Fifty years of the mitochondrial pyruvate carrier: New insights into its structure, function, and inhibition

Abstract

The mitochondrial pyruvate carrier (MPC) resides in the mitochondrial inner membrane, where it links cytosolic and mitochondrial metabolism by transporting pyruvate produced in glycolysis into the mitochondrial matrix. Due to its central metabolic role, it has been proposed as a potential drug target for diabetes, non-alcoholic fatty liver disease, neurodegeneration, and cancers relying on mitochondrial metabolism. Little is known about the structure and mechanism of MPC, as the proteins involved were only identified a decade ago and technical difficulties concerning their purification and stability have hindered progress in functional and structural analyses. The functional unit of MPC is a hetero-dimer comprising two small homologous membrane proteins, MPC1/MPC2 in humans, with the alternative complex MPC1L/MPC2 forming in the testis, but MPC proteins are found throughout the tree of life. The predicted topology of each protomer consists of an amphipathic helix followed by three transmembrane helices. An increasing number of inhibitors are being identified, expanding MPC pharmacology and providing insights into the inhibitory mechanism. Here, we provide critical insights on the composition, structure, and function of the complex and we summarize the different classes of small molecule inhibitors and their potential in therapeutics.

Keywords: metabolism; mitochondria; pyruvate transport; small molecule inhibitors; transport mechanism.

FIGURE 1

Biochemical pathways involved in glycolysis (red), tricarboxylic acid cycle (TCA) (red), respiratory chain (cyan), gluconeogenesis (blue), malate–aspartate shuttle (black), de novo lipogenesis and beta oxidation (red) are shown schematically. Conversion of malate to pyruvate is shown in purple. The MPC dimer is shown in red/orange and other mitochondrial carriers in yellow (aspartate/glutamate carrier (citrin), ADP/ATP carrier (AAC), carnitine/acylcarnitine (CAC), phosphate carrier (PIC), and oxoglutarate carrier (OGC)). The respiratory chain complexes 1–4 (CI–CIV) and acyl‐CoA dehydrogenases (FI), electron transfer flavoprotein (FII), and ETF‐ubiquinone oxidoreductase (FIII) are shown in green and the dimer of ATP synthase in blue. The voltage‐gated anion channel (VDAC) in the outer membrane is shown in gray. Key metabolites, such as phosphate (Pi, purple), ADP (orange), ATP (red) and ubiquinone (Q, brown) are shown, and protons are black circles with a plus sign.

FIGURE 2

Schematic representation of the functional yeast (A) and human (B) mitochondrial pyruvate carrier (MPC) dimers. Pyruvate transport activity measured in yeast Mpc1/Mpc3 (C) or human MPC1L/MPC2 (D) proteoliposomes in comparison with empty liposomes at a ΔpH of 1.6. Oligomeric state analysis for the purified yeast Mpc1/Mpc3 (E) and human MPC1L/MPC2 (F) by SEC‐MALLS. In (E) and (F), the masses of the protein–detergent–lipid complex (PDL, gray), the detergent–lipid micelle (DL, blue), and the protein (P, red) are indicated.

FIGURE 3

(A) Sequence alignment of human and yeast MPC proteins, generated by Jalview. The residues are labeled according to the Zappo coloring scheme, where aliphatic, polar, aromatic, positively charged, negatively charged, Pro/Gly, and Cys, are colored pink, green, orange, blue, red, magenta, and yellow, respectively. (B) Average hydropathy score for each position across MPC proteins from different species; MPC1 (n = 96), MPC2 (n = 132), and MPC1L (n = 60). Sequences were aligned using Jalview and each residue was given a hydropathy score following the Kyte–Doolittle matrix. Moving averages across 16 residues were used to calculate the final score for each position, after which all three plots were aligned according to position of the predicted transmembrane helices. Portions after the final transmembrane helix were truncated in each alignment. The secondary structure predictions given are based on AlphaFold structural models, as well as the hydropathy plots.

FIGURE 4

Comparison of MPC with the SemiSWEET transporter. (A) Vibrio sp SemiSweet transporter protomer structure (PDB: 4QND). (B–G) Structural predictions of each human and yeast MPC protomer. AlphaFold predictions, are shown in bold colors, while predictions from trRosetta, are shown in lighter shades of the same color‐scheme. In (H, I), the homo‐dimer of the SemiSWEET is shown, as well as a predicted structure of the hMPC1/MPC2 hetero‐dimer. In all panels, transmembrane helices TM1, TM2, and TM3 are shown in blue, yellow, and red, respectively, with the N‐terminal amphipathic helix shown in white. The C‐terminal extensions in yeast MPC proteins, predicted to be helical, are shown in gray. Root mean square deviation values, comparing the predictions by AlphaFold and trRosetta, are indicated.

FIGURE 5

Common chemical properties of key MPC inhibitors. 2D models of pyruvate and selected MPC inhibitors are shown with key chemical properties highlighted. Polar groups mimicking pyruvate are shown in red, while hydrophobic ring features are shown in yellow spheres. IC₅₀ measurements are also stated for compounds, where available.