In this study, a new regenerative strategy to treat several neurodegenerative diseases is suggested by theuse of a multitarget approach induced by our small molecule, MC111. Considering the importance of Pgpand BCRP expression on stem cell differentiation and the involvement of TLR4 on neurodegenerationprocesses, we investigated the effect of MC111, belonging to our library of P-gp active compounds on:(i) TLR4 signaling; (ii) P-gp and BCRP activity and expression; (iii) neurite sprouting. The observed findingsexerted by MC111, open a new scenario for a multitarget and regenerative approach inneurodegenerative diseases encouraging the in vivo evaluation of MC111 as new tool in neuroreparativemedicine.

The role of hydrophobic residues of the mitochondrial carnitine/acylcarnitine carrier (CAC) in the inhibition by acylcarnitines has been investigated by site-directed mutagenesis. According to the homology model of CAC in cytosolic opened conformation (c-state), L14, G17, G21, V25, P78, V82, M85, C89, F93, A276, A279, C283, F287 are located in the 1st (H1), 2nd (H2) and 6th (H6) transmembrane ?-helices and exposed in the central cavity, forming a hydrophobic half shell. These residues have been substituted with A (or G) and in some cases with M. Mutants have been assayed for transport activity measured as [3H]carnitine/carnitine antiport in proteoliposomes. With the exception of G17A and G21M, mutants exhibited activity from 20% to 100% of WT. Among the active mutants only G21A, V25M, P78A and P78M showed Vmax lower than half and/or Km more than two fold respect to WT. Acylcarnitines competitively inhibited carnitine antiport. The extent of inhibition of the mutants by acylcarnitines with acyl chain length of 2, 4, 8, 12, 14 and 16 has been compared with the WT. V25A, P78A, P78M and A279G showed reduced extent of inhibition by all the acylcarnitines; V25M showed reduced inhibition by shorter acylcarnitines; V82A, V82M, M85A, C89A and A276G showed reduced inhibition by longer acylcarnitines, respect to WT. C283A showed increased extent of inhibition by acylcarnitines. Variations of Ki of mutants for acylcarnitines reflected variations of the inhibition profiles. The data demonstrated that V25, P78, V82, M85 and C89 are involved in the acyl chain binding to the CAC in c-state.

The mitochondrial carnitine/acylcarnitine carrier catalyzes the transport of carnitine and acylcarnitines by antiport as well as by uniport with a rate slower than the rate of antiport. The mechanism of antiport resulting from coupling of two opposed uniport reactions was investigated by site-directed mutagenesis. The transport reaction was followed as [3H]carnitine uptake in or efflux from proteoliposomes reconstituted with the wild type or mutants, in the presence or absence of a countersubstrate. The ratio between the antiport and uniport rates for the wild type was 3.0 or 2.5, using the uptake or efflux procedure, respectively. This ratio did not vary substantially in mutants H29A, K35R, G121A, E132A, K135A, R178A, D179E, E191A, K194A, K234A, and E288A. A ratio of 1.0 was measured for mutant K35A, indicating a loss of antiport function by this mutant. Ratios of >1.0 but significantly lower than that of the wild type were measured for mutants D32A, K97A, and D231A, indicating the involvement of these residues in the antiport mechanism. To investigate the role of the countersubstrate in the conformational changes underlying the transport reaction, the m-state of the transporter (opened toward the matrix side) was specifically labeled with N-ethylmaleimide while the c-state of the carrier (opened toward the cytosolic side) was labeled with fluorescein maleimide. The labeling results indicated that the addition of an external substrate, on one hand, reduced the amount of protein in the m-state and, on the other, increased the protein fraction in the c-state. This substrate-induced conformational change was abolished in the protein lacking K35, pointing to the role of this residue as a sensor in the mechanism of the antiport reaction.

H2O2 inhibits the [3H]carnitine/carnitine antiport catalysed by the mitochondrial carnitine/acylcarnitinetransporter reconstituted in proteoliposomes. The inhibition was reversed by dithioerythritol, N-acetylcysteineand L-cysteine. Inhibition time-dependence revealed a faster and a slower reaction stages withorders of reaction of 1.0 and 1.9, respectively. Inhibition was tested on mutants in which one or more ofthe six Cys residues had been substituted with Ser or with Val. The four replacement mutant C23S/C58S/C89S/C283S containing C136 and C155 was inhibited as the wild-type. Mutants C23V/C58V/C155V/C89S/C283S and C23V/C58V/C136V/C89S/C283S containing only C136 or C155, respectively, were inhibited ata much lower extent respect to the wild-type, while the mutant C136S/C155S in which the two Cys weresubstituted and the C-less protein were virtually insensitive to inhibition. DTE reversed the inhibition ofthe H2O2 sensitive proteins except that in the case of the mutants containing only C136 or C155 afterlong time of incubation with H2O2. The IC50 values obtained by dose-response curves of H2O2 inhibitionwere 0.17 mM for the wild-type, 0.39 mM for the four replacement mutant containing C136 and C155,2.23 or 1.8 mM in the five replacement mutants containing the single C136 or C155, respectively.Carnitine and acetylcarnitine protected the protein from the inhibition by H2O2. Inhibition kineticsshowed a competitive behaviour of H2O2 respect to carnitine. All the data concur to demonstrate thatH2O2 interacts with C136 and C155 and completely inactivates the transporter by inducing the formationof a disulphide.

The carnitine/acylcarnitine transporter is atransport system whose function is essential for the mitochondrialb-oxidation of fatty acids. Here, the presence ofcarnitine/acylcarnitine carrier (CACT) in nervous tissueand its sub-cellular localization in dorsal root ganglia(DRG) neurons have been investigated. Western blot analysisusing a polyclonal anti-CACT antibody produced inour laboratory revealed the presence of CACT in all thenervous tissue extracts analyzed. Confocal microscopyexperiments performed on fixed and permeabilized DRGneurons co-stained with the anti-CACT antibody and themitochondrial marker MitoTracker Red clearly showed amitochondrial localization for the carnitine/acylcarnitinetransporter. The transport activity of CACT from DRGextracts reconstituted into liposomes was about 50 % inrespect to liver extracts. The experimental data herereported represent the first direct evidence of the expressionof the carnitine/acylcarnitine transporter in sensoryneurons, thus supporting the existence of the b-oxidationpathway in these cells

The mitochondrial carnitine/acylcarnitine translocase has been identified, purified and reconstituted in liposomes in 1990. Since that time it has been object of studies aimed to characterize its function and to define the molecular determinants of the translocation pathway. Thanks to these tenacious studies the molecular map of the amino acids involved in the catalysis has been constructed and the roles of critical residues in the translocation pathway have been elucidated. This has been possible through the combination of transport assay in reconstituted liposomes, site-directed mutagenesis, chemical labeling and bioinformatics. Recently some molecules which modulate CACT activity have been identified, such as glutathione and hydrogen peroxide, constituting some of the few cases of control mechanisms of mitochondrial carriers. The vast knowledge on the carnitine/acylcarnitine translocase is essential both as a progress in basic science and as instrument to foresee therapeutic or toxic effects of xenobiotics and drugs. Such studies have been already started pointing out the inhibitory action of drugs such as K⁺/H⁺-ATPase inhibitors (omeprazole) or antibiotics (ß-lactams) on the carnitine/acylcarnitine translocase, which can explain some of their adverse effects.

The effect of Hg²⁺ and CH<inf>3</inf>Hg⁺ on the mitochondrial carnitine/acylcarnitine transporter (CACT) has been studied on the recombinant protein and on the CACT extracted from HeLa cells or Zebrafish and reconstituted in proteoliposomes. Transport was abolished upon treatment of the recombinant CACT in proteoliposomes by Hg²⁺ or CH<inf>3</inf>Hg⁺. Inhibition was reversed by the SH reducing agent 1,4-dithioerythritol, GSH, and N-acetylcysteine. IC<inf>50</inf> for Hg²⁺ and CH<inf>3</inf>Hg⁺ of 90 nM and 137 nM, respectively, were measured by dose-response analyses. Inhibition was abolished in the C-less CACT mutant. Strong reduction of inhibition by both reagents was observed in the C136A and some reduction in the C155A mutants. Inhibition similar to that of the WT was observed in the C23V/C58V/C89S/C155V/C283S mutant, containing only C136. Optimal inhibition by Hg²⁺was found in the four replacement mutants C23V/C58V/C89S/C283S containing both C136 and C155 indicating cross-reaction of Hg²⁺ with the two Cys residues. Inhibition kinetic analysis showed mixed inhibition by Hg²⁺ or competitive inhibition by CH<inf>3</inf>Hg⁺. HeLa cells or Zebrafish were treated with the more potent inhibitor. Ten micromolar HgCl<inf>2</inf> caused clear impairment of viability of HeLa cells. The transport assay in proteoliposomes with CACT extracted from treated cells showed that the transporter was inactivated and that DTE rescued the activity. Nearly identical results were observed with Zebrafish upon extraction of the CACT from the liver of the treated animals that, indeed, showed accumulation of the mercurial compound. (Chemical Presented).

The mitochondrial carnitine/acylcarnitine transporter (CACT) catalyses carnitine/acylcarnitine antiport. Its function has been defined mainly in proteoliposome experimental models. Despite CACT represents a putative site of?-oxidation regulation, few data are available about its modulation.Lysineacetylationisapost-translationalmodification(PTM) of a huge number of proteins. It has been shown that iper-acetylation of longchainacylCoAdehydrogenase(LCAD)impairsitsenzymaticactivity. It could be hypothesized that other components of the same pathway, suchasCACT,couldberegulatedbyasimilarmechanism.Indeed,CACTis partially acetylated in rat liver as revealed by WB analysis using an antiacetyl-Lys antibody. Acetylation can be reversed by the mitochondrial deacetylase SIRT3. After treatment of the mitochondrial extract with SIRT3, the CACT activity, assayed in proteoliposomes, increases with respect to the untreated control. The half-saturation constant is not influenced, while the Vmax is increased. The kinetic data suggests that steric hindrance of acetyl groups impairs conformational changes, rather than substrate binding. Recently, it was shown that acetylation of mitochondrial proteins also occur by a non-enzymatic pathway under conditionsofreducedAcetyl-CoAbuffering[1].RecombinantCACTwhich is not acetylated was incubated with acetyl-CoA and then subjected to WB withanti-acetyl-Lys antibodyand transportassay. Datashowed that non-enzymatic acetylation of CACT occurs and impairs its activity. In conclusion,CACTisregulatedbyacetylationrepresentingacontrolsiteof beta-oxidation pathway togetherwith LCAD.

The effect of omeprazole on the mitochondrial carnitine/acylcarnitine transporter has been studied in proteoliposomes.Externally added omeprazole inhibited the carnitine/carnitine antiport catalysed by the transporter. The inhibition waspartially reversed by DTE indicating that it was caused by the covalent reaction of omeprazole with Cys residue(s). Inhibitionof the C-less mutant transporter indicated also the occurrence of an alternative non-covalent mechanism. The IC50 of theinhibition of the WT and the C-less CACT by omeprazole were 5.4 mM and 29 mM, respectively. Inhibition kinetics showednon competitive inhibition of the WT and competitive inhibition of the C-less. The presence of carnitine or acylcarnitinesduring the incubation of the proteoliposomes with omeprazole increased the inhibition. Using site-directed Cys mutants itwas demonstrated that C283 and C136 were essential for covalent inhibition. Molecular docking of omeprazole with CACTindicated the formation of both covalent interactions with C136 and C283 and non-covalent interactions in agreement withthe experimental data.

S-nitrosylation of the mitochondrial carnitine/acylcarnitine transporter (CACT) has been investigated on the native and the recombinant proteins reconstituted in proteoliposomes, and on intact mitochondria. The widely-used NO-releasing compound, GSNO, strongly inhibited the antiport measured in proteoliposomes reconstituted with the native CACT from rat liver mitochondria or the recombinant rat CACT over-expressed in E. coli. Inhibition was reversed by the reducing agent dithioerythritol, indicating a reaction mechanism based on nitrosylation of Cys residues of the CACT. The half inhibition constant (IC50) was very similar for the native and recombinant proteins, i.e., 74 and 71 ?M, respectively. The inhibition resulted to be competitive with respect the substrate, carnitine. NO competed also with NEM, correlating well with previous data showing interference of NEM with the substrate transport path. Using a site-directed mutagenesis approach on Cys residues of the recombinant CACT, the target of NO was identified. C136 plays a major role in the reaction mechanism. The occurrence of S-nitrosylation was demonstrated in intact mitochondria after treatment with GSNO, immunoprecipitation and immunostaining of CACT with a specific anti NO-Cys antibody. In parallel samples, transport activity of CACT measured in intact mitochondria, was strongly inhibited after GSNO treatment. The possible physiological and pathological implications of the post-translational modification of CACT are discussed.

The carnitine/acylcarnitine transporter (CACT; SLC25A20) mediates an antiport reaction allowing entry of acyl moieties in the form of acylcarnitines into the mitochondrial matrix and exit of free carnitine. The transport function of CACT is crucial for the beta-oxidation pathway. In this work, it has been found that CACT is partially acetylated in rat liver mitochondria as demonstrated by anti-acetyl-lys antibody immunostaining. Acetylation was reversed by the deacetylase Sirtuin 3 in the presence of NAD(+). After treatment of the mitochondrial extract with the deacetylase, the CACT activity, assayed in proteoliposomes, increased. The half-saturation constant of the CACT was not influenced, while the V max was increased by deacetylation. Sirtuin 3 was not able to deacetylate the CACT when incubation was performed in intact mitoplasts, indicating that the acetylation sites are located in the mitochondrial matrix. Prediction on the localization of acetylated residues by bioinformatics correlates well with the experimental data. Recombinant CACT treated with acetyl-CoA was partially acetylated by non-enzymatic mechanism with a corresponding decrease of transport activity. The experimental data indicate that acetylation of CACT inhibits its transport activity, and thus may contribute to the regulation of the mitochondrial beta-oxidation pathway.

Proteoliposomes represent a suitable and up to date tool for studying membranetransporters which physiologically mediate absorption, excretion, trafficking andreabsorption of nutrients and metabolites. Using recently developed reconstitutionstrategies, transporters can be inserted in artificial bilayers with the same orientation as inthe cell membranes and in the absence of other interfering molecular systems. Thesemethodologies are very suitable for studying kinetic parameters and molecularmechanisms. After the first applications on mitochondrial transporters, in the last decade,proteoliposomes obtained with optimized methodologies have been used for studyingplasma membrane transporters and defining their functional and kinetic properties andstructure/function relationships. A lot of information has been obtained which has clarifiedand completed the knowledge on several transporters among which the OCTN sub-familymembers, transporters for neutral amino acid, B0AT1 and ASCT2, and others.Transporters can mediate absorption of substrate-like derivatives or drugs, improving theirbioavailability or can interact with these compounds or other xenobiotics, leading toside/toxic effects. Therefore, proteoliposomes have recently been used for studying theinteraction of some plasma membrane and mitochondrial transporters with toxiccompounds, such as mercurials, H2O2 and some drugs. Several mechanisms have beendefined and in some cases the amino acid residues responsible for the interaction have been identified. The data obtained indicate proteoliposomes as a novel and potentially importanttool in drug discovery.

The structure/function relationships of charged residues of the human mitochondrial carnitine/acylcarnitine carrier, which are conserved in the carnitine/acylcarnitine carrier subfamily and exposed to the water-filled cavity of carnitine/acylcarnitine carrier in the c-state, have been investigated by site-directed mutagenesis. The mutants were expressed in Escherichia coil, purified and reconstituted in liposomes, and their transport activity was measured as (3)H-carnitine/carnitine antiport. The mutants K35A, E132A, D179A and R275A were nearly inactive with transport activities between 5 and 10% of the wild-type carnitine/acylcarnitine carrier. R178A, K234A and D231A showed transport function of about 15% of the wild-type carnitine/acylcarnitine carrier. The substitutions of the other residues with alanine had little or no effect on the carnitine/acylcarnitine carrier activity. Marked changes in the kinetic parameters with three-fold higher Km and lower Vmax values with respect to the wild-type carnitine/acylcarnitine carrier were found when replacing Lys-35, Glu-132, Asp-179 and Arg-275 with alanine. Double mutants exhibited transport activities and kinetic parameters reflecting those of the single mutants; however, lack of D179A activity was partially rescued by the additional mutation R178A. The results provide evidence that Arg-275, Asp-179 and Arg-178, which protrude into the carrier's internal cavity at about the midpoint of the membrane, are the critical binding sites for carnitine. Furthermore, Lys-35 and Glu-132, which are very probably involved in the salt-bridge network located at the bottom of the cavity, play a major role in opening and closing the matrix gate.

Transport systems are hydrophobic proteins localized in cell membranes where they mediate transmembrane flow of nutrients, ions and any other compounds essential for cell metabolism. More than 400 transporters of the SoLuteCarrier (SLC) group are present in human cells. Transporters take contacts also with xenobiotics, thus mediating absorption and/or interaction with these exogenous compounds. Since drugs belong to xenobiotics, transporters gained interest also in drug discovery. Transporters differentially expressed in pathological conditions are exploited as drug targets. Among the methodologies for defining drug interactions, in silico ligand screening and intact cell transport assay were the most diffused, so far. The first is a predictive methodology based on docking chemicals to transporters. It presents limitations due to the small number of human transporter 3D structures that have to be constructed by homology modeling. Intact cells are used for testing effects of drugs and for validating predictions. The challenges of handling this very complex experimental system, are the interferences caused by other transporters and/or intracellular enzymes. Thus, methodologies with lower complexity are welcome. One of the most updated is the proteoliposome nanotechnology consisting in a cell mimicking phospholipid membrane in which a purified transporter is inserted. In this system, drug-transporter interaction can be studied defining kinetics and mechanisms. Several data have been obtained by proteoliposome nanotechnology. An overview of data obtained on the organic cation transporters OCTN1, OCTN2 and on the amino acid transporters ASCT2 and B0AT1 will be presented. Standardized procedures, expected to be pointed out, will enlarge the assay to High Throughput Screenings.

The carnitine/acylcarnitine carrier (CAC) is a transport protein of the inner mitochondrial membrane that belongs to the mitochondrial carrier protein family. In its cytosolic conformation the carrier consists of a bundle of six transmembrane alpha-helices, which delimit a water filled cavity opened towards the cytosol and closed towards the matrix by a network of interacting charged residues. Most of the functional data on this transporter come from studies performed with the protein purified from rat liver mitochondria or recombinant proteins from different sources incorporated into phospholipid vesicles (liposomes). The carnitine/acylcarnitine carrier transports carnitine and acylcarnitines with acyl chains of various lengths from 2 to 18 carbon atoms. The mammalian transporter exhibits higher affinity for acylcarnitines with longer carbon chains. The functional data indicate that CAC plays the important function of catalyzing transport of acylcarnitines into the mitochondria in exchange for intramitochondrial free carnitine. This results in net transport of fatty acyl units into the mitochondrial matrix where they are oxidized by the beta-oxidation enzymes. The essential role of the transporter in cell metabolism is demonstrated by the fact that alterations of the human gene SLC25A20 coding for CAC are associated with a severe disease known as carnitine carrier dificiency. This autosomal recessive disorder is characterized by life-threatening episodes of coma induced by fasting, cardiomyopathy, liver dysfunction, muscle weakness, respiratory distress and seizures. Until now 35 different mutations of CAC gene have been identified in carnitine carrier deficient patients. Some missense mutations concern residues of the signature motif present in all mitochondrial carriers. Diagnosis of carnitine carrier deficiency required biochemical and genetic tests; treatment is essentially limited to important dietetic measures. Recently, a pharmacological approach based on the used of statins and/or fibrates for the treatment of CAC-deficient patients with mild phenotype has been proposed.