Fardini Yann, Perez-Cervera Yobana, Camoin Luc, Pagesy Patrick, Lefebvre Tony, Issad Tarik
INSERM, U1016, Institut Cochin, Paris, France; CNRS, UMR8104, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
Structural and Functional Glycobiology Unit, Lille 1 University, CNRS (UMR 8576), IFR 117, Villeneuve d'Ascq, France; Facultad de Odontología, Universidad Autónoma Benito Juárez de Oaxaca, Oaxaca, Mexico.
Biochem Biophys Res Commun. 2015 Jun 26;462(2):151-8. doi: 10.1016/j.bbrc.2015.04.114. Epub 2015 May 2.
O-GlcNAcylation is a reversible post-translational modification that regulates cytosolic and nuclear proteins. We and others previously demonstrated that FoxO1 is O-GlcNAcylated in different cell types, resulting in an increase in its transcriptional activity. Four O-GlcNAcylation sites were identified in human FOXO1 but directed mutagenesis of each site individually had modest (T317) or no effect (S550, T648, S654) on its O-GlcNAcylation status and transcriptional activity. Moreover, the consequences of mutating all four sites had not been investigated. In the present work, we mutated these sites in the mouse Foxo1 and found that mutation of all four sites did not decrease Foxo1 O-GlcNAcylation status and transcriptional activity, and would even tend to increase them. In an attempt to identify other O-GlcNAcylation sites, we immunoprecipitated wild-type O-GlcNAcylated Foxo1 and analysed the tryptic digest peptides by mass spectrometry using High-energy Collisional Dissociation. We identified T646 as a new O-GlcNAcylation site on Foxo1. However, site directed mutagenesis of this site individually or together with all four previously identified residues did not impair Foxo1 O-GlcNAcylation and transcriptional activity. These results suggest that residues important for the control of Foxo1 activity by O-GlcNAcylation still remain to be identified.
O-连接的N-乙酰葡糖胺化（O-GlcNAcylation）是一种可逆的翻译后修饰，可调节胞质和核蛋白。我们和其他人之前证明，FoxO1在不同细胞类型中发生O-GlcNAcylation修饰，导致其转录活性增加。在人类FOXO1中鉴定出四个O-GlcNAcylation位点，但对每个位点进行单独的定向诱变对其O-GlcNAcylation状态和转录活性影响不大（T317）或没有影响（S550、T648、S654）。此外，尚未研究突变所有四个位点的后果。在本研究中，我们在小鼠Foxo1中对这些位点进行了突变，发现突变所有四个位点并没有降低Foxo1的O-GlcNAcylation状态和转录活性，甚至有增加的趋势。为了鉴定其他O-GlcNAcylation位点，我们免疫沉淀了野生型O-GlcNAcylated Foxo1，并使用高能碰撞解离通过质谱分析胰蛋白酶消化肽。我们将T646鉴定为Foxo1上一个新的O-GlcNAcylation位点。然而，对该位点单独或与之前鉴定的所有四个残基一起进行位点定向诱变并不损害Foxo1的O-GlcNAcylation和转录活性。这些结果表明，对于通过O-GlcNAcylation控制Foxo1活性重要的残基仍有待确定。
