蛋白酶体
亚细胞定位
突变体
突变蛋白
化学
蛋白质降解
突变
细胞生物学
体内
野生型
体外
丙氨酸
线粒体
赖氨酸
生物化学
分子生物学
细胞质
生物
基因
氨基酸
遗传学
作者
Tanmoy Dutta,Kavitha Sasidharan,Ester Ciociola,Grazia Pennisi,Francesca Rita Noto,Lohitesh Kovooru,Tobias Kroon,Anna Lindblom,Yiqin Du,Mohammad Pirmoradian,Simonetta Wallin,Rosellina Margherita Mancina,Daniel Lindén,Stefano Romeo
摘要
Abstract 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.
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