Mitochondrial F₀F₁‐ATP synthase is a molecular target of 3‐iodothyronamine, an endogenous metabolite of thyroid hormone

Abstract

BACKGROUND AND PURPOSE3-iodothyronamine (T1AM) is a metabolite of thyroid hormone acting as a signalling molecule via non-genomic effectors and can reach intracellular targets. Because of the importance of mitochondrial F0F1-ATP synthase as a drug target, here we evaluated interactions of T1AM with this enzyme. EXPERIMENTAL APPROACHKinetic analyses were performed on F0F1-ATP synthase in sub-mitochondrial particles and soluble F1-ATPase. Activity assays and immunodetection of the inhibitor protein IF1 were used and combined with molecular docking analyses. Effects of T1AM on H9c2 cardiomyocytes were measured by in situ respirometric analysis. KEY RESULTST1AM was a non-competitive inhibitor of F0F1-ATP synthase whose binding was mutually exclusive with that of the inhibitors IF1 and aurovertin B. Both kinetic and docking analyses were consistent with two different binding sites for T1AM. At low nanomolar concentrations, T1AM bound to a high-affinity region most likely located within the IF1 binding site, causing IF1 release. At higher concentrations, T1AM bound to a low affinity-region probably located within the aurovertin binding cavity and inhibited enzyme activity. Low nanomolar concentrations of T1AM increased ADP-stimulated mitochondrial respiration in cardiomyocytes, indicating activation of F0F1-ATP synthase consistent with displacement of endogenous IF1,, reinforcing the in vitro results. CONCLUSIONS AND IMPLICATIONSEffects of T1AM on F0F1-ATP synthase were twofold: IF1 displacement and enzyme inhibition. By targeting F0F1-ATP synthase within mitochondria, T1AM might affect cell bioenergetics with a positive effect on mitochondrial energy production at low, endogenous, concentrations. T1AM putative binding locations overlapping with IF1 and aurovertin binding sites are described. AbbreviationsDmH + , proton motive force; ASp, AS particles;; IF1, F0F1-ATP synthase inhibitor protein; Ki inhibition constant; SMP, Mg-ATP sub-mitochondrial particles; T1AM, 3-iodothyronamine BJP British Journal of Pharmacology

Trace Amines and Their Receptors

“…This has subsequently been verified by numerous laboratories (Mühlhaus et al, 2014;Cöster et al, 2015;Chiellini et al, 2017), with 3IT also shown to act as an agonist at TAAR2 (Babusyte et al, 2013;Cichero and Tonelli, 2017) and as an inverse agonist at TAAR5 (Dinter et al, 2015c). 3IT is promiscuous, however, and also interacts with high affinity at a 2 -adrenoceptors (Regard et al, 2007;Dinter et al, 2015b), b-adrenergic receptors (Meyer and Hesch, 1983;Kleinau et al, 2011;Dinter et al, 2015a), muscarinic acetylcholine receptors (Laurino et al, 2016), transient receptor potential cation channel subfamily M member 8 ion channels (Khajavi et al, 2015;Lucius et al, 2016), various monoamine and organic anion transporters (Snead et al, 2007;Panas et al, 2010), and molecular target(s) within mitochondria, including the F 1 -F 0 ATP synthase (Cumero et al, 2012) and possibly complex III (Venditti et al, 2011). 3IT also tightly binds to the plasma protein apolipoprotein B-100 .…”

Uptake and metabolic effects of 3-iodothyronamine in hepatocytes

“…In vitro studies provided evidence that T 1 AM can activate with high-affinity G protein-coupled receptors, including trace amine-associated receptor 1 (Scanlan et al 2004, Zucchi et al 2006) and possibly a2-adrenoceptors (Regard et al 2007). In addition to these receptors, T 1 AM also interacts with plasma membrane and vesicular biogenic amine transporters (Snead et al 2007) as well as different mitochondrial targets (Venditti et al 2011, Cumero et al 2012.…”

3-Iodothyronamine: A High Potency Metabolic Hormone and its Potential for Therapeutic Applications

“…This is underscored by a recent study examining mitochondria effects of T1AM whereby low physiologic levels in cardiomyocytes enhanced ADP-stimulated mitochondrial respiration while higher levels inhibited F o F 1 -ATP synthase activity [39]. T1AM is also observed to interact with Complex III via antimycin A in mitochondria, decreasing O 2 consumption and increasing H 2 O 2 release in mitochondria of cultured hepatocytes, which may partially explain observed decrease in O 2 consumption in vivo [39]. Most recently, Ghanian et al [4] found that T1AM is able to modulate cellular redox state (NADH/FAD ratio) in mouse tissue-specifically and in a dose specific manner [4]; interestingly this effect was divergent between dosage and tissue type.…”

“…T1AM affects a broad range of physiological processes and organ systems. The physiology of T1AM appears to be complex as its effects at similar dosages can have opposing effects in different organs [4] or opposing effects on the same system at different dosages [39]. The specific physiologic response appears to be further complicated by differing responses to duration and dosage pattern; acute administration of high dosages of T1AM (50 mg/kg) results in rapid induction of hypothermia [1] and a drop in metabolic rate [36], but chronic low dosages of T1AM (10 mg/kg repeated over 8 days) results in elevated lipolysis and sustained reduction in body mass [3].…”