Adrenocortical Mitochondria-Associated Membranes: Isolation, Characterization, and Lipidoproteomic Response to Mitotane

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Mitotane is an inhibitor of sterol O-acyltransferase 1 (SOAT1) approved for the treatment of adrenocortical carcinoma (ACC). In cells, mitotane increases reactive oxygen species, lipid peroxidation, and ultimately cell death. This mechanism is similar but distinct from ferroptosis, a cell death mechanism adrenal cortex cells are endogenously predisposed to. Both Acyl-CoA-Synthetase 4 (ACSL4), essential for ferroptosis, and SOAT1 are localized in mitochondria-associated membranes (MAM), specialized contact sites between mitochondria and endoplasmic reticulum (ER). Here, we used protein and lipid mass spectrometry to explore the role of MAMs in adrenocortical cell death. MAMs were isolated from NCI-H295S cells treated with mitotane, the ferroptosis inducer RSL3, or control. Western blotting of marker proteins was used for quality control prior to lipid and protein mass spectrometry. MAM fractions showed strong enrichment of SOAT1 and FATE1 (fetal and adult testis expressed 1) marker proteins, contained ACSL4, and were depleted from mitochondrial MTCO2 independent of treatment condition. Protein mass spectrometry identified IRE1alpha/ERN1, and PERK/EIF2AK3 implicated in the response to mitotane. Proteins involved in ER- and mitochondria-related processes were functionally enriched. We discovered the guanosine nucleotide exchange factor GRIPAP1 in MAMs of mitotane but not RSL3- or control-treated samples. In NCI-H295S cells mitotane upregulated GRIPAP1 expression. Mitotane but not RSL3 pronouncedly reduced the quantity of ubiquinone (Q10) and heme B in MAMs. In conclusion, locally reduced Q10 in MAM may contribute to impaired respiratory chain activity and free radical excess induced by mitotane. Recruitment of GRIPAP1 protein to MAMs may transduce cell death.

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