The lysosome degrades and recycles macromolecules, signals to the cytosol and nucleus, and is implicated in many diseases. Here we describe a method for the rapid isolation of mammalian lysosomes and use it to quantitatively profile lysosomal metabolites under various cell states. Under nutrient replete conditions, many lysosomal amino acids are in rapid exchange with those in the cytosol. Loss of lysosomal acidification through inhibition of the vacuolar H+ATPase (V-ATPase) increased the luminal concentrations of most metabolites, but had no effect on those of the majority of essential amino acids. Instead, nutrient starvation regulates the lysosomal concentrations of these amino acids, an effect we traced to regulation of the mTOR pathway. Inhibition of mTOR strongly reduced the lysosomal efflux of most essential amino acids, converting the lysosome into a cellular depot for them. These results reveal the dynamic nature of lysosomal metabolites and that V-ATPase- and mTOR-dependent mechanisms exist for controlling lysosomal amino acid efflux.

The lysosome degrades and recycles macromolecules, signals to the master growth regulator mTORC1 [mechanistic target of rapamycin (mTOR) complex 1], and is associated with human disease. We performed quantitative proteomic analyses of rapidly isolated lysosomes and found that nutrient levels and mTOR dynamically modulate the lysosomal proteome. Upon mTORC1 inhibition, NUFIP1 (nuclear fragile X mental retardation-interacting protein 1) redistributes from the nucleus to autophagosomes and lysosomes. Upon these conditions, NUFIP1 interacts with ribosomes and delivers them to autophagosomes by directly binding to microtubule-associated proteins 1A/1B light chain 3B (LC3B). The starvation-induced degradation of ribosomes via autophagy (ribophagy) depends on the capacity of NUFIP1 to bind LC3B and promotes cell survival. We propose that NUFIP1 is a receptor for the selective autophagy of ribosomes.

The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase regulates growth in response to nutrients and growth factors. Nutrients promote its translocation to the lysosomal surface, where its Raptor subunit interacts with the Rag guanosine triphosphatase (GTPase)–Ragulator complex. Nutrients switch the heterodimeric Rag GTPases among four different nucleotide-binding states, only one of which (RagA/B•GTP–RagC/D•GDP) permits mTORC1 association. We used cryo–electron microscopy to determine the structure of the supercomplex of Raptor with Rag-Ragulator at a resolution of 3.2 angstroms. Our findings indicate that the Raptor α-solenoid directly detects the nucleotide state of RagA while the Raptor “claw” threads between the GTPase domains to detect that of RagC. Mutations that disrupted Rag-Raptor binding inhibited mTORC1 lysosomal localization and signaling. By comparison with a structure of mTORC1 bound to its activator Rheb, we developed a model of active mTORC1 docked on the lysosome.

Macrophage (M⌽) activation must be tightly controlled to preclude overzealous responses that cause self-damage. MicroRNAs promote classical M⌽ activation by blocking antiinflammatory signals and transcription factors but also can prevent excessive TLR signaling. In contrast, the microRNA profile associated with alternatively activated M⌽ and their role in regulating wound healing or antihelminthic responses has not been described. By using an in vivo model of alternative activation in which adult Brugia malayi nematodes are implanted surgically in the peritoneal cavity of mice, we identified differential expression of miR125b-5p, miR-146a-5p, miR-199b-5p, and miR-378-3p in helminth-induced M⌽. In vitro experiments demonstrated that miR-378-3p was specifically induced by IL-4 and revealed the IL-4-receptor/PI3K/Aktsignaling pathway as a target. Chemical inhibition of this pathway showed that intact Akt signaling is an important enhancement factor for alternative activation in vitro and in vivo and is essential for IL-4-driven M⌽ proliferation in vivo. Thus, identification of miR-378-3p as an IL-4R␣-induced microRNA led to the discovery that Akt regulates the newly discovered mechanism of IL-4-driven macrophage proliferation. Together, the data suggest that negative regulation of Akt signaling via microRNAs might play a central role in limiting M⌽ expansion and alternative activation during type 2 inflammatory settings. (Blood. 2012;120(11): 2307-2316) IntroductionMacrophages (M⌽) are involved centrally in recognizing and containing pathogens. Subsequently, they ensure the efficient induction and upkeep of a protective adaptive immune response. M⌽ also help to limit the ensuing immune reaction as well as clear apoptotic cells and other debris. 1 The adaption of M⌽ to these diverse roles is reflected in the multitude of activation phenotypes that have been described. 2 Classical (or M1) and IL-4R␣-driven alternative (or M2) activation represent the 2 most divergent phenotypes, with the former thought to be proinflammatory and important for the clearance of microbial pathogens, whereas the latter are predominantly found during helminth infections and are associated with wound healing and immunosuppression. [3][4][5][6] In either case, M⌽ activation must be closely controlled because excessive activation can lead to tissue destruction or fibrosis, respectively. 6,7 Control is achieved by external signals, including cytokines (eg,9 ) and hormones (eg, glucocorticoids 10 ) but also by M⌽-intrinsic mechanisms. For example, classically activated M⌽ become unresponsive to secondary stimulation with lipopolysaccharide (LPS) as the result, at least in part, of the induction of negative feedback loops blocking or limiting activating signaling cascades. 11 The possibility that microRNAs may mediate such feedback mechanisms has recently attracted considerable interest. 11 MicroRNAs (miRNA) are short (18-24 nt), noncoding RNAs that influence the translation of specific genes by binding to the 3Ј-untranslated region (3ЈUTR) of t...

Although the functional parameters of microRNAs (miRNAs) have been explored in some depth, the roles of these molecules in viral infections remain elusive. Here we report a general method for global analysis of miRNA function that compares the significance of both overexpressing and inhibiting each mouse miRNA on the growth properties of different viruses. Our comparative analysis of representatives of all three herpesvirus subfamilies identified host miRNAs with broad anti-and proviral properties which extend to a singlestranded RNA virus. Specifically, we demonstrate the broad antiviral capacity of miR-199a-3p and illustrate that this individual hostencoded miRNA regulates multiple pathways required and/or activated by viruses, including PI3K/AKT and ERK/MAPK signaling, oxidative stress signaling, and prostaglandin synthesis. Global miRNA expression analysis further demonstrated that the miR-199a/miR-214 cluster is down-regulated in both murine and human cytomegalovirus infection and manifests similar antiviral properties in mouse and human cells. Overall, we report a general strategy for examining the contributions of individual host miRNAs in viral infection and provide evidence that these molecules confer broad inhibitory potential against multiple viruses.RNAi | herpesvirus | RNA virus | RNA processing | phosphatidylinositol-3-kinase-Akt signalling S ince the discovery of the first microRNA (miRNA) in Caenorhabditis elegans, research in diverse organisms has illuminated the role of this class of small RNA in a wide range of cellular processes (reviewed in ref.

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Significance Neurodegenerative diseases are poorly understood and difficult to treat. One common hallmark is lysosomal dysfunction leading to the accumulation of aggregates and other undegradable materials, which cause damage to brain resident cells. Lysosomes are acidic organelles responsible for breaking down biomolecules and recycling their constitutive parts. In this work, we find that the antiinflammatory and neuroprotective compound, discovered via a phenotypic screen, imparts its beneficial effects by targeting the lysosome and restoring its function. This is established using a genome-wide CRISPRi target identification screen and then confirmed using a variety of lysosome-targeted studies. The resulting small molecule from this study represents a potential treatment for neurodegenerative diseases as well as a research tool for the study of lysosomes in disease.

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