bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021‒10‒31
forty papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing


  1. Cell Syst. 2021 Oct 21. pii: S2405-4712(21)00382-3. [Epub ahead of print]
      Pancreatic cancer cells with limited access to free amino acids can grow by scavenging extracellular protein. In a murine model of pancreatic cancer, we performed a genome-wide CRISPR screen for genes required for scavenging-dependent growth. The screen identified key mediators of macropinocytosis, peripheral lysosome positioning, endosome-lysosome fusion, lysosomal protein catabolism, and translational control. The top hit was GCN2, a kinase that suppresses translation initiation upon amino acid depletion. Using isotope tracers, we show that GCN2 is not required for protein scavenging. Instead, GCN2 prevents ribosome stalling but without slowing protein synthesis; cells still use all of the limiting amino acids as they emerge from lysosomes. GCN2 also adapts gene expression to the nutrient-poor environment, reorienting protein synthesis away from ribosomes and toward lysosomal hydrolases, such as cathepsin L. GCN2, cathepsin L, and the other genes identified in the screen are potential therapeutic targets in pancreatic cancer.
    Keywords:  Cathepsin L; GCN2; PDAC; lysosomes; macropinocytosis; protein scavenging; protein synthesis; translation
    DOI:  https://doi.org/10.1016/j.cels.2021.09.014
  2. Autophagy. 2021 Oct 25.
      Organelle-specific autophagy directs degradation of eukaryotic organelles under certain conditions. Like other organelles, peroxisomes are subject to autophagic turnover at lysosomes. However, peroxisome autophagy (pexophagy) has yet to be analyzed in a live-animal system, limiting knowledge on its regulation during an animal's life. Here, we generated a tandem-fluorophore reporter that enabled real-time tracking of pexophagy in live Caenorhabditis elegans. We observed that pexophagy occurred at a population of non-canonical, tubular lysosomes specifically during starvation and aging. Remarkably, in these contexts, tubular lysosomes were the predominant type of lysosome in the intestine, transforming from vesicles. Though we found that peroxisomes were largely eliminated in early adulthood, they appeared restored in new generations. We identified peroxisomal genes that regulated age-dependent peroxisome loss and demonstrated that modifying this process altered animal lifespan. These findings reveal new facets of peroxisome homeostasis relevant to aging and challenge the prevailing perception of lysosome homogeneity in autophagy.
    Keywords:  fluorescent reporters; lysosome morphology; markers of aging; peroxisomes; pexophagy; spinster; transgenerational rejuvenation; tubular lysosomes
    DOI:  https://doi.org/10.1080/15548627.2021.1990647
  3. J Mol Biol. 2021 Oct 22. pii: S0022-2836(21)00563-5. [Epub ahead of print] 167326
      The budding yeast Sch9 kinase (functional orthologue of the mammalian S6 kinase) is a major effector of the Target of Rapamycin Complex 1 (TORC1) complex in the regulation of cell growth in response to nutrient availability and stress. Sch9 is partially localized at the vacuolar surface, where it is phosphorylated by TORC1. The recruitment of Sch9 on the vacuole is mediated by direct interaction between phospholipids of the vacuolar membrane and the region of Sch9 encompassing amino acid residues 1-390, which contains a C2 domain. Since many C2 domains mediate phospholipid binding, it had been suggested that the C2 domain of Sch9 mediates its vacuolar recruitment. However, the in vivo requirement of the C2 domain for Sch9 localization had not been demonstrated, and the phenotypic consequences of Sch9 delocalization remained unknown. Here, by examining cellular localization, phosphorylation state and growth phenotypes of Sch9 truncation mutants, we show that deletion of the N-terminal domain of Sch9 (aa 1-182), but not the C2 domain (aa 183-399), impairs vacuolar localization and TORC1-dependent phosphorylation of Sch9, while causing growth defects similar to those observed in sch9Δ cells. These defects can be reversed either via artificial tethering of the protein to the vacuole, or by introducing phosphomimetic mutations at the TORC1 target sites, suggesting that Sch9 localization on the vacuole is needed for the TORC1-dependent activation of the kinase. Our study uncovers a key role for the N-terminal domain of Sch9 and provides new mechanistic insight into the regulation of a major TORC1 signaling branch.
    Keywords:  C2 domain; Cell growth; Saccharomyces cerevisiae; TOR signaling; kinase
    DOI:  https://doi.org/10.1016/j.jmb.2021.167326
  4. Cell Mol Life Sci. 2021 Oct 30.
      Lysosomes are single membrane-bound organelles containing acid hydrolases responsible for the degradation of cellular cargo and maintenance of cellular homeostasis. Lysosomes could originate from pre-existing endolysosomes or autolysosomes, acting as a critical juncture between autophagy and endocytosis. Stress that triggers lysosomal membrane permeabilization can be altered by ESCRT complexes; however, irreparable damage to the membrane results in the induction of a selective lysosomal degradation pathway, specifically lysophagy. Lysosomes play an indispensable role in different types of autophagy, including microautophagy, macroautophagy, and chaperone-mediated autophagy, and various cell death pathways such as lysosomal cell death, apoptotic cell death, and autophagic cell death. In this review, we discuss lysosomal reformation, maintenance, and degradation pathways following the involvement of the lysosome in autophagy and cell death, which are related to several pathophysiological conditions observed in humans.
    Keywords:  Autolysosome; Autophagic cell death; Autophagic lysosome reformation; Autophagy; Lysosome
    DOI:  https://doi.org/10.1007/s00018-021-03988-3
  5. PLoS One. 2021 ;16(10): e0247211
      Gaucher disease (GD) is caused by deficiency of the lysosomal membrane enzyme glucocerebrosidase (GCase) and the subsequent accumulation of its substrate, glucosylceramide (GC). Mostly missense mutations of the glucocerebrosidase gene (GBA) cause GCase misfolding and inhibition of proper lysosomal trafficking. The accumulated GC leads to lysosomal dysfunction and impairs the autophagy pathway. GD types 2 and 3 (GD2-3), or the neuronopathic forms, affect not only the Central Nervous System (CNS) but also have severe systemic involvement and progressive bone disease. Enzyme replacement therapy (ERT) successfully treats the hematologic manifestations; however, due to the lack of equal distribution of the recombinant enzyme in different organs, it has no direct impact on the nervous system and has minimal effect on bone involvement. Small molecules have the potential for better tissue distribution. Ambroxol (AMB) is a pharmacologic chaperone that partially recovers the mutated GCase activity and crosses the blood-brain barrier. Eliglustat (EGT) works by inhibiting UDP-glucosylceramide synthase, an enzyme that catalyzes GC biosynthesis, reducing GC influx load into the lysosome. Substrate reduction therapy (SRT) using EGT is associated with improvement in GD bone marrow burden score and bone mineral density parallel with the improvement in hematological parameters. We assessed the effects of EGT and AMB on GCase activity and autophagy-lysosomal pathway (ALP) in primary cell lines derived from patients with GD2-3 and compared to cell lines from healthy controls. We found that EGT, same as AMB, enhanced GCase activity in control cells and that an individualized response, that varied with GBA mutations, was observed in cells from patients with GD2-3. EGT and AMB enhanced the formation of lysosomal/late endosomal compartments and improved autophagy, independent of GBA mutations. Both AMB and EGT increased mitochondrial mass and density in GD2-3 fibroblasts, suggesting enhancement of mitochondrial function by activating the mitochondrial membrane potential. These results demonstrate that EGT and AMB, with different molecular mechanisms of action, enhance GCase activity and improve autophagy-lysosome dynamics and mitochondrial functions.
    DOI:  https://doi.org/10.1371/journal.pone.0247211
  6. Psychopharmacology (Berl). 2021 Oct 30.
      RATIONALE: Accumulating evidence indicates critical involvement of mammalian target of rapamycin (mTOR) in the treatment of depressive disorders, epilepsy, and neurodegenerative disorders through its signal transduction mechanisms related to protein translation, autophagy, and synaptic remodeling. Electroconvulsive seizure (ECS) treatment is a potent antidepressive, anti-convulsive, and neuroprotective therapeutic modality; however, its effects on mTOR signaling have not yet been clarified.METHODS: The effect of ECS on the mTOR complex 1 (mTORC1) pathway was investigated in the rat frontal cortex. ECS or sham treatment was administered once per day for 10 days (E10X or sham), and compound C was administered through the intracerebroventricular cannula. Changes in mTORC1-associated signaling molecules and their interactions were analyzed.
    RESULTS: E10X reduced phosphorylation of mTOR downstream substrates, including p70S6K, S6, and 4E-BP1, and increased inhibitory phosphorylation of mTOR at Thr2446 compared to the sham group in the rat frontal cortex, indicating E10X-induced inhibition of mTORC1 activity. Akt and ERK1/2, upstream kinases that activate mTORC1, were not inhibited; however, AMPK, which can inhibit mTORC1, was activated. AMPK-responsive phosphorylation of Raptor at Ser792 and TSC2 at Ser1387 inhibiting mTORC1 was increased by E10X. Moreover, intrabrain inhibition of AMPK restored E10X-induced changes in the phosphorylation of S6, Raptor, and TSC2, indicating mediation of AMPK in E10X-induced mTOR inhibition.
    CONCLUSIONS: Repeated ECS treatments inhibit mTORC1 signaling by interactive crosstalk between mTOR and AMPK pathways, which could play important roles in the action of ECS via autophagy induction.
    Keywords:  AMP-activated protein kinase; Electroconvulsive therapy; Mammalian target of rapamycin
    DOI:  https://doi.org/10.1007/s00213-021-06015-2
  7. Natl Sci Rev. 2019 Nov;6(6): 1149-1162
      The mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr protein kinase with essential cellular function via processing various extracellular and intracellular inputs. Two distinct multi-protein mTOR complexes (mTORC), mTORC1 and mTORC2, have been identified and well characterized in eukaryotic cells from yeast to human. Sin1, which stands for Sty1/Spc1-interacting protein1, also known as mitogen-activated protein kinase (MAPK) associated protein (MAPKAP)1, is an evolutionarily conserved adaptor protein. Mammalian Sin1 interacts with many cellular proteins, but it has been widely studied as an essential component of mTORC2, and it is crucial not only for the assembly of mTORC2 but also for the regulation of its substrate specificity. In this review, we summarize our current knowledge of the structure and functions of Sin1, focusing specifically on its protein interaction network and its roles in the mTOR pathway that could account for various cellular functions of mTOR in growth, metabolism, immunity and cancer.
    Keywords:  AGC kinases; Akt; Sin1; mTOR complex; metabolism and immune response
    DOI:  https://doi.org/10.1093/nsr/nwz171
  8. Bone. 2021 Oct 22. pii: S8756-3282(21)00403-8. [Epub ahead of print]154 116237
      Mucopolysaccharidosis (MPS) I is a lysosomal storage disease characterized by deficient activity of the enzyme alpha-L-iduronidase, leading to abnormal accumulation of heparan and dermatan sulfate glycosaminoglycans in cells and tissues. Patients commonly exhibit progressive skeletal abnormalities, in part due to failures of endochondral ossification during postnatal growth. Previously, using the naturally-occurring canine model, we showed that bone and cartilage cells in MPS I exhibit elevated lysosomal storage from an early age and that animals subsequently exhibit significantly diminished vertebral trabecular bone formation. Wnts are critical regulators of endochondral ossification that depend on glycosaminoglycans for signaling. The objective of this study was to examine whether lithium, a glycogen synthase kinase-3 inhibitor and stimulator of Wnt/beta-catenin signaling, administered during postnatal growth could attenuate progression of vertebral trabecular bone disease in MPS I. MPS I dogs were treated orally with therapeutic levels of lithium carbonate from 14 days to 6 months-of-age. Untreated heterozygous and MPS I dogs served as controls. Serum was collected at 3 and 6 months for assessment of bone turnover markers. At the study end point, thoracic vertebrae were excised and assessed using microcomputed tomography and histology. Lithium-treated animals exhibited significantly improved trabecular spacing, number and connectivity density, and serum bone-specific alkaline phosphatase levels compared to untreated animals. Growth plates from lithium-treated animals exhibited increased numbers of hypertrophic chondrocytes relative to both untreated MPS I and heterozygous animals. These findings suggest that bone and cartilage cells in MPS I are still capable of responding to exogenous osteogenic signals even in the presence of significant lysosomal storage, and that targeted osteogenic therapies may represent a promising approach for attenuating bone disease progression in MPS I.
    Keywords:  Bone; Cartilage; Growth plate; Hurler Syndrome; Mucopolysaccharidosis I; Spine; lithium
    DOI:  https://doi.org/10.1016/j.bone.2021.116237
  9. Dev Cell. 2021 Oct 11. pii: S1534-5807(21)00807-8. [Epub ahead of print]
      Viral entry and egress are important determinants of virus infectivity and pathogenicity. β-coronaviruses, including the COVID-19 virus SARS-CoV-2 and mouse hepatitis virus (MHV), exploit the lysosomal exocytosis pathway for egress. Here, we show that SARS-CoV-2 ORF3a, but not SARS-CoV ORF3a, promotes lysosomal exocytosis. SARS-CoV-2 ORF3a facilitates lysosomal targeting of the BORC-ARL8b complex, which mediates trafficking of lysosomes to the vicinity of the plasma membrane, and exocytosis-related SNARE proteins. The Ca2+ channel TRPML3 is required for SARS-CoV-2 ORF3a-mediated lysosomal exocytosis. Expression of SARS-CoV-2 ORF3a greatly elevates extracellular viral release in cells infected with the coronavirus MHV-A59, which itself lacks ORF3a. In SARS-CoV-2 ORF3a, Ser171 and Trp193 are critical for promoting lysosomal exocytosis and blocking autophagy. When these residues are introduced into SARS-CoV ORF3a, it acquires the ability to promote lysosomal exocytosis and inhibit autophagy. Our results reveal a mechanism by which SARS-CoV-2 interacts with host factors to promote its extracellular egress.
    Keywords:  COVID-19; ORF3a; SARS-CoV; SARS-CoV-2; lysosomal exocytosis
    DOI:  https://doi.org/10.1016/j.devcel.2021.10.006
  10. J Biol Chem. 2021 Oct 26. pii: S0021-9258(21)01153-4. [Epub ahead of print] 101347
      The cellular specificity, potency, and modular nature of bacterial protein toxins enable their application for targeted cytosolic delivery of therapeutic cargo. Efficient endosomal escape is a critical step in the design of bacterial toxin-inspired drug delivery (BTIDD) vehicles to avoid lysosomal degradation and promote optimal cargo delivery. The cytotoxic necrotizing factor (CNF) family of modular toxins represents a useful model for investigating cargo-delivery mechanisms due to the availability of many homologs with high sequence identity, their flexibility in swapping domains, and their differential activity profiles. Previously, we found that CNFy is more sensitive to endosomal acidification inhibitors than CNF1 and CNF2. Here, we report that CNF3 is even less sensitive than CNF1/2. We identified two amino acid residues within the putative translocation domain (E374 and E412 in CNFy, Q373 and S411 in CNF3) that differentiate between these two toxins. Swapping these corresponding residues in each toxin changed the sensitivity to endosomal acidification and efficiency of cargo-delivery to be more similar to the other toxin. Results suggested that trafficking to the more acidic late endosome is required for cargo delivery by CNFy but not CNF3. This model was supported by results from toxin treatment of cells in the presence of NH4Cl, which blocks endosomal acidification, and of small molecule inhibitors EGA, which blocks trafficking to late endosomes, and ABMA, which blocks endosomal escape and trafficking to the lysosomal degradative pathway. These findings suggest that it is possible to fine tune endosomal escape and cytosolic cargo delivery efficiency in designing BTIDD platforms.
    Keywords:  bacterial toxin; drug delivery system; fusion protein; molecular evolution; protein chimera; protein deamidation; protein engineering; protein translocation; small GTPase; structure-function
    DOI:  https://doi.org/10.1016/j.jbc.2021.101347
  11. J Biomed Sci. 2021 Oct 27. 28(1): 72
      BACKGROUND: During autophagy defense against invading microbes, certain lipid types are indispensable for generating specialized membrane-bound organelles. The lipid composition of autophagosomes remains obscure, as does the issue of how specific lipids and lipid-associated enzymes participate in autophagosome formation and maturation. Helicobacter pylori is auxotrophic for cholesterol and converts cholesterol to cholesteryl glucoside derivatives, including cholesteryl 6'-O-acyl-α-D-glucoside (CAG). We investigated how CAG and its biosynthetic acyltransferase assist H. pylori to escape host-cell autophagy.METHODS: We applied a metabolite-tagging method to obtain fluorophore-containing cholesteryl glucosides that were utilized to understand their intracellular locations. H. pylori 26695 and a cholesteryl glucosyltransferase (CGT)-deletion mutant (ΔCGT) were used as the standard strain and the negative control that contains no cholesterol-derived metabolites, respectively. Bacterial internalization and several autophagy-related assays were conducted to unravel the possible mechanism that H. pylori develops to hijack the host-cell autophagy response. Subcellular fractions of H. pylori-infected AGS cells were obtained and measured for the acyltransferase activity.
    RESULTS: The imaging studies of fluorophore-labeled cholesteryl glucosides pinpointed their intracellular localization in AGS cells. The result indicated that CAG enhances the internalization of H. pylori in AGS cells. Particularly, CAG, instead of CG and CPG, is able to augment the autophagy response induced by H. pylori. How CAG participates in the autophagy process is multifaceted. CAG was found to intervene in the degradation of autophagosomes and reduce lysosomal biogenesis, supporting the idea that intracellular H. pylori is harbored by autophago-lysosomes in favor of the bacterial survival. Furthermore, we performed the enzyme activity assay of subcellular fractions of H. pylori-infected AGS cells. The analysis showed that the acyltransferase is mainly distributed in autophago-lysosomal compartments.
    CONCLUSIONS: Our results support the idea that the acyltransferase is mainly distributed in the subcellular compartment consisting of autophagosomes, late endosomes, and lysosomes, in which the acidic environment is beneficial for the maximal acyltransferase activity. The resulting elevated level of CAG can facilitate bacterial internalization, interfere with the autophagy flux, and causes reduced lysosomal biogenesis.
    Keywords:  Autophagosomes; Autophagy; Autophagy flux; Bacterial internalization; Cholesteryl glucosides; H. pylori; Lipid-raft clustering; Lysosome biogenesis; Lysosomes
    DOI:  https://doi.org/10.1186/s12929-021-00768-w
  12. iScience. 2021 Nov 19. 24(11): 103177
      The mammalian target of rapamycin (mTOR) is a serine-threonine kinase involved in cellular innate immunity, metabolism, and senescence. FK506-binding protein 12 (FKBP12) inhibits mTOR kinase activity via direct association. The FKBP12-mTOR association can be strengthened by the immunosuppressant rapamycin, but the underlying mechanism remains elusive. We show here that the FKBP12-mTOR association is tightly regulated by an acetylation-deacetylation cycle. FKBP12 is acetylated on the lysine cluster (K45/K48/K53) by CREB-binding protein (CBP) in mammalian cells in response to nutrient treatment. Acetyl-FKBP12 associates with CBP acetylated Rheb. Rapamycin recruits SIRT2 with a high affinity for FKBP12 association and deacetylation. SIRT2-deacetylated FKBP12 then switches its association from Rheb to mTOR. Nutrient-activated mTOR phosphorylates IRF3S386 for the antiviral response. In contrast, rapamycin strengthening FKBP12-mTOR association blocks mTOR antiviral activity by recruiting SIRT2 to deacetylate FKBP12. Hence, on/off mTOR activity in response to environmental nutrients relies on FKBP12 acetylation and deacetylation status in mammalian cells.
    Keywords:  Biochemistry; Molecular biology; Protein
    DOI:  https://doi.org/10.1016/j.isci.2021.103177
  13. J Hepatol. 2021 Oct 25. pii: S0168-8278(21)02151-6. [Epub ahead of print]
      BACKGROUND & AIMS: Either activation of mTORC1 due to loss of Tsc1 (Tuberous Sclerosis Complex 1) or defective hepatic autophagy due to loss of Atg5 leads to spontaneous liver tumorigenesis in mice. The purpose of this study was to investigate the mechanisms of how autophagy impacts mTORC1 activation-mediated liver metabolic changes and tumorigenesis.METHODS: Atg5 Flox/Flox (Atg5F/F) and Tsc1F/F mice were crossed with albumin Cre mice to generate liver-specific Atg5 knockout (L-Atg5 KO), L-Tsc1 KO and L-Atg5/Tsc1 double KO (DKO) mice. These mice were crossed with p62/Sqstm1F/F (p62) and whole body Nrf2 KO mice to generate L-Atg5/Tsc1/p62 and L-Atg5/Tsc1, Nrf2 triple KO (TKO) mice. These mice were housed for various time points up to 12 months, and blood and liver tissues were harvested for biochemical and histological analysis RESULTS: Deletion of Atg5 in L-Tsc1 KO mice inhibited liver tumorigenesis, but increased mortality of L-Tsc1 KO mice accompanied by drastically enhanced hepatic ductular reaction (DR), hepatocyte degeneration and metabolic reprogramming. Deletion of p62 reversed DR, hepatocyte degeneration and metabolic reprogramming as well as the mortality of L-Atg5/Tsc1 DKO mice, but unexpectedly promoted liver tumorigenesis via activation of a group of oncogenic signaling pathways. Nrf2 ablation markedly improved DR with increased hepatocyte population and improved metabolic reprogramming and survival of the L- Atg5/Tsc1 DKO mice without tumor formation. Decreased p62 and increased mTOR activity was also found in a subset of human hepatocellular carcinoma.
    CONCLUSIONS: These results reveal previously undescribed functions of hepatic p62 in suppressing tumorigenesis and regulating liver cell repopulation and metabolic reprogramming resulting from persistent mTORC1 activation and defective autophagy.
    LAY SUMMARY: Metabolic liver disease and viral hepatitis are common chronic liver diseases and risk factors of hepatocellular carcinoma, which are often associated with impaired hepatic autophagy with increased mTOR activation. Using multiple genetic engineered mice that are defective of hepatic autophagy with persistent mTOR activation, we dissected the complex interplay among mTOR, autophagy, p62 and Nrf2 on liver cell repopulation, metabolic reprogramming and redox homeostasis in liver tumorigenesis. Our results uncovered an unexpected novel tumor suppressor function of p62/Sqstm1 by regulating liver cell repopulation, ductular reaction and metabolic reprogramming in liver tumorigenesis.
    Keywords:  Atg5; HCC; Nrf2; Tsc1; fibrosis; p62
    DOI:  https://doi.org/10.1016/j.jhep.2021.10.014
  14. Sci Rep. 2021 Oct 26. 11(1): 21119
      Transcription factor EB (TFEB) is a master regulator of the autophagy-lysosomal pathway (ALP). Here, we cloned a novel splicing variant of TFEB, comprising 281 amino acids (hereafter referred to as small TFEB), and lacking the helix-loop-helix (HLH) and leucine zipper (LZ) motifs present in the full-length TFEB (TFEB-L). The TFEB variant is widely expressed in several tissues, including the brain, although its expression level is considerably lower than that of TFEB-L. Intriguingly, in cells stably expressing small TFEB, the expression profile of genes was inverted compared to that in cells ectopically expressing TFEB-L. In addition, fisetin-induced luciferase activity of promoter containing either coordinated lysosomal expression and regulation (CLEAR) element or antioxidant response element (ARE) was significantly repressed by co-transfection with small TFEB. Moreover, fisetin-mediated clearance of phosphorylated tau or α-synuclein was attenuated in the presence of small TFEB. Taken together, the results suggest that small TFEB is a novel splicing variant of TFEB that might act as a negative regulator of TFEB-L, thus fine tuning the activity of ALP during cellular stress.
    DOI:  https://doi.org/10.1038/s41598-021-00613-y
  15. Anal Chem. 2021 Oct 28.
      Lysosomal acidification is essential for its degradative function, and the flux of H+ correlated with that of K+ in lysosomes. However, there is little research on their correlation due to the lack of probes that can simultaneously image these two ions. To deeply understand the role of K+ in lysosomal acidification, here, we designed and fabricated a nanodevice using a K+-aptamer and two pH-triggered nanoswitches incorporated into a DNA triangular prism (DTP) as a dual signal response platform to simultaneously visualize K+ and pH in lysosomes by a fluorescence method. This strategy could conveniently integrate two signal recognition modules into one probe, so as to achieve the goal of sensitive detection of two kinds of signals in the same time and space, which is suitable for the detection of various signals with the correlation of concentration. By co-imaging both K+ and H+ in lysosomes, we found that the efflux of K+ was accompanied by a decrease of pH, which is of great value in understanding lysosomal acidification. Moreover, this strategy also has broad prospects as a versatile optical sensing platform for multiplexed analysis of other biomolecules in living cells.
    DOI:  https://doi.org/10.1021/acs.analchem.1c04056
  16. Autophagy. 2021 Oct 27.
      Mice deficient for GHR (growth hormone receptor; ghr KO) have a dramatic lifespan extension, and elevated levels of hepatic chaperone-mediated autophagy (CMA). Using quantitative proteomics to identify protein changes in purified liver lysosomes and whole liver lysates, we provide evidence that elevated CMA in ghr KO mice downregulates proteins involved in ribosomal structure, translation initiation and elongation, and nucleocytosolic acetyl-coA production. Following up on these initial proteomics findings, we used a cell culture approach to show that CMA is necessary and sufficient to regulate the abundance of ACLY and ACSS2, the two enzymes that produce nucleocytosolic (but not mitochondrial) acetyl-coA. Inhibition of CMA in NIH3T3 cells has been shown to lead to aberrant accumulation of lipid droplets. We show that this lipid droplet phenotype is rescued by knocking down ACLY or ACSS2, suggesting that CMA regulates lipid droplet formation by controlling ACLY and ACSS2. This evidence leads to a model of how constitutive activation of CMA can shape specific metabolic pathways in long-lived endocrine mutant mice.
    Keywords:  aging; autophagy; growth hormone; metabolism; proteomics
    DOI:  https://doi.org/10.1080/15548627.2021.1990670
  17. Antiviral Res. 2021 Oct 20. pii: S0166-3542(21)00183-2. [Epub ahead of print]195 105193
      Transient receptor potential mucolipin 2 and 3 (TRPML2 and TRPML3), as key channels in the endosomal-lysosomal system, are associated with many different cellular processes, including ion release, membrane trafficking and autophagy. In particular, they can also facilitate viral entry into host cells and enhance viral infection. We previously identified that two selective TRPML agonists, ML-SA1 and SN-2, that showed antiviral activities against dengue virus type 2 (DENV2) and Zika virus (ZIKV) in vitro, but their antiviral mechanisms are still elusive. Here, we reported that ML-SA1 could inhibit DENV2 replication by downregulating the expression of both TRPML2 and TRPML3, while the other TRPML activator, SN-2, suppressed DENV2 infection by reducing only TRPML3 expression. Consistently, the channel activities of both TRPML2 and TRPML3 were also found to be associated with the antiviral activity of ML-SA1 on DENV2 and ZIKV, but SN-2 relied only on TRPML3 channel activity. Further mechanistic experiments revealed that ML-SA1 and SN-2 decreased the expression of the late endosomal marker Rab7, dependent on TRPML2 and TRPML3, indicating that these two compounds likely inhibit viral infection by promoting vesicular trafficking from late endosomes to lysosomes and then accelerating lysosomal degradation of the virus. As expected, neither ML-SA1 nor SN-2 inhibited herpes simplex virus type I (HSV-1), whose entry is independent of the endolysosomal network. Together, our work reveals the antiviral mechanisms of ML-SA1 and SN-2 in targeting TRPML channels, possibly leading to the discovery of new drug candidates to inhibit endocytosed viruses.
    Keywords:  DENV2; ML-SA1; SN-2; TRPML2 and TRPML3; Trafficking
    DOI:  https://doi.org/10.1016/j.antiviral.2021.105193
  18. Elife. 2021 Oct 26. pii: e73240. [Epub ahead of print]10
      Yeast vacuolar membrane fusion has been reconstituted with R, Qa, Qb, and Qc-family SNAREs, Sec17/αSNAP, Sec18/NSF, and the hexameric HOPS complex. HOPS tethers membranes and catalyzes SNARE assembly into RQaQbQc trans-complexes which zipper through their SNARE domains to promote fusion. Previously, we demonstrated that Sec17 and Sec18 can bypass the requirement of complete zippering for fusion (Song et al., 2021), but it has been unclear whether this activity of Sec17 and Sec18 is directly coupled to HOPS. HOPS can be replaced for fusion by a synthetic tether when the three Q-SNAREs are pre-assembled. We now report that fusion intermediates with arrested SNARE zippering, formed with a synthetic tether but without HOPS, support Sec17/Sec18-triggered fusion. This zippering-bypass fusion is thus a direct result of Sec17 and Sec18 interactions: with each other, with the platform of partially zippered SNAREs, and with the apposed tethered membranes. As these fusion elements are shared among all exocytic and endocytic traffic, Sec17 and Sec18 may have a general role in directly promoting fusion.
    Keywords:  S. cerevisiae; biochemistry; chemical biology
    DOI:  https://doi.org/10.7554/eLife.73240
  19. Mol Genet Metab Rep. 2021 Dec;29 100811
      Mucopolysaccharidosis type IIIA (MPS IIIA) is characterised by a progressive neurological decline leading to early death. It is caused by bi-allelic loss-of-function mutations in SGSH encoding sulphamidase, a lysosomal enzyme required for heparan sulphate glycosaminoglycan (HS GAG) degradation, that results in the progressive build-up of HS GAGs in multiple tissues most notably the central nervous system (CNS). Skin fibroblasts from two MPS IIIA patients who presented with an intermediate and a severe clinical phenotype, respectively, were reprogrammed into induced pluripotent stem cells (iPSCs). The intermediate MPS IIIA iPSCs were then differentiated into neural progenitor cells (NPCs) and subsequently neurons. The patient derived fibroblasts, iPSCs, NPCs and neurons all displayed hallmark biochemical characteristics of MPS IIIA including reduced sulphamidase activity and increased accumulation of an MPS IIIA HS GAG biomarker. Proliferation of MPS IIIA iPSC-derived NPCs was reduced compared to control, but could be partially rescued by reintroducing functional sulphamidase enzyme, or by doubling the concentration of the mitogen fibroblast growth factor 2 (FGF2). Whilst both control heparin, and MPS IIIA HS GAGs had a similar binding affinity for FGF2, only the latter inhibited FGF signalling, suggesting accumulated MPS IIIA HS GAGs disrupt the FGF2:FGF2 receptor:HS signalling complex. Neuronal differentiation of MPS IIIA iPSC-derived NPCs was associated with a reduction in the expression of neuronal cell marker genes βIII-TUBULIN, NF-H and NSE, revealing reduced neurogenesis compared to control. A similar result was achieved by adding MPS IIIA HS GAGs to the culture medium during neuronal differentiation of control iPSC-derived NPCs. This study demonstrates the generation of MPS IIIA iPSCs, and NPCs, the latter of which display reduced proliferation and neurogenic capacity. Reduced NPC proliferation can be explained by a model in which soluble MPS IIIA HS GAGs compete with cell surface HS for FGF2 binding. The mechanism driving reduced neurogenesis remains to be determined but appears downstream of MPS IIIA HS GAG accumulation.
    Keywords:  CNS, central nervous system; FGF, Fibroblast growth factor; Fibroblast growth factor 2; GAG, glycosaminoglycan; HS, heparan sulphate; Heparan sulphate glycosaminoglycan; Induced pluripotent stem cells; MEF, mouse embryonic fibroblast; MPS IIIA; MPS IIIA, mucopolysaccharidosis type IIIA; NPC, neural progenitor cell; Neurogenesis; iPSC, induced pluripotent stem cell
    DOI:  https://doi.org/10.1016/j.ymgmr.2021.100811
  20. Exp Gerontol. 2021 Oct 22. pii: S0531-5565(21)00380-6. [Epub ahead of print]156 111598
      Cellular senescence is caused by a wide range of intracellular and extracellular stimuli and influences physiological functions, leading to the progression of age-related diseases. Many studies have shown that cellular senescence is related to phosphatase and tension homolog deleted on chromosome ten (PTEN) loss and mammalian target of rapamycin (mTOR) activation. Although it has been reported that mTOR complex 1 (mTORC1) is major anti-aging target in several cell types, the functions and mechanisms of mTOR complex 2 (mTORC2) during aging have not been elucidated in vascular smooth muscle cells (VSMCs). Therefore, the aim of this study was to reveal the relationship between PTEN and mTORC2 during VSMC senescence. We found adriamycin-induced VSMC senescence was accompanied by reduced PTEN protein expression and upregulation of the mTORC2-Akt (Ser 473) pathway and that fisetin treatment reduced VSMC senescence by increasing PTEN and decreasing mTORC2 protein levels. Furthermore, PTEN played a primary role in the anti-aging effect of fisetin, and fisetin-activated PTEN directly regulated the mTORC2-Akt (Ser 473) signaling pathway, and attenuated senescence phenotypes such as senescence-associated β-galactosidase (SA-β-gal) and the p53-p21 signaling pathway in VSMCs. In mouse aortas, fisetin delayed aging by regulating the PTEN-mTORC2-Akt (Ser473) signaling pathway. These results suggest PTEN and mTORC2 are associated with cellular senescence in VSMCs and that the mTORC2-Akt (Ser 473) signaling pathway be considered a new target for preventing senescence-related diseases.
    Keywords:  Fisetin; PTEN; Senescence; Vascular smooth muscle cell; mTOR complex 2
    DOI:  https://doi.org/10.1016/j.exger.2021.111598
  21. Stem Cells Int. 2021 ;2021 9981589
      Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is characterized by pulmonary microvascular endothelial barrier dysfunction. Mesenchymal stem cell-secreted hepatocyte growth factor (HGF) has positive effects of lipopolysaccharide- (LPS-) induced pulmonary endothelial barrier. Studies have exhibited the mammalian TORC1 (mTORC1) signaling is of potent angiogenesis effects. The mTOR protein kinase has two distinct multiprotein complexes mTORC1 and mTORC2 that regulate different branches of the mTOR network. However, detailed mTORC2 mechanisms of HGF protective effects remain poorly defined. Therefore, the aim of this study was to determine whether mTORC2 mediated protective effects of MSC-secreted HGF against LPS-induced pulmonary microvascular endothelial barrier dysfunction activated like mTORC1 activation. We introduced MSC-PMVEC coculture transwell system and recombinant murine HGF on LPS-induced endothelial cell barrier dysfunction in vitro and then explored potential mechanisms by lentivirus vector-mediated HGF, mTORC1 (raptor), and mTORC2 (rictor) gene knockdown modification. Endothelial paracellular and transcellular permeability, adherent junction protein (VE-Cadherin), cell proliferation, apoptosis, and mTOR-associated proteins were tested. These revealed that HGF could promote quick reestablishment of adherent junction VE-cadherin and decrease endothelial paracellular and transcellular permeability during LSP-induced endothelial dysfunction with the involvement of mTORC2 (rictor) and mTORC1 (raptor) pathways. Raptor and rictor knockdown in LPS-induced PMEVECs with stimulation of HGF increased apoptosis ratio, activated Cleaved-Caspase-3 expression, and downregulated cell proliferation. Moreover, mTORC2/Akt but not mTORC2/PKC had significance on HGF endothelial protective effects. Taken together, these highlight activation mTORC2 pathway could also contribute to vascular endothelial barrier recovery by MSC-secreted HGF in LPS stimulation.
    DOI:  https://doi.org/10.1155/2021/9981589
  22. Sci Rep. 2021 Oct 28. 11(1): 21268
      Non-alcoholic fatty liver disease (NAFLD) is the most frequent liver disease worldwide and can progress to non-alcoholic steatohepatitis (NASH), which is characterized by triglyceride accumulation, inflammation, and fibrosis. No pharmacological agents are currently approved to treat these conditions, but it is clear now that modulation of lipid synthesis and autophagy are key biological mechanisms that could help reduce or prevent these liver diseases. The folliculin (FLCN) protein has been recently identified as a central regulatory node governing whole body energy homeostasis, and we hypothesized that FLCN regulates highly metabolic tissues like the liver. We thus generated a liver specific Flcn knockout mouse model to study its role in liver disease progression. Using the methionine- and choline-deficient diet to mimic liver fibrosis, we demonstrate that loss of Flcn reduced triglyceride accumulation, fibrosis, and inflammation in mice. In this aggressive liver disease setting, loss of Flcn led to activation of transcription factors TFEB and TFE3 to promote autophagy, promoting the degradation of intracellular lipid stores, ultimately resulting in reduced hepatocellular damage and inflammation. Hence, the activity of FLCN could be a promising target for small molecule drugs to treat liver fibrosis by specifically activating autophagy. Collectively, these results show an unexpected role for Flcn in fatty liver disease progression and highlight new potential treatment strategies.
    DOI:  https://doi.org/10.1038/s41598-021-99958-7
  23. Mol Cell. 2021 Oct 15. pii: S1097-2765(21)00800-5. [Epub ahead of print]
      Cell state changes are associated with proteome remodeling to serve newly emergent cell functions. Here, we show that NGN2-driven conversion of human embryonic stem cells to induced neurons (iNeurons) is associated with increased PINK1-independent mitophagic flux that is temporally correlated with metabolic reprogramming to support oxidative phosphorylation. Global multiplex proteomics during neurogenesis revealed large-scale remodeling of functional modules linked with pluripotency, mitochondrial metabolism, and proteostasis. Differentiation-dependent mitophagic flux required BNIP3L and its LC3-interacting region (LIR) motif, and BNIP3L also promoted mitophagy in dopaminergic neurons. Proteomic analysis of ATG12-/- iNeurons revealed accumulation of endoplasmic reticulum, Golgi, and mitochondria during differentiation, indicative of widespread organelle remodeling during neurogenesis. This work reveals broad organelle remodeling of membrane-bound organelles during NGN2-driven neurogenesis via autophagy, identifies BNIP3L's central role in programmed mitophagic flux, and provides a proteomic resource for elucidating how organelle remodeling and autophagy alter the proteome during changes in cell state.
    Keywords:  autophagy; iNeurons; mitophagy; proteomics
    DOI:  https://doi.org/10.1016/j.molcel.2021.10.001
  24. Mol Cell. 2021 Oct 19. pii: S1097-2765(21)00831-5. [Epub ahead of print]
      Mutations in ATP13A2, also known as PARK9, cause a rare monogenic form of juvenile-onset Parkinson's disease named Kufor-Rakeb syndrome and other neurodegenerative diseases. ATP13A2 encodes a neuroprotective P5B P-type ATPase highly enriched in the brain that mediates selective import of spermine ions from lysosomes into the cytosol via an unknown mechanism. Here we present three structures of human ATP13A2 bound to an ATP analog or to spermine in the presence of phosphomimetics determined by cryoelectron microscopy. ATP13A2 autophosphorylation opens a lysosome luminal gate to reveal a narrow lumen access channel that holds a spermine ion in its entrance. ATP13A2's architecture suggests physical principles underlying selective polyamine transport and anticipates a "pump-channel" intermediate that could function as a counter-cation conduit to facilitate lysosome acidification. Our findings establish a firm foundation to understand ATP13A2 mutations associated with disease and bring us closer to realizing ATP13A2's potential in neuroprotective therapy.
    Keywords:  ATP13A2; P-type ATPase; PARK9; Parkinson’s disease; cryo-EM; gating mechanism; ion channel; ion selectivity; ion transport; polyamine
    DOI:  https://doi.org/10.1016/j.molcel.2021.10.002
  25. J Neural Transm (Vienna). 2021 Oct 23.
      Protein homeostasis, or proteostasis, is essential for cell function and viability. Unwanted, damaged, misfolded and aggregated proteins are degraded by the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. Growing evidence indicates that alterations in these major proteolytic mechanisms lead to a demise in proteostasis, contributing to the onset and development of distinct diseases. Indeed, dysregulation of the UPS or autophagy is linked to several neurodegenerative, infectious and inflammatory disorders as well as cancer. Thus, modulation of protein clearance pathways is a promising approach for therapeutics. In this review, we discuss recent findings and open questions on how targeting proteolytic mechanisms could be applied for disease intervention.
    Keywords:  Aging; Alzheimer’s disease; Amyotrophic lateral sclerosis; Autophagy; Cancer; Huntington’s disease; Parkinson’s disease; Proteasome; Proteostasis
    DOI:  https://doi.org/10.1007/s00702-021-02431-y
  26. Cell Mol Neurobiol. 2021 Oct 28.
      Tuberous sclerosis complex (TSC) is a monogenic disorder caused by mutations in either the TSC1 or TSC2 gene, two key regulators of the mechanistic target of the rapamycin complex pathway. Phenotypically, this leads to growth and formation of hamartomas in several organs, including the brain. Subependymal giant cell astrocytomas (SEGAs) are low-grade brain tumors commonly associated with TSC. Recently, gene expression studies provided evidence that the immune system, the MAPK pathway and extracellular matrix organization play an important role in SEGA development. However, the precise mechanisms behind the gene expression changes in SEGA are still largely unknown, providing a potential role for DNA methylation. We investigated the methylation profile of SEGAs using the Illumina Infinium HumanMethylation450 BeadChip (SEGAs n = 42, periventricular control n = 8). The SEGA methylation profile was enriched for the adaptive immune system, T cell activation, leukocyte mediated immunity, extracellular structure organization and the ERK1 & ERK2 cascade. More interestingly, we identified two subgroups in the SEGA methylation data and show that the differentially expressed genes between the two subgroups are related to the MAPK cascade and adaptive immune response. Overall, this study shows that the immune system, the MAPK pathway and extracellular matrix organization are also affected on DNA methylation level, suggesting that therapeutic intervention on DNA level could be useful for these specific pathways in SEGA. Moreover, we identified two subgroups in SEGA that seem to be driven by changes in the adaptive immune response and MAPK pathway and could potentially hold predictive information on target treatment response.
    Keywords:  Low-grade glioma; Methylation; RNA-sequencing; SEGA; TSC
    DOI:  https://doi.org/10.1007/s10571-021-01157-5
  27. Mol Cell. 2021 Oct 21. pii: S1097-2765(21)00684-5. [Epub ahead of print]
      Polyamines are small, organic polycations that are ubiquitous and essential to all forms of life. Currently, how polyamines are transported across membranes is not understood. Recent studies have suggested that ATP13A2 and its close homologs, collectively known as P5B-ATPases, are polyamine transporters at endo-/lysosomes. Loss-of-function mutations of ATP13A2 in humans cause hereditary early-onset Parkinson's disease. To understand the polyamine transport mechanism of ATP13A2, we determined high-resolution cryoelectron microscopy (cryo-EM) structures of human ATP13A2 in five distinct conformational intermediates, which together, represent a near-complete transport cycle of ATP13A2. The structural basis of the polyamine specificity was revealed by an endogenous polyamine molecule bound to a narrow, elongated cavity within the transmembrane domain. The structures show an atypical transport path for a water-soluble substrate, in which polyamines may exit within the cytosolic leaflet of the membrane. Our study provides important mechanistic insights into polyamine transport and a framework to understand the functions and mechanisms of P5B-ATPases.
    Keywords:  P-type ATPase; P5B-ATPase; Parkinson's disease; cryo-EM; lysosome; membrane protein; polyamine; spermine; transporter
    DOI:  https://doi.org/10.1016/j.molcel.2021.08.017
  28. Sci Rep. 2021 Oct 27. 11(1): 21224
      Skeletal muscle mass is critical for good quality of life. Mesenchymal stem cells (MSCs) are multipotent stem cells distributed across various tissues. They are characterized by the capacity to secrete growth factors and differentiate into skeletal muscle cells. These capabilities suggest that MSCs might be beneficial for muscle growth. Nevertheless, little is known regarding the effects on muscle protein anabolic and catabolic systems of intramuscular injection of MSCs into skeletal muscle. Therefore, in the present study, we measured changes in mechanistic target of rapamycin complex 1 (mTORC1) signaling, the ubiquitin-proteasome system, and autophagy-lysosome system-related factors after a single intramuscular injection of MSCs with green fluorescence protein (GFP) into mouse muscles. The intramuscularly-injected MSCs were retained in the gastrocnemius muscle for 7 days after the injection, indicated by detection of GFP and expression of platelet-derived growth factor receptor-alpha. The injection of MSCs increased the expression of satellite cell-related genes, activated mTORC1 signaling and muscle protein synthesis, and increased protein ubiquitination and autophagosome formation (indicated by the expression of microtubule-associated protein 1 light chain 3-II). These results suggest that the intramuscular injection of MSCs activated muscle anabolic and catabolic systems and accelerated muscle protein turnover.
    DOI:  https://doi.org/10.1038/s41598-021-00627-6
  29. Nature. 2021 Oct 27.
      Glutathione (GSH) is a small-molecule thiol that is abundant in all eukaryotes and has key roles in oxidative metabolism1. Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions2. GSH is synthesized exclusively in the cytosol, yet the molecular machinery involved in mitochondrial GSH import remains unknown. Here, using organellar proteomics and metabolomics approaches, we identify SLC25A39, a mitochondrial membrane carrier of unknown function, as a regulator of GSH transport into mitochondria. Loss of SLC25A39 reduces mitochondrial GSH import and abundance without affecting cellular GSH levels. Cells lacking both SLC25A39 and its paralogue SLC25A40 exhibit defects in the activity and stability of proteins containing iron-sulfur clusters. We find that mitochondrial GSH import is necessary for cell proliferation in vitro and red blood cell development in mice. Heterologous expression of an engineered bifunctional bacterial GSH biosynthetic enzyme (GshF) in mitochondria enables mitochondrial GSH production and ameliorates the metabolic and proliferative defects caused by its depletion. Finally, GSH availability negatively regulates SLC25A39 protein abundance, coupling redox homeostasis to mitochondrial GSH import in mammalian cells. Our work identifies SLC25A39 as an essential and regulated component of the mitochondrial GSH-import machinery.
    DOI:  https://doi.org/10.1038/s41586-021-04025-w
  30. Cell Rep. 2021 Oct 26. pii: S2211-1247(21)01369-3. [Epub ahead of print]37(4): 109899
      Although commonly associated with autophagosomes, LC3 can also be recruited to membranes by covalent lipidation in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. We report a subtractive CRISPR screen that identifies factors required for non-canonical LC3 lipidation. As well as the enzyme complexes directly responsible for LC3 lipidation in all contexts, we show the RALGAP complex is important for M2-induced, but not ionophore drug-induced, LC3 lipidation. In contrast, ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation. Identification of a vacuolar ATPase subunit in the screen suggests a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug-induced LC3 lipidation lead to association of the vacuolar ATPase and ATG16L1 and can be antagonized by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens.
    Keywords:  ATG16L1; ATG4D; RALGAP; autophagy; influenza; v-ATPase
    DOI:  https://doi.org/10.1016/j.celrep.2021.109899
  31. Mol Neurobiol. 2021 Oct 27.
      The cell-to-cell transmission of pathological α-synuclein (α-syn) has been proposed to be a critical event in the development of synucleinopathies. Recent studies have begun to reveal the underlying molecular mechanism of α-syn propagation. As one of the central steps, α-syn secretion is reported to be Ca2+-dependent and mediated by unconventional exocytosis. However, the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) requirement and vesicle identity of α-syn secretion remain elusive. Here we found that α-syn secretion is SNARE-dependent by systematically knocking down Q-SNAREs and R-SNAREs in exocytosis pathways. α-Syn secretion was mainly mediated by syntaxin 4 (STX4) and synaptosomal-associated protein 23 (SNAP23), but did not require STX1 and SNAP25, in differentiated SH-SY5Y cells. On the other hand, vesicle-associated membrane protein 3 (VAMP3), VAMP7, and VAMP8 were all involved in α-syn secretion, most likely in overlapping pathways. Application of super-resolution microscopy revealed localization of both endogenous and overexpressed α-syn in endosomes, lysosomes, and autophagosomes in rat primary cortical neurons. α-Syn co-localized with microtubule-associated protein 1 light chain 3 (LC3) most extensively, suggesting its tight association with the autophagy pathway. Consistently, α-syn secretion was regulated by the autophagy-lysosome pathway. Collectively, our data suggest that α-syn secretion is SNARE-dependent and is mediated by multiple vesicular pathways including exocytosis of recycling endosomes, multivesicular bodies, autophagosomes, and lysosomes.
    Keywords:  Autophagosome; Endosome; Exocytosis; Lysosome; SNARE; α-Synuclein
    DOI:  https://doi.org/10.1007/s12035-021-02599-0
  32. JHEP Rep. 2021 Dec;3(6): 100359
      Background & Aims: Phosphatidylinositides-3 kinases (PI3Ks) are promising drug targets for cancer therapy, but blockage of PI3K-AKT signalling causes hyperglycaemia, hyperinsulinaemia, and liver damage in patients, and hepatocellular carcinoma (HCC) in mice. There are 4 PI3Ks: PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ. The role of PI3Kγ in HCC is unknown.Methods: We performed histopathological, metabolic, and molecular phenotyping of mice with genetic ablation of PI3Kγ using models where HCC was initiated by the carcinogen diethylnitrosamine (DEN) and promoted by dietary or genetic obesity (ob/ob). The role of PI3Kγ in leucocytes was investigated in mice lacking PI3Kγ in haematopoietic and endothelial cells.
    Results: Loss of PI3Kγ had no effects on the development of DEN-induced HCC in lean mice. However, in mice injected with DEN and placed on an obesogenic diet, PI3Kγ ablation reduced tumour growth, which was associated with reduced insulinaemia, steatosis, and expression of inflammatory cytokines. ob/ob mice lacking PI3Kγ, and mice with diet-induced obesity lacking PI3Kγ in leucocytes and endothelial cells did not display improved insulin sensitivity, steatosis, metabolic inflammation, or reduced tumour growth. However, these mice showed a reduced number of tumours, reduced liver infiltration by neutrophils, and reduced hepatocyte proliferation acutely induced by DEN.
    Conclusions: Loss of PI3Kγ reduces tumour development in obesity-promoted HCC through multiple cell types and mechanisms that include improved insulinaemia, steatosis, and metabolic inflammation as well as the regulation of acute neutrophil infiltration and compensatory hepatocyte proliferation. PI3Kγ-selective inhibition may represent a novel therapeutic approach to reduce HCC initiation and slow HCC progression.
    Lay summary: Class-1 phosphatidylinositides-3 kinases (PI3Ks) are critical targets in cancer therapy, but complete inhibition of all isoforms causes liver damage, hyperglycaemia, and insulinaemia. Here we show that selective ablation of the PI3Kγ isoform dampens tumour initiation and growth in a mouse model of carcinogen-initiated and obesity-promoted hepatocellular carcinoma (HCC). The effect of PI3Kγ ablation on reduced tumour growth was explained by reduced tumour cell proliferation, which was associated with reduced insulin levels, liver lipids, and reduced expression of tumour-promoting cytokines. PI3Kγ ablation in leucocytes of obese mice had no effects on tumour size. However, it reduced tumour number in association with reduced carcinogen-induced neutrophil infiltration and hepatocyte proliferation in livers of obese mice. Inhibition of PI3Kγ may thus reduce HCC initiation and growth in obese subjects by a mechanism involving reduced metabolic stress and insulinaemia and reduced carcinogen-induced neutrophil infiltration to the fatty liver.
    Keywords:  AKT; AST, aspartate aminotransferase; BMDM, bone marrow-derived macrophages; DEN, diethylnitrosamine; GTT, glucose tolerance test; HCC, hepatocellular carcinoma; HFD, high-fat diet; ITT, insulin tolerance test; Insulin; NAFLD; NASH; PI3K, phosphatidylinositides-3 kinase; PTEN, phosphatase and tensin homolog; RT, room temperature; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labelling; WT, wild-type; mTOR
    DOI:  https://doi.org/10.1016/j.jhepr.2021.100359
  33. Front Mol Neurosci. 2021 ;14 695937
      In recent years, gene therapy has been raising hopes toward viable treatment strategies for rare genetic diseases for which there has been almost exclusively supportive treatment. We here review this progress at the pre-clinical and clinical trial levels as well as market approvals within diseases that specifically affect the brain and spinal cord, including degenerative, developmental, lysosomal storage, and metabolic disorders. The field reached an unprecedented milestone when Zolgensma® (onasemnogene abeparvovec) was approved by the FDA and EMA for in vivo adeno-associated virus-mediated gene replacement therapy for spinal muscular atrophy. Shortly after EMA approved Libmeldy®, an ex vivo gene therapy with lentivirus vector-transduced autologous CD34-positive stem cells, for treatment of metachromatic leukodystrophy. These successes could be the first of many more new gene therapies in development that mostly target loss-of-function mutation diseases with gene replacement (e.g., Batten disease, mucopolysaccharidoses, gangliosidoses) or, less frequently, gain-of-toxic-function mutation diseases by gene therapeutic silencing of pathologic genes (e.g., amyotrophic lateral sclerosis, Huntington's disease). In addition, the use of genome editing as a gene therapy is being explored for some diseases, but this has so far only reached clinical testing in the treatment of mucopolysaccharidoses. Based on the large number of planned, ongoing, and completed clinical trials for rare genetic central nervous system diseases, it can be expected that several novel gene therapies will be approved and become available within the near future. Essential for this to happen is the in depth characterization of short- and long-term effects, safety aspects, and pharmacodynamics of the applied gene therapy platforms.
    Keywords:  central nervous system; clinical trials; gene therapy; personalized medicine; rare diseases; spinal cord; spinal muscular atrophy; viral vectors
    DOI:  https://doi.org/10.3389/fnmol.2021.695937
  34. Biochem J. 2021 Oct 27. pii: BCJ20210585. [Epub ahead of print]
      Metabolic reprogramming in cancer necessitates increased amino acid uptake, which is accomplished by upregulation of specific amino acid transporters. However, not all tumors rely on any single amino acid transporter for this purpose. Here we report on the differential upregulation of the amino acid transporter SLC38A5 in triple-negative breast cancer (TNBC). The upregulation is evident in TNBC tumors, conventional and patient-derived xenograft TNBC cell lines, and a mouse model of spontaneous TNBC mammary tumor. The upregulation is confirmed by functional assays. SLC38A5 is an amino acid-dependent Na+/H+ exchanger which transports Na+ and amino acids into cells coupled with H+ efflux. Since cell-surface Na+/H+ exchanger is an established inducer of macropinocytosis, an endocytic process for cellular uptake of bulk fluid and its components, we examined the impact of SLC38A5 on macropinocytosis in TNBC cells. We found that the transport function of SLC38A5 is coupled to induction of macropinocytosis. Surprisingly, the transport function of SLC38A5 is inhibited by amilorides, the well-known inhibitors of Na+/H+ exchanger. Downregulation of SLC38A5 in TNBC cells attenuates serine-induced macropinocytosis and reduces cell proliferation significantly as assessed by multiple methods, but does not induce cell death. The Cancer Genome Atlas database corroborates SLC38A5 upregulation in TNBC. This represents the first report on the selective expression of SLC38A5 in TNBC and its role as an inducer of macropinocytosis, thus revealing a novel, hitherto unsuspected, function for an amino acid transporter that goes beyond amino acid delivery but is still relevant to cancer cell nutrition and proliferation.
    Keywords:  breast cancer; cell proliferation; glutamine addiction; intracellular alkalinization; macropinocytosis; one-carbon metabolism
    DOI:  https://doi.org/10.1042/BCJ20210585
  35. Front Genome Ed. 2020 ;2 8
      In mammals over 65% of the total body iron is located within erythrocytes in the heme moieties of hemoglobin. Iron homeostasis requires iron absorbed from the diet by the gut as well as recycling of iron after the destruction of senescent erythrocytes. Senescent erythrocytes are engulfed by reticuloendothelial system macrophages where hemoglobin is broken down in the lysosomes, releasing heme for iron recovery in the cytoplasm. We recently showed that the SLC48A1 protein is responsible for transporting heme from the lysosome to the cytoplasm. CRISPR generated SLC48A1-deficient mice accumulate heme in their reticuloendothelial system macrophages as hemozoin crystals. Here we describe additional features of SLC48A1-deficient mice. We show that visible hemozoin first appears in the reticuloendothelial system macrophages of SLC48A1-deficient mice at 8 days of age, indicating the onset of erythrocyte recycling. Evaluation of normal and SLC48A1-deficient mice on iron-controlled diets show that SLC48A1-mediated iron recycling is equivalent to at least 10 parts per million of dietary iron. We propose that mutations in human SLC48A1 could contribute to idiopathic iron disorders.
    Keywords:  anemia; erythrocyte phagocytosis; erythropoiesis; gene editing (CRISPR-Cas9); mouse model
    DOI:  https://doi.org/10.3389/fgeed.2020.00008
  36. Neuromuscul Disord. 2021 Jul 31. pii: S0960-8966(21)00204-2. [Epub ahead of print]
      Danon disease is typically lethal by the mid-twenties in male patients due to cardiomyopathy. This report aims to describe two unrelated male patients showing mild manifestations of the disease. A 39-year-old man presented with a 10-year history of elevated serum creatine kinase levels with slowly progressive muscle weakness. Muscle pathology showed autophagic vacuoles with sarcolemmal features. Genetic testing revealed a hemizygous mutation in exon 9b, an alternatively spliced exon, of lysosome-associated membrane protein-2 (LAMP-2) (c.1097_1098delAA). Cardiac testing showed asymptomatic mild left ventricular hypertrophy. He had borderline intelligence. Early stage of retinopathy was detected. Another male patient, currently 53-year-old, had asymptomatic supraventricular extrasystole and muscle weakness but no intellectual disability, harboring the same mutation. He also had retinopathy. The present patients commonly carry a mutation in exon 9b of LAMP-2, suggesting that mutations in the exon are associated with a mild form of Danon disease.
    Keywords:  Alternative splicing; Autophagic vacuolar myopathy; Autophagy; Cardiomyopathy; Genotype-phenotype correlation; Lysosome-associated membrane protein-2 (LAMP-2)
    DOI:  https://doi.org/10.1016/j.nmd.2021.07.017
  37. Nat Commun. 2021 Oct 26. 12(1): 6176
      Serine is a non-essential amino acid that is critical for tumour proliferation and depletion of circulating serine results in reduced tumour growth and increased survival in various cancer models. While many cancer cells cultured in a standard tissue culture medium depend on exogenous serine for optimal growth, here we report that these cells are less sensitive to serine/glycine depletion in medium containing physiological levels of metabolites. The lower requirement for exogenous serine under these culture conditions reflects both increased de novo serine synthesis and the use of hypoxanthine (not present in the standard medium) to support purine synthesis. Limiting serine availability leads to increased uptake of extracellular hypoxanthine, sparing available serine for other pathways such as glutathione synthesis. Taken together these results improve our understanding of serine metabolism in physiologically relevant nutrient conditions and allow us to predict interventions that may enhance the therapeutic response to dietary serine/glycine limitation.
    DOI:  https://doi.org/10.1038/s41467-021-26395-5
  38. Mol Ther Methods Clin Dev. 2021 Dec 10. 23 128-134
      Transformative results of adeno-associated virus (AAV) gene therapy in patients with spinal muscular atrophy and Leber's congenital amaurosis led to approval of the first two AAV products in the United States to treat these diseases. These extraordinary results led to a dramatic increase in the number and type of AAV gene-therapy programs. However, the field lacks non-invasive means to assess levels and duration of therapeutic protein function in patients. Here, we describe a new magnetic resonance imaging (MRI) technology for real-time reporting of gene-therapy products in the living animal in the form of an MRI probe that is activated in the presence of therapeutic protein expression. For the first time, we show reliable tracking of enzyme expression after a now in-human clinical trial AAV gene therapy (ClinicalTrials.gov: NTC03952637) encoding lysosomal acid beta-galactosidase (βgal) using a self-immolative βgal-responsive MRI probe. MRI enhancement in AAV-treated enzyme-deficient mice (GLB-1-/-) correlates with βgal activity in central nervous system and peripheral organs after intracranial or intravenous AAV gene therapy, respectively. With >1,800 gene therapies in phase I/II clinical trials (ClinicalTrials.gov), development of a non-invasive method to track gene expression over time in patients is crucial to the future of the gene-therapy field.
    Keywords:  AAV gene therapy; GM1 gangliosidosis; MR tracking of enzyme expression; enzyme-activated contrast agent probes; magnetic resonance imaging
    DOI:  https://doi.org/10.1016/j.omtm.2021.08.003
  39. EMBO J. 2021 Oct 27. e108069
      Brown and beige fat are specialized for energy expenditure by dissipating energy from glucose and fatty acid oxidation as heat. While glucose and fatty acid metabolism have been extensively studied in thermogenic adipose tissues, the involvement of amino acids in regulating adaptive thermogenesis remains little studied. Here, we report that asparagine supplementation in brown and beige adipocytes drastically upregulated the thermogenic transcriptional program and lipogenic gene expression, so that asparagine-fed mice showed better cold tolerance. In mice with diet-induced obesity, the asparagine-fed group was more responsive to β3-adrenergic receptor agonists, manifesting in blunted body weight gain and improved glucose tolerance. Metabolomics and 13 C-glucose flux analysis revealed that asparagine supplement spurred glycolysis to fuel thermogenesis and lipogenesis in adipocytes. Mechanistically, asparagine stimulated the mTORC1 pathway, which promoted expression of thermogenic genes and key enzymes in glycolysis. These findings show that asparagine bioavailability affects glycolytic and thermogenic activities in adipose tissues, providing a possible nutritional strategy for improving systemic energy homeostasis.
    Keywords:  asparagine; brown adipocytes; glycolysis; mTORC1; thermogenesis
    DOI:  https://doi.org/10.15252/embj.2021108069
  40. J Biol Chem. 2021 Oct 21. pii: S0021-9258(21)01143-1. [Epub ahead of print] 101337
      The extracellular domain (ED) of the membrane-spanning sialoglycoprotein, mucin-1 (MUC1), is an in vivo substrate for the lysosomal sialidase, neuraminidase-1 (NEU1). Engagement of the MUC1-ED by its cognate ligand, Pseudomonas aeruginosa-expressed flagellin, increases NEU1-MUC1 association and NEU1-mediated MUC1-ED desialylation to unmask cryptic binding sites for its ligand. However, the mechanism(s) through which intracellular NEU1 might physically interact with its surface-expressed MUC1-ED substrate are unclear. Using reciprocal co-immunoprecipitation and in vitro binding assays in a human airway epithelial cell system, we show here that NEU1 associates with the MUC1-cytoplasmic domain (CD), but not with the MUC1-ED. Prior pharmacologic inhibition of NEU1 catalytic activity using the NEU1-selective sialidase inhibitor, C9-BA-DANA, did not diminish NEU1-MUC1-CD association. In addition, glutathione S-transferase (GST) pull-down assays utilizing deletion mutants of the MUC1-CD mapped the NEU1-binding site to the membrane-proximal 36 amino acids of the MUC1-CD. In a cell-free system, we found that purified NEU1 interacted with immobilized GST-MUC1-CD, and purified MUC1-CD associated with immobilized 6XHis-NEU1, indicating that the NEU1-MUC1-CD interaction was direct and independent of its chaperone protein, protective protein/cathepsin A. However, the NEU1-MUC1-CD interaction was not required for NEU1-mediated MUC1-ED desialylation. Finally, we demonstrated that overexpression of either wild-type NEU1 or a catalytically-dead NEU1 G68V mutant diminished association of the established MUC1-CD binding partner, phosphoinositide 3-kinase (PI3K), to MUC1-CD and reduced downstream Akt kinase phosphorylation. These results indicate that NEU1 associates with the juxtamembranous region of the MUC1-CD to inhibit PI3K-Akt signaling independent of NEU1 catalytic activity.
    Keywords:  Akt PBK; cell surface associated (MUC1); mucin 1; neuraminidase-1; phosphatidylinositide 3‐kinase (PI 3‐kinase); sialic acid; sialidase
    DOI:  https://doi.org/10.1016/j.jbc.2021.101337