Résumé
BackgroundGNPTAB codes for the cis-Golgi enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-PTase), a key enzyme in the synthesis of mannose-6-phosphate (M6P) signals that are required for acid hydrolases sorting to lysosomes. To be active, the GlcNAc-1-PTase undergoes a proteolytic maturation mediated by the Site-1 protease (S1P).
Mutations in GNPTAB cause Mucolipidosis II/III, i.e. lysosomal storage diseases characterized by acid hydrolases hypersecretion. A recent report has also highlighted that the frequency of GNPTAB variants is increased in breast and uterine tumors. Since acid hydrolases hypersecretion can promote cancer cell migration and invasion, a GlcNAc-1-PTase deficiency could have important consequences on cancer development and progression.
Aims
To investigate whether GNPTAB mutations found in breast cancers impair GlcNAc-1-PTase expression, maturation and acid hydrolase trafficking to lysosomes; to set up a cell model in which GlcNac-1-Ptase is inactivated by pharmacological inhibition of S1P.
Methods
Seven mutations found in breast tumors were inserted in a GNPTAB cDNA and expressed in HeLa or BT549 cells prior to analyzing GlcNAc-1-PTase expression and maturation (by western blotting), localization (by fluorescence microscopy), and involvement in acid hydrolases sorting (using activity assays on cell lysates and media). We also evaluated how S1P inhibition with PF-429242 affects acid hydrolases sorting and lysosomal morphology in HeLa cells.
Results
All GNPTAB mutants tested in immunofluorescence assays reached the cis-Golgi apparatus and five out of seven exhibited decreased expression at the protein level. In addition, mutation S1022Ydecreased GlcNAc-1-PTase maturation, similarly to the Y916C mutant characterized in a previous master thesis. Transient transfection of all mutants in GNPTAB KO HeLa cells corrected their acid hydrolase hypersecretion phenotype (to the same extent as WT GNPTAB). Save for Y916C, correction was also observed, at least to some extent, after transfection of the mutants in BT549 cells. These cells contain the GNPTAB-Y916C mutation in their genome and hyper-secrete their acid hydrolases.
We also found that pharmacological inhibition of S1P caused a concentration-dependent mis-sorting of acid hydrolases, expansion/enlargement of the lysosomal population and nuclear translocation of TFEB, in HeLa cells.
Conclusion
Five of the tested mutations decrease GlcNAc-1-PTase expression level, suggesting that they affect the synthesis and/or stability of the enzyme. Moreover, two mutations impair its S1P-mediated maturation: Y916C and S1022Y. Since, when over-expressed, all mutants could correct acid hydrolase missorting (in BT549 and/or HeLa cells), we conclude that none of the mutations completely inactivate the enzyme. Additional analyses will be needed to identify if these mutations affect M6P synthesis to some extent (including when the enzyme is not overexpressed). In the meantime, the pharmacological inhibition of S1P will be useful to initiate the study of the consequences of a GlcNac-1-PTase inactivation on cancer cell behav
| la date de réponse | janv. 2022 |
|---|---|
| langue originale | Anglais |
| L'institution diplômante |
|
| Superviseur | Marielle Boonen (Promoteur) & Michel Jadot (Promoteur) |