bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2022–10–30
forty-two papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing
  1. Ferroptosis inhibition by lysosome-dependent catabolism of extracellular protein.
  2. Lysosomal positioning diseases: beyond substrate storage.
  3. PITTching in for lysosome repair.
  4. Insulin-induced mTOR signaling and gluconeogenesis in renal proximal tubules: A mini-review of current evidence and therapeutic potential.
  5. mTORC1 beyond anabolic metabolism: Regulation of cell death.
  6. Tsc2 shapes olfactory bulb granule cell molecular and morphological characteristics.
  7. A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling.
  8. Autophagic perturbation caused by reduced lysosomal activity positively regulates cell competition.
  9. Intracerebroventricular dosing of N-sulfoglucosamine sulfohydrolase in mucopolysaccharidosis IIIA mice reduces markers of brain lysosomal dysfunction.
  10. mTOR gets greasy: lysosomal sensing of cholesterol.
  11. Lysosomes and Their Role in Regulating the Metabolism of Hematopoietic Stem Cells.
  12. GATOR2-dependent mTORC1 activity is a therapeutic vulnerability in FOXO1 fusion positive rhabdomyosarcoma.
  13. Lgd regulates ESCRT-III complex accumulation at multivesicular endosomes to control intralumenal vesicle formation.
  14. mTOR as a Potential Target for the Treatment of Microbial Infections, Inflammatory Bowel Diseases, and Colorectal Cancer.
  15. Species-specific differences in NPC1 protein trafficking govern therapeutic response in Niemann-Pick type C disease.
  16. New mechanism of metformin action mediated by lysosomal presenilin enhancer 2.
  17. RUFY1 binds Arl8b and mediates endosome-to-TGN CI-M6PR retrieval for cargo sorting to lysosomes.
  18. Impaired autophagic flux and dedifferentiation in podocytes lacking Asah1 gene: Role of lysosomal TRPML1 channel.
  19. mTOR Contributes to the Proteome Diversity through Transcriptome-Wide Alternative Splicing.
  20. Phospholipid imbalance impairs autophagosome completion.
  21. Control of infection by LC3-associated phagocytosis, CASM, and detection of raised vacuolar pH by the V-ATPase-ATG16L1 axis.
  22. PTP4A2 promotes lysophagy by dephosphorylation of VCP/p97 at Tyr805.
  23. Cryo-EM structure of the SEA complex.
  24. Dual roles of mTORC1-dependent activation of the ubiquitin-proteasome system in muscle proteostasis.
  25. IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy.
  26. The PITT pathway: Keeping lysosomes young.
  27. mTOR- and LARP1-dependent regulation of TOP mRNA poly(A) tail and ribosome loading.
  28. An Overview of Molecular Mechanisms in Fabry Disease.
  29. Genome-wide CRISPR screen reveals v-ATPase as a drug target to lower levels of ALS protein ataxin-2.
  30. Digest it all: the lysosomal turnover of cytoplasmic aggregates.
  31. Mechanism of 4-aminopyridine inhibition of the lysosomal channel TMEM175.
  32. Non-canonical β-adrenergic activation of ERK at endosomes.
  33. PTEN directs developmental and metabolic signaling for innate-like T cell fate and tissue homeostasis.
  34. The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to bacterial infection.
  35. All Roads Lead to Cathepsins: The Role of Cathepsins in Non-Alcoholic Steatohepatitis-Induced Hepatocellular Carcinoma.
  36. The evolution of organellar calcium mapping technologies.
  37. A method for the isolation and characterization of autophagic bodies from yeast provides a key tool to investigate cargos of autophagy.
  38. Multi‑faceted roles of cathepsins in ischemia reperfusion injury (Review).
  39. A Caenorhabditis elegans model of autosomal dominant adult-onset neuronal ceroid lipofuscinosis identifies ethosuximide as a potential therapeutic.
  40. Many roads lead to CASM: Diverse stimuli of noncanonical autophagy share a unifying molecular mechanism.
  41. The mechanisms and roles of selective autophagy in mammals.
  42. Fundamental roles for inter-organelle communication in aging.
  1. Cell Chem Biol. 2022 Oct 24. pii: S2451-9456(22)00360-9. [Epub ahead of print]
    Ferroptosis inhibition by lysosome-dependent catabolism of extracellular protein.
    David A Armenta, Nouf N Laqtom, Grace Alchemy, Wentao Dong, Danielle Morrow, Carson D Poltorack, David A Nathanson, Monther Abu-Remalieh, Scott J Dixon.
      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.
    Keywords:  ROS; albumin; cancer; cathepsin; cell death; cysteine; ferroptosis; glutathione; lysosome; mTOR
    DOI:  https://doi.org/10.1016/j.chembiol.2022.10.006
  2. Open Biol. 2022 Oct;12(10): 220155
    Lysosomal positioning diseases: beyond substrate storage.
    Gianluca Scerra, Valeria De Pasquale, Melania Scarcella, Maria Gabriella Caporaso, Luigi Michele Pavone, Massimo D'Agostino.
      Lysosomal storage diseases (LSDs) comprise a group of inherited monogenic disorders characterized by lysosomal dysfunctions due to undegraded substrate accumulation. They are caused by a deficiency in specific lysosomal hydrolases involved in cellular catabolism, or non-enzymatic proteins essential for normal lysosomal functions. In LSDs, the lack of degradation of the accumulated substrate and its lysosomal storage impairs lysosome functions resulting in the perturbation of cellular homeostasis and, in turn, the damage of multiple organ systems. A substantial number of studies on the pathogenesis of LSDs has highlighted how the accumulation of lysosomal substrates is only the first event of a cascade of processes including the accumulation of secondary metabolites and the impairment of cellular trafficking, cell signalling, autophagic flux, mitochondria functionality and calcium homeostasis, that significantly contribute to the onset and progression of these diseases. Emerging studies on lysosomal biology have described the fundamental roles of these organelles in a variety of physiological functions and pathological conditions beyond their canonical activity in cellular waste clearance. Here, we discuss recent advances in the knowledge of cellular and molecular mechanisms linking lysosomal positioning and trafficking to LSDs.
    Keywords:  lysosomal storage diseases; lysosome; membrane contact sites; microtubule tracks; positioning; trafficking
    DOI:  https://doi.org/10.1098/rsob.220155
  3. Dev Cell. 2022 Oct 24. pii: S1534-5807(22)00684-0. [Epub ahead of print]57(20): 2347-2349
    PITTching in for lysosome repair.
    Claire S Goul, Roberto Zoncu.
      Lysosomes, guardians of cell health, can sustain physical damage from biological, mechanical, and chemical stressors, necessitating dedicated mechanisms for their upkeep. In a recent issue of Nature, Tan and Finkel report the discovery of a lysosomal repair pathway controlled by phosphoinositides, which operates via bulk transport of lipids across ER-lysosome contacts.
    DOI:  https://doi.org/10.1016/j.devcel.2022.09.014
  4. Front Pharmacol. 2022 ;13 1015204
    Insulin-induced mTOR signaling and gluconeogenesis in renal proximal tubules: A mini-review of current evidence and therapeutic potential.
    Motonobu Nakamura, Nobuhiko Satoh, Shoko Horita, Masaomi Nangaku.
      Energy is continuously expended in the body, and gluconeogenesis maintains glucose homeostasis during starvation. Gluconeogenesis occurs in the liver and kidneys. The proximal tubule is the primary location for renal gluconeogenesis, accounting for up to 25% and 60% of endogenous glucose production during fasting and after a meal, respectively. The mechanistic target of rapamycin (mTOR), which exists downstream of the insulin pathway, plays an important role in regulating proximal tubular gluconeogenesis. mTOR is an atypical serine/threonine kinase present in two complexes. mTORC1 phosphorylates substrates that enhance anabolic processes such as mRNA translation and lipid synthesis and catabolic processes such as autophagy. mTORC2 regulates cytoskeletal dynamics and controls ion transport and proliferation via phosphorylation of SGK1. Therefore, mTOR signaling defects have been implicated in various pathological conditions, including cancer, cardiovascular disease, and diabetes. However, concrete elucidations of the associated mechanisms are still unclear. This review provides an overview of mTOR and describes the relationship between mTOR and renal.
    Keywords:  diabetes; gluconeogenesis; insulin resistance; mTOR; proximal tubules
    DOI:  https://doi.org/10.3389/fphar.2022.1015204
  5. J Cell Biol. 2022 Dec 05. pii: e202208103. [Epub ahead of print]221(12):
    mTORC1 beyond anabolic metabolism: Regulation of cell death.
    Jiajun Zhu, Hua Wang, Xuejun Jiang.
      The mechanistic target of rapamycin complex 1 (mTORC1), a multi-subunit protein kinase complex, interrogates growth factor signaling with cellular nutrient and energy status to control metabolic homeostasis. Activation of mTORC1 promotes biosynthesis of macromolecules, including proteins, lipids, and nucleic acids, and simultaneously suppresses catabolic processes such as lysosomal degradation of self-constituents and extracellular components. Metabolic regulation has emerged as a critical determinant of various cellular death programs, including apoptosis, pyroptosis, and ferroptosis. In this article, we review the expanding knowledge on how mTORC1 coordinates metabolic pathways to impinge on cell death regulation. We focus on the current understanding on how nutrient status and cellular signaling pathways connect mTORC1 activity with ferroptosis, an iron-dependent cell death program that has been implicated in a plethora of human diseases. In-depth understanding of the principles governing the interaction between mTORC1 and cell death pathways can ultimately guide the development of novel therapies for the treatment of relevant pathological conditions.
    DOI:  https://doi.org/10.1083/jcb.202208103
  6. Front Mol Neurosci. 2022 ;15 970357
    Tsc2 shapes olfactory bulb granule cell molecular and morphological characteristics.
    Victoria A Riley, Jennie C Holmberg, Aidan M Sokolov, David M Feliciano.
      Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations that inactivate TSC1 or TSC2. Hamartin and tuberin are encoded by TSC1 and TSC2 which form a GTPase activating protein heteromer that inhibits the Rheb GTPase from activating a growth promoting protein kinase called mammalian target of rapamycin (mTOR). Growths and lesions occur in the ventricular-subventricular zone (V-SVZ), cortex, olfactory tract, and olfactory bulbs (OB) in TSC. A leading hypothesis is that mutations in inhibitory neural progenitor cells cause brain growths in TSC. OB granule cells (GCs) are GABAergic inhibitory neurons that are generated through infancy by inhibitory progenitor cells along the V-SVZ. Removal of Tsc1 from mouse OB GCs creates cellular phenotypes seen in TSC lesions. However, the role of Tsc2 in OB GC maturation requires clarification. Here, it is demonstrated that conditional loss of Tsc2 alters GC development. A mosaic model of TSC was created by performing neonatal CRE recombinase electroporation into inhibitory V-SVZ progenitors yielded clusters of ectopic cytomegalic neurons with hyperactive mTOR complex 1 (mTORC1) in homozygous Tsc2 mutant but not heterozygous or wild type mice. Similarly, homozygous Tsc2 mutant GC morphology was altered at postnatal days 30 and 60. Tsc2 mutant GCs had hypertrophic dendritic arbors that were established by postnatal day 30. In contrast, loss of Tsc2 from mature GCs had negligible effects on mTORC1, soma size, and dendrite arborization. OB transcriptome profiling revealed a network of significantly differentially expressed genes following loss of Tsc2 during development that altered neural circuitry. These results demonstrate that Tsc2 has a critical role in regulating neural development and shapes inhibitory GC molecular and morphological characteristics.
    Keywords:  granule cell; mammalian target of rapamycin (mTOR); olfactory bulb (OB); tuberous sclerosis complex (TSC); tuberous sclerosis complex 1 (TSC1); tuberous sclerosis complex 2 (TSC2)
    DOI:  https://doi.org/10.3389/fnmol.2022.970357
  7. J Biol Chem. 2022 Oct 20. pii: S0021-9258(22)01072-9. [Epub ahead of print] 102629
    A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling.
    Johanna B Brüggenthies, Alessandra Fiore, Marion Russier, Christina Bitsina, Julian Brötzmann, Susanne Kordes, Sascha Menninger, Alexander Wolf, Elena Conti, Jan E Eickhoff, Peter J Murray.
      mTORC1 and GCN2 are serine/threonine kinases that control how cells adapt to amino acid availability. mTORC1 responds to amino acids to promote translation and cell growth while GCN2 senses limiting amino acids to hinder translation via eIF2α phosphorylation. GCN2 is an appealing target for cancer therapies because malignant cells can harness the GCN2 pathway to temper the rate of translation during rapid amino acid consumption. To isolate new GCN2 inhibitors, we created cell-based, amino acid limitation reporters via genetic manipulation of Ddit3 (encoding the transcription factor CHOP). CHOP is strongly induced by limiting amino acids and in this context, GCN2-dependent. Using leucine starvation as a model for essential amino acid sensing, we unexpectedly discovered ATP-competitive PI3 kinase-related kinase inhibitors, including ATR and mTOR inhibitors like torins, completely reversed GCN2 activation in a time-dependent way. Mechanistically, via inhibiting mTORC1-dependent translation, torins increased intracellular leucine, which was sufficient to reverse GCN2 activation and the downstream integrated stress response including stress-induced transcriptional factor ATF4 expression. Strikingly, we found that general translation inhibitors mirrored the effects of torins. Therefore, we propose that mTOR kinase inhibitors concurrently inhibit different branches of amino acid sensing by a dual mechanism involving direct inhibition of mTOR and indirect suppression of GCN2 that are connected by effects on the translation machinery. Collectively, our results highlight distinct ways of regulating GCN2 activity.
    Keywords:  GCN2; amino acid starvation; integrated stress response; mTORC1; torins
    DOI:  https://doi.org/10.1016/j.jbc.2022.102629
  8. Autophagy. 2022 Oct 26.
    Autophagic perturbation caused by reduced lysosomal activity positively regulates cell competition.
    Eilma Akter, Shunsuke Kon.
      Newly emerging transformed epithelial cells are recognized and apically removed by surrounding normal cells through a biological event termed "cell competition". However, little is known about the mechanisms underlying this process. In a recent study, we describe that RASG12V/RasV12-transformed cells surrounded by normal cells exhibit decreased lysosomal activity accompanied with accumulation of autophagosomes. Restoration of low lysosomal activity or inhibition of autophagosome formation significantly antagonizes apical extrusion of RASG12V cells, suggesting that non-degradable autophagosomes are required for cell competition. Notably, analysis of a cell competition mouse model demonstrates that macroautophagy/autophagy-ablated RASG12V cells are less readily eliminated by cell competition, and remaining transformed cells destroy ductal integrity, leading to chronic pancreatitis. Thus, our findings illuminate a critical role for non-degradable autophagosomes in cell competition and reveal a homeostasis-preserving role of autophagy upon emergence of transformed cells.
    Keywords:  cell competition; hindered autophagic flux; lysosomal dysfunction; non-degradable autophagosomes; pancreatic cancer
    DOI:  https://doi.org/10.1080/15548627.2022.2140559
  9. J Biol Chem. 2022 Oct 25. pii: S0021-9258(22)01068-7. [Epub ahead of print] 102625
    Intracerebroventricular dosing of N-sulfoglucosamine sulfohydrolase in mucopolysaccharidosis IIIA mice reduces markers of brain lysosomal dysfunction.
    Jenna Magat, Samantha Jones, Brian Baridon, Vishal Agrawal, Hio Wong, Alexander Giaramita, Linley Mangini, Britta Handyside, Catherine Vitelli, Monica Parker, Natasha Yeung, Yu Zhou, Erno Pungor, Ilya Slabodkin, Olivia Gorostiza, Allora Aguilera, Melanie J Lo, Saida Alcozie, Terri M Christianson, Pascale M N Tiger, Jon Vincelette, Sylvia Fong, Geuncheol Gil, Chuck Hague, Roger Lawrence, Daniel J Wendt, Jonathan H Lebowitz, Stuart Bunting, Sherry Bullens, Brett E Crawford, Sushmita M Roy, Josh C Woloszynek.
      Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by N-sulfoglucosamine sulfohydrolase (SGSH) deficiency. SGSH removes the sulfate from N-sulfoglucosamine residues on the non-reducing end of heparan sulfate (HS-NRE) within lysosomes. Enzyme deficiency results in accumulation of partially degraded heparan sulfate (HS) within lysosomes throughout the body, leading to a progressive severe neurological disease. Enzyme replacement therapy has been proposed, but further evaluation of the treatment strategy is needed. Here, we used Chinese hamster ovary (CHO) cells to produce a highly soluble and fully active recombinant human sulfamidase (rhSGSH). We discovered that rhSGSH utilizes both the CI-MPR and LRP1 receptors for uptake into patient fibroblasts. A single intracerebroventricular (ICV) injection of rhSGSH in MPS IIIA mice resulted in a tissue half-life of nine days and widespread distribution throughout the brain. Following a single ICV dose, both total HS and the MPS IIIA disease-specific HS-NRE were dramatically reduced, reaching a nadir two weeks post dose. The durability of effect for reduction of both substrate and protein markers of lysosomal dysfunction and a neuro-immune response lasted through the 56 days tested. Furthermore, seven weekly 148 μg doses ICV reduced those markers to near normal and produced a 99.5% reduction in HS-NRE levels. A pilot study utilizing every other week dosing in two animals supports further evaluation of less frequent dosing. Finally, our dose-response study also suggests lower doses may be efficacious. Our findings show that rhSGSH can normalize lysosomal HS storage and markers of a neuro-immune response when delivered ICV.
    Keywords:  Sanfilippo; enzyme replacement therapy (ERT); lysosomal storage disease (LSD); mucopolysaccharidosis (MPS); rhSGSH
    DOI:  https://doi.org/10.1016/j.jbc.2022.102625
  10. Cell Res. 2022 Oct 25.
    mTOR gets greasy: lysosomal sensing of cholesterol.
    Liron Bar-Peled, Dudley W Lamming.
      
    DOI:  https://doi.org/10.1038/s41422-022-00740-9
  11. Biology (Basel). 2022 Sep 27. pii: 1410. [Epub ahead of print]11(10):
    Lysosomes and Their Role in Regulating the Metabolism of Hematopoietic Stem Cells.
    Tasleem Arif.
      Hematopoietic stem cells (HSCs) have the capacity to renew blood cells at all stages of life and are largely quiescent at a steady state. It is essential to understand the processes that govern quiescence in HSCs to enhance bone marrow transplantation. It is hypothesized that in their quiescent state, HSCs primarily use glycolysis for energy production rather than mitochondrial oxidative phosphorylation (OXPHOS). In addition, the HSC switch from quiescence to activation occurs along a continuous developmental path that is driven by metabolism. Specifying the metabolic regulation pathway of HSC quiescence will provide insights into HSC homeostasis for therapeutic application. Therefore, understanding the metabolic demands of HSCs at a steady state is key to developing innovative hematological therapeutics. Lysosomes are the major degradative organelle in eukaryotic cells. Catabolic, anabolic, and lysosomal function abnormalities are connected to an expanding list of diseases. In recent years, lysosomes have emerged as control centers of cellular metabolism, particularly in HSC quiescence, and essential regulators of cell signaling have been found on the lysosomal membrane. In addition to autophagic processes, lysosomal activities have been shown to be crucial in sustaining quiescence by restricting HSCs access to a nutritional reserve essential for their activation into the cell cycle. Lysosomal activity may preserve HSC quiescence by altering glycolysis-mitochondrial biogenesis. The understanding of HSC metabolism has significantly expanded over the decade, revealing previously unknown requirements of HSCs in both their dividing (active) and quiescent states. Therefore, understanding the role of lysosomes in HSCs will allow for the development of innovative treatment methods based on HSCs to fight clonal hematopoiesis and HSC aging.
    Keywords:  HSCs; glycolysis; lysosomes; metabolism; mitochondria; quiescence
    DOI:  https://doi.org/10.3390/biology11101410
  12. JCI Insight. 2022 Oct 25. pii: e162207. [Epub ahead of print]
    GATOR2-dependent mTORC1 activity is a therapeutic vulnerability in FOXO1 fusion positive rhabdomyosarcoma.
    Jacqueline Morales, David V Allegakoen, José A Garcia, Kristen Kwong, Pushpendra K Sahu, Drew A Fajardo, Yue Pan, Max A Horlbeck, Jonathan S Weissman, W Clay Gustafson, Trever G Bivona, Amit J Sabnis.
      Oncogenic FOXO1 gene fusions drive a subset of rhabdomyosarcoma (RMS) with poor survival and to date these cancer drivers are therapeutically intractable. To identify new therapies for this disease, we undertook an isogenic CRISPR-interference screen to define PAX3-FOXO1 specific genetic dependencies and identified genes in the GATOR2 complex. GATOR2 loss in RMS abrogated amino acid-induced lysosomal localization of mTORC1 and consequent downstream signaling, slowing G1-S cell cycle transition. In vivo suppression of GATOR2 impaired the growth of tumor xenografts and favored the outgrowth of cells lacking PAX3-FOXO1. Loss of a subset of GATOR2 members can be compensated by direct genetic activation of mTORC1. RAS mutations are also sufficient to decouple mTORC1 activation from GATOR2, and indeed fusion negative RMS harboring such mutations exhibit amino acid-independent mTORC1 activity. A bi-steric, mTORC1-selective small molecule induced tumor regressions in fusion positive patient-derived tumor xenografts. These findings highlight a vulnerability in FOXO1 fusion positive RMS and provide rationale for the clinical evaluation of bi-steric mTORC1 inhibitors, currently in phase 1 testing, to treat this disease. Isogenic genetic screens can thus identify potentially exploitable vulnerabilities in fusion driven pediatric cancers which otherwise remain mostly undruggable.
    Keywords:  Cancer; Drug therapy; Genetics; Oncology; Signal transduction
    DOI:  https://doi.org/10.1172/jci.insight.162207
  13. Mol Biol Cell. 2022 Oct 26. mbcE22080342
    Lgd regulates ESCRT-III complex accumulation at multivesicular endosomes to control intralumenal vesicle formation.
    Aryel L Clarke, Molly M Lettman, Anjon Audhya.
      Membrane remodeling mediated by heteropolymeric filaments composed of ESCRT-III subunits is an essential process that occurs at a variety of organelles to maintain cellular homeostasis. Members of the evolutionarily conserved Lgd/CC2D1 protein family have been suggested to regulate ESCRT-III polymer assembly, although their specific roles, particularly in vivo, remain unclear. Using the Caenorhabditis elegans early embryo as a model system, we show that Lgd/CC2D1 localizes to endosomal membranes, and its loss impairs endolysosomal cargo sorting and degradation. At the ultrastructural level, the absence of Lgd/CC2D1 results in the accumulation of enlarged endosomal compartments that contain a reduced number of intralumenal vesicles (ILVs). However, unlike aberrant endosome morphology caused by depletion of other ESCRT components, ILV size is only modestly altered in embryos lacking Lgd/CC2D1. Instead, loss of Lgd/CC2D1 impairs normal accumulation of ESCRT-III on endosomal membranes, likely slowing the kinetics of ILV formation. Together, our findings suggest a role for Lgd/CC2D1 in the recruitment and/or stable assembly of ESCRT-III subunits on endosomal membranes to facilitate efficient ILV biogenesis. [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E22-08-0342
  14. Int J Mol Sci. 2022 Oct 18. pii: 12470. [Epub ahead of print]23(20):
    mTOR as a Potential Target for the Treatment of Microbial Infections, Inflammatory Bowel Diseases, and Colorectal Cancer.
    Obaid Afzal, Abdulmalik S A Altamimi, Bismillah Mubeen, Sami I Alzarea, Waleed Hassan Almalki, Salwa D Al-Qahtani, Eman M Atiya, Fahad A Al-Abbasi, Fatima Ali, Inam Ullah, Muhammad Shahid Nadeem, Imran Kazmi.
      The mammalian target of rapamycin (mTOR) is the major controller of a number of important cellular activities, including protein synthesis, cell expansion, multiplication, autophagy, lysosomal function, and cellular metabolism. When mTOR interacts with specific adaptor proteins, it forms two complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The mTOR signaling system regulates gene transcription and protein manufacturing to control proliferation of cell, differentiation of immune cell, and tumor metabolism. Due to its vital role in case of microbial infections, inflammations and cancer development and progression, mTOR has been considered as a key therapeutic target for the development of targeted medication. As autophagy dysfunction is linked to changes in both innate and adaptive immune responses, bacterial clearance defects, and goblet and Paneth cell malfunction, all of these changes are linked to inflammatory bowel diseases (IBD) and colorectal cancer (CRC) pathogenesis. Preclinical and clinical data have shown that the inhibition and induction of autophagy have significant potential to be translated into the clinical applications. In IBD and several CRC models, mTORC1 inhibitors have been found effective. In the recent years, a number of novel mTOR inhibitors have been investigated in clinical trials, and a number of drugs have shown considerably enhanced efficacy when combined with mTOR inhibitors. The future developments in the mTOR targeting medications can benefit patients in individualized therapy. Advanced and innovative medicines that are more effective and have lower drug resistance are still in high demand. New findings could be relevant in medicine development, pharmacological modification, or future mTOR inhibitor research. Therefore, the goal of this review is to present a comprehensive account of current developments on the mTOR pathway and its inhibitors, with an emphasis on the management of microbial infections, the treatment of inflammatory bowel disease, and the management of colon cancer.
    Keywords:  colorectal cancer (CRC); inflammatory bowel diseases (IBD); mammalian target of rapamycin (mTOR)
    DOI:  https://doi.org/10.3390/ijms232012470
  15. JCI Insight. 2022 Oct 27. pii: e160308. [Epub ahead of print]
    Species-specific differences in NPC1 protein trafficking govern therapeutic response in Niemann-Pick type C disease.
    Mark L Schultz, Kylie J Schache, Ruth D Azaria, Esmée Q Kuiper, Steven Erwood, Evgueni A Ivakine, Nicole Y Farhat, Forbes D Porter, Koralege C Pathmasiri, Stephanie M Cologna, Michael D Uhler, Andrew P Lieberman.
      The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, these drug development efforts have been unsuccessful in mouse models. Here, we demonstrate unexpected differences in trafficking through the medial Golgi between mouse and human I1061T-NPC1, a common disease-causing mutant. We establish that these distinctions are governed by differences in the NPC1 protein sequence rather than by variations in the ER folding environment. Moreover, we demonstrate direct effects of mutant protein trafficking on the response to small molecules that modulate the endoplasmic reticulum folding environment by affecting Ca++ concentration. Finally, we develop a panel of isogenic human NPC1 iNeurons expressing wild type, I1061T-, and R934L-NPC1 and demonstrate their utility in testing these candidate therapeutics. Our findings identify important rules governing mutant NPC1's response to proteostatic modulators and highlight the importance of species- and mutation-specific responses for therapy development.
    Keywords:  Lysosomes; Neuroscience; Protein misfolding; Protein traffic; Therapeutics
    DOI:  https://doi.org/10.1172/jci.insight.160308
  16. J Diabetes Investig. 2022 Oct 28.
    New mechanism of metformin action mediated by lysosomal presenilin enhancer 2.
    Kenji Sugawara, Wataru Ogawa.
      Formation of the PEN2-ATP6AP1 complex induced by the binding of metformin to PEN2 results in the inhibition of v-ATPase activity and in the recruitment of AXIN/LKB1 to lysosomes, which in turn results in the phosphorylation and activation of AMPK.
    DOI:  https://doi.org/10.1111/jdi.13925
  17. J Cell Biol. 2023 Jan 02. pii: e202108001. [Epub ahead of print]222(1):
    RUFY1 binds Arl8b and mediates endosome-to-TGN CI-M6PR retrieval for cargo sorting to lysosomes.
    Shalini Rawat, Dhruba Chatterjee, Rituraj Marwaha, Gitanjali Charak, Gaurav Kumar, Shrestha Shaw, Divya Khatter, Sheetal Sharma, Cecilia de Heus, Nalan Liv, Judith Klumperman, Amit Tuli, Mahak Sharma.
      Arl8b, an Arf-like GTP-binding protein, regulates cargo trafficking and positioning of lysosomes. However, it is unknown whether Arl8b regulates lysosomal cargo sorting. Here, we report that Arl8b binds to the Rab4 and Rab14 interaction partner, RUN and FYVE domain-containing protein (RUFY) 1, a known regulator of cargo sorting from recycling endosomes. Arl8b determines RUFY1 endosomal localization through regulating its interaction with Rab14. RUFY1 depletion led to a delay in CI-M6PR retrieval from endosomes to the TGN, resulting in impaired delivery of newly synthesized hydrolases to lysosomes. We identified the dynein-dynactin complex as an RUFY1 interaction partner, and similar to a subset of activating dynein adaptors, the coiled-coil region of RUFY1 was required for interaction with dynein and the ability to mediate dynein-dependent organelle clustering. Our findings suggest that Arl8b and RUFY1 play a novel role on recycling endosomes, from where this machinery regulates endosomes to TGN retrieval of CI-M6PR and, consequently, lysosomal cargo sorting.
    DOI:  https://doi.org/10.1083/jcb.202108001
  18. Biochim Biophys Acta Mol Cell Res. 2022 Oct 24. pii: S0167-4889(22)00178-1. [Epub ahead of print] 119386
    Impaired autophagic flux and dedifferentiation in podocytes lacking Asah1 gene: Role of lysosomal TRPML1 channel.
    Guangbi Li, Dandan Huang, Yao Zou, Jason Kidd, Todd W B Gehr, Ningjun Li, Joseph K Ritter, Pin-Lan Li.
      Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain (Asah1fl/fl/Podocre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether Ac deficiency impairs autophagic flux in podocytes through blockade of transient receptor potential mucolipin 1 (TRPML1) channel as a potential pathogenic mechanism of podocytopathy in Asah1fl/fl/Podocre mice. We first demonstrated that impairment of autophagic flux occurred in podocytes lacking Asah1 gene, which was evidenced by autophagosome accumulation and reduced lysosome-autophagosome interaction. TRPML1 channel agonists recovered lysosome-autophagosome interaction and attenuated autophagosome accumulation in podocytes from Asah1fl/fl/Podocre mice, while TRPML1 channel inhibitors impaired autophagic flux in WT/WT podocytes and worsened autophagic deficiency in podocytes lacking Asah1 gene. The effects of TRPML1 channel agonist were blocked by dynein inhibitors, indicating a critical role of dynein activity in the control of lysosome movement due to TRPML1 channel-mediated Ca2+ release. It was also found that there is an enhanced phenotypic transition to dedifferentiation status in podocytes lacking Asah1 gene in vitro and in vivo. Such podocyte phenotypic transition was inhibited by TRPML1 channel agonists but enhanced by TRPML1 channel inhibitors. Moreover, we found that TRPML1 gene silencing induced autophagosome accumulation and dedifferentiation in podocytes. Based on these results, we conclude that Ac activity is essential for autophagic flux and maintenance of differentiated status of podocytes. Dysfunction or deficiency of Ac may impair autophagic flux and induce podocyte dedifferentiation, which may be an important pathogenic mechanism of podocytopathy and associated nephrotic syndrome.
    Keywords:  Acid ceramidase; Autophagy; Dedifferentiation; Lysosome; Podocyte; TRPML1 channel
    DOI:  https://doi.org/10.1016/j.bbamcr.2022.119386
  19. Int J Mol Sci. 2022 Oct 17. pii: 12416. [Epub ahead of print]23(20):
    mTOR Contributes to the Proteome Diversity through Transcriptome-Wide Alternative Splicing.
    Sze Cheng, Naima Ahmed Fahmi, Meeyeon Park, Jiao Sun, Kaitlyn Thao, Hsin-Sung Yeh, Wei Zhang, Jeongsik Yong.
      The mammalian target of rapamycin (mTOR) pathway is crucial in energy metabolism and cell proliferation. Previously, we reported transcriptome-wide 3'-untranslated region (UTR) shortening by alternative polyadenylation upon mTOR activation and its impact on the proteome. Here, we further interrogated the mTOR-activated transcriptome and found that hyperactivation of mTOR promotes transcriptome-wide exon skipping/exclusion, producing short isoform transcripts from genes. This widespread exon skipping confers multifarious regulations in the mTOR-controlled functional proteomics: AS in coding regions widely affects the protein length and functional domains. They also alter the half-life of proteins and affect the regulatory post-translational modifications. Among the RNA processing factors differentially regulated by mTOR signaling, we found that SRSF3 mechanistically facilitates exon skipping in the mTOR-activated transcriptome. This study reveals a role of mTOR in AS regulation and demonstrates that widespread AS is a multifaceted modulator of the mTOR-regulated functional proteome.
    Keywords:  alternative splicing; functional proteome; mTOR signaling; post-transcriptional gene regulation
    DOI:  https://doi.org/10.3390/ijms232012416
  20. EMBO J. 2022 Oct 27. e110771
    Phospholipid imbalance impairs autophagosome completion.
    Alexandra Polyansky, Oren Shatz, Milana Fraiberg, Eyal Shimoni, Tali Dadosh, Muriel Mari, Fulvio M Reggiori, Chao Qin, Xianlin Han, Zvulun Elazar.
      Autophagy, a conserved eukaryotic intracellular catabolic pathway, maintains cell homeostasis by lysosomal degradation of cytosolic material engulfed in double membrane vesicles termed autophagosomes, which form upon sealing of single-membrane cisternae called phagophores. While the role of phosphatidylinositol 3-phosphate (PI3P) and phosphatidylethanolamine (PE) in autophagosome biogenesis is well-studied, the roles of other phospholipids in autophagy remain rather obscure. Here we utilized budding yeast to study the contribution of phosphatidylcholine (PC) to autophagy. We reveal for the first time that genetic loss of PC biosynthesis via the CDP-DAG pathway leads to changes in lipid composition of autophagic membranes, specifically replacement of PC by phosphatidylserine (PS). This impairs closure of the autophagic membrane and autophagic flux. Consequently, we show that choline-dependent recovery of de novo PC biosynthesis via the CDP-choline pathway restores autophagosome formation and autophagic flux in PC-deficient cells. Our findings therefore implicate phospholipid metabolism in autophagosome biogenesis.
    Keywords:  autophagosome biogenesis; autophagy; phagophore; phospholipids
    DOI:  https://doi.org/10.15252/embj.2022110771
  21. Sci Adv. 2022 Oct 28. 8(43): eabn3298
    Control of infection by LC3-associated phagocytosis, CASM, and detection of raised vacuolar pH by the V-ATPase-ATG16L1 axis.
    Yingxue Wang, Maria Ramos, Matthew Jefferson, Weijiao Zhang, Naiara Beraza, Simon Carding, Penny P Powell, James P Stewart, Ulrike Mayer, Thomas Wileman.
      The delivery of pathogens to lysosomes for degradation provides an important defense against infection. Degradation is enhanced when LC3 is conjugated to endosomes and phagosomes containing pathogens to facilitate fusion with lysosomes. In phagocytic cells, TLR signaling and Rubicon activate LC3-associated phagocytosis (LAP) where stabilization of the NADPH oxidase leads to sustained ROS production and raised vacuolar pH. Raised pH triggers the assembly of the vacuolar ATPase on the vacuole membrane where it binds ATG16L1 to recruit the core LC3 conjugation complex (ATG16L1:ATG5-12). This V-ATPase-ATG16L1 axis is also activated in nonphagocytic cells to conjugate LC3 to endosomes containing extracellular microbes. Pathogens provide additional signals for recruitment of LC3 when they raise vacuolar pH with pore-forming toxins and proteins, phospholipases, or specialized secretion systems. Many microbes secrete virulence factors to inhibit ROS production and/or the V-ATPase-ATG16L1 axis to slow LC3 recruitment and avoid degradation in lysosomes.
    DOI:  https://doi.org/10.1126/sciadv.abn3298
  22. Autophagy. 2022 Oct 27.
    PTP4A2 promotes lysophagy by dephosphorylation of VCP/p97 at Tyr805.
    Yunpeng Bai, Guimei Yu, Hong-Ming Zhou, Ovini Amarasinghe, Yuan Zhou, Peipei Zhu, Qinglin Li, Lujuan Zhang, Frederick Nguele Meke, Yiming Miao, Eli Chapman, W Andy Tao, Zhong-Yin Zhang.
      Overexpression of PTP4A phosphatases are associated with advanced cancers, but their biological functions are far from fully understood due to limited knowledge about their physiological substrates. VCP is implicated in lysophagy via collaboration with specific cofactors in the ELDR complex. However, how the ELDR complex assembly is regulated has not been determined. Moreover, the functional significance of the penultimate and conserved Tyr805 phosphorylation in VCP has not been established. Here, we use an unbiased substrate trapping and mass spectrometry approach and identify VCP/p97 as a bona fide substrate of PTP4A2. Biochemical studies show that PTP4A2 dephosphorylates VCP at Tyr805, enabling the association of VCP with its C-terminal cofactors UBXN6/UBXD1 and PLAA, which are components of the ELDR complex responsible for lysophagy, the autophagic clearance of damaged lysosomes. Functionally, PTP4A2 is required for cellular homeostasis by promoting lysophagy through facilitating ELDR-mediated K48-linked ubiquitin conjugate removal and autophagosome formation on the damaged lysosomes. Deletion of Ptp4a2 in vivo compromises the recovery of glycerol-injection induced acute kidney injury due to impaired lysophagy and sustained lysosomal damage. Taken together, our data establish PTP4A2 as a critical regulator of VCP and uncover an important role for PTP4A2 in maintaining lysosomal homeostasis through dephosphorylation of VCP at Tyr805. Our study suggests that PTP4A2 targeting could be a potential therapeutic approach to treat cancers and other degenerative diseases by modulating lysosomal homeostasis and macroautophagy/autophagy.
    Keywords:  ELDR complex; PLAA; PRL phosphatase; PTP4A2; UBXN6; VCP; autophagy; dephosphorylation; lysosome
    DOI:  https://doi.org/10.1080/15548627.2022.2140558
  23. Nature. 2022 Oct 26.
    Cryo-EM structure of the SEA complex.
    Lucas Tafur, Kerstin Hinterndorfer, Caroline Gabus, Chiara Lamanna, Ariane Bergmann, Yashar Sadian, Farzad Hamdi, Fotis L Kyrilis, Panagiotis L Kastritis, Robbie Loewith.
      The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in yeast1. Functionally, the SEAC has been divided into two subcomplexes: SEACIT, which has GAP activity and inhibits TORC1, and SEACAT, which regulates SEACIT2. This system is conserved in mammals: the GATOR complex, consisting of GATOR1 (SEACIT) and GATOR2 (SEACAT), transmits amino acid3 and glucose4 signals to mTORC1. Despite its importance, the structure of SEAC/GATOR, and thus molecular understanding of its function, is lacking. Here, we solve the cryo-EM structure of the native eight-subunit SEAC. The SEAC has a modular structure in which a COPII-like cage corresponding to SEACAT binds two flexible wings, which correspond to SEACIT. The wings are tethered to the core via Sea3, which forms part of both modules. The GAP mechanism of GATOR1 is conserved in SEACIT, and GAP activity is unaffected by SEACAT in vitro. In vivo, the wings are essential for recruitment of the SEAC to the vacuole, primarily via the EGO complex. Our results indicate that rather than being a direct inhibitor of SEACIT, SEACAT acts as a scaffold for the binding of TORC1 regulators.
    DOI:  https://doi.org/10.1038/s41586-022-05370-0
  24. Commun Biol. 2022 Oct 27. 5(1): 1141
    Dual roles of mTORC1-dependent activation of the ubiquitin-proteasome system in muscle proteostasis.
    Marco S Kaiser, Giulia Milan, Daniel J Ham, Shuo Lin, Filippo Oliveri, Kathrin Chojnowska, Lionel A Tintignac, Nitish Mittal, Christian E Zimmerli, David J Glass, Mihaela Zavolan, Markus A Rüegg.
      Muscle size is controlled by the PI3K-PKB/Akt-mTORC1-FoxO pathway, which integrates signals from growth factors, energy and amino acids to activate protein synthesis and inhibit protein breakdown. While mTORC1 activity is necessary for PKB/Akt-induced muscle hypertrophy, its constant activation alone induces muscle atrophy. Here we show that this paradox is based on mTORC1 activity promoting protein breakdown through the ubiquitin-proteasome system (UPS) by simultaneously inducing ubiquitin E3 ligase expression via feedback inhibition of PKB/Akt and proteasome biogenesis via Nuclear Factor Erythroid 2-Like 1 (Nrf1). Muscle growth was restored by reactivation of PKB/Akt, but not by Nrf1 knockdown, implicating ubiquitination as the limiting step. However, both PKB/Akt activation and proteasome depletion by Nrf1 knockdown led to an immediate disruption of proteome integrity with rapid accumulation of damaged material. These data highlight the physiological importance of mTORC1-mediated PKB/Akt inhibition and point to juxtaposed roles of the UPS in atrophy and proteome integrity.
    DOI:  https://doi.org/10.1038/s42003-022-04097-y
  25. Mol Ther Methods Clin Dev. 2022 Dec 08. 27 109-130
    IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy.
    Qiushi Liang, Fabio Catalano, Eva C Vlaar, Joon M Pijnenburg, Merel Stok, Yvette van Helsdingen, Arnold G Vulto, Ans T van der Ploeg, Niek P van Til, W W M Pim Pijnappel.
      Pompe disease is caused by deficiency of acid α-glucosidase (GAA), resulting in glycogen accumulation in various tissues, including cardiac and skeletal muscles and the central nervous system (CNS). Enzyme replacement therapy (ERT) improves cardiac, motor, and respiratory functions but is limited by poor cellular uptake and its inability to cross the blood-brain barrier. Previously, we showed that hematopoietic stem cell (HSPC)-mediated lentiviral gene therapy (LVGT) with codon-optimized GAA (LV-GAAco) caused glycogen reduction in heart, skeletal muscles, and partially in the brain at high vector copy number (VCN). Here, we fused insulin-like growth factor 2 (IGF2) to a codon-optimized version of GAA (LV-IGF2.GAAco) to improve cellular uptake by the cation-independent mannose 6-phosphate/IGF2 (CI-M6P/IGF2) receptor. In contrast to LV-GAAco, LV-IGF2.GAAco was able to completely normalize glycogen levels, pathology, and impaired autophagy at a clinically relevant VCN of 3 in heart and skeletal muscles. LV-IGF2.GAAco was particularly effective in treating the CNS, as normalization of glycogen levels and neuroinflammation was achieved at a VCN between 0.5 and 3, doses at which LV-GAAco was largely ineffective. These results identify IGF2.GAA as a candidate transgene for future clinical development of HSPC-LVGT for Pompe disease.
    Keywords:  GAA; IGF2; Pompe; gene therapy; glycogen storage disease; hematopoietic; lentiviral
    DOI:  https://doi.org/10.1016/j.omtm.2022.09.010
  26. Clin Transl Med. 2022 Oct;12(10): e1097
    The PITT pathway: Keeping lysosomes young.
    Haoxiang Yang, Jay Xiaojun Tan.
      
    DOI:  https://doi.org/10.1002/ctm2.1097
  27. Cell Rep. 2022 Oct 25. pii: S2211-1247(22)01404-8. [Epub ahead of print]41(4): 111548
    mTOR- and LARP1-dependent regulation of TOP mRNA poly(A) tail and ribosome loading.
    Koichi Ogami, Yuka Oishi, Kentaro Sakamoto, Mayu Okumura, Ryota Yamagishi, Takumi Inoue, Masaya Hibino, Takuto Nogimori, Natsumi Yamaguchi, Kazuya Furutachi, Nao Hosoda, Hiroto Inagaki, Shin-Ichi Hoshino.
      Translation of 5' terminal oligopyrimidine (TOP) mRNAs encoding the protein synthesis machinery is strictly regulated by an amino-acid-sensing mTOR pathway. However, its regulatory mechanism remains elusive. Here, we demonstrate that TOP mRNA translation positively correlates with its poly(A) tail length under mTOR active/amino-acid-rich conditions, suggesting that TOP mRNAs are post-transcriptionally controlled by poly(A) tail-length regulation. Consistent with this, the tail length of TOP mRNAs dynamically fluctuates in response to amino acid availability. The poly(A) tail shortens under mTOR active/amino-acid-rich conditions, whereas the long-tailed TOP mRNAs accumulate under mTOR inactive/amino-acid-starved (AAS) conditions. An RNA-binding protein, LARP1, is indispensable for the process. LARP1 interacts with non-canonical poly(A) polymerases and induces post-transcriptional polyadenylation of the target. Our findings illustrate that LARP1 contributes to the selective accumulation of TOP mRNAs with long poly(A) tails under AAS, resulting in accelerated ribosomal loading onto TOP mRNAs for the resumption of translation after AAS.
    Keywords:  CP: Molecular biology; LARP1; TOP mRNA; amino acid starvation; deadenylation; mTOR; poly(A) tail; polyadenylation; ribosome; translation
    DOI:  https://doi.org/10.1016/j.celrep.2022.111548
  28. Biomolecules. 2022 Oct 12. pii: 1460. [Epub ahead of print]12(10):
    An Overview of Molecular Mechanisms in Fabry Disease.
    Federica Amodio, Martina Caiazza, Emanuele Monda, Marta Rubino, Laura Capodicasa, Flavia Chiosi, Vincenzo Simonelli, Francesca Dongiglio, Fabio Fimiani, Nicola Pepe, Cristina Chimenti, Paolo Calabrò, Giuseppe Limongelli.
      Fabry disease (FD) (OMIM #301500) is a rare genetic lysosomal storage disorder (LSD). LSDs are characterized by inappropriate lipid accumulation in lysosomes due to specific enzyme deficiencies. In FD, the defective enzyme is α-galactosidase A (α-Gal A), which is due to a mutation in the GLA gene on the X chromosome. The enzyme deficiency leads to a continuous deposition of neutral glycosphingolipids (globotriaosylceramide) in the lysosomes of numerous tissues and organs, including endothelial cells, smooth muscle cells, corneal epithelial cells, renal glomeruli and tubules, cardiac muscle and ganglion cells of the nervous system. This condition leads to progressive organ failure and premature death. The increasing understanding of FD, and LSD in general, has led in recent years to the introduction of enzyme replacement therapy (ERT), which aims to slow, if not halt, the progression of the metabolic disorder. In this review, we provide an overview of the main features of FD, focusing on its molecular mechanism and the role of biomarkers.
    Keywords:  Fabry disease; GLA gene; biomarkers; mutations; α-galactosidase A
    DOI:  https://doi.org/10.3390/biom12101460
  29. Cell Rep. 2022 10 25. pii: S2211-1247(22)01358-4. [Epub ahead of print]41(4): 111508
    Genome-wide CRISPR screen reveals v-ATPase as a drug target to lower levels of ALS protein ataxin-2.
    Garam Kim, Lisa Nakayama, Jacob A Blum, Tetsuya Akiyama, Steven Boeynaems, Meenakshi Chakraborty, Julien Couthouis, Eduardo Tassoni-Tsuchida, Caitlin M Rodriguez, Michael C Bassik, Aaron D Gitler.
      Mutations in the ataxin-2 gene (ATXN2) cause the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). A therapeutic strategy using antisense oligonucleotides targeting ATXN2 has entered clinical trial in humans. Additional ways to decrease ataxin-2 levels could lead to cheaper or less invasive therapies and elucidate how ataxin-2 is normally regulated. Here, we perform a genome-wide fluorescence-activated cell sorting (FACS)-based CRISPR-Cas9 screen in human cells and identify genes encoding components of the lysosomal vacuolar ATPase (v-ATPase) as modifiers of endogenous ataxin-2 protein levels. Multiple FDA-approved small molecule v-ATPase inhibitors lower ataxin-2 protein levels in mouse and human neurons, and oral administration of at least one of these drugs-etidronate-is sufficient to decrease ataxin-2 in the brains of mice. Together, we propose v-ATPase as a drug target for ALS and SCA2 and demonstrate the value of FACS-based screens in identifying genetic-and potentially druggable-modifiers of human disease proteins.
    Keywords:  ALS; CP: Neuroscience; FACS; SCA2; TDP-43; ataxin-2; bisphosphonate; etidronate; genetic screens; small-molecule therapy; v-ATPase
    DOI:  https://doi.org/10.1016/j.celrep.2022.111508
  30. Trends Biochem Sci. 2022 Oct 21. pii: S0968-0004(22)00271-7. [Epub ahead of print]
    Digest it all: the lysosomal turnover of cytoplasmic aggregates.
    Mario Mauthe, Harm H Kampinga, Mark S Hipp, Fulvio Reggiori.
      Aggrephagy describes the selective lysosomal transport and turnover of cytoplasmic protein aggregates by macro-autophagy. In this process, protein aggregates and conglomerates are polyubiquitinated and then sequestered by autophagosomes. Soluble selective autophagy receptors (SARs) are central to aggrephagy and physically bind to both ubiquitin and the autophagy machinery, thus linking the cargo to the forming autophagosomal membrane. Because the accumulation of protein aggregates is associated with cytotoxicity in several diseases, a better molecular understanding of aggrephagy might provide a conceptual framework to develop therapeutic strategies aimed at delaying the onset of these pathologies by preventing the buildup of potentially toxic aggregates. We review recent advances in our knowledge about the mechanism of aggrephagy.
    Keywords:  cellular protein quality control; chaperone-mediated autophagy; macro-autophagy; micro-autophagy; p62 bodies; selective autophagy receptors
    DOI:  https://doi.org/10.1016/j.tibs.2022.09.012
  31. Proc Natl Acad Sci U S A. 2022 Nov;119(44): e2208882119
    Mechanism of 4-aminopyridine inhibition of the lysosomal channel TMEM175.
    SeCheol Oh, Robyn Stix, Wenchang Zhou, José D Faraldo-Gómez, Richard K Hite.
      Transmembrane protein 175 (TMEM175) is an evolutionarily distinct lysosomal cation channel whose mutation is associated with the development of Parkinson's disease. Here, we present a cryoelectron microscopy structure and molecular simulations of TMEM175 bound to 4-aminopyridine (4-AP), the only known small-molecule inhibitor of TMEM175 and a broad K+ channel inhibitor, as well as a drug approved by the Food and Drug Administration against multiple sclerosis. The structure shows that 4-AP, whose mode of action had not been previously visualized, binds near the center of the ion conduction pathway, in the open state of the channel. Molecular dynamics simulations reveal that this binding site is near the middle of the transmembrane potential gradient, providing a rationale for the voltage-dependent dissociation of 4-AP from TMEM175. Interestingly, bound 4-AP rapidly switches between three predominant binding poses, stabilized by alternate interaction patterns dictated by the twofold symmetry of the channel. Despite this highly dynamic binding mode, bound 4-AP prevents not only ion permeation but also water flow. Together, these studies provide a framework for the rational design of novel small-molecule inhibitors of TMEM175 that might reveal the role of this channel in human lysosomal physiology both in health and disease.
    Keywords:  4-AP; Parkinson’s disease; T MEM175; lysosomal channel
    DOI:  https://doi.org/10.1073/pnas.2208882119
  32. Nature. 2022 Oct 26.
    Non-canonical β-adrenergic activation of ERK at endosomes.
    Yonghoon Kwon, Sohum Mehta, Mary Clark, Geneva Walters, Yanghao Zhong, Ha Neul Lee, Roger K Sunahara, Jin Zhang.
      G-protein-coupled receptors (GPCRs), the largest family of signalling receptors, as well as important drug targets, are known to activate extracellular-signal-regulated kinase (ERK)-a master regulator of cell proliferation and survival1. However, the precise mechanisms that underlie GPCR-mediated ERK activation are not clearly understood2-4. Here we investigated how spatially organized β2-adrenergic receptor (β2AR) signalling controls ERK. Using subcellularly targeted ERK activity biosensors5, we show that β2AR signalling induces ERK activity at endosomes, but not at the plasma membrane. This pool of ERK activity depends on active, endosome-localized Gαs and requires ligand-stimulated β2AR endocytosis. We further identify an endosomally localized non-canonical signalling axis comprising Gαs, RAF and mitogen-activated protein kinase kinase, resulting in endosomal ERK activity that propagates into the nucleus. Selective inhibition of endosomal β2AR and Gαs signalling blunted nuclear ERK activity, MYC gene expression and cell proliferation. These results reveal a non-canonical mechanism for the spatial regulation of ERK through GPCR signalling and identify a functionally important endosomal signalling axis.
    DOI:  https://doi.org/10.1038/s41586-022-05343-3
  33. Nat Cell Biol. 2022 Oct 27.
    PTEN directs developmental and metabolic signaling for innate-like T cell fate and tissue homeostasis.
    Daniel Bastardo Blanco, Nicole M Chapman, Jana L Raynor, Chengxian Xu, Wei Su, Anil Kc, Wei Li, Seon Ah Lim, Stefan Schattgen, Hao Shi, Isabel Risch, Yu Sun, Yogesh Dhungana, Yunjung Kim, Jun Wei, Sherri Rankin, Geoffrey Neale, Paul G Thomas, Kai Yang, Hongbo Chi.
      Phosphatase and tensin homologue (PTEN) is frequently mutated in human cancer, but its roles in lymphopoiesis and tissue homeostasis remain poorly defined. Here we show that PTEN orchestrates a two-step developmental process linking antigen receptor and IL-23-Stat3 signalling to type-17 innate-like T cell generation. Loss of PTEN leads to pronounced accumulation of mature IL-17-producing innate-like T cells in the thymus. IL-23 is essential for their accumulation, and ablation of IL-23 or IL-17 signalling rectifies the reduced survival of female PTEN-haploinsufficient mice that model human patients with PTEN mutations. Single-cell transcriptome and network analyses revealed the dynamic regulation of PTEN, mTOR and metabolic activities that accompanied type-17 cell programming. Furthermore, deletion of mTORC1 or mTORC2 blocks PTEN loss-driven type-17 cell accumulation, and this is further shaped by the Foxo1 and Stat3 pathways. Collectively, our study establishes developmental and metabolic signalling networks underpinning type-17 cell fate decisions and their functional effects at coordinating PTEN-dependent tissue homeostasis.
    DOI:  https://doi.org/10.1038/s41556-022-01011-w
  34. EMBO J. 2022 Oct 25. e108970
    The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to bacterial infection.
    Orsolya Bilkei-Gorzo, Tiaan Heunis, José Luis Marín-Rubio, Francesca Romana Cianfanelli, Benjamin Bernard Armando Raymond, Joseph Inns, Daniela Fabrikova, Julien Peltier, Fiona Oakley, Ralf Schmid, Anetta Härtlova, Matthias Trost.
      Phagocytosis is a key process in innate immunity and homeostasis. After particle uptake, newly formed phagosomes mature by acquisition of endolysosomal enzymes. Macrophage activation by interferon gamma (IFN-γ) increases microbicidal activity, but delays phagosomal maturation by an unknown mechanism. Using quantitative proteomics, we show that phagosomal proteins harbour high levels of typical and atypical ubiquitin chain types. Moreover, phagosomal ubiquitylation of vesicle trafficking proteins is substantially enhanced upon IFN-γ activation of macrophages, suggesting a role in regulating phagosomal functions. We identified the E3 ubiquitin ligase RNF115, which is enriched on phagosomes of IFN-γ activated macrophages, as an important regulator of phagosomal maturation. Loss of RNF115 protein or ligase activity enhanced phagosomal maturation and increased cytokine responses to bacterial infection, suggesting that both innate immune signalling from the phagosome and phagolysosomal trafficking are controlled through ubiquitylation. RNF115 knock-out mice show less tissue damage in response to S. aureus infection, indicating a role of RNF115 in inflammatory responses in vivo. In conclusion, RNF115 and phagosomal ubiquitylation are important regulators of innate immune functions during bacterial infections.
    Keywords:  E3 ligase; RNF115; macrophage; phagosome; ubiquitin
    DOI:  https://doi.org/10.15252/embj.2021108970
  35. Biomedicines. 2022 Sep 21. pii: 2351. [Epub ahead of print]10(10):
    All Roads Lead to Cathepsins: The Role of Cathepsins in Non-Alcoholic Steatohepatitis-Induced Hepatocellular Carcinoma.
    Hester van Mourik, Mengying Li, Sabine Baumgartner, Jan Theys, Ronit Shiri-Sverdlov.
      Cathepsins are lysosomal proteases that are essential to maintain cellular physiological homeostasis and are involved in multiple processes, such as immune and energy regulation. Predominantly, cathepsins reside in the lysosomal compartment; however, they can also be secreted by cells and enter the extracellular space. Extracellular cathepsins have been linked to several pathologies, including non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). NASH is an increasingly important risk factor for the development of HCC, which is the third leading cause of cancer-related deaths and poses a great medical and economic burden. While information regarding the involvement of cathepsins in NASH-induced HCC (NASH-HCC) is limited, data to support the role of cathepsins in either NASH or HCC is accumulating. Since cathepsins play a role in both NASH and HCC, it is likely that the role of cathepsins is more significant in NASH-HCC compared to HCC derived from other etiologies. In the current review, we provide an overview on the available data regarding cathepsins in NASH and HCC, argue that cathepsins play a key role in the transition from NASH to HCC, and shed light on therapeutic options in this context.
    Keywords:  HCC; NASH; cancer hallmarks; cathepsins
    DOI:  https://doi.org/10.3390/biomedicines10102351
  36. Cell Calcium. 2022 Oct 11. pii: S0143-4160(22)00131-2. [Epub ahead of print]108 102658
    The evolution of organellar calcium mapping technologies.
    Matthew Zajac, Souvik Modi, Yamuna Krishnan.
      Intracellular Ca2+ fluxes are dynamically controlled by the co-involvement of multiple organellar pools of stored Ca2+. Endolysosomes are emerging as physiologically critical, yet underexplored, sources and sinks of intracellular Ca2+. Delineating the role of organelles in Ca2+ signaling has relied on chemical fluorescent probes and electrophysiological strategies. However, the acidic endolysosomal environment presents unique issues, which preclude the use of traditional chemical reporter strategies to map lumenal Ca2+. Here, we broadly address the current state of knowledge about organellar Ca2+ pools. We then outline the application of traditional probes, and their sensing paradigms. We then discuss how a new generation of probes overcomes the limitations of traditional Ca2+probes, emphasizing their ability to offer critical insights into endolysosomal Ca2+, and its feedback with other organellar pools.
    DOI:  https://doi.org/10.1016/j.ceca.2022.102658
  37. J Biol Chem. 2022 Oct 25. pii: S0021-9258(22)01084-5. [Epub ahead of print] 102641
    A method for the isolation and characterization of autophagic bodies from yeast provides a key tool to investigate cargos of autophagy.
    Tomoko Kawamata, Shiho Makino, Yoko Kagohashi, Michiko Sasaki, Yoshinori Ohsumi.
      Autophagy is a major cellular degradation pathway that is highly conserved among eukaryotes. The identification of cargos captured by autophagosomes is critical to our understanding of the physiological significance of autophagy in cells, but these studies can be challenging because autophagosomes disintegrate easily. In the yeast S. cerevisiae, cells deficient in the vacuolar lipase Atg15 accumulate autophagic bodies (ABs) within the vacuole following the induction of autophagy. As ABs contain cytosolic components including proteins, RNAs, and lipids, their purification allows the identification of material targeted by autophagy for degradation. In this study, we demonstrate a method to purify intact ABs using isolated vacuoles from atg15Δ cells. Taking advantage of the size discrepancy between the vacuoles and ABs, the vacuolar membrane was disrupted by filtration to release ABs. Filtered vacuolar lysates were subjected to density gradient centrifugation to obtain AB fractions. Purified ABs retain membrane integrity and contain autophagic cargos. This technique offers a valuable tool for the identification of the cargos of autophagy, examination of autophagic cargo selectivity, and biochemical characterization of autophagosome membranes.
    Keywords:  autophagic bodies; autophagy; degradation; starvation; yeast
    DOI:  https://doi.org/10.1016/j.jbc.2022.102641
  38. Mol Med Rep. 2022 Dec;pii: 368. [Epub ahead of print]26(6):
    Multi‑faceted roles of cathepsins in ischemia reperfusion injury (Review).
    Jaime Huertas, H Thomas Lee.
      Cathepsins are one of the most abundant proteases within the lysosomes with diverse physiological effects ranging from immune responses, cell death and intracellular protein degradation. Cathepsins are involved in extracellular and systemic functions such as systemic inflammation and extracellular matrix degradation. Ischemia reperfusion (IR) injury is responsible for numerous diseases including myocardial infarction, acute kidney injury, stroke and acute graft failure after transplant surgery. Inflammation plays a major role in the reperfusion phase of IR injury and previous research has shown that cathepsins are key mediators of the inflammation cascade as well as apoptosis. Taken together, cathepsins modulation could provide potential therapeutic approaches to attenuate IR injury. The present review summarized the current understanding of various cathepsin subtypes, their major physiologic functions, their roles in multi‑organ IR injury and detailed selective cathepsin inhibitors with therapeutic potential.
    Keywords:  apoptosis; cell death; inflammation; lysosome; necrosis
    DOI:  https://doi.org/10.3892/mmr.2022.12885
  39. Hum Mol Genet. 2022 Oct 25. pii: ddac263. [Epub ahead of print]
    A Caenorhabditis elegans model of autosomal dominant adult-onset neuronal ceroid lipofuscinosis identifies ethosuximide as a potential therapeutic.
    Eleanor Barker, Alan Morgan, Jeff W Barclay.
      Autosomal dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is a rare neurodegenerative disorder characterised by progressive dementia and premature death. Four ANCL-causing mutations have been identified, all mapping to the DNAJC5 gene that encodes cysteine string protein α (CSPα). Here, using Caenorhabditis elegans, we describe an animal model of ANCL in which disease-causing mutations are introduced into their endogenous chromosomal locus, thereby mirroring the human genetic disorder. This was achieved through CRISPR/Cas9 mediated gene editing of dnj-14, the C. elegans ortholog of DNAJC5. The resultant homozygous ANCL mutant worms exhibited reduced lifespans and severely impaired chemotaxis, similar to isogenic dnj-14 null mutants. Importantly, these phenotypes were also seen in balanced heterozygotes carrying one wild-type and one ANCL mutant dnj-14 allele, mimicking the heterozygosity of ANCL patients. We observed a more severe chemotaxis phenotype in heterozygous ANCL mutant worms compared to haploinsufficient worms lacking one copy of CSP, consistent with a dominant-negative mechanism of action. Additionally, we provide evidence of CSP haploinsufficiency in longevity, as heterozygous null mutants exhibited significantly shorter lifespan than wild-type controls. The chemotaxis phenotype of dnj-14 null mutants was fully rescued by transgenic human CSPα, confirming the translational relevance of the worm model. Finally, a focused compound screen revealed that the anti-epileptic drug ethosuximide could restore chemotaxis in dnj-14 ANCL mutants to wild-type levels. This suggests that ethosuximide may have therapeutic potential for ANCL and demonstrates the utility of this C. elegans model for future larger-scale drug screening.
    DOI:  https://doi.org/10.1093/hmg/ddac263
  40. Sci Adv. 2022 Oct 28. 8(43): eabo1274
    Many roads lead to CASM: Diverse stimuli of noncanonical autophagy share a unifying molecular mechanism.
    Joanne Durgan, Oliver Florey.
      Autophagy is a fundamental catabolic process coordinated by a network of autophagy-related (ATG) proteins. These ATG proteins also perform an important parallel role in "noncanonical" autophagy, a lysosome-associated signaling pathway with key functions in immunity, inflammation, cancer, and neurodegeneration. While the noncanonical autophagy pathway shares the common ATG machinery, it bears key mechanistic and functional distinctions, and is characterized by conjugation of ATG8 to single membranes (CASM). Here, we review the diverse, and still expanding, collection of stimuli and processes now known to harness the noncanonical autophagy pathway, including engulfment processes, drug treatments, TRPML1 and STING signaling, viral infection, and other pathogenic factors. We discuss the multiple associated routes to CASM and assess their shared and distinctive molecular features. By integrating these findings, we propose an updated and unifying mechanism for noncanonical autophagy, centered on ATG16L1 and V-ATPase.
    DOI:  https://doi.org/10.1126/sciadv.abo1274
  41. Nat Rev Mol Cell Biol. 2022 Oct 27.
    The mechanisms and roles of selective autophagy in mammals.
    Jose Norberto S Vargas, Maho Hamasaki, Tsuyoshi Kawabata, Richard J Youle, Tamotsu Yoshimori.
      Autophagy is a process that targets various intracellular elements for degradation. Autophagy can be non-selective - associated with the indiscriminate engulfment of cytosolic components - occurring in response to nutrient starvation and is commonly referred to as bulk autophagy. By contrast, selective autophagy degrades specific targets, such as damaged organelles (mitophagy, lysophagy, ER-phagy, ribophagy), aggregated proteins (aggrephagy) or invading bacteria (xenophagy), thereby being importantly involved in cellular quality control. Hence, not surprisingly, aberrant selective autophagy has been associated with various human pathologies, prominently including neurodegeneration and infection. In recent years, considerable progress has been made in understanding mechanisms governing selective cargo engulfment in mammals, including the identification of ubiquitin-dependent selective autophagy receptors such as p62, NBR1, OPTN and NDP52, which can bind cargo and ubiquitin simultaneously to initiate pathways leading to autophagy initiation and membrane recruitment. This progress opens the prospects for enhancing selective autophagy pathways to boost cellular quality control capabilities and alleviate pathology.
    DOI:  https://doi.org/10.1038/s41580-022-00542-2
  42. Biochem Soc Trans. 2022 Oct 28. pii: BST20220519. [Epub ahead of print]
    Fundamental roles for inter-organelle communication in aging.
    Eric K F Donahue, Elizabeth M Ruark, Kristopher Burkewitz.
      Advances in public health have nearly doubled life expectancy over the last century, but this demographic shift has also changed the landscape of human illness. Today, chronic and age-dependent diseases dominate the leading causes of morbidity and mortality worldwide. Targeting the underlying molecular, genetic and cell biological drivers of the aging process itself appears to be an increasingly viable strategy for developing therapeutics against these diseases of aging. Towards this end, one of the most exciting developments in cell biology over the last decade is the explosion of research into organelle contact sites and related mechanisms of inter-organelle communication. Identification of the molecular mediators of inter-organelle tethering and signaling is now allowing the field to investigate the consequences of aberrant organelle interactions, which frequently seem to correlate with age-onset pathophysiology. This review introduces the major cellular roles for inter-organelle interactions, including the regulation of organelle morphology, the transfer of ions, lipids and other metabolites, and the formation of hubs for nutrient and stress signaling. We explore how these interactions are disrupted in aging and present findings that modulation of inter-organelle communication is a promising avenue for promoting longevity. Through this review, we propose that the maintenance of inter-organelle interactions is a pillar of healthy aging. Learning how to target the cellular mechanisms for sensing and controlling inter-organelle communication is a key next hurdle for geroscience.
    Keywords:  aging; endoplasmic reticulum; inter-organelle; longevity; mitochondria
    DOI:  https://doi.org/10.1042/BST20220519
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