MCJ (Methylation-Controlled J protein), an endogenous repressor of the mitochondrial respiratory chain, is upregulated in multiple liver diseases but little is known about how it is regulated. S-adenosylmethionine (SAMe), the biological methyl donor, is frequently depleted in chronic liver diseases. Here, we show that SAMe negatively regulates MCJ in the liver. While deficiency in methionine adenosyltransferase alpha 1 (MATα1), enzyme that catalyzes SAMe biosynthesis, leads to hepatic MCJ upregulation, MAT1A overexpression and SAMe treatment reduced MCJ expression. We found that MCJ is methylated at lysine residues and that it interacts with MATα1 in liver mitochondria, likely to facilitate its methylation. Lastly, we observed that MCJ is upregulated in alcohol-associated liver disease, a condition characterized by reduced MAT1A expression and SAMe levels along with mitochondrial injury. MCJ silencing protected against alcohol-induced mitochondrial dysfunction and lipid accumulation. Our study demonstrates a new role of MATα1 and SAMe in reducing hepatic MCJ expression.

(DDA-MS) Data-dependent acquisitions and data-independent acquisition mass spectrometry (DIA-MS) acquisitions were performed on an Orbitrap LUMOS Fusion mass spectrometer equipped with an EasySpray ion source and connected to an Ultimate 3000 nano LC system with a 60-min gradient. Peptides were loaded onto a PepMap RSLC C18 column (2 µm, 100 Å, 150 µm i.d. × 15 cm, Thermo) using a flow rate of 1.4 µL/min for 7 min at 1%B (mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile) after which point, they were separated with a linear gradient of 5–20%B for 45 min, 20–35%B for 15 min, 35–85%B for 3 min, holding at 85%B for 5 min, and re-equilibrating at 1%B for 5 min. Each sample was followed by a blank injection to both clean the column and re-equilibrate at 1%B. The nano-source capillary temperature was set to 300 °C and the spray voltage was set to 1.8 kV. For DIA analysis, MS1 scans were acquired in the Orbitrap at a resolution of 60,000 Hz from mass range 400–1000 m/z. For MS1 scans the AGC target was set to 3 × 105 ions with a max fill time of 50 ms. DIA MS2 scans were acquired in the Orbitrap at a resolution of 15,000 Hz with fragmentation in the HCD cell at a normalized CE of 30. The MS2 AGC was set to 5e4 target ions and a max fill time of 22 ms. DIA was performed using 4 Da (150 scan events) windows over the precursor mass range of 400–1000 m/z and the MS2 mass range was set from 100 to 1500 m/z. For DDA analysis MS1 scans the AGC target was set to 4 × 105 ions with a max fill time of 50 ms. MS2 spectra were acquired using the TopSpeed method with a total cycle time of 3 s and an AGC target of 5 × 104 and a max fill time of 22 ms, and an isolation width of 1.6 Da in the quadrapole. Precursor ions were fragmented using HCD with a normalized collision energy of 30% and analyzed in the Orbitrap at 15,000 K resolution. Monoisotopic precursor selection was enabled and only MS1 signals exceeding 50,000 counts triggered the MS2 scans, with +1 and unassigned charge states not being selected for MS2 analysis. Dynamic exclusion was enabled with a repeat count of 1 and exclusion duration of 15 s. For MCJ methylation analysis, first, spectra were obtained from DDA-MS acquisitions of a MCJ immunoprecipitation. Then, Raw DIA-MS data was imported into Skyline (Skyline Daily, version 19.1.1.309), which was then used to visualize and manually validate the co-eluting precursor and fragment trace of a peptide containing Lys83 methylation.