The mitochondrial ADP/ATP carrier (AAC, ANT), a member of the SLC25 family of solute carriers, plays a critical role in transporting purine nucleotides (ATP and ADP) as well as protons across the inner mitochondrial membrane. However, the precise mechanism and physiological significance of proton transport by ADP/ATP carrier remain unclear. Notably, the presence of uncouplers-such as long-chain fatty acids (FA) or artificial compounds like dinitrophenol (DNP)-is essential for this process. We explore two potential mechanisms that describe ADP/ATP carrier as either (i) a proton carrier that functions in the presence of FA or DNP, or (ii) an anion transporter (FA- or DNP). In the latter case, the proton is translocated by the neutral form of FA, which carries it from the matrix to the intermembrane space (FA-cycling hypothesis). Our recent results support this hypothesis. We describe a four-step mechanism for the "sliding" of the FA anion from the matrix to the mitochondrial intermembrane space and discuss a possible generalization of this mechanism to other SLC25 carriers.

• Klíčová slova
• MD simulations, bilayer lipid membranes, membrane proteins, mitochondrial transporter, reconstituted protein, uncoupling protein,
• MeSH
• 2,4-dinitrofenol farmakologie   metabolismus   MeSH
• iontový transport MeSH
• lidé MeSH
• mastné kyseliny metabolismus   MeSH
• mitochondriální ADP/ATP-translokasy * metabolismus   chemie   MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• protony * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• 2,4-dinitrofenol MeSH
• mastné kyseliny MeSH
• mitochondriální ADP/ATP-translokasy * MeSH
• protony * MeSH

Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein-lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane.

• Klíčová slova
• AAC, ADP/ATP carrier, arachidonic acid, fatty acid cycling hypothesis, fatty acids anion transport, proton transport, uncoupling proteins,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• anionty metabolismus   MeSH
• lipidové dvojvrstvy * MeSH
• mastné kyseliny metabolismus   MeSH
• mitochondriální ADP/ATP-translokasy metabolismus   MeSH
• protony * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• anionty MeSH
• lipidové dvojvrstvy * MeSH
• mastné kyseliny MeSH
• mitochondriální ADP/ATP-translokasy MeSH
• protony * MeSH

Oxidative stress and ROS are important players in the pathogenesis of numerous diseases. In addition to directly altering proteins, ROS also affects lipids with negative intrinsic curvature such as phosphatidylethanolamine (PE), producing PE adducts and lysolipids. The formation of PE adducts potentiates the protonophoric activity of mitochondrial uncoupling proteins, but the molecular mechanism remains unclear. Here, we linked the ROS-mediated change in lipid shape to the mechanical properties of the membrane and the function of uncoupling protein 1 (UCP1) and adenine nucleotide translocase 1 (ANT1). We show that the increase in the protonophoric activity of both proteins occurs due to the decrease in bending modulus in lipid bilayers in the presence of lysophosphatidylcholines (OPC and MPC) and PE adducts. Moreover, MD simulations showed that modified PEs and lysolipids change the lateral pressure profile of the membrane in the same direction and by the similar amplitude, indicating that modified PEs act as lipids with positive intrinsic curvature. Both results indicate that oxidative stress decreases stored curvature elastic stress (SCES) in the lipid bilayer membrane. We demonstrated that UCP1 and ANT1 sense SCES and proposed a novel regulatory mechanism for the function of these proteins. The new findings should draw the attention of the scientific community to this important and unexplored area of redox biochemistry.

• Klíčová slova
• bending moduli, lateral pressure profile, lipid shape, lipid–protein interaction, mitochondrial membrane protein, protonophoric function, reactive aldehydes, stored curvature elastic stress,
• Publikační typ
• časopisecké články MeSH

2,4-Dinitrophenol (DNP) is a classic uncoupler of oxidative phosphorylation in mitochondria which is still used in "diet pills", despite its high toxicity and lack of antidotes. DNP increases the proton current through pure lipid membranes, similar to other chemical uncouplers. However, the molecular mechanism of its action in the mitochondria is far from being understood. The sensitivity of DNP's uncoupling action in mitochondria to carboxyatractyloside, a specific inhibitor of adenine nucleotide translocase (ANT), suggests the involvement of ANT and probably other mitochondrial proton-transporting proteins in the DNP's protonophoric activity. To test this hypothesis, we investigated the contribution of recombinant ANT1 and the uncoupling proteins UCP1-UCP3 to DNP-mediated proton leakage using the well-defined model of planar bilayer lipid membranes. All four proteins significantly enhanced the protonophoric effect of DNP. Notably, only long-chain free fatty acids were previously shown to be co-factors of UCPs and ANT1. Using site-directed mutagenesis and molecular dynamics simulations, we showed that arginine 79 of ANT1 is crucial for the DNP-mediated increase of membrane conductance, implying that this amino acid participates in DNP binding to ANT1.

• Klíčová slova
• artificial membranes, membrane potential, mitochondrial uncoupler, molecular dynamics simulations, proton conductance, protonophore,
• MeSH
• 2,4-dinitrofenol farmakologie   MeSH
• jaterní mitochondrie metabolismus   MeSH
• krysa rodu Rattus MeSH
• lipidové dvojvrstvy metabolismus   MeSH
• membránové potenciály účinky léků   MeSH
• mitochondriální ADP/ATP-translokasy metabolismus   MeSH
• mitochondriální odpřahující proteiny metabolismus   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2,4-dinitrofenol MeSH
• lipidové dvojvrstvy MeSH
• mitochondriální ADP/ATP-translokasy MeSH
• mitochondriální odpřahující proteiny MeSH

Adenine nucleotide translocase (ANT) is a well-known mitochondrial exchanger of ATP against ADP. In contrast, few studies have shown that ANT also mediates proton transport across the inner mitochondrial membrane. The results of these studies are controversial and lead to different hypotheses about molecular transport mechanisms. We hypothesized that the H+-transport mediated by ANT and uncoupling proteins (UCP) has a similar regulation pattern and can be explained by the fatty acid cycling concept. The reconstitution of purified recombinant ANT1 in the planar lipid bilayers allowed us to measure the membrane current after the direct application of transmembrane potential ΔΨ, which would correspond to the mitochondrial states III and IV. Experimental results reveal that ANT1 does not contribute to a basal proton leak. Instead, it mediates H+ transport only in the presence of long-chain fatty acids (FA), as already known for UCPs. It depends on FA chain length and saturation, implying that FA's transport is confined to the lipid-protein interface. Purine nucleotides with the preference for ATP and ADP inhibited H+ transport. Specific inhibitors of ATP/ADP transport, carboxyatractyloside or bongkrekic acid, also decreased proton transport. The H+ turnover number was calculated based on ANT1 concentration determined by fluorescence correlation spectroscopy and is equal to 14.6 ± 2.5 s-1. Molecular dynamic simulations revealed a large positively charged area at the protein/lipid interface that might facilitate FA anion's transport across the membrane. ANT's dual function-ADP/ATP and H+ transport in the presence of FA-may be important for the regulation of mitochondrial membrane potential and thus for potential-dependent processes in mitochondria. Moreover, the expansion of proton-transport modulating drug targets to ANT1 may improve the therapy of obesity, cancer, steatosis, cardiovascular and neurodegenerative diseases.

• Klíčová slova
• ADP/ATP carrier protein, arachidonic acid, fatty acid anion transport, long-chain fatty acids, mitochondrial transporter, proton transport,
• MeSH
• iontový transport MeSH
• konformace proteinů MeSH
• mastné kyseliny metabolismus   MeSH
• membránový potenciál mitochondrií MeSH
• mitochondrie metabolismus   MeSH
• myši MeSH
• protony * MeSH
• translokátor adeninových nukleotidů 1 chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mastné kyseliny MeSH
• protony * MeSH
• translokátor adeninových nukleotidů 1 MeSH

Molecular dynamics (MD) simulations of uncoupling proteins (UCP), a class of transmembrane proteins relevant for proton transport across inner mitochondrial membranes, represent a complicated task due to the lack of available structural data. In this work, we use a combination of homology modelling and subsequent microsecond molecular dynamics simulations of UCP2 in the DOPC phospholipid bilayer, starting from the structure of the mitochondrial ATP/ADP carrier (ANT) as a template. We show that this protocol leads to a structure that is impermeable to water, in contrast to MD simulations of UCP2 structures based on the experimental NMR structure. We also show that ATP binding in the UCP2 cavity is tight in the homology modelled structure of UCP2 in agreement with experimental observations. Finally, we corroborate our results with conductance measurements in model membranes, which further suggest that the UCP2 structure modeled from ANT protein possesses additional key functional elements, such as a fatty acid-binding site at the R60 region of the protein, directly related to the proton transport mechanism across inner mitochondrial membranes.

• Klíčová slova
• conductance measurements in model membranes, long-chain fatty acid, membrane protein, proton transfer, purine nucleotide, uncoupling,
• MeSH
• adenosintrifosfát chemie   metabolismus   MeSH
• iontový transport MeSH
• konformace proteinů * MeSH
• mastné kyseliny chemie   metabolismus   MeSH
• membránové proteiny chemie   MeSH
• mitochondriální proteiny chemie   metabolismus   MeSH
• myši MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky * MeSH
• stabilita proteinů MeSH
• uncoupling protein 2 chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• mastné kyseliny MeSH
• membránové proteiny MeSH
• mitochondriální proteiny MeSH
• uncoupling protein 2 MeSH

The major role of mitochondria is to provide cells with energy, but no less important are their roles in responding to various stress factors and the metabolic changes and pathological processes that might occur inside and outside the cells. The post-translational modification of proteins is a fast and efficient way for cells to adapt to ever changing conditions. Phosphorylation is a post-translational modification that signals these changes and propagates these signals throughout the whole cell, but it also changes the structure, function and interaction of individual proteins. In this review, we summarize the influence of kinases, the proteins responsible for phosphorylation, on mitochondrial biogenesis under various cellular conditions. We focus on their role in keeping mitochondria fully functional in healthy cells and also on the changes in mitochondrial structure and function that occur in pathological processes arising from the phosphorylation of mitochondrial proteins.

• Klíčová slova
• disease, kinases, mitochondria, phosphorylation,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH