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1.
Biomolecules ; 10(9)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32872229

RESUMO

The Wnt Inhibitory Factor 1 (Wif1), known to inhibit Wnt signaling pathways, is composed of a WIF domain and five EGF-like domains (EGF-LDs) involved in protein interactions. Despite the presence of a potential O-fucosylation site in its EGF-LDs III and V, the O-fucose sites occupancy has never been demonstrated for WIF1. In this study, a phylogenetic analysis on the distribution, conservation and evolution of Wif1 proteins was performed, as well as biochemical approaches focusing on O-fucosylation sites occupancy of recombinant mouse WIF1. In the monophyletic group of gnathostomes, we showed that the consensus sequence for O-fucose modification by Pofut1 is highly conserved in Wif1 EGF-LD III while it was more divergent in EGF-LD V. Using click chemistry and mass spectrometry, we demonstrated that mouse WIF1 was only modified with a non-extended O-fucose on its EGF-LD III. In addition, a decreased amount of mouse WIF1 in the secretome of CHO cells was observed when the O-fucosylation site in EGF-LD III was mutated. Based on sequence comparison and automated protein modeling, we suggest that the absence of O-fucose on EGF-LD V of WIF1 in mouse and probably in most gnathostomes, could be related to EGF-LD V inability to interact with POFUT1.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência Consenso , Fator de Crescimento Epidérmico/química , Evolução Molecular , Fucose/metabolismo , Animais , Fator de Crescimento Epidérmico/metabolismo , Fucosiltransferases/metabolismo , Camundongos , Modelos Moleculares , Filogenia , Domínios Proteicos , Proteínas Recombinantes/metabolismo
2.
Open Biol ; 6(9)2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27628322

RESUMO

Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O-fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1(cax/cax) (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1(cax/cax) mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1(cax/cax) SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7(+)/MYOD(-) progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O-fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice.


Assuntos
Fucosiltransferases/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Mioblastos/fisiologia , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Membrana Celular/metabolismo , Células Cultivadas , Fucosiltransferases/genética , Expressão Gênica , Hipertrofia , Ligantes , Camundongos , Camundongos Endogâmicos C3H , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos/citologia , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Receptores Notch/genética , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/fisiologia , Células-Tronco
3.
Mol Cell Biol ; 35(2): 391-405, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25384974

RESUMO

The Notch signaling pathway plays a crucial role in skeletal muscle regeneration in mammals by controlling the transition of satellite cells from quiescence to an activated state, their proliferation, and their commitment toward myotubes or self-renewal. O-fucosylation on Notch receptor epidermal growth factor (EGF)-like repeats is catalyzed by the protein O-fucosyltransferase 1 (Pofut1) and primarily controls Notch interaction with its ligands. To approach the role of O-fucosylation in myogenesis, we analyzed a murine myoblastic C2C12 cell line downregulated for Pofut1 expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown of Pofut1 affected the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7(+)/MyoD(-) cells and earlier myogenic program entrance were observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue of Pofut1 expression in knockdown cells restored Notch signaling activation and a normal course in C2C12 differentiation. Our results establish the critical role of Pofut1 on Notch pathway activation during myogenic differentiation.


Assuntos
Fucosiltransferases/metabolismo , Mioblastos/citologia , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Técnicas de Silenciamento de Genes/métodos , Camundongos , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Fator de Transcrição PAX7/metabolismo
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