Stefania Contessi

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

BACKGROUND AND PURPOSE 3‐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.

Mitochondrial and cell-surface F0F1ATPsynthase in innate and acquired cardioprotection

Mitochondria are central to heart function and dysfunction, and the pathways activated by different cardioprotective interventions mostly converge on mitochondria. In a context of perspectives in innate and acquired cardioprotection, we review some recent advances in F0F1ATPsynthase structure/function and regulation in cardiac cells. We focus on three topics regarding the mitochondrial F0F1ATPsynthase and the plasma membrane enzyme, i.e.: i) the crucial role of cardiac mitochondrial F0F1ATPsynthase regulation by the inhibitory protein IF1 in heart preconditioning strategies; ii) the structure and function of mitochondrial F0F1ATPsynthase oligomers in mammalian myocardium as possible endogenous factors of mitochondria resistance to ischemic insult; iii) the external location and characterization of plasma membrane F0F1 ATP synthase in search for possible actors of its regulation, such as IF1 and calmodulin, at cell surface.

Stoichiometry and Topology of the Complex of the Endogenous ATP Synthase Inhibitor Protein IF1 with Calmodulin

IF(1), the natural inhibitor protein of F(O)F(1)ATP synthase able to regulate the ATP hydrolytic activity of both mitochondrial and cell surface enzyme, exists in two oligomeric states depending on pH: an inactive, highly helical, tetrameric form above pH 6.7 and an active, inhibitory, dimeric form below pH 6.7 [ Cabezon , E. , Butler , P. J. , Runswick , M. J. , and Walker , J. E. ( 2000 ) J. Biol. Chem. 275 , 25460 -25464 ]. IF(1) is known to interact in vitro with the archetypal EF-hand calcium sensor calmodulin (CaM), as well to colocalize with CaM on the plasma membrane of cultured cells. Low resolution structural data were herein obtained in order to get insights into the molecular interaction between IF(1) and CaM. A combined structural proteomic strategy was used which integrates limited proteolysis and chemical cross-linking with mass spectrometric analysis. Specifically, chemical cross-linking data clearly indicate that the C-terminal lobe of CaM molecule contacts IF(1) within the inhibitory, flexible N-terminal region that is not involved in the dimeric interface in IF(1). Nevertheless, native mass spectrometry analysis demonstrated that in the micromolar range the stoichiometry of the IF(1)-CaM complex is 1:1, thereby indicating that binding to CaM promotes IF(1) dimer dissociation without directly interfering with the intersubunit contacts of the IF(1) dimer. The relevance of the finding that only the C-terminal lobe of CaM is involved in the interaction is two fold: (i) the IF(1)-CaM complex can be included in the category of noncanonical structures of CaM complexes; (ii) it can be inferred that the N-terminal region of CaM might have the opportunity to bind to a second target.

Effects of Fe(III) binding to the nucleotide‐independent site of F1‐ATPase: enzyme thermostability and response to activating anions

Mitochondrial F1‐ATPase was induced in different conformations by binding of specific ligands, such as nucleotides. Then, Fourier transform infrared spectroscopy (FT‐IR) and kinetic analyses were run to evaluate the structural and functional effects of Fe(III) binding to the nucleotide‐independent site. Binding of one equivalent of Fe(III) induced a localised stabilising effect on the F1‐ATPase structure destabilised by a high concentration of NaCl, through rearrangements of the ionic network essential for the maintenance of enzyme tertiary and/or quaternary structure. Concomitantly, a lower response of ATPase activity to activating anions was observed. Both FT‐IR and kinetic data were in accordance with the hypothesis of the Fe(III) site location near one of the catalytic sites, i.e. at the α/β subunit interface.

Effect of inhibitor binding to β subunits of F1ATPase on enzyme thermostability: a kinetic and FT-IR spectroscopic analysis

FT‐IR analysis shows that treatment of F1ATPase with the inhibitors DCCD and Nbf‐Cl, in the presence of saturating concentrations of ADP and AMP‐PNP and in the absence of Mg2+, does not modify the secondary structure of the enzyme, but significantly modifies its compactness and thermal stability, although to different extents. Nbf‐Cl causes a significant increase in stabilisation, in addition to that induced by nucleotides, while DCCD is less effective in this regard. Determination by HPLC of the exchange rate, in the absence of Mg2+, of tightly bound nucleotides of F1ATPase treated with the two inhibitors shows that DCCD does not significantly affect the exchange rate of ADP with AMP‐PNP and vice versa in catalytic and non‐catalytic tight sites, while Nbf‐Cl selectively reduces the enzymes capacity to exchange ADP bound in the tight catalytic site. It is suggested that the effects of DCCD, unlike those of Nbf‐Cl, are closely related to the presence or absence of Mg2+.

Mitochondrial F0F1-ATP synthase is a molecular target of 3-iodothyronamine, an endogenous metabolite of thyroid hormone: 3-iodothyronamine binding to F0F1-ATP synthase

BACKGROUND AND PURPOSE 3‐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.

F(0) F(1) -Atpsynthase as a Molecular Target of 3-Iodothyronamine

BACKGROUND AND PURPOSE 3‐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.