Summary Autophagy is a process of cellular self-digestion induced by various forms of starvation. The mechanisms by which metabolic deficiencies are sensed by a cell to regulate autophagy remain unclear. While nitrogen deficit is a common trigger, yeast cells induce autophagy upon switch from a rich to minimal media without nitrogen starvation. We show that the amino acid methionine is sufficient to inhibit such non-nitrogen starvation (NNS)-induced autophagy. Methionine boosts synthesis of the methyl donor, S-adenosylmethionine (SAM). SAM inhibits autophagy and promotes growth through the action of the methyltransferase Ppm1p, which methylates the catalytic subunit of PP2A in tune with SAM levels. Methylated PP2A promotes dephosphorylation of Npr2p, a component of a conserved complex that regulates NNS-autophagy and other growth-related processes. Thus, methionine and SAM levels represent a critical gauge of amino acid availability that is sensed via this distinctive methylation modification of PP2A to reciprocally regulate cell growth and autophagy.
SUMMARY Protein translation is an energetically demanding process that must be regulated in response to changes in nutrient availability. Herein, we report that the thiolation status of wobble-uridine (U34) nucleotides present on lysine, glutamine or glutamate tRNAs reflects intracellular methionine and cysteine availability, and regulates cellular translational capacity and metabolic homeostasis. tRNA thiolation is important for growth under nutritionally challenging environments and required for efficient translation of genes enriched in lysine, glutamine, and glutamate codons, which frequently encode proteins important for translation and growth-specific processes. tRNA thiolation is down-regulated during sulfur starvation in order to decrease sulfur consumption and growth, and its absence leads to a compensatory increase in enzymes involved in methionine, cysteine, and lysine biosynthesis. Thus, tRNA thiolation enables cells to modulate translational capacity according to the availability of sulfur amino acids, establishing a functional significance for this conserved tRNA nucleotide modification in cell growth control.
Many organisms, including species from all kingdoms of life, can survive desiccation by entering a state with no detectable metabolism. To survive, C. elegans dauer larvae and stationary phase S. cerevisiae require elevated amounts of the disaccharide trehalose. We found that dauer larvae and stationary phase yeast switched into a gluconeogenic mode in which metabolism was reoriented toward production of sugars from non-carbohydrate sources. This mode depended on full activity of the glyoxylate shunt (GS), which enables synthesis of trehalose from acetate. The GS was especially critical during preparation of worms for harsh desiccation (preconditioning) and during the entry of yeast into stationary phase. Loss of the GS dramatically decreased desiccation tolerance in both organisms. Our results reveal a novel physiological role for the GS and elucidate a conserved metabolic rewiring that confers desiccation tolerance on organisms as diverse as worm and yeast.DOI: http://dx.doi.org/10.7554/eLife.13614.001
African sleeping sickness is a disease caused by Trypanosoma brucei. T. brucei proliferate rapidly in the mammalian bloodstream as long, slender forms, but at higher population densities they transform into nondividing, short, stumpy forms. This is thought to be a mechanism adopted by T. brucei to establish a stable hostparasite relationship and to allow a transition into the insect stage of its life cycle. Earlier studies have suggested a role for cAMP in mediating this transformation. In this study, using membranepermeable nucleotide analogs, we show that it is not the cAMP analogs themselves but rather the hydrolyzed products of membrane-permeable cAMP analogs that prevent proliferation of T. brucei. The metabolic products are more potent than the cAMP analogs, and hydrolysis-resistant cAMP analogs are not antiproliferative. We further show that the antiproliferative effect of these membrane-permeable adenosine analogs is caused by transformation into forms resembling short, stumpy bloodstream forms. These data suggest that the slender-to-stumpy transformation of T. brucei may not be mediated directly by cAMP and also raise the possibility of using such adenosine analogs as antitrypanosomal drugs.adenosine ͉ EPACs ͉ phosphodiesterases ͉ trypanosomes
The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are elevated in maple syrup urine disease, heart failure, obesity, and type 2 diabetes. BCAA homeostasis is controlled by the mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC), which is negatively regulated by the specific BCKD kinase (BDK). Here, we used structure-based design to develop a BDK inhibitor, (S)-α-chloro-phenylpropionic acid [(S)-CPP]. Crystal structures of the BDK-(S)-CPP complex show that (S)-CPP binds to a unique allosteric site in the N-terminal domain, triggering helix movements in BDK. These conformational changes are communicated to the lipoyl-binding pocket, which nullifies BDK activity by blocking its binding to the BCKDC core. Administration of (S)-CPP to mice leads to the full activation and dephosphorylation of BCKDC with significant reduction in plasma BCAA concentrations. The results buttress the concept of targeting mitochondrial BDK as a pharmacological approach to mitigate BCAA accumulation in metabolic diseases and heart failure.branched-chain α-ketoacid dehydrogenase kinase inhibitor | structure-based inhibitor design | allosteric mechanisms | kinase-inhibitor complex structures | in vivo kinase inhibitor studies T he branched-chain amino acids (BCAA) leucine, isoleucine, and valine comprise 40% of essential amino acids in daily dietary intake (1). The catabolic pathways of BCAA begin with transamination by branched-chain aminotransferases giving rise to corresponding branch-chain α-ketoacids (BCKA). The second common step, the irreversible oxidative decarboxylation of BCKA, is catalyzed by the single mitochondrial branch-chain α-ketoacid dehydrogenase complex (BCKDC). The homeostasis of BCAA and BCKA in vivo is critical for health. The accumulation of BCAA and BCKA secondary to inherited BCKDC deficiency produces maple syrup urine disease (MSUD), which can lead to fatal acidosis, neurological derangement, and mental retardation (2, 3). In large-scale high-throughput metabolic profiling studies, high blood BCAA concentrations were found to be highly associated with the development of insulin resistance (4, 5) and can serve as useful metabolic markers in type 2 diabetes risk assessment (6, 7). Pathologic stresses produced by the accumulated BCKA are linked to congenital heart diseases and heart failure (8). The above findings underscore the pivotal role of aberrant BCAA metabolism in the pathogenesis of metabolic, cardiac, and neurological diseases.The 4.5-MDa human BCKDC consists of three catalytic components: a heterotetrameric (α 2 β 2 ) branched-chain α-ketoacid decarboxylase (E1), a homo-24 meric dihydrolipoyltransacylase (E2), and a homodimeric dihydrolipoamide dehydrogenase (E3). In addition, human BCKDC contains two regulatory enzymes: BCKD kinase (BDK) and BCKD phosphatase (BDP), the latter also called PP2Cm phosphatase; these enzymes tightly regulate activity of BCKDC through the phosphorylation (inactivation)/ dephosphorylation (activation) of the E1α subunits (9, 10). BCKDC is or...
Methionine availability during overall amino acid limitation metabolically reprograms cells to support proliferation, the underlying basis for which remains unclear. Here we construct the organization of this methionine-mediated anabolic program using yeast. Combining comparative transcriptome analysis and biochemical and metabolic flux-based approaches, we discover that methionine rewires overall metabolic outputs by increasing the activity of a key regulatory node. This comprises the pentose phosphate pathway (PPP) coupled with reductive biosynthesis, the glutamate dehydrogenase (GDH)-dependent synthesis of glutamate/glutamine, and pyridoxal-5-phosphate (PLP)-dependent transamination capacity. This PPP-GDH-PLP node provides the required cofactors and/or substrates for subsequent rate-limiting reactions in the synthesis of amino acids and therefore nucleotides. These rate-limiting steps in amino acid biosynthesis are also induced in a methionine-dependent manner. This thereby results in a biochemical cascade establishing a hierarchically organized anabolic program. For this methionine-mediated anabolic program to be sustained, cells co-opt a “starvation stress response” regulator, Gcn4p. Collectively, our data suggest a hierarchical metabolic framework explaining how methionine mediates an anabolic switch.
Cells must appropriately sense and integrate multiple metabolic resources to commit to proliferation. Here, we report that S. cerevisiae cells regulate carbon and nitrogen metabolic homeostasis through tRNA U34-thiolation. Despite amino acid sufficiency, tRNA-thiolation deficient cells appear amino acid starved. In these cells, carbon flux towards nucleotide synthesis decreases, and trehalose synthesis increases, resulting in a starvation-like metabolic signature. Thiolation mutants have only minor translation defects. However, in these cells phosphate homeostasis genes are strongly down-regulated, resulting in an effectively phosphate-limited state. Reduced phosphate enforces a metabolic switch, where glucose-6-phosphate is routed towards storage carbohydrates. Notably, trehalose synthesis, which releases phosphate and thereby restores phosphate availability, is central to this metabolic rewiring. Thus, cells use thiolated tRNAs to perceive amino acid sufficiency, balance carbon and amino acid metabolic flux and grow optimally, by controlling phosphate availability. These results further biochemically explain how phosphate availability determines a switch to a ‘starvation-state’.
The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/ cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branchedchain ␣-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-␣2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis.
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