Recent genome-wide association studies have identified a missense variant p.A165T in mitochondrial amidoxime-reducing component 1 (mARC1) that is strongly associated with protection from all-cause cirrhosis and improved prognosis in nonalcoholic steatohepatitis. The precise mechanism of this protective effect is unknown. Substitution of alanine 165 with threonine is predicted to affect mARC1 protein stability and to have deleterious effects on its function. To investigate the mechanism, we have generated a knock-in mutant mARC1 A165T and a catalytically dead mutant C273A (as a control) in human hepatoma HepG2 cells, enabling characterization of protein subcellular distribution, stability, and biochemical functions of the mARC1 mutant protein expressed from its endogenous locus. Compared to WT mARC1, we found that the A165T mutant exhibits significant mislocalization outside of its traditional location anchored in the mitochondrial outer membrane and reduces protein stability, resulting in lower basal levels. We evaluated the involvement of the ubiquitin proteasome system in mARC1 A165T degradation and observed increased ubiquitination and faster degradation of the A165T variant. In addition, we have shown that HepG2 cells carrying the MTARC1 p.A165T variant exhibit lower N-reductive activity on exogenously added amidoxime substrates in vitro. The data from these biochemical and functional assays suggest a mechanism by which the MTARC1 p.A165T variant abrogates enzyme function which may contribute to its protective effect in liver disease.

OBJECTIVE: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health concern with no effective and specific drug treatment available. The rs2642438 minor allele in mitochondrial amidoxime-reducing component 1 (MARC1) results in an aminoacidic substitution (p.Ala165Thr) and associates with protection against MASLD. However, the mechanisms behind this protective effect are unknown. In this study, we examined the consequences of this aminoacidic substitution on protein stability and subcellular localization.
METHODS: We overexpressed the human MARC1 A165 (wild-type) or 165T (mutant) in vivo in mice and in vitro in human hepatoma cells (HepG2 and HuH-7), generated several mutants at position 165 by in situ mutagenesis and then examined protein levels. We also generated HepG2 cells stably overexpressing MARC1 A165 or 165T to test the effect of this substitution on MARC1 subcellular localization.
RESULTS: MARC1 165T overexpression resulted in lower protein levels than A165 both in vivo and in vitro. Similarly, any mutant at position 165 showed lower protein levels compared to the wild-type protein. We showed that the 165T mutant protein is polyubiquitinated and its degradation is accelerated through lysine-48 ubiquitin-mediated proteasomal degradation. We also showed that the 165T substitution does not affect the MARC1 subcellular localization.
CONCLUSIONS: This study shows that alanine at position 165 in MARC1 is crucial for protein stability, and the threonine substitution at this position leads to a hypomorphic protein variant due to lower protein levels. Our result supports the notion that lowering hepatic MARC1 protein level may be a successful therapeutic strategy for treating MASLD.

The identification of genetic variants associated with fatty liver disease (FLD) from genome-wide association studies started in 2008 when single nucleotide polymorphisms in PNPLA3, the gene encoding patatin-like phospholipase domain-containing 3, were found to be associated with altered hepatic fat content. Since then, several genetic variants associated with protection from, or an increased risk of, FLD have been identified. The identification of these variants has provided insight into the metabolic pathways that cause FLD and enabled the identification of potential therapeutic targets. In this mini-review, we will examine the therapeutic opportunities derived from genetically validated targets in FLD, including oligonucleotide-based therapies targeting PNPLA3 and HSD17B13 that are currently being evaluated in clinical trials for the treatment of NASH (non-alcoholic steatohepatitis).

A coding variant in MTARC1 (rs2642438; p.Ala165Thr) was recently associated with protection from cirrhosis in European individuals. However, its impact on overall and cause-specific mortality remained elusive.
Using a genome-first approach, we explored a range of metabolic phenotypes and outcomes associated with MTARC1 p.Ala165Thr in the UKBiobank and the Penn-Medicine BioBank.
MTARC1 p.Ala165Thr was significantly associated with higher triglycerides, lower total cholesterol, lower LDL-C, lower ApoB, lower HDL-C, lower ApoA-I and higher IGF-1. Per each minor allele, the risk of NAFLD was reduced by ~15%. The ALT-lowering and NAFLD-protective effect of MTARC1 p.Ala165Thr was amplified by obesity, diabetes mellitus and presence of PNPLA3 rs738409:G. In African-American and Black-British individuals, the allele frequency of MTARC1 p.Ala165Thr was lower, but carriers showed the same distinctive lipid phenotype. Importantly, MTARC1 p.Ala165Thr carriers did not show higher cardiovascular disease burden as evidenced by cardiac MRI and carotid ultrasound. In prospective analyses, the homozygous minor allele was associated with up to 39% lower rates of liver-related mortality, while no risk of increased overall or cardiovascular death could be observed. Strikingly, liver-related mortality was more than 50% reduced in diabetic participants or carriers of PNPLA3 rs738409:G.
Together these data highlight MTARC1 as an important liver disease modifier that influences plasma lipids in an allele-dose-dependent manner without increasing cardiovascular outcomes. Our results point toward potential mechanisms and reveal a remarkable association with liver-related mortality calling for future studies exploring its therapeutic potential.
This study was funded by the German Research Foundation (DFG).