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.
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.
摘要：
目的：代谢功能障碍相关的脂肪变性肝病（MASLD）是一个全球性的健康问题，目前尚无有效的特异性药物治疗方法。线粒体胺肟还原成分1（MARC1）中的rs2642438次要等位基因导致氨基酸取代（p。Ala165Thr）并与MASLD防护相关。然而,这种保护作用背后的机制是未知的。在这项研究中,我们研究了这种氨基酸取代对蛋白质稳定性和亚细胞定位的影响。
方法：我们在小鼠体内和体外在人肝癌细胞（HepG2和HuH-7）中过表达人MARC1A165（野生型）或165T（突变体），通过原位诱变在165位产生了几个突变体，然后检查了蛋白质水平。我们还产生了稳定过表达MARC1A165或165T的HepG2细胞，以测试这种取代对MARC1亚细胞定位的影响。
结果：MARC1165T过表达导致体内和体外的蛋白质水平低于A165。同样，与野生型蛋白相比,165位的任何突变体均显示出更低的蛋白水平.我们表明，165T突变蛋白是多泛素化的，并且通过赖氨酸48泛素介导的蛋白酶体降解加速了其降解。我们还显示165T取代不影响MARC1亚细胞定位。
结论：这项研究表明，在MARC1中165位的丙氨酸对蛋白质的稳定性至关重要，由于蛋白质水平较低，在该位置的苏氨酸取代导致低态蛋白质变体。我们的结果支持以下观点：降低肝脏MARC1蛋白水平可能是治疗MASLD的成功治疗策略。
方法：我们在小鼠体内和体外在人肝癌细胞（HepG2和HuH-7）中过表达人MARC1A165（野生型）或165T（突变体），通过原位诱变在165位产生了几个突变体，然后检查了蛋白质水平。我们还产生了稳定过表达MARC1A165或165T的HepG2细胞，以测试这种取代对MARC1亚细胞定位的影响。
结果：MARC1165T过表达导致体内和体外的蛋白质水平低于A165。同样，与野生型蛋白相比,165位的任何突变体均显示出更低的蛋白水平.我们表明，165T突变蛋白是多泛素化的，并且通过赖氨酸48泛素介导的蛋白酶体降解加速了其降解。我们还显示165T取代不影响MARC1亚细胞定位。
结论：这项研究表明，在MARC1中165位的丙氨酸对蛋白质的稳定性至关重要，由于蛋白质水平较低，在该位置的苏氨酸取代导致低态蛋白质变体。我们的结果支持以下观点：降低肝脏MARC1蛋白水平可能是治疗MASLD的成功治疗策略。
