Quantitative analysis of amino acid metabolism in liver cancer links glutamate excretion to nucleotide synthesis

Many cancer cells consume glutamine at high rates; counterintuitively, they simultaneously excrete glutamate, the first intermediate in glutamine metabolism. Glutamine consumption has been linked to replenishment of tricarboxylic acid cycle (TCA) intermediates and synthesis of adenosine triphosphate (ATP), but the reason for glutamate excretion is unclear. Here, we dynamically profile the uptake and excretion fluxes of a liver cancer cell line (HepG2) and use genome-scale metabolic modeling for in-depth analysis. We find that up to 30% of the glutamine is metabolized in the cytosol, primarily for nucleotide synthesis, producing cytosolic glutamate. We hypothesize that excreting glutamate helps the cell to increase the nucleotide synthesis rate to sustain growth. Indeed, we show experimentally that partial inhibition of glutamate excretion reduces cell growth. Our integrative approach thus links glutamine addiction to glutamate excretion in cancer and points toward potential drug targets.

A Novel Corynebacterium glutamicum L-Glutamate Exporter

ABSTRACT Besides metabolic pathways and regulatory networks, transport systems are also pivotal for cellular metabolism and hyperproduction of biochemicals using microbial cell factories. The identification and characterization of transporters are therefore of great significance for the understanding and engineering of transport reactions. Herein, a novel l -glutamate exporter, MscCG2, which exists extensively in Corynebacterium glutamicum strains but is distinct from the only known l -glutamate exporter, MscCG, was discovered in an industrial l -glutamate-producing C. glutamicum strain. MscCG2 was predicted to possess three transmembrane helices in the N-terminal region and located in the cytoplasmic membrane, which are typical structural characteristics of the mechanosensitive channel of small conductance. MscCG2 has a low amino acid sequence identity (23%) to MscCG and evolved separately from MscCG with four transmembrane helices. Despite the considerable differences between MscCG2 and MscCG in sequence and structure, gene deletion and complementation confirmed that MscCG2 also functioned as an l -glutamate exporter and an osmotic safety valve in C. glutamicum . Besides, transcriptional analysis showed that MscCG2 and MscCG genes were transcribed in similar patterns and not induced by l -glutamate-producing conditions. It was also demonstrated that MscCG2-mediated l -glutamate excretion was activated by biotin limitation or penicillin treatment and that constitutive l -glutamate excretion was triggered by a gain-of-function mutation of MscCG2 (A151V). Discovery of MscCG2 will enrich the understanding of bacterial amino acid transport and provide additional targets for exporter engineering. IMPORTANCE The exchange of matter, energy, and information with surroundings is fundamental for cellular metabolism. Therefore, studying transport systems that are essential for these processes is of great significance. Besides, transport systems of bacterial cells are usually related to product excretion as well as product reuptake, making transporter engineering a useful strategy for strain improvement. The significance of our research is in identifying and characterizing a novel l -glutamate exporter from the industrial workhorse Corynebacterium glutamicum , which will enrich the understanding of l -glutamate excretion and provide a new target for studying bacterial amino acid transport and engineering transport reactions.