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SILAC APMS analysis of GFP tagged TSC2 in Hela Cells

ABSTRACT: HSP90 and its co-chaperon R2TP are involved to assemble and stabilize many macro-molecular complexes involved in cell proliferation. R2TP complex is composed of RUVBL1 and RUVBL2 AAA+ ATPases and two adapter proteins RPAP3 and PIH1D1. The N-terminal domain of PIH1D1 was described to possess a basic pocket that binds phosphorylated peptides on some of the R2TP clients. Subunits of TSC complex (Tuberous SClerosis) have previously been described in proteomic analysis of proteins immune-precipitated with PIH1D1 N-terminal domain. TSC complex is a regulator of mTOR activity. It is composed of TSC1, TSC2 and TBC1D7 subunits. Here, we show that (i) TSC1 and TSC2 are both substrates of HSP90, (ii) the HSP90/R2TP system has multiple interaction with TSC complex subunits and (iii) it is functionally involved in the assembly of the TSC complex. We found a direct interaction of TSC1 with the phospho-binding pocket of PIH1D1 N-terminal domain regardless of phosphorylationbut in a non-canonical phosphorylation independent manner[XM1] . TSC2 and TBC1D7 would also interact with R2TP but independently of PIH1D1. Taken together, these data suggest that R2TP acts as a platform for the independent recruitment of TSC subunits before the assembly of the TSC complex

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Franck Vandermoere

LAB HEAD: Celine Verheggen

PROVIDER: PXD030402 | Pride | 2024-11-12

REPOSITORIES: Pride

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			Action	DRS
	PHAXRNASE_171006_1_E171010_TSC2_A01.raw	Raw
	PHAXRNASE_171006_1_E171010_TSC2_A02.raw	Raw
	PHAXRNASE_171006_1_E171010_TSC2_A03.raw	Raw
	PHAXRNASE_171006_1_E171010_TSC2_A04.raw	Raw
	PHAXRNASE_171006_1_E171010_TSC2_A05.raw	Raw

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Project description:Tuberous sclerosis complex (TSC) is an inherited multi-system disorder caused by mutations in the TSC1 or TSC2 gene. TSC patients are often diagnosed with a range of neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD), intellectual disability (ID), anxiety and mood disorders. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) kinase signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using neural progenitor cells (NPCs) from patient-derived induced pluripotent stem cells (iPSCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in CRISPR-modified TSC1-null NPCs, which were unaffected by mTORC1 inhibition by rapamycin,the only approved therapy for TSC1. Here, to assess TSC1-dependent gene expression programs in NPCs, we used polysome-profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level. In addition to changes in mRNA abundance, this revealed numerous TSC1-dependent alterations in translational efficiencies. To assess the relevance of these gene expression alterations we performed polysome-profiling in post-mortem brains originating from ASD donors and matched controls. Strikingly, TSC1-dependent alterations in mRNA translation observed in NPCs were largely recapitulated in human brains. Furthermore, although polysome-profiling revealed a partial reversal of TSC1-associated gene expression alterations following rapamycin treatment, most genes related to neural activity/synaptic regulation or ASD that showed TSC1-dependent translation were rapamycin-insensitive. Therefore, we also examined whether early ND rapamycin-insensitive phenotypes in TSC1-null NPCs could be rescued by a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272 (Revolution Medicines, Inc.). Unlike rapamycin, RMC-6272 strongly inhibited translation and reversed TSC1-associated proliferation and neurite outgrowth phenotypes. In summary, we reveal ample translational alterations in TSC1 patient-derived NPCs recapitulating human brain expression profiles and potential implications for treatment of TAND.

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SILAC APMS analysis of GFP tagged TSC2 in Hela Cells

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