Variants in Human ATP Synthase Mitochondrial Genes: Biochemical Dysfunctions, Associated Diseases, and Therapies

Abstract

Mitochondrial ATP synthase (Complex V) catalyzes the last step of oxidative phosphorylation and provides most of the energy (ATP) required by human cells. The mitochondrial genes MT-ATP6 and MT-ATP8 encode two subunits of the multi-subunit Complex V. Since the discovery of the first MT-ATP6 variant in the year 1990 as the cause of Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) syndrome, a large and continuously increasing number of inborn variants in the MT-ATP6 and MT-ATP8 genes have been identified as pathogenic. Variants in these genes correlate with various clinical phenotypes, which include several neurodegenerative and multisystemic disorders. In the present review, we report the pathogenic variants in mitochondrial ATP synthase genes and highlight the molecular mechanisms underlying ATP synthase deficiency that promote biochemical dysfunctions. We discuss the possible structural changes induced by the most common variants found in patients by considering the recent cryo-electron microscopy structure of human ATP synthase. Finally, we provide the state-of-the-art of all therapeutic proposals reported in the literature, including drug interventions targeting mitochondrial dysfunctions, allotopic gene expression- and nuclease-based strategies, and discuss their potential translation into clinical trials.

Keywords: ATP synthase; ATP6; ATP8; F1Fo-ATPase modeling; mitochondria; mt-DNA; mutations; therapy.

Figure 1

(A) Organization of the human mitochondrial genome. These include the non-coding control region (D-loop) (purple); 37 genes encoding 2 rRNAs (yellow); 22 tRNAs (grey); and the 13 polypeptides belonging to CI (light blue), CIII (orange), CIV (green), and CV (red and pink). An enlargement of MT-ATP6/MT-ATP8 genes is shown, highlighting the sequence overlap (violet). Multiple alignments of ATP8 (B) and ATP6 (C) from a selection of mammalian species: Homo sapiens, Bos taurus, and Ovis aries. Conserved amino acids are in blue, and, at the bottom, transmembrane α-helices are indicated with pink lines according the PROMOTIF analysis performed on the human PDB structure. The three amino acid residues that are more frequently mutated in patients and are studied in detail in this review are highlighted in pink, whereas other MT-ATP6/MT-ATP8 variants that are also shown in Table 1 are in bold and indicated by the upper red lines.

Figure 2

(A) Schematic representation of the monomeric mammalian ATP synthase, according to [16,17]. (B) Structure of human ATP synthase in state 1 (PDB id: 8H9S) ([16]) bound to the inhibitor protein IF₁. The α- and β-subunits of the F₁-catalytic domain are in different shades of red and yellow with a different labelling if the subunit is empty (αE, βE) or bound to ADP (αDP, βDP) or ATP (αTP, βTP). The γ-, δ-, and ε-subunits of the F₁-catalytic domain are in sienna brown, sandy brown, and tan, respectively. The central stalk formed by subunits γ, δ, and ε is in contact with the c8-ring (different shades of light blue) that is part of the membrane domain and in contact with subunit a (or ATP6, olive drab). The peripheral stalk subunits OSCP, b, d, and F6 are in dim gray, violet, violet red, and black, respectively, and the A6L subunit (or ATP8) is in coral. The e, f, and g subunits in the membrane domain are forest green, pale green, and olive, respectively. The 6.8 kDa proteolipid (6.8PL) is in green yellow, and the IF₁ inhibitor is in purple. The DAPIT subunit in yellow is not reported in (B) because it is not present in the cryo-EM structure in [16].

Figure 3

Detail of the region comprising Leu156 of ATP6 in the human structure of ATP synthase (state 1). (A,B) The native ATP6 and c subunits are reported in ribbons colored as in Figure 2. Residues labels are colored as the corresponding subunits. Leu156 is in ball-and-stick, while other residues in the vicinity of Leu156 or proposed to be part of the proton translocation process are in stick. The side chains are colored according to the atom type. The interaction between Arg159 in ATP6 and Glu58 in c₈-ring is shown. The orientation of panel (B) is clockwise rotated by 90° around the vertical axis with respect to the orientation in panel (A). Panels (C,D) reports the model structures of the p.Leu156Arg and p.Leu156Pro variants, respectively (see Appendix A for details).

Figure 4

Detail of the region comprising Leu217 from ATP6 in the human structure of ATP synthase in state 1 (A), state 2 and 3a (B), and 3b (C). The native ATP6 and c subunits are reported in ribbons colored as in Figure 2. Residue labels are colored as the corresponding subunits. Leu217 is shown as ball-and-stick, while other residues in the vicinity of Leu217 or proposed to be part of the proton translocation process are shown as a stick. The side chains are colored according to the atom type. In the left panels, the wild-type protein is reported, while the models of Leu217Arg and Leu217Pro variants are reported in the central and right panels, respectively (see Appendix A for details). H-bonds are indicated using dashed red lines.

Figure 5

Detail of the region comprising Leu220 from ATP6 in the human structure of ATP synthase in states 1 and 3b (A) and states 2 and 3a (B). The native ATP6 and c subunits are reported in ribbons colored as in Figure 2. Residue labels are colored as the corresponding subunits. Leu220 is shown as ball-and-stick, while other residues in the vicinity of Leu220 or proposed to be part of the proton translocation process are shown as a stick. The side chains are colored according to the atom type. In the left panels, the wild-type protein is reported, while the models of Leu220Pro variants are in the right panels (see Appendix A for details).

Figure 6

Overview of the therapeutic options proposed for the treatment of pathologies associated with MT-ATP6/MT-ATP8 variants. (A) Drugs (blue square) targeting mitochondrial dysfunctions in mutated cells: compounds that limit an abnormally increased process (red square) or that boost a pathological reduced pathway (green square). Black arrows indicate positive regulation, while red arrows indicate negative regulation. Antioxidants or Avanafil have been reported to reduce high ROS production and mitochondrial membrane potential (Δψm), respectively. Supplementation of α-ketoglutarate and aspartate (α-KG/Asp) contributes to increasing ATP levels through reactions of the tricarboxylic acid (TCA) cycle. The limitation of ATP deficit is induced by Epicatechin or by Rapamycin. Epicatechin, by blocking the ATP hydrolytic activity of CV, increases cellular energy availability, whereas Rapamycin, by reducing the enhanced activity of mTORC1, limits the related energy-consuming processes. (B) Allotopic expression is aimed at replacing the mutant protein (ATP6 or ATP8) with the wild-type counterpart, restoring the ATP synthase function. (C) A third approach uses nucleases (black scissors) that selectively cleave the mutant mt-DNA molecules (red chains), inducing their degradation. The consequent replication of the wild-type mitochondrial genome (green chains) to maintain the copy number will increase its percentage and lead to a shift of mt-DNA heteroplasmy.