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Mucins are functionally implicated in a range of human pathologies, including cystic fibrosis, influenza, bacterial endocarditis, gut dysbiosis, and cancer. These observations have motivated the study of mucin biosynthesis as well as the development of strategies for inhibition of mucin glycosylation. Mammalian pathways for mucin catabolism, however, have remained underexplored. The canonical view, derived from analysis of N -glycoproteins in human lysosomal storage disorders, is that glycan degradation and proteolysis occur sequentially. Here, we challenge this view by providing genetic and biochemical evidence supporting mammalian proteolysis of heavily O -glycosylated mucin domains without prior deglycosylation. Using activity screening coupled with mass spectrometry, we ascribed mucin-degrading activity in murine liver to the lysosomal protease cathepsin D. Glycoproteomics of substrates digested with purified human liver lysosomal cathepsin D provided direct evidence for proteolysis within densely O -glycosylated domains. Finally, knockout of cathepsin D in a murine model of the human lysosomal storage disorder neuronal ceroid lipofuscinosis 10 resulted in accumulation of mucins in liver-resident macrophages. Our findings imply that mucin-degrading activity is a component of endogenous pathways for glycoprotein catabolism in mammalian tissues.

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.

Cancer cells need a steady supply of nutrients to evade cell death and proliferate. Depriving cancer cells of the amino acid cystine can trigger the non-apoptotic cell death process of ferroptosis. Here, we report that cancer cells can evade cystine deprivation-induced ferroptosis by uptake and catabolism of the cysteine-rich extracellular protein albumin. This protective mechanism is enhanced by mTORC1 inhibition and involves albumin degradation in the lysosome, predominantly by cathepsin B (CTSB). CTSB-dependent albumin breakdown followed by export of cystine from the lysosome via the transporter cystinosin fuels the synthesis of glutathione, which suppresses lethal lipid peroxidation. When cancer cells are grown under non-adherent conditions as spheroids, mTORC1 pathway activity is reduced, and albumin supplementation alone affords considerable protection against ferroptosis. These results identify the catabolism of extracellular protein within the lysosome as a mechanism that can inhibit ferroptosis in cancer cells.

Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus

Complex regulation The protein kinase mTORC1 controls cellular growth in response to external signals. In the presence of nutrients, it localizes on the surface of the lysosome, where it is activated. The Raptor domain of mTORC1 binds to a complex comprising the protein Ragulator and a heterodimer of the Rag guanosine triphosphatase, which can adopt four different nucleotide conformations depending on nutrient availability. Rogala et al. determined the structure of the Raptor-Rag-Ragulator complex at 3.2-angstrom resolution by cryo–electron microscopy. The structure shows why Raptor binds only to a specific nucleotide conformation of the Rag heterodimer and suggests a model for how mTORC1 would dock onto the lysosomal surface, which is a key step in its activation. Science , this issue p. 468

B-cell lymphoma 11A, a transcriptional repressor, is highly expressed in triple negative breast cancer. The in vitro studies and animal models provide initial evidence suggesting that the knockdown of B-cell lymphoma 11A has a therapeutic eff ect on breast cancer. Defining the regulators driving the high expression of B-cell lymphoma 11A is important to understand its cancer-related functions. Among these regulators, transcription factors and microRNAs are critical for gene expression and associated with expression perturbations. Firstly, weidentifi ed the transcription factors that potentially interact with B-cell lymphoma 11A promoter. Based on bioinformatics prediction and multiple Omics datasets, two upregulated transcriptional activators Zinc Finger BED-Type Containing 4 and E2F Transcription Factor 1 in triple negative breast cancer were found to have seven sites within B-cell lymphoma 11A promoter. Secondly, we aimed to determine a putative set of microRNA that can mediate the post-transcriptional repression of B-cell lymphoma 11A. miR-513a-5p, miR-139-5p, miR-1179, miR-140-5p, and miR-542-3p, harboring at least one site of interaction with B-cell lymphoma 11A 3 untranslated region, were found inhibited in triple negative breast cancer. Taken together, the combinatorial regulation by transcription factors and microRNAs provide valuable information for further investigation on controlling the expression level of B-cell lymphoma 11A in triple negative breast cancer.

Deregulation of microRNAs contributes to multiple processes in cancer growth and progression. miR-199a-3p is decreased in highly metastatic breast cancer cells, MDA-MB-231, and its ectopic expression has a potent antimetastatic effect on these cells. However, the mechanism by which miR-199a-3p mediates its antimetastatic function has yet to be elucidated. Because miR-199a-3p reduces the expression levels of its target genes, it is likely to observe an inverse association between miR-199a-3p and its prometastatic target genes at the expression level. The current work determines that the Vesicleassociated membrane protein 3 (VAMP3) expression is increased in highly metastatic breast cancer cells compared to less metastatic cells, Michigan Cancer Foundation-7. The ectopic expression of miR-199a-3p strongly inhibits VAMP3 Messenger RNA and protein in vitro. Herein, it is confirmed that two sites within the 3'-untranslated sequence of VAMP3 Messenger RNA are actively targeted by miR-199a- 3p, discovering a new regulatory mechanism for VAMP3 expression. Functional studies reveal that the suppression of VAMP3 contributes to miR-199a-3p antimetastatic effect, particularly cellular migration in vitro. In conclusion, these results indicate that miR-199a-3p targeting of VAMP3 possesses a significant potential impact in preventing or curing metastatic breast cancers.

Regulated lysosomal efflux of amino acids A new technique allows rapid purification of lysosomes and metabolic profiling by liquid chromatography and mass spectrometry. Abu-Remaileh et al. engineered cultured human cells to produce a protein tag on lysosomal membranes that could be used to rapidly precipitate purified lysosomes on magnetic beads. Analysis of their contents under various conditions showed that efflux from the lysosome of most essential amino acids (but not that of most other amino acids) is a regulated process. Amino acid transport was inhibited under conditions of nutrient depletion as a result of inhibition of the mTOR (mechanistic target of rapamycin) protein kinase complex. Science , this issue p. 807

Macrophage (MΦ) activation must be tightly controlled to preclude overzealous responses that cause self-damage. MicroRNAs promote classical MΦ activation by blocking antiinflammatory signals and transcription factors but also can prevent excessive TLR signaling. In contrast, the microRNA profile associated with alternatively activated MΦ 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 miR-125b-5p, miR-146a-5p, miR-199b-5p, and miR-378-3p in helminth-induced MΦ. In vitro experiments demonstrated that miR-378-3p was specifically induced by IL-4 and revealed the IL-4–receptor/PI3K/Akt-signaling 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 MΦ proliferation in vivo. Thus, identification of miR-378-3p as an IL-4Rα–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 MΦ expansion and alternative activation during type 2 inflammatory settings.

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 single-stranded RNA virus. Specifically, we demonstrate the broad antiviral capacity of miR-199a-3p and illustrate that this individual host-encoded 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.

Human Cytomegalovirus (HCMV) affects 50 to 80% of the global population and establishes life-long infections. Besides causing morbidity and mortality in immunocompromised patients, HCMV is also a leading cause of congenital infections. The virus has evolved diverse mechanisms to modify the cellular environment to be beneficial to its replication as well as spread of infection, involving both proteins and non-coding RNAs. microRNAs (miRNAs) are one class of small non-coding RNAs (18~22 nucleotides in length) which negatively influence the stability and translational efficiency of specific target messenger RNAs (mRNAs). We have previously reported that a cluster of host-encoded miRNAs, miR-199a/214, is down-regulated in both murine CMV (MCMV) and HCMV-infected cells. Consistent with this, a member of this cluster, miR-199a-3p, manifests broad antiviral properties against CMVs as well as other herpseviruses when over-expressed in vitro. However, the molecular mechanisms involved in miR-199a/214 cluster down regulation at 24 hours post infection, as well as the impact of miR-199a-3p down regulation on host gene targets has not been previously reported. We demonstrate the transcriptional down regulation of the miR-199a/214 cluster by 4 hours post infection. Reporter assays demonstrate that the promoter of the pri-miRNA transcript is repressed by 4 hours post infection, which correlates with a rapid decrease in the pri-miRNA transcript level. Using viral deletion mutants, we demonstrate that the expression of immediate early viral genes is critical for the suppression of the miR-199a/214 promoter. We further show that miR-199-3p suppresses AKT phosphorylation, and this at least in part mediates its antiviral properties. Using luciferase reporter assays and qRT-PCR, we validate several of the targets of this miRNA that are associated with AKT phosphorylation including PI3KCB, ITGA6, and ITGA3. These genes are upregulated at 24 hours post infection, when the miRNAs are reduced. Taken together, our results suggest that viral gene expression suppress the transcription of miR-199a/214 cluster to enhance its replication, and this is at least partly mediated by enhanced AKT phosphorylation and ITGA6.