Effect of Copper on the Mitochondrial Carnitine/Acylcarnitine Carrier Via Interaction with Cys136 and Cys155. Possible Implications in Pathophysiology.

Nicola Giangregorio,Mario Prejanò,Tiziana Marino,Cesare Indiveri,Nino Russo,Annamaria Tonazzi,Lara Console

doi:10.3390/molecules25040820

Abstract

The effect of copper on the mitochondrial carnitine/acylcarnitine carrier (CAC) was studied. Transport function was assayed as [3H]carnitine/carnitine antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Cu2+ (as well as Cu+) strongly inhibited the native transporter. The inhibition was reversed by GSH (reduced glutathione) or by DTE (dithioerythritol). Dose-response analysis of the inhibition of the native protein was performed from which an IC50 of 1.6 µM for Cu2+ was derived. The mechanism of inhibition was studied by using the recombinant WT or Cys site-directed mutants of CAC. From the dose-response curve of the effect of Cu2+ on the recombinant protein, an IC50 of 0.28 µM was derived. Inhibition kinetics revealed a non-competitive type of inhibition by Cu2+. However, a substrate protection experiment indicated that the interaction of Cu2+ with the protein occurred in the vicinity of the substrate-binding site. Dose-response analysis on Cys mutants led to much higher IC50 values for the mutants C136S or C155S. The highest value was obtained for the C136/155S double mutant, indicating the involvement of both Cys residues in the interaction with Cu2+. Computational analysis performed on the WT CAC and on Cys mutants showed a pattern of the binding energy mostly overlapping the binding affinity derived from the dose-response analysis. All the data concur with bridging of Cu2+ with the two Cys residues, which blocks the conformational changes required for transport cycle.

Highlights

Copper is an essential cofactor of many cellular molecular systems
After removal of the unreacted compounds, mitochondria were solubilized with detergents, and the extracted carnitine/acylcarnitine carrier (CAC) was reconstituted in proteoliposomes for transport assay as [3 H]carnitine/carnitine antiport
Since it is known that copper can exist as Cu+ in cells, we have tested the monovalent cation in some experiments

Summary

Introduction

Copper is an essential cofactor of many cellular molecular systems. it needs to be absorbed with diet and distributed to the various regions of the body for reaching the final destination and assembly into proteins. In the intermembrane space (IMS), copper may give side interactions with other molecular components, which possess amino acid groups reactive towards copper [6] In this framework, it was previously demonstrated that the ornithine/citrulline transporter (SLC25A15) can be targeted by copper. The interaction causes alterations of the transport functions that result in inactivation of the physiological transport activity or in the conversion of the transporter into an unspecific pore [7] Another member of the mitochondrial carrier family, which has a strong propensity to react with heavy metals, is the carnitine acylcarnitine carrier (CAC, SLC25A20) [8].

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Conclusion