovarian follicles and identification of binding partners

By Gabriel Benoit (3515561), Aly Warma (6407729), Jacques G. Lussier (6407732) and Kalidou Ndiaye (3515552)

Abstract

<div><p>Ankyrin-repeat and SOCS-box protein 9 (ASB9) is a member of the large SOCS-box containing proteins family and acts as the specific substrate recognition component of E3 ubiquitin ligases in the process of ubiquitination and proteasomal degradation. We previously identified ASB9 as a differentially expressed gene in granulosa cells (GC) of bovine ovulatory follicles. This study aimed to further investigate ASB9 mRNA and protein regulation, identify binding partners in GC of bovine ovulatory follicles, and study its function. GC were obtained from small follicles (SF: 2–4 mm), dominant follicles at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 hours following hCG injection (OF). Analyses by RT-PCR showed a 104-fold greater expression of <i>ASB9</i> in GC of OF than in DF. Steady-state levels of <i>ASB9</i> in follicular walls (granulosa and theca cells) analyzed at 0, 6, 12, 18 and 24 hours after hCG injection showed a significant induction of ASB9 expression at 12 and 18 hours, reaching a maximum induction of 10.2-fold at 24 hours post-hCG as compared to 0 hour. These results were confirmed in western blot analysis showing strongest ASB9 protein amounts in OF. Yeast two-hybrid screening of OF-cDNAs library resulted in the identification of 10 potential ASB9 binding partners in GC but no interaction was found between ASB9 and creatine kinase B (CKB) in these GC. Functional studies using CRISPR-Cas9 approach revealed that ASB9 inhibition led to increased GC proliferation and modulation of target genes expression. Overall, these results support a physiologically relevant role of ASB9 in the ovulatory follicle by targeting specific proteins likely for degradation, contributing to reduced GC proliferation, and could be involved in the final GC differentiation into luteal cells.</p></div

Topics: Biophysics, Biochemistry, Microbiology, Cell Biology, Genetics, Molecular Biology, Physiology, Developmental Biology, Cancer, Biological Sciences not elsewhere classified, Chemical Sciences not elsewhere classified, CKB, ASB 9, RT-PCR, ASB 9 mRNA, ASB 9 protein amounts, DF, ASB 9 binding partners, ASB 9 inhibition, E 3 ubiquitin ligases, GC proliferation, hCG injection, Yeast two-hybrid screening, SOCS-box protein 9, target genes expression, creatine kinase B, ASB 9 expression, 24 hours, SF, binding partners Ankyrin-repeat, ovulatory follicles, substrate recognition component, CRISPR-Cas 9 approach, 24 hours post-hCG
Year: 2019
DOI identifier: 10.1371/journal.pone.0212571
OAI identifier: oai:figshare.com:article/7779986
Provided by: FigShare
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