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. 2024 Apr 25;25(9):4655.
doi: 10.3390/ijms25094655.

Peptides Targeting the IF1-ATP Synthase Complex Modulate the Permeability Transition Pore in Cancer HeLa Cells

Martina Grandi  1 , Simone Fabbian  2 , Giancarlo Solaini  1 , Alessandra Baracca  1 , Massimo Bellanda  2   3 , Valentina Giorgio  1
Affiliations

Peptides Targeting the IF1-ATP Synthase Complex Modulate the Permeability Transition Pore in Cancer HeLa Cells

Martina Grandi et al. Int J Mol Sci. .

Abstract

The mitochondrial protein IF1 is upregulated in many tumors and acts as a pro-oncogenic protein through its interaction with the ATP synthase and the inhibition of apoptosis. We have recently characterized the molecular nature of the IF1-Oligomycin Sensitivity Conferring Protein (OSCP) subunit interaction; however, it remains to be determined whether this interaction could be targeted for novel anti-cancer therapeutic intervention. We generated mitochondria-targeting peptides to displace IF1 from the OSCP interaction. The use of one selective peptide led to displacement of the inhibitor IF1 from ATP synthase, as shown by immunoprecipitation. NMR spectroscopy analysis, aimed at clarifying whether these peptides were able to directly bind to the OSCP protein, identified a second peptide which showed affinity for the N-terminal region of this subunit overlapping the IF1 binding region. In situ treatment with the membrane-permeable derivatives of these peptides in HeLa cells, that are silenced for the IF1 inhibitor protein, showed significant inhibition in mitochondrial permeability transition and no effects on mitochondrial respiration. These peptides mimic the effects of the IF1 inhibitor protein in cancer HeLa cells and confirm that the IF1-OSCP interaction inhibits apoptosis. A third peptide was identified which counteracts the anti-apoptotic role of IF1, showing that OSCP is a promising target for anti-cancer therapies.

Keywords: ATP synthase; cancer; inhibitor protein IF1; mitochondria; permeability transition pore; therapy.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Amino acid sequences of mitochondria-targeting peptides and predicted binding sites on IF1 or OSCP proteins. In (A), a cartoon representation of the C-terminal coiled-coil domain from bovine IF1 (PDB code 1HF9) showing the region targeted by peptide IF1-O.1, colored in magenta. In (B), a cartoon representation of the OSCP subunit from the cryo-EM structure of Homo Sapiens ATP synthase (PDB code 8H9V) in state 3 conformation of the rotary catalysis showing the regions targeted by the peptide IF1-O.2 and peptide IF1-O.3, colored in orange and green, respectively. In (C), multiple alignments are shown of the human amino acid sequences (harboring the MTS) for (i) IF1 (residues 51–106) and (ii) OSCP subunit (residues 26–75 and 181–213) with their equivalent sequences from ox, pig, mouse and rat. The predicted sequences that are targeted by peptide IF1-O.1, peptide IF1-O.2 and peptide IF1-O.3 are shown in magenta, orange and green, respectively. The UniProt accession numbers for proteins from Bos taurus, Sus scrofa, Homo sapiens, Mus musculus, Rattus norvegicus are shown on the left of each sequence. An asterisk (*) indicates positions which have a single, fully conserved residue; a colon (:) indicates conservation between amino acid groups or similar properties.
Figure 2
Figure 2
Peptide IF1-O.1 displaces IF1 from ATP synthase and IF1-O.2 interacts with the IF1 binding site on the OSCP subunit. In (A), mitochondria isolated from HeLa wild-type cells are incubated in a buffer promoting state 3 respiration in the presence or absence of 30 μM TAT-IF1-O.1, TAT-IF1-O.2 and TAT-IF1-O.3 peptides and are solubilized in a 1% (w/v) digitonin-containing buffer. Mitochondrial extracts are subjected to immunoprecipitation of ATP synthase. Western blotting shows (i) β subunit and IF1 protein in the immunoprecipitated fractions. In (ii), the mean of IF1/β pixel ratio is shown (expressed as % of control). Data are mean ± SEM of three independent experiments. In (B), 1H-15N SOFAST HMQC spectra of OSCP-NT (residues R6-G114) in the absence (red) and in presence (black) of a 10-fold molar excess of peptide IF1-O.2. The expanded plots show a region of the spectrum containing some of the residues with a pronounced chemical shift perturbation, which are labeled.
Figure 3
Figure 3
The OSCP amino acidic regions that are shifted upon the IF1-O.2 peptide- or IF1–OSCP interaction and their localization on the human ATP synthase. In (A), comparison between chemical shift perturbation (Δδ) calculated from 1H-15N SOFAST HMQC spectra of OSCP-NT (residues R6-G114) in the absence or presence of 10-fold molar excess of peptide IF1-O.2 or 20-fold molar excess of unlabeled IF1-NT (residues G1-E40). Blue, yellow, red and pink bars indicate residues that belong, respectively, to helix 1 (H1), helix 4 (H4), both helix 5 and the loop region between helices 5 and 6 (H5–H6), and helix 6 (H6), showing a deviation larger than one standard deviation (σ, dashed line). Residues with Δδ larger than two standard deviations are above the continuous line. In (B), a schematic representation of OSCP-NT secondary structures is reported. In (C), a surface representation of OSCP-NT from the cryo-EM structure of Homo Sapiens ATP synthase in state 3 (PDB code 8H9V), (i,iii) and in state 1 (PDB code 8H9S), (ii,iv) each showing two different orientations. The OSCP subunit is colored in black. Residues of OSCP-NT affected by the interaction with IF1-O.2 (with Δδ > σ) are colored as described above. Only residues accessible on the surface of the protein are visible.
Figure 4
Figure 4
Knocking down the IF1 inhibitor protein does not alter respiration nor OXPHOS complex levels in mitochondria. In (A), Western blotting shows β subunit and IF1 protein levels in both control (plko) and IF1 KD HeLa cell lysates (i) and the mean of the IF1/β subunit pixel ratio (ii), (expressed as % of control). Data are mean ± SEM of four independent experiments. p value is **** p < 0.0001; Student’s t test. In (B), representative oxygen consumption rate (OCR) traces of adherent control and IF1 KD HeLa cells in situ. OCR is measured before (basal) and after treatment with oligomycin (oligo), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), rotenone (Rot) and antimycin A (AA). In (C), Western blotting (i) is shown of the indicated OXPHOS complexes, GAPDH (glyceraldehyde dehydrogenase as loading control) in control and IF1 KD HeLa cells. The molecular marker is indicated on the left. In (ii), the mean ratio is analyzed of band pixels between each complex subunit and GAPDH (mean ± SEM of four independent experiments).
Figure 5
Figure 5
Effects of peptides IF1-O.1, IF1-O.2 and IF1-O.3 on mitochondrial respiration. Traces are shown of the oxygen consumption rate (OCR) by adherent plko (i) and IF1 KD (ii) HeLa cells, treated with or without 30 μM of membrane-permeable peptides. OCR is measured before (basal) and after treatment with oligomycin (oligo), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), rotenone (Rot) and antimycin A (AA). In (A), TAT-IF1-O.1; in (B), TAT-IF1-O.2 and in (C), TAT-IF1-O.3 are used for a 30 min treatment before measurements. Data are mean ± SEM of three independent experiments.
Figure 6
Figure 6
Effects of peptides IF1-O.1, IF1-O.2 and IF1-O.3 on mitochondrial permeability transition pore opening. Ca2+ retention capacity (CRC) is assessed in permeabilized control (plko) and IF1 KD HeLa cells in an ADP-regenerating buffer containing respiratory substrates and the membrane-impermeable Ca2+ sensor, Ca2+ Green-5N. Ca2+ Green-5N fluorescence was monitored following repeated additions of 5 μM Ca2+ pulses (bottom arrows). A return of Ca2+ Green-5N fluorescence to baseline reflects an uptake of Ca2+ by mitochondria, whereas a sudden increase in fluorescence is indicative of PTP opening. In ((A(i)), one representative experiment is shown out of 4 for plko and IF1 KD HeLa cells. Ca2+ Green-5N fluorescence baselines are graphically shifted upward to avoid trace overlap. The histogram (A(ii)) represents nmols of Ca2+ per μg of protein retained by plko and IF1 KD cells. Data represent the mean ± SEM (4 independent experiments), the p value is * p = 0.018, Student’s t test. In (B–D), CRC is assessed as above in plko and IF1 KD permeabilized HeLa cells in the presence of 0–50 μM of the indicated membrane-permeable peptides (TAT-IF1-O.1; TAT-IF1-O.2 and TAT-IF1-O.3). Data are mean (expressed as % of controls) ± SEM of 3 independent experiments. p values are * p ≤ 0.05, ** p ≤ 0.01, *** p = 0.0001; two-way ANOVA.
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