Conference Agenda

The pro-oncogenic role of IF₁, the endogenous inhibitor of mitochondrial F₁F_o-ATPase (ATP synthase) has been ascribed to several actions [1]. The present work concerns the possibility that IF₁ can express its pro-oncogenic action by inhibiting the ATP synthesis activity of F₁F_o-ATPase and oxidative phosphorylation, thus challenging the reprogramming of energy metabolism under normoxia proposed by Cuezva and coworkers [2].

We prepared stably IF₁-silenced clones and compared their bioenergetics with that of the three parental IF₁-expressing cancer cell lines (143B, HCT116, HeLa). All functional parameters: respiration rate, ATP synthesis rate (OXPHOS), and mitochondrial membrane potential were similar in IF₁-silenced and control cells. Since Cuezva and coworkers [3] have proposed a possible regulatory mechanism that could hinder the action of IF₁ in cancer cells, we investigated whether the claimed PKA-dependent phosphorylation of a serine in IF₁ prevents its binding and action on the F₁F_o-ATPase. However, we found that, regardless of the presence of IF₁, activation or inhibition of PKA similarly affected OXPHOS, but no effect on ATP synthase activity was observed when either succinate or glutamate/malate fueled respiration [4]. Therefore, this study rules out that IF₁ inhibits the ATP synthase in cancer cells when the enzyme works physiologically, synthesizing ATP.

References

[1] G. Solaini, G. Sgarbi, A. Baracca, The F1Fo-ATPase inhibitor, IF1, is a critical regulator of energy metabolism in cancer cells, Biochem. Soc. Trans. 49 (2021) 815–827.

[2] L. Sánchez-Cenizo, L. Formentini, M. Aldea, A.D. Ortega, et al., Up-regulation of the ATPase inhibitory factor 1 (IF1) of the mitochondrial H+-ATP synthase in human tumors mediates the metabolic shift of Cancer cells to a Warburg phenotype, J Biol Chem 285 (2010) 25308-313.

[3] J. Garcia-Bermudez, M. Sánchez-Aragó, B. Soldevilla, A. Del Arco, et al., PKA phosphorylates the ATPase inhibitory factor 1 and inactivates its capacity to bind and inhibit the mitochondrial H+-ATP synthase, Cell Rep. 12 (2015) 2143–55.

[4] G. Sgarbi, R. Righetti, V. Del Dotto, S. Grillini, et al., The pro-oncogenic protein IF1 does not contribute to the Warburg effect and is not regulated by PKA in cancer cells, BBA - Mol. Basis Dis. 1870 (2024) 166879-89.

Embryos of the brine shrimp Artemia franciscana survive metabolic depression during anoxia and diapause for months to years during which metabolic rates diminish to <1% of the active state [1,2]. While a drop in embryo ATP occurs during diapause, a substantial fraction remains. Proton leak by mitochondria from diapause embryos is not downregulated. Thus, mitochondrial ΔΨ is compromised because respiration of embryos decreases during diapause far below that required to compensate for leak. Under such conditions, the F₁F_o-ATP synthase could reverse and fully deplete cellular ATP. The synthase may be blocked during diapause by the regulatory inhibitor protein IF₁. The F₁F_o ATP synthase from A. franciscana was purified by affinity chromatography and 16 subunits (α, β, γ, b, OSCP, d, a, δ, f, g, e, A6L, F6, DAPIT, ε, c) identified by MALDI and Orbitrap mass spectrometry and comparison to A. franciscana genomic sequences. Interaction of A. franciscana F₁F_o with the bovine monomeric fragment I(1-60)His was characterized at multiple molar ratios of I(1-60)His:F₁F_o. Bovine I(1-60)His inhibits purified A. franciscana F₁F_o, and inhibition strongly increases as pH is lowered across the range experienced during metabolic arrest [values for K_i (µM^-1) x 10^-2 are 22.0 (pH 7.9), 11.9 (pH 7.1), and 1.65 (pH 6.3)]. In contrast, I(1-60)His inhibition of bovine F₁ ATPase is pH-independent across the same range. Thus, pH-dependent inhibition of activity by I(1-60)His is specific to the F₁F_o synthase from A. franciscana. The sequence for IF₁ protein from A. franciscana is noticeably conserved across the F₁ binding domain compared to bovine. We document the capacity of brine shrimp F₁F_o to dimerize and report sequences for small subunits in the F_o domain. [NSF grant IOS-1457061/IOS-1456809 to SCH and intramural programme funds from the MRC to JEW].

[1] S. Hand, D. Denlinger, E. Podrabsky, R. Roy, Mechanisms of animal diapause: Recent developments from nematodes, crustaceans, insects and fish, Amer. J. Physiol. 310 (2016) R1193-R1211.

[2] Y. Patil, E. Gnaiger, A. Landry, Z. Leno, S. Hand, OXPHOS capacity is diminished and the phosphorylation system inhibited during diapause in an extremophile, embryos of Artemia franciscana. J. Exp. Biol. 227 (2024) jeb245828.

In response to elevated Ca²⁺ levels, mitochondria undergo an increase permeation of the inner membrane, also called the permeability transition (PT), a process discovered more than 50 years ago that still awaits a molecular definition. Disclosing the identity of the PT pore (PTP) has long intrigued this field of research. Recent progress highlighted a critical role of the ATP synthase, which can generate in reconstituted systems Ca²⁺-dependent channels matching those of the PTP. Interestingly, mitochondria with a defective ATP synthase still undergo PT, which is however mediated by the adenine nucleotide translocator (ANT), one of the historical candidates for PTP formation. We are currently exploring two main aspects: i) the molecular conversion of the ATP synthase into a channel and ii) a possible cross-talk with ANT. According to the “death finger” hypothesis, pore formation occurs within the ATP synthase c-ring upon a Ca²⁺-dependent removal of the lipid plug by the peripheral domain, which contacts lipids through subunit e C-terminus. Our findings support a key role of subunit e C-terminus, since HeLa cells ablated for this domain undergo PT that is no longer mediated by ATP synthase but rather by ANT. Thus, ANT might represent an additional permeation pathway, which contributes to the PT when associated to the ATP synthase. Interestingly, ANT mediates PT in response to Benzodiazepine-423 which targets ATP synthase peripheral stalk, suggesting an intimate connection. Consistently, co-immunoprecipitation studies reveal specific interaction between ANT and ATP synthase subunit g and c. Altogether, our data help to define the mechanism of channel formation by the ATP synthase and shed light on its relationship with ANT.

Mitochondria are evolutionarily conserved organelles that mediate cell survival by conferring energetic plasticity and adaptive potential. As such, modifying the fraction of useful energy converted to work has grave implications on cellular fitness. I will discuss emerging evidence in mice and humans linking the fine-tuning of bioenergetic efficiency to the progression of diseases related to uncontrolled cellular expansion such as obesity and cancer. More specifically, I will focus on the role of bioenergetic efficiency in the progression of metabolic dysfunction-associated steatotic liver disease to liver cancer. Collectively, these data support continued development of small molecule and bioactive compounds to restrict bioenergetic efficiency to treat obesity-related diseases.