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.
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•CASTOR1 interacts with GATOR2 to inhibit mTORC1 upon arginine deprivation•CASTOR1 homodimerizes and can also heterodimerize with a related protein, CASTOR2•Arginine binds to CASTOR1 to disrupt the CASTOR1-GATOR2 complex•Arginine must bind to CASTOR1 in order for arginine to activate mTORC1 in cells
Arginine stimulation of cells activates mTORC1, a major growth regulator, but how it is sensed is not well understood. Chantranupong et al. now identify CASTOR1 as an arginine sensor and show that binding with CASTOR1 is required for arginine to activate the mTORC1 pathway.
Thousands of interactions assemble proteins into modules that impart spatial and functional organization to the cellular proteome. Through affinity-purification mass spectrometry, we have created two ...proteome-scale, cell-line-specific interaction networks. The first, BioPlex 3.0, results from affinity purification of 10,128 human proteins—half the proteome—in 293T cells and includes 118,162 interactions among 14,586 proteins. The second results from 5,522 immunoprecipitations in HCT116 cells. These networks model the interactome whose structure encodes protein function, localization, and complex membership. Comparison across cell lines validates thousands of interactions and reveals extensive customization. Whereas shared interactions reside in core complexes and involve essential proteins, cell-specific interactions link these complexes, “rewiring” subnetworks within each cell’s interactome. Interactions covary among proteins of shared function as the proteome remodels to produce each cell’s phenotype. Viewable interactively online through BioPlexExplorer, these networks define principles of proteome organization and enable unknown protein characterization.
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•Two protein interaction networks built from 15,650 pull-downs in two human cell lines•Extensive network remodeling reflects specialized biology of each cell line•Shared interactions form core complexes with essential, conserved functions•Networks reveal biological context for thousands of uncharacterized proteins
Comparative analysis of large-scale protein-protein interactions across two cell lines highlights context-specific interactions and proteome-scale shifts in how functional networks are arranged.
Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally ...characterize the human ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that recognizes diglycine (diGly)-containing isopeptides following trypsin digestion. We identify ∼19,000 diGly-modified lysine residues within ∼5000 proteins. Using quantitative proteomics we monitored temporal changes in diGly site abundance in response to both proteasomal and translational inhibition, indicating both a dependence on ongoing translation to observe alterations in site abundance and distinct dynamics of individual modified lysines in response to proteasome inhibition. Further, we demonstrate that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitin ligases. Interrogation of the ubiquitinome allows for not only a quantitative assessment of alterations in protein homeostasis fidelity, but also identification of substrates for individual ubiquitin pathway enzymes.
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► α-diGly was used to identify/quantify 19,000 ubiquitylation sites in 5,000 proteins ► Kinetics of ubiquitylation allows for classification of distinct substrate types ► Ubiquitinome formation largely requires ongoing protein synthesis ► diGly proteomics can be used to identify cullin-RING ligase substrates
Angiogenin is a stress-activated ribonuclease that cleaves tRNA within anticodon loops to produce tRNA-derived stress-induced fragments (tiRNAs). Transfection of natural or synthetic tiRNAs inhibits ...protein synthesis and triggers the phospho-eIF2α-independent assembly of stress granules (SGs), essential components of the stress response program. We show that selected tiRNAs inhibit protein synthesis by displacing eIF4G/eIF4A from uncapped > capped RNAs. tiRNAs also displace eIF4F, but not eIF4E:4EBP1, from isolated m
7G cap. We identify a terminal oligoguanine motif that is required to displace the eIF4F complex, inhibit translation, and induce SG assembly. We show that the tiRNA-associated translational silencer YB-1 contributes to angiogenin-, tiRNA-, and oxidative stress-induced translational repression. Our data reveal some of the mechanisms by which stress-induced tRNA cleavage inhibits protein synthesis and activates a cytoprotective stress response program.
► Selected 5′-tiRNAs target eIF4G/A to inhibit translation in reticulocyte lysates ► A terminal oligoguanine motif is required for tiRNA-induced translational repression ► 5′-tiRNAs cooperate with YB-1 to promote stress granule assembly ► YB-1 is required for optimal 5′-tiRNA- and arsenite-induced translational repression
Protein interactions form a network whose structure drives cellular function and whose organization informs biological inquiry. Using high-throughput affinity-purification mass spectrometry, we ...identify interacting partners for 2,594 human proteins in HEK293T cells. The resulting network (BioPlex) contains 23,744 interactions among 7,668 proteins with 86% previously undocumented. BioPlex accurately depicts known complexes, attaining 80%–100% coverage for most CORUM complexes. The network readily subdivides into communities that correspond to complexes or clusters of functionally related proteins. More generally, network architecture reflects cellular localization, biological process, and molecular function, enabling functional characterization of thousands of proteins. Network structure also reveals associations among thousands of protein domains, suggesting a basis for examining structurally related proteins. Finally, BioPlex, in combination with other approaches, can be used to reveal interactions of biological or clinical significance. For example, mutations in the membrane protein VAPB implicated in familial amyotrophic lateral sclerosis perturb a defined community of interactors.
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•2,594 AP-MS experiments provide 23,744 interactions involving 7,668 proteins•The network subdivides into complexes and clusters of functionally related proteins•Network architecture reveals subcellular localization and PFAM domain associations•The network offers a roadmap for characterization of poorly studied proteins
An interaction network for human proteins developed from affinity purification-mass spectrometry analyses provides a basis for understanding the architecture of protein complexes and for functional characterization of over 2,500 proteins.
Growth factors and nutrients enhance protein synthesis and suppress overall protein degradation by activating the protein kinase mammalian target of rapamycin (mTOR). Conversely, nutrient or serum ...deprivation inhibits mTOR and stimulates protein breakdown by inducing autophagy, which provides the starved cells with amino acids for protein synthesis and energy production. However, it is unclear whether proteolysis by the ubiquitin proteasome system (UPS), which catalyzes most protein degradation in mammalian cells, also increases when mTOR activity decreases. Here we show that inhibiting mTOR with rapamycin or Torin1 rapidly increases the degradation of long-lived cell proteins, but not short-lived ones, by stimulating proteolysis by proteasomes, in addition to autophagy. This enhanced proteasomal degradation required protein ubiquitination, and within 30 min afterm TOR inhibition, the cellular content of K48-linked ubiquitinated proteins increased without any change in proteasome content or activity. This rapid increase in UPS-mediated proteolysis continued for many hours and resulted primarily from inhibition of mTORC1 (not mTORC2), but did not require new protein synthesis or key mTOR targets: S6Ks, 4E-BPs, or Ulks. These findings do not support the recent report that mTORC1 inhibition reduces proteolysis by suppressing proteasome expression Zhang Y, et al. (2014) Nature 513(7518):440–443. Several growth-related proteins were identified that were ubiquitinated and degraded more rapidly after mTOR inhibition, including HMG-CoA synthase, whose enhanced degradation probably limits cholesterol biosynthesis upon insulin deficiency. Thus, mTOR inhibition coordinately activates the UPS and autophagy, which provide essential amino acids and, together with the enhanced ubiquitination of anabolic proteins, help slow growth.
Exercise provides many health benefits, including improved metabolism, cardiovascular health, and cognition. We have shown previously that FNDC5, a type I transmembrane protein, and its circulating ...form, irisin, convey some of these benefits in mice. However, recent reports questioned the existence of circulating human irisin both because human FNDC5 has a non-canonical ATA translation start and because of claims that many human irisin antibodies used in commercial ELISA kits lack required specificity. In this paper we have identified and quantitated human irisin in plasma using mass spectrometry with control peptides enriched with heavy stable isotopes as internal standards. This precise state-of-the-art method shows that human irisin is mainly translated from its non-canonical start codon and circulates at ∼3.6 ng/ml in sedentary individuals; this level is increased to ∼4.3 ng/ml in individuals undergoing aerobic interval training. These data unequivocally demonstrate that human irisin exists, circulates, and is regulated by exercise.
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•Detection and quantitation of human plasma irisin by quantitative mass spectrometry•Irisin is mainly translated from its non-canonical start codon•Irisin circulates at ∼3.6 ng/ml in sedentary individuals•Irisin levels significantly increase in individuals undergoing aerobic training
Irisin is an exercise-induced myokine with beneficial metabolic functions. Its detection in human plasma has, however, been problematic. Here, Jedrychowski et al. use state-of-the-art quantitative mass spectrometry to precisely detect and quantify circulating irisin and show that it goes up in individuals undergoing aerobic interval training.
Mass spectrometry-based proteomics enables the global identification and quantification of proteins and their posttranslational modifications in complex biological samples. However, proteomic ...analysis requires a complete and accurate reference set of proteins and is therefore largely restricted to model organisms with sequenced genomes.
Here, we demonstrate the feasibility of deep genome-free proteomics by using a reference proteome derived from heterogeneous mRNA data. We identify more than 11,000 proteins with 99% confidence from the unfertilized Xenopus laevis egg and estimate protein abundance with approximately 2-fold precision. Our reference database outperforms the provisional gene models based on genomic DNA sequencing and references generated by other methods. Surprisingly, we find that many proteins in the egg lack mRNA support and that many of these proteins are found in blood or liver, suggesting that they are taken up from the blood plasma, together with yolk, during oocyte growth and maturation, potentially contributing to early embryogenesis.
To facilitate proteomics in nonmodel organisms, we make our platform available as an online resource that converts heterogeneous mRNA data into a protein reference set. Thus, we demonstrate the feasibility and power of genome-free proteomics while shedding new light on embryogenesis in vertebrates.
•Genome-free proteomics identifies more than 11,000 proteins in the Xenopus laevis egg•Each protein’s expression level is predicted with approximately 2-fold precision•Many blood plasma proteins are taken up from oocyte during growth in the ovary•Web tool generates proteomic reference sets from mRNA data for any organism
Wühr et al. demonstrate the feasibility of deep proteomics, without the use of a sequenced genome, using the example of the egg of the African clawed frog Xenopus laevis. The authors identify more than 11,000 proteins and can predict each protein’s expression level with approximately 2-fold precision.
The NAD
+-dependent deacetylase SIRT1 is an evolutionarily conserved metabolic sensor of the Sirtuin family that mediates homeostatic responses to certain physiological stresses such as nutrient ...restriction. Previous reports have implicated fluctuations in intracellular NAD
+ concentrations as the principal regulator of SIRT1 activity. However, here we have identified a cAMP-induced phosphorylation of a highly conserved serine (S434) located in the SIRT1 catalytic domain that rapidly enhanced intrinsic deacetylase activity independently of changes in NAD
+ levels. Attenuation of SIRT1 expression or the use of a nonphosphorylatable SIRT1 mutant prevented cAMP-mediated stimulation of fatty acid oxidation and gene expression linked to this pathway. Overexpression of SIRT1 in mice significantly potentiated the increases in fatty acid oxidation and energy expenditure caused by either pharmacological β-adrenergic agonism or cold exposure. These studies support a mechanism of Sirtuin enzymatic control through the cAMP/PKA pathway with important implications for stress responses and maintenance of energy homeostasis.
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► Stimulation of the cAMP/PKA pathway results in phosphorylation of SIRT1 serine 434 ► SIRT1 S434 phosphorylation increases intrinsic deacetylase activity ► SIRT1 activation by S434 phosphorylation is rapid and independent of changes in NAD
+ ► S434 phosphorylation induces PGC-1α deacetylation and increased fatty acid oxidation
Autophagy, the process by which proteins and organelles are sequestered in double-membrane structures called autophagosomes and delivered to lysosomes for degradation, is critical in diseases such as ...cancer and neurodegeneration. Much of our understanding of this process has emerged from analysis of bulk cytoplasmic autophagy, but our understanding of how specific cargo, including organelles, proteins or intracellular pathogens, are targeted for selective autophagy is limited. Here we use quantitative proteomics to identify a cohort of novel and known autophagosome-enriched proteins in human cells, including cargo receptors. Like known cargo receptors, nuclear receptor coactivator 4 (NCOA4) was highly enriched in autophagosomes, and associated with ATG8 proteins that recruit cargo-receptor complexes into autophagosomes. Unbiased identification of NCOA4-associated proteins revealed ferritin heavy and light chains, components of an iron-filled cage structure that protects cells from reactive iron species but is degraded via autophagy to release iron through an unknown mechanism. We found that delivery of ferritin to lysosomes required NCOA4, and an inability of NCOA4-deficient cells to degrade ferritin led to decreased bioavailable intracellular iron. This work identifies NCOA4 as a selective cargo receptor for autophagic turnover of ferritin (ferritinophagy), which is critical for iron homeostasis, and provides a resource for further dissection of autophagosomal cargo-receptor connectivity.
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