Marcos Aldea

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

The H+-ATP synthase is a reversible engine of mitochondria that synthesizes or hydrolyzes ATP upon changes in cell physiology. ATP synthase dysfunction is involved in the onset and progression of diverse human pathologies. During ischemia, the ATP hydrolytic activity of the enzyme is inhibited by the ATPase inhibitory factor 1 (IF1). The expression of IF1 in human tissues and its participation in the development of human pathology are unknown. Here, we have developed monoclonal antibodies against human IF1 and determined its expression in paired normal and tumor biopsies of human carcinomas. We show that the relative mitochondrial content of IF1 increases significantly in carcinomas, suggesting the participation of IF1 in oncogenesis. The expression of IF1 varies significantly in cancer cell lines. To investigate the functional activity of IF1 in cancer, we have manipulated its cellular content. Overexpression of IF1 or of its pH-insensitive H49K mutant in cells that express low levels of IF1 triggers the up-regulation of aerobic glycolysis and the inhibition of oxidative phosphorylation with concurrent mitochondrial hyperpolarization. Treatment of the cells with the H+-ATP synthase inhibitor oligomycin mimicked the effects of IF1 overexpression. Conversely, small interfering RNA-mediated silencing of IF1 in cells that express high levels of IF1 promotes the down-regulation of aerobic glycolysis and the increase in oxidative phosphorylation. Overall, these findings support that the mitochondrial content of IF1 controls the activity of oxidative phosphorylation mediating the shift of cancer cells to an enhanced aerobic glycolysis, thus supporting an oncogenic role for the de-regulated expression of IF1 in cancer.

The tumor suppressor function of mitochondria: Translation into the clinics

Recently, the inevitable metabolic reprogramming experienced by cancer cells as a result of the onset of cellular proliferation has been added to the list of hallmarks of the cancer cell phenotype. Proliferation is bound to the synchronous fluctuation of cycles of an increased glycolysis concurrent with a restrained oxidative phosphorylation. Mitochondria are key players in the metabolic cycling experienced during proliferation because of their essential roles in the transduction of biological energy and in defining the life-death fate of the cell. These two activities are molecularly and functionally integrated and are both targets of commonly altered cancer genes. Moreover, energetic metabolism of the cancer cell also affords a target to develop new therapies because the activity of mitochondria has an unquestionable tumor suppressor function. In this review, we summarize most of these findings paying special attention to the opportunity that translation of energetic metabolism into the clinics could afford for the management of cancer patients. More specifically, we emphasize the role that mitochondrial beta-F1-ATPase has as a marker for the prognosis of different cancer patients as well as in predicting the tumor response to therapy.

Expression, regulation and clinical relevance of the ATPase inhibitory factor 1 in human cancers.

Recent findings in colon cancer cells indicate that inhibition of the mitochondrial H+-adenosine triphosphate (ATP) synthase by the ATPase inhibitory factor 1 (IF1) promotes aerobic glycolysis and a reactive oxygen species (ROS)-mediated signal that enhances proliferation and cell survival. Herein, we have studied the expression, biological relevance, mechanism of regulation and potential clinical impact of IF1 in some prevalent human carcinomas. We show that IF1 is highly overexpressed in most (>90%) of the colon (n=64), lung (n=30), breast (n=129) and ovarian (n=10) carcinomas studied as assessed by different approaches in independent cohorts of cancer patients. The expression of IF1 in the corresponding normal tissues is negligible. By contrast, the endometrium, stomach and kidney show high expression of IF1 in the normal tissue revealing subtle differences by carcinogenesis. The overexpression of IF1 also promotes the activation of aerobic glycolysis and a concurrent ROS signal in mitochondria of the lung, breast and ovarian cancer cells mimicking the activity of oligomycin. IF1-mediated ROS signaling activates cell-type specific adaptive responses aimed at preventing death in these cell lines. Remarkably, regulation of IF1 expression in the colon, lung, breast and ovarian carcinomas is exerted at post-transcriptional levels. We demonstrate that IF1 is a short-lived protein (t1/2 ∼100 min) strongly implicating translation and/or protein stabilization as main drivers of metabolic reprogramming and cell survival in these human cancers. Analysis of tumor expression of IF1 in cohorts of breast and colon cancer patients revealed its relevance as a predictive marker for clinical outcome, emphasizing the high potential of IF1 as therapeutic target.

Role of the N‐terminal α‐helix in biogenesis of α7 nicotinic receptors

We studied the role of the α‐helix present at the N‐terminus of nicotinic acetylcholine receptor (nAChR) subunits in the expression of functional channels. Deletion of this motif in α7 subunits abolished expression of nAChRs at the membrane of Xenopus oocytes. The same effect was observed upon substitution by homologous motifs of other ligand‐gated receptors. When residues from Gln4 to Tyr15 were individually mutated to proline, receptor expression strongly decreased or was totally abolished. Equivalent substitutions to alanine were less harmful, suggesting that proline‐induced break of the α‐helix is responsible for the low expression. Steady‐state levels of wild‐type and mutant subunits were similar but the formation of pentameric receptors was impaired in the latter. In addition, those mutants that reached the membrane showed a slightly increased internalization rate. Expression of α7 nAChRs in neuroblastoma cells confirmed that mutant subunits, although stable, were unable to reach the cell membrane. Analogous mutations in heteromeric nAChRs (α3β4 and α4β2) and 5‐HT3A receptors also abolished their expression at the membrane. We conclude that the N‐terminal α‐helix of nAChRs is an important requirement for receptor assembly and, therefore, for membrane expression.

A choline-evoked [Ca2+]c signal causes catecholamine release and hyperpolarization of chromaffin cells

In bovine chromaffin cells fast‐superfused with Krebs‐HEPES solution containing 1–2 mM Ca2+, 5 s pulses of choline (1–10 mM), elicited catecholamine secretory responses that were only ~10% of those evoked by ACh (0.01–0.1 mM). However, in high‐Ca2+ solutions (10–20 mM) the size of the choline secretory responses approached those of ACh. The choline responses (10 mM choline in 20 mM Ca2+, 10Cho/20Ca2+) tended to decline upon repetitive pulsing, whereas those of ACh were well maintained. The confocal [Ca2+]c increases evoked by 10Cho/20Ca2+ were similar to those of ACh. Whereas 10Cho/20Ca2+ caused mostly hyperpolarization of chromaffin cells, 0.1ACh/20 Ca2+ caused first depolarization and then hyperpolarization; in regular solutions (2 mM Ca2+), the hyperpolarizing responses did not show up. In Xenopus oocytes injected with mRNA for bovine α7 nicotinic receptors (nAChRs), 10Cho/20 Ca2+ fully activated an inward current; in oocytes expressing α3β4, however, the inward current elicited by choline amounted to only 4% of the size of α7 current. Our results suggest that choline activates the entry of Ca2+ through α7 nAChRs; this leads to a cytosolic concentration of calcium ([Ca2+]c) rise that causes the activation of nearby Ca2+‐dependent K+ channels and the hyperpolarization of the chromaffin cell. This response, which could be unmasked provided that cells were stimulated with high‐Ca2+ solutions, may be the underlying mechanism through which choline exerts a modulatory effect on the electrical activity of the chromaffin cell and on neurotransmitter release at cholinergic synapses.

Cytoplasmic regions adjacent to the M3 and M4 transmembrane segments influence expression and function of α7 nicotinic acetylcholine receptors. A study with single amino acid mutants

We studied the role of the cytoplasmic regions adjacent to the M3 and M4 transmembrane segments of α7 nicotinic receptors in the expression of functional channels. For this purpose, a total of 50 amino acids were mutated throughout the mentioned regions. Mutants close to M3, from Arg294 to Leu321, showed slight modifications in the levels of α‐bungarotoxin binding sites and acetylcholine‐evoked currents. Exceptions were mutants located at two clusters (His296 to Pro300 and Ile312 to Trp316), which exhibited low expression levels. In addition, some mutants showed altered functional responses. Many mutants close to M4 showed increased receptor expression, especially the ones located at the hydrophobic face of a putative amphipathic helix. This effect seems to be the consequence of a combination of increased receptor biosynthesis, higher transport efficiency and delayed degradation, such that we postulate that elements in the amphipathic domain strongly influence receptor stability. Finally, some mutants in this region showed altered functional responses: elimination of positively charged residues (Arg424 and Arg426) increased currents, whereas the opposite was observed upon suppression of negatively charged ones (Glu430 and Glu432). These results suggest that the cytoplasmic regions close to M3 and M4 play important structural and functional roles.

Non-charged amino acids from three different domains contribute to link agonist binding to channel gating in α7 nicotinic acetylcholine receptors

Binding of agonists to nicotinic acetylcholine receptors results in channel opening. Previously, we have shown that several charged residues at three different domains of the α7 nicotinic receptor are involved in coupling binding and gating, probably through a network of electrostatic interactions. This network, however, could also be integrated by other residues. To test this hypothesis, non‐charged amino acids were mutated and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Asn47 and Gln48 (loop 2), Ile130, Trp134, and Gln140 (loop 7), and Thr264 (M2–M3 linker) showed poor or null functional responses, despite significant membrane expression. By contrast, mutants F137A and S265A exhibited a gain of function effect. In all cases, changes in dose–response relationships were small, EC50 values being between threefold smaller and fivefold larger, arguing against large modifications of agonist binding. Peak currents decayed at the same rate in all receptors except two, excluding large effects on desensitization. Thus, the observed changes could be mostly caused by alterations of the gating characteristics. Moreover, analysis of double mutants showed an interconnection between some residues in these domains, especially Gln48 with Ile130, suggesting a potential coupling between agonist binding and channel gating through these amino acids.

Role of the extracellular transmembrane domain interface in gating and pharmacology of a heteromeric neuronal nicotinic receptor

J. Neurochem. (2010) 113, 1036–1045.

Interactions between loop 5 and β-strand β6′ are involved in α7 nicotinic acetylcholine receptors channel gating

Binding of agonists to nicotinic acetylcholine receptors (nAChR) is coupled to channel opening through local rearrangements of different domains of the protein. Recent structural data suggest that two of these regions could be the loop 5 (L5) and the β‐strand β6′, both forming the inner part of the N‐terminal domain. Amino acids in these domains were mutated in α7 nAChRs, and expression levels and functional responses of mutant receptors were measured. Mutations located at the putative apex of L5, Asp97 and Glu98, and also at Phe100, gave receptors with smaller currents, showing qualitative differences with respect to muscle nAChRs. In contrast, mutations in the β‐strand β6′ (at Phe124 and Lys125) showed increased functional responses. Mutations affected equally the responses to acetylcholine and dimethylphenylpiperazinium, except in Phe100 where the latter was sevenfold less effective than in wild‐type. Currents in mutants decayed with almost the same kinetics, ruling out large effects on desensitization. Analysis of double mutants demonstrated a functional coupling among the three electrically charged amino acids Asp97, Glu98, and Lys125, and also between Phe100 and Phe124. The results are compatible with the involvement of functional interactions between L5 and β‐strand β6′ during nAChR activation.