Characterization of a new mutation of mitochondrial ND6 gene in hepatocellular carcinoma and its effects on respiratory complex I

Abstract

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, which often arises from previous liver pathologies such as HBV, HCV, and alcohol abuse. It is typically associated with an enlarged cirrhotic organ. In this study, we analyzed tumor and distal tissues from a patient who underwent liver resection for HCC with no previous pathologies and whose liver showed normal function without signs of cirrhosis. Genetic analysis of mitochondrial DNA (mtDNA) revealed a novel variant of the gene encoding the NADH dehydrogenase subunit 6 (ND6) protein in the tumor tissue. The deletion of a thymidine generated an early stop codon, resulting in a truncated form of the protein (ΔND6) with 50% of the C-terminal primary sequence missing. ND6 is a subunit of the NADH dehydrogenase complex, also known as Complex I, the largest complex in the electron transport chain. Previous studies have linked mtDNA Complex I mutations to mitochondrial disorders and cancer. Through biochemical analyses, we characterized this new mutation and showed that the expression of ΔND6 negatively affects the stability and functionality of Complex I. Data were confirmed by molecular dynamics simulations suggesting conformational rearrangements, overall revealing a leading role of ND6 in the assembly of Complex I.

Keywords: Hepatocellular carcinoma; Mitochondria; Mitochondrial DNA; Molecular dynamics simulations.; ND6 gene mutation; Respiratory complex I assembly.

Fig. 1

Assembly and activity of Complex I are reduced in tumor tissue of case-study patients expressing ΔND6 form. (A) Western blot analyses of 40 µg of the distal (Dist.) and tumoral (Tum.) isolated mitochondria. ND6 protein was detected using two different anti-ND6 antibodies: one recognizing an epitope located in the N-terminal part of the protein (ND6 N-term), which detects both the wild-type (ND6) (black arrow) and the truncated (ΔND6) forms (red arrow). A second antibody recognizes an epitope at the C-terminus of the protein (ND6 C-term), which is not present in the truncated form. NDUFS1 and ND5 antibodies were used as loading controls. (B) Representative images of BN-PAGE gel followed by Coomassie staining (left) and Western blot (right) with anti-NDUFS1 antibody of the distal and tumoral liver-isolated mitochondria of the case-study patient (Pt_ΔND6) and a control patient (Pt_ND6-WT) without ND6 mutation. Expression of ΔND6 resulted in a significant reduction of Complex I in tumor mitochondria. In the graph is reported the BN-PAGE densitometric analysis of Complex I (mtCx-I) from three independent replicates (***: p ≤ 0.001). (C) Representative image of Complex I in-gel activity assay of the distal and tumoral liver-isolated mitochondria of the case-study patient (Pt_ΔND6) and a control patient (Pt_ND6-WT) without ND6 mutation. In the graph is reported the densitometric analysis of mtCx-I activity from three independent replicates (**: p ≤ 0.01).

Fig. 2

Molecular dynamics simulations of ND6-WT and ΔND6 reveal conformational changes upon loss of the C-terminal region. (A) Ribbon representations of the ND6-WT (green), ΔND6 (blue), and the missing C-terminal fragment (violet). (B) Comparison of the Residual Mean Square Fluctuation (RMSF) indicates increased movement in the N-terminal region of ΔND6 compared to WT. (C) Solvent Accessible Surface Area (SASA) analysis reveals that the N-terminal region of ΔND6 adopts a more compact conformation than ND6-WT. (D) Analysis of native contacts in ΔND6 shows that only a quarter of the original ND6-WT molecular contacts are present; however, the retained native contacts remain stable. Overall, the MDS suggests that ΔND6 undergoes conformational rearrangements, resulting in a relatively stable, non-native structure that resists unfolding and degradation.

Fig. 3

mtDNA-encoded Complex I proteins are overexpressed but not efficiently assembled in tumor mitochondria. (A) Representative Western blot analysis of 40 µg of mitochondrial extract from distal and tumor tissues separated on SDS-PAGE. The expression of nuclear DNA (nDNA)-encoded proteins reduces the mitochondrial DNA-encoded proteins that are upregulated in tumor mitochondria. In the graph are reported the means and standard deviations of three independent replicates expressed as relative to distal tissue (*** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05). (B) Normalized amounts of BN-PAGE resolved Complex I were isolated and separated on SDS-PAGE to evaluate the levels of mtDNA-encoded proteins for both Pt_ND6-WT and Pt_ΔND6.

Fig. 4

Patient ΔND6 developed HCC in the right liver lobe without any evident triggering factor. The mtDNA genetic analysis revealed a mutation on the ND6 gene present only in the tumor with a heteroplasmy level of around 70%. The m.14,423 > - mutation leads to an early stop codon and, consequently, to a truncated form of the protein, which loses the C-terminal alpha helices (green). The presence of a truncated form of ND6 negatively affects Complex I assembly, decreasing the levels of the holo-complex and its activity.