Key features of inhibitor binding to the human mitochondrial pyruvate carrier hetero-dimer

The mitochondrial pyruvate carrier (MPC) has emerged as a promising drug target for metabolic disorders, including non-alcoholic steatohepatitis and diabetes, metabolically dependent cancers and neurodegenerative diseases. Human MPC is a protein complex, but the composition of its active form is debated and the mechanisms of transport and inhibition are not resolved. We have recombinantly expressed and purified the human hetero-complex MPC1L/MPC2 and demonstrate that it is a functional hetero-dimer, like the yeast MPC hetero-dimers. Unlike the latter, human MPC1L/MPC2 binds the known inhibitors with high potencies. We identify the essential chemical features shared between these structurally diverse inhibitors and demonstrate that high affinity binding is not attributed to covalent bond formation with MPC cysteines, as previously thought. We also identify 14 new inhibitors of MPC, one outperforming the most potent compound UK5099 by tenfold. Two of them are the commonly prescribed drugs entacapone and nitrofurantoin, suggesting possible off-target mechanisms associated with their adverse effects. This work advances our understanding of MPC inhibition and will accelerate the development of clinically relevant MPC modulators.

Peroxisomal support of mitochondrial respiratory efficiency promotes ER stress survival

Endoplasmic reticulum stress (ERS) occurs when cellular demand for protein folding exceeds the capacity of the organelle. Adaptation and cell survival in response to ERS requires a critical contribution by mitochondria and peroxisomes. During ERS response, mitochondrial respiration increases to ameliorate reactive oxygen species (ROS) accumulation; we now show in yeast that peroxisome abundance also increases to promote an adaptive response. In pox1▵ cells, defective in peroxisomal ß oxidation of fatty acids, respiratory response to ERS is impaired, and ROS accrues. However, respiratory response to ERS is rescued, and ROS production is mitigated in pox1▵ cells by overexpression of Mpc1, the mitochondrial pyruvate carrier that provides another source of acetyl CoA to fuel the TCA cycle and oxidative phosphorylation. Using proteomics, select mitochondrial proteins were identified that undergo upregulation by ERS to remodel respiratory machinery. Several peroxisome-based proteins were also increased, corroborating the peroxisomal role in ERS adaptation. Finally, ERS stimulates assembly of respiratory complexes into higher order supercomplexes, underlying increased electron transfer efficiency. Our results highlight peroxisomal and mitochondrial support for ERS adaptation to favor cell survival.

Immunometabolic hijacking of immune cells by a Pseudomonas aeruginosa quorum-sensing signal

Macrophages utilize metabolic pathways to generate energy and metabolites that may be vulnerable to pathogen hijacking to favor pathogen survival and persistence. It is unclear how bacterial pathogens alter metabolic pathways in immune cells for their benefit and persistence in the infected host. We have shown that the Pseudomonas aeruginosa quorum sensing (QS) signal molecule 2-aminoacetophenone (2-AA) allows pathogen persistence in host tissues by triggering host tolerization via histone deacetylase (HDAC)1-mediated epigenetic reprogramming. Here, we provide strong evidence that 2-AA-meditated persistence is linked to specific metabolic pathway alterations that reduce energy availability and biosynthetic macromolecules involved in host immune responses. 2-AA promotes a Warburg-like metabolic reprogramming effect, thereby increasing levels of lactate, which repressed inflammatory signaling in macrophages. Moreover, it interferes with pyruvate translocation to mitochondria, reducing mitochondrial (mt)-oxidative phosphorylation (OXPHOS) due to down-regulation of estrogen-regulated receptor (ERR)α and mitochondrial pyruvate carrier (MPC)-1. This metabolic reprogramming dampened energy production, reduced the acetyl-CoA pool, and generated an anti-inflammatory milieu that favors P. aeruginosa persistence. These findings provide evidence of first-in-class metabolic reprogramming in immune cells mediated by a QS signaling molecule. The specific metabolic programs affected provide insights that may guide the design and development of therapeutics and protective interventions against pathogen-induced immunometabolic alterations and persistence factors.

Progenitor-intrinsic Metabolic Sensing Promotes Hematopoietic Homeostasis

Hematopoietic homeostasis is maintained by stem and progenitor cells in part by extrinsic feedback cues triggered by mature cell loss. We demonstrate a different mechanism by which hematopoietic progenitors intrinsically anticipate and prevent the loss of mature progeny through metabolic switches. We examined hematopoiesis in mice conditionally deficient in long-chain fatty acid oxidation (carnitine palmitoyltransferase 2, Cpt2), glutaminolysis (glutaminase, Gls), or mitochondrial pyruvate import (mitochondrial pyruvate carrier 2, Mpc2). While genetic ablation of Cpt2 or Gls minimally impacted most blood lineages, deletion of Mpc2 led to a sharp decline in mature myeloid cells. However, MPC2-deficient myeloid cells rapidly recovered due to a transient increase in myeloid progenitor proliferation. Competitive bone marrow chimera and stable isotope tracing experiments demonstrated that this proliferative burst was intrinsic to MPC2-deficient progenitors and accompanied by a metabolic switch to glutaminolysis. Thus, hematopoietic progenitors intrinsically adjust to metabolic perturbations independently of feedback from downstream mature cells to maintain homeostasis.

UK5099 inhibits macrophage activation independent of mitochondrial pyruvate carrier mediated metabolism

Glycolysis is essential for the classical activation of macrophages (M1), but how glycolytic pathway metabolites engage in this process remains to be elucidated. Glycolysis culminates in the production of pyruvate, which can be transported into the mitochondria by the mitochondrial pyruvate carrier (MPC) followed by conversion to citrate and utilization in the TCA cycle. Alternatively, pyruvate can be metabolized to lactate under aerobic conditions, which had been considered to be the dominant route in the setting of classical macrophage activation. However, based on studies that used UK5099 as a MPC inhibitor and showed reduction in key inflammatory cytokines, the mitochondrial route has been considered to be of significance for M1 activation as well. Herein, using a genetic depletion model, we found that MPC is dispensable for metabolic reprogramming and the activation of M1. While UK5099 reaches maximal MPC inhibitory capacity at approximately 2–5µM, higher concentrations are required to inhibit inflammatory cytokine production in M1 and this is independent of MPC expression. Apart from MPC inhibition, UK5099 at high doses impairs glutamate oxidation, mitochondrial membrane potential and HIF-1α stabilization. Taken together, UK5099 inhibits inflammatory responses in M1 macrophages due to effects other than MPC inhibition.

A non-canonical convergence of carbohydrate and glutamine metabolism is required after metabolic rewiring in 3D environment

The fate of pyruvate, which is modulated mitochondrial pyruvate carrier (MPC) activity, is a defining metabolic feature in many cancers. Diffuse large B-cell lymphomas (DLBCLs) are a genetically and metabolically heterogenous cancer. Although MPC expression and activity differed between DLBCL subgroups, mitochondrial pyruvate oxidation was uniformly minimal. Mitochondrial pyruvate was instead robustly consumed by glutamate pyruvate transaminase 2 to support α-ketoglutarate production as part of glutamine catabolism. This led us to discover that glutamine exceeds pyruvate as a carbon source for the TCA cycle, but, MPC function is required to enable GPT2-mediated glutamine catabolism. Furthermore, we found that MPC inhibition only decreased DLBCL proliferation in a solid culture environment, but not in a suspension environment. Thus, the non-canonical connection between the consumption and assimilation of carbohydrates and glutamine in DLBCLs enables their proliferation in a solid 3D environment.