Deterioration of liver fibrosis by ALR deficiency is associated with reducing MICU1/2 heterodimerization and mitochondrial Ca2+ imbalance in hepatic stellate cells

Abstract

Hepatic stellate cells (HSCs) activation is a pivotal event in the pathogenesis of liver fibrosis (LF). Mitochondrial calcium (mCa²⁺) dyshomeostasis is a known driver for HSCs activation. Although we previously demonstrated that augmenter of liver regeneration (ALR) inhibits HSCs activation potentially through regulation of mitochondrial calcium uniporter (MCU) and prevention of mCa²⁺ overload, the precise molecular mechanisms remain poorly understood. In this study we revealed that hepatic ALR expression was continuously reduced with LF progression in both patients and mice, which coincided with a loss of MICU1/2 dimerization. Similarly, Alr deletion (Alr-KO) could exacerbate LF progression in mice fed a choline-deficient high-fat diet, subjected to bile duct ligation or developing spontaneous LF. In vitro studies showed that Alr-KO disrupted the dimerization of mitochondrial calcium uptake 1 and 2 (MICU1/2), core regulatory components of MCU complex, deteriorating mCa²⁺ overload and promoting HSCs activation. Protein-binding assays revealed that the regulation of MICU1/2 dimerization by ALR appeared indirect; instead, it potentially relied on coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4), a member of the mitochondrial disulfide relay system (DRS). Further data analyses demonstrated that ALR interacted with CHCHD4 at cysteine residues C4, C53 and C55 via modulating its redox status. Alr knockdown or mutation of these cysteine residues in CHCHD4 restrained ALR-CHCHD4 interaction, reduced dimerized MICU1/2 levels, and consequently led to mCa²⁺ overload and HSCs activation. Overall, this study provides mechanistic insights into how ALR attenuates liver fibrosis by promoting MICU1/2 dimerization and maintaining mCa²⁺ homeostasis in HSCs.

Keywords: Augmenter of liver regeneration; Coiled-coil-helix-coiled-coil-helix domain-containing protein 4; Hepatic stellate cells; Liver fibrosis; Mitochondrial calcium.