RagA, but Not RagB, Is Essential for Embryonic Development and Adult Mice

The mechanistic target of rapamycin complex 1 (mTORC1) integrates cues from growth factors and nutrients to control metabolism. In contrast to the growth factor input, genetic disruption of nutrient-dependent activation of mTORC1 in mammals remains unexplored. We engineered mice lacking RagA and RagB genes, which encode the GTPases responsible for mTORC1 activation by nutrients. RagB has limited expression, and its loss shows no effects on mammalian physiology. RagA deficiency leads to E10.5 embryonic death, loss of mTORC1 activity, and severe growth defects. Primary cells derived from these mice exhibit no regulation of mTORC1 by nutrients and maintain high sensitivity to growth factors. Deletion of RagA in adult mice is lethal. Upon RagA loss, a myeloid population expands in peripheral tissues. RagA-specific deletion in liver increases cellular responses to growth factors. These results show the essentiality of nutrient sensing for mTORC1 activity in mice and its suppression of PI3K/Akt signaling.United States. National Institutes of Health (R01 CA129105)United States. National Institutes of Health (R01 CA103866)United States. National Institutes of Health (R01 AI047389)United States. National Institutes of Health (R21 AG042876)American Federation for Aging ResearchStarr FoundationDavid H. Koch Institute for Integrative Cancer Research at MIT. Frontier Research ProgramEllison Medical FoundationUnited States. National Institutes of Health (AG041765)National Cancer Institute (U.S.) (F31CA167872

A Latent Pro-survival Function for the Mir-290-295 Cluster in Mouse Embryonic Stem Cells

MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir-290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target.National Institutes of Health (U.S.) (NIH grant RO1-GM34277)National Cancer Institute (U.S.) (NCI grant PO1-CA42063)National Cancer Institute (U.S.) (NCI Cancer Center Support (core) grant P30-CA14051

Enzymatic Blockade of the Ubiquitin-Proteasome Pathway

Ubiquitin-dependent processes control much of cellular physiology. We show that expression of a highly active, Epstein-Barr virus-derived deubiquitylating enzyme (EBV-DUB) blocks proteasomal degradation of cytosolic and ER-derived proteins by preemptive removal of ubiquitin from proteasome substrates, a treatment less toxic than the use of proteasome inhibitors. Recognition of misfolded proteins in the ER lumen, their dislocation to the cytosol, and degradation are usually tightly coupled but can be uncoupled by the EBV-DUB: a misfolded glycoprotein that originates in the ER accumulates in association with cytosolic chaperones as a deglycosylated intermediate. Our data underscore the necessity of a DUB activity for completion of the dislocation reaction and provide a new means of inhibition of proteasomal proteolysis with reduced cytotoxicity.National Institutes of Health (U.S.)EMBO (long term Fellowship 2008-379)Boehringer Ingelheim Fond

Ubiquitin-Dependent Control of Class II MHC Localization Is Dispensable for Antigen Presentation and Antibody Production

Controlled localization of class II MHC molecules is essential for proper class II MHC-restricted antigen presentation and the subsequent initiation of an adaptive immune response. Ubiquitination of class II MHC molecules on cytosolic lysine (K225) of the β-chain has been shown to affect localization of the complex. We generated mice in which the endogenous β-chain locus is replaced with a GFP tagged mutant version that lacks the cytosolic lysine residue (I-A-β-K225R-EGFP). These mice have elevated levels of class II MHC as compared to I-A-β-EGFP mice, and immature bone marrow-derived dendritic cells show redistribution of class II MHC to the cell surface. Nonetheless, in these same cells efficiency of antigen presentation is unaffected in I-A-β-K225R-EGFP mice, as assayed for presentation of ovalbumin to appropriately specific T cells. The I-A-β-K225R-EGFP animals have normal CD4 T cell populations and are capable of generating antigen-specific antibody in response to model antigens and viral infection. We therefore conclude that in our experimental system modulation of trafficking by ubiquitination of residue K225 of the β-chain is not essential for the function of class II MHC products in antigen presentation or antibody production

Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival

The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates organismal growth in response to many environmental cues, including nutrients and growth factors. Cell-based studies showed that mTORC1 senses amino acids through the RagA–D family of GTPases (also known as RRAGA, B, C and D), but their importance in mammalian physiology is unknown. Here we generate knock-in mice that express a constitutively active form of RagA (RagA[superscript GTP]) from its endogenous promoter. RagA[superscript GTP/GTP] mice develop normally, but fail to survive postnatal day 1. When delivered by Caesarean section, fasted RagA[superscript GTP/GTP] neonates die almost twice as rapidly as wild-type littermates. Within an hour of birth, wild-type neonates strongly inhibit mTORC1, which coincides with profound hypoglycaemia and a decrease in plasma amino-acid concentrations. In contrast, mTORC1 inhibition does not occur in RagA[superscript GTP/GTP] neonates, despite identical reductions in blood nutrient amounts. With prolonged fasting, wild-type neonates recover their plasma glucose concentrations, but RagA[superscript GTP/GTP] mice remain hypoglycaemic until death, despite using glycogen at a faster rate. The glucose homeostasis defect correlates with the inability of fasted RagA[superscript GTP/GTP] neonates to trigger autophagy and produce amino acids for de novo glucose production. Because profound hypoglycaemia does not inhibit mTORC1 in RagA[superscript GTP/GTP] neonates, we considered the possibility that the Rag pathway signals glucose as well as amino-acid sufficiency to mTORC1. Indeed, mTORC1 is resistant to glucose deprivation in RagA[superscript GTP/GTP] fibroblasts, and glucose, like amino acids, controls its recruitment to the lysosomal surface, the site of mTORC1 activation. Thus, the Rag GTPases signal glucose and amino-acid concentrations to mTORC1, and have an unexpectedly key role in neonates in autophagy induction and thus nutrient homeostasis and viability.National Institutes of Health (U.S.) (Grant R01 CA129105)National Institutes of Health (U.S.) (Grant R01 CA103866)National Institutes of Health (U.S.) (Grant R37 AI047389)American Federation for Aging ResearchStarr Cancer ConsortiumDavid H. Koch Institute for Integrative Cancer Research at MIT (Frontier Research Program)Ellison Medical Foundatio

Synthesis and maturation of class II MHC is normal in I-A-β-K225R-EGFP mice.

<p><i>A</i>, Splenocytes were pulsed with [³⁵S]-cystine/methionine for 30 minutes and chased for the indicated times. Immunoprecipitations were performed with the I-Aα-specific antibody JV1, precipitates were eluted at room temperature (left panel) or boiled (right panel). <i>B</i>, Experiments were performed as in (A), immunoprecipitates were boiled to elute samples. <i>C and D</i>, Quantification of the β-EGFP and Ii-p31 data in (B). Closed circles: I-A-β-EGFP, open circles: I-A-β-K225R-EGFP. These experiments were performed three separate times; representative data is shown.</p

CD8+ T Cells from Mice Transnuclear for a TCR that Recognizes a Single H-2Kb-Restricted MHV68 Epitope Derived from gB-ORF8 Help Control Infection

To study the CD8+ T cell response against a mouse γ-herpes virus, we generated Kb-MHV-68-ORF8604–612RAG−/− CD8+ T cell receptor transnuclear (TN) mice as a source of virus-specific CD8+ T cells. Kb-ORF8-Tet+ CD8+ T cells, expanded in the course of a resolving MHV-68 infection, served as a source of nucleus donors. Various in vivo and ex vivo assay criteria demonstrated the fine specificity and functionality of TN cells. TN cells proliferated extensively in response to viral infection, helped control viral burden, and exhibited a phenotype similar to that of endogenous Kb-ORF8-Tet+ cells. When compared to OT-1 cells, TN cells displayed distinct properties in response to lymphopenia and cognate antigen stimulation, which may be attributable to the affinity of the TCR expressed by the TN cells. The availability of MHV-68-specific CD8+ TCR TN mice provides a new tool for investigating aspects of host-pathogen interactions unique to γ-herpes viruses

Generation of I-A-β-K225R-EGFP mice.

<p><i>A</i>, Schematic of the approach used to target the I-A^b-β locus. The exon structure of the I-A^b-β locus is shown with the locations of the signal peptide (SP), extracellular domain (Extracellular), transmembrane domain (TM), and cytoplasmic tail (Cytoplasmic) indicated. The targeting construct consisted of a long arm of homology (LAH) consisting of exons 2–4, the K225R mutation introduced into exon 4, exons 5 and 6 fused to each other and to EGFP, a floxed self-excisable neomycin resistance cassette and a short arm of homology (SAH) consisting of the 3′UTR. Not drawn to scale. <i>B</i>, Splenocytes from I-A-β-EGFP or I-A-β-K225R-EGFP mice were stained with antibodies to detect B cell (B220), dendritic cell (CD11c) and T cell (CD3) populations and were analyzed by flow cytometry. Class II MHC, as detected from EGFP fluorescence, was present in B cells and dendritic cells, and class II MHC levels were increased in cells isolated from I-A-β-K225R-EGFP mice. <i>C</i>, Total splenocytes were isolated from heterozygous I-A-β-EGFP/HA-Ub and I-A-β-K225R-EGFP/HA-Ub mice and subjected to immunoprecipitation with an antibody against the MHC II α chain and subsequently to immunoprecipitation with an antibody against GFP. Total lysates were subjected to SDS-PAGE and immunoblot analysis with anti-GFP and anti-HA. All experiments were performed at least twice; representative experiments are shown.</p