A Loss of Function of the Mitochondrial Branched-Chain Aminotransferase (BCATm) Leads to Increased Glycolytic and Oxidative Metabolism in Activated CD4+ T Cells

Objectives T cells use the amino acid leucine to respond to their increased biosynthetic demands during activation. However, once inside T cells, leucine is subjected to degradation, which is initiated by the mitochondrial branched-chain aminotransferase (BCATm) that catalyzes the reversible transamination of leucine. We hypothesized that if BCATm is absent from T cells, this would provide more intracellular leucine to stimulate T cell metabolism. Methods To explore the dependence of T cells on BCATm function, we isolated CD4+ T cells from spleens of wild type (WT) and BCATm global knockout (KO) mice, and after cell activation with anti-CD3 and anti-CD28 for 24 h, we measured leucine transamination, glycolysis, mitochondrial respiration and ATP synthesis, the activity of the mammalian target of rapamycin (mTOR) pathway, and the release of IFN-γ. Results The global deletion of BCATm resulted in a 1.8-fold reduction in leucine transamination and a 1.2-fold increase in the intracellular leucine concentrations in activated CD4+ T cells from BCATmKO mice. These T cells demonstrated 4.0– and 5.0-fold increases in glycolysis and glycolytic capacity, along with 1.8– and 2-0-fold increases in the maximal respiration and spare respiratory capacity when compared to WT T cells after 24 h of activation. In addition, mTOR signaling was more active in BCATmKO T cells and their IFN-γ release was increased by 2.1-fold relative to WT T cells. Conclusions The results suggested that leucine catabolism at the BCATm step negatively affects T cell metabolism by limiting glycolytic intermediates for biosynthetic needs and mitochondrial respiration for energy. Thus, leucine catabolism is regarded as a metabolic checkpoint of T cells that may prove useful for therapeutic purposes. Funding Sources Des Moines University, (IOER-112-3705 to EAS), the National Institute of Health (DK 34,738 to SMH).

More Than One HMG-CoA Lyase: The Classical Mitochondrial Enzyme Plus the Peroxisomal and the Cytosolic Ones

There are three human enzymes with HMG-CoA lyase activity that are able to synthesize ketone bodies in different subcellular compartments. The mitochondrial HMG-CoA lyase was the first to be described, and catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetate and acetyl-CoA, the common final step in ketogenesis and leucine catabolism. This protein is mainly expressed in the liver and its function is metabolic, since it produces ketone bodies as energetic fuels when glucose levels are low. Another isoform is encoded by the same gene for the mitochondrial HMG-CoA lyase (HMGCL), but it is located in peroxisomes. The last HMG-CoA lyase to be described is encoded by a different gene, HMGCLL1, and is located in the cytosolic side of the endoplasmic reticulum membrane. Some activity assays and tissue distribution of this enzyme have shown the brain and lung as key tissues for studying its function. Although the roles of the peroxisomal and cytosolic HMG-CoA lyases remain unknown, recent studies highlight the role of ketone bodies in metabolic remodeling, homeostasis, and signaling, providing new insights into the molecular and cellular function of these enzymes.

An essential bifunctional enzyme in Mycobacterium tuberculosis for itaconate dissimilation and leucine catabolism

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for ∼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in l-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional β-hydroxyacyl-CoA lyase and that deletion of the rv2498c gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an l-leucine catabolic pathway that proceeds via an unprecedented (R)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature.

Discovery of a novel stereospecific β-hydroxyacyl-CoA lyase/thioesterase shared by three metabolic pathways in Mycobacterium tuberculosis

The vast number of poorly characterised enzymes in Mycobacterium tuberculosis (Mtb) is one of the key barriers precluding a better understanding of the biology that underpins pathogenesis. Here, we investigated the Mtb orphan enzyme Rv2498c to delineate its physiological role. Our results from in vitro enzymatic assays, phylogenetic analysis, X-ray crystallography and in vivo Mtb experiments, de-orphan Rv2498c as a multi-functional β-hydroxyacyl-CoA lyase/thioesterase (β-HAClyase/thioesterase) that participates in three different metabolic pathways: L-leucine catabolism, itaconate dissimilation, and glyoxylate shunt. Moreover, the deletion of the rv2498c gene from the Mtb genome resulted in attenuation in the mouse model compared to infection with the parent strain. To the best of our knowledge, this is the first report of an (R)-3-hydroxyl-3-methylglutaryl-CoA for leucine catabolism and an itaconate-specific resistance mechanism in Mtb.

An asymptomatic mother diagnosed with 3-methylcrotonyl-CoA carboxylase deficiency after newborn screening

Abstract3-Methylcrotonyl-CoA carboxylase (3-MCC) deficiency is an autosomal recessively inherited disease of leucine catabolism. It is the most commonly observed organic acidemia where tandem mass spectrometry can be performed in newborn screening. The clinical phenotypes may differ from neurological involvement in newborns to asymptomatic adults. Diagnosis is made by increased 3-hydroxyisovaleric acid in blood and 3-methylcrotonylglycine in urine.We would like to present an interesting case of a 32-year-old asymptomatic mother, who was investigated metabolically and diagnosed with 3-MCC deficiency, after a 7-day-old healthy baby referred to our unit with the preliminary diagnosis of organic academia during her extended newborn screening.All of the metabolic findings of the baby were normal except for very low carnitine levels. Her mother’s total and free carnitine levels were also extremely low. Urine organic acid analysis revealed excessively increased 3-methylcrotonylglycine and 3-hydroxyisovaleric acid. Acylcarnitine profile showed markedly elevated C5 hydroxy 3 hydroxyisovalerylcarnitine and decreased C2 acetylcarnitine. In order to confirm the diagnosis of 3-methylcrotonylglycinuria, molecular analysis was done, and IVS3-1G>C/p.T556I compound heterozygote mutation was detected. p.T556I is a novel mutation.We would like to emphasize performing extended metabolic investigations in case of suspicion of metabolic disease in order to diagnose metabolic diseases both in babies and in asymptomatic mothers.