AAA+ RuvBL1 charges liver metabolism: from mitochondria to circadian rhythm.

Hepatocellular carcinoma (HCC) is currently a major challenge in medicine for its poor prognosis and lack of effective treatments. The RuvBL1 is a AAA+ ATPase whose expression correlates with a worse prognosis in HCC patients, however its mechanistic role in HCC is still unknown. It associates with several multiprotein complexes regulating essential biological processes such as cell proliferation, chromatin remodeling, DNA repair and mTOR pathway activity. In a previous work of our group, RuvBL1 liver specific haploinsufficient mice revealed an impairment of liver metabolism, mainly of insulin signalling and glucose homeostasis. Furthermore, metabolism and circadian rhythms were amongst the most significantly altered biological functions in heterozygous mice. Therefore, we aimed to dissect the mechanistic role of RuvBL1 in the regulation of liver metabolism, focusing on mitochondrial biology and core clock gene regulation. Untargeted GC/MS metabolome analysis of RuvBL1-silenced Huh7 cells highlighted a change in intermediates of TCA and amino acids metabolism and a significant association of modulated metabolites with cancer metabolic pathways. Seahorse functional analysis revealed that targeting RuvBL1 by either silencing or by its specific ATPase inhibitor CB-6644 reduced mitochondrial respiration and ATP production by OXPHOS in normal (primary isolated hepatocytes, AML-12) or tumoral cells (Hepa 1-6, HepG2, Hep3B, Huh7). Moreover, treatment with CB-6644 caused structural alterations of mitochondria both in primary hepatocytes and cultured cell lines; moreover; the analysis of intact RuvBL1hep-/- liver samples by TEM revealed a reduced number of mitochondria and ultrastructural defects such as the absence of cristae. Then, by super-resolution STED microscopy we detected RuvBL1 in the mitochondria of liver cell lines, which was confirmed by immunogold labelling and electron microscopy also in mouse liver samples. By 2D-DIGE and IP/MS analysis, we found RuvBL1 physically interacting with key proteins involved in ETC, energy metabolism, mito-proteome homeostasis and stress response. In-silico correlative analysis found out an enrichment of mitochondria-related terms in HCC patients with high RUVBL1 expression and a positive correlation between RUVBL1 and several molecular chaperones, including the master regulator of stress response HSF1. Thus, we explored the RuvBL1-HSF1 axis identifying RuvBL1 as a modulator of the transcriptional activation of HSF1 in stressful conditions, suggesting that this ATPase is recruited at HSE response element of HSF1 target genes. We then addressed the impact of RuvBL1 in the regulation of the liver circadian clock. qPCR analysis showed a clear derangement in the circadian expression of metabolic and core clock genes in RuvBL1hep+/-mice. NGS analysis of the hepatic circadian transcriptome revealed that genes with a significantly different oscillatory expression are majorly involved in mitochondria organization and function, amino acid metabolism, protein folding and stress response. Mechanistically, by promoter reporter experiments we identified ARNTL (BMAL), CRY2 and DBP as the most consistently RuvBL1-modulated genes in AML-12 and Huh7 cell lines, with CRY2 and ARNTL being strongly induced by RuvBL1 inhibition. Interestingly, CRY2 showed the higher inverse correlation with RUVBL1 expression in the TCGA-LIHC cohort. We assumed that stress response orchestrated by the HSF1-RUVBL1 axis could modulate the liver circadian rhythm. Indeed, we showed that inhibition of RuvBL1 altered the rhythmic response of clock genes in heat shocked Huh7, AML-12 and Hepa 1-6 cells. In conclusion our work unravels the role of RuvBL1 in the regulation of HCC cell metabolism and identified previously unrecognized roles of this ATPase at the crossroad of mitochondria biology, stress response and liver circadian rhythms.