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Neurons communicate by the activity-dependent release of small-molecule neurotransmitters packaged into synaptic vesicles (SVs). Although many molecules have been identified as neurotransmitters, technical limitations have precluded a full metabolomic analysis of synaptic vesicle content. Here, we present a workflow to rapidly isolate SVs and to interrogate their metabolic contents at a high-resolution using mass spectrometry. We validated the enrichment of glutamate in SVs of primary cortical neurons using targeted polar metabolomics. Unbiased and extensive global profiling of SVs isolated from these neurons revealed that the only detectable polar metabolites they contain are the established neurotransmitters glutamate and GABA. Finally, we adapted the approach to enable quick capture of SVs directly from brain tissue and determined the neurotransmitter profiles of diverse brain regions in a cell-type specific manner. The speed, robustness, and precision of this method to interrogate SV contents will facilitate novel insights into the chemical basis of neurotransmission.

From sensing leucine to metabolic control The mTORC1 protein kinase complex plays central roles in regulating cell growth and metabolism and is implicated in common human diseases such as diabetes and cancer. The level of the amino acid leucine tells an organism a lot about its physiological state, including how much food is available, how much insulin is going to be needed, and whether new muscle mass can be made (see the Perspective by Buel and Blenis). Wolfson et al. identified a biochemical sensor of leucine, Sestrin2, which connects the concentration of leucine to the control of organismal metabolism and growth. When leucine bound to Sestrin2, it was released from a complex with the mTORC1 regulatory factor GATOR2, activating the mTORC1 complex. Saxton et al. describe the crystal structure of Sestrin2 and show how it specifically detects leucine. Aylett et al. determined the structure of human mTORC1 by cryoelectron microscopy and the crystal structure of a regulatory subunit, Raptor. The results reveal the structural basis for the function and intricate regulation of this important enzyme, which is also a strategic drug target. Science , this issue p. 43 , p. 48 , p. 53 ; see also p. 25

Sensing amino acids at the lysosome The mTORC1 protein kinase is a complex of proteins that functions to regulate growth and metabolism. Activity of mTORC1 is sensitive to the abundance of amino acids, but how the sensing of amino acids is coupled to the control of mTORC1 has been unclear. Wang et al. searched for predicted membrane proteins that interacted with regulators of mTORC1. They identified a protein currently known only as SLC38A9. Interaction of SLC38A9 with mTORC1 regulators was sensitive to the presence of amino acids. SLC38A9 has sequence similarity to amino acid transporters. Effects of modulation of SLC38A9 in cultured human cells indicate that it may be the sensor that connects the abundance of arginine and leucine to mTORC1 activity. Science , this issue p. 188

Nutrients like amino acids and glucose signal through the heterodimeric Rag GTPases to activate mTORC1. Several protein complexes regulate the Rag GTPases, each serving as the effector of a distinct sensing branch of the pathway. One such regulator is GATOR1, which consists of Depdc5, Nprl2, and Nprl3, and is a GTPase Activating Protein (GAP) for RagA. Loss of GATOR1 renders mTORC1 signaling insensitive to nutrient starvation. Despite its central role in mTORC1 signaling, none of the GATOR1 components have sequence homology to other proteins, so the function of GATOR1 at the molecular level is unknown. Here we used Cryo-EM to solve two structures: GATOR1 alone and GATOR1 bound to the Rag GTPases. GATOR1 adopts an extended architecture with a cavity in the middle. Nprl2 serves as a link between Depdc5 and Nprl3, and Depdc5, the largest GATOR1 subunit, contacts the Rag heterodimer. Biochemical analyses reveal that our GATOR1-Rag structure represents an inhibitory state and that at least two binding modes must exist between the Rag GTPases and GATOR1. The direct interaction of Depdc5 with RagA inhibits the capacity of GATOR1 to stimulate GTP hydrolysis by RagA, while a weaker interaction between the Nprl2-Nprl3 heterodimer and RagA executes the GAP activity. These data reveal the structure of a critical component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase.

The mTORC1 kinase is a master growth regulator that senses numerous environmental cues, including amino acids. The Rag GTPases interact with mTORC1 and signal amino acid sufficiency by promoting the translocation of mTORC1 to the lysosomal surface, its site of activation. The Rags are unusual GTPases in that they function as obligate heterodimers, which consist of RagA or B bound to RagC or D. While the loading of RagA/B with GTP initiates amino acid signaling to mTORC1, the role of RagC/D is unknown. Here, we show that RagC/D is a key regulator of the interaction of mTORC1 with the Rag heterodimer and that, unexpectedly, RagC/D must be GDP bound for the interaction to occur. We identify FLCN and its binding partners, FNIP1/2, as Rag-interacting proteins with GAP activity for RagC/D, but not RagA/B. Thus, we reveal a role for RagC/D in mTORC1 activation and a molecular function for the FLCN tumor suppressor., United States. National Institutes of Health (CA103866), United States. National Institutes of Health (AI47389), United States. Department of Defense (W81XWH-07-0448), National Cancer Institute (U.S.) (F30CA180754)

Limiting mTORC1 The mTORC1 protein kinase complex has important functions linking metabolism to cell growth and its functions are disrupted in common diseases, including cancer and diabetes. Bar-Peled et al. (p. 1100 ; see the Perspective by Shaw ) discovered regulatory components that help turn down signaling by mTORC1 when cells are deprived of amino acids. Two complexes of proteins, GATOR1 and GATOR2, have opposite effects on activity and cellular localization of mTORC1. Components of the GATOR1 complex negatively regulate mTORC1 and appear to function as tumor supressors. Cancers with loss of GATOR1 function may be particularly amenable to therapeutic strategies that limit activity of mTORC1.

Arginases catalyze the divalent cation-dependent hydrolysis of l-arginine to urea and l-ornithine. There is significant interest in using arginase as a therapeutic anti-neogenic agent against l-arginine auxotrophic tumors and in enzyme replacement therapy for treating hyperargininemia. Both therapeutic applications require enzymes with sufficient stability under physiological conditions. To explore sequence elements that contribute to arginase stability we used SCHEMA-guided recombination to design a library of chimeric enzymes composed of sequence fragments from the two human isozymes Arginase I and II. We then developed a novel active learning algorithm that selects sequences from this library that are both highly informative and functional. Using high-throughput gene synthesis and our two-step active learning algorithm, we were able to rapidly create a small but highly informative set of seven enzymatically active chimeras that had an average variant distance of 40 mutations from the closest parent arginase. Within this set of sequences, linear regression was used to identify the sequence elements that contribute to the long-term stability of human arginase under physiological conditions. This approach revealed a striking correlation between the isoelectric point and the long-term stability of the enzyme to deactivation under physiological conditions.

Systemic L-arginine depletion following intravenous administration of l-arginine hydrolyzing enzymes has been shown to selectively impact tumors displaying urea cycle defects including a large fraction of hepatocellular carcinomas, metastatic melanomas and small cell lung carcinomas. However, the human arginases display poor serum stability (t(1/2)=4.8h) whereas a bacterial arginine deiminase evaluated in phase II clinical trials was reported to be immunogenic, eliciting strong neutralizing antibody responses. Recently, we showed that substitution of the Mn(2+) metal center in human Arginase I with Co(2+) (Co-hArgI) results in an enzyme that displays 10-fold higher catalytic efficiency for L-Arg hydrolysis, 12-15 fold reduction in the IC(50) towards a variety of malignant cell lines and, importantly a t(1/2)=22h in serum. To investigate the utility of Co-hArgI for L-Arg depletion therapy in cancer we systematically investigated three strategies for enhancing the persistence of the enzyme in circulation: (i) site specific conjugation of Co-hArgI engineered with an accessible N-terminal Cys residue to 20kDa PEG-maleimide (Co-hArgI-C(PEG-20K)); (ii) engineering of the homotrimeric Co-hArgI into a linked, monomeric 110kDa polypeptide (Co-hArgI x3) and (iii) lysyl conjugation of 5kDa PEG-N-hydroxysuccinimide (NHS) ester (Co-hArgI-K(PEG-5K)). Surprisingly, even though all three formulations resulted in proteins with a predicted hydrodynamic radius larger than the cut-off for renal filtration, only Co-hArgI amine conjugated to 5kDa PEG remained in circulation for sufficiently long durations. Using Co-hArgI-K(PEG-5K) labeled with an end-terminal fluorescein for easy detection, we demonstrated that following intraperitoneal administration at 6mg/kg weight, a well tolerated dose, the circulation t(1/2) of the protein in Balb/c mice is 63±10h. Very low levels of serum L-Arg (5μM) could be sustained for over 75h after injection, representing a 9-fold increase in pharmacodynamic efficacy relative to similarly prepared Mn(2+)-containing hArgI conjugated to 5kDa PEG-NHS ester (Mn-hArgI-K(PEG-5K)). The favorable pharmacokinetic and pharmacodynamic properties of Co-hArgI-K(PEG-5K) reported here, coupled with its human origin which should reduce the likelihood of adverse immune responses, make it a promising candidate for cancer therapy.

The active sites of eukaryotic arginase enzymes are strictly conserved, especially the first- and second-shell ligands that coordinate the two divalent metal cations that generate a hydroxide molecule for nucleophilic attack on the guanidinium carbon of l-arginine and the subsequent production of urea and l-ornithine. Here by using comprehensive pairwise saturation mutagenesis of the first- and second-shell metal ligands in human arginase I, we demonstrate that several metal binding ligands are actually quite tolerant to amino acid substitutions. Of >2800 double mutants of first- and second-shell residues analyzed, we found more than 80 unique amino acid substitutions, of which four were in first-shell residues. Remarkably, certain second-shell mutations could modulate the binding of both the nucleophilic water/hydroxide molecule and substrate or product ligands, resulting in activity greater than that of the wild-type enzyme. The data presented here constitute the first comprehensive saturation mutagenesis analysis of a metallohydrolase active site and reveal that the strict conservation of the second-shell metal binding residues in eukaryotic arginases does not reflect kinetic optimization of the enzyme during the course of evolution.

Herein we report the bacterial expression, purification, and enzymatic characterization of the human asparaginase-like protein 1 (hASRGL1). We present evidence that hASRGL1 exhibits beta-aspartyl peptidase activity consistent with enzymes designated as plant-type asparaginases, which had thus far been found in only plants and bacteria. Similar to nonmammalian plant-type asparaginases, hASRGL1 is shown to be an Ntn hydrolase for which Thr168 serves as the essential N-terminal nucleophile for intramolecular processing and catalysis, corroborated in part by abolishment of both activities through the Thr168Ala point mutation. In light of the activity profile reported here, ASRGL1s may act synergistically with protein l-isoaspartyl methyl transferase to relieve accumulation of potentially toxic isoaspartyl peptides in mammalian brain and other tissues.

Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ∼30 μM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway., United States. National Institutes of Health (R01CA103866), United States. National Institutes of Health (AI47389), United States. Department of Energy (W81XWH-07-0448), United States. National Institutes of Health (F31 CA180271), United States. National Institutes of Health (F31 CA189437)

From sensing leucine to metabolic control The mTORC1 protein kinase complex plays central roles in regulating cell growth and metabolism and is implicated in common human diseases such as diabetes and cancer. The level of the amino acid leucine tells an organism a lot about its physiological state, including how much food is available, how much insulin is going to be needed, and whether new muscle mass can be made (see the Perspective by Buel and Blenis). Wolfson et al. identified a biochemical sensor of leucine, Sestrin2, which connects the concentration of leucine to the control of organismal metabolism and growth. When leucine bound to Sestrin2, it was released from a complex with the mTORC1 regulatory factor GATOR2, activating the mTORC1 complex. Saxton et al. describe the crystal structure of Sestrin2 and show how it specifically detects leucine. Aylett et al. determined the structure of human mTORC1 by cryoelectron microscopy and the crystal structure of a regulatory subunit, Raptor. The results reveal the structural basis for the function and intricate regulation of this important enzyme, which is also a strategic drug target. Science , this issue p. 43 , p. 48 , p. 53 ; see also p. 25

Replacing the two Mn(2+) ions normally present in human Arginase I with Co(2+) resulted in a significantly lowered K(M) value without a concomitant reduction in k(cat). In addition, the pH dependence of the reaction was shifted from a pK(a) of 8.5 to a pK(a) of 7.5. The combination of these effects led to a 10-fold increase in overall catalytic activity (k(cat)/K(M)) at pH 7.4, close to the pH of human serum. Just as important for therapeutic applications, Co(2+) substitution lead to significantly increased serum stability of the enzyme. Our data can be explained by direct coordination of l-Arg to one of the Co(2+) ions during reaction, consistent with previously reported model studies. In vitro cytotoxicity experiments verified that the Co(2+)-substituted human Arg I displays an approximately 12- to 15-fold lower IC(50) value for the killing of human hepatocellular carcinoma and melanoma cell lines and thus constitutes a promising new candidate for the treatment of l-Arg auxotrophic tumors.