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GPRC Function in autophagy control: a systematic approach of chemical intervention

ABSTRACT: Autophagy facilitates the degradation of cellular content via the lysosome and is involved in cellular homeostasis and stress response pathways. As such, malfunction of autophagy is linked to a variety of diseases ranging from organ specific illnesses like cardiomyopathy to systemic illnesses such as cancer or metabolic syndromes. Given the variety of autophagic functions within a cell and tissue, regulation of autophagy is complex and contains numerous positive and negative feedback loops. While our knowledge of mechanisms for cargo selectivity has significantly improved over the last decade, our understanding of signaling routes activating individual autophagy pathways remains rather sparce. In this resource study, we report on a well-characterized chemical library assembled within the IMI initiative EUbOPEN containing 77 GPCR-targeting ligands that was used to systematically analyze LC3B-based autophagy as well as ER-phagy flux upon compound treatment. The global impact of hit compounds TC-G 1004, BAY 60-6583, PSNCBAM-1, TC-G 1008, LPA2 Antagonist 1, ML154, JTC-801 and ML-290 targeting Adenosine receptor A2a (ADORA2A), Adenosine receptor A2b (ADORA2B), Cannabinoid receptor 1 (CNR1), G-protein coupled receptor 39 (GPR39), Lysophosphatidic acid receptor 2 (LPAR2), neuropeptide S receptor 1 (NPSR1), NOP (OPRL1), and Relaxin receptor 1 (RXFP1) were subsequently analyzed and compared by TMT-based mass spectrometry. Our results indicate the differential impact of targeted GPCRs on known autophagy-associated proteins.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Bone Marrow

SUBMITTER: Pablo Sanz Martinez

LAB HEAD: Dr.Alexandra Stolz

PROVIDER: PXD047645 | Pride | 2024-07-18

REPOSITORIES: Pride

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			Action	DRS
	20231116_LU_LC2_MAA_PS_034_1_WCP_400ng_F.msf	Msf
	20231116_LU_LC2_MAA_PS_034_1_WCP_400ng_F.msfView	Msf
	20231116_LU_LC2_MAA_PS_034_1_WCP_400ng_F.pdResult	Other
	20231116_LU_LC2_MAA_PS_034_1_WCP_400ng_F.pdResultView	Other
	20231116_LU_LC2_MAA_PS_034_1_WCP_400ng_F.xlsx	Xlsx

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Project description:Although malignant gliomas frequently show aberrant activation of the mammalian target of rapamycin (mTOR), mTOR inhibitors have performed poorly in clinical trials. Besides regulating cell growth and translation, mTOR controls the initiation of autophagy. By recycling cellular components, autophagy can mobilize energy resources, and has thus been attributed cancer-promoting effects. Here, we asked whether the activation of autophagy represents an escape mechanism to pharmacological mTOR inhibition, and explored co-treatment with mTOR and autophagy inhibitors as a therapeutic strategy. Mimicking conditions of the glioma microenvironment, glioma cells were exposed to nutrient starvation and hypoxia. Following treatment with mTOR inhibitor torin2 or rapamycin, and autophagy inhibitors bafilomycin A1 or MRT68921, we analyzed autophagic activity, cell growth, viability and oxygen consumption. Changes in global proteome were quantified by mass spectrometry. In the context of hypoxia and starvation, autophagy was strongly induced in glioma cells, and further increased by mTOR inhibition. While torin2 enhanced glioma cell survival, co-treatment with torin2 and bafilomycin A1 failed to promote cell death. Importantly, treatment with bafilomycin A1 alone also protected glioma cells from cell death. Mechanistically, both compounds significantly reduced glioma growth and oxygen consumption. Mass spectrometry analysis showed that bafilomycin A1 induced broad changes in the cellular proteome. More specifically, proteins downregulated by bafilomycin A1 were associated with the mitochondrial respiratory chain and ATP synthesis. Taken together, our results show that activation of autophagy does not account for the cytoprotective effects of mTOR inhibition in our in vitro model of the glioma microenvironment. Our proteomic findings suggest that the pharmacological inhibition of autophagy induces extensive changes in the cellular proteome that can support glioma cell survival under nutrient-deplete and hypoxic conditions. These findings provide a novel perspective on the complex role of autophagy in gliomas and may have implications for the design of future trials.

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GPRC Function in autophagy control: a systematic approach of chemical intervention

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