Oxoglutarate dehydrogenase coordinates myofibril growth by maintaining amino acid homeostasis

Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin filaments. The actin and myosin filaments are organized into repeated units called sarcomeres, which form the myofibril cables. Muscle contraction is achieved by the simultaneous shortening of sarcomeres and for a highly coordinated contraction to occur all sarcomeres should have the same size. Muscles have evolved a variety of ways to ensure sarcomere homogeneity, one example being the controlled oligomerization of Zasp proteins that sets the diameter of the myofibril. To understand how Zasp proteins effect myofibril growth, we looked for Zasp-binding proteins at the Z-disc. We found that the E1 subunit of the oxoglutarate dehydrogenase complex is recruited to the Z-disc by Zasp52 and is required to sustain myofibril growth. By making specific mutants, we show that its enzymatic activity is important for myofibril growth, and that the other two subunits of the complex are also required for myofibril formation. Using super resolution microscopy, we revealed the overall organization of the complex at the Z-disc. Then, using metabolomic analysis, we uncovered an amino acid balance defect affecting protein synthesis, that we also confirmed by genetic tools. In summary, we show that Zasp controls the local amino acid pool responsible for myofibril growth by recruiting the OGDH complex to the Z-disc.

A non-canonical role for glutamate decarboxylase 1 in cancer cell amino acid homeostasis, independent of the GABA shunt

Glutamate decarboxylase 1 (GAD1) is best known for its role in producing the neurotransmitter γ-amino butyric acid (GABA) as part of the “GABA shunt” metabolic pathway, an alternative mechanism of glutamine anaplerosis for TCA cycle metabolism (Yogeeswari et al., 2005). However, understanding of the metabolic function of GAD1 in non-neuronal tissues has remained limited. Here, we show that GAD1 supports cancer cell proliferation independent of the GABA shunt. Despite its elevated expression in lung cancer tissue, GAD1 is not engaged in the GABA shunt in proliferating non-small cell lung cancer (NSCLC) cells, but rather is required for regulating amino acid homeostasis. Silencing GAD1 promotes a broad deficiency in amino acid uptake, leading to reduced glutamine-dependent TCA cycle metabolism and defects in serum- and amino acid-stimulated mTORC1 activation. Mechanistically, GAD1 regulates amino acid uptake through ATF4-dependent amino acid transporter expression including SLC7A5 (LAT1), an amino acid transporter required for branched chain amino acid (BCAA) uptake. Overexpression of LAT1 rescues the proliferative and mTORC1 signalling defects of GAD1-deficient tumor cells. Our results, therefore, define a non-canonical role for GAD1, independent of its characterised role in GABA metabolism, whereby GAD1 regulates amino acid homeostasis to maintain tumor cell proliferation.

A unified model of amino acid homeostasis in mammalian cells

Homeostasis is one of the fundamental concepts in physiology. Despite remarkable progress in our molecular understanding of amino acid transport, metabolism and signalling, it remains unclear by what mechanisms cytosolic amino acid concentrations are maintained. We propose that amino acid transporters are the primary determinants of intracellular amino acid levels. We show that a cell’s endowment with amino acid transporters can be deconvoluted by a logical series of experiments. This was used to computationally simulate amino acid translocation across the plasma membrane. For two different cancer cell lines and human myotubes, transport simulation generates cytosolic amino acid concentrations that are close to those observed in vitro. Perturbations of the system were replicated in silico and could be applied to systems where only transcriptomic data are available. The methodology developed in this study is widely applicable to other transport processes and explain amino acid homeostasis at the systems-level.

Benchmark examination of blood amino acids patterns in phenylketonuric neonates and young children on phenylalanine-restricted dietary treatment

Background Phe-restricted diets have been the basis of therapy for phenylketonurics; however, little is known how this treatment effects homeostasis of other important amino acids. This study aimed to describe blood amino acid patterns in neonates with phenylketonuria (PKU) and identify any effects of Phe restriction on these patterns in young children.Methods Neonate group (age 0-4 weeks): 45 PKU patients, 45 age-/sex-matched controls without PKU; 1-4 year-old group: 27 diet-treated PKU patients, 27 age-/sex-matched children without PKU. Concentrations of 11 amino acids were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) performed on dried blood spots.Results Elevated blood phenylalanine (Phe), arginine (Arg), citrulline (Cit), valine (Val) and methionine (Met) concentrations were observed in PKU neonates relative to controls (Phe, Arg, Cit, Val: P < 0.001; Met: P < 0.05), of which Phe, Arg, and Met levels could be either partially or completely restored with dietary intervention. Diet had no effect on elevated Cit and Val. Decreased blood tyrosine (Tyr) and proline (Pro) concentrations were observed in PKU neonates compared to controls (P < 0.001). Both amino acids could be near completely restored to normal with dietary treatment. No significant differences in alanine (Ala), leucine (Leu), ornithine (Orn) and glycine (Gly) concentrations were found in the PKU neonates and 1-4 year-old groups (P > 0.05).Conclusions Blood amino acid homeostasis is disrupted in neonates and young children with PKU. Although dietary intervention adjusts amino acid homeostasis in the direction of a healthy equilibrium, complete restoration is not achieved. This persistent disruption may represent a clinically significant barrier to achieving the best possible therapy for those with PKU. Use of laboratory technologies such as LC-MS/MS enable characterization of persistent blood amino acid disequilibrium in the treated phenylketonuric. Testing of this kind presents opportunity for customized treatment feedback that may allow even greater optimization of therapy for neonates and children with PKU.

Induction of GLUTAMINE DUMPER1 reveals a link between amino acid export, abscisic acid, and immune responses

ABSTRACTAmino acid homeostasis in plants is finely tuned to match developmental needs and response to adverse environments. Over-expression of the single-transmembrane domain protein GLUTAMINE DUMPER1 (GDU1) leads to increased amino acid export, reduced growth and constitutive induction of immune responses. We used an inducible gene expression system to tease apart the primary and secondary effects caused by GDU1, and demonstrated that the primary effect is increasing amino acid export, followed by increased amino acid content and abscisic acid (ABA) response, and a subsequent activation of defense responses. The GDU1-mediated hypersensitivity to ABA partially depended on the E3 ubiquitin ligase LOSS-OF-GDU1 2 (LOG2), a known GDU1 interactor. More importantly, the lysine catabolite pipecolic acid played a pivotal role in the GDU1-induced defense responses. This work unravels a novel relationship between amino acid transport, ABA and defense responses, potentially mediated by the GDU1-LOG2 complex, critical for understanding how plants respond to amino acid imbalance.ONE SENTENCE SUMMARYGenetically induced disturbance of amino acid homeostasis sequentially triggers responses to abiotic stresses and plant defenses to pathogens in Arabidopsis through undefined sensing mechanisms