Acute ACAT1/SOAT1 blockade increases MAM cholesterol and strengthens ER-mitochondria connectivity
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Eclipse
ORGANISM(S): Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
Ta-Yuan Chang
PROVIDER: MSV000091335 | MassIVE | Tue Feb 21 11:06:00 GMT 2023
SECONDARY ACCESSION(S): PXD040312
REPOSITORIES: MassIVE
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Project description: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.
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1Department of Physiology & 3Department of Neurology, Northwestern University Feinberg School of Medicine; 2Departments of Neurology and Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 & 4Department of Neurobiology, Weinberg College of Arts and Sciences, Northwestern University, Chicago, IL 60611
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Project description:In this work, we have explored the subcellular localization of Bcl2, a major antiapoptotic protein. In U251 glioma cells, we found that Bcl2 is localized mainly in the ER and is translocated to MAM and mitochondria upon induction of apoptosis; this mitochondrial transfer was not restricted to the demonstrator cell line, even if cell-specific modulations exist. We found that the Bcl2/mitochondria interaction is controlled by TOM20, a protein that belongs to the protein import machinery of the mitochondrial outer membrane. The expression of a small domain of interaction of TOM20 with Bcl2 potentiates its anti-apoptotic properties, which suggests that the Bcl2-TOM20 interaction is proapoptotic. The role of MAM and TOM20 in Bcl2 apoptotic mitochondrial localization and function has been confirmed in a yeast model in which the ER-mitochondria encounter structure (ERMES) complex (required for MAM stability in yeast) has been disrupted. Bcl2-TOM20 interaction is thus an additional player in the control of apoptosis.
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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.
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Project description:The mitochondria-associated ER membrane (MAM) plays a critical role in cellular energetics and calcium homeostasis; however, how MAM is affected under diabetic condition remains elusive. This study presented a comprehensive proteome profiling of isolated brain MAM from long-term type 2 diabetic mice vs. non-diabetic controls. MAM protein was extracted efficiently by a surfactant-aided precipitation/on-pellet digestion (SOD) method, and MAM proteome was quantified by an ion-current-based MS1 method combined with nanoLC-MS/MS. A total of 1,313 non-redundant proteins of MAM were identified, among which 144 proteins were found significantly altered by diabetes. In-depth IPA analysis identified multiple disease-relevant signaling pathways associated with the MAM proteome changes in diabetes, most significantly the unfolded protein response (UPR), p53, hypoxia-related transcription factors, and methyl CpG binding protein 2. Using immunofluorescence labeling we confirmed the activation of three UPR branches and increased ERp29 and calreticulin in diabetic retinas. Moreover, we found GRP75, a key MAM tethering protein, was drastically reduced by long-term diabetes. In vitro, acute high glucose treatment reduces ER-mitochondrial contact in retinal endothelial cells. This study provides first insight into the significant alterations in MAM proteome associated with activation of the UPR in diabetes, which may serve as novel benchmarks for the future studies of diabetic complications.
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Project description:Abnormal tau protein impairs mitochondrial function, including transport, dynamics, and bioenergetics. Mitochondria interact with the endoplasmic reticulum (ER) via mitochondria-associated ER membranes (MAMs), which coordinate and modulate many cellular functions, including mitochondrial cholesterol metabolism. Here, we show that abnormal tau loosens the association between the ER and mitochondria in vivo and in vitro. Especially, ER-mitochondria interactions via vesicle-associated membrane protein-associated protein (VAPB)-protein tyrosine phosphatase-interacting protein 51 (PTPIP51) are decreased in the presence of abnormal tau. Disruption of MAMs in cells with abnormal tau alters the levels of mitochondrial cholesterol and pregnenolone, indicating that conversion of cholesterol into pregnenolone is impaired. Opposite effects are observed in the absence of tau. Besides, targeted metabolomics reveals overall alterations in cholesterol-related metabolites by tau. The inhibition of GSK3β decreases abnormal tau hyperphosphorylation and increases VAPB-PTPIP51 interactions, restoring mitochondrial cholesterol and pregnenolone levels. This study is the first to highlight a link between tau-induced impairments in the ER-mitochondria interaction and cholesterol metabolism.
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