The 3D crystallization of the bovine heart ANT monomer with carboxyatractyloside (CAT) established a structural organization based on a threefold repeat of about 100 amino acids, 6 transmembrane helices with a depression at the IMS surface, and a short mitochondrial carrier sequence (RRRMMM) at the bottom (Pebay-Peyroula et al. 2003). Each ANT monomer has turned out to associate with several cardiolipin molecules, correlating with functional studies indicating that ANT activity depends on this lipid. CAT binding excluded that of adenosine nucleotides (i.e., ADP and ATP), suggesting that the residues of ANT that are involved in the nucleotide binding site are also important for the physical association with CAT. A close-open conformation switch has been suggested to facilitate ADP/ATP antiport and to result from the cooperative binding of the nucleotides. However, the exact molecular and enzymatic mechanisms that account for ADP/ATP exchange by ANT are still under investigation (Brüschweiler et al. 2015, Curcio et al. (2016), Pietropaolo et al. 2016; Lunetti et al. 2016).
A Vital Mitochondrial ADP/ATP Carrier
The enzymatic function of ANT as an ADP/ATP exchanger has been first characterized in isolated mitochondria from rodent livers, in yeast, and in native ANT-containing proteoliposomes. These studies have benefited from two specific inhibitors, namely, CAT and bongkrekic acid (BA), which bind different sites within the protein and favor the c- and m-conformation of the protein, respectively (Klingenberg 2008). The efficacy of ANT in exchanging ADP with ATP is moderate, and the relatively high abundance of the carrier at the IM might reflect an adaptation to the high intracellular demand for mitochondrial ATP export (Klingenberg 2008). It is still unclear whether in cellula ANT can function independently, as a monomer and/or an homodimer, or whether (at least in some tissues) it requires the physical binding of a partner such as the voltage-dependent anion channel (VDAC), cyclophilin D (CYPD), the mitochondrial phosphate carrier protein (PiC), the FOF1-ATP synthase (within the so-called ATP synthasome (Ko et al. 2003), and/or the mitochondrial creatine kinase (MTCK1). An association with such mitochondrial proteins might improve the channeling of ATP to hexokinase and of ADP to the Fo F1-ATP synthase in response to increased ATP demands and might be particularly relevant for the metabolic adaptation of mitochondria in cancer cells.
The enzymatic characterization of ANT can be carried out with radiolabeled nucleotides. Briefly, isolated mitochondria are loaded with tritiated ATP ([3H]ATP) in a suitable energizing buffer, at constant temperature allowing for oxidative phosphorylation and for the maintenance of a mitochondrial transmembrane potential (ΔΨm). Upon the elimination of extramitochondrial nucleotides, ADP/ATP exchange is initiated by addition of exogenous ADP. Then, the import reaction is blocked by CAT, mitochondria are centrifuged and separated from their supernatant, and the exported radioactive ATP is quantified. This methodology has been successfully used to estimate the Vmax and Km of ANT from various origins (Pfaff and Klingenberg 1968). Alternatively, the addition of an extramitochondrial ATP detection system (containing NADP+, hexokinase, glucose-6-phosphate dehydrogenase, and glucose) to mitochondria suspended in energizing buffer avoids the use of radioactivity and is suitable for the high-throughput screening of molecules that inhibits ANT antiporter activity (Belzacq-Casagrande et al. 2009).
A Lethal Channel
In response to various stimuli, ANT can be converted into a poorly selective cation channel (Fig. 1b and c). Single-channel current measurements in giant bovine ANT proteoliposomes revealed the reversible opening of a Ca2+-dependent channel (Brustovetsky and Klingenberg 1996). This might be caused by the binding of Ca2+ to cardiolipin, in turn triggering a conformational change that results in pore opening. The ANT channel has also been studied upon purification from rat heart, reconstitution of ANT-containing proteoliposomes, and measurement of the release of a fluorescent probe (Rück et al. 1998; Marzo et al. 1998) and in black lipid membranes (Brenner et al. 2000). Several classes of natural or synthetic molecules have been shown to activate or inhibit the channel function of ANT, suggesting that ANT can constitute a pharmacological target (for review, Halestrap and Brenner 2003). In support of this hypothesis, inhibition of ANT by Nelfinavir, a small molecule used in antiretroviral therapy, has been shown to inhibit pathological manifestations linked to massive cell death in mice undergoing septic shock, cerebral ischemia-reperfusion, and fulminant hepatitis (Weaver et al. 2005).
Based on channel conductance and sensitivity to pharmacological inhibitors, ANT has been proposed to constitute the IM channel of the permeability transition pore complex (PTPC), a multiprotein structure assembled at the interface between mitochondrial membranes that is involved in both mitochondrial homeostasis and cell death (Brenner and Moulin 2012) (Fig. 1b). However, knock-down of ANT does not prevent PTPC opening but attenuated the response to Ca2+ and ANT ligands, indicating that ANT can behave as a regulator of PTPC (Kokoszka et al. 2004 and below).
Four ANT Isoforms
The human ANT gene family is composed of four homologues, ANT1 to ANT4. Primary sequence homology ranges from 68% to 88% (Dolce et al. 2005). The expression of distinct ANT isoforms is highly regulated and exhibits tissue specificity, suggesting a differential role for each isoform. Accordingly, the promoter region of ANT genes contains elements that can be bound by multiple transcription factors including (though presumably not limited to) members of the OXBOX-REBOX family, GRBOX, SP1, and AP2. ANT1 is mainly expressed in nonproliferating tissues, including skeletal muscle and brain. In contrast, ANT2 is upregulated during growth, and ANT2 constitutes the prevalent ANT isoform that is expressed in hepatocytes, fibroblasts, and lymphocytes. The ANT3 gene is transcribed ubiquitously at a comparatively lower level. In humans, ANT4 can be detected mainly in testis, liver, and brain. In mice, ANT4 is exclusively expressed in embryonic stem cells (Dolce et al. 2005). The genetic invalidation of Ant1 and Ant2 in the mouse liver enhances the capacity of mitochondria to accumulate Ca2+ ions, decreases the probability of PTPC opening, and yet does not compromise cell death, suggesting that ANT1 and 2 are dispensable for apoptosis or that other proteins (e.g., ANT4, other members of the mitochondrial carrier protein family as well as other IM protein) may substitute for the lethal functions of ANT1 and 2 in their absence. However, genetic strategies to modulate the expression levels of various ANT isoforms in human cancer cell lines revealed that ANT1 and 3 overexpression favors apoptosis (Bauer et al. 1999), whereas increased levels of ANT2 and 4 augment the resistance of cancer cells against death induction (Le Bras et al. 2006; Gallerne et al. 2010).
ANT1 in Cardiopathy
ANT1 has been associated with several cardiac diseases (Dörner and Schultheiss 2007). In particular, five-point mutations of ANT1 have been identified in patients with autosomal dominant progressive external ophthalmoplegia (adPEO), an adult-onset pathology characterized by weakening of external eye muscles, generalized myopathy, exercise intolerance, and multiorgan disorders including cardiomyopathy. Muscle biopsies of individuals affected by adPEO revealed common intracellular features, including deficient oxidative phosphorylation, scattered ragged red fibers, and multiple mitochondrial DNA deletions. When introduced in yeast, these mutations decrease oxidative growth and lead to diminished ADP/ATP exchange.
Cardiac ischemia/reperfusion and alteration of the myocardium have also been associated with reduced ANT function. It has been speculated that oxidative stress and/or alterations of the lipid metabolism can negatively affect ANT ADP/ATP exchange activity, inhibit energy supplies, and also, can favor PTPC opening and apoptosis.
Thus, transgenic mice that overexpress ANT1 in the heart are protected from diabetic cardiomyopathy and cardiac ischemia (Wang et al. 2009; Klumpe et al. 2016), correlating with improved mitochondrial ANT functionality and oxidative phosphorylation. In contrast to in vitro studies, no signs of enhanced apoptosis were detected in ANT-transgenic animals. In addition, these animals showed features of athlete’s hearts, including enlarged end-diastolic and end-systolic volumes and raised heart rate with unchanged ejection fraction, but no signs of abnormal dilatation.
ANT2 in Cancer
According to the Expressed Sequence Tags database (EST, Unigene), cancer patients often show the deregulated expression of ANT isoforms. In particular, the ANT2 isoform is overexpressed in biopsies from distinct types of cancers as well as in several human tumor cell lines. Changes in the expression levels of ANT2 neither have major effects on cell metabolism and morphology nor negatively affect cell viability or proliferation, suggesting compensatory effects from other ANT isoforms (Le Bras et al. 2006). However, ANT2 overexpression decreases the sensitivity of cancer to some pro-apoptotic stimuli. Accordingly, ANT2 silencing by RNA interference (RNAi) induced apoptosis in breast cancer cells in vitro and tumor regression in mice. Overexpression of ANT4 also protects cancer cells from apoptosis (Gallerne et al. 2010), but the antiapoptotic potential of ANT4 in vivo awaits confirmation.
Mitochondria, which are the cell’s powerhouse but also centralize lethal pathways, constitute a crossroad for numerous critical intracellular signaling pathways. Mitochondrial dysfunction is frequently associated to severe human diseases, including cancer, neurodegenerative diseases, and cardiovascular diseases. Some of these pathologies are closely associated to deregulation of apoptosis and energy metabolism failure. The adenine nucleotide translocase (ANT), which possesses two opposite functions (as a vital ADP/ATP antiporter and as a lethal channel), appears to participate to the large protein network that controls the cell fate. It is now clearly established that both ANT expression and function can be modulated by pharmacological agents (i.e., small molecules, natural compounds and RNAi). However, a more detailed knowledge of the specific pathophysiological role of each isoform is still required in validated preclinical models.
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