Cellular Amino Acid Levels Research Articles

AMPK regulates phagophore-to-autophagosome maturation.

Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example, by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK-mediated inhibition of phagophore tethering to donor membrane. Our results clarify AMPKs regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.

Mitochondrial Hyperfusion Via Metabolic Sensing of Regulatory Amino Acids

Here, we aimed to understand the relationship between nutrient starvation and mitophagy. We found that amino acid starvation of 4T1 breast cancer cells led to clear mitochondrial fusion, entirely consistent with the established model in which elongating mitochondria evade autophagosomes to sustain cellular bioenergetics. As we further dissected amino acid signals, we surprisingly found that supplementation of regulatory amino acids glutamine, leucine and arginine did not reverse, but led to strong mitochondrial hyperfusion. This hyperfusion response to Gln+Leu+Arg appeared distinct from other known remodelling mechanisms, but was dependent upon mitochondrial fusion proteins Mfn1 and Opa1. However, amino-acid dependent hyperfusion did not involve phosphorylation and inhibition of the mitochondrial fission protein Drp1. The ability of Gln+Leu+Arg supplementation to promote mitochondrial hyperfusion was independent of MTORC1, suggesting that the amino acids were being sensed by the mitochondria directly. Accordingly, metabolite profiling confirmed that Gln+Leu+Arg addback led to metabolic reprogramming and replenishment of cell amino acid and nucleotide pools. We found that loss of function in fumarate hydratase blocked Gln+Leu+Arg-dependent mitochondrial hyperfusion, suggesting that TCA-mediated metabolism of supplemented amino acids was driving remodelling. In addition, inhibition of inosine monophosphate dehydrogenase blocked Gln+Leu+Arg-dependent mitochondrial hyperfusion, suggesting a critical role of purine biosynthesis. Amino acid-dependent hyperfusion, like the previously reported stress-induced mitochondrial hyperfusion response, led to a decreased sensitivity towards apoptosis. Thus, we describe here a novel metabolic mechanism for the direct sensing of cellular amino acid levels that is able to control mitochondrial fusion and cell fate.

GATORopathies: The role of amino acid regulatory gene mutations in epilepsy and cortical malformations.

The mechanistic target of rapamycin (mTOR) pathway has been implicated in a growing number of malformations of cortical development (MCD) associated with intractable epilepsy. Mutations in single genes encoding mTOR pathway regulatory proteins have been linked to MCD such as focal cortical dysplasia (FCD) types IIa and IIb, hemimegalencephaly (HME), and megalencephaly. Recent studies have demonstrated that the GATOR1 protein complex, comprised of DEPDC5, NPRL3, and NPRL2, plays a pivotal role in regulating mTOR signaling in response to cellular amino acid levels and that mutations in DEPDC5, NPRL3, or NPRL2 are linked to FCD, HME, and seizures. Histopathological analysis of FCD and HME tissue specimens resected from individuals harboring DEPDC5, NPRL3, or NPRL2 gene mutations reveals hyperactivation of mTOR pathway signaling. Family pedigrees carrying mutations in either DEPDC5 or NPRL3 share clinical phenotypes of epilepsy and MCD, as well as intellectual and neuropsychiatric disabilities. Interestingly, some individuals with seizures associated with DEPDC5, NPRL3, or NPRL2 variants exhibit normal brain imaging suggesting either occult MCD or a role for these genes in non-lesional neocortical epilepsy. Mouse models resulting from knockdown or knockout of either Depdc5 or Nprl3 exhibit altered cortical lamination, neuronal dysmorphogenesis, and enhanced neuronal excitability as reported in models resulting from direct mTOR activation through expression of its canonical activator RHEB. The role of the GATOR1 proteins in regulating mTOR signaling suggest plausible options for mTOR inhibition in the treatment of epilepsy associated with mutations in DEPDC5, NPRL3, or NPRL2.

Hyperammonemia and proteostasis in cirrhosis.

Skeletal muscle loss or sarcopenia is a frequent complication of cirrhosis that adversely affects clinical outcomes. As skeletal muscle is the largest store of proteins in the body, proteostasis or protein homeostasis is required for maintenance of muscle mass. This review will focus on disordered skeletal muscle proteostasis in liver disease. Increased skeletal muscle uptake of ammonia initiates responses that result in disordered proteostasis including impaired protein synthesis and increased autophagy. The cellular response to the stress of hyperammonemia (hyperammonemic stress response, HASR) involves the coordinated action of diverse signaling pathways targeting the molecular mechanisms of regulation of protein synthesis. Transcriptional upregulation of myostatin, a TGFβ superfamily member, results in impaired mTORC1 signaling. Phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) also relates to decreased global protein synthesis rates and mTORC1 signaling. Ammonia also causes mitochondrial and bioenergetic dysfunction because of cataplerosis of α-ketoglutarate. Lowering ammonia, targeting components of HASR and regulating cellular amino acid levels can potentially restore proteostasis. Signaling via myostatin and eIF2α phosphorylation causes decreases in protein synthesis and mTORC1 activity with a parallel mitochondrial dysfunction and increased autophagy contributing to proteostasis perturbations during skeletal muscle hyperammonemia of liver disease.

Braking bad: stopping translation in hard times.

Braking bad: stopping translation in hard times.

Rapid Nutritional Remodeling of the Host Cell upon Attachment of Legionella pneumophila

Upon entry of Legionella pneumophila into amoebas and macrophages, host-mediated farnesylation of the AnkB effector enables its anchoring to the Legionella-containing vacuole (LCV) membrane. On the LCV, AnkB triggers docking of K(48)-linked polyubiquitinated proteins that are degraded by the host proteasomes to elevate cellular levels of amino acids needed for intracellular proliferation. Interference with AnkB function triggers L. pneumophila to exhibit a starvation response and differentiate into the nonreplicative phase in response to the basal levels of cellular amino acids that are not sufficient to power intracellular proliferation of L. pneumophila. Therefore, we have determined whether the biological function of AnkB is temporally and spatially triggered upon bacterial attachment to the host cell to circumvent a counterproductive bacterial differentiation into the nonreplicative phase upon bacterial entry. Here, we show that upon attachment of L. pneumophila to human monocyte-derived macrophages (hMDMs), the host farnesylation and ubiquitination machineries are recruited by the Dot/Icm system to the plasma membrane exclusively beneath sites of bacterial attachment. Transcription and injection of ankB is triggered by attached extracellular bacteria followed by rapid farnesylation and anchoring of AnkB to the cytosolic side of the plasma membrane beneath bacterial attachment, where K(48)-linked polyubiquitinated proteins are assembled and degraded by the proteasomes, leading to a rapid rise in the cellular levels of amino acids. Our data represent a novel strategy by an intracellular pathogen that triggers rapid nutritional remodeling of the host cell upon attachment to the plasma membrane, and as a result, a gratuitous surplus of cellular amino acids is generated to support proliferation of the incoming pathogen.

Abstract LB-129: Evidence from expression profiling, proteomics, metabolomics and FDG-PET of an estrogen-dependent metabolic switch in tuberin-null cells.

Abstract Lymphangioleiomyomatosis (LAM) is a female predominant lung disease characterized by widely-distributed nodules of ‘LAM’ cells and progressive cyst formation that leads to respiratory failure. LAM cells typically have mutation or inactivation of TSC2, leading to mTORC1 activation. We previously demonstrated that estrogen promotes the survival of tuberin-deficient ELT3 cells both in vitro and in vivo, and that estrogen treatment of mice bearing ELT3 cells xenograft tumors promotes lung metastasis. This estrogen-induced cell survival and metastasis was inhibited in vivo by the MEK inhibitor CI-1040. To elucidate pro-survival events mediated by estrogen in tuberin-null cells, we compared Tsc2-null ELT3 cell xenograft tumors from estrogen or placebo-treated mice using expression profiling and proteomic analysis . Expression profiling of ELT3 cell xenograft tumors revealed that estrogen upregulates the expression of genes in lipid and amino acid metabolism pathways. Among these genes, the estrogen-induced expression of amino adipatetransferase (AADAT), which is involved in lysine degradation, has been confirmed by immunoblotting of tumor lysates. Using iTRAQ proteomics from ELT3 xenograft tumors, we identified 40 proteins that are significantly regulated by estrogen, including proteins involved in carbohydrate metabolism (creatine kinase), amino acid degradation (aspartate aminotransferase) and lipid metabolism (isocitrate dehydrogenase). Metabolic profiling by LC/MS/MS of TSC2-null angiomyolipoma-derived cells from a LAM patient (621-101 cells) showed a temporal effect of rapamycin (10 nM) on the accumulation of pentose phosphate pathway intermediates and glycolytic metabolites. Cellular amino acid levels were reduced after rapamycin treatment. Consistent with a metabolic switch occurring in LAM, we found, using IHC staining, that the tumor-associated PKM2 is abundantly expressed in ELT3 cell xenograft tumors, lung metastatic lesions from estrogen-treated mice bearing ELT3 cell xenograft tumors, and in human LAM cell nodules. Finally, using FDG-PET, we found that xenograft ELT3 cell tumors in estrogen-treated mice exhibited higher levels of uptake compared with placebo-treated mice. In conclusion, gene expression profiling indicated that estrogen enhances the expression of genes which products regulate glucose and amino acid metabolism. Proteomic analysis showed estrogen-regulated candidates involved in glycolysis, amino acid, and lipid metabolisms. Metabolomic screening revealed that cells lacking tuberin have a metabolic response to rapamycin treatment. FDG-PET imaging showed enhanced uptake in estrogen-treated xenograft tumors. Collectively, these data indicate that cellular metabolic alterations may contribute to the pathogenesis of LAM. Targeting metabolic regulators might have therapeutic benefit for LAM. Citation Format: Yang Sun, Chenggang Li, Kevin Marks, Erik Zhang, Tasha Morrison, Mi-Ae Park, Shuyan Wang, Simon Dillon, Manoj Bhasin, Towia Libermann, Edward Driggers, Victor Gerbaudo, Elizabeth Petri Henske, Jane Yu. Evidence from expression profiling, proteomics, metabolomics and FDG-PET of an estrogen-dependent metabolic switch in tuberin-null cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-129. doi:10.1158/1538-7445.AM2013-LB-129

Nutritional virulence of Francisella tularensis

Nutritional virulence of Francisella tularensis

Drosophila TRPML Is Required for TORC1 Activation

Loss-of-function mutations in TRPML1 (transient receptor potential mucolipin 1) cause the lysosomal storage disorder, mucolipidosis type IV (MLIV). Here, we report that flies lacking the TRPML1 homolog displayed incomplete autophagy and reduced viability during the pupal period--a phase when animals rely on autophagy for nutrients. We show that TRPML was required for fusion of amphisomes with lysosomes, and its absence led to accumulation of vesicles of significantly larger volume and higher luminal Ca(2+). We also found that trpml(1) mutant cells showed decreased TORC1 (target of rapamycin complex 1) signaling and a concomitant upregulation of autophagy induction. Both of these defects in the mutants were reversed by genetically activating TORC1 or by feeding the larvae a high-protein diet. The high-protein diet also reduced the pupal lethality and the increased volume of acidic vesicles. Conversely, further inhibition of TORC1 activity by rapamycin exacerbated the mutant phenotypes. Finally, TORC1 exerted reciprocal control on TRPML function. A high-protein diet caused cortical localization of TRPML, and this effect was blocked by rapamycin. Our findings delineate the interrelationship between the TRPML and TORC1 pathways and raise the intriguing possibility that a high-protein diet might reduce the severity of MLIV.

Blood amino acid compartmentalization during pregnancy and lactation in the rat.

The blood amino acid compartmentalization during pregnancy and lactation of the rat have been studied. Cellular amino acid levels are similar to those of the plasmatic fraction. Important generalized decreases are detected on days 12 and 21 of the pregnancy; Glu + Gln, Ala and essential and semiessential amino acids being the most affected. During lactation, after a transient phase on day 1 after delivery, there is a generalized increase in blood levels, especially in the plasma fraction, that affects the whole essential values. The different patterns shown by amino acids during pregnancy and lactation confirm that the measurement of plasmatic levels underestimates the actual capacity of whole blood to transport amino acids.

The Effect of Fluoride on the Cellular Uptake and Pool of Amino Acids

Fluoride decreased the initial uptake of 2-aminoisobutyric acid (AIB) in LS cells only in high (supralethal) concentrations and after several hours of exposure. The steady state level of AIB seemed somewhat more sensitive as it was halved after 23 hours exposure to 6 mM-NaF. The cellular levels of some natural amino acids were also reduced by fluoride. However, cells resistant to fluoride showed equally lowered levels of amino acids, indicating that the observed effect of fluoride on cellular amino acid levels may be of little importance.

Regulation of amino acid transport activity and growth rate of animal cells in culture.

Balb/3T3 mouse cells grown in cell culture contain the A and L systems for neutral amino acid transport. L-Alanine was used as a test amino acid for the A system and L-leucine for the L system. Transport activities for both transport systems are altered as the growth conditions for the cells are altered. When cell growth was arrested by allowing cells to approach confluency, or by the removal of serum or by the addition of protein synthesis inhibitors, System A transport activity decreased and System L activity increased significantly. The content of the cellular amino acids increased 2- to 3-fold whenever cell growth was arrested by the conditions stated above. Increased cellular amino acids, therefore, are associated with decreased transport activity of System A and increased activity of System L. Altering cellular amino acids by loading or depleting the cells produced the predicted responses for the rapidly exchanging L-system but System A was less responsive. It is possible that regulatory mechanisms operate for the A system other than the direct action of alterations in cellular amino acid levels. The implications of the amino acid transport activity changes observed here on various studies on the role of transport in cell growth regulation and malignant transformation are discussed.

Influence of alterations in intracellular levels of amino-acids on protein-synthesizing activity of isolated ribosomes.

EVIDENCE has been presented which strongly suggests that the rate of protein synthesis in vivo1,2 and in vitro3,4 may be very dependent on intracellular concentrations of amino-acids. Of particular significance to the present investigations was the finding that protein turn-over in surviving liver tissue was stimulated in the presence of elevated levels of a single essential ammo-acid3,4. The catabolic as well as the anabolic phase of protein turnover appeared to be augmented in these circumstances. Interpretation of the results obtained was partially obscured by the fact that intracellular compartmentation of amino-acids may exist, with the result that exogenous and endogenous amino-acids are differentially metabolized5,6. The experiments recorded here were performed in an attempt to provide definitive data on the influence of cellular levels of amino-acids on the activity of protein-synthesizing systems. The results revealed that the amino-acid incorporating activity of ribosomes isolated from liver slices incubated with a single essential amino-acid varied with the concentration of that amino-acid.

INFLUENCE OF AMINO-ACID LEVELS ON PROTEIN SYNTHESIS IN VITRO.

ALTHOUGH protein synthesis is obviously dependent on availability of amino-acids1, the relationship of alterations in intracellular levels of amino-acids to rates of protein synthesis and breakdown remains obscure. Thus, enhanced protein synthesis has been observed in the presence of normal2 as well as elevated3 cellular levels of amino-acids. A maximum effective cellular concentration may exist for each amino-acid beyond which net protein synthesis cannot be further stimulated4,5. However, above this concentration alterations in gross protein synthesis may occur which are masked by concomitant variations in protein breakdown. The investigations recorded here were designed to investigate this possibility. The data indicate that a primary increase in extracellular concentration of one essential amino-acid may result in stimulation of protein synthesis and protein breakdown in surviving liver cells.

Insulin and incorporation of amino acids into protein of muscle. Cellular amino acid levels and aminoisobutyric acid uptake.

Research Article| October 01 1961 Insulin and incorporation of amino acids into protein of muscle. Cellular amino acid levels and aminoisobutyric acid uptake KL MANCHESTER KL MANCHESTER Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1961) 81 (1): 135–147. https://doi.org/10.1042/bj0810135 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation KL MANCHESTER; Insulin and incorporation of amino acids into protein of muscle. Cellular amino acid levels and aminoisobutyric acid uptake. Biochem J 1 October 1961; 81 (1): 135–147. doi: https://doi.org/10.1042/bj0810135 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1961 The Biochemical Society1961 Article PDF first page preview Close Modal You do not currently have access to this content.