biomolecules Review The Mitochondrial Carnitine Acyl-carnitine Carrier (SLC25A20): Molecular Mechanisms of Transport, Role in Redox Sensing and Interaction with Drugs Annamaria Tonazzi 1,† , Nicola Giangregorio 1,† , Lara Console 2 , Ferdinando Palmieri 1,3,* and Cesare Indiveri 1,2,* 1 Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), National Research Council, Via Orabona 4, 70126 Bari, Italy; [email protected] (A.T.); [email protected] (N.G.) 2 Unit of Biochemistry and Molecular Biotechnology, Department DiBEST (Biologia, Ecologia, Scienze della Terra), University of Calabria, Via P. Bucci 4C, 87036 Arcavacata di Rende, Italy; [email protected] 3 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, 70125 Bari, Italy * Correspondence: [email protected] (F.P.); [email protected] (C.I.); Tel.: +39-080-544-3323 (F.P.); +39-0984-492939 (C.I.) † These authors contributed equally to this work. Abstract: The SLC25A20 transporter, also known as carnitine acyl-carnitine carrier (CAC), catalyzes the transport of short, medium and long carbon chain acyl-carnitines across the mitochondrial inner membrane in exchange for carnitine. The 30-year story of the protein responsible for this Citation: Tonazzi, A.; function started with its purification from rat liver mitochondria. Even though its 3D structure Giangregorio, N.; Console, L.; is not yet available, CAC is one of the most deeply characterized transport proteins of the inner Palmieri, F.; Indiveri, C. The mitochondrial membrane. Other than functional, kinetic and mechanistic data, post-translational Mitochondrial Carnitine modifications regulating the transport activity of CAC have been revealed. CAC interactions with Acyl-carnitine Carrier (SLC25A20): Molecular Mechanisms of Transport, drugs or xenobiotics relevant to human health and toxicology and the response of the carrier function Role in Redox Sensing and to dietary compounds have been discovered. Exploiting combined approaches of site-directed Interaction with Drugs. Biomolecules mutagenesis with chemical targeting and bioinformatics, a large set of data on structure/function 2021, 11, 521. https://doi.org/ relationships have been obtained, giving novel information on the molecular mechanism of the 10.3390/biom11040521 transport catalyzed by this protein. Academic Editor: Vladimir Keywords: carnitine; carnitine acyl-carnitine carrier; carnitine acyl-carnitine translocase; membrane N. Uversky transport; mitochondria; mitochondrial carrier; mitochondrial transporter; post-translational modifi- cation; solute carrier family 25; SLC25A20 Received: 8 March 2021 Accepted: 26 March 2021 Published: 31 March 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in The mitochondrial carnitine acyl-carnitine carrier (CAC) is the member A20 of the published maps and institutional affil- SLC25 protein family, including 53 solute transporters in humans [1–3], the majority of iations. which are localized in the inner mitochondrial membrane. Until now, only one family member has been found in the peroxisomal membrane [4]. Furthermore, approximately one-third of them are still orphans, i.e., their transported substrates are unknown. This family members share a peculiar structural fold of six transmembrane segments charac- terized by 3-fold repeated couples of hydrophobic α-helices. Each couple is connected Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. by a hydrophilic loop and contains the SLC25 sequence motif PX[D/E]XX[K/R] at about α This article is an open access article the boundary of the odd -helix and the loop. The structural information on the SLC25 distributed under the terms and proteins derives mainly from the ADP/ATP carrier, which has been crystallized in both the conditions of the Creative Commons outwards and inward open conformations [5,6]. All the other carrier structures have been Attribution (CC BY) license (https:// predicted by homology modeling, including CAC, whose structure has been corroborated creativecommons.org/licenses/by/ by site-directed mutagenesis and chemical targeting approaches. CAC is a key component 4.0/). of the carnitine shuttle [7], which is crucial for the mitochondrial β-oxidation pathway. Biomolecules 2021, 11, 521. https://doi.org/10.3390/biom11040521 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, x 2 of 21 Biomolecules 2021, 11, 521 2 of 21 this shuttle (Figure 1), fatty acids are activated by the cytosolic acyl-CoA synthetase In(ACSL) this shuttle to fatty (Figure acyl-CoAs1), fatty thioesters acids are [8,9]. activated Since bythe themitochondrial cytosolic acyl-CoA inner membrane synthetase is not (ACSL)permeable to fatty to acyl-CoAs, acyl-CoAs thioestersacyl groups [8,9 are]. Since transferred the mitochondrial from CoA inner to carnitine membrane by the is not action permeableof “carnitine to acyl-CoAs,palmitoyltransferase-1a acyl groups are and transferred b” (CPT-1a; from CoA CPT-1b), to carnitine an integral by the actionouter ofmem- “carnitinebrane enzyme palmitoyltransferase-1a [10]. The acyl-carnitines and b” cro (CPT-1a;ss the outer CPT-1b), mitochondrial an integral outer membrane membrane through enzymean almost [10 unspecific]. The acyl-carnitines pore constituted cross theby outerthe voltage-dependent mitochondrial membrane anion channel through (VDAC) an almost[11] and, unspecific then, are pore specifically constituted translocated by the voltage-dependent across the inner anion mitochondrial channel (VDAC) membrane [11] by and,the action then, areof CAC. specifically In the mitochondrial translocated across matr theix, the inner enzyme mitochondrial carnitine membrane palmitoyltransferase by the action2 (CPT-2) of CAC. catalyzes In the the mitochondrial trans-esterification matrix, of the the enzyme acyl groups carnitine from palmitoyltransferase carnitine to mitochon- 2drial (CPT-2) CoA catalyzes with the the release trans-esterification of free carnitine, of the thereby acyl groups providing from carnitine acyl-CoA to mitochon-substrates for drialfatty CoAacid β with-oxidation. the release CAC of and free CPT-2 carnitine, form thereby a supramolecular providing acyl-CoA complex substratesin the inner for mito- β fattychondrial acid membrane,-oxidation. CACdevoted and to CPT-2 acyl-carnitine form a supramolecular channeling from complex the carrier in the inner to the mito- enzyme chondrial membrane, devoted to acyl-carnitine channeling from the carrier to the enzyme (Figure 1) [12]. The carnitine released in this reaction is translocated backward to the cy- (Figure1)[ 12]. The carnitine released in this reaction is translocated backward to the cy- tosol by the same carrier via an acyl-carnitine/carnitine antiport reaction. The β-oxidation tosol by the same carrier via an acyl-carnitine/carnitine antiport reaction. The β-oxidation pathway isis activeactive in in many many tissues, tissues, especially especially those those characterized characterized by higherby higher metabolic metabolic ex- ex- penditure. ItIt provides provides a a large large portion portion of of the th energye energy required required by heartby heart muscle, muscle, kidneys kidneys and and also skeletalskeletal muscle, muscle, when when glycogen glycogen has has been been consumed consumed [13,14 [13,14].]. This pathwayThis pathway is also activeis also ac- intive hepatocytes in hepatocytes where where fatty acid fatty oxidation acid oxidatio providesn provides acetyl-CoA acetyl-CoA for ketone for body ketone synthesis body syn- duringthesis during prolonged prolonged fasting fasting conditions, conditions, in which in glycogen which glycogen stores have stores been have depleted been depleted [15]. Neurons[15]. Neurons also perform also perform fatty acid fatty oxidation acid oxidatio even thoughn even atthough a very at low a very rate. Indeed,low rate. CAC Indeed, alsoCAC has also been has describedbeen described in brain in [brain16–18 ].[16–18]. The crucial The crucial role of CACrole of in CAC energy in metabolismenergy metabo- waslism demonstratedwas demonstrated by the by discovery the discover of inheritedy of inherited defects defects of its of gene its geneSLC25A20 SLC25A20causing causing secondary carnitinecarnitine deficiency deficiency [19 [19––2424],], a syndromea syndrome that that arises arises in the in the very very first first stage stage of life of life as aa life-threateninglife-threatening pathology. pathology. In In this this altered altere metabolicd metabolic condition, condition, acyl-carnitines acyl-carnitines fail tofail to reach thethe mitochondrialmitochondrial matrix matrix with with consequent consequent strong strong impairment impairment of theof theβ-oxidation. β-oxidation. This syndromesyndrome isis moremore severe severe than than the the primary primary carnitine carnitine deficiency deficiency caused caused by by defects defects of of the plasma membrane transporter OCTN2 (SLC22A5) [25–27]. Recent findings have the plasma membrane transporter OCTN2 (SLC22A5) [25–27]. Recent findings have corre- correlated alterations of CAC expression or regulation with diabetes [28,29]. lated alterations of CAC expression or regulation with diabetes [28,29]. Figure 1. Role of the carnitine shuttle in the mitochondrial β-oxidation pathway. The shuttle is constitutedFigure 1. Role by carnitineof the carnitine palmitoyltransferase shuttle in the 1mitochondrial (CPT1) that converts β-oxidation acyl-CoAs