USP9X deubiquitylating enzyme maintains RAPTOR protein levels, mTORC1 signalling and proliferation in neural progenitors.
USP9X, is highly expressed in neural progenitors and, essential for neural development in mice. In humans, mutations in USP9X are associated with neurodevelopmental disorders. To understand USP9X's role in neural progenitors, we studied the effects of altering its expression in both the human neural progenitor cell line, ReNcell VM, as well as neural stem and progenitor cells derived from Nestin-cre conditionally deleted Usp9x mice. Decreasing USP9X resulted in ReNcell VM cells arresting in G0 cell cycle phase, with a concomitant decrease in mTORC1 signalling, a major regulator of G0/G1 cell cycle progression. Decreased mTORC1 signalling was also observed in Usp9x-null neurospheres and embryonic mouse brains. Further analyses revealed, (i) the canonical mTORC1 protein, RAPTOR, physically associates with Usp9x in embryonic brains, (ii) RAPTOR protein level is directly proportional to USP9X, in both loss- and gain-of-function experiments in cultured cells and, (iii) USP9X deubiquitlyating activity opposes the proteasomal degradation of RAPTOR. EdU incorporation assays confirmed Usp9x maintains the proliferation of neural progenitors similar to Raptor-null and rapamycin-treated neurospheres. Interestingly, loss of Usp9x increased the number of sphere-forming cells consistent with enhanced neural stem cell self-renewal. To our knowledge, USP9X is the first deubiquitylating enzyme shown to stabilize RAPTOR.
Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia.
Caitlin R
CR
Bridges
Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia.
Men-Chee
MC
Tan
Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia.
Susitha
S
Premarathne
Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia.
Devathri
D
Nanayakkara
Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia.
Bernadette
B
Bellette
Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia.
Dusan
D
Zencak
School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia.
Deepti
D
Domingo
School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia.|Robinson Institute, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, South Australia, Australia.
Jozef
0000-0002-7884-6861
J
Gecz
Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia.
Mariyam
M
Murtaza
Robinson Institute, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, South Australia, Australia. lachlan.jolly@adelaide.edu.au.
Lachlan A
LA
Jolly
Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia. s.wood@griffith.edu.au.
Stephen A
0000-0002-2576-7457
SA
Wood
D000818:Animals; D059447:Cell Cycle Checkpoints; D000066673:Cell Self Renewal; D010450:Endopeptidases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1 / Q000378:metabolism*; D051379:Mice; D058953:Neural Stem Cells / Q000378:metabolism*; D059748:Proteolysis; D000076223:Regulatory-Associated Protein of mTOR / Q000378:metabolism*; D015398:Signal Transduction; D043222:Ubiquitin Thiolesterase / Q000378:metabolism*
C463659:RPTOR protein, human; D000076223:Regulatory-Associated Protein of mTOR; C540162:Rptor protein, mouse; C506234:USP9X protein, human; D000076222:Mechanistic Target of Rapamycin Complex 1; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase; C404328:Usp9x protein, mouse