Inhibition of the carnitine acylcarnitine carrier by carbon monoxide reveals a novel mechanism of action with non-metal-containing proteins

Tonazzi, Annamaria; Giangregorio, Nicola; Console, Lara; Calvano, Cosima Damiana; Prejanò, Mario; Scalise, Mariafrancesca; Incampo, Giovanna; Marino, Tiziana; Russo, Nino; Cataldi, Tommaso R. I.; Indiveri, Cesare

doi:10.1016/j.freeradbiomed.2022.06.244

Cited by 3 publications

(1 citation statement)

References 44 publications

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“…Then, the reactivity of the transporter toward alkylating and mercury-related compounds was characterized in a proteoliposome experimental model harboring the transporter purified from rat liver mitochondria [ 7 ]. In the last ten years, by using integrated approaches of intact mitochondria, cell lines, site-directed mutagenesis, and bioinformatics, the CAC has been shown to sense thiol-reactive physiological agents such as hydrogen peroxide, nitric oxide, hydrogen sulfide, glutathione and, very recently, carbon monoxide [ 8 , 9 , 10 , 11 , 12 ]. The response to these physiological effectors is mediated by a crucial couple of Cys resides, i.e., C136 and C155, located in a peculiar microenvironment that makes these two residues highly sensitive to thiol-reactive compounds giving the CAC a specific relevance to oxidative and/or inflammatory processes [ 9 , 10 , 11 ].…”

Section: Introductionmentioning

confidence: 99%

Inhibition of the Mitochondrial Carnitine/Acylcarnitine Carrier by Itaconate through Irreversible Binding to Cysteine 136: Possible Pathophysiological Implications

et al. 2023

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Background: The carnitine/acylcarnitine carrier (CAC) represents the route of delivering acyl moieties to the mitochondrial matrix for accomplishing the fatty acid β-oxidation. The CAC has a couple of Cys residues (C136 and C155) most reactive toward ROS and redox signaling compounds such as GSH, NO, and H2S. Among physiological compounds reacting with Cys, itaconate is produced during inflammation and represents the connection between oxidative metabolism and immune responses. The possible interaction between the CAC and itaconate has been investigated. Methods: the modulatory effects of itaconate on the transport activity of the native and recombinant CAC were tested using the proteoliposome experimental model together with site-directed mutagenesis and computational analysis. Results: Itaconate reacts with the CAC causing irreversible inhibition. Dose–response experiment performed with the native and recombinant protein showed IC50 for itaconate of 11 ± 4.6 mM and 8.4 ± 2.9 mM, respectively. The IC50 decreased to 3.8 ± 1.0 mM by lowering the pH from pH 7.0 to pH 6.5. Inhibition kinetics revealed a non-competitive type of inhibition. C136 is the main target of itaconate, as demonstrated by the increased IC50 of mutants in which this Cys was substituted by Val. The central role of C136 was confirmed by covalent docking. Administration of dimethyl itaconate to HeLa cells inhibited the CAC transport activity, suggesting that itaconate could react with the CAC also in intact cells.

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Section: Introductionmentioning

confidence: 99%

Inhibition of the Mitochondrial Carnitine/Acylcarnitine Carrier by Itaconate through Irreversible Binding to Cysteine 136: Possible Pathophysiological Implications

et al. 2023

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Pd-catalyzed oxidative carbonylation of sulfurated substrates: Theoretical mechanistic outcomes

Prejanò,

Mancuso,

Marino

et al. 2023

Journal of Catalysis

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The Mycotoxin Patulin Inhibits the Mitochondrial Carnitine/Acylcarnitine Carrier (SLC25A20) by Interaction with Cys136 Implications for Human Health

Giangregorio

Tonazzi

Calvano

et al. 2023

IJMS

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The effect of mycotoxin patulin (4-hydroxy-4H-furo [3,2c] pyran-2 [6H] -one) on the mitochondrial carnitine/acylcarnitine carrier (CAC, SLC25A20) was investigated. Transport function was measured as [3H]-carnitineex/carnitinein antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Patulin (PAT) inhibited both the mitochondrial native and recombinant transporters. The inhibition was not reversed by physiological and sulfhydryl-reducing reagents, such as glutathione (GSH) or dithioerythritol (DTE). The IC50 derived from the dose–response analysis indicated that PAT inhibition was in the range of 50 µM both on the native and on rat and human recombinant protein. The kinetics process revealed a competitive type of inhibition. A substrate protection experiment confirmed that the interaction of PAT with the protein occurred within a protein region, including the substrate-binding area. The mechanism of inhibition was identified using the site-directed mutagenesis of CAC. No inhibition was observed on Cys mutants in which only the C136 residue was mutated. Mass spectrometry studies and in silico molecular modeling analysis corroborated the outcomes derived from the biochemical assays.

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Inhibition of the carnitine acylcarnitine carrier by carbon monoxide reveals a novel mechanism of action with non-metal-containing proteins

Cited by 3 publications

References 44 publications

Inhibition of the Mitochondrial Carnitine/Acylcarnitine Carrier by Itaconate through Irreversible Binding to Cysteine 136: Possible Pathophysiological Implications

Inhibition of the Mitochondrial Carnitine/Acylcarnitine Carrier by Itaconate through Irreversible Binding to Cysteine 136: Possible Pathophysiological Implications

Pd-catalyzed oxidative carbonylation of sulfurated substrates: Theoretical mechanistic outcomes

The Mycotoxin Patulin Inhibits the Mitochondrial Carnitine/Acylcarnitine Carrier (SLC25A20) by Interaction with Cys136 Implications for Human Health

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