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

Nicola Giangregorio; Annamaria Tonazzi; Lara Console; Mariafrancesca Scalise; Cesare Indiveri

doi:10.3390/biom13060993

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

Biomolecules. 2023 Jun 15;13(6):993. doi: 10.3390/biom13060993.

Authors

Nicola Giangregorio¹, Annamaria Tonazzi¹, Lara Console², Mariafrancesca Scalise², Cesare Indiveri^{1

2}

Affiliations

¹ National Research Council (CNR), Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), Via Amendola 122/O, 70126 Bari, Italy.
² Unit of Biochemistry and Molecular Biotechnology, Department DiBEST (Biologia, Ecologia, Scienze della Terra), University of Calabria, Via Bucci 4C, 87036 Arcavacata di Rende, Italy.

Abstract

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.

Keywords: ROS; SLC25A20; carnitine; carrier; cysteines; itaconate; mitochondria.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Carnitine / metabolism
Cysteine / metabolism
HeLa Cells / drug effects
Humans
Membrane Transport Modulators / pharmacology
Membrane Transport Proteins* / drug effects
Membrane Transport Proteins* / genetics
Membrane Transport Proteins* / metabolism
Mitochondria* / drug effects
Mitochondria* / metabolism

Substances

acylcarnitine
Carnitine
Cysteine
itaconic acid
Membrane Transport Proteins
SLC25A20 protein, human
Membrane Transport Modulators

Grants and funding

“SI.F.I.PA.CRO.DE.—Sviluppo e industrializzazione farmaci innovativi per terapia molecolare personalizzata PA.CRO.DE.” PON ARS01_00568. We acknowledge co-funding from Next Generation EU, in the context of the National Recovery and Resilience Plan, Investment PE8—Project Age-It: “Ageing Well in an Ageing Society”. This resource was co-financed by the Next Generation EU [DM 1557 11.10.2022]. The views and opinions expressed are those of the authors only and do not necessarily reflect those of the European Union or the European Commission. Neither the European Union nor the European Commission can be held responsible for them.