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USP9X deubiquitylating enzyme maintains RAPTOR protein levels, mTORC1 signalling and proliferation in neural progenitors.

ABSTRACT: 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.

SUBMITTER: Bridges CR

PROVIDER: S-EPMC5427856 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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USP9X deubiquitylating enzyme maintains RAPTOR protein levels, mTORC1 signalling and proliferation in neural progenitors.

Bridges Caitlin R CR Tan Men-Chee MC Premarathne Susitha S Nanayakkara Devathri D Bellette Bernadette B Zencak Dusan D Domingo Deepti D Gecz Jozef J Murtaza Mariyam M Jolly Lachlan A LA Wood Stephen A SA

Scientific reports 20170324 1

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 c ...[more]

PMID: 28341829

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Project description:Glioblastoma is a very aggressive form of brain tumor with limited therapeutic options. Usually, glioblastoma is treated with ionizing radiation (IR) and chemotherapy after surgical removal. However, radiotherapy is frequently unsuccessful, among others owing to resistance mechanisms the tumor cells have developed. Antiapoptotic B-cell leukemia (Bcl)-2 family members can contribute to radioresistance by interfering with apoptosis induction in response to IR. Bcl-2 and the closely related Bcl-xL and Mcl-1 are often overexpressed in glioblastoma cells. In contrast to Bcl-2 and Bcl-xL, Mcl-1 is a short-lived protein whose stability is closely regulated by ubiquitylation-dependent proteasomal degradation. Although ubiquitin ligases facilitate degradation, the deubiquitylating enzyme ubiquitin-specific protease 9x (USP9x) interferes with degradation by removing polyubiquitin chains from Mcl-1, thereby stabilizing this protein. Thus, an inability to downregulate Mcl-1 by enhanced USP9x activity might contribute to radioresistance. Here we analyzed the impact of USP9x on Mcl-1 levels and radiosensitivity in glioblastoma cells. Correlating Mcl-1 and USP9x expressions were significantly higher in human glioblastoma than in astrocytoma. Downregulation of Mcl-1 correlated with apoptosis induction in established glioblastoma cell lines. Although Mcl-1 knockdown by siRNA increased apoptosis induction after irradiation in all glioblastoma cell lines, USP9x knockdown significantly improved radiation-induced apoptosis in one of four cell lines and slightly increased apoptosis in another cell line. In the latter two cell lines, USP9x knockdown also increased radiation-induced clonogenic death. The massive downregulation of Mcl-1 and apoptosis induction in A172 cells transfected with USP9x siRNA shows that the deubiquitinase regulates cell survival by regulating Mcl-1 levels. In contrast, USP9x regulated radiosensitivity in Ln229 cells without affecting Mcl-1 levels. We conclude that USP9x can control survival and radiosensitivity in glioblastoma cells by Mcl-1-dependent and Mcl-1-independent mechanisms.

Dataset Information

USP9X deubiquitylating enzyme maintains RAPTOR protein levels, mTORC1 signalling and proliferation in neural progenitors.

Publications

USP9X deubiquitylating enzyme maintains RAPTOR protein levels, mTORC1 signalling and proliferation in neural progenitors.

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