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Acute ACAT1/SOAT1 Blockade Increases MAM Cholesterol and Strengthens ER-Mitochondria Connectivity.

ABSTRACT: Cholesterol is a key component of all mammalian cell membranes. Disruptions in cholesterol metabolism have been observed in the context of various diseases, including neurodegenerative disorders such as Alzheimer's disease (AD). The genetic and pharmacological blockade of acyl-CoA:cholesterol acyltransferase 1/sterol O-acyltransferase 1 (ACAT1/SOAT1), a cholesterol storage enzyme found on the endoplasmic reticulum (ER) and enriched at the mitochondria-associated ER membrane (MAM), has been shown to reduce amyloid pathology and rescue cognitive deficits in mouse models of AD. Additionally, blocking ACAT1/SOAT1 activity stimulates autophagy and lysosomal biogenesis; however, the exact molecular connection between the ACAT1/SOAT1 blockade and these observed benefits remain unknown. Here, using biochemical fractionation techniques, we observe cholesterol accumulation at the MAM which leads to ACAT1/SOAT1 enrichment in this domain. MAM proteomics data suggests that ACAT1/SOAT1 inhibition strengthens the ER-mitochondria connection. Confocal and electron microscopy confirms that ACAT1/SOAT1 inhibition increases the number of ER-mitochondria contact sites and strengthens this connection by shortening the distance between these two organelles. This work demonstrates how directly manipulating local cholesterol levels at the MAM can alter inter-organellar contact sites and suggests that cholesterol buildup at the MAM is the impetus behind the therapeutic benefits of ACAT1/SOAT1 inhibition.

SUBMITTER: Harned TC

PROVIDER: S-EPMC10059652 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Acute ACAT1/SOAT1 Blockade Increases MAM Cholesterol and Strengthens ER-Mitochondria Connectivity.

Harned Taylor C TC Stan Radu V RV Cao Ze Z Chakrabarti Rajarshi R Higgs Henry N HN Chang Catherine C Y CCY Chang Ta Yuan TY

International journal of molecular sciences 20230314 6

Cholesterol is a key component of all mammalian cell membranes. Disruptions in cholesterol metabolism have been observed in the context of various diseases, including neurodegenerative disorders such as Alzheimer's disease (AD). The genetic and pharmacological blockade of acyl-CoA:cholesterol acyltransferase 1/sterol O-acyltransferase 1 (ACAT1/SOAT1), a cholesterol storage enzyme found on the endoplasmic reticulum (ER) and enriched at the mitochondria-associated ER membrane (MAM), has been shown ...[more]

PMID: 36982602

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Project description:Background: Control of ER-mitochondrial Ca2+ fluxes is a critical checkpoint to determine cell fate under stress. The 75-kDa glucose-regulated protein (GRP75) is a key tether protein facilitating mitochondria-associated ER membrane (MAM) formation through the IP3R-GRP75-VDAC1 complex. Although GRP75 contributes to cisplatin (CP)-resistance of ovarian cancer (OC), the underlying mechanisms are not clear. Methods: CP-resistant and -sensitive OC cell lines with GRP75 stable modulation were established. Confocal, PLA, co-IP, and TEM analysis were utilized to detect MAM integrity. Live cell Ca2+ imaging, intracellular ATP, ROS, and NAD+ assays were utilized to investigate ER-to-mitochondrial Ca2+ transfer and mitochondrial bioenergetics. Western blot, flow cytometry, CCK-8, Δψm, and mPTP assays were utilized to examine apoptotic cell death. Bioinformatics, patient's specimens, and immunohistochemistry were conducted to obtain the clinical relevance for GRP75-facilitated MAM formation. Results: GRP75-faciliated MAM formation was enriched in CP-resistant OC cells. CP-exposure only increased MAM formation in CP-sensitive OC cells, and enrichment of GRP75 and VDAC1 at MAMs is indispensable to CP-resistance. Diminishing MAM integrity by GRP75-deficiency reduced ER-to-mitochondria Ca2+ transfer, accelerated CP-induced mitochondrial dysfunction, provoked catastrophic ROS, and enhanced CP-triggered apoptotic cell death in OC cells. Clinical investigations confirmed the enrichment of GRP75-faciliated MAM formation in relapsed OC patients, and such enrichment was associated with the CP-resistance phenotype. Conclusion: GRP75-overexpression confers CP-resistance by distinctively managing MAM-facilitated Ca2+ fluxes and the pro-survival ROS signal, whereas GRP75-deficiency induces cell death via bioenergetic crisis and apoptotic ROS accumulation in OC cells. Our results show that GRP75-faciliated MAM formation is a potential target to overcome CP-resistance of OC.

Dataset Information

Acute ACAT1/SOAT1 Blockade Increases MAM Cholesterol and Strengthens ER-Mitochondria Connectivity.

Publications

Acute ACAT1/SOAT1 Blockade Increases MAM Cholesterol and Strengthens ER-Mitochondria Connectivity.

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