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Sample records for a3 facilitates lysosomal

  1. Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin.

    PubMed

    Zhou, Xiaolai; Sun, Lirong; Bastos de Oliveira, Francisco; Qi, Xiaoyang; Brown, William J; Smolka, Marcus B; Sun, Ying; Hu, Fenghua

    2015-09-14

    Mutations in the progranulin (PGRN) gene have been linked to two distinct neurodegenerative diseases, frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests a critical role of PGRN in lysosomes. However, how PGRN is trafficked to lysosomes is still not clear. Here we report a novel pathway for lysosomal delivery of PGRN. We found that prosaposin (PSAP) interacts with PGRN and facilitates its lysosomal targeting in both biosynthetic and endocytic pathways via the cation-independent mannose 6-phosphate receptor and low density lipoprotein receptor-related protein 1. PSAP deficiency in mice leads to severe PGRN trafficking defects and a drastic increase in serum PGRN levels. We further showed that this PSAP pathway is independent of, but complementary to, the previously identified PGRN lysosomal trafficking mediated by sortilin. Collectively, our results provide new understanding on PGRN trafficking and shed light on the molecular mechanisms behind FTLD and NCL caused by PGRN mutations.

  2. Enhancing astrocytic lysosome biogenesis facilitates Aβ clearance and attenuates amyloid plaque pathogenesis.

    PubMed

    Xiao, Qingli; Yan, Ping; Ma, Xiucui; Liu, Haiyan; Perez, Ronaldo; Zhu, Alec; Gonzales, Ernesto; Burchett, Jack M; Schuler, Dorothy R; Cirrito, John R; Diwan, Abhinav; Lee, Jin-Moo

    2014-07-16

    In sporadic Alzheimer's disease (AD), impaired Aβ removal contributes to elevated extracellular Aβ levels that drive amyloid plaque pathogenesis. Extracellular proteolysis, export across the blood-brain barrier, and cellular uptake facilitate physiologic Aβ clearance. Astrocytes can take up and degrade Aβ, but it remains unclear whether this function is insufficient in AD or can be enhanced to accelerate Aβ removal. Additionally, age-related dysfunction of lysosomes, the major degradative organelles wherein Aβ localizes after uptake, has been implicated in amyloid plaque pathogenesis. We tested the hypothesis that enhancing lysosomal function in astrocytes with transcription factor EB (TFEB), a master regulator of lysosome biogenesis, would promote Aβ uptake and catabolism and attenuate plaque pathogenesis. Exogenous TFEB localized to the nucleus with transcriptional induction of lysosomal biogenesis and function in vitro. This resulted in significantly accelerated uptake of exogenously applied Aβ42, with increased localization to and degradation within lysosomes in C17.2 cells and primary astrocytes, indicating that TFEB is sufficient to coordinately enhance uptake, trafficking, and degradation of Aβ. Stereotactic injection of adeno-associated viral particles carrying TFEB driven by a glial fibrillary acidic protein promoter was used to achieve astrocyte-specific expression in the hippocampus of APP/PS1 transgenic mice. Exogenous TFEB localized to astrocyte nuclei and enhanced lysosome function, resulting in reduced Aβ levels and shortened half-life in the brain interstitial fluid and reduced amyloid plaque load in the hippocampus compared with control virus-injected mice. Therefore, activation of TFEB in astrocytes is an effective strategy to restore adequate Aβ removal and counter amyloid plaque pathogenesis in AD. Copyright © 2014 the authors 0270-6474/14/349607-14$15.00/0.

  3. Enhancing Astrocytic Lysosome Biogenesis Facilitates Aβ Clearance and Attenuates Amyloid Plaque Pathogenesis

    PubMed Central

    Xiao, Qingli; Yan, Ping; Ma, Xiucui; Liu, Haiyan; Perez, Ronaldo; Zhu, Alec; Gonzales, Ernesto; Burchett, Jack M.; Schuler, Dorothy R.; Cirrito, John R.

    2014-01-01

    In sporadic Alzheimer's disease (AD), impaired Aβ removal contributes to elevated extracellular Aβ levels that drive amyloid plaque pathogenesis. Extracellular proteolysis, export across the blood–brain barrier, and cellular uptake facilitate physiologic Aβ clearance. Astrocytes can take up and degrade Aβ, but it remains unclear whether this function is insufficient in AD or can be enhanced to accelerate Aβ removal. Additionally, age-related dysfunction of lysosomes, the major degradative organelles wherein Aβ localizes after uptake, has been implicated in amyloid plaque pathogenesis. We tested the hypothesis that enhancing lysosomal function in astrocytes with transcription factor EB (TFEB), a master regulator of lysosome biogenesis, would promote Aβ uptake and catabolism and attenuate plaque pathogenesis. Exogenous TFEB localized to the nucleus with transcriptional induction of lysosomal biogenesis and function in vitro. This resulted in significantly accelerated uptake of exogenously applied Aβ42, with increased localization to and degradation within lysosomes in C17.2 cells and primary astrocytes, indicating that TFEB is sufficient to coordinately enhance uptake, trafficking, and degradation of Aβ. Stereotactic injection of adeno-associated viral particles carrying TFEB driven by a glial fibrillary acidic protein promoter was used to achieve astrocyte-specific expression in the hippocampus of APP/PS1 transgenic mice. Exogenous TFEB localized to astrocyte nuclei and enhanced lysosome function, resulting in reduced Aβ levels and shortened half-life in the brain interstitial fluid and reduced amyloid plaque load in the hippocampus compared with control virus-injected mice. Therefore, activation of TFEB in astrocytes is an effective strategy to restore adequate Aβ removal and counter amyloid plaque pathogenesis in AD. PMID:25031402

  4. The permeability of rat liver lysosomes to sugars. Evidence for carrier-mediated facilitated diffusion.

    PubMed

    Docherty, K; Brenchley, G V; Hales, C N

    1979-02-15

    1. By the osmotic-protection method, the penetration of sugars through the rat liver lysosomal membranes was studied with a view of determining whether sugar uptake was by facilitated diffusion. 2. The following criteria for this type of transport were established: sugar specificity, the order of uptake being 2-deoxy-D-glucose less than D-glucose less than D-mannose less than D-galactose less than D-ribose less than 2-deoxy-D-ribose; stereospecificity, the uptake of L-glucose and L-ribose being 50% slower than their D-stereoisomers; inhibition by 1 MM-phlorrhizin and 1 M-cytochalastin B; competition between sugars for uptake, and a Q10 (rate difference over a 10 degrees C temperature range) for uptake of approx. 2.8. 3. It is proposed that sugar uptake into lysosomes from rat liver is by facilitated diffusion.

  5. Cellular Cholesterol Accumulation Facilitates Ubiquitination and Lysosomal Degradation of Cell Surface-Resident ABCA1.

    PubMed

    Mizuno, Tadahaya; Hayashi, Hisamitsu; Kusuhara, Hiroyuki

    2015-06-01

    By excreting cellular cholesterol to apolipoprotein A-I, ATP-binding cassette transporter A1 (ABCA1) mediates the biogenesis of high-density lipoprotein in hepatocytes and prevents foam cell formation from macrophages. We recently showed that cell surface-resident ABCA1 (csABCA1) undergoes ubiquitination and later lysosomal degradation through the endosomal sorting complex required for transport system. Herein, we investigated the relevance of this degradation pathway to the turnover of csABCA1 in hypercholesterolemia. Immunoprecipitation and cell surface-biotinylation studies with HepG2 cells and mouse peritoneal macrophages showed that the ubiquitination level and degradation of csABCA1 were facilitated by treatment with a liver X receptor (LXR) agonist and acetylated low-density lipoprotein. The effects of an LXR agonist and acetylated low-density lipoprotein on the degradation of csABCA1 were repressed completely by treatment with bafilomycin, an inhibitor of lysosomal degradation, and by depletion of tumor susceptibility gene 101, a major component of endosomal sorting complex required for transport-I. RNAi analysis indicated that LXRβ inhibited the accelerated lysosomal degradation of csABCA1 by the LXR agonist, regardless of its transcriptional activity. Cell surface coimmunoprecipitation with COS1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that LXRβ interacted with csABCA1 and inhibited the ubiquitination of csABCA1. Immunoprecipitates with anti-ABCA1 antibodies from the liver plasma membranes showed less LXRβ and a higher ubiquitination level of ABCA1 in high-fat diet-fed mice than in normal chow-fed mice. Under conditions of high cellular cholesterol content, csABCA1 became susceptible to ubiquitination by dissociation of LXRβ from csABCA1, which facilitated the lysosomal degradation of csABCA1 through the endosomal sorting complex required for transport system. © 2015 American Heart Association, Inc.

  6. SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm.

    PubMed

    Hamblett, Kevin J; Jacob, Allison P; Gurgel, Jesse L; Tometsko, Mark E; Rock, Brooke M; Patel, Sonal K; Milburn, Robert R; Siu, Sophia; Ragan, Seamus P; Rock, Dan A; Borths, Christopher J; O'Neill, Jason W; Chang, Wesley S; Weidner, Margaret F; Bio, Matthew M; Quon, Kim C; Fanslow, William C

    2015-12-15

    Antibody-drug conjugates (ADC) target cytotoxic drugs to antigen-positive cells for treating cancer. After internalization, ADCs with noncleavable linkers are catabolized to amino acid-linker-warheads within the lysosome, which then enter the cytoplasm by an unknown mechanism. We hypothesized that a lysosomal transporter was responsible for delivering noncleavable ADC catabolites into the cytoplasm. To identify candidate transporters, we performed a phenotypic shRNA screen with an anti-CD70 maytansine-based ADC. This screen revealed the lysosomal membrane protein SLC46A3, the genetic attenuation of which inhibited the potency of multiple noncleavable antibody-maytansine ADCs, including ado-trastuzumab emtansine. In contrast, the potencies of noncleavable ADCs carrying the structurally distinct monomethyl auristatin F were unaffected by SLC46A3 attenuation. Structure-activity experiments suggested that maytansine is a substrate for SLC46A3. Notably, SLC46A3 silencing led to relative increases in catabolite concentrations in the lysosome. Taken together, our results establish SLC46A3 as a direct transporter of maytansine-based catabolites from the lysosome to the cytoplasm, prompting further investigation of SLC46A3 as a predictive response marker in breast cancer specimens. ©2015 American Association for Cancer Research.

  7. Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus

    PubMed Central

    Monteith, Andrew J.; Kang, SunAh; Scott, Eric; Hillman, Kai; Rajfur, Zenon; Jacobson, Ken; Costello, M. Joseph; Vilen, Barbara J.

    2016-01-01

    Defects in clearing apoptotic debris disrupt tissue and immunological homeostasis, leading to autoimmune and inflammatory diseases. Herein, we report that macrophages from lupus-prone MRL/lpr mice have impaired lysosomal maturation, resulting in heightened ROS production and attenuated lysosomal acidification. Impaired lysosomal maturation diminishes the ability of lysosomes to degrade apoptotic debris contained within IgG–immune complexes (IgG-ICs) and promotes recycling and the accumulation of nuclear self-antigens at the membrane 72 h after internalization. Diminished degradation of IgG-ICs prolongs the intracellular residency of nucleic acids, leading to the activation of Toll-like receptors. It also promotes phagosomal membrane permeabilization, allowing dsDNA and IgG to leak into the cytosol and activate AIM2 and TRIM21. Collectively, these events promote the accumulation of nuclear antigens and activate innate sensors that drive IFNα production and heightened cell death. These data identify a previously unidentified defect in lysosomal maturation that provides a mechanism for the chronic activation of intracellular innate sensors in systemic lupus erythematosus. PMID:27035940

  8. Mucolipin 1 positively regulates TLR7 responses in dendritic cells by facilitating RNA transportation to lysosomes.

    PubMed

    Li, Xiaobing; Saitoh, Shin-Ichiroh; Shibata, Takuma; Tanimura, Natsuko; Fukui, Ryutaro; Miyake, Kensuke

    2015-02-01

    Toll-like receptor 7 (TLR7) and TLR9 sense microbial single-stranded RNA (ssRNA) and ssDNA in endolysosomes. Nucleic acid (NA)-sensing in endolysosomes is thought to be important for avoiding TLR7/9 responses to self-derived NAs. Aberrant self-derived NA transportation to endolysosomes predisposes to autoimmune diseases. To restrict NA-sensing in endolysosomes, TLR7/9 trafficking is tightly controlled by a multiple transmembrane protein Unc93B1. In contrast to TLR7/9 trafficking, little is known about a mechanism underlying NA transportation. We here show that Mucolipin 1 (Mcoln1), a member of the transient receptor potential (TRP) cation channel gene family, has an important role in ssRNA trafficking into lysosomes. Mcoln1(-/-) dendritic cells (DCs) showed impaired TLR7 responses to ssRNA. A mucolipin agonist specifically enhanced TLR7 responses to ssRNAs. The channel activity of Mcoln1 is activated by a phospholipid phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), which is generated by a class III lipid kinase PIKfyve. A PIKfyve inhibitor completely inhibited TLR7 responses to ssRNA in DCs. Confocal analyses showed that ssRNA transportation to lysosomes in DCs was impaired by PIKfyve inhibitor as well as by the lack of Mcoln1. Transportation of TLR9 ligands was also impaired by the PIKfyve inhibitor. These results demonstrate that the PtdIns(3,5)P2-Mcoln1 axis has an important role in ssRNA transportation into lysosomes in DCs.

  9. Human copper transporter 2 is localized in late endosomes and lysosomes and facilitates cellular copper uptake

    PubMed Central

    vandenBerghe, Peter V. E.; Folmer, Dineke E.; Malingré, Helga E. M.; vanBeurden, Ellen; Klomp, Adriana E. M.; vandeSluis, Bart; Merkx, Maarten; Berger, Ruud; Klomp, Leo W. J.

    2007-01-01

    High-affinity cellular copper uptake is mediated by the CTR (copper transporter) 1 family of proteins. The highly homologous hCTR (human CTR) 2 protein has been identified, but its function in copper uptake is currently unknown. To characterize the role of hCTR2 in copper homoeostasis, epitope-tagged hCTR2 was transiently expressed in different cell lines. hCTR2–vsvG (vesicular-stomatitis-virus glycoprotein) predominantly migrated as a 17 kDa protein after imunoblot analysis, consistent with its predicted molecular mass. Chemical cross-linking resulted in the detection of higher-molecular-mass complexes containing hCTR2–vsvG. Furthermore, hCTR2–vsvG was co-immunoprecipitated with hCTR2–FLAG, suggesting that hCTR2 can form multimers, like hCTR1. Transiently transfected hCTR2–eGFP (enhanced green fluorescent protein) was localized exclusively to late endosomes and lysosomes, and was not detected at the plasma membrane. To functionally address the role of hCTR2 in copper metabolism, a novel transcription-based copper sensor was developed. This MRE (metal-responsive element)–luciferase reporter contained four MREs from the mouse metallothionein 1A promoter upstream of the firefly luciferase open reading frame. Thus the MRE–luciferase reporter measured bioavailable cytosolic copper. Expression of hCTR1 resulted in strong activation of the reporter, with maximal induction at 1 μM CuCl2, consistent with the Km of hCTR1. Interestingly, expression of hCTR2 significantly induced MRE–luciferase reporter activation in a copper-dependent manner at 40 and 100 μM CuCl2. Taken together, these results identify hCTR2 as an oligomeric membrane protein localized in lysosomes, which stimulates copper delivery to the cytosol of human cells at relatively high copper concentrations. This work suggests a role for endosomal and lysosomal copper pools in the maintenance of cellular copper homoeostasis. PMID:17617060

  10. [Cestode lysosomes].

    PubMed

    Smirnov, L P; Bogdan, V V

    1989-01-01

    By differential centrifugation method a lysosomal fraction was obtained from five species of cestodes, which possesses the highest specific activity of acidic phosphatases as compared to other subcellular fractions. By isopyknic centrifugation in the density gradient of saccharose the lysosomal fraction is divided into primary and secondary lysosomes. Lysosomes of cestodes are similar to those of vertebrate animals in the character of fractional distribution of acidic phosphatase, sedimentation abilities and sensitivity of membranes to triton X-100.

  11. The proteome of lysosomes.

    PubMed

    Schröder, Bernd A; Wrocklage, Christian; Hasilik, Andrej; Saftig, Paul

    2010-11-01

    Lysosomes are organelles of eukaryotic cells that are critically involved in the degradation of macromolecules mainly delivered by endocytosis and autophagocytosis. Degradation is achieved by more than 60 hydrolases sequestered by a single phospholipid bilayer. The lysosomal membrane facilitates interaction and fusion with other compartments and harbours transport proteins catalysing the export of catabolites, thereby allowing their recycling. Lysosomal proteins have been addressed in various proteomic studies that are compared in this review regarding the source of material, the organelle/protein purification scheme, the proteomic methodology applied and the proteins identified. Distinguishing true constituents of an organelle from co-purifying contaminants is a central issue in subcellular proteomics, with additional implications for lysosomes as being the site of degradation of many cellular and extracellular proteins. Although many of the lysosomal hydrolases were identified by classical biochemical approaches, the knowledge about the protein composition of the lysosomal membrane has remained fragmentary for a long time. Using proteomics many novel lysosomal candidate proteins have been discovered and it can be expected that their functional characterisation will help to understand functions of lysosomes at a molecular level that have been characterised only phenomenologically so far and to generally deepen our understanding of this indispensable organelle.

  12. Progranulin regulates lysosomal function and biogenesis through acidification of lysosomes.

    PubMed

    Tanaka, Yoshinori; Suzuki, Genjiro; Matsuwaki, Takashi; Hosokawa, Masato; Serrano, Geidy; Beach, Thomas G; Yamanouchi, Keitaro; Hasegawa, Masato; Nishihara, Masugi

    2017-01-10

    Progranulin (PGRN) haploinsufficiency resulting from loss-of-function mutations in the PGRN gene causes frontotemporal lobar degeneration accompanied by TDP-43 accumulation, and patients with homozygous mutations in the PGRN gene present with neuronal ceroid lipofuscinosis. Although it remains unknown why PGRN deficiency causes neurodegenerative diseases, there is increasing evidence that PGRN is implicated in lysosomal functions. Here, we show PGRN is a secretory lysosomal protein that regulates lysosomal function and biogenesis by controlling the acidification of lysosomes. PGRN gene expression and protein levels increased concomitantly with the increase of lysosomal biogenesis induced by lysosome alkalizers or serum starvation. Down-regulation or insufficiency of PGRN led to the increased lysosomal gene expression and protein levels, while PGRN overexpression led to the decreased lysosomal gene expression and protein levels. In particular, the level of mature cathepsin D (CTSDmat) dramatically changed depending upon PGRN levels. The acidification of lysosomes was facilitated in cells transfected with PGRN. Then, this caused degradation of CTSDmat by cathepsin B. Secreted PGRN is incorporated into cells via sortilin or cation-independent mannose 6-phosphate receptor, and facilitated the acidification of lysosomes and degradation of CTSDmat Moreover, the change of PGRN levels led to a cell-type-specific increase of insoluble TDP-43. In the brain tissue of FTLD-TDP patients with PGRN deficiency, CTSD and phosphorylated TDP-43 accumulated in neurons. Our study provides new insights into the physiological function of PGRN and the role of PGRN insufficiency in the pathogenesis of neurodegenerative diseases.

  13. Lysosomal sialidase (neuraminidase-1) is targeted to the cell surface in a multiprotein complex that facilitates elastic fiber assembly.

    PubMed

    Hinek, Aleksander; Pshezhetsky, Alexey V; von Itzstein, Mark; Starcher, Barry

    2006-02-10

    We have established previously that the 67-kDa elastin-binding protein (EBP), identical to the spliced variant of beta-galactosidase, acts as a recyclable chaperone that facilitates secretion of tropoelastin. (Hinek, A., Keeley, F. W., and Callahan, J. W. (1995) Exp. Cell Res. 220, 312-324). We now demonstrate that EBP also forms a cell surface-targeted molecular complex with protective protein/cathepsin A and sialidase (neuraminidase-1), and provide evidence that this sialidase activity is a prerequisite for the subsequent release of tropoelastin. We found that treatment with sialidase inhibitors repressed assembly of elastic fibers in cultures of human skin fibroblasts, aortic smooth muscle cells, and ear cartilage chondrocytes and caused impaired elastogenesis in developing chick embryos. Fibroblasts derived from patients with congenital sialidosis (primary deficiency of neuraminidase-1) and galactosialidosis (secondary deficiency of neuraminidase-1) demonstrated impaired elastogenesis, which could be reversed after their transduction with neuraminidase-1 cDNA or after treatment with bacterial sialidase, which has a similar substrate specificity to human neuraminidase-1. We postulate that neuraminidase-1 catalyzes removal of the terminal sialic acids from carbohydrate chains of microfibrillar glycoproteins and other adjacent matrix glycoconjugates, unmasking their penultimate galactosugars. In turn, the exposed galactosugars interact with the galectin domain of EBP, thereby inducing the release of transported tropoelastin molecules and facilitating their subsequent assembly into elastic fibers.

  14. Deficiency of Neuronal p38α MAPK Attenuates Amyloid Pathology in Alzheimer Disease Mouse and Cell Models through Facilitating Lysosomal Degradation of BACE1.

    PubMed

    Schnöder, Laura; Hao, Wenlin; Qin, Yiren; Liu, Shirong; Tomic, Inge; Liu, Xu; Fassbender, Klaus; Liu, Yang

    2016-01-29

    Amyloid β (Aβ) damages neurons and triggers microglial inflammatory activation in the Alzheimer disease (AD) brain. BACE1 is the primary enzyme in Aβ generation. Neuroinflammation potentially up-regulates BACE1 expression and increases Aβ production. In Alzheimer amyloid precursor protein-transgenic mice and SH-SY5Y cell models, we specifically knocked out or knocked down gene expression of mapk14, which encodes p38α MAPK, a kinase sensitive to inflammatory and oxidative stimuli. Using immunological and biochemical methods, we observed that reduction of p38α MAPK expression facilitated the lysosomal degradation of BACE1, decreased BACE1 protein and activity, and subsequently attenuated Aβ generation in the AD mouse brain. Inhibition of p38α MAPK also enhanced autophagy. Blocking autophagy by treating cells with 3-methyladenine or overexpressing dominant-negative ATG5 abolished the deficiency of the p38α MAPK-induced BACE1 protein reduction in cultured cells. Thus, our study demonstrates that p38α MAPK plays a critical role in the regulation of BACE1 degradation and Aβ generation in AD pathogenesis.

  15. GNeosomes: Highly Lysosomotropic Nanoassemblies for Lysosomal Delivery.

    PubMed

    Wexselblatt, Ezequiel; Esko, Jeffrey D; Tor, Yitzhak

    2015-01-01

    GNeosomes, lysosomotropic lipid vesicles decorated with guanidinoneomycin, can encapsulate and facilitate the cellular internalization and lysosomal delivery of cargo ranging from small molecules to high molecular weight proteins, in a process that is exclusively dependent on cell surface glycosaminoglycans. Their cellular uptake mechanism and co-localization with lysosomes, as well as the delivery, release, and activity of internalized cargo, are quantified. GNeosomes are proposed as a universal platform for lysosomal delivery with potential as a basic research tool and a therapeutic vehicle.

  16. Brief exposure to copper activates lysosomal exocytosis.

    PubMed

    Peña, Karina; Coblenz, Jessica; Kiselyov, Kirill

    2015-04-01

    Copper (Cu) is essential mineral, but its toxicity necessitates existence of powerful machinery responsible for the extraction of excess Cu from the cell. Cu exposure was recently shown to induce the translocation of Cu pump ATP7B to the lysosomes followed by lysosomal exocytosis. Here we sought to investigate the mechanisms underlying the effect of Cu on lysosomal exocytosis. We found that brief exposure to Cu activates lysosomal exocytosis, which was measured as a release of the lysosomal digestive enzyme β-hexosaminidase (β-hex) into the extracellular medium and by the presence lysosomal protein LAMP1 at the plasma membrane. Such release depends on calcium (Ca) and on the lysosomal SNARE VAMP7. ATP7B knockdown using RNAi suppressed the basal lysosomal exocytosis, but did not affect the ability of Cu to activate it. ATP7B knockdown was associated with sustained oxidative stress. The removal of Ca from the extracellular medium suppressed the Cu-dependent component of the lysosomal exocytosis. We propose that Cu promotes lysosomal exocytosis by facilitating a Ca-dependent step of the lysosomal exocytosis.

  17. Nitric Oxide Interacts with Caveolin-1 to Facilitate Autophagy-Lysosome-Mediated Claudin-5 Degradation in Oxygen-Glucose Deprivation-Treated Endothelial Cells

    PubMed Central

    Liu, Jie; Weaver, John; Jin, Xinchun; Zhang, Yuan; Xu, Ji; Liu, Ke J.; Li, Weiping; Liu, Wenlan

    2017-01-01

    Using in vitro oxygen-glucose deprivation (OGD) model, we have previously demonstrated that 2-h OGD induces rapid, caveolin-1-mediated dissociation of claudin-5 from the cellular cytoskeletal framework and quick endothelial barrier disruption. In this study, we further investigated the fate of translocated claudin-5 and the mechanisms by which OGD promotes caveolin-1 translocation. Exposure of bEND3 cells to 4-h OGD, but not 2-h OGD plus 2-h reoxygenation, resulted in claudin-5 degradation. Inhibition of autophagy or the fusion of autophagosome with lysosome, but not proteasome, blocked OGD-induced claudin-5 degradation. Moreover, knockdown of caveolin-1 with siRNA blocked OGD-induced claudin-5 degradation. Western blot analysis showed a transient colocalization of caveolin-1, claudin-5, and LC3B in autolysosome or lipid raft fractions at 2-h OGD. Of note, inhibiting autophagosome and lysosome fusion sustained the colocalization of caveolin-1, claudin-5, and LC3B throughout the 4-h OGD exposure. EPR spin trapping showed increased nitric oxide (NO) generation in 2-h OGD-treated cells, and inhibiting NO with its scavenger C-PTIO or inducible nitric oxide synthase (iNOS) inhibitor 1400W prevented OGD-induced caveolin-1 translocation and claudin-5 degradation. Taken together, our data provide a novel mechanism underlying endothelial barrier disruption under prolonged ischemic conditions, in which NO promotes caveolin-1-mediated delivery of claudin-5 to the autophagosome for autophagy-lysosome-dependent degradation. PMID:26515186

  18. Nitric Oxide Interacts with Caveolin-1 to Facilitate Autophagy-Lysosome-Mediated Claudin-5 Degradation in Oxygen-Glucose Deprivation-Treated Endothelial Cells.

    PubMed

    Liu, Jie; Weaver, John; Jin, Xinchun; Zhang, Yuan; Xu, Ji; Liu, Ke J; Li, Weiping; Liu, Wenlan

    2016-11-01

    Using in vitro oxygen-glucose deprivation (OGD) model, we have previously demonstrated that 2-h OGD induces rapid, caveolin-1-mediated dissociation of claudin-5 from the cellular cytoskeletal framework and quick endothelial barrier disruption. In this study, we further investigated the fate of translocated claudin-5 and the mechanisms by which OGD promotes caveolin-1 translocation. Exposure of bEND3 cells to 4-h OGD, but not 2-h OGD plus 2-h reoxygenation, resulted in claudin-5 degradation. Inhibition of autophagy or the fusion of autophagosome with lysosome, but not proteasome, blocked OGD-induced claudin-5 degradation. Moreover, knockdown of caveolin-1 with siRNA blocked OGD-induced claudin-5 degradation. Western blot analysis showed a transient colocalization of caveolin-1, claudin-5, and LC3B in autolysosome or lipid raft fractions at 2-h OGD. Of note, inhibiting autophagosome and lysosome fusion sustained the colocalization of caveolin-1, claudin-5, and LC3B throughout the 4-h OGD exposure. EPR spin trapping showed increased nitric oxide (NO) generation in 2-h OGD-treated cells, and inhibiting NO with its scavenger C-PTIO or inducible nitric oxide synthase (iNOS) inhibitor 1400W prevented OGD-induced caveolin-1 translocation and claudin-5 degradation. Taken together, our data provide a novel mechanism underlying endothelial barrier disruption under prolonged ischemic conditions, in which NO promotes caveolin-1-mediated delivery of claudin-5 to the autophagosome for autophagy-lysosome-dependent degradation.

  19. Lysosomal solute carrier transporters gain momentum in research.

    PubMed

    Bissa, B; Beedle, A M; Govindarajan, R

    2016-11-01

    Emerging evidence indicates that lysosome function extends beyond macromolecular degradation. Genetic and functional defects in components of the lysosomal transport machinery cause lysosomal storage disorders implicating the lysosomal solute carrier (SLC) transporters as essential to vital cell processes. The pathophysiology and therapeutic potential of lysosomal SLC transporters are highlighted here, focusing on recent discoveries in autophagic amino acid sensing (SLC38A9), phagocytic regulation in macrophages (SLC29A3, SLC15A3/A4), adenosine triphosphate (ATP) exocytosis in neurotransmission (SLC17A9), and lysosomal transport of maytansine catabolites into the cytoplasm (SLC46A3).

  20. Lysosomal solute carrier transporters gain momentum in research

    PubMed Central

    Beedle, AM; Govindarajan, R

    2016-01-01

    Emerging evidence indicates that lysosome function extends beyond macromolecular degradation. Genetic and functional defects in components of the lysosomal transport machinery cause lysosomal storage disorders implicating the lysosomal solute carrier (SLC) transporters as essential to vital cell processes. The pathophysiology and therapeutic potential of lysosomal SLC transporters are highlighted here, focusing on recent discoveries in autophagic amino acid sensing (SLC38A9), phagocytic regulation in macrophages (SLC29A3, SLC15A3/A4), adenosine triphosphate (ATP) exocytosis in neurotransmission (SLC17A9), and lysosomal transport of maytansine catabolites into the cytoplasm (SLC46A3). PMID:27530302

  1. Synergy of 2-deoxy-D-glucose combined with berberine in inducing the lysosome/autophagy and transglutaminase activation-facilitated apoptosis.

    PubMed

    Halicka, H Dorota; Garcia, Jorge; Li, Jiangwei; Zhao, Hong; Darzynkiewicz, Zbigniew

    2017-02-01

    Utilizing a variety of flow cytometric methods evidence was obtained indicating that a combination of the glucose analog 2-deoxy-D-glucose (2-dG) and the plant alkaloid berberine (BRB) produces synergistic effect in the induction of apoptosis in human lymphoblastoid TK6 cells. The synergistic effect is seen at concentrations of the drugs at which each of them alone shows no cytotoxicity at all. The data suggest that the combination of these drugs, which are known in terms of their overall toxicity, side effects and pharmacokinetics may be considered for further studies as chemopreventive and cancer treatment modalities. Of interest are results indicating that rapamycin, which similarly to BRB, suppresses mTOR signaling, when combined with 2-dG shows no synergistic properties. Metformin, on other hand, requires much higher concentration to show the synergy with 2-dG. Also of interest are the findings pertaining to the methodology of the present study. Specifically, dynamic assessment of cellular viability was performed by using the DRAQ7 cell exclusion fluorochrome present in cultures from 0 to 72 h. Concurrent measurement of lysosomal proton pump using acridine orange as the probe shows activation of lysosomes in the cells treated with 2-dG or BRB alone as well as with the drugs combined. Apoptosis was assessed by measuring DNA fragmentation, cell cycle, activation of caspase-3 and tissue transglutaminase (Tgase). A novel cytometric method was developed based on analysis of lysosomal (acidic vesicles) proton pump in live cells followed by cell lysis with detergent and fluorochrome labeling of proteins and DNA to analyze Tgase activation concurrently with cell cycle, in same population of cells. The data show that the cell subpopulation undergoing apoptosis has increased side (right-angle) light scatter likely due to the presence of the crosslinked (solid state) proteins, the consequence Tgase activation.

  2. Hepoxilin A3 Facilitates Neutrophilic Breach of Lipoxygenase-expressing Airway Epithelial Barriers1

    PubMed Central

    Tamang, David L.; Pirzai, Waheed; Priebe, Gregory P.; Traficante, David C.; Pier, Gerald B.; Falck, J. R.; Morisseau, Christophe; Hammock, Bruce D.; McCormick, Beth A.; Gronert, Karsten; Hurley, Bryan P.

    2012-01-01

    A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A3 is a neutrophil chemo-attractant produced by pathogen infected epithelial cells hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with P. aeruginosa, we explored the role of hepoxilin A3 in neutrophil trans-epithelial migration. Pharmacological inhibitors of enzymatic pathways necessary to generate hepoxilin A3, including phospholipase A2 and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil trans-epithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate hepoxilin A3 precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing hepoxilin A3 are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased hepoxilin A3 and lipid derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with hepoxilin A3 serving a chemotactic role. Stable inhibitory structural analogues of hepoxilin A3 are capable of impeding P. aeruginosa-induced neutrophil trans-epithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace as well as increased concentration of the 12-lipoxygenase metabolites hepoxilin A3 and 12-HETE. Data generated from multiple models herein provide further evidence that hepoxilin A3 is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers. PMID:23045615

  3. Regulated lysosomal exocytosis mediates cancer progression

    PubMed Central

    Machado, Eda; White-Gilbertson, Shai; van de Vlekkert, Diantha; Janke, Laura; Moshiach, Simon; Campos, Yvan; Finkelstein, David; Gomero, Elida; Mosca, Rosario; Qiu, Xiaohui; Morton, Christopher L.; Annunziata, Ida; d’Azzo, Alessandra

    2015-01-01

    Understanding how tumor cells transition to an invasive and drug-resistant phenotype is central to cancer biology, but the mechanisms underlying this transition remain unclear. We show that sarcomas gain these malignant traits by inducing lysosomal exocytosis, a ubiquitous physiological process. During lysosomal exocytosis, the movement of exocytic lysosomes along the cytoskeleton and their docking at the plasma membrane involve LAMP1, a sialylated membrane glycoprotein and target of the sialidase NEU1. Cleavage of LAMP1 sialic acids by NEU1 limits the extent of lysosomal exocytosis. We found that by down-regulation of NEU1 and accumulation of oversialylated LAMP1, tumor cells exacerbate lysosomal exocytosis of soluble hydrolases and exosomes. This facilitates matrix invasion and propagation of invasive signals, and purging of lysosomotropic chemotherapeutics. In Arf−⁄− mice, Neu1 haploinsufficiency fostered the development of invasive, pleomorphic sarcomas, expressing epithelial and mesenchymal markers, and lysosomal exocytosis effectors, LAMP1 and Myosin-11. These features are analogous to those of metastatic, pleomorphic human sarcomas, where low NEU1 levels correlate with high expression of lysosomal exocytosis markers. In a therapeutic proof of principle, we demonstrate that inhibiting lysosomal exocytosis reversed invasiveness and chemoresistance in aggressive sarcoma cells. Thus, we reveal that this unconventional, lysosome-regulated pathway plays a primary role in tumor progression and chemoresistance. PMID:26824057

  4. Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase-MTORC1 signaling

    PubMed Central

    Valapala, Mallika; Wilson, Christine; Hose, Stacey; Bhutto, Imran A; Grebe, Rhonda; Dong, Aling; Greenbaum, Seth; Gu, Limin; Sengupta, Samhita; Cano, Marisol; Hackett, Sean; Xu, Guotong; Lutty, Gerard A; Dong, Lijin; Sergeev, Yuri; Handa, James T; Campochiaro, Peter; Wawrousek, Eric; Zigler, Jr, J Samuel; Sinha, Debasish

    2014-01-01

    In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V0-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the βA3- and βA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis. PMID:24468901

  5. Salinomycin co-treatment enhances tamoxifen cytotoxicity in luminal A breast tumor cells by facilitating lysosomal degradation of receptor tyrosine kinases.

    PubMed

    Sommer, Ann-Katrin; Hermawan, Adam; Mickler, Frauke Martina; Ljepoja, Bojan; Knyazev, Pjotr; Bräuchle, Christoph; Ullrich, Axel; Wagner, Ernst; Roidl, Andreas

    2016-08-02

    Luminal A breast cancer is the most common breast cancer subtype which is usually treated with selective estrogen receptor modulators (SERMS) like tamoxifen. Nevertheless, one third of estrogen receptor positive breast cancer patients initially do not respond to endocrine therapy and about 40% of luminal A breast tumors recur in five years. In this study, we investigated an alternative treatment approach by combining tamoxifen and salinomycin in luminal A breast cancer cell lines. We have found that salinomycin induces an additional cytotoxic effect by inhibiting the ligand independent activation of ERα. Thereby salinomycin increases the intracellular calcium level. This leads to a premature fusion of endosomes with lysosomes and thus to the degradation of Egfr family members. Since this process is essential for luminal A breast cancer cells to circumvent tamoxifen treatment, the combination of both drugs induces cytotoxicity in tamoxifen sensitive as well as resistant luminal A breast cancer cell lines.

  6. Salinomycin co-treatment enhances tamoxifen cytotoxicity in luminal A breast tumor cells by facilitating lysosomal degradation of receptor tyrosine kinases

    PubMed Central

    Sommer, Ann-Katrin; Hermawan, Adam; Mickler, Frauke Martina; Ljepoja, Bojan; Knyazev, Pjotr; Bräuchle, Christoph; Ullrich, Axel; Wagner, Ernst; Roidl, Andreas

    2016-01-01

    Luminal A breast cancer is the most common breast cancer subtype which is usually treated with selective estrogen receptor modulators (SERMS) like tamoxifen. Nevertheless, one third of estrogen receptor positive breast cancer patients initially do not respond to endocrine therapy and about 40% of luminal A breast tumors recur in five years. In this study, we investigated an alternative treatment approach by combining tamoxifen and salinomycin in luminal A breast cancer cell lines. We have found that salinomycin induces an additional cytotoxic effect by inhibiting the ligand independent activation of ERα. Thereby salinomycin increases the intracellular calcium level. This leads to a premature fusion of endosomes with lysosomes and thus to the degradation of Egfr family members. Since this process is essential for luminal A breast cancer cells to circumvent tamoxifen treatment, the combination of both drugs induces cytotoxicity in tamoxifen sensitive as well as resistant luminal A breast cancer cell lines. PMID:27409163

  7. Lysosomes in apoptosis.

    PubMed

    Ivanova, Saska; Repnik, Urska; Bojic, Lea; Petelin, Ana; Turk, Vito; Turk, Boris

    2008-01-01

    Lysosomes are specialized organelles for protein recycling and as such are involved in the terminal steps of autophagy. However, it has become evident that lysosomes also play an important role in the progression of apoptosis. This latter function seems to be dependent on lysosomal proteases, which need to be released into the cytosol for apoptosis to be efficient. Among the lysosomal proteases, the most abundant are the cysteine cathepsins and the aspartic protease cathepsin D, which seem to be the major apoptosis mediators. This chapter reviews the methods used to study lysosomes and lysosomal proteases.

  8. Lysosomes as mediators of drug resistance in cancer.

    PubMed

    Zhitomirsky, Benny; Assaraf, Yehuda G

    2016-01-01

    Drug resistance remains a leading cause of chemotherapeutic treatment failure and cancer-related mortality. While some mechanisms of anticancer drug resistance have been well characterized, multiple mechanisms remain elusive. In this respect, passive ion trapping-based lysosomal sequestration of multiple hydrophobic weak-base chemotherapeutic agents was found to reduce the accessibility of these drugs to their target sites, resulting in a markedly reduced cytotoxic effect and drug resistance. Recently we have demonstrated that lysosomal sequestration of hydrophobic weak base drugs triggers TFEB-mediated lysosomal biogenesis resulting in an enlarged lysosomal compartment, capable of enhanced drug sequestration. This study further showed that cancer cells with an increased number of drug-accumulating lysosomes are more resistant to lysosome-sequestered drugs, suggesting a model of drug-induced lysosome-mediated chemoresistance. In addition to passive drug sequestration of hydrophobic weak base chemotherapeutics, other mechanisms of lysosome-mediated drug resistance have also been reported; these include active lysosomal drug sequestration mediated by ATP-driven transporters from the ABC superfamily, and a role for lysosomal copper transporters in cancer resistance to platinum-based chemotherapeutics. Furthermore, lysosomal exocytosis was suggested as a mechanism to facilitate the clearance of chemotherapeutics which highly accumulated in lysosomes, thus providing an additional line of resistance, supplementing the organelle entrapment of chemotherapeutics away from their target sites. Along with these mechanisms of lysosome-mediated drug resistance, several approaches were recently developed for the overcoming of drug resistance or exploiting lysosomal drug sequestration, including lysosomal photodestruction and drug-induced lysosomal membrane permeabilization. In this review we explore the current literature addressing the role of lysosomes in mediating cancer drug

  9. Lysosomes, cholesterol and atherosclerosis

    PubMed Central

    Jerome, W Gray

    2011-01-01

    Cholesterol-engorged macrophage foam cells are a critical component of the atherosclerotic lesion. Reducing the sterol deposits in lesions reduces clinical events. Sterol accumulations within lysosomes have proven to be particularly hard to mobilize out of foam cells. Moreover, excess sterol accumulation in lysosomes has untoward effects, including a complete disruption of lysosome function. Recently, we demonstrated that treatment of sterol-engorged macrophages in culture with triglyceride-containing particles can reverse many of the effects of cholesterol on lysosomes and dramatically reduce the sterol burden in these cells. This article describes what is known about lysosomal sterol engorgement, discusses the possible mechanisms by which triglyceride could produce its effects, and evaluates the possible positive and negative effects of reducing the lysosomal cholesterol levels in foam cells. PMID:21643524

  10. Uncovering the role of Snapin in regulating autophagy-lysosomal function.

    PubMed

    Cai, Qian; Sheng, Zu-Hang

    2011-04-01

    The autophagy-lysosomal system is the major degradation pathway essential for the maintenance and survival of neurons. This process requires efficient late endocytic transport from distal processes to the soma, in which lysosomes are predominantly localized. However, it is not clear how late endocytic transport has an impact upon neuronal autophagy-lysosomal function. We recently revealed that Snapin acts as a dynein motor adaptor and coordinates retrograde transport and late endosomal-lysosomal trafficking, thus maintaining efficient autophagy-lysosomal function in neurons. Snapin(-/-) neurons display impaired retrograde transport and clustering of late endosomes along neuronal processes, aberrant accumulation of immature lysosomes, and impaired clearance of autolysosomes. Snapin deficiency leads to reduced neuron viability, neurodegeneration, and developmental defects in the central nervous system. Reintroducing the snapin transgene rescues these phenotypes by enhancing the delivery of endosomal cargos to lysosomes and by facilitating autophagy-lysosomal function. Our study suggests that Snapin is a candidate molecular target for autophagy-lysosomal regulation.

  11. TFEB regulates lysosomal proteostasis.

    PubMed

    Song, Wensi; Wang, Fan; Savini, Marzia; Ake, Ashley; di Ronza, Alberto; Sardiello, Marco; Segatori, Laura

    2013-05-15

    Loss-of-function diseases are often caused by destabilizing mutations that lead to protein misfolding and degradation. Modulating the innate protein homeostasis (proteostasis) capacity may lead to rescue of native folding of the mutated variants, thereby ameliorating the disease phenotype. In lysosomal storage disorders (LSDs), a number of highly prevalent alleles have missense mutations that do not impair the enzyme's catalytic activity but destabilize its native structure, resulting in the degradation of the misfolded protein. Enhancing the cellular folding capacity enables rescuing the native, biologically functional structure of these unstable mutated enzymes. However, proteostasis modulators specific for the lysosomal system are currently unknown. Here, we investigate the role of the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and function, in modulating lysosomal proteostasis in LSDs. We show that TFEB activation results in enhanced folding, trafficking and lysosomal activity of a severely destabilized glucocerebrosidase (GC) variant associated with the development of Gaucher disease (GD), the most common LSD. TFEB specifically induces the expression of GC and of key genes involved in folding and lysosomal trafficking, thereby enhancing both the pool of mutated enzyme and its processing through the secretory pathway. TFEB activation also rescues the activity of a β-hexosaminidase mutant associated with the development of another LSD, Tay-Sachs disease, thus suggesting general applicability of TFEB-mediated proteostasis modulation to rescue destabilizing mutations in LSDs. In summary, our findings identify TFEB as a specific regulator of lysosomal proteostasis and suggest that TFEB may be used as a therapeutic target to rescue enzyme homeostasis in LSDs.

  12. Attenuation of the lysosomal death pathway by lysosomal cholesterol accumulation.

    PubMed

    Appelqvist, Hanna; Nilsson, Cathrine; Garner, Brett; Brown, Andrew J; Kågedal, Katarina; Ollinger, Karin

    2011-02-01

    In the past decade, lysosomal membrane permeabilization (LMP) has emerged as a significant component of cell death signaling. The mechanisms by which lysosomal stability is regulated are not yet fully understood, but changes in the lysosomal membrane lipid composition have been suggested to be involved. Our aim was to investigate the importance of cholesterol in the regulation of lysosomal membrane permeability and its potential impact on apoptosis. Treatment of normal human fibroblasts with U18666A, an amphiphilic drug that inhibits cholesterol transport and causes accumulation of cholesterol in lysosomes, rescued cells from lysosome-dependent cell death induced by the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), staurosporine (STS), or cisplatin. LMP was decreased by pretreating cells with U18666A, and there was a linear relationship between the cholesterol content of lysosomes and their resistance to permeabilization induced by MSDH. U18666A did not induce changes in expression or localization of 70-kDa heat shock proteins (Hsp70) or antiapoptotic Bcl-2 proteins known to protect the lysosomal membrane. Induction of autophagy also was excluded as a contributor to the protective mechanism. By using Chinese hamster ovary (CHO) cells with lysosomal cholesterol overload due to a mutation in the cholesterol transporting protein Niemann-Pick type C1 (NPC1), the relationship between lysosomal cholesterol accumulation and protection from lysosome-dependent cell death was confirmed. Cholesterol accumulation in lysosomes attenuates apoptosis by increasing lysosomal membrane stability.

  13. Lysosome dysfunction in the pathogenesis of kidney diseases

    PubMed Central

    Surendran, Kameswaran; Vitiello, Seasson P.; Pearce, David A.

    2013-01-01

    The lysosome, an organelle central to macromolecule degradation and recycling, plays a pivotal role in normal cell processes, ranging from autophagy to redox regulation. Not surprisingly, lysosomes are an integral part of the renal epithelial molecular machinery that facilitates normal renal physiology. Two inherited diseases that manifest as kidney dysfunction are Fabry’s disease and cystinosis, each of which is caused by a primary biochemical defect at the lysosome resulting from loss of function mutations in genes that encode lysosomal proteins. The functions of the lysosomes in the kidney and how lysosomal dysfunction might contribute to Fabry’s disease and cystinosis are discussed. Unlike most other pediatric renal diseases, therapies are available for Fabry’s disease and cystinosis, but require early diagnosis. Recent analysis of ceroid neuronal lipofuscinosis type 3 (Cln3) null mice, a mouse model of lysosomal disease that is primarily associated with neurological deficits, revealed renal functional abnormalities. As current and future therapeutics increase the life-span of those suffering from diseases like neuronal ceroid lipofuscinosis, it remains a distinct possibility that many more lysosomal disorders that primarily manifest as infant and juvenile neurodegenerative diseases may also include renal disease phenotypes. PMID:24217784

  14. Lysosomal membrane glycoproteins bind cholesterol and contribute to lysosomal cholesterol export

    PubMed Central

    Li, Jian; Pfeffer, Suzanne R

    2016-01-01

    LAMP1 and LAMP2 proteins are highly abundant, ubiquitous, mammalian proteins that line the lysosome limiting membrane, and protect it from lysosomal hydrolase action. LAMP2 deficiency causes Danon’s disease, an X-linked hypertrophic cardiomyopathy. LAMP2 is needed for chaperone-mediated autophagy, and its expression improves tissue function in models of aging. We show here that human LAMP1 and LAMP2 bind cholesterol in a manner that buries the cholesterol 3β-hydroxyl group; they also bind tightly to NPC1 and NPC2 proteins that export cholesterol from lysosomes. Quantitation of cellular LAMP2 and NPC1 protein levels suggest that LAMP proteins represent a significant cholesterol binding site at the lysosome limiting membrane, and may signal cholesterol availability. Functional rescue experiments show that the ability of human LAMP2 to facilitate cholesterol export from lysosomes relies on its ability to bind cholesterol directly. DOI: http://dx.doi.org/10.7554/eLife.21635.001 PMID:27664420

  15. Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

    PubMed Central

    Sreetama, S C; Takano, T; Nedergaard, M; Simon, S M; Jaiswal, J K

    2016-01-01

    Astrocytes are known to facilitate repair following brain injury; however, little is known about how injured astrocytes repair themselves. Repair of cell membrane injury requires Ca2+-triggered vesicle exocytosis. In astrocytes, lysosomes are the main Ca2+-regulated exocytic vesicles. Here we show that astrocyte cell membrane injury results in a large and rapid calcium increase. This triggers robust lysosome exocytosis where the fusing lysosomes release all luminal contents and merge fully with the plasma membrane. In contrast to this, receptor stimulation produces a small sustained calcium increase, which is associated with partial release of the lysosomal luminal content, and the lysosome membrane does not merge into the plasma membrane. In most cells, lysosomes express the synaptotagmin (Syt) isoform Syt VII; however, this isoform is not present on astrocyte lysosomes and exogenous expression of Syt VII on lysosome inhibits their exocytosis. Deletion of one of the most abundant Syt isoform in astrocyte – Syt XI – suppresses astrocyte lysosome exocytosis. This identifies lysosome as Syt XI-regulated exocytic vesicle in astrocytes. Further, inhibition of lysosome exocytosis (by Syt XI depletion or Syt VII expression) prevents repair of injured astrocytes. These results identify the lysosomes and Syt XI as the sub-cellular and molecular regulators, respectively of astrocyte cell membrane repair. PMID:26450452

  16. Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis.

    PubMed

    Sreetama, S C; Takano, T; Nedergaard, M; Simon, S M; Jaiswal, J K

    2016-04-01

    Astrocytes are known to facilitate repair following brain injury; however, little is known about how injured astrocytes repair themselves. Repair of cell membrane injury requires Ca(2+)-triggered vesicle exocytosis. In astrocytes, lysosomes are the main Ca(2+)-regulated exocytic vesicles. Here we show that astrocyte cell membrane injury results in a large and rapid calcium increase. This triggers robust lysosome exocytosis where the fusing lysosomes release all luminal contents and merge fully with the plasma membrane. In contrast to this, receptor stimulation produces a small sustained calcium increase, which is associated with partial release of the lysosomal luminal content, and the lysosome membrane does not merge into the plasma membrane. In most cells, lysosomes express the synaptotagmin (Syt) isoform Syt VII; however, this isoform is not present on astrocyte lysosomes and exogenous expression of Syt VII on lysosome inhibits their exocytosis. Deletion of one of the most abundant Syt isoform in astrocyte--Syt XI--suppresses astrocyte lysosome exocytosis. This identifies lysosome as Syt XI-regulated exocytic vesicle in astrocytes. Further, inhibition of lysosome exocytosis (by Syt XI depletion or Syt VII expression) prevents repair of injured astrocytes. These results identify the lysosomes and Syt XI as the sub-cellular and molecular regulators, respectively of astrocyte cell membrane repair.

  17. The biogenesis of lysosomes and lysosome-related organelles.

    PubMed

    Luzio, J Paul; Hackmann, Yvonne; Dieckmann, Nele M G; Griffiths, Gillian M

    2014-09-02

    Lysosomes were once considered the end point of endocytosis, simply used for macromolecule degradation. They are now recognized to be dynamic organelles, able to fuse with a variety of targets and to be re-formed after fusion events. They are also now known to be the site of nutrient sensing and signaling to the cell nucleus. In addition, lysosomes are secretory organelles, with specialized machinery for regulated secretion of proteins in some cell types. The biogenesis of lysosomes and lysosome-related organelles is discussed, taking into account their dynamic nature and multiple roles. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  18. Prostaglandins, Lysosomes, and Radiation Injury

    DTIC Science & Technology

    1980-01-01

    SCIENTIFIC REPORT Prostaglandins, lysosomes, and radiation injury 7 P. J. Trocha <G. N. Catravas DTI.C A DEFENSE NUCLEAR AGENCY -LL ARMED FORCES...been shown to be altered with tissue injury . Yet no studies have been performed to monitor lysosomal enzyme activi- ties and PG levels simultaneously...nd R. Pi olm. Raven Pra& New Yort- D 980. Prostaglandins, Lysosomes, and Radiation Injury Paul J. Trocha and George N. Catravas Armed Forces

  19. Vacuolar ATPase in Phagosome-Lysosome Fusion

    PubMed Central

    Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S.; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

    2015-01-01

    The vacuolar H+-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. PMID:25903133

  20. Kinetics of lysosomal storage of indigestible matter.

    PubMed Central

    Hurley, J; Alward, J

    1975-01-01

    In lysosomal storage diseases and in accumulation of lipofusion in the lysosomes there is a gradual eroding of the lysosomal system due to overloading the lysosomes by molecules which cannot be digested or expelled. The kinetics of this accumulation is examined for tissue cultures in terms of the cell growth rate, lysosomal production rate, and of generation of the indigestible element. PMID:1125388

  1. BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases

    PubMed Central

    Zhong, Xi Zoë; Sun, Xue; Cao, Qi; Dong, Gaofeng; Schiffmann, Raphael; Dong, Xian-Ping

    2016-01-01

    Efficient lysosomal Ca2+ release plays an essential role in lysosomal trafficking. We have recently shown that lysosomal big conductance Ca2+-activated potassium (BK) channel forms a physical and functional coupling with the lysosomal Ca2+ release channel Transient Receptor Potential Mucolipin-1 (TRPML1). BK and TRPML1 forms a positive feedback loop to facilitate lysosomal Ca2+ release and subsequent lysosome membrane trafficking. However, it is unclear whether the positive feedback mechanism is common for other lysosomal storage diseases (LSDs) and whether BK channel agonists rescue abnormal lysosomal storage in LSDs. In this study, we assessed the effect of BK agonist, NS1619 and NS11021 in a number of LSDs including NPC1, mild cases of mucolipidosis type IV (ML4) (TRPML1-F408∆), Niemann-Pick type A (NPA) and Fabry disease. We found that TRPML1-mediated Ca2+ release was compromised in these LSDs. BK activation corrected the impaired Ca2+ release in these LSDs and successfully rescued the abnormal lysosomal storage of these diseases by promoting TRPML1-mediated lysosomal exocytosis. Our study suggests that BK channel activation stimulates the TRPML1-BK positive reinforcing loop to correct abnormal lysosomal storage in LSDs. Drugs targeting BK channel represent a potential therapeutic approach for LSDs. PMID:27670435

  2. Lysosomal adaptation: how the lysosome responds to external cues.

    PubMed

    Settembre, Carmine; Ballabio, Andrea

    2014-05-05

    Recent evidence indicates that the importance of the lysosome in cell metabolism and organism physiology goes far beyond the simple disposal of cellular garbage. This dynamic organelle is situated at the crossroad of the most important cellular pathways and is involved in sensing, signaling, and transcriptional mechanisms that respond to environmental cues, such as nutrients. Two main mediators of these lysosomal adaptation mechanisms are the mTORC1 kinase complex and the transcription factor EB (TFEB). These two factors are linked in a lysosome-to-nucleus signaling pathway that provides the lysosome with the ability to adapt to extracellular cues and control its own biogenesis. Modulation of lysosomal function by acting on TFEB has a profound impact on cellular clearance and energy metabolism and is a promising therapeutic target for a large variety of disease conditions. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  3. Genetics Home Reference: lysosomal acid lipase deficiency

    MedlinePlus

    ... Home Health Conditions lysosomal acid lipase deficiency lysosomal acid lipase deficiency Printable PDF Open All Close All ... to view the expand/collapse boxes. Description Lysosomal acid lipase deficiency is an inherited condition characterized by ...

  4. LYSOSOMAL DISRUPTION BY BACTERIAL TOXINS

    PubMed Central

    Bernheimer, Alan W.; Schwartz, Lois L.

    1964-01-01

    Bernheimer, Alan W. (New York University School of Medicine, New York), and Lois L. Schwartz. Lysosomal disruption by bacterial toxins. J. Bacteriol. 87:1100–1104. 1964.—Seventeen bacterial toxins were examined for capacity (i) to disrupt rabbit leukocyte lysosomes as indicated by decrease in turbidity of lysosomal suspensions, and (ii) to alter rabbit liver lysosomes as measured by release of β-glucuronidase and acid phosphatase. Staphylococcal α-toxin, Clostridium perfringens α-toxin, and streptolysins O and S affected lysosomes in both systems. Staphylococcal β-toxin, leucocidin and enterotoxin, Shiga neurotoxin, Serratia endotoxin, diphtheria toxin, tetanus neurotoxin, C. botulinum type A toxin, and C. perfringens ε-toxin were not active in either system. Staphylococcal δ-toxin, C. histolyticum collagenase, crude C. perfringens β-toxin, and crude anthrax toxin caused lysosomal damage in only one of the test systems. There is a substantial correlation between the hemolytic property of a toxin and its capacity to disrupt lysosomes, lending support to the concept that erythrocytes and lysosomes are bounded by similar membranes. PMID:5874534

  5. Robot-Assisted Rehabilitation of Ankle Plantar Flexors Spasticity: A 3-Month Study with Proprioceptive Neuromuscular Facilitation.

    PubMed

    Zhou, Zhihao; Sun, Yao; Wang, Ninghua; Gao, Fan; Wei, Kunlin; Wang, Qining

    2016-01-01

    In this paper, we aim to investigate the effect of proprioceptive neuromuscular facilitation (PNF)-based rehabilitation for ankle plantar flexors spasticity by using a Robotic Ankle-foot Rehabilitation System (RARS). A modified robot-assisted system was proposed, and seven poststroke patients with hemiplegic spastic ankles participated in a 3-month robotic PNF training. Their impaired sides were used as the experimental group, while their unimpaired sides as the control group. A robotic intervention for the experimental group started from a 2-min passive stretching to warming-up or relaxing the soleus and gastrocnemius muscles and also ended with the same one. Then a PNF training session including 30 trials was activated between them. The rehabilitation trainings were carried out three times a week as an addition to their regular rehabilitation exercise. Passive range of motion, resistance torque, and stiffness were measured in both ankles before and after the interventions. The changes in Achilles tendon length, walking speed, and lower limb function were also evaluated by the same physician or physiotherapist for each participant. Biomechanical measurements before interventions showed significant difference between the experimental group and the control group due to ankle spasticity. For the control group, there was no significant difference in the 3 months with no robotic intervention. But for the experimental group, passive dorsiflexion range of motion increased (p < 0.01), resistance torque under different dorsiflexion angle levels (0°, 10°, and 20°) decreased (p < 0.05, p < 0.001, and p < 0.001, respectively), and quasi-static stiffness under different dorsiflexion angle levels (0°, 10°, and 20°) also decreased (p < 0.01, p < 0.001, and p < 0.001, respectively). Achilles's tendon length shortened (p < 0.01), while its thickness showed no significant change (p > 0.05). The robotic rehabilitation also improved the

  6. Robot-Assisted Rehabilitation of Ankle Plantar Flexors Spasticity: A 3-Month Study with Proprioceptive Neuromuscular Facilitation

    PubMed Central

    Zhou, Zhihao; Sun, Yao; Wang, Ninghua; Gao, Fan; Wei, Kunlin; Wang, Qining

    2016-01-01

    In this paper, we aim to investigate the effect of proprioceptive neuromuscular facilitation (PNF)-based rehabilitation for ankle plantar flexors spasticity by using a Robotic Ankle–foot Rehabilitation System (RARS). A modified robot-assisted system was proposed, and seven poststroke patients with hemiplegic spastic ankles participated in a 3-month robotic PNF training. Their impaired sides were used as the experimental group, while their unimpaired sides as the control group. A robotic intervention for the experimental group started from a 2-min passive stretching to warming-up or relaxing the soleus and gastrocnemius muscles and also ended with the same one. Then a PNF training session including 30 trials was activated between them. The rehabilitation trainings were carried out three times a week as an addition to their regular rehabilitation exercise. Passive range of motion, resistance torque, and stiffness were measured in both ankles before and after the interventions. The changes in Achilles tendon length, walking speed, and lower limb function were also evaluated by the same physician or physiotherapist for each participant. Biomechanical measurements before interventions showed significant difference between the experimental group and the control group due to ankle spasticity. For the control group, there was no significant difference in the 3 months with no robotic intervention. But for the experimental group, passive dorsiflexion range of motion increased (p < 0.01), resistance torque under different dorsiflexion angle levels (0°, 10°, and 20°) decreased (p < 0.05, p < 0.001, and p < 0.001, respectively), and quasi-static stiffness under different dorsiflexion angle levels (0°, 10°, and 20°) also decreased (p < 0.01, p < 0.001, and p < 0.001, respectively). Achilles’s tendon length shortened (p < 0.01), while its thickness showed no significant change (p > 0.05). The robotic rehabilitation also improved the

  7. Impaired prosaposin lysosomal trafficking in frontotemporal lobar degeneration due to progranulin mutations

    PubMed Central

    Zhou, Xiaolai; Sun, Lirong; Bracko, Oliver; Choi, Ji Whae; Jia, Yan; Nana, Alissa L.; Brady, Owen Adam; Hernandez, Jean C. Cruz; Nishimura, Nozomi; Seeley, William W.; Hu, Fenghua

    2017-01-01

    Haploinsufficiency of progranulin (PGRN) due to mutations in the granulin (GRN) gene causes frontotemporal lobar degeneration (FTLD), and complete loss of PGRN leads to a lysosomal storage disorder, neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests that PGRN is essential for proper lysosomal function, but the precise mechanisms involved are not known. Here, we show that PGRN facilitates neuronal uptake and lysosomal delivery of prosaposin (PSAP), the precursor of saposin peptides that are essential for lysosomal glycosphingolipid degradation. We found reduced levels of PSAP in neurons both in mice deficient in PGRN and in human samples from FTLD patients due to GRN mutations. Furthermore, mice with reduced PSAP expression demonstrated FTLD-like pathology and behavioural changes. Thus, our data demonstrate a role of PGRN in PSAP lysosomal trafficking and suggest that impaired lysosomal trafficking of PSAP is an underlying disease mechanism for NCL and FTLD due to GRN mutations. PMID:28541286

  8. Neuropathic Lysosomal Storage Disorders

    PubMed Central

    Pastores, Gregory M.; Maegawa, Gustavo H.B.

    2014-01-01

    The lysosomal storage disorders (LSDs) are a clinically heterogeneous group of inborn errors of metabolism, associated with the accumulation of incompletely degraded macromolecules within several cellular sites. Affected individuals present with a broad range of clinical problems, including hepatosplenomegaly and skeletal dysplasia. Onset of symptoms may range from birth to adulthood. The majority are associated with neurological features, including developmental delay, behavioral/psychiatric disturbances, seizures, acroparesthesia, motor weakness, cerebrovascular ischemic events and extra-pyramidal signs. It should be noted that later-onset forms are often misdiagnosed as symptoms, which might include psychiatric manifestations, are slowly progressive and may precede other neurologic or systemic features. Inheritance is primarily autosomal recessive. For all subtypes, diagnosis can be confirmed using a combination of biochemical and/or molecular assays. In a few LSDs, treatment with either hematopoietic stem cell transplantation, enzyme replacement or substrate reduction therapy is available. Genetic counseling is important, so patients and their families can be informed of reproductive risks, disease prognosis and therapeutic options. Investigations of disease mechanisms are providing insights into potential therapeutic approaches. Symptomatic care, which remains the mainstay for most subtypes, can lead to significant improvement in quality of life. PMID:24176423

  9. Lysosomal Dysfunction and α-Synuclein Aggregation in Parkinson's Disease: Diagnostic Links.

    PubMed

    Moors, Tim; Paciotti, Silvia; Chiasserini, Davide; Calabresi, Paolo; Parnetti, Lucilla; Beccari, Tommaso; van de Berg, Wilma D J

    2016-06-01

    Lysosomal impairment is increasingly recognized as a central event in the pathophysiology of PD. Genetic associations between lysosomal storage disorders, including Gaucher disease and PD, highlight common risk factors and pathological mechanisms. Because the autophagy-lysosomal system is involved in the intralysosomal hydrolysis of dysfunctional proteins, lysosomal impairment may contribute to α-synuclein aggregation in PD. The degradation of α-synuclein is a complex process involving different proteolytic mechanisms depending on protein burden, folding, posttranslational modifications, and yet unknown factors. In this review, evidence for lysosomal dysfunction in PD and its intimate relationship with α-synuclein aggregation are discussed, after which the question of whether lysosomal proteins may serve as diagnostic biomarkers for PD is addressed. Changes in lysosomal enzymes, such as reduced glucocerebrosidase and cathepsin levels, have been observed in affected brain regions in PD patients. The detection of lysosomal proteins in CSF may provide a read-out of lysosomal dysfunction in PD and holds promise for the development of diagnostic PD biomarkers. Initial PD biomarker studies demonstrated altered lysosomal enzyme activities in CSF of PD patients when compared with controls. However, CSF lysosomal enzyme activities alone could not discriminate between PD patients and controls. The combination of CSF lysosomal markers with α-synuclein species and indicators of mitochondrial dysfunction, inflammation, and other pathological proteins in PD may be able to facilitate a more accurate diagnosis of PD. Further CSF biomarker studies are needed to investigate the utility of CSF lysosomal proteins as measures of disease state and disease progression in PD. © 2016 International Parkinson and Movement Disorder Society.

  10. Reporter assay for endo/lysosomal escape of toxin-based therapeutics.

    PubMed

    Gilabert-Oriol, Roger; Thakur, Mayank; von Mallinckrodt, Benedicta; Bhargava, Cheenu; Wiesner, Burkhard; Eichhorst, Jenny; Melzig, Matthias F; Fuchs, Hendrik; Weng, Alexander

    2014-05-22

    Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters-horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)-were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates-saporin-HRP, (Alexa)saporin and saporin-KQ-RTA-were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of (Alexa)saporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10-1000 nM.

  11. Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics

    PubMed Central

    Gilabert-Oriol, Roger; Thakur, Mayank; von Mallinckrodt, Benedicta; Bhargava, Cheenu; Wiesner, Burkhard; Eichhorst, Jenny; Melzig, Matthias F.; Fuchs, Hendrik; Weng, Alexander

    2014-01-01

    Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters—horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)—were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates—saporin-HRP, Alexasaporin and saporin-KQ-RTA—were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of Alexasaporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10–1000 nM. PMID:24859158

  12. Toxicological findings in cases of alleged drug-facilitated sexual assault in the United Kingdom over a 3-year period.

    PubMed

    Scott-Ham, Michael; Burton, Fiona C

    2005-08-01

    This paper outlines the toxicology results from 1014 cases of claimed drug-facilitated sexual assault (DFSA) analysed at the Forensic Science Service, London Laboratory between January 2000 and December 2002. Where appropriate, either a whole blood sample and/or a urine sample was analysed for alcohol, common drugs of abuse and potentially stupefying drugs. The results were interpreted with respect to the number of drugs detected and an attempt was made to distinguish between voluntary and involuntary ingestion from information supplied. Alcohol (either alone or with an illicit and/or medicinal drug) was detected in 470 of all cases (46%). Illicit drugs were detected in 344 cases (34%), with cannabis being the most commonly detected (26% of cases), followed by cocaine (11%). In 21 cases (2%), a sedative or disinhibiting drug was detected which had not been admitted and could therefore be an instance of deliberate spiking. This included three cases in which complainants were allegedly given Ecstasy (MDMA) without their knowledge. Other drugs detected included gammahydroxybutyrate (GHB) and the benzodiazepine drugs diazepam and temazepam. Another nine cases (1%) involved the complainant being either given or forced to ingest pharmaceutical tablets or an illicit drug.

  13. Lysosomal cell death mechanisms in aging.

    PubMed

    Gómez-Sintes, Raquel; Ledesma, María Dolores; Boya, Patricia

    2016-12-01

    Lysosomes are degradative organelles essential for cell homeostasis that regulate a variety of processes, from calcium signaling and nutrient responses to autophagic degradation of intracellular components. Lysosomal cell death is mediated by the lethal effects of cathepsins, which are released into the cytoplasm following lysosomal damage. This process of lysosomal membrane permeabilization and cathepsin release is observed in several physiopathological conditions and plays a role in tissue remodeling, the immune response to intracellular pathogens and neurodegenerative diseases. Many evidences indicate that aging strongly influences lysosomal activity by altering the physical and chemical properties of these organelles, rendering them more sensitive to stress. In this review we focus on how aging alters lysosomal function and increases cell sensitivity to lysosomal membrane permeabilization and lysosomal cell death, both in physiological conditions and age-related pathologies.

  14. Palmitoylation-dependent association with CD63 targets the Ca2+ sensor synaptotagmin VII to lysosomes

    PubMed Central

    Flannery, Andrew R.; Czibener, Cecilia

    2010-01-01

    Syt VII is a Ca2+ sensor that regulates lysosome exocytosis and plasma membrane repair. Because it lacks motifs that mediate lysosomal targeting, it is unclear how Syt VII traffics to these organelles. In this paper, we show that mutations or inhibitors that abolish palmitoylation disrupt Syt VII targeting to lysosomes, causing its retention in the Golgi complex. In macrophages, Syt VII is translocated simultaneously with the lysosomal tetraspanin CD63 from tubular lysosomes to nascent phagosomes in a Ca2+-dependent process that facilitates particle uptake. Mutations in Syt VII palmitoylation sites block trafficking of Syt VII, but not CD63, to lysosomes and phagosomes, whereas tyrosine replacement in the lysosomal targeting motif of CD63 causes both proteins to accumulate on the plasma membrane. Complexes of CD63 and Syt VII are detected only when Syt VII palmitoylation sites are intact. These findings identify palmitoylation-dependent association with the tetraspanin CD63 as the mechanism by which Syt VII is targeted to lysosomes. PMID:21041449

  15. A fluorescence resonance energy transfer-based approach for investigating late endosome-lysosome retrograde fusion events.

    PubMed

    Kaufmann, A M; Goldman, S D B; Krise, J P

    2009-03-01

    Traditionally, lysosomes have been considered to be a terminal endocytic compartment. Recent studies suggest that lysosomes are quite dynamic, being able to fuse with other late endocytic compartments as well as with the plasma membrane. Here we describe a quantitative fluorescence energy transfer (FRET)-based method for assessing rates of retrograde fusion between terminal lysosomes and late endosomes in living cells. Late endosomes were specifically labeled with 800-nm latex beads that were conjugated with streptavidin and Alexa Fluor 555 (FRET donor). Terminal lysosomes were specifically labeled with 10,000-MW dextran polymers conjugated with biotin and Alexa Fluor 647 (FRET acceptor). Following late endosome-lysosome fusion, the strong binding affinity between streptavidin and biotin brought the donor and acceptor fluorophore molecules into close proximity, thereby facilitating the appearance of a FRET emission signal. Because apparent size restrictions in the endocytic pathway do not permit endocytosed latex beads from reaching terminal lysosomes in an anterograde fashion, the appearance of the FRET signal is consistent with retrograde transport of lysosomal cargo back to late endosomes. We assessed the efficiency of this transport step in fibroblasts affected by different lysosome storage disorders-Niemann-Pick type C, mucolipidosis type IV, and Sandhoff's disease, all of which have a similar lysosomal lipid accumulation phenotype. We report here, for the first time, that these disorders can be distinguished by their rate of transfer of lysosome cargos to late endosomes, and we discuss the implications of these findings for developing new therapeutic strategies.

  16. Lysosomal degradation of membrane lipids.

    PubMed

    Kolter, Thomas; Sandhoff, Konrad

    2010-05-03

    The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. Characterization of Drosophila Saposin-related mutants as a model for lysosomal sphingolipid storage diseases

    PubMed Central

    Schulze, Heike; Paradis, Marie; Gosejacob, Dominic; Papan, Cyrus; Shevchenko, Andrej; Psathaki, Olympia Ekaterina; Thielisch, Melanie; Sandhoff, Konrad

    2017-01-01

    ABSTRACT Sphingolipidoses are inherited diseases belonging to the class of lysosomal storage diseases (LSDs), which are characterized by the accumulation of indigestible material in the lysosome caused by specific defects in the lysosomal degradation machinery. While some LSDs can be efficiently treated by enzyme replacement therapy (ERT), this is not possible if the nervous system is affected due to the presence of the blood-brain barrier. Sphingolipidoses in particular often present as severe, untreatable forms of LSDs with massive sphingolipid and membrane accumulation in lysosomes, neurodegeneration and very short life expectancy. The digestion of intralumenal membranes within lysosomes is facilitated by lysosomal sphingolipid activator proteins (saposins), which are cleaved from a prosaposin precursor. Prosaposin mutations cause some of the severest forms of sphingolipidoses, and are associated with perinatal lethality in mice, hampering studies on disease progression. We identify the Drosophila prosaposin orthologue Saposin-related (Sap-r) as a key regulator of lysosomal lipid homeostasis in the fly. Its mutation leads to a typical spingolipidosis phenotype with an enlarged endolysosomal compartment and sphingolipid accumulation as shown by mass spectrometry and thin layer chromatography. Sap-r mutants show reduced viability with ∼50% survival to adulthood, allowing us to study progressive neurodegeneration and analyze their lipid profile in young and aged flies. Additionally, we observe a defect in sterol homeostasis with local sterol depletion at the plasma membrane. Furthermore, we find that autophagy is increased, resulting in the accumulation of mitochondria in lysosomes, concomitant with increased oxidative stress. Together, we establish Drosophila Sap-r mutants as a lysosomal storage disease model suitable for studying the age-dependent progression of lysosomal dysfunction associated with lipid accumulation and the resulting pathological signaling

  18. High lumenal chloride in the lysosome is critical for lysosome function

    PubMed Central

    Chakraborty, Kasturi; Leung, KaHo; Krishnan, Yamuna

    2017-01-01

    Lysosomes are organelles responsible for the breakdown and recycling of cellular machinery. Dysfunctional lysosomes give rise to lysosomal storage disorders as well as common neurodegenerative diseases. Here, we use a DNA-based, fluorescent chloride reporter to measure lysosomal chloride in Caenorhabditis elegans as well as murine and human cell culture models of lysosomal diseases. We find that the lysosome is highly enriched in chloride, and that chloride reduction correlates directly with a loss in the degradative function of the lysosome. In nematodes and mammalian cell culture models of diverse lysosomal disorders, where previously only lysosomal pH dysregulation has been described, massive reduction of lumenal chloride is observed that is ~103 fold greater than the accompanying pH change. Reducing chloride within the lysosome impacts Ca2+ release from the lysosome and impedes the activity of specific lysosomal enzymes indicating a broader role for chloride in lysosomal function. DOI: http://dx.doi.org/10.7554/eLife.28862.001 PMID:28742019

  19. Structure Dependence of Lysosomal Transit of Chitosan-Based Polyplexes for Gene Delivery.

    PubMed

    Thibault, Marc; Lavertu, Marc; Astolfi, Mélina; Buschmann, Michael D

    2016-10-01

    Chitosan-based polyplexes are known to traffic through lysosomes for a relatively long time, independent of the degree of deacetylation (DDA) and the number average molecular weight (Mn) of the polymer, even though both of these parameters have profound effects on polyplex stability and transfection efficiency. A better understanding of the lysosomal barrier is paramount to the rational design of vectors capable of overcoming obstacles to transgene expression. The aim of the present study was to investigate if lysosomal transit affects chitosan-based polyplex transfection efficiency in a structure-dependent (DDA, Mn) manner. Toward this end, we analyzed the effects of intracellular trafficking modifying agents on transfection efficiency and intracellular vesicular trafficking of polyplexes with different structural properties and stabilities or nucleic acid binding affinity. The use of agents that modify endosome/lysosome acidification and transit processes by distinct mechanisms and their effect on cell viability, polyplex uptake, vesicular trafficking, and transfection efficiency revealed novel and strong chitosan structure-dependent consequences of lysosomal transit. Inhibiting lysosomal transit using chloroquine significantly increased the efficiency of unstable polyplexes, while having minimal effects for polyplexes with intermediate or high stability. In parallel, specifically inhibiting the acidification of vesicles abrogated transfection for all formulations, suggesting that vesicular acidification is essential to promote transfection, most probably by facilitating lysosomal escape. These results provide novel insights into the structure-performance relationship of chitosan-based gene delivery systems.

  20. Deacetylation of TFEB promotes fibrillar Aβ degradation by upregulating lysosomal biogenesis in microglia.

    PubMed

    Bao, Jintao; Zheng, Liangjun; Zhang, Qi; Li, Xinya; Zhang, Xuefei; Li, Zeyang; Bai, Xue; Zhang, Zhong; Huo, Wei; Zhao, Xuyang; Shang, Shujiang; Wang, Qingsong; Zhang, Chen; Ji, Jianguo

    2016-06-01

    Microglia play a pivotal role in clearance of Aβ by degrading them in lysosomes, countering amyloid plaque pathogenesis in Alzheimer's disease (AD). Recent evidence suggests that lysosomal dysfunction leads to insufficient elimination of toxic protein aggregates. We tested whether enhancing lysosomal function with transcription factor EB (TFEB), an essential regulator modulating lysosomal pathways, would promote Aβ clearance in microglia. Here we show that microglial expression of TFEB facilitates fibrillar Aβ (fAβ) degradation and reduces deposited amyloid plaques, which are further enhanced by deacetylation of TFEB. Using mass spectrometry analysis, we firstly confirmed acetylation as a previously unreported modification of TFEB and found that SIRT1 directly interacted with and deacetylated TFEB at lysine residue 116. Subsequently, SIRT1 overexpression enhanced lysosomal function and fAβ degradation by upregulating transcriptional levels of TFEB downstream targets, which could be inhibited when TFEB was knocked down. Furthermore, overexpression of deacetylated TFEB at K116R mutant in microglia accelerated intracellular fAβ degradation by stimulating lysosomal biogenesis and greatly reduced the deposited amyloid plaques in the brain slices of APP/PS1 transgenic mice. Our findings reveal that deacetylation of TFEB could regulate lysosomal biogenesis and fAβ degradation, making microglial activation of TFEB a possible strategy for attenuating amyloid plaque deposition in AD.

  1. Regulators of Lysosome Function and Dynamics in Caenorhabditis elegans

    PubMed Central

    Gee, Kevin; Zamora, Danniel; Horm, Teresa; George, Laeth; Upchurch, Cameron; Randall, Justin; Weaver, Colby; Sanford, Caitlin; Miller, Austin; Hernandez, Sebastian; Dang, Hope; Fares, Hanna

    2017-01-01

    Lysosomes, the major membrane-bound degradative organelles, have a multitude of functions in eukaryotic cells. Lysosomes are the terminal compartments in the endocytic pathway, though they display highly dynamic behaviors, fusing with each other and with late endosomes in the endocytic pathway, and with the plasma membrane during regulated exocytosis and for wound repair. After fusing with late endosomes, lysosomes are reformed from the resulting hybrid organelles through a process that involves budding of a nascent lysosome, extension of the nascent lysosome from the hybrid organelle, while remaining connected by a membrane bridge, and scission of the membrane bridge to release the newly formed lysosome. The newly formed lysosomes undergo cycles of homotypic fusion and fission reactions to form mature lysosomes. In this study, we used a forward genetic screen in Caenorhabditis elegans to identify six regulators of lysosome biology. We show that these proteins function in different steps of lysosome biology, regulating lysosome formation, lysosome fusion, and lysosome degradation. PMID:28122949

  2. A cation counterflux supports lysosomal acidification

    PubMed Central

    Steinberg, Benjamin E.; Huynh, Kassidy K.; Brodovitch, Alexandre; Jabs, Sabrina; Stauber, Tobias; Jentsch, Thomas J.

    2010-01-01

    The profound luminal acidification essential for the degradative function of lysosomes requires a counter-ion flux to dissipate an opposing voltage that would prohibit proton accumulation. It has generally been assumed that a parallel anion influx is the main or only counter-ion transport that enables acidification. Indeed, defective anion conductance has been suggested as the mechanism underlying attenuated lysosome acidification in cells deficient in CFTR or ClC-7. To assess the individual contribution of counter-ions to acidification, we devised means of reversibly and separately permeabilizing the plasma and lysosomal membranes to dialyze the cytosol and lysosome lumen in intact cells, while ratiometrically monitoring lysosomal pH. Replacement of cytosolic Cl− with impermeant anions did not significantly alter proton pumping, while the presence of permeant cations in the lysosomal lumen supported acidification. Accordingly, the lysosomes were found to acidify to the same pH in both CFTR- and ClC-7–deficient cells. We conclude that cations, in addition to chloride, can support lysosomal acidification and defects in lysosomal anion conductance cannot explain the impaired microbicidal capacity of CF phagocytes. PMID:20566682

  3. Prodigiosins uncouple lysosomal vacuolar-type ATPase through promotion of H+/Cl- symport.

    PubMed Central

    Ohkuma, S; Sato, T; Okamoto, M; Matsuya, H; Arai, K; Kataoka, T; Nagai, K; Wasserman, H H

    1998-01-01

    We reported previously [Kataoka, Muroi, Ohkuma, Waritani, Magae, Takatsuki, Kondo, Yamasaki and Nagai (1995) FEBS Lett. 359, 53-59] that prodigiosin 25-C (one of the red pigments of the prodigiosin group produced by micro-organisms like Streptomyces and Serratia) uncoupled vacuolar H+-ATPase, inhibited vacuolar acidification and affected glycoprotein processing. In the present study we show that prodigiosin, metacycloprodigiosin and prodigiosin 25-C, all raise intralysosomal pH through inhibition of lysosomal acidification driven by vacuolar-type (V-)ATPase without inhibiting ATP hydrolysis in a dose-dependent manner with IC50 values of 30-120 pmol/mg of protein. The inhibition against lysosomal acidification was quick and reversible, showing kinetics of simple non-competitive (for ATP) inhibition. However, the prodigiosins neither raised the internal pH of isolated lysosomes nor showed ionophoric activity against H+ or K+ at concentrations where they strongly inhibited lysosomal acidification. They required Cl- for their acidification inhibitory activity even when driven in the presence of K+ and valinomycin, suggesting that their target is not anion (chloride) channel(s). In fact, the prodigiosins inhibited acidification of proteoliposomes devoid of anion channels that were reconstituted from lysosomal vacuolar-type (V-)ATPase and Escherichia coli phospholipids. However, they did not inhibit the formation of an inside-positive membrane potential driven by lysosomal V-ATPase. Instead, they caused quick reversal of acidified pH driven by lysosomal V-ATPase and, in acidic buffer, produced quick acidification of lysosomal pH, both only in the presence of Cl-. In addition, they induced swelling of liposomes and erythrocytes in iso-osmotic ammonium salt of chloride but not of gluconate, suggesting the promotion of Cl- entry by prodigiosins. These results suggest that prodigiosins facilitate the symport of H+ with Cl- (or exchange of OH- with Cl-) through lysosomal

  4. Curcumin targets the TFEB-lysosome pathway for induction of autophagy

    PubMed Central

    Xu, Jian; Lu, Yuanqiang; Jiang, Jiukun; Wang, Liming; Shen, Han-Ming; Xia, Dajing

    2016-01-01

    Curcumin is a hydrophobic polyphenol derived from the herb Curcumalonga and its wide spectrum of pharmacological activities has been widely studied. It has been reported that Curcumin can induce autophagy through inhibition of the Akt-mTOR pathway. However, the effect of Curcumin on lysosome remains largely elusive. In this study, we first found that Curcumin treatment enhances autophagic flux in both human colon cancer HCT116 cells and mouse embryonic fibroblasts (MEFs). Moreover, Curcumin treatment promotes lysosomal function, evidenced by the increased lysosomal acidification and enzyme activity. Second, Curcumin is capable of suppressing the mammalian target of rapamycin (mTOR). Interestingly, Curcumin fails to inhibit mTOR and to activate lysosomal function in Tsc2−/−MEFs with constitutive activation of mTOR, indicating that Curcumin-mediated lysosomal activation is achieved via suppression of mTOR. Third, Curcumin treatment activates transcription factor EB (TFEB), a key nuclear transcription factor in control of autophagy and lysosome biogenesis and function, based on the following observations: (i) Curcumin directly binds to TFEB, (ii) Curcumin promotes TFEB nuclear translocation; and (iii) Curcumin increases transcriptional activity of TFEB. Finally, inhibition of autophagy and lysosome leads to more cell death in Curcumin-treated HCT116 cells, suggesting that autophagy and lysosomal activation serves as a cell survival mechanism to protect against Curcumin-mediated cell death. Taken together, data from our study provide a novel insight into the regulatory mechanisms of Curcumin on autophagy and lysosome, which may facilitate the development of Curcumin as a potential cancer therapeutic agent. PMID:27689333

  5. LYSOSOMAL ACTIVITY ASSOCIATED WITH DEVELOPMENTAL AXON PRUNING

    PubMed Central

    Song, Jae W.; Misgeld, Thomas; Kang, Hyuno; Knecht, Sharm; Lu, Ju; Cao, Yi; Cotman, Susan L.; Bishop, Derron L.; Lichtman, Jeff W.

    2009-01-01

    Clearance of cellular debris is a critical feature of the developing nervous system, as evidenced by the severe neurological consequences of lysosomal storage diseases in children. An important developmental process, that generates considerable cellular debris, is synapse elimination in which many axonal branches are pruned. The fate of these pruned branches is not known. Here, we investigate the role of lysosomal activity in neurons and glia in the removal of axon branches during early postnatal life. Using a probe for lysosomal activity, we observed robust staining associated with retreating motor axons. Lysosomal function was involved in axon removal because retreating axons were cleared more slowly in a mouse model of a lysosomal storage disease. In addition, we found lysosomal activity in the cerebellum at the time of, and at sites where, climbing fibers are eliminated. We propose that lysosomal activity is a central feature of synapse elimination. Moreover, staining for lysosomal activity may serve as a marker for regions of the developing nervous system undergoing axon pruning. PMID:18768693

  6. A Molecular Mechanism to Regulate Lysosome Motility for Lysosome Positioning and Tubulation

    PubMed Central

    Li, Xinran; Rydzewski, Nicholas; Hider, Ahmad; Zhang, Xiaoli; Yang, Junsheng; Wang, Wuyang; Gao, Qiong; Cheng, Xiping; Xu, Haoxing

    2016-01-01

    To mediate the degradation of bio-macromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca2+ channel TRPML1 cause lysosome storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca2+-dependent centripetal movement of lysosomes towards the perinuclear region, where autophagosomes accumulate, upon autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca2+ sensor that associates physically with the minus-end directed dynactin-dynein motor, while PI(3,5)P2, a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PI(3,5)P2-TRPML1-ALG-2-dynein signaling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Collectively, Ca2+ release from lysosomes provides an on-demand mechanism regulating lysosome motility, positioning, and tubulation. PMID:26950892

  7. [Lysosomal system in hormonal mechanisms. Review].

    PubMed

    Duran Reyes, G; González Macías, G; Hicks, J J

    1995-02-01

    The role of lysosomes in the intracellular mechanism of action of several steroid an proteic hormones has been demonstrated. In presence of the specific hormone the target cell induce membranal changes and the lysosomes are moved toward the nucleus; after this the lysosomal enzymes are released in the perinuclear space. For the moment it is not possible to know the biochemical role of this enzymatic activities upon the nucleic acids function and des-repretion process of specific genes, but the inhibition of lysosomes movement utilizing hormone antagonist or dexamethasone inhibits some reproductive process like the implantation of the mammalian egg. We present herein a review related with the mode action of some hormones through the lysosomes in reproductive processes.

  8. Monitoring Autophagy in Lysosomal Storage Disorders

    PubMed Central

    Raben, Nina; Shea, Lauren; Hill, Victoria; Plotz, Paul

    2009-01-01

    Lysosomes are the final destination of the autophagic pathway. It is in the acidic milieu of the lysosomes that autophagic cargo is metabolized and recycled. One would expect that diseases with primary lysosomal defects would be among the first systems in which autophagy would be studied. In reality, this is not the case. Lysosomal storage diseases, a group of more than 60 diverse inherited disorders, have only recently become a focus of autophagic research. Studies of these clinically severe conditions promise not only to clarify pathogenic mechanisms, but also to expand our knowledge of autophagy itself. In this chapter, we will describe the lysosomal storage diseases in which autophagy has been explored, and present the approaches used to evaluate this essential cellular pathway. PMID:19216919

  9. Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury

    PubMed Central

    Maejima, Ikuko; Takahashi, Atsushi; Omori, Hiroko; Kimura, Tomonori; Takabatake, Yoshitsugu; Saitoh, Tatsuya; Yamamoto, Akitsugu; Hamasaki, Maho; Noda, Takeshi; Isaka, Yoshitaka; Yoshimori, Tamotsu

    2013-01-01

    Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy-dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis. PMID:23921551

  10. Podocytes Degrade Endocytosed Albumin Primarily in Lysosomes

    PubMed Central

    Carson, John M.; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B.; Blaine, Judith

    2014-01-01

    Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and

  11. Endosome-lysosomes and neurodegeneration.

    PubMed

    Mayer, R J; Tipler, C; Laszlo, L; Arnold, J; Lowe, J; Landon, M

    1994-01-01

    A number of the major human and animal neurodegenerative diseases, such as Alzheimer's disease and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins. Although our knowledge concerning these diseases is increasing, they remain largely untreatable. Recently, attention has focussed on the mechanisms of production of different types of amyloid and the likely involvement within cells of acid compartments called endosome-lysosomes. These organelles may be 'bioreactor' sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials. These subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities. Common features of the disease processes give new direction to therapeutic intervention.

  12. Recent advances in gene therapy for lysosomal storage disorders.

    PubMed

    Rastall, David Pw; Amalfitano, Andrea

    2015-01-01

    Lysosomal storage disorders (LSDs) are a group of genetic diseases that result in metabolic derangements of the lysosome. Most LSDs are due to the genetic absence of a single catabolic enzyme, causing accumulation of the enzyme's substrate within the lysosome. Over time, tissue-specific substrate accumulations result in a spectrum of symptoms and disabilities that vary by LSD. LSDs are promising targets for gene therapy because delivery of a single gene into a small percentage of the appropriate target cells may be sufficient to impact the clinical course of the disease. Recently, there have been several significant advancements in the potential for gene therapy of these disorders, including the first human trials. Future clinical trials will build upon these initial attempts, with an improved understanding of immune system responses to gene therapy, the obstacle that the blood-brain barrier poses for neuropathic LSDs, as well other biological barriers that, when overcome, may facilitate gene therapy for LSDs. In this manuscript, we will highlight the recent innovations in gene therapy for LSDs and discuss the clinical limitations that remain to be overcome, with the goal of fostering an understanding and further development of this important field.

  13. Screening for lysosomal storage disorders--a clinical perspective.

    PubMed

    Fletcher, Janice M

    2006-01-01

    The availability of therapies for lysosomal storage diseases (LSDs) and clear documentation from animal studies that optimal therapy depends on early diagnosis have set the scene for newborn screening for LSDs. The combined incidence of this group of conditions is approximately 1 in 7000, well within the feasible range for newborn screening programmes. The availability of multiplex technology has facilitated the technical aspects of initial screening. The scientific challenge is to predict disease severity early enough to influence choice of therapy. LSD screening is discussed from the point of view of the scientists, the families affected by these conditions, the community and clinicians.

  14. Methods for the quantification of lysosomal membrane permeabilization: a hallmark of lysosomal cell death.

    PubMed

    Aits, Sonja; Jäättelä, Marja; Nylandsted, Jesper

    2015-01-01

    Lysosomal cell death is triggered by lysosomal membrane permeabilization (LMP) and subsequent release of lysosomal hydrolases from the lysosomal lumen into the cytosol. Once released into the cytosol, the lysosomal cathepsin proteases act as executioner proteases for the subsequent cell death-either autonomously without caspase activation or in concert with the classical apoptotic machinery. Lysosomal cell death usually remains functional in apoptosis-resistant cancer cells and thus holds great potential as a therapeutic strategy for circumventing apoptosis deficiency in cancers. Notably, lysosomal cell death also plays an important role in normal physiology, e.g., during the regression of the mammary gland. Here we present four complementary methods for the quantification and visualization of LMP during the onset of death: (1) enzymatic activity measurements of released lysosomal hydrolases in the cytosol after digitonin extraction, (2) direct visualization of LMP by monitoring the release of fluorescent dextran from lysosomes into the cytosol, (3) immunocytochemistry to detect cathepsins released into the cytosol, and (4) detection of the translocation of galectins to damaged lysosomes. The methods presented here can ideally be combined as needed to provide solid evidence for LMP after a given cytotoxic stimuli.

  15. Nanoparticles restore lysosomal acidification defects: Implications for Parkinson and other lysosomal-related diseases

    PubMed Central

    Bourdenx, Mathieu; Daniel, Jonathan; Genin, Emilie; Soria, Federico N.; Blanchard-Desce, Mireille; Bezard, Erwan; Dehay, Benjamin

    2016-01-01

    ABSTRACT Lysosomal impairment causes lysosomal storage disorders (LSD) and is involved in pathogenesis of neurodegenerative diseases, notably Parkinson disease (PD). Strategies enhancing or restoring lysosomal-mediated degradation thus appear as tantalizing disease-modifying therapeutics. Here we demonstrate that poly(DL-lactide-co-glycolide) (PLGA) acidic nanoparticles (aNP) restore impaired lysosomal function in a series of toxin and genetic cellular models of PD, i.e. ATP13A2-mutant or depleted cells or glucocerebrosidase (GBA)-mutant cells, as well as in a genetic model of lysosomal-related myopathy. We show that PLGA-aNP are transported to the lysosome within 24 h, lower lysosomal pH and rescue chloroquine (CQ)-induced toxicity. Re-acidification of defective lysosomes following PLGA-aNP treatment restores lysosomal function in different pathological contexts. Finally, our results show that PLGA-aNP may be detected after intracerebral injection in neurons and attenuate PD-related neurodegeneration in vivo by mechanisms involving a rescue of compromised lysosomes. PMID:26761717

  16. Ubiquitination and dynactin regulate TMEPAI lysosomal trafficking

    PubMed Central

    Luo, Shenheng; Jing, Lei; Zhao, Tian; Li, Yuyin; Liu, Zhenxing; Diao, Aipo

    2017-01-01

    The transmembrane prostate androgen-induced protein (TMEPAI) has been reported to be elevated in various tumor cells, is localized to the lysosome and promotes lysosome stability. The molecular mechanism of TMEPAI trafficking however to the lysosome is unknown. Here we report that clathrin and CI-M6PR mediate TMEPAI transport from the Golgi directly into the endo-lysosomal pathway. TMEPAI is ubiquitinated at its C-terminal region and ubiquitination modification of TMEPAI is a signal for its lysosomal trafficking. Moreover, TMEPAI binds the ubiquitin binding proteins Hrs and STAM which is required for its lysosomal transport. In addition, TMEPAI interacts with the dynactin pointed-end complex subunits dynactin 5 and dynactin 6. The aa 132–155 domain is essential for specific TMEPAI binding and deletion of this binding site leads to mis-trafficking of TMEPAI to the plasma membrane. These results reveal the pathway and mechanism regulating transport of TMEPAI to the lysosome, which helps to further understand the role of TMEPAI in tumorigenesis. PMID:28218281

  17. Lysosomal exoglycosidases in nasal polyps.

    PubMed

    Chojnowska, Sylwia; Minarowska, Alina; Knaś, Małgorzata; Niemcunowicz-Janica, Anna; Kołodziejczyk, Paweł; Zalewska-Szajda, Beata; Kępka, Alina; Minarowski, Łukasz; Waszkiewicz, Napoleon; Zwierz, Krzysztof; Szajda, Sławomir Dariusz

    2013-01-01

    Nasal polyps are smooth outgrowths assuming a shape of grapes, formed from the nasal mucosa, limiting air flow by projecting into a lumen of a nasal cavity. Up to now the surgical resection is the best method of their treatment, but etiology and pathogenesis of the nasal polyps is not yet fully established. The aim of the study was the assessment of the selected lysosomal exoglycosidases activity in the nasal polyps. In this study the activity of β-galactosidase, α-mannosidase and α-fucosidase was determined in the tissue of the nasal polyps obtained from 40 patients (10F, 30M) and control tissues derived from mucosa of lower nasal conchas obtained during mucotomy from 20 patients (10F, 10M). We observed significant lower values of GAL, FUC and tendency to decrease of MAN and GLU concentration in nasal polyps (P) in comparison to control healthy nasal mucosa (C). In nasal polyp tissue (P) no differences of GAL, MAN and FUC specific activity in comparison to control mucosa (C) were found. Our research supports bioelectrical theory of the nasal polyps pathogenesis and directs attention at research on glycoconjugates and glycosidases of the nasal mucosa extracellular matrix. Copyright © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z.o.o. All rights reserved.

  18. Presenilin 1 maintains lysosomal Ca2+ homeostasis by regulating vATPase-mediated lysosome acidification

    PubMed Central

    Lee, Ju-Hyun; McBrayer, Mary Kate; Wolfe, Devin M.; Haslett, Luke J.; Kumar, Asok; Sato, Yutaka; Lie, Pearl P. Y.; Mohan, Panaiyur; Coffey, Erin E.; Kompella, Uday; Mitchell, Claire H.; Lloyd-Evans, Emyr; Nixon, Ralph A.

    2015-01-01

    Summary Presenilin-1 (PS1) deletion or Alzheimer’s Disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in PS1KO cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss of function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism. PMID:26299959

  19. Lysosomal Storage Disorders in the Newborn

    PubMed Central

    Staretz-Chacham, Orna; Lang, Tess C.; LaMarca, Mary E.; Krasnewich, Donna; Sidransky, Ellen

    2009-01-01

    Lysosomal storage disorders are rare inborn errors of metabolism, with a combined incidence of 1 in 1500 to 7000 live births. These relatively rare disorders are seldom considered when evaluating a sick newborn. A significant number of the >50 different lysosomal storage disorders, however, do manifest in the neonatal period and should be part of the differential diagnosis of several perinatal phenotypes. We review the earliest clinical features, diagnostic tests, and treatment options for lysosomal storage disorders that can present in the newborn. Although many of the lysosomal storage disorders are characterized by a range in phenotypes, the focus of this review is on the specific symptoms and clinical findings that present in the perinatal period, including neurologic, respiratory, endocrine, and cardiovascular manifestations, dysmorphic features, hepatosplenomegaly, skin or ocular involvement, and hydrops fetalis/congenital ascites. A greater awareness of these features may help to reduce misdiagnosis and promote the early detection of lysosomal storage disorders. Implementing therapy at the earliest stage possible is crucial for several of the lysosomal storage disorders; hence, an early appreciation of these disorders by physicians who treat newborns is essential. PMID:19336380

  20. [Application of lysosomal detection in marine pollution monitoring: research progress].

    PubMed

    Weng, You-Zhu; Fang, Yong-Qiang; Zhang, Yu-Sheng

    2013-11-01

    Lysosome is an important organelle existing in eukaryotic cells. With the development of the study on the structure and function of lysosome in recent years, lysosome is considered as a target of toxic substances on subcellular level, and has been widely applied abroad in marine pollution monitoring. This paper summarized the biological characteristics of lysosomal marker enzyme, lysosome-autophagy system, and lysosomal membrane, and introduced the principles and methods of applying lysosomal detection in marine pollution monitoring. Bivalve shellfish digestive gland and fish liver are the most sensitive organs for lysosomal detection. By adopting the lysosomal detection techniques such as lysosomal membrane stability (LMS) test, neutral red retention time (NRRT) assay, morphological measurement (MM) of lysosome, immunohistochemical (Ih) assay of lysosomal marker enzyme, and electron microscopy (EM), the status of marine pollution can be evaluated. It was suggested that the lysosome could be used as a biomarker for monitoring marine environmental pollution. The advantages and disadvantages of lysosomal detection and some problems worthy of attention were analyzed, and the application prospects of lysosomal detection were discussed.

  1. AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway

    PubMed Central

    Dores, Michael R.; Paing, May M.; Lin, Huilan; Montagne, William A.; Marchese, Adriano; Trejo, JoAnn

    2012-01-01

    The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting. PMID:22833563

  2. Role of lysosomal and cytosolic pH in the regulation of macrophage lysosomal enzyme secretion.

    PubMed Central

    Tapper, H; Sundler, R

    1990-01-01

    Rapid and parallel secretion of lysosomal beta-N-acetylglucosaminidase and preloaded fluorescein-labelled dextran was initiated in macrophages by agents affecting intracellular pH (methylamine, chlorpromazine, and the ionophores monensin and nigericin). In order to evaluate the relative role of changes in lysosomal and cytosolic pH, these parameters were monitored by using pH-sensitive fluorescent probes [fluorescein-labelled dextran or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein]. All agents except chlorpromazine caused large increases in lysosomal pH under conditions where they induced secretion. By varying extracellular pH and ion composition, the changes in lysosomal and cytosolic pH could be dissociated. Secretion was then found to be significantly modulated by changes in cytosolic pH, being enhanced by alkalinization and severely inhibited by cytosolic acidification. However, changes in cytosolic pH in the absence of stimulus were unable to initiate secretion. Dissociation of the effects on lysosomal and cytosolic pH was also achieved by combining stimuli with either nigericin or acetate. Further support for a role of intracellular pH in the control of lysosomal enzyme secretion was provided by experiments where bicarbonate was included in the medium. The present study demonstrates that an increase in lysosomal pH is sufficient to initiate lysosomal enzyme secretion in macrophages and provides evidence for a significant regulatory role of cytosolic pH. PMID:2268269

  3. Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay

    PubMed Central

    Aits, Sonja; Kricker, Jennifer; Liu, Bin; Ellegaard, Anne-Marie; Hämälistö, Saara; Tvingsholm, Siri; Corcelle-Termeau, Elisabeth; Høgh, Søren; Farkas, Thomas; Holm Jonassen, Anna; Gromova, Irina; Mortensen, Monika; Jäättelä, Marja

    2015-01-01

    Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP. LGALS1/galectin-1 and LGALS3/galectin-3 are best suited for this purpose due to their widespread expression, rapid translocation to leaky lysosomes and availability of high-affinity antibodies. Galectin staining marks individual leaky lysosomes early during lysosomal cell death and is useful when defining whether LMP is a primary or secondary cause of cell death. This sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. Importantly, galectin staining detects individual leaky lysosomes also in paraffin-embedded tissues allowing us to demonstrate LMP in tumor xenografts in mice treated with cationic amphiphilic drugs and to identify a subpopulation of lysosomes that initiates LMP in involuting mouse mammary gland. The use of ectopic fluorescent galectins renders the galectin puncta assay suitable for automated screening and visualization of LMP in live cells and animals. Thus, the lysosomal galectin puncta assay opens up new possibilities to study LMP in cell death and its role in other cellular processes such as autophagy, senescence, aging, and inflammation. PMID:26114578

  4. Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

    PubMed Central

    Fraldi, Alessandro; Annunziata, Fabio; Lombardi, Alessia; Kaiser, Hermann-Josef; Medina, Diego Luis; Spampanato, Carmine; Fedele, Anthony Olind; Polishchuk, Roman; Sorrentino, Nicolina Cristina; Simons, Kai; Ballabio, Andrea

    2010-01-01

    The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol-enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders. PMID:20871593

  5. Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay.

    PubMed

    Aits, Sonja; Kricker, Jennifer; Liu, Bin; Ellegaard, Anne-Marie; Hämälistö, Saara; Tvingsholm, Siri; Corcelle-Termeau, Elisabeth; Høgh, Søren; Farkas, Thomas; Holm Jonassen, Anna; Gromova, Irina; Mortensen, Monika; Jäättelä, Marja

    2015-01-01

    Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP. LGALS1/galectin-1 and LGALS3/galectin-3 are best suited for this purpose due to their widespread expression, rapid translocation to leaky lysosomes and availability of high-affinity antibodies. Galectin staining marks individual leaky lysosomes early during lysosomal cell death and is useful when defining whether LMP is a primary or secondary cause of cell death. This sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. Importantly, galectin staining detects individual leaky lysosomes also in paraffin-embedded tissues allowing us to demonstrate LMP in tumor xenografts in mice treated with cationic amphiphilic drugs and to identify a subpopulation of lysosomes that initiates LMP in involuting mouse mammary gland. The use of ectopic fluorescent galectins renders the galectin puncta assay suitable for automated screening and visualization of LMP in live cells and animals. Thus, the lysosomal galectin puncta assay opens up new possibilities to study LMP in cell death and its role in other cellular processes such as autophagy, senescence, aging, and inflammation.

  6. Differential regulation of amyloid-β endocytic trafficking and lysosomal degradation by apolipoprotein E isoforms.

    PubMed

    Li, Jie; Kanekiyo, Takahisa; Shinohara, Mitsuru; Zhang, Yunwu; LaDu, Mary Jo; Xu, Huaxi; Bu, Guojun

    2012-12-28

    Aggregation of amyloid-β (Aβ) peptides leads to synaptic disruption and neurodegeneration in Alzheimer disease (AD). A major Aβ clearance pathway in the brain is cellular uptake and degradation. However, how Aβ traffics through the endocytic pathway and how AD risk factors regulate this event is unclear. Here we show that the majority of endocytosed Aβ in neurons traffics through early and late endosomes to the lysosomes for degradation. Overexpression of Rab5 or Rab7, small GTPases that function in vesicle fusion for early and late endosomes, respectively, significantly accelerates Aβ endocytic trafficking to the lysosomes. We also found that a portion of endocytosed Aβ traffics through Rab11-positive recycling vesicles. A blockage of this Aβ recycling pathway with a constitutively active Rab11 mutant significantly accelerates cellular Aβ accumulation. Inhibition of lysosomal enzymes results in Aβ accumulation and aggregation. Importantly, apolipoprotein E (apoE) accelerates neuronal Aβ uptake, lysosomal trafficking, and degradation in an isoform-dependent manner with apoE3 more efficiently facilitating Aβ trafficking and degradation than apoE4, a risk factor for AD. Taken together, our results demonstrate that Aβ endocytic trafficking to lysosomes for degradation is a major Aβ clearance pathway that is differentially regulated by apoE isoforms. A disturbance of this pathway can lead to accumulation and aggregation of cellular Aβ capable of causing neurotoxicity and seeding amyloid.

  7. Effect of temperature on lysosomal enzyme activity during preparation and storage of dried blood spots.

    PubMed

    Supriya, Manjunath; De, Tanima; Christopher, Rita

    2017-03-27

    The use of dried blood spots (DBS) for the assay of lysosomal enzymes has facilitated the implementation of pilot studies for newborn screening for lysosomal storage disorders in various developed countries. The aim of the study was to determine the influence of ambient temperature during DBS preparation and storage on lysosomal enzyme activity in a developing, tropical country. Blood samples from 12 healthy subjects collected on a S&S 903 filter paper were dried and stored at different temperatures for different periods of time. Activities of five lysosomal enzymes (acid α-glucosidase, acid α-galactosidase, acid β-glucocerebrosidase, acid sphingomyelinase, and galactocerebrosidase) were determined by tandem mass spectrometric and fluorimetric (acid α-glucosidase and acid β-glucocerebrosidase only) assays. The mean activities of all five enzymes decreased significantly when DBS was dried at temperatures above 24°C (P<.0001). DBS stored at 4°C, 24°C, 30°C, 37°C, and 45°C for 10 days and more, also showed significant reduction in activities of all five enzymes (P<.0001). The results highlight the importance of maintaining the correct ambient temperature during DBS preparation and storage to avoid false positive results when screening for lysosomal storage disorders. © 2017 Wiley Periodicals, Inc.

  8. Lysosomal adaptation: How cells respond to lysosomotropic compounds.

    PubMed

    Lu, Shuyan; Sung, Tae; Lin, Nianwei; Abraham, Robert T; Jessen, Bart A

    2017-01-01

    Lysosomes are acidic organelles essential for degradation and cellular homoeostasis and recently lysosomes have been shown as signaling hub to respond to the intra and extracellular changes (e.g. amino acid availability). Compounds including pharmaceutical drugs that are basic and lipophilic will become sequestered inside lysosomes (lysosomotropic). How cells respond to the lysosomal stress associated with lysosomotropism is not well characterized. Our goal is to assess the lysosomal changes and identify the signaling pathways that involve in the lysosomal changes. Eight chemically diverse lysosomotropic drugs from different therapeutic areas were subjected to the evaluation using the human adult retinal pigmented epithelium cell line, ARPE-19. All lysosomotropic drugs tested triggered lysosomal activation demonstrated by increased lysosotracker red (LTR) and lysosensor green staining, increased cathepsin activity, and increased LAMP2 staining. However, tested lysosomotropic drugs also prompted lysosomal dysfunction exemplified by intracellular and extracellular substrate accumulation including phospholipid, SQSTM1/p62, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and opsin. Lysosomal activation observed was likely attributed to lysosomal dysfunction, leading to compensatory responses including nuclear translocation of transcriptional factors TFEB, TFE3 and MITF. The adaptive changes are protective to the cells under lysosomal stress. Mechanistic studies implicate calcium and mTORC1 modulation involvement in the adaptive changes. These results indicate that lysosomotropic compounds could evoke a compensatory lysosomal biogenic response but with the ultimate consequence of lysosomal functional impairment. This work also highlights a pathway of response to lysosomal stress and evidences the role of TFEB, TFE3 and MITF in the stress response.

  9. Gastroprotection and lysosomal membrane stabilization by sulglicotide.

    PubMed

    Porta, R; Niada, R; Pescador, R; Mantovani, M; Prino, G

    1986-07-01

    Well-known agents that induce gastric ulcers cause a decrease in lysosomal stability, with release of lytic enzymes. Some antiulcer and cytoprotective agents have lysosomal membrane stabilizing activity when tested in vitro and ex vivo. Sulglicotide (Gliptide), a polysulfated glycopeptide with antiulcer and cytoprotective activities, was able to stabilize lysosomal membranes in vitro at concentrations between 9 and 36 micrograms/ml. The ratio of potency of sulglicotide to that of carbenoxolone was 12.2. In ex vivo experiments in rats, it was found that sulglicotide stabilized lysosomes after oral treatment. The effect was dose-dependent after intravenous treatment. Carbenoxolone, injected i.v. under the same experimental conditions, was less active (potency ratio 0.65). 16,16-dimethyl-PGE2, administered at a dose of 10 micrograms/kg orally or intravenously, had an activity equivalent to that of sulglicotide at a dose of 12.5 mg/kg i.v. or 200 mg/kg p.o. Sulglicotide (200-400 mg/kg p.o.) was also able to prevent the release of acid phosphatase from stomachs challenged for 10 min or 3 h with absolute ethanol. The same result was obtained with 200 mg/kg p.o. of carbenoxolone. These data show that sulglicotide is a potent lysosomal membrane stabilizer in vitro and ex vivo, and could explain the cytoprotective activity of this compound in different experimental models of ulcer.

  10. Lysosomal Storage Diseases—Regulating Neurodegeneration

    PubMed Central

    Onyenwoke, Rob U.; Brenman, Jay E.

    2015-01-01

    Autophagy is a complex pathway regulated by numerous signaling events that recycles macromolecules and can be perturbed in lysosomal storage diseases (LSDs). The concept of LSDs, which are characterized by aberrant, excessive storage of cellular material in lysosomes, developed following the discovery of an enzyme deficiency as the cause of Pompe disease in 1963. Great strides have since been made in better understanding the biology of LSDs. Defective lysosomal storage typically occurs in many cell types, but the nervous system, including the central nervous system and peripheral nervous system, is particularly vulnerable to LSDs, being affected in two-thirds of LSDs. This review provides a summary of some of the better characterized LSDs and the pathways affected in these disorders. PMID:27081317

  11. Immunomodulatory gene therapy in lysosomal storage disorders.

    PubMed

    Koeberl, Dwight D; Kishnani, Priya S

    2009-12-01

    Significant advances in therapy for lysosomal storage disorders have occurred with an accelerating pace over the past decade. Although enzyme replacement therapy has improved the outcome of lysosomal storage disorders, antibody responses have occurred and sometimes prevented efficacy, especially in cross-reacting immune material negative patients with Pompe disease. Preclinical gene therapy experiments have revealed the relevance of immune responses to long-term efficacy. The choice of regulatory cassette played a critical role in evading humoral and cellular immune responses to gene therapy in knockout mouse models, at least in adult animals. Liver-specific regulatory cassettes prevented antibody formation and enhanced the efficacy of gene therapy. Regulatory T cells prevented transgene directed immune responses, as shown by adoptive transfer of antigen-specific immune tolerance to enzyme therapy. Immunomodulatory gene therapy with a very low vector dose could enhance the efficacy of enzyme therapy in Pompe disease and other lysosomal storage disorders.

  12. Immunomodulatory gene therapy in lysosomal storage disorders

    PubMed Central

    Koeberl, D.D.; Kishnani, P.S.

    2010-01-01

    Significant advances in therapy for lysosomal storage disorders have occurred with an accelerating pace over the past decade. Although enzyme replacement therapy has improved the outcome of lysosomal storage disorders, antibody responses have occurred and sometimes prevented efficacy, especially in cross-reacting immune material negative patients with Pompe disease. Preclinical gene therapy experiments have revealed the relevance of immune responses to long-term efficacy. The choice of regulatory cassette played a critical role in evading humoral and cellular immune responses to gene therapy in knockout mouse models, at least in adult animals. Liver-specific regulatory cassettes prevented antibody formation and enhanced the efficacy of gene therapy. Regulatory T cells prevented transgene directed immune responses, as shown by adoptive transfer of antigen-specific immune tolerance to enzyme therapy. Immunomodulatory gene therapy with a very low vector dose could enhance the efficacy of enzyme therapy in Pompe disease and other lysosomal storage disorders. PMID:19807648

  13. Secretion from Myeloid Cells: Secretory Lysosomes.

    PubMed

    Griffiths, Gillian M

    2016-08-01

    Many cells of the myeloid lineage use an unusual secretory organelle to deliver their effector mechanisms. In these cells, the lysosomal compartment is often modified not only to fulfill the degradative functions of a lysosome but also as a mechanism for secreting additional proteins that are found in the lysosomes of each specialized cell type. These extra proteins vary from one cell type to another according to the specialized function of the cell. For example, mast cells package histamine; cytotoxic T cells express perforin; azurophilic granules in neutrophils express antimicrobial peptides, and platelets von Willebrand factor. Upon release, these very different proteins can trigger inflammation, cell lysis, microbial death, and clotting, respectively, and hence deliver the very different effector mechanisms of these different myeloid cells.

  14. Endo-Lysosomal Dysfunction in Human Proximal Tubular Epithelial Cells Deficient for Lysosomal Cystine Transporter Cystinosin

    PubMed Central

    Van Den Heuvel, Lambertus; Pastore, Anna; Dijkman, Henry; De Matteis, Maria Antonietta; Levtchenko, Elena N.

    2015-01-01

    Nephropathic cystinosis is a lysosomal storage disorder caused by mutations in the CTNS gene encoding cystine transporter cystinosin that results in accumulation of amino acid cystine in the lysosomes throughout the body and especially affects kidneys. Early manifestations of the disease include renal Fanconi syndrome, a generalized proximal tubular dysfunction. Current therapy of cystinosis is based on cystine-lowering drug cysteamine that postpones the disease progression but offers no cure for the Fanconi syndrome. We studied the mechanisms of impaired reabsorption in human proximal tubular epithelial cells (PTEC) deficient for cystinosin and investigated the endo-lysosomal compartments of cystinosin-deficient PTEC by means of light and electron microscopy. We demonstrate that cystinosin-deficient cells had abnormal shape and distribution of the endo-lysosomal compartments and impaired endocytosis, with decreased surface expression of multiligand receptors and delayed lysosomal cargo processing. Treatment with cysteamine improved surface expression and lysosomal cargo processing but did not lead to a complete restoration and had no effect on the abnormal morphology of endo-lysosomal compartments. The obtained results improve our understanding of the mechanism of proximal tubular dysfunction in cystinosis and indicate that impaired protein reabsorption can, at least partially, be explained by abnormal trafficking of endosomal vesicles. PMID:25811383

  15. Principles of lysosomal membrane degradation: Cellular topology and biochemistry of lysosomal lipid degradation.

    PubMed

    Schulze, Heike; Kolter, Thomas; Sandhoff, Konrad

    2009-04-01

    Cellular membranes enter the lysosomal compartment by endocytosis, phagocytosis, or autophagy. Within the lysosomal compartment, membrane components of complex structure are degraded into their building blocks. These are able to leave the lysosome and can then be utilized for the resynthesis of complex molecules or can be further degraded. Constitutive degradation of membranes occurs on the surface of intra-endosomal and intra-lysosomal membrane structures. Many integral membrane proteins are sorted to the inner membranes of endosomes and lysosome after ubiquitinylation. In the lysosome, proteins are degraded by proteolytic enzymes, the cathepsins. Phospholipids originating from lipoproteins or cellular membranes are degraded by phospholipases. Water-soluble glycosidases sequentially cleave off the terminal carbohydrate residues of glycoproteins, glycosaminoglycans, and glycosphingolipids. For glycosphingolipids with short oligosaccharide chains, the additional presence of membrane-active lysosomal lipid-binding proteins is required. The presence of lipid-binding proteins overcomes the phase problem of water soluble enzymes and lipid substrates by transferring the substrate to the degrading enzyme or by solubilizing the internal membranes. The lipid composition of intra-lysosomal vesicles differs from that of the plasma membrane. To allow at least glycosphingolipid degradation by hydrolases and activator proteins, the cholesterol content of these intraorganellar membranes decreases during endocytosis and the concentration of bis(monoacylglycero)phosphate, a stimulator of sphingolipid degradation, increases. A considerable part of our current knowledge about mechanism and biochemistry of lysosomal lipid degradation is derived from a class of human diseases, the sphingolipidoses, which are caused by inherited defects within sphingolipid and glycosphingolipid catabolism.

  16. Lysosomal membrane permeability stimulates protein aggregate formation in neurons of a lysosomal disease.

    PubMed

    Micsenyi, Matthew C; Sikora, Jakub; Stephney, Gloria; Dobrenis, Kostantin; Walkley, Steven U

    2013-06-26

    Protein aggregates are a common pathological feature of neurodegenerative diseases and several lysosomal diseases, but it is currently unclear what aggregates represent for pathogenesis. Here we report the accumulation of intraneuronal aggregates containing the macroautophagy adapter proteins p62 and NBR1 in the neurodegenerative lysosomal disease late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). CLN2 disease is caused by a deficiency in the lysosomal enzyme tripeptidyl peptidase I, which results in aberrant lysosomal storage of catabolites, including the subunit c of mitochondrial ATP synthase (SCMAS). In an effort to define the role of aggregates in CLN2, we evaluated p62 and NBR1 accumulation in the CNS of Cln2(-/-) mice. Although increases in p62 and NBR1 often suggest compromised degradative mechanisms, we found normal ubiquitin-proteasome system function and only modest inefficiency in macroautophagy late in disease. Importantly, we identified that SCMAS colocalizes with p62 in extra-lysosomal aggregates in Cln2(-/-) neurons in vivo. This finding is consistent with SCMAS being released from lysosomes, an event known as lysosomal membrane permeability (LMP). We predicted that LMP and storage release from lysosomes results in the sequestration of this material as cytosolic aggregates by p62 and NBR1. Notably, LMP induction in primary neuronal cultures generates p62-positive aggregates and promotes p62 localization to lysosomal membranes, supporting our in vivo findings. We conclude that LMP is a previously unrecognized pathogenic event in CLN2 disease that stimulates cytosolic aggregate formation. Furthermore, we offer a novel role for p62 in response to LMP that may be relevant for other diseases exhibiting p62 accumulation.

  17. Drug induced phospholipidosis: an acquired lysosomal storage disorder.

    PubMed

    Shayman, James A; Abe, Akira

    2013-03-01

    There is a strong association between lysosome enzyme deficiencies and monogenic disorders resulting in lysosomal storage disease. Of the more than 75 characterized lysosomal proteins, two thirds are directly linked to inherited diseases of metabolism. Only one lysosomal storage disease, Niemann-Pick disease, is associated with impaired phospholipid metabolism. However, other phospholipases are found in the lysosome but remain poorly characterized. A recent exception is lysosomal phospholipase A2 (group XV phospholipase A2). Although no inherited disorder of lysosomal phospholipid metabolism has yet been associated with a loss of function of this lipase, this enzyme may be a target for an acquired form of lysosomal storage, drug induced phospholipidosis. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

  18. Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion.

    PubMed

    Zhou, Jing; Tan, Shi-Hao; Nicolas, Valérie; Bauvy, Chantal; Yang, Nai-Di; Zhang, Jianbin; Xue, Yuan; Codogno, Patrice; Shen, Han-Ming

    2013-04-01

    Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.

  19. Lysosomal protease expression in mature enamel.

    PubMed

    Tye, Coralee E; Lorenz, Rachel L; Bartlett, John D

    2009-01-01

    The enamel matrix proteins (amelogenin, enamelin and ameloblastin) are degraded by matrix metalloproteinase-20 and kallikrein-4 during enamel development and mature enamel is virtually protein free. The precise mechanism of removal and degradation of the enamel protein cleavage products from the matrix, however, remains poorly understood. It has been proposed that receptor-mediated endocytosis allows for the cleaved proteins to be removed from the matrix during enamel formation and then transported to the lysosome for further degradation. This study aims to identify lysosomal proteases that are present in maturation-stage enamel organ. RNA from first molars of 11-day-old mice was collected and expression was initially assessed by RT-PCR and then quantified by qPCR. The pattern of expression of selected proteases was assessed by immunohistochemical staining of demineralized mouse incisors. With the exception of cathepsin G, all lysosomal proteases assessed were expressed in maturation-stage enamel organ. Identified proteases included cathepsins B, D, F, H, K, L, O, S and Z. Tripeptidyl peptidases I and II as well as dipeptidyl peptidases I, II, III and IV were also found to be expressed. Immunohistochemical staining confirmed that the maturation-stage ameloblasts express cathepsins L and S and tripeptidyl peptidase II. Our results suggest that the ameloblasts are enriched by a large number of lysosomal proteases at maturation that are likely involved in the degradation of the organic matrix.

  20. Lysosomal proteolysis: effects of aging and insulin.

    PubMed

    Gromakova, I A; Konovalenko, O A

    2003-07-01

    Age-related characteristics of the effect of insulin on the activity of lysosomal proteolytic enzymes were studied. The relationship between the insulin effect on protein degradation and insulin degradation was analyzed. The effect of insulin on the activities of lysosomal enzymes was opposite in young and old rats (inhibitory in 3-month-old and stimulatory in 24-month-old animals). The activities of proteolytic enzymes were regulated by insulin in a glucose-independent manner: similar hypoglycemic effects of insulin in animals of different ages were accompanied by opposite changes in the activities of lysosomal enzymes. The inhibition of lysosomal enzymes by insulin in 3-month-old rats is consistent with a notion on the inhibitory effect of insulin on protein degradation. An opposite insulin effect in 24-month-old rats (i.e., stimulation of proteolytic activity by insulin) may be partly associated with attenuation of the degradation of insulin, resulting in disturbances in signaling that mediates the regulatory effects of insulin on protein degradation.

  1. Using the Technology Acceptance Model to explore community dwelling older adults' perceptions of a 3D interior design application to facilitate pre-discharge home adaptations.

    PubMed

    Money, Arthur G; Atwal, Anita; Young, Katherine L; Day, Yasmin; Wilson, Lesley; Money, Kevin G

    2015-08-26

    In the UK occupational therapy pre-discharge home visits are routinely carried out as a means of facilitating safe transfer from the hospital to home. Whilst they are an integral part of practice, there is little evidence to demonstrate they have a positive outcome on the discharge process. Current issues for patients are around the speed of home visits and the lack of shared decision making in the process, resulting in less than 50 % of the specialist equipment installed actually being used by patients on follow-up. To improve practice there is an urgent need to examine other ways of conducting home visits to facilitate safe discharge. We believe that Computerised 3D Interior Design Applications (CIDAs) could be a means to support more efficient, effective and collaborative practice. A previous study explored practitioners perceptions of using CIDAs; however it is important to ascertain older adult's views about the usability of technology and to compare findings. This study explores the perceptions of community dwelling older adults with regards to adopting and using CIDAs as an assistive tool for the home adaptations process. Ten community dwelling older adults participated in individual interactive task-focused usability sessions with a customised CIDA, utilising the think-aloud protocol and individual semi-structured interviews. Template analysis was used to carry out both deductive and inductive analysis of the think-aloud and interview data. Initially, a deductive stance was adopted, using the three pre-determined high-level themes of the technology acceptance model (TAM): Perceived Usefulness (PU), Perceived Ease of Use (PEOU), Actual Use (AU). Inductive template analysis was then carried out on the data within these themes, from which a number of sub-thmes emerged. Regarding PU, participants believed CIDAs served as a useful visual tool and saw clear potential to facilitate shared understanding and partnership in care delivery. For PEOU, participants were

  2. [Fabry disease and cystinosis, two lysosomal diseases: similarities and differences].

    PubMed

    Grünfeld, J-P; Servais, A

    2010-12-01

    Fabry disease and cystinosis are both lysosomal diseases. Some clinical features (such as renal and corneal involvement) are shared by both diseases whereas many other features are different (mode of inheritance, rate of progression, mechanism of lysosomal storage, therapeutic modalities etc.). Intermediary mechanisms that lead from lysosomal overload to lesions and disease are still incompletely understood.

  3. Reactivation of Lysosomal Ca2+ Efflux Rescues Abnormal Lysosomal Storage in FIG4-Deficient Cells.

    PubMed

    Zou, Jianlong; Hu, Bo; Arpag, Sezgi; Yan, Qing; Hamilton, Audra; Zeng, Yuan-Shan; Vanoye, Carlos G; Li, Jun

    2015-04-29

    Loss of function of FIG4 leads to Charcot-Marie-Tooth disease Type 4J, Yunis-Varon syndrome, or an epilepsy syndrome. FIG4 is a phosphatase with its catalytic specificity toward 5'-phosphate of phosphatidylinositol-3,5-diphosphate (PI3,5P2). However, the loss of FIG4 decreases PI3,5P2 levels likely due to FIG4's dominant effect in scaffolding a PI3,5P2 synthetic protein complex. At the cellular level, all these diseases share similar pathology with abnormal lysosomal storage and neuronal degeneration. Mice with no FIG4 expression (Fig4(-/-)) recapitulate the pathology in humans with FIG4 deficiency. Using a flow cytometry technique that rapidly quantifies lysosome sizes, we detected an impaired lysosomal fission, but normal fusion, in Fig4(-/-) cells. The fission defect was associated with a robust increase of intralysosomal Ca(2+) in Fig4(-/-) cells, including FIG4-deficient neurons. This finding was consistent with a suppressed Ca(2+) efflux of lysosomes because the endogenous ligand of lysosomal Ca(2+) channel TRPML1 is PI3,5P2 that is deficient in Fig4(-/-) cells. We reactivated the TRPML1 channels by application of TRPML1 synthetic ligand, ML-SA1. This treatment reduced the intralysosomal Ca(2+) level and rescued abnormal lysosomal storage in Fig4(-/-) culture cells and ex vivo DRGs. Furthermore, we found that the suppressed Ca(2+) efflux in Fig4(-/-) culture cells and Fig4(-/-) mouse brains profoundly downregulated the expression/activity of dynamin-1, a GTPase known to scissor organelle membranes during fission. This downregulation made dynamin-1 unavailable for lysosomal fission. Together, our study revealed a novel mechanism explaining abnormal lysosomal storage in FIG4 deficiency. Synthetic ligands of the TRPML1 may become a potential therapy against diseases with FIG4 deficiency.

  4. General lysosomal hydrolysis can process prorenin accurately.

    PubMed

    Xa, Lucie K; Lacombe, Marie-Josée; Mercure, Chantal; Lazure, Claude; Reudelhuber, Timothy L

    2014-09-01

    Renin, an aspartyl protease that catalyzes the rate-limiting step of the renin-angiotensin system, is first synthesized as an inactive precursor, prorenin. Prorenin is activated by the proteolytic removal of an amino terminal prosegment in the dense granules of the juxtaglomerular (JG) cells of the kidney by one or more proteases whose identity is uncertain but commonly referred to as the prorenin-processing enzyme (PPE). Because several extrarenal tissues secrete only prorenin, we tested the hypothesis that the unique ability of JG cells to produce active renin might be explained by the existence of a PPE whose expression is restricted to JG cells. We found that inducing renin production by the mouse kidney by up to 20-fold was not associated with the concomitant induction of candidate PPEs. Because the renin-containing granules of JG cells also contain several lysosomal hydrolases, we engineered mouse Ren1 prorenin to be targeted to the classical vesicular lysosomes of cultured HEK-293 cells, where it was accurately processed and stored. Furthermore, we found that HEK cell lysosomes hydrolyzed any artificial extensions placed on the protein and that active renin was extraordinarily resistant to proteolytic degradation. Altogether, our results demonstrate that accurate processing of prorenin is not restricted to JG cells but can occur in classical vesicular lysosomes of heterologous cells. The implication is that renin production may not require a specific PPE but rather can be achieved by general hydrolysis in the lysosome-like granules of JG cells. Copyright © 2014 the American Physiological Society.

  5. Neuroinflammatory paradigms in lysosomal storage diseases

    PubMed Central

    Bosch, Megan E.; Kielian, Tammy

    2015-01-01

    Lysosomal storage diseases (LSDs) include approximately 70 distinct disorders that collectively account for 14% of all inherited metabolic diseases. LSDs are caused by mutations in various enzymes/proteins that disrupt lysosomal function, which impairs macromolecule degradation following endosome-lysosome and phagosome-lysosome fusion and autophagy, ultimately disrupting cellular homeostasis. LSDs are pathologically typified by lysosomal inclusions composed of a heterogeneous mixture of various proteins and lipids that can be found throughout the body. However, in many cases the CNS is dramatically affected, which may result from heightened neuronal vulnerability based on their post-mitotic state. Besides intrinsic neuronal defects, another emerging factor common to many LSDs is neuroinflammation, which may negatively impact neuronal survival and contribute to neurodegeneration. Microglial and astrocyte activation is a hallmark of many LSDs that affect the CNS, which often precedes and predicts regions where eventual neuron loss will occur. However, the timing, intensity, and duration of neuroinflammation may ultimately dictate the impact on CNS homeostasis. For example, a transient inflammatory response following CNS insult/injury can be neuroprotective, as glial cells attempt to remove the insult and provide trophic support to neurons. However, chronic inflammation, as seen in several LSDs, can promote neurodegeneration by creating a neurotoxic environment due to elevated levels of cytokines, chemokines, and pro-apoptotic molecules. Although neuroinflammation has been reported in several LSDs, the cellular basis and mechanisms responsible for eliciting neuroinflammatory pathways are just beginning to be defined. This review highlights the role of neuroinflammation in select LSDs and its potential contribution to neuron loss. PMID:26578874

  6. Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

    PubMed Central

    Aizawa, Shu; Fujiwara, Yuuki; Contu, Viorica Raluca; Hase, Katsunori; Takahashi, Masayuki; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

    2016-01-01

    ABSTRACT Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy. PMID:27046251

  7. A study of blood and urine alcohol concentrations in cases of alleged drug-facilitated sexual assault in the United Kingdom over a 3-year period.

    PubMed

    Scott-Ham, Michael; Burton, Fiona C

    2006-04-01

    This paper details the alcohol concentrations found in a selection of 1,014 cases of claimed drug-facilitated sexual assault analysed at The Forensic Science Service, London Laboratory between January 2000 and December 2002. Where appropriate, either a whole blood sample and/or a urine sample was analysed for alcohol, common drugs of abuse and potentially stupefying drugs. The samples were collected from a complainant within 12 h of an alleged incident in 391 of the 1014 cases analysed. Of these, the majority (81%) contained alcohol. The presence of alcohol itself was not surprising as most of the alleged incidents were associated with social situations such as at a public house, bar, night-club or party, where it is expected that alcohol would have been consumed. However, 233 (60%) of the 391 cases had a high back-calculated figure, where high is defined as greater than 150 milligrams per 100 millilitres (150 mg%). Some of these samples were also found to contain illicit drugs. This is the first paper to our knowledge which discusses in detail the significance of the alcohol concentrations found in cases of this type.

  8. Mechanisms of Dendritic Cell Lysosomal Killing of Cryptococcus

    NASA Astrophysics Data System (ADS)

    Hole, Camaron R.; Bui, Hoang; Wormley, Floyd L.; Wozniak, Karen L.

    2012-10-01

    Cryptococcus neoformans is an opportunistic pulmonary fungal pathogen that disseminates to the CNS causing fatal meningitis in immunocompromised patients. Dendritic cells (DCs) phagocytose C. neoformans following inhalation. Following uptake, cryptococci translocate to the DC lysosomal compartment and are killed by oxidative and non-oxidative mechanisms. DC lysosomal extracts kill cryptococci in vitro; however, the means of antifungal activity remain unknown. Our studies determined non-oxidative antifungal activity by DC lysosomal extract. We examined DC lysosomal killing of cryptococcal strains, anti-fungal activity of purified lysosomal enzymes, and mechanisms of killing against C. neoformans. Results confirmed DC lysosome fungicidal activity against all cryptococcal serotypes. Purified lysosomal enzymes, specifically cathepsin B, inhibited cryptococcal growth. Interestingly, cathepsin B combined with its enzymatic inhibitors led to enhanced cryptococcal killing. Electron microscopy revealed structural changes and ruptured cryptococcal cell walls following treatment. Finally, additional studies demonstrated that osmotic lysis was responsible for cryptococcal death.

  9. Denaturation of circular or linear DNA facilitates targeted integrative transformation of Streptomyces coelicolor A3(2): possible relevance to other organisms.

    PubMed Central

    Oh, S H; Chater, K F

    1997-01-01

    Using Streptomyces coelicolor A3(2) protoplasts, the number of transformants obtained by homologous recombination of incoming double-stranded circular DNA with the recipient chromosome was greatly stimulated by simple denaturation of the donor DNA. This procedure was very effective with inserts over a ca. 100-fold size range, the largest tested being ca. 40-kb inserts in cosmids. These observations led to transformation experiments with linearized cloned DNA and randomly sheared genomic DNA. In both cases, DNA denaturation led to significant levels of transformation. Most of the transformants had resulted from the predicted homologous recombination events. A number of genetic manipulations will be made easier or possible by these procedures. PMID:8981988

  10. Lysosomal-associated transmembrane protein 5 (LAPTM5) is a molecular partner of CD1e.

    PubMed

    Angénieux, Catherine; Waharte, François; Gidon, Alexandre; Signorino-Gelo, François; Wurtz, Virginie; Hojeij, Rim; Proamer, Fabienne; Gachet, Christian; Van Dorsselaer, Alain; Hanau, Daniel; Salamero, Jean; de la Salle, Henri

    2012-01-01

    The CD1e protein participates in the presentation of lipid antigens in dendritic cells. Its transmembrane precursor is transported to lysosomes where it is cleaved into an active soluble form. In the presence of bafilomycin, which inhibits vacuolar ATPase and consequently the acidification of endosomal compartments, CD1e associates with a 27 kD protein. In this work, we identified this molecular partner as LAPTM5. The latter protein and CD1e colocalize in trans-Golgi and late endosomal compartments. The quantity of LAPTM5/CD1e complexes increases when the cells are treated with bafilomycin, probably due to the protection of LAPTM5 from lysosomal proteases. Moreover, we could demonstrate that LAPTM5/CD1e association occurs under physiological conditions. Although LAPTM5 was previously shown to act as a platform recruiting ubiquitin ligases and facilitating the transport of receptors to lysosomes, we found no evidence that LATPM5 controls either CD1e ubiquitination or the generation of soluble lysosomal CD1e proteins. Notwithstanding these last observations, the interaction of LAPTM5 with CD1e and their colocalization in antigen processing compartments both suggest that LAPTM5 might influence the role of CD1e in the presentation of lipid antigens.

  11. Lysosomal adaptation: How cells respond to lysosomotropic compounds

    PubMed Central

    Lu, Shuyan; Sung, Tae; Lin, Nianwei; Abraham, Robert T.; Jessen, Bart A.

    2017-01-01

    Lysosomes are acidic organelles essential for degradation and cellular homoeostasis and recently lysosomes have been shown as signaling hub to respond to the intra and extracellular changes (e.g. amino acid availability). Compounds including pharmaceutical drugs that are basic and lipophilic will become sequestered inside lysosomes (lysosomotropic). How cells respond to the lysosomal stress associated with lysosomotropism is not well characterized. Our goal is to assess the lysosomal changes and identify the signaling pathways that involve in the lysosomal changes. Eight chemically diverse lysosomotropic drugs from different therapeutic areas were subjected to the evaluation using the human adult retinal pigmented epithelium cell line, ARPE-19. All lysosomotropic drugs tested triggered lysosomal activation demonstrated by increased lysosotracker red (LTR) and lysosensor green staining, increased cathepsin activity, and increased LAMP2 staining. However, tested lysosomotropic drugs also prompted lysosomal dysfunction exemplified by intracellular and extracellular substrate accumulation including phospholipid, SQSTM1/p62, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and opsin. Lysosomal activation observed was likely attributed to lysosomal dysfunction, leading to compensatory responses including nuclear translocation of transcriptional factors TFEB, TFE3 and MITF. The adaptive changes are protective to the cells under lysosomal stress. Mechanistic studies implicate calcium and mTORC1 modulation involvement in the adaptive changes. These results indicate that lysosomotropic compounds could evoke a compensatory lysosomal biogenic response but with the ultimate consequence of lysosomal functional impairment. This work also highlights a pathway of response to lysosomal stress and evidences the role of TFEB, TFE3 and MITF in the stress response. PMID:28301521

  12. Circulating lysosomal enzymes and acute hepatic necrosis.

    PubMed Central

    Gove, C D; Wardle, E N; Williams, R

    1981-01-01

    The activities of the lysosomal enzymes acid and neutral protease, N-acetylglucosaminidase, and acid phosphatase were measured in the serum of patients with fulminant hepatic failure. Acid protease (cathepsin D) activity was increased about tenfold in patients who died and nearly fourfold in those who survived fulminant hepatic failure after paracetamol overdose, whereas activities were increased equally in patients with fulminant hepatic failure due to viral hepatitis whether or not they survived. A correlation was found between serum acid protease activity and prothrombin time, and the increase in cathepsin D activity was sustained over several days compared with aspartate aminotransferase, which showed a sharp early peak and then a fall. Circulating lysosomal proteases can damage other organs, and measurement of their activity may therefore be of added value in assessing prognosis in this condition. PMID:7007443

  13. Activity of lysosomal exoglycosidases in human gliomas.

    PubMed

    Wielgat, P; Walczuk, U; Szajda, S; Bień, M; Zimnoch, L; Mariak, Z; Zwierz, K

    2006-12-01

    There is a lot of data suggesting that modifications of cell glycoconjugates may be important in progression of cancer. In the present work we studied activities of lysosomal exoglycosidases: beta-hexosaminidase and its isoenzymes A and B, beta-galactosidase and alpha-mannosidase, in human gliomas. Enzyme activity was determined spectrophotometrically based on the release of p-nitrophenol from p-nitrophenyl-derivative of appropriate sugars. The activities of the exoglycosidases tested were significantly higher in malignant glial tumors than in control tissue (normal brain tissue) and non-glial tumors. The highest activities of exoglycosidases were observed in high-grade gliomas, and a positive correlation of enzyme activities and degree of malignancy was noted. Our results suggest that lysosomal exoglycosidases may participate in the progression and dynamical development of glial tumors.

  14. Intracellular sphingosine releases calcium from lysosomes

    PubMed Central

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-01-01

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC. DOI: http://dx.doi.org/10.7554/eLife.10616.001 PMID:26613410

  15. Newborn Screening for Lysosomal Storage Diseases

    PubMed Central

    Gelb, Michael H.; Scott, C. Ronald; Turecek, Frantisek

    2015-01-01

    BACKGROUND There is worldwide interest in newborn screening for lysosomal storage diseases because of the development of treatment options that give better results when carried out early in life. Screens with high differentiation between affected and nonaffected individuals are critical because of the large number of potential false positives. CONTENT This review summarizes 3 screening methods: (a) direct assay of enzymatic activities using tandem mass spectrometry or fluorometry, (b) immunocapture-based measurement of lysosomal enzyme abundance, and (c) measurement of biomarkers. Assay performance is compared on the basis of small-scale studies as well as on large-scale pilot studies of mass spectrometric and fluorometric screens. SUMMARY Tandem mass spectrometry and fluorometry techniques for direct assay of lysosomal enzymatic activity in dried blood spots have emerged as the most studied approaches. Comparative mass spectrometry vs fluorometry studies show that the former better differentiates between nonaffected vs affected individuals. This in turn leads to a manageable number of screen positives that can be further evaluated with second-tier methods. PMID:25477536

  16. TRP-ML1 regulates lysosomal pH and acidic lysosomal lipid hydrolytic activity.

    PubMed

    Soyombo, Abigail A; Tjon-Kon-Sang, Sandra; Rbaibi, Youssef; Bashllari, Enkelejda; Bisceglia, Jill; Muallem, Shmuel; Kiselyov, Kirill

    2006-03-17

    Mucolipidosis type IV (MLIV) is caused by mutations in the ion channel mucolipin 1 (TRP-ML1). MLIV is typified by accumulation of lipids and membranous materials in intracellular organelles, which was hypothesized to be caused by the altered membrane fusion and fission events. How mutations in TRP-ML1 lead to aberrant lipolysis is not known. Here we present evidence that MLIV is a metabolic disorder that is not associated with aberrant membrane fusion/fission events. Thus, measurement of lysosomal pH revealed that the lysosomes in TRP-ML1(-/-) cells obtained from the patients with MLIV are over-acidified. TRP-ML1 can function as a H(+) channel, and the increased lysosomal acidification in TRP-ML1(-/-) cells is likely caused by the loss of TRP-ML1-mediated H(+) leak. Measurement of lipase activity using several substrates revealed a marked reduction in lipid hydrolysis in TRP-ML1(-/-) cells, which was rescued by the expression of TRP-ML1. Cell fractionation indicated specific loss of acidic lipase activity in TRP-ML1(-/-) cells. Furthermore, dissipation of the acidic lysosomal pH of TRP-ML1(-/-) cells by nigericin or chloroquine reversed the lysosomal storage disease phenotype. These findings provide a new mechanism to account for the pathogenesis of MLIV.

  17. Lysosomal Re-acidification Prevents Lysosphingolipid-Induced Lysosomal Impairment and Cellular Toxicity

    PubMed Central

    Pröschel, Christoph; Mayer-Pröschel, Margot; Noble, Mark

    2016-01-01

    Neurodegenerative lysosomal storage disorders (LSDs) are severe and untreatable, and mechanisms underlying cellular dysfunction are poorly understood. We found that toxic lipids relevant to three different LSDs disrupt multiple lysosomal and other cellular functions. Unbiased drug discovery revealed several structurally distinct protective compounds, approved for other uses, that prevent lysosomal and cellular toxicities of these lipids. Toxic lipids and protective agents show unexpected convergence on control of lysosomal pH and re-acidification as a critical component of toxicity and protection. In twitcher mice (a model of Krabbe disease [KD]), a central nervous system (CNS)-penetrant protective agent rescued myelin and oligodendrocyte (OL) progenitors, improved motor behavior, and extended lifespan. Our studies reveal shared principles relevant to several LSDs, in which diverse cellular and biochemical disruptions appear to be secondary to disruption of lysosomal pH regulation by specific lipids. These studies also provide novel protective strategies that confer therapeutic benefits in a mouse model of a severe LSD. PMID:27977664

  18. Involvement of lysosomes in the early stages of axon degeneration.

    PubMed

    Zheng, Jin; Yan, Tingting; Feng, Yan; Zhai, Qiwei

    2010-02-01

    Axon degeneration is a common hallmark of many neurodegenerative diseases, and the underlying mechanism remains largely unknown. Lysosomes are involved in some neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Whether lysosomes are involved in axon degeneration is yet to be elucidated. In this study, we found only about 10% lysosomes remained in axons of cultured superior cervical ganglia (SCGs) after transection for 4h when stained with LysoTracker. Furthermore, we found that lysosomal disruption occurred earlier than morphological changes and loss of mitochondrial membrane potential. In addition, the well-known axon-protective protein Wld(S) delayed injury-induced axon degeneration from both morphological changes and lysosomal disruption. Lysosomal inhibitors including chloroquine and ammonium chloride induced axon degeneration in cultured SCGs, and Wld(S) also slowed down the axon degeneration induced by lysosomal inhibitors. All these data suggest that lysosomal disruption is an early marker of axon degeneration, and inhibition of lysosome induces axon degeneration in a Wld(S)-protectable way. Thus, maintenance of normal lysosomal function might be an important approach to delay axon degeneration in neurodegenerative diseases.

  19. A phenotypic compound screening assay for lysosomal storage diseases.

    PubMed

    Xu, Miao; Liu, Ke; Swaroop, Manju; Sun, Wei; Dehdashti, Seameen J; McKew, John C; Zheng, Wei

    2014-01-01

    The lysosome is a vital cellular organelle that primarily functions as a recycling center for breaking down unwanted macromolecules through a series of hydrolases. Functional deficiencies in lysosomal proteins due to genetic mutations have been found in more than 50 lysosomal storage diseases that exhibit characteristic lipid/macromolecule accumulation and enlarged lysosomes. Recently, the lysosome has emerged as a new therapeutic target for drug development for the treatment of lysosomal storage diseases. However, a suitable assay for compound screening against the diseased lysosomes is currently unavailable. We have developed a Lysotracker staining assay that measures the enlarged lysosomes in patient-derived cells using both fluorescence intensity readout and fluorescence microscopic measurement. This phenotypic assay has been tested in patient cells obtained from several lysosomal storage diseases and validated using a known compound, methyl-β-cyclodextrin, in primary fibroblast cells derived from Niemann Pick C disease patients. The results demonstrate that the Lysotracker assay can be used in compound screening for the identification of lead compounds that are capable of reducing enlarged lysosomes for drug development.

  20. Regulation of lysosomal ion homeostasis by channels and transporters.

    PubMed

    Xiong, Jian; Zhu, Michael X

    2016-08-01

    Lysosomes are the major organelles that carry out degradation functions. They integrate and digest materials compartmentalized by endocytosis, phagocytosis or autophagy. In addition to more than 60 hydrolases residing in the lysosomes, there are also ion channels and transporters that mediate the flux or transport of H(+), Ca(2+), Na(+), K(+), and Cl(-) across the lysosomal membranes. Defects in ionic exchange can lead to abnormal lysosome morphology, defective vesicle trafficking, impaired autophagy, and diseases such as neurodegeneration and lysosomal storage disorders. The latter are characterized by incomplete lysosomal digestion and accumulation of toxic materials inside enlarged intracellular vacuoles. In addition to degradation, recent studies have revealed the roles of lysosomes in metabolic pathways through kinases such as mechanistic target of rapamycin (mTOR) and transcriptional regulation through calcium signaling molecules such as transcription factor EB (TFEB) and calcineurin. Owing to the development of new approaches including genetically encoded fluorescence probes and whole endolysosomal patch clamp recording techniques, studies on lysosomal ion channels have made remarkable progress in recent years. In this review, we will focus on the current knowledge of lysosome-resident ion channels and transporters, discuss their roles in maintaining lysosomal function, and evaluate how their dysfunction can result in disease.

  1. The Role of Microscopy in Understanding Atherosclerotic Lysosomal Lipid Metabolism

    NASA Astrophysics Data System (ADS)

    Gray Jerome, W.; Yancey, Patricia G.

    2003-02-01

    Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with modified lipoproteins suggests this abnormal metabolism occurs in two steps. Initially, hydrolysis of lipoprotein cholesteryl esters occurs normally, but the resultant free cholesterol cannot exit the lysosome. Further lysosomal cholesterol accumulation inhibits hydrolysis, producing a mixture of cholesterol and cholesteryl esters within swollen lysosomes. Various lipoprotein modifications can produce this lysosomal engorgement in vitro and it remains to be seen which modifications are most important in vivo.

  2. Regulation of lysosomal ion homeostasis by channels and transporters

    PubMed Central

    Xiong, Jian; Zhu, Michael X.

    2016-01-01

    Lysosomes are the major organelles that carry out degradation functions. They integrate and digest materials compartmentalized by endocytosis, phagocytosis or autophagy. In addition to more than 60 hydrolases residing in the lysosomes, there are also ion channels and transporters that mediate the flux or transport of H+, Ca2+, Na+, K+, and Cl− across the lysosomal membranes. Defects in ionic exchange can lead to abnormal lysosome morphology, defective vesicle trafficking, impaired autophagy, and diseases such as neurodegeneration and lysosomal storage disorders. The latter are characterized by incomplete lysosomal digestion and accumulation of toxic materials inside enlarged intracellular vacuoles. In addition to degradation, recent studies have revealed the roles of lysosomes in metabolic pathways through kinases such as mechanistic target of rapamycin (mTOR) and transcriptional regulation through calcium signaling molecules such as transcription factor EB (TFEB) and calcineurin. Owing to the development of new approaches including genetically encoded fluorescence probes and whole endolysosomal patch clamp recording techniques, studies on lysosomal ion channels have made remarkable progress in recent years. In this review, we will focus on the current knowledge of lysosome-resident ion channels and transporters, discuss their roles in maintaining lysosomal function, and evaluate how their dysfunction can result in disease. PMID:27430889

  3. Biphasic regulation of lysosomal exocytosis by oxidative stress.

    PubMed

    Ravi, Sreeram; Peña, Karina A; Chu, Charleen T; Kiselyov, Kirill

    2016-11-01

    Oxidative stress drives cell death in a number of diseases including ischemic stroke and neurodegenerative diseases. A better understanding of how cells recover from oxidative stress is likely to lead to better treatments for stroke and other diseases. The recent evidence obtained in several models ties the process of lysosomal exocytosis to the clearance of protein aggregates and toxic metals. The mechanisms that regulate lysosomal exocytosis, under normal or pathological conditions, are only beginning to emerge. Here we provide evidence for the biphasic effect of oxidative stress on lysosomal exocytosis. Lysosomal exocytosis was measured using the extracellular levels of the lysosomal enzyme beta-hexosaminidase (ß-hex). Low levels or oxidative stress stimulated lysosomal exocytosis, but inhibited it at high levels. Deletion of the lysosomal ion channel TRPML1 eliminated the stimulatory effect of low levels of oxidative stress. The inhibitory effects of oxidative stress appear to target the component of lysosomal exocytosis that is driven by extracellular Ca(2+). We propose that while moderate oxidative stress promotes cellular repair by stimulating lysosomal exocytosis, at high levels oxidative stress has a dual pathological effect: it directly causes cell damage and impairs damage repair by inhibiting lysosomal exocytosis. Harnessing these adaptive mechanisms may point to pharmacological interventions for diseases involving oxidative proteotoxicity or metal toxicity.

  4. BAX channel activity mediates lysosomal disruption linked to Parkinson disease.

    PubMed

    Bové, Jordi; Martínez-Vicente, Marta; Dehay, Benjamin; Perier, Celine; Recasens, Ariadna; Bombrun, Agnes; Antonsson, Bruno; Vila, Miquel

    2014-05-01

    Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP). However, the molecular mechanisms underlying PD-linked LMP and subsequent lysosomal defects remain virtually unknown, thereby precluding their potential therapeutic targeting. Here we show that the pro-apoptotic protein BAX (BCL2-associated X protein), which permeabilizes mitochondrial membranes in PD models and is activated in PD patients, translocates and internalizes into lysosomal membranes early following treatment with the parkinsonian neurotoxin MPTP, both in vitro and in vivo, within a time-frame correlating with LMP, lysosomal disruption, and autophagosome accumulation and preceding mitochondrial permeabilization and dopaminergic neurodegeneration. Supporting a direct permeabilizing effect of BAX on lysosomal membranes, recombinant BAX is able to induce LMP in purified mouse brain lysosomes and the latter can be prevented by pharmacological blockade of BAX channel activity. Furthermore, pharmacological BAX channel inhibition is able to prevent LMP, restore lysosomal levels, reverse AP accumulation, and attenuate mitochondrial permeabilization and overall nigrostriatal degeneration caused by MPTP, both in vitro and in vivo. Overall, our results reveal that PD-linked lysosomal impairment relies on BAX-induced LMP, and point to small molecules able to block BAX channel activity as potentially beneficial to attenuate both lysosomal defects and neurodegeneration occurring in PD.

  5. The release of lysosomal arylsulfatase from liver lysosomes exposed to 2-chloroethylethyl sulfide.

    PubMed

    Shin, S; Choi, D S; Kim, Y B; Cha, S H; Sok, D E

    1995-08-18

    Treatment of a lysosome-rich fraction from liver with 2-chloroethylethyl sulfide resulted in a dose-dependent release of arylsulfatase. The inclusion of Ca2+ enhanced the enzyme release by approximately 2.3-fold. The enhancing effect of Ca2+, showing an EC50 value of 30 mM, was mimicked by neither Mg2+ nor Mn2+. Studies on a structural requirement and a time-dependent release suggest that the Ca(2+)-dependent release proceeds via a specific process involving the alkylation of lysosomal membranes by 2-chloroethylethyl sulfide. Furthermore, the Ca(2+)-dependent process was prevented partially by either leupeptin or gentamycin, but neither pepstatin nor PMSF, implying that the enzyme release may be partially mediated by lysosomal cysteine-protease or phospholipase. Meanwhile, the Ca(2+)-independent release seems to be expressed non-specifically by various compounds.

  6. Characterization of lysosomes and lysosomal enzymes from Chediak-Higashi-syndrome cultured fibroblasts.

    PubMed Central

    Miller, A L; Stein, R; Sundsmo, M; Yeh, R Y

    1986-01-01

    Chediak-Higashi-syndrome cultured skin fibroblasts were used to study the possible involvement of lysosomal enzymes and lysosomal dysfunction in this disorder. Our evidence indicated that Chediak-Higashi fibroblasts displayed a significant decrease in the specific activity of the acidic alpha-D-mannosidase (pH 4.2) compared with normal controls. Additional studies revealed a small, but significant, decrease in the rate of degradation of 125I-labelled beta-D-glucosidase that had been endocytosed into Chediak-Higashi cells. PMID:3099770

  7. Cysteine cathepsins are essential in lysosomal degradation of α-synuclein.

    PubMed

    McGlinchey, Ryan P; Lee, Jennifer C

    2015-07-28

    A cellular feature of Parkinson's disease is cytosolic accumulation and amyloid formation of α-synuclein (α-syn), implicating a misregulation or impairment of protein degradation pathways involving the proteasome and lysosome. Within lysosomes, cathepsin D (CtsD), an aspartyl protease, is suggested to be the main protease for α-syn clearance; however, the protease alone only generates amyloidogenic C terminal-truncated species (e.g., 1-94, 5-94), implying that other proteases and/or environmental factors are needed to facilitate degradation and to avoid α-syn aggregation in vivo. Using liquid chromatography-mass spectrometry, to our knowledge, we report the first peptide cleavage map of the lysosomal degradation process of α-syn. Studies of purified mouse brain and liver lysosomal extracts and individual human cathepsins demonstrate a direct involvement of cysteine cathepsin B (CtsB) and L (CtsL). Both CtsB and CtsL cleave α-syn within its amyloid region and circumvent fibril formation. For CtsD, only in the presence of anionic phospholipids can this protease cleave throughout the α-syn sequence, suggesting that phospholipids are crucial for its activity. Taken together, an interplay exists between α-syn conformation and cathepsin activity with CtsL as the most efficient under the conditions examined. Notably, we discovered that CtsL efficiently degrades α-syn amyloid fibrils, which by definition are resistant to broad spectrum proteases. This work implicates CtsB and CtsL as essential in α-syn lysosomal degradation, establishing groundwork to explore mechanisms to enhance their cellular activity and levels as a potential strategy for clearance of α-syn.

  8. Glucolipotoxicity diminishes cardiomyocyte TFEB and inhibits lysosomal autophagy during obesity and diabetes.

    PubMed

    Trivedi, Purvi C; Bartlett, Jordan J; Perez, Lester J; Brunt, Keith R; Legare, Jean Francois; Hassan, Ansar; Kienesberger, Petra C; Pulinilkunnil, Thomas

    2016-12-01

    Impaired cardiac metabolism in the obese and diabetic heart leads to glucolipotoxicity and ensuing cardiomyopathy. Glucolipotoxicity causes cardiomyocyte injury by increasing energy insufficiency, impairing proteasomal-mediated protein degradation and inducing apoptosis. Proteasome-evading proteins are degraded by autophagy in the lysosome, whose metabolism and function are regulated by master regulator transcription factor EB (TFEB). Limited studies have examined the impact of glucolipotoxicity on intra-lysosomal signaling proteins and their regulators. By utilizing a mouse model of diet-induced obesity, type-1 diabetes (Akita) and ex-vivo model of glucolipotoxicity (H9C2 cells and NRCM, neonatal rat cardiomyocyte), we examined whether glucolipotoxicity negatively targets TFEB and lysosomal proteins to dysregulate autophagy and cause cardiac injury. Despite differential effects of obesity and diabetes on LC3B-II, expression of proteins facilitating autophagosomal clearance such as TFEB, LAMP-2A, Hsc70 and Hsp90 were decreased in the obese and diabetic heart. In-vivo data was recapitulated in H9C2 and NRCM cells, which exhibited impaired autophagic flux and reduced TFEB content when exposed to a glucolipotoxic milieu. Notably, overloading myocytes with a saturated fatty acid (palmitate) but not an unsaturated fatty acid (oleate) depleted cellular TFEB and suppressed autophagy, suggesting a fatty acid specific regulation of TFEB and autophagy in the cardiomyocyte. The effect of glucolipotoxicity to reduce TFEB content was also confirmed in heart tissue from patients with Class-I obesity. Therefore, during glucolipotoxicity, suppression of lysosomal autophagy was associated with reduced lysosomal content, decreased cathepsin-B activity and diminished cellular TFEB content likely rendering myocytes susceptible to cardiac injury. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Evaluating the roles of autophagy and lysosomal trafficking defects in intracellular distribution-based drug-drug interactions involving lysosomes.

    PubMed

    Logan, Randall; Kong, Alex; Krise, Jeffrey P

    2013-11-01

    Many currently approved drugs possess weakly basic properties that make them substrates for extensive sequestration in acidic intracellular compartments such as lysosomes through an ion trapping-type mechanism. Lysosomotropic drugs often have unique pharmacokinetic properties that stem from the extensive entrapment in lysosomes, including an extremely large volume of distribution and a long half-life. Accordingly, pharmacokinetic drug-drug interactions can occur when one drug modifies lysosomal volume such that the degree of lysosomal sequestration of secondarily administered drugs is significantly altered. In this work, we have investigated potential mechanisms for drug-induced alterations in lysosomal volume that give rise to drug-drug interactions involving lysosomes. We show that eight hydrophobic amines, previously characterized as perpetrators in this type of drug-drug interaction, cause a significant expansion in lysosomal volume that was correlated with both the induction of autophagy and with decreases in the efficiency of lysosomal egress. We also show that well-known chemical inducers of autophagy caused an increase in apparent lysosomal volume and an increase in secondarily administered lysosomotropic drugs without negatively impacting vesicle-mediated lysosomal egress. These results could help rationalize how the induction of autophagy could cause variability in the pharmacokinetic properties of lysosomotropic drugs.

  10. A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB.

    PubMed

    Settembre, Carmine; Zoncu, Roberto; Medina, Diego L; Vetrini, Francesco; Erdin, Serkan; Erdin, SerpilUckac; Huynh, Tuong; Ferron, Mathieu; Karsenty, Gerard; Vellard, Michel C; Facchinetti, Valeria; Sabatini, David M; Ballabio, Andrea

    2012-03-07

    The lysosome plays a key role in cellular homeostasis by controlling both cellular clearance and energy production to respond to environmental cues. However, the mechanisms mediating lysosomal adaptation are largely unknown. Here, we show that the Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, colocalizes with master growth regulator mTOR complex 1 (mTORC1) on the lysosomal membrane. When nutrients are present, phosphorylation of TFEB by mTORC1 inhibits TFEB activity. Conversely, pharmacological inhibition of mTORC1, as well as starvation and lysosomal disruption, activates TFEB by promoting its nuclear translocation. In addition, the transcriptional response of lysosomal and autophagic genes to either lysosomal dysfunction or pharmacological inhibition of mTORC1 is suppressed in TFEB-/- cells. Interestingly, the Rag GTPase complex, which senses lysosomal amino acids and activates mTORC1, is both necessary and sufficient to regulate starvation- and stress-induced nuclear translocation of TFEB. These data indicate that the lysosome senses its content and regulates its own biogenesis by a lysosome-to-nucleus signalling mechanism that involves TFEB and mTOR.

  11. [Lysosomal enzyme activity in white blood cells in leukemias].

    PubMed

    Rybakova, L P; Kharchenko, M F

    1996-01-01

    Total enzyme activity of acidic hydrolases and total neutral proteinase were compared in the post-nuclear fraction of leukocytes from healthy subjects and leukemia patients. The levels of acidic phosphotase and neutral proteinase in lymphoid cells of healthy donors were 11 and 7 times lower than those in myeloid cells, respectively. Patients suffering chronic myeloid leukemia revealed enhanced levels of beta-glucuronidase and neutral proteinases whereas B-chronic lymphoid leukemia involved acidic hydrolase concentrations lower than normal. As chronic myeloid leukemia advanced, neutral proteinase activity dropped dramatically (2.5 times); an aggressive course of B-chronic lymphoid leukemia was accompanied by a 3-fold decrease in acidic hydrolase level. The results may be used as indirect evidence of differences in the role of lysosomal enzymes in the mechanism of protein processing involved in myeloid and lymphoid proliferative pathologies.

  12. Lysosomal Disorders Drive Susceptibility to Tuberculosis by Compromising Macrophage Migration

    PubMed Central

    Berg, Russell D.; Levitte, Steven; O’Sullivan, Mary P.; O’Leary, Seónadh M.; Cambier, C.J.; Cameron, James; Takaki, Kevin K.; Moens, Cecilia B.; Tobin, David M.; Keane, Joseph; Ramakrishnan, Lalita

    2016-01-01

    Summary A zebrafish genetic screen for determinants of susceptibility to Mycobacterium marinum identified a hypersusceptible mutant deficient in lysosomal cysteine cathepsins that manifests hallmarks of human lysosomal storage diseases. Under homeostatic conditions, mutant macrophages accumulate undigested lysosomal material, which disrupts endocytic recycling and impairs their migration to, and thus engulfment of, dying cells. This causes a buildup of unengulfed cell debris. During mycobacterial infection, macrophages with lysosomal storage cannot migrate toward infected macrophages undergoing apoptosis in the tuberculous granuloma. The unengulfed apoptotic macrophages undergo secondary necrosis, causing granuloma breakdown and increased mycobacterial growth. Macrophage lysosomal storage similarly impairs migration to newly infecting mycobacteria. This phenotype is recapitulated in human smokers, who are at increased risk for tuberculosis. A majority of their alveolar macrophages exhibit lysosomal accumulations of tobacco smoke particulates and do not migrate to Mycobacterium tuberculosis. The incapacitation of highly microbicidal first-responding macrophages may contribute to smokers’ susceptibility to tuberculosis. PMID:27015311

  13. Pulmonary arterial hypertension associated with impaired lysosomal endothelin-1 degradation.

    PubMed

    Recla, Sabine; Hahn, Andreas; Apitz, Christian

    2015-04-01

    We report on a boy with severe pulmonary arterial hypertension associated with mucolipidosis, a rare lysosomal storage disorder. During diagnostic catheterisation, we found increased endothelin-1 levels, but normal big endothelin-1-levels (the precursor form of endothelin-1), which suggests impaired degradation of endothelin-1 rather than increased synthesis. As endothelin-1 degradation takes place in the lysosome, it appears likely that lysosomal dysfunction caused by the underlying disease contributes to the development of pulmonary arterial hypertension in this patient.

  14. Phosphatidic acid osmotically destabilizes lysosomes through increased permeability to K+ and H+.

    PubMed

    Yi, Y-P; Wang, X; Zhang, G; Fu, T-S; Zhang, G-J

    2006-06-01

    Lysosomal destabilization is a critical event not only for the organelle but also for living cells. However, what factors can affect lysosomal stability is not fully studied. In this work, the effects of phosphatidic acid (PA) on the lysosomal integrity were investigated. Through the measurements of lysosomal beta-hexosaminidase free activity, intralysosomal pH, leakage of lysosomal protons and lysosomal latency loss in hypotonic sucrose medium, we established that PA could increase the lysosomal permeability to K+ and H+, and enhance the lysosomal osmotic sensitivity. Treatment of lysosomes with PA promoted entry of K+ into the organelle via K+/H+ exchange, which could produce osmotic stresses and osmotically destabilize the lysosomes. In addition, PA-induced increase in the lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shocks. The results suggest that PA may play a role in the lysosomal destabilization.

  15. Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network

    PubMed Central

    1994-01-01

    Newly synthesized lysosomal enzymes bind to mannose 6-phosphate receptors (MPRs) in the TGN, and are carried to prelysosomes, where they are released. MPRs then return to the TGN for another round of transport. Rab9 is a ras-like GTPase which facilitates MPR recycling to the TGN in vitro. We show here that a dominant negative form of rab9, rab9 S21N, strongly inhibited MPR recycling in living cells. The block was specific in that the rates of biosynthetic protein transport, fluid phase endocytosis and receptor-mediated endocytosis were unchanged. Expression of rab9 S21N was accompanied by a decrease in the efficiency of lysosomal enzyme sorting. Cells compensated for the presence of the mutant protein by inducing the synthesis of both soluble and membrane- associated lysosomal enzymes, and by internalizing lysosomal enzymes that were secreted by default. These data show that MPRs are limiting in the secretory pathway of cells expressing rab9 S21N and document the importance of MPR recycling and the rab9 GTPase for efficient lysosomal enzyme delivery. PMID:7909812

  16. Lysosome acidification by photoactivated nanoparticles restores autophagy under lipotoxicity

    PubMed Central

    Trudeau, Kyle M.; Colby, Aaron H.; Zeng, Jialiu; Las, Guy; Feng, Jiazuo H.; Shirihai, Orian S.

    2016-01-01

    In pancreatic β-cells, liver hepatocytes, and cardiomyocytes, chronic exposure to high levels of fatty acids (lipotoxicity) inhibits autophagic flux and concomitantly decreases lysosomal acidity. Whether impaired lysosomal acidification is causally inhibiting autophagic flux and cellular functions could not, up to the present, be determined because of the lack of an approach to modify lysosomal acidity. To address this question, lysosome-localizing nanoparticles are described that, upon UV photoactivation, enable controlled acidification of impaired lysosomes. The photoactivatable, acidifying nanoparticles (paNPs) demonstrate lysosomal uptake in INS1 and mouse β-cells. Photoactivation of paNPs in fatty acid–treated INS1 cells enhances lysosomal acidity and function while decreasing p62 and LC3-II levels, indicating rescue of autophagic flux upon acute lysosomal acidification. Furthermore, paNPs improve glucose-stimulated insulin secretion that is reduced under lipotoxicity in INS1 cells and mouse islets. These results establish a causative role for impaired lysosomal acidification in the deregulation of autophagy and β-cell function under lipotoxicity. PMID:27377248

  17. Cell biology in China: Focusing on the lysosome.

    PubMed

    Yang, Chonglin; Wang, Xiaochen

    2017-06-01

    The view that lysosomes are merely the recycling bins of the cell has changed greatly during recent years. Lysosomes are now known to play a central role in signal transduction, cellular adaptation, plasma membrane repair, immune responses and many other fundamental cellular processes. In conjunction with the seminal discoveries made by international colleagues, many important questions regarding lysosomes are being addressed by Chinese scientists. In this review, we briefly summarize recent exciting findings in China on lysosomal signaling, biogenesis, integrity and physiological functions. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Biochemical characterization of lysosomes in unfertilized eggs of Xenopus laevis.

    PubMed

    Decroly, M; Goldfinger, M; Six-Tondeur, N

    1979-11-01

    Relations between lysosomes and yolk platelets of amphibian eggs have been suggested. This work demonstrates the presence of acid hydrolases in oocytes induced to ovulate in vitro. About 40% of the acid hydrolases are found in a sedimentable fraction, and, in accordance with the lysosomal concept, they display structural latency. Biochemical data did not indicate any association between lysosomal enzymes and yolk platelets. The mechanism of yolk resorption is discussed and it is suggested that the fusion of lysosomes and yolk platelets might be one of the mechanisms involved in yolk digestion.

  19. A lysosome-centered view of nutrient homeostasis.

    PubMed

    Mony, Vinod K; Benjamin, Shawna; O'Rourke, Eyleen J

    2016-01-01

    Lysosomes are highly acidic cellular organelles traditionally viewed as sacs of enzymes involved in digesting extracellular or intracellular macromolecules for the regeneration of basic building blocks, cellular housekeeping, or pathogen degradation. Bound by a single lipid bilayer, lysosomes receive their substrates by fusing with endosomes or autophagosomes, or through specialized translocation mechanisms such as chaperone-mediated autophagy or microautophagy. Lysosomes degrade their substrates using up to 60 different soluble hydrolases and release their products either to the cytosol through poorly defined exporting and efflux mechanisms or to the extracellular space by fusing with the plasma membrane. However, it is becoming evident that the role of the lysosome in nutrient homeostasis goes beyond the disposal of waste or the recycling of building blocks. The lysosome is emerging as a signaling hub that can integrate and relay external and internal nutritional information to promote cellular and organismal homeostasis, as well as a major contributor to the processing of energy-dense molecules like glycogen and triglycerides. Here we describe the current knowledge of the nutrient signaling pathways governing lysosomal function, the role of the lysosome in nutrient mobilization, and how lysosomes signal other organelles, distant tissues, and even themselves to ensure energy homeostasis in spite of fluctuations in energy intake. At the same time, we highlight the value of genomics approaches to the past and future discoveries of how the lysosome simultaneously executes and controls cellular homeostasis.

  20. Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors

    PubMed Central

    Sukhai, Mahadeo A.; Prabha, Swayam; Hurren, Rose; Rutledge, Angela C.; Lee, Anna Y.; Sriskanthadevan, Shrivani; Sun, Hong; Wang, Xiaoming; Skrtic, Marko; Seneviratne, Ayesh; Cusimano, Maria; Jhas, Bozhena; Gronda, Marcela; MacLean, Neil; Cho, Eunice E.; Spagnuolo, Paul A.; Sharmeen, Sumaiya; Gebbia, Marinella; Urbanus, Malene; Eppert, Kolja; Dissanayake, Dilan; Jonet, Alexia; Dassonville-Klimpt, Alexandra; Li, Xiaoming; Datti, Alessandro; Ohashi, Pamela S.; Wrana, Jeff; Rogers, Ian; Sonnet, Pascal; Ellis, William Y.; Corey, Seth J.; Eaves, Connie; Minden, Mark D.; Wang, Jean C.Y.; Dick, John E.; Nislow, Corey; Giaever, Guri; Schimmer, Aaron D.

    2012-01-01

    Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML. PMID:23202731

  1. Autophagosome-lysosome fusion triggers a lysosomal response mediated by TLR9 and controlled by OCRL

    PubMed Central

    Vicinanza, Mariella; Luciani, Alessandro; Carissimo, Annamaria; Mutarelli, Margherita; Di Campli, Antonella; Polishchuk, Elena; Di Tullio, Giuseppe; Morra, Valentina; Levtchenko, Elena; Oltrabella, Francesca; Starborg, Tobias; Santoro, Michele; Di Bernardo, Diego; Devuyst, Olivier; Lowe, Martin; Medina, Diego L.; Ballabio, Andrea; De Matteis, Maria Antonietta

    2016-01-01

    Phosphoinositides (PIs) control fundamental cell processes, and inherited defects of PI kinases or phosphatases cause severe human diseases including Lowe syndrome due to mutations in OCRL that encodes a PI(4,5)P2 5-phosphatase. Here we unveil a lysosomal response to the arrival of autophagosomal cargo where OCRL plays a key role. We identify mitochondrial DNA and TLR9 as the cargo and the receptor that triggers and mediates, respectively, this response. This lysosome-cargo response is required to sustain the autophagic flux and involves a local increase in PI(4,5)P2 that is confined in space and time by OCRL. Depleting or inhibiting OCRL leads to an accumulation of lysosomal PI(4,5)P2, an inhibitor of the calcium channel mucolipin-1 that controls autophagosome-lysosome fusion. Hence, autophagosomes accumulate in OCRL-depleted cells and in the kidneys of Lowe syndrome patients. Importantly, boosting the activity of mucolipin-1 with selective agonists restores the autophagic flux in cells from Lowe syndrome patients. PMID:27398910

  2. Animal models for lysosomal storage disorders.

    PubMed

    Pastores, G M; Torres, P A; Zeng, B-J

    2013-07-01

    The lysosomal storage disorders (LSD) represent a heterogeneous group of inherited diseases characterized by the accumulation of non-metabolized macromolecules (by-products of cellular turnover) in different tissues and organs. LSDs primarily develop as a consequence of a deficiency in a lysosomal hydrolase or its co-factor. The majority of these enzymes are glycosidases and sulfatases, which in normal conditions participate in degradation of glycoconjugates: glycoproteins, glycosaminoproteoglycans, and glycolipids. Significant insights have been gained from studies of animal models, both in understanding mechanisms of disease and in establishing proof of therapeutic concept. These studies have led to the introduction of therapy for certain LSD subtypes, primarily by enzyme replacement or substrate reduction therapy. Animal models have been useful in elucidating molecular changes, particularly prior to onset of symptoms. On the other hand, it should be noted certain animal (mouse) models may have the underlying biochemical defect, but not show the course of disease observed in human patients. There is interest in examining therapeutic options in the larger spontaneous animal models that may more closely mimic the brain size and pathology of humans. This review will highlight lessons learned from studies of animal models of disease, drawing primarily from publications in 2011-2012.

  3. Enhanced lysosomal activity by overexpressed aminopeptidase Y in Saccharomyces cerevisiae.

    PubMed

    Yoon, Jihee; Sekhon, Simranjeet Singh; Kim, Yang-Hoon; Min, Jiho

    2016-06-01

    Saccharomyces cerevisiae contains vacuoles corresponding to lysosomes in higher eukaryotes. Lysosomes are dynamic (not silent) organelles in which enzymes can be easily integrated or released when exposed to stressful conditions. Changes in lysosomal enzymes have been observed due to oxidative stress, resulting in an increased function of lysosomes. The protein profiles from H2O2- and NH4Cl-treated lysosomes showed different expression patterns, observed with two-dimensional gel electrophoresis. The aminopeptidase Y protein (APE3) that conspicuously enhanced antimicrobial activity than other proteins was selected for further studies. The S. cerevisiae APE3 gene was isolated and inserted into pYES2.0 expression vector. The GFP gene was inserted downstream to the APE3 gene for confirmation of APE3 targeting to lysosomes, and S. cerevisiae was transformed to pYES2::APE3::GFP. The APE3 did not enter in lysosomes and formed an inclusion body at 30 °C, but it inserted to lysosomes as shown by the merger of GFP with lysosomes at 28 °C. Antimicrobial activity of the cloned S. cerevisiae increased about 5 to 10 % against eight strains, compared to normal cells, and galactose induction is increased more two folds than that of normal cells. Therefore, S. cerevisiae was transformed to pYES2::APE3::GFP, accumulating a large amount of APE3, resulting in increased lysosomal activity. Increase in endogenous levels of lysosomes and their activity following genetic modification can lead to its use in applications such as antimicrobial agents and apoptosis-inducing materials for cancer cells, and consequently, it may also be possible to use the organelles for improving in vitro functions.

  4. Lysosomal and tissue-level biomarkers in mussels cross-transplanted among four estuaries with different pollution levels.

    PubMed

    Lekube, Xabier; Izagirre, Urtzi; Soto, Manu; Marigómez, Ionan

    2014-02-15

    A 3-4 wk cross-transplantation experiment was carried out in order to investigate the sensitivity, rapidity, durability and reversibility of lysosomal and tissue-level biomarkers in the digestive gland of mussels. Four localities in the Basque coast with different levels of chemical pollution and environmental stress were selected. Lysosomal membrane stability (LP) and lysosomal structural changes (VvL; S/VL; NvL) and changes in cell-type composition in digestive gland epithelium (VvBAS) were investigated to determine short (2d) and mid-term (3-4 wk) responses after cross-transplantation. Mussels from Txatxarramendi presented VvBAS<0.1 μm(3)/μm(3) (unstressed) whilst VvBAS>0.12 μm(3)/μm(3) was recorded in mussels from Plentzia (moderate stress) and VvBAS>0.2 μm(3)/μm(3) in Arriluze and Muskiz (high stress). Accordingly, LP<10 min (high stress) was recorded in mussels from Muskiz and Arriluze and LP~15 min (low-to-moderate stress) in those from Plentzia and Txatxarramendi. According to the VvL, S/VL and NvL data, a certain lysosomal enlargement was envisaged in mussels from Arriluze in comparison with those from Txatxarramendi and Plentzia. Mussels from Muskiz exhibited a peculiar endo-lysosomal system made of abundant tiny lysosomes (low VvL and high S/VL and NvL values). Lysosomal and tissue-level biomarkers were responsive after 2d cross-transplantation between the reference and the polluted localities, which indicated that these biomarkers were quickly induced and, to a large extent, reversible. Moreover, the tissue-level biomarker values were maintained during the entire period (3-4 wk) of cross-transplantation, which evidenced the durability of the responsiveness. In contrast, comparisons in the mid-term were unfeasible for lysosomal biomarkers as these exhibited a seasonal winter attenuation resulting from low food availability and low temperatures. In conclusion, lysosomal enlargement and membrane stability and changes in cell-type composition were

  5. Effects of phospholipase A2 on the lysosomal ion permeability and osmotic sensitivity.

    PubMed

    Wang, Jiong-Wei; Sun, Lin; Hu, Jin-Shan; Li, Ying-Bin; Zhang, Guo-Jiang

    2006-01-01

    In this study, we investigated the mechanism of PLA(2)-induced lysosomal destabilization. Through the measurements of lysosomal beta-hexosaminidase free activity, their membrane potential, the intra-lysosomal pH and the lysosomal latency loss in hypotonic sucrose medium, we established that PLA(2) could increase the lysosomal membrane permeability to both potassium ions and protons. The enzyme could also enhance the organelle osmotic sensitivity. The increases in the lysosomal ion permeability promoted influx of potassium ions into the lysosomes via K(+)/H(+) exchange. The resulted osmotic imbalance across the lysosomal membranes osmotically destabilized the lysosomes. In addition, the enhancement of the lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in the osmotic stress. The results explain how PLA(2) destabilized the lysosomes.

  6. [The blood-brain barrier and neurodegenerative lysosomal storage diseases].

    PubMed

    Urayama, Akihiko

    2013-02-01

    Enzyme replacement therapy has been a very effective treatment for several lysosomal storage diseases. However, correcting central nervous system (CNS) storage has been challenging due to the presence of the blood-brain barrier (BBB), which hampers the entry of circulating lysosomal enzymes into the brain. In our previous studies, we discovered that luminally expressed cation-independent mannose 6-phosphate (M6P) receptor is a universal transporter for lysosomal enzymes that contain M6P moieties on the enzyme molecule. This receptor-mediated transport of lysosomal enzymes showed developmental down-regulation that resulted in a failure of delivery of lysosomal enzymes across the BBB in the adult brain. Conceptually, if one can re-induce M6P receptor-mediated transport of lysosomal enzymes in adult BBB, this could provide a novel brain targeting approach for treating abnormal storage in the CNS, regardless of the age of subjects. We found that systemic adrenergic stimuli restored functional transport of β-glucuronidase across the adult BBB. The concept of manipulating BBB transport activity by endogenous characteristics has also been demonstrated by another group who showed effective treatment in a Pompe disease model animal in vivo. It is intriguing that lysosomal enzymes utilize multiple mechanisms for their transport across the BBB. This review explores pharmacological manipulations for the delivery of lysosomal enzymes into the CNS, and the mechanisms of their transport across the BBB, based on existing evidence from studies of β-glucuronidase, sulfamidase, acid α-glucosidase, and arylsulfatase A.

  7. Lysosome/lipid droplet interplay in metabolic diseases.

    PubMed

    Dugail, Isabelle

    2014-01-01

    Lysosomes and lipid droplets are generally considered as intracellular compartments with divergent roles in cell metabolism, lipid droplets serving as lipid reservoirs in anabolic pathways, whereas lysosomes are specialized in the catabolism of intracellular components. During the last few years, new insights in the biology of lysosomes has challenged this view by providing evidence for the importance of lysosome recycling as a sparing mechanism to maintain cellular fitness. On the other hand the understanding of lipid droplets has evolved from an inert intracellular deposit toward the status of an intracellular organelle with dynamic roles in cellular homeostasis beyond storage. These unrelated aspects have also recently converged in the finding of unexpected lipid droplet/lysosome communication through autophagy, and the discovery of lysosome-mediated lipid droplet degradation called lipopagy. Furthermore, adipocytes which are professional cells for lipid droplet formation were also shown to be active in peptide antigen presentation a pathway requiring lysosomal activity. The potential importance of lipid droplet/lysosome interplay is discussed in the context of metabolic diseases and the setting of chronic inflammation.

  8. Expanding Newborn Screening for Lysosomal Disorders: Opportunities and Challenges

    ERIC Educational Resources Information Center

    Waggoner, Darrel J.; Tan, Christopher A.

    2011-01-01

    Newborn screening (NBS), since its implementation in the 1960s, has traditionally been successful in reducing mortality and disability in children with a range of different conditions. Lysosomal storage disorders (LSD) are a heterogeneous group of inherited metabolic diseases that result from lysosomal dysfunction. Based on available treatment and…

  9. Expanding Newborn Screening for Lysosomal Disorders: Opportunities and Challenges

    ERIC Educational Resources Information Center

    Waggoner, Darrel J.; Tan, Christopher A.

    2011-01-01

    Newborn screening (NBS), since its implementation in the 1960s, has traditionally been successful in reducing mortality and disability in children with a range of different conditions. Lysosomal storage disorders (LSD) are a heterogeneous group of inherited metabolic diseases that result from lysosomal dysfunction. Based on available treatment and…

  10. Fasting-induced hormonal regulation of lysosomal function

    PubMed Central

    Chen, Liqun; Wang, Ke; Long, Aijun; Jia, Liangjie; Zhang, Yuanyuan; Deng, Haiteng; Li, Yu; Han, Jinbo; Wang, Yiguo

    2017-01-01

    Lysosomes are centers for nutrient sensing and recycling that allow mammals to adapt to starvation. Regulation of lysosome dynamics by internal nutrient signaling is well described, but the mechanisms by which external cues modulate lysosomal function are unclear. Here, we describe an essential role of the fasting-induced hormone fibroblast growth factor 21 (FGF21) in lysosome homeostasis in mice. Fgf21 deficiency impairs hepatic lysosomal function by blocking transcription factor EB (TFEB), a master regulator of lysosome biogenesis and autophagy. FGF21 induces mobilization of calcium from the endoplasmic reticulum, which activates the transcriptional repressor downstream regulatory element antagonist modulator (DREAM), and thereby inhibits expression of Mid1 (encoding the E3 ligase Midline-1). Protein phosphatase PP2A, a substrate of MID1, accumulates and dephosphorylates TFEB, thereby upregulating genes involved in lysosome biogenesis, autophagy and lipid metabolism. Thus, an FGF21-TFEB signaling axis links lysosome homeostasis with extracellular hormonal signaling to orchestrate lipid metabolism during fasting. PMID:28374748

  11. Metallothionein protects against oxidative stress-induced lysosomal destabilization

    PubMed Central

    Baird, Sarah K.; Kurz, Tino; Brunk, Ulf T.

    2005-01-01

    The introduction of apo-ferritin or the iron chelator DFO (desferrioxamine) conjugated to starch into the lysosomal compartment protects cells against oxidative stress, lysosomal rupture and ensuing apoptosis/necrosis by binding intralysosomal redox-active iron, thus preventing Fenton-type reactions and ensuing peroxidation of lysosomal membranes. Because up-regulation of MTs (metallothioneins) also generates enhanced cellular resistance to oxidative stress, including X-irradiation, and MTs were found to be capable of iron binding in an acidic and reducing lysosomal-like environment, we propose that these proteins might similarly stabilize lysosomes following autophagocytotic delivery to the lysosomal compartment. Here, we report that Zn-mediated MT up-regulation, assayed by Western blotting and immunocytochemistry, results in lysosomal stabilization and decreased apoptosis following oxidative stress, similar to the protection afforded by fluid-phase endocytosis of apo-ferritin or DFO. In contrast, the endocytotic uptake of an iron phosphate complex destabilized lysosomes against oxidative stress, but this was suppressed in cells with up-regulated MT. It is suggested that the resistance against oxidative stress, known to occur in MT-rich cells, may be a consequence of autophagic turnover of MT, resulting in reduced iron-catalysed intralysosomal peroxidative reactions. PMID:16236025

  12. Artesunate induces cell death in human cancer cells via enhancing lysosomal function and lysosomal degradation of ferritin.

    PubMed

    Yang, Nai-Di; Tan, Shi-Hao; Ng, Shukie; Shi, Yin; Zhou, Jing; Tan, Kevin Shyong Wei; Wong, Wai-Shiu Fred; Shen, Han-Ming

    2014-11-28

    Artesunate (ART) is an anti-malaria drug that has been shown to exhibit anti-tumor activity, and functional lysosomes are reported to be required for ART-induced cancer cell death, whereas the underlying molecular mechanisms remain largely elusive. In this study, we aimed to elucidate the molecular mechanisms underlying ART-induced cell death. We first confirmed that ART induces apoptotic cell death in cancer cells. Interestingly, we found that ART preferably accumulates in the lysosomes and is able to activate lysosomal function via promotion of lysosomal V-ATPase assembly. Furthermore, we found that lysosomes function upstream of mitochondria in reactive oxygen species production. Importantly, we provided evidence showing that lysosomal iron is required for the lysosomal activation and mitochondrial reactive oxygen species production induced by ART. Finally, we showed that ART-induced cell death is mediated by the release of iron in the lysosomes, which results from the lysosomal degradation of ferritin, an iron storage protein. Meanwhile, overexpression of ferritin heavy chain significantly protected cells from ART-induced cell death. In addition, knockdown of nuclear receptor coactivator 4, the adaptor protein for ferritin degradation, was able to block ART-mediated ferritin degradation and rescue the ART-induced cell death. In summary, our study demonstrates that ART treatment activates lysosomal function and then promotes ferritin degradation, subsequently leading to the increase of lysosomal iron that is utilized by ART for its cytotoxic effect on cancer cells. Thus, our data reveal a new mechanistic action underlying ART-induced cell death in cancer cells.

  13. Artesunate Induces Cell Death in Human Cancer Cells via Enhancing Lysosomal Function and Lysosomal Degradation of Ferritin*

    PubMed Central

    Yang, Nai-Di; Tan, Shi-Hao; Ng, Shukie; Shi, Yin; Zhou, Jing; Tan, Kevin Shyong Wei; Wong, Wai-Shiu Fred; Shen, Han-Ming

    2014-01-01

    Artesunate (ART) is an anti-malaria drug that has been shown to exhibit anti-tumor activity, and functional lysosomes are reported to be required for ART-induced cancer cell death, whereas the underlying molecular mechanisms remain largely elusive. In this study, we aimed to elucidate the molecular mechanisms underlying ART-induced cell death. We first confirmed that ART induces apoptotic cell death in cancer cells. Interestingly, we found that ART preferably accumulates in the lysosomes and is able to activate lysosomal function via promotion of lysosomal V-ATPase assembly. Furthermore, we found that lysosomes function upstream of mitochondria in reactive oxygen species production. Importantly, we provided evidence showing that lysosomal iron is required for the lysosomal activation and mitochondrial reactive oxygen species production induced by ART. Finally, we showed that ART-induced cell death is mediated by the release of iron in the lysosomes, which results from the lysosomal degradation of ferritin, an iron storage protein. Meanwhile, overexpression of ferritin heavy chain significantly protected cells from ART-induced cell death. In addition, knockdown of nuclear receptor coactivator 4, the adaptor protein for ferritin degradation, was able to block ART-mediated ferritin degradation and rescue the ART-induced cell death. In summary, our study demonstrates that ART treatment activates lysosomal function and then promotes ferritin degradation, subsequently leading to the increase of lysosomal iron that is utilized by ART for its cytotoxic effect on cancer cells. Thus, our data reveal a new mechanistic action underlying ART-induced cell death in cancer cells. PMID:25305013

  14. Lysosomal Dysfunction Caused by Cellular Accumulation of Silica Nanoparticles*

    PubMed Central

    Schütz, Irene; Lopez-Hernandez, Tania; Gao, Qi; Puchkov, Dmytro; Jabs, Sabrina; Nordmeyer, Daniel; Schmudde, Madlen; Rühl, Eckart; Graf, Christina M.; Haucke, Volker

    2016-01-01

    Nanoparticles (NPs) are widely used as components of drugs or cosmetics and hold great promise for biomedicine, yet their effects on cell physiology remain poorly understood. Here we demonstrate that clathrin-independent dynamin 2-mediated caveolar uptake of surface-functionalized silica nanoparticles (SiNPs) impairs cell viability due to lysosomal dysfunction. We show that internalized SiNPs accumulate in lysosomes resulting in inhibition of autophagy-mediated protein turnover and impaired degradation of internalized epidermal growth factor, whereas endosomal recycling proceeds unperturbed. This phenotype is caused by perturbed delivery of cargo via autophagosomes and late endosomes to SiNP-filled cathepsin B/L-containing lysosomes rather than elevated lysosomal pH or altered mTOR activity. Given the importance of autophagy and lysosomal protein degradation for cellular proteostasis and clearance of aggregated proteins, these results raise the question of beneficial use of NPs in biomedicine and beyond. PMID:27226546

  15. Mitochondrial respiration controls lysosomal function during inflammatory T cell responses

    PubMed Central

    Baixauli, Francesc; Acín-Pérez, Rebeca; Villarroya-Beltrí, Carolina; Mazzeo, Carla; Nuñez-Andrade, Norman; Gabandé-Rodriguez, Enrique; Dolores Ledesma, Maria; Blázquez, Alberto; Martin, Miguel Angel; Falcón-Pérez, Juan Manuel; Redondo, Juan Miguel; Enríquez, Jose Antonio; Mittelbrunn, Maria

    2016-01-01

    Summary The endolysosomal system is critical for the maintenance of cellular homeostasis. However, how endolysosomal compartment is regulated by mitochondrial function is largely unknown. We have generated a mouse model with defective mitochondrial function in CD4+ T lymphocytes by genetic deletion of the mitochondrial transcription factor A (Tfam). Mitochondrial respiration-deficiency impairs lysosome function, promotes p62 and sphingomyelin accumulation and disrupts endolysosomal trafficking pathways and autophagy, thus linking a primary mitochondrial dysfunction to a lysosomal storage disorder. The impaired lysosome function in Tfam-deficient cells subverts T cell differentiation toward pro-inflammatory subsets and exacerbates the in vivo inflammatory response. Restoration of NAD+ levels improves lysosome function and corrects the inflammatory defects in Tfam-deficient T cells. Our results uncover a mechanism by which mitochondria regulate lysosome function to preserve T cell differentiation and effector functions, and identify novel strategies for intervention in mitochondrial-related diseases. PMID:26299452

  16. Mitochondrial Respiration Controls Lysosomal Function during Inflammatory T Cell Responses.

    PubMed

    Baixauli, Francesc; Acín-Pérez, Rebeca; Villarroya-Beltrí, Carolina; Mazzeo, Carla; Nuñez-Andrade, Norman; Gabandé-Rodriguez, Enrique; Ledesma, Maria Dolores; Blázquez, Alberto; Martin, Miguel Angel; Falcón-Pérez, Juan Manuel; Redondo, Juan Miguel; Enríquez, Jose Antonio; Mittelbrunn, Maria

    2015-09-01

    The endolysosomal system is critical for the maintenance of cellular homeostasis. However, how endolysosomal compartment is regulated by mitochondrial function is largely unknown. We have generated a mouse model with defective mitochondrial function in CD4(+) T lymphocytes by genetic deletion of the mitochondrial transcription factor A (Tfam). Mitochondrial respiration deficiency impairs lysosome function, promotes p62 and sphingomyelin accumulation, and disrupts endolysosomal trafficking pathways and autophagy, thus linking a primary mitochondrial dysfunction to a lysosomal storage disorder. The impaired lysosome function in Tfam-deficient cells subverts T cell differentiation toward proinflammatory subsets and exacerbates the in vivo inflammatory response. Restoration of NAD(+) levels improves lysosome function and corrects the inflammatory defects in Tfam-deficient T cells. Our results uncover a mechanism by which mitochondria regulate lysosome function to preserve T cell differentiation and effector functions, and identify strategies for intervention in mitochondrial-related diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Genetic Analysis of Lysosomal Trafficking in Caenorhabditis elegans

    PubMed Central

    Hermann, Greg J.; Schroeder, Lena K.; Hieb, Caroline A.; Kershner, Aaron M.; Rabbitts, Beverley M.; Fonarev, Paul; Grant, Barth D.; Priess, James R.

    2005-01-01

    The intestinal cells of Caenorhabditis elegans embryos contain prominent, birefringent gut granules that we show are lysosome-related organelles. Gut granules are labeled by lysosomal markers, and their formation is disrupted in embryos depleted of AP-3 subunits, VPS-16, and VPS-41. We define a class of gut granule loss (glo) mutants that are defective in gut granule biogenesis. We show that the glo-1 gene encodes a predicted Rab GTPase that localizes to lysosome-related gut granules in the intestine and that glo-4 encodes a possible GLO-1 guanine nucleotide exchange factor. These and other glo genes are homologous to genes implicated in the biogenesis of specialized, lysosome-related organelles such as melanosomes in mammals and pigment granules in Drosophila. The glo mutants thus provide a simple model system for the analysis of lysosome-related organelle biogenesis in animal cells. PMID:15843430

  18. Genetic analysis of lysosomal trafficking in Caenorhabditis elegans.

    PubMed

    Hermann, Greg J; Schroeder, Lena K; Hieb, Caroline A; Kershner, Aaron M; Rabbitts, Beverley M; Fonarev, Paul; Grant, Barth D; Priess, James R

    2005-07-01

    The intestinal cells of Caenorhabditis elegans embryos contain prominent, birefringent gut granules that we show are lysosome-related organelles. Gut granules are labeled by lysosomal markers, and their formation is disrupted in embryos depleted of AP-3 subunits, VPS-16, and VPS-41. We define a class of gut granule loss (glo) mutants that are defective in gut granule biogenesis. We show that the glo-1 gene encodes a predicted Rab GTPase that localizes to lysosome-related gut granules in the intestine and that glo-4 encodes a possible GLO-1 guanine nucleotide exchange factor. These and other glo genes are homologous to genes implicated in the biogenesis of specialized, lysosome-related organelles such as melanosomes in mammals and pigment granules in Drosophila. The glo mutants thus provide a simple model system for the analysis of lysosome-related organelle biogenesis in animal cells.

  19. Autophagic/lysosomal dysfunction in Alzheimer’s disease

    PubMed Central

    2013-01-01

    Autophagy serves as the sole catabolic mechanism for degrading organelles and protein aggregates. Increasing evidence implicates autophagic dysfunction in Alzheimer’s disease (AD) and other neurodegenerative diseases associated with protein misprocessing and accumulation. Under physiologic conditions, the autophagic/lysosomal system efficiently recycles organelles and substrate proteins. However, reduced autophagy function leads to the accumulation of proteins and autophagic and lysosomal vesicles. These vesicles contain toxic lysosomal hydrolases as well as the proper cellular machinery to generate amyloid-beta, the major component of AD plaques. Here, we provide an overview of current research focused on the relevance of autophagic/lysosomal dysfunction in AD pathogenesis as well as potential therapeutic targets aimed at restoring autophagic/lysosomal pathway function. PMID:24171818

  20. FIG4 regulates lysosome membrane homeostasis independent of phosphatase function

    PubMed Central

    Bharadwaj, Rajnish; Cunningham, Kathleen M.; Zhang, Ke; Lloyd, Thomas E.

    2016-01-01

    FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS. PMID:26662798

  1. FIG4 regulates lysosome membrane homeostasis independent of phosphatase function.

    PubMed

    Bharadwaj, Rajnish; Cunningham, Kathleen M; Zhang, Ke; Lloyd, Thomas E

    2016-02-15

    FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS.

  2. Misrouting of v-ATPase subunit V0a1 dysregulates lysosomal acidification in a neurodegenerative lysosomal storage disease model

    PubMed Central

    Bagh, Maria B.; Peng, Shiyong; Chandra, Goutam; Zhang, Zhongjian; Singh, Satya P.; Pattabiraman, Nagarajan; Liu, Aiyi; Mukherjee, Anil B.

    2017-01-01

    Defective lysosomal acidification contributes to virtually all lysosomal storage disorders (LSDs) and to common neurodegenerative diseases like Alzheimer's and Parkinson's. Despite its fundamental importance, the mechanism(s) underlying this defect remains unclear. The v-ATPase, a multisubunit protein complex composed of cytosolic V1-sector and lysosomal membrane-anchored V0-sector, regulates lysosomal acidification. Mutations in the CLN1 gene, encoding PPT1, cause a devastating neurodegenerative LSD, INCL. Here we report that in Cln1−/− mice, which mimic INCL, reduced v-ATPase activity correlates with elevated lysosomal pH. Moreover, v-ATPase subunit a1 of the V0 sector (V0a1) requires palmitoylation for interacting with adaptor protein-2 (AP-2) and AP-3, respectively, for trafficking to the lysosomal membrane. Notably, treatment of Cln1−/− mice with a thioesterase (Ppt1)-mimetic, NtBuHA, ameliorated this defect. Our findings reveal an unanticipated role of Cln1 in regulating lysosomal targeting of V0a1 and suggest that varying factors adversely affecting v-ATPase function dysregulate lysosomal acidification in other LSDs and common neurodegenerative diseases. PMID:28266544

  3. Activation of peroxisome proliferator-activated receptor α induces lysosomal biogenesis in brain cells: implications for lysosomal storage disorders.

    PubMed

    Ghosh, Arunava; Jana, Malabendu; Modi, Khushbu; Gonzalez, Frank J; Sims, Katherine B; Berry-Kravis, Elizabeth; Pahan, Kalipada

    2015-04-17

    Lysosomes are ubiquitous membrane-enclosed organelles filled with an acidic interior and are central to the autophagic, endocytic, or phagocytic pathway. In contrast to its classical function as the waste management machinery, lysosomes are now considered to be an integral part of various cellular signaling processes. The diverse functionality of this single organelle requires a very complex and coordinated regulation of its activity with transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, at its core. However, mechanisms by which TFEB is regulated are poorly understood. This study demonstrates that gemfibrozil, an agonist of peroxisome proliferator-activated receptor (PPAR) α, alone and in conjunction with all-trans-retinoic acid is capable of enhancing TFEB in brain cells. We also observed that PPARα, but not PPARβ and PPARγ, is involved in gemfibrozil-mediated up-regulation of TFEB. Reporter assay and chromatin immunoprecipitation studies confirmed the recruitment of retinoid X receptor α, PPARα, and PGC1α on the PPAR-binding site on the Tfeb promoter as well. Subsequently, the drug-mediated induction of TFEB caused an increase in lysosomal protein and the lysosomal abundance in cell. Collectively, this study reinforces the link between lysosomal biogenesis and lipid metabolism with TFEB at the crossroads. Furthermore, gemfibrozil may be of therapeutic value in the treatment of lysosomal storage disorders in which autophagy-lysosome pathway plays an important role.

  4. PRESENILIN-NULL CELLS HAVE ALTERED TWO-PORE CALCIUM CHANNEL EXPRESSION AND LYSOSOMAL CALCIUM; IMPLICATIONS FOR LYSOSOMAL FUNCTION

    PubMed Central

    Kayala, Kara M Neely; Dickinson, George D; Minassian, Anet; Walls, Ken C; Green, Kim N; LaFerla, Frank M

    2012-01-01

    Presenilins are necessary for calcium homeostasis and also for efficient proteolysis through the autophagy/lysosome system. Presenilin regulates both endoplasmic reticulum calcium stores and autophagic proteolysis in a γ-secretase independent fashion. The endo-lysosome system can also act as a calcium store, with calcium efflux channels being recently identified as two-pore channels 1 and 2. Here we investigated lysosomal calcium content and the channels that mediate calcium release from these acidic stores in presenilin knockout cells. We report that presenilin loss leads to a lower total lysosomal calcium store despite the buildup of lysosomes found in these cells. Additionally, we find alterations in two-pore calcium channel protein expression, with loss of presenilin preventing the formation of a high molecular weight species of TPC1 and TPC2. Finally, we find that treatments that disturb lysosomal calcium release lead to a reduction in autophagy function yet lysosomal inhibitors do not alter two-pore calcium channel expression. These data indicate that alterations in lysosomal calcium in the absence of presenilins might be leading to disruptions in autophagy. PMID:23103503

  5. Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content

    PubMed Central

    Mundy, Dorothy I.; Li, Wei Ping; Luby-Phelps, Katherine; Anderson, Richard G. W.

    2012-01-01

    Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intralysosomal pH, since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions, however, live-cell imaging shows cavicles actively docking with lysosomes, suggesting that these structures might be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally, and the degradation rate of caveolin-1 is not altered by any of these conditions, indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking. PMID:22238363

  6. The clinical presentation of lysosomal storage disorders.

    PubMed

    Wraith, James E

    2004-09-01

    Lysosomal storage disorders (LSDs) are present from conception and produce a clinical phenotype that evolves with time. The introduction of new therapies has made early diagnosis a priority. Clues to the clinical diagnosis of a LSD can be found in the tempo of the illness especially if the central nervous system is involved. Loss of a previously acquired skill (regression) is very characteristic of this group of disorders. Other clinical clues can include a dysmorphic appearance or the presence of characteristic skeletal involvement (dysostosis multiplex), but in some disorders such as Pompe disease or Krabbe disease, these do not occur. The approach to diagnosis has to involve "screening" as there can be considerable overlap in clinical presentation (e.g. Gaucher disease and Niemann-Pick B). Both urine and blood testing are necessary and the majority of diagnoses can now be confirmed at a molecular level. Prenatal diagnosis is possible for all.

  7. Optogenetic Acidification of Synaptic Vesicles and Lysosomes

    PubMed Central

    Grauel, M. Katharina; Wozny, Christian; Bentz, Claudia; Blessing, Anja; Rosenmund, Tanja; Jentsch, Thomas J.; Schmitz, Dietmar; Hegemann, Peter; Rosenmund, Christian

    2016-01-01

    Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes. PMID:26551543

  8. Treatment perspectives for the lysosomal storage diseases.

    PubMed

    Grabowski, Gregory A

    2008-03-01

    The therapy of the lysosomal storage diseases (LSDs) was developed by supplying adequate amounts of the needed enzyme to affected individuals. This approach in Gaucher disease provided a prototype for the basic and clinical sciences, and the economic foundation for other ultra-orphan diseases. Using the success of enzyme therapy for Gaucher disease, the challenges are highlighted for alternative bioproduction systems, and substrate reduction and molecular chaperone approaches for treatment of Gaucher disease and other ultra-orphan diseases. Literature review provided insight into the current status of enzyme therapies for LSDs, the proposed mechanisms of alternative approaches to therapy, and the obstacles in a competitive marketplace for treatment of ultra-rare diseases. These developments are placed in the contexts of finding rare patients with LSDs, their marked phenotypic spectrum, potential markets, and new orphan drug costs. The confluence of these challenges has led to a competitive environment with the potential for multiple, alternative, expensive treatments for orphan diseases.

  9. Lysosomal Cathepsin A Plays a Significant Role in the Processing of Endogenous Bioactive Peptides

    PubMed Central

    Timur, Zehra Kevser; Akyildiz Demir, Secil; Seyrantepe, Volkan

    2016-01-01

    Lysosomal serine carboxypeptidase Cathepsin A (CTSA) is a multifunctional enzyme with distinct protective and catalytic function. CTSA present in the lysosomal multienzyme complex to facilitate the correct lysosomal routing, stability and activation of with beta–galactosidase and alpha-neuraminidase. Beside CTSA has role in inactivation of bioactive peptides including bradykinin, substances P, oxytocin, angiotensin I and endothelin-I by cleavage of 1 or 2 amino acid(s) from C-terminal ends. In this study, we aimed to elucidate the regulatory role of CTSA on bioactive peptides in knock-in mice model of CTSAS190A. We investigated the level of bradykinin, substances P, oxytocin, angiotensin I and endothelin-I in the kidney, liver, lung, brain and serum from CTSAS190A mouse model at 3- and 6-months of age. Our results suggest CTSA selectively contributes to processing of bioactive peptides in different tissues from CTSAS190A mice compared to age matched WT mice. PMID:27826550

  10. Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

    PubMed

    Fernandes, Ana Clara; Uytterhoeven, Valerie; Kuenen, Sabine; Wang, Yu-Chun; Slabbaert, Jan R; Swerts, Jef; Kasprowicz, Jaroslaw; Aerts, Stein; Verstreken, Patrik

    2014-11-24

    Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans.

  11. Mucolipidosis type IV: the effect of increased lysosomal pH on the abnormal lysosomal storage.

    PubMed

    Kogot-Levin, Aviram; Zeigler, Marsha; Ornoy, Asher; Bach, Gideon

    2009-06-01

    Mucolipidosis type IV (MLIV) is a neurodegenerative channelopathy that is caused by the deficiency of TRPML1 activity, a nonselective cation channel. TRPML1 is a lysosomal membrane protein, and thus, MLIV is a lysosomal storage disorder. The basic, specific function of TRPML1 has not been yet clarified. A recent report (Soyombo AA, Tjon-Kon-Sang S, Rbaibi Y, Bashllari E, Bisceglia J, Muallem S, Kiselyov K: J Biol Chem 281:7294-7301, 2006) indicated that TRPML1 functions as an outwardly proton channel whose function is the prevention of overacidification of these organelles. Thus, in MLIV the lysosomal pH is lower than normal. Furthermore, attempts by these investigators to increase slightly the lysososmal pH with either Nigericin or Chloroquine suggested corrective effect of the abnormal storage in MLIV cells. We investigated this approach using these agents with cultured fibroblasts from severely affected and milder patients. Our data indicated that there was no reduction in the total number of storage vesicles by either agent, although Nigericin resulted in a change in the nature of the storage materials, reducing the presence of lamellated substances (lipids) so that the storage vesicles contained predominantly granulated substances. On the other hand, transfection with the normal MCOLN1 cDNA (the gene coding for TRPML1) resulted in the removal of almost all the storage materials.

  12. The unconventional myosin-VIIa associates with lysosomes

    PubMed Central

    Soni, Lily E.; Warren, Carmen M.; Bucci, Cecilia; Orten, Dana J.; Hasson, Tama

    2005-01-01

    Mutations in the myosin-VIIa (MYO7a) gene cause human Usher disease, characterized by hearing impairment and progressive retinal degeneration. In the retina, myosin-VIIa is highly expressed in the retinal pigment epithelium, where it plays a role in the positioning of melanosomes and other digestion organelles. Using a human cultured retinal pigmented epithelia cell line, ARPE-19, as a model system, we have found that a population of myosin-VIIa is associated with cathepsin D- and Rab7-positive lysosomes. Association of myosin-VIIa with lysosomes was Rab7 independent, as dominant negative and dominant active versions of Rab7 did not disrupt myosin-VIIa recruitment to lysosomes. Association of myosin-VIIa with lysosomes was also independent of the actin and microtubule cytoskeleton. Myosin-VIIa copurified with lysosomes on density gradients, and fractionation and extraction experiments suggested that it was tightly associated with the lysosome surface. These studies suggest that myosin-VIIa is a lysosome motor. PMID:16001398

  13. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity.

    PubMed

    Stern, Stephan T; Adiseshaiah, Pavan P; Crist, Rachael M

    2012-06-14

    The study of the potential risks associated with the manufacture, use, and disposal of nanoscale materials, and their mechanisms of toxicity, is important for the continued advancement of nanotechnology. Currently, the most widely accepted paradigms of nanomaterial toxicity are oxidative stress and inflammation, but the underlying mechanisms are poorly defined. This review will highlight the significance of autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Most endocytic routes of nanomaterial cell uptake converge upon the lysosome, making the lysosomal compartment the most common intracellular site of nanoparticle sequestration and degradation. In addition to the endo-lysosomal pathway, recent evidence suggests that some nanomaterials can also induce autophagy. Among the many physiological functions, the lysosome, by way of the autophagy (macroautophagy) pathway, degrades intracellular pathogens, and damaged organelles and proteins. Thus, autophagy induction by nanoparticles may be an attempt to degrade what is perceived by the cell as foreign or aberrant. While the autophagy and endo-lysosomal pathways have the potential to influence the disposition of nanomaterials, there is also a growing body of literature suggesting that biopersistent nanomaterials can, in turn, negatively impact these pathways. Indeed, there is ample evidence that biopersistent nanomaterials can cause autophagy and lysosomal dysfunctions resulting in toxicological consequences.

  14. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity

    PubMed Central

    2012-01-01

    The study of the potential risks associated with the manufacture, use, and disposal of nanoscale materials, and their mechanisms of toxicity, is important for the continued advancement of nanotechnology. Currently, the most widely accepted paradigms of nanomaterial toxicity are oxidative stress and inflammation, but the underlying mechanisms are poorly defined. This review will highlight the significance of autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Most endocytic routes of nanomaterial cell uptake converge upon the lysosome, making the lysosomal compartment the most common intracellular site of nanoparticle sequestration and degradation. In addition to the endo-lysosomal pathway, recent evidence suggests that some nanomaterials can also induce autophagy. Among the many physiological functions, the lysosome, by way of the autophagy (macroautophagy) pathway, degrades intracellular pathogens, and damaged organelles and proteins. Thus, autophagy induction by nanoparticles may be an attempt to degrade what is perceived by the cell as foreign or aberrant. While the autophagy and endo-lysosomal pathways have the potential to influence the disposition of nanomaterials, there is also a growing body of literature suggesting that biopersistent nanomaterials can, in turn, negatively impact these pathways. Indeed, there is ample evidence that biopersistent nanomaterials can cause autophagy and lysosomal dysfunctions resulting in toxicological consequences. PMID:22697169

  15. New form of acid phosphatase during lysosome biogenesis.

    PubMed Central

    Rao, G R; Aithal, H N; Toback, F G; Getz, G S

    1981-01-01

    Lysosome formation was induced in cells of the renal medulla by feeding rats on a K+-deficient diet. The role of the endoplasmic reticulum in the production of acid phosphatase, a typical lysosomal enzyme, was examined. Lysosomal and microsomal fractions were prepared for study by differential centrifugation of homogenates of renal papilla and inner stripe of red medulla. Acid phosphatase activity in the microsomal fraction was distinguished from the activity in the lysosomal fraction in normal tissue by differences in pH optima, tartrate inhibition, distribution of multiple forms after polyacrylamide-gel electrophoresis and detergent-sensitivity. During progressive K+ depletion, acid phosphatase activity in both microsomal and lysosomal fractions of the tissue increased 3-fold. In the lysosomes, K+ depletion was associated with the appearance of a new band of acid phosphatase. The neuraminidase-sensitivity of this band on polyacrylamide-gel electrophoresis indicated that the enzyme protein had been modified by the addition of sialic acid residues. K+ depletion also altered the lysosomal enzyme so that thiol compounds were able to stimulate its activity. Images Fig. 4. PMID:7326004

  16. Zinc finger E-box binding homeobox-1 (Zeb1) drives anterograde lysosome trafficking and tumor cell invasion via upregulation of Na+/H+ Exchanger-1 (NHE1).

    PubMed

    Dykes, Samantha S; Gao, ChongFeng; Songock, William K; Bigelow, Rebecca L; Woude, George Vande; Bodily, Jason M; Cardelli, James A

    2017-02-01

    Tumor cell invasion through the extracellular matrix is facilitated by the secretion of lysosome-associated proteases. As a common mechanism for secretion, lysosomes must first traffic to the cell periphery (anterograde trafficking), consistent with invasive cells often containing lysosomes closer to the plasma membrane compared to non-invasive cells. Epithelial to mesenchymal transition (EMT) is a transcriptionally driven program that promotes an invasive phenotype, and Zeb1 is one transcription factor that activates the mesenchymal gene expression program. The role of lysosome trafficking in EMT-driven invasion has not been previously investigated. We found that cells with increased levels of Zeb1 displayed lysosomes located closer to the cell periphery and demonstrated increased protease secretion and invasion in 3-dimensional (3D) cultures compared to their epithelial counterparts. Additionally, preventing anterograde lysosome trafficking via pharmacological inhibition of Na+/H+ exchanger 1 (NHE1) or shRNA depletion of ADP-ribosylation like protein 8b (Arl8b) reversed the invasive phenotype of mesenchymal cells, thus supporting a role for lysosome positioning in EMT-mediated tumor cell invasion. Immunoblot revealed that expression of Na+/H+ exchanger 1 correlated with Zeb1 expression. Furthermore, we found that the transcription factor Zeb1 binds to the Na+/H+ exchanger 1 promoter, suggesting that Zeb1 directly controls Na+/H+ transcription. Collectively, these results provide insight into a novel mechanism regulating Na+/H+ exchanger 1 expression and support a role for anterograde lysosome trafficking in Zeb1-driven cancer progression. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Mediated calcium transport by isolated human fibroblast lysosomes

    SciTech Connect

    Lemons, R.M.; Thoene, J.G. )

    1991-08-05

    Lysosomes purified by Percoll gradient from normal human fibroblasts (GM0010A) show uptake of Ca2+ in a mediated manner. The uptake is linear over the first 1.5 min and approaches a steady state by 10 min. Uptake is saturable, displaying a Vmax of about 10 pmol/min/unit hexosaminidase at 20 mM Ca2+ (7 nmol/min/mg protein), and a Km of 5.7 mM. Ca2+ uptake increases with increasing extralysosomal pH from 5.0 to 8.5. The Q10 is 1.6, and Ea 8.7 kcal/mol. Uptake of 0.1 mM Ca2+ was inhibited to the extent indicated by 1.0 mM of the following: Cd2+, 100%; Hg2+, 100%; Zn2+, 89%; Mg2+, 77%; Ba2+, 60%; Sr2+, 37%; Fe2+, 20%; Cu2+, 0%. Mono- and trivalent cations had no effect. ATP (1.0 mM) inhibited uptake by 80%, and chloroquine (0.1 mM) inhibited by 60%, as did 1.0 mM L-cystine. Cysteamine, N-ethylmaleimide, and the anions Cl-, SO(2-)4, and acetate had no effect. The calcium ionophore A23187 augmented uptake by 10-fold at 10 microM. Surprisingly, Pb2+ greatly augmented lysosomal Ca2+ uptake in a concentration-dependent manner. Pb2+, however, adversely affected lysosomal latency. Lysosomal calcium uptake was not affected by inositol 1,4,5-triphosphate, and calcium-induced calcium release from lysosomes was not observed. A role for lysosomes in cellular calcium homeostasis has not been previously suggested. This work shows that Ca2+ can be transported into and out of lysosomes and could assist in lysosomal proteolysis. The extent of further lysosomal participation in cellular calcium regulation is unclear.

  18. Systems biology of the autophagy-lysosomal pathway.

    PubMed

    Jegga, Anil G; Schneider, Lonnie; Ouyang, Xiaosen; Zhang, Jianhua

    2011-05-01

    The mechanisms of the control and activity of the autophagy-lysosomal protein degradation machinery are emerging as an important theme for neurodevelopment and neurodegeneration. However, the underlying regulatory and functional networks of known genes controlling autophagy and lysosomal function and their role in disease are relatively unexplored. We performed a systems biology-based integrative computational analysis to study the interactions between molecular components and to develop models for regulation and function of genes involved in autophagy and lysosomal function. Specifically, we analyzed transcriptional and microRNA-based post-transcriptional regulation of these genes and performed functional enrichment analyses to understand their involvement in nervous system-related diseases and phenotypes. Transcriptional regulatory network analysis showed that binding sites for transcription factors, SREBP1, USF, AP-1 and NFE2, are common among autophagy and lysosomal genes. MicroRNA enrichment analysis revealed miR-130, 98, 124, 204 and 142 as the putative post-transcriptional regulators of the autophagy-lysosomal pathway genes. Pathway enrichment analyses revealed that the mTOR and insulin signaling pathways are important in the regulation of genes involved in autophagy. In addition, we found that glycosaminoglycan and glycosphingolipid pathways also make a major contribution to lysosomal gene regulation. The analysis confirmed the known contribution of the autophagy-lysosomal genes to Alzheimer and Parkinson diseases and also revealed potential involvement in tuberous sclerosis, neuronal ceroidlipofuscinoses, sepsis and lung, liver and prostatic neoplasms. To further probe the impact of autophagy-lysosomal gene deficits on neurologically-linked phenotypes, we also mined the mouse knockout phenotype data for the autophagylysosomal genes and found them to be highly predictive of nervous system dysfunction. Overall this study demonstrates the utility of systems

  19. Aneuploidy triggers a TFEB-mediated lysosomal stress response.

    PubMed

    Santaguida, Stefano; Amon, Angelika

    2015-01-01

    Aneuploidy, defined as an alteration in chromosome number that is not a multiple of the haploid complement, severely affects cellular physiology. Changes in chromosome number lead to imbalances in cellular protein composition, thus disrupting cellular processes and causing proteins to misfold and aggregate. We recently reported that in mammalian cells protein aggregates are readily encapsulated within autophagosomes but are not degraded by lysosomes. This leads to a lysosomal stress response in which the transcription factor TFEB induces expression of factors needed for macroautophagy-mediated protein degradation. Our studies uncover lysosomal degradation defects as a feature of the aneuploid state, and a role for the transcription factor TFEB in the response thereto.

  20. NLRP3 inflammasome signaling is activated by low-level lysosome disruption but inhibited by extensive lysosome disruption: roles for K+ efflux and Ca2+ influx.

    PubMed

    Katsnelson, Michael A; Lozada-Soto, Kristen M; Russo, Hana M; Miller, Barbara A; Dubyak, George R

    2016-07-01

    Nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) is a cytosolic protein that nucleates assembly of inflammasome signaling platforms, which facilitate caspase-1-mediated IL-1β release and other inflammatory responses in myeloid leukocytes. NLRP3 inflammasomes are assembled in response to multiple pathogen- or environmental stress-induced changes in basic cell physiology, including the destabilization of lysosome integrity and activation of K(+)-permeable channels/transporters in the plasma membrane (PM). However, the quantitative relationships between lysosome membrane permeabilization (LMP), induction of increased PM K(+) permeability, and activation of NLRP3 signaling are incompletely characterized. We used Leu-Leu-O-methyl ester (LLME), a soluble lysosomotropic agent, to quantitatively track the kinetics and extent of LMP in relation to NLRP3 inflammasome signaling responses (ASC oligomerization, caspase-1 activation, IL-1β release) and PM cation fluxes in murine bone marrow-derived dendritic cells (BMDCs). Treatment of BMDCs with submillimolar (≤1 mM) LLME induced slower and partial increases in LMP that correlated with robust NLRP3 inflammasome activation and K(+) efflux. In contrast, supramillimolar (≥2 mM) LLME elicited extremely rapid and complete collapse of lysosome integrity that was correlated with suppression of inflammasome signaling. Supramillimolar LLME also induced dominant negative effects on inflammasome activation by the canonical NLRP3 agonist nigericin; this inhibition correlated with an increase in NLRP3 ubiquitination. LMP elicited rapid BMDC death by both inflammasome-dependent pyroptosis and inflammasome-independent necrosis. LMP also triggered Ca(2+) influx, which attenuated LLME-stimulated NLRP3 inflammasome signaling but potentiated LLME-induced necrosis. Taken together, these studies reveal a previously unappreciated signaling network that defines the coupling between LMP, changes

  1. Two Pore Channel 2 (TPC2) Inhibits Autophagosomal-Lysosomal Fusion by Alkalinizing Lysosomal pH*

    PubMed Central

    Lu, Yingying; Hao, Bai-Xia; Graeff, Richard; Wong, Connie W. M.; Wu, Wu-Tian; Yue, Jianbo

    2013-01-01

    Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca2+ mobilizing messengers, elicits Ca2+ release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca2+ signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression. PMID:23836916

  2. Two pore channel 2 (TPC2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH.

    PubMed

    Lu, Yingying; Hao, Bai-Xia; Graeff, Richard; Wong, Connie W M; Wu, Wu-Tian; Yue, Jianbo

    2013-08-16

    Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca(2+) mobilizing messengers, elicits Ca(2+) release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca(2+) signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.

  3. Cathepsin‐L Ameliorates Cardiac Hypertrophy Through Activation of the Autophagy–Lysosomal Dependent Protein Processing Pathways

    PubMed Central

    Sun, Mei; Ouzounian, Maral; de Couto, Geoffrey; Chen, Manyin; Yan, Ran; Fukuoka, Masahiro; Li, Guohua; Moon, Mark; Liu, Youan; Gramolini, Anthony; Wells, George J.; Liu, Peter P.

    2013-01-01

    Background Autophagy is critical in the maintenance of cellular protein quality control, the final step of which involves the fusion of autophagosomes with lysosomes. Cathepsin‐L (CTSL) is a key member of the lysosomal protease family that is expressed in the murine and human heart, and it may play an important role in protein turnover. We hypothesized that CTSL is important in regulating protein processing in the heart, particularly under pathological stress. Methods and Results Phenylephrine‐induced cardiac hypertrophy in vitro was more pronounced in CTSL‐deficient neonatal cardiomyocytes than in in controls. This was accompanied by a significant accumulation of autophagosomes, increased levels of ubiquitin‐conjugated protein, as well as impaired protein degradation and decreased cell viability. These effects were partially rescued with CTSL1 replacement via adeno‐associated virus–mediated gene transfer. In the in vivo murine model of aortic banding (AB), a deficiency in CTSL markedly exacerbated cardiac hypertrophy, worsened cardiac function, and increased mortality. Ctsl−/− AB mice demonstrated significantly decreased lysosomal activity and increased sarcomere‐associated protein aggregation. Homeostasis of the endoplasmic reticulum was also altered by CTSL deficiency, with increases in Bip and GRP94 proteins, accompanied by increased ubiquitin–proteasome system activity and higher levels of ubiquitinated proteins in response to AB. These changes ultimately led to a decrease in cellular ATP production, enhanced oxidative stress, and increased cellular apoptosis. Conclusions Lysosomal CTSL attenuates cardiac hypertrophy and preserves cardiac function through facilitation of autophagy and proteasomal protein processing. PMID:23608608

  4. Action of polystyrene nanoparticles of different sizes on lysosomal function and integrity

    PubMed Central

    2012-01-01

    Background Data from environmental exposure to nanoparticles (NPs) suggest that chronic exposure may increase the incidence of lung, cardiovascular and neurodegenerative diseases. Impairment of cell function by intracellular accumulation of NPs is also suspected. Many types of NPs have been detected in the endosomal-lysosomal system and, upon repeated exposure, alterations of the endosomal-lysosomal system may occur. To identify such effects we compared the effect of carboxyl polystyrene particles (CPS) of different sizes (20-500 nm) on lysosomes of the endothelial cell line EAhy926 after short (24h) and long (72h-96h) exposure times. Lysosomal localization of CPS, as well as lysosomal pH, lysosomal membrane integrity, morphology of the endosomal-lysosomal system and activities of the lysosomal enzymes,cathepsin B and sulfatases, upon exposure to CPS were recorded. Results CPS in sizes ≤100 nm showed high co-localization with lysosomes already after 4h, larger CPS after 24h. None of the particles at non-cytotoxic concentrations caused marked changes in lysosomal pH or destroyed lysosomal membrane integrity. At 24h of exposure, 20 nm CPS induced significant dilatation of the endosomal-lysosomal system and reduced activity of lysosomal sulfatases. After 72h, these alterations were less pronounced. Conclusions Despite accumulation in lysosomes CPS induced only small changes in lysosomes. Upon longer contact, these changes are even less pronounced. The presented panel of assays may serve to identify effects on lysosomes also for other NPs. PMID:22789069

  5. Stabilization of lysosomes in anoxic myocardium by propranolol.

    PubMed Central

    Welman, E

    1979-01-01

    1. Isolated hearts of guinea-pigs were perfused aerobically and anoxically for 60 min. (+/-)-Propranolol was added to the perfusion medium in concentrations ranging from 10 ng to 10 microgram/ml. 2. Lysosome stability was assessed by measurements of latent acid hydrolase activity in homogenates of left ventricular tissue. 3. In the absence of propranolol, the integrity of the myocardial lysosomes was considerably reduced after 60 min anoxia. Lysosome stability was enhanced by the presence of (+/-)-propranolol. The optimal concentration was found to be 0.1 microgram/ml. This concentration of the (+)-isomer alone was less effective. 4. It was concluded that beta-adrenoceptor blockade was the major mechanism by which lysosome disruption was prevented but that some direct membrane effect of propranolol may also be involved. PMID:427322

  6. Sequential vs. simultaneous photokilling by mitochondrial and lysosomal photodamage

    NASA Astrophysics Data System (ADS)

    Kessel, David

    2017-02-01

    We previously reported that a low level of lysosomal photoda mage can markedly promote the subsequent efficacy of PDT directed at mitochondria. This involves release of Ca2+ from photo damaged lysosomes, cleavage of the autophagy-associated protein ATG5 after activation of calpain and an interaction between the ATG5 fragment and mitochondria resulting in enhanced apoptosis. Inhibition of calpain activity abolished th is effect. We examined permissible irradiation sequences. Lysosomal photodamage must occur first with the `enhancement' effect showing a short half-life ( 15 min), presumably reflecting the survival of the ATG5 fragment. Simultaneous photo damage to both loci was found to be as effective as the sequential protocol. Since Photofrin can target both lysosomes and mitochondria for photo damage, this broad spectrum of photo damage may explain the efficacy of this photo sensitizing agent in spite of a sub-optimal absorbance profile at a sub- optimal wavelength for tissue transparency.

  7. The Yeast Lysosome-like Vacuole: Endpoint and Crossroads

    PubMed Central

    Li, Sheena Claire; Kane, Patricia M.

    2009-01-01

    Summary Fungal vacuoles are acidic organelles with degradative and storage capabilities that have many similarities to mammalian lysosomes and plant vacuoles. In the past several years, well-developed genetic, genomic, biochemical and cell biological tools in S. cerevisiae have provided fresh insights into vacuolar protein sorting, organelle acidification, ion homeostasis, autophagy, and stress-related functions of the vacuole, and these insights have often found parallels in mammalian lysosomes. This review provides a broad overview of the defining features and functions of S. cerevisiae vacuoles and compares these features to mammalian lysosomes. Recent research challenges the traditional view of vacuoles and lysosomes as simply the terminal compartment of biosynthetic and endocytic pathways (i.e. the “garbage dump” of the cell), and suggests instead that these compartments are unexpectedly dynamic and highly regulated. PMID:18786576

  8. What lysosomes actually tell us about Parkinson's disease?

    PubMed

    Bourdenx, Mathieu; Dehay, Benjamin

    2016-12-01

    Parkinson's disease is a common neurodegenerative disorder of unknown origin mainly characterized by the loss of neuromelanin-containing dopaminergic neurons in the substantia nigra pars compacta and the presence of intraneuronal proteinaceous inclusions called Lewy bodies. Lysosomes are dynamic organelles that degrade, in a controlled manner, cellular components delivered via the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways. Increasing amounts of evidence suggest a central role of lysosomal impairment in PD aetiology. This review provides an update on how genetic evidence support this connection and highlights how the neuropathologic and mechanistic evidence might relate to the disease process in sporadic forms of Parkinson's disease. Finally, we discuss the influence of ageing on lysosomal impairment and PD aetiology and therapeutic strategies targeting lysosomal function.

  9. Lysosomal storage disorders: A review of the musculoskeletal features.

    PubMed

    James, Rebecca A; Singh-Grewal, Davinder; Lee, Senq-J; McGill, Jim; Adib, Navid

    2016-03-01

    The lysosomal storage disorders are a collection of progressive, multisystem disorders that frequently present in childhood. Their timely diagnosis is paramount as they are becoming increasingly treatable. Musculoskeletal manifestations often occur early in the disease course, hence are useful as diagnostics clues. Non-inflammatory joint stiffness or pain, carpal tunnel syndrome, trigger fingers, unexplained pain crises and short stature should all prompt consideration of a lysosomal storage disorder. Recurrent ENT infections, hepatosplenomegaly, recurrent hernias and visual/hearing impairment - especially when clustered together - are important extra-skeletal features. As diagnostic and therapeutic options continue to evolve, children with lysosomal storage disorders and their families are facing more sophisticated options for screening and treatment. The aim of this article is to highlight the paediatric presentations of lysosomal storage disorders, with an emphasis on the musculoskeletal features.

  10. Autophagy in astrocytes: a novel culprit in lysosomal storage disorders.

    PubMed

    Di Malta, Chiara; Fryer, John D; Settembre, Carmine; Ballabio, Andrea

    2012-12-01

    Neurodegeneration is a prominent feature of lysosomal storage disorders (LSDs). Emerging data identify autophagy dysfunction in neurons as a major component of the phenotype. However, the autophagy pathway in the CNS has been studied predominantly in neurons, whereas in other cell types it has been largely unexplored. We studied the lysosome-autophagic pathway in astrocytes from a murine model of multiple sulfatase deficiency (MSD), a severe form of LSD. Similar to what was observed in neurons, we found that lysosomal storage in astrocytes impairs autophagosome maturation and this, in turn, has an impact upon the survival of cortical neurons and accounts for some of the neurological features found in MSD. Thus, our data indicate that lysosomal/autophagic dysfunction in astrocytes is an important component of neurodegeneration in LSDs.

  11. Endosome-lysosomes, ubiquitin and neurodegeneration.

    PubMed

    Mayer, R J; Tipler, C; Arnold, J; Laszlo, L; Al-Khedhairy, A; Lowe, J; Landon, M

    1996-01-01

    Before the advent of ubiquitin immunochemistry and immunogold electron microscopy, there was no known intracellular molecular commonality between neurodegenerative diseases. The application of antibodies which primarily detect ubiquitin protein conjugates has shown that all of the human and animal idiopathic and transmissible chronic neurodegenerative diseases, (including Alzheimer's disease (AD), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jakob disease (CJD) and scrapie) are related by some form of intraneuronal inclusion which contains ubiquitin protein conjugates. In addition, disorders such as Alzheimer's disease, CJD and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins which may be associated with cytoskeletal reorganisation. Although our knowledge about these diseases is increasing, they remain largely untreatable. Recently, attention has focused on the mechanisms of production of different types of amyloid and the likely involvement within cells of the endosome-lysosome system, organelles which are immuno-positive for ubiquitin protein conjugates. These organelles may be 'bioreactor' sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials or their precursors which subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities. Such common features of the disease processes give new direction to therapeutic intervention.

  12. Demonstration of adenosine deaminase activity in human fibroblast lysosomes.

    PubMed Central

    Lindley, E R; Pisoni, R L

    1993-01-01

    Human fibroblast lysosomes, purified on Percoll density gradients, contain an adenosine deaminase (ADA) activity that accounts for approximately 10% of the total ADA activity in GM0010A human fibroblasts. In assays of lysosomal ADA, the conversion of [3H]adenosine into [3H]inosine was proportional to incubation time and the amount of lysosomal material added to reaction mixtures. Maximal activity was observed between pH 7 and 8, and lysosomal ADA displayed a Km of 37 microM for adenosine at 25 degrees C and pH 5.5. Lysosomal ADA was completely inhibited by 2.5 mM Cu2+ or Hg2+ salts, but not by other bivalent cations (Ba2+, Cd2+, Ca2+, Fe2+, Mg2+, Mn2+ and Zn2+). Coformycin (2.5 mM), deoxycoformycin (0.02 mM), 2'-deoxyadenosine (2.5 mM), 6-methylaminopurine riboside (2.5 mM), 2'-3'-isopropylidene-adenosine (2.5 mM) and erythro-9-(2-hydroxy-3-nonyl)adenine (0.2 mM) inhibited lysosomal ADA by > 97%. In contrast, 2.5 mM S-adenosyl-L-homocysteine and cytosine were poor inhibitors. Nearly all lysosomal ADA activity is eluted as a high-molecular-mass protein (> 200 kDa) just after the void volume on a Sephacryl S-200 column, and is very heat-stable, retaining 70% of its activity after incubation at 65 degrees C for 80 min. We speculate that compartmentalization of ADA within lysosomes would allow deamination of adenosine to occur without competition by adenosine kinase, which could assist in maintaining cellular energy requirements under conditions of nutritional deprivation. PMID:8452534

  13. Acidic Nanoparticles Are Trafficked to Lysosomes and Restore an Acidic Lysosomal pH and Degradative Function to Compromised ARPE-19 Cells

    PubMed Central

    Baltazar, Gabriel C.; Guha, Sonia; Lu, Wennan; Lim, Jason; Boesze-Battaglia, Kathleen; Laties, Alan M.; Tyagi, Puneet; Kompella, Uday B.; Mitchell, Claire H.

    2012-01-01

    Lysosomal enzymes function optimally in acidic environments, and elevation of lysosomal pH can impede their ability to degrade material delivered to lysosomes through autophagy or phagocytosis. We hypothesize that abnormal lysosomal pH is a key aspect in diseases of accumulation and that restoring lysosomal pH will improve cell function. The propensity of nanoparticles to end up in the lysosome makes them an ideal method of delivering drugs to lysosomes. This study asked whether acidic nanoparticles could traffic to lysosomes, lower lysosomal pH and enhance lysosomal degradation by the cultured human retinal pigmented epithelial cell line ARPE-19. Acidic nanoparticles composed of poly (DL-lactide-co-glycolide) (PLGA) 502 H, PLGA 503 H and poly (DL-lactide) (PLA) colocalized to lysosomes of ARPE-19 cells within 60 min. PLGA 503 H and PLA lowered lysosomal pH in cells compromised by the alkalinizing agent chloroquine when measured 1 hr. after treatment, with acidification still observed 12 days later. PLA enhanced binding of Bodipy-pepstatin-A to the active site of cathepsin D in compromised cells. PLA also reduced the cellular levels of opsin and the lipofuscin-like autofluorescence associated with photoreceptor outer segments. These observations suggest the acidification produced by the nanoparticles was functionally effective. In summary, acid nanoparticles lead to a rapid and sustained lowering of lysosomal pH and improved degradative activity. PMID:23272048

  14. Effects of arachidonic acid on the lysosomal ion permeability and osmotic stability.

    PubMed

    Zhang, Gu; Yi, Ya-Ping; Zhang, Guo-Jiang

    2006-02-01

    In this study, we investigated the effects of arachidonic acid, a PLA2-produced lipid metabolite, on the lysosomal permeability, osmotic sensitivity and stability. Through the measurements of lysosomal beta-hexosaminidase free activity, membrane potential, intralysosomal pH, and lysosomal latency loss in hypotonic sucrose medium, we established that arachidonic acid could increase the lysosomal permeability to both potassium ions and protons, and enhance the lysosomal osmotic sensitivity. As a result, the fatty-acid-promoted entry of potassium ions into the lysosomes via K+/H+ exchange, which could produce osmotic imbalance across their membranes and osmotically destabilize the lysosomes. In addition, the enhancement of lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shock. The results suggest that arachidonic acid may play a role in the lysosomal destabilization.

  15. Lysosome and calcium dysregulation in Alzheimer's disease: partners in crime.

    PubMed

    McBrayer, MaryKate; Nixon, Ralph A

    2013-12-01

    Early-onset FAD (familial Alzheimer's disease) is caused by mutations of PS1 (presenilin 1), PS2 (presenilin 2) and APP (amyloid precursor protein). Beyond the effects of PS1 mutations on proteolytic functions of the γ-secretase complex, mutant or deficient PS1 disrupts lysosomal function and Ca2+ homoeostasis, both of which are considered strong pathogenic factors in FAD. Loss of PS1 function compromises assembly and proton-pumping activity of the vacuolar-ATPase on lysosomes, leading to defective lysosomal acidification and marked impairment of autophagy. Additional dysregulation of cellular Ca2+ by mutant PS1 in FAD has been ascribed to altered ion channels in the endoplasmic reticulum; however, rich stores of Ca2+ in lysosomes are also abnormally released in PS1-deficient cells secondary to the lysosomal acidification defect. The resultant rise in cytosolic Ca2+ activates Ca2+-dependent enzymes, contributing substantially to calpain overactivation that is a final common pathway leading to neurofibrillary degeneration in all forms of AD (Alzheimer's disease). In the present review, we discuss the close inter-relationships among deficits of lysosomal function, autophagy and Ca2+ homoeostasis as a pathogenic process in PS1-related FAD and their relevance to sporadic AD.

  16. Impact of Lysosome Status on Extracellular Vesicle Content and Release

    PubMed Central

    Eitan, Erez; Suire, Caitlin; Zhang, Shi; Mattson, Mark P.

    2016-01-01

    Extracellular vesicles (EVs) are nanoscale size bubble-like membranous structures released from cells. EVs contain RNA, lipids and proteins and are thought to serve various roles including intercellular communication and removal of misfolded proteins. The secretion of misfolded and aggregated proteins in EVs may be a cargo disposal alternative to the autophagy-lysosomal and ubiquitin-proteasome pathways. In this review we will discuss the importance of lysosome functionality for the regulation of EV secretion and content. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies (MVB) with the plasma membrane. MVBs can also fuse with lysosomes, and the trafficking pathway of MVBs can therefore determine whether or not exosomes are released from cells. Here we summarize data from studies of the effects of lysosome inhibition on the secretion of EVs and on the possibility that cells compensate for lysosome malfunction by disposal of potentially toxic cargos in EVs. A better understanding of the molecular mechanisms that regulate trafficking of MVBs to lysosomes and the plasma membrane may advance an understanding of diseases in which pathogenic proteins, lipids or infectious agents accumulate within or outside of cells. PMID:27238186

  17. Lysosomal enzymes and their receptors in invertebrates: an evolutionary perspective.

    PubMed

    Kumar, Nadimpalli Siva; Bhamidimarri, Poorna M

    2015-01-01

    Lysosomal biogenesis is an important process in eukaryotic cells to maintain cellular homeostasis. The key components that are involved in the biogenesis such as the lysosomal enzymes, their modifications and the mannose 6-phosphate receptors have been well studied and their evolutionary conservation across mammalian and non-mammalian vertebrates is clearly established. Invertebrate lysosomal biogenesis pathway on the other hand is not well studied. Although, details on mannose 6-phosphate receptors and enzymes involved in lysosomal enzyme modifications were reported earlier, a clear cut pathway has not been established. Recent research on the invertebrate species involving biogenesis of lysosomal enzymes suggests a possible conserved pathway in invertebrates. This review presents certain observations based on these processes that include biochemical, immunological and functional studies. Major conclusions include conservation of MPR-dependent pathway in higher invertebrates and recent evidence suggests that MPR-independent pathway might have been more prominent among lower invertebrates. The possible components of MPR-independent pathway that may play a role in lysosomal enzyme targeting are also discussed here.

  18. Subcellular Trafficking of Mammalian Lysosomal Proteins: An Extended View

    PubMed Central

    Staudt, Catherine; Puissant, Emeline; Boonen, Marielle

    2016-01-01

    Lysosomes clear macromolecules, maintain nutrient and cholesterol homeostasis, participate in tissue repair, and in many other cellular functions. To assume these tasks, lysosomes rely on their large arsenal of acid hydrolases, transmembrane proteins and membrane-associated proteins. It is therefore imperative that, post-synthesis, these proteins are specifically recognized as lysosomal components and are correctly sorted to this organelle through the endosomes. Lysosomal transmembrane proteins contain consensus motifs in their cytosolic regions (tyrosine- or dileucine-based) that serve as sorting signals to the endosomes, whereas most lysosomal acid hydrolases acquire mannose 6-phosphate (Man-6-P) moieties that mediate binding to two membrane receptors with endosomal sorting motifs in their cytosolic tails. These tyrosine- and dileucine-based motifs are tickets for boarding in clathrin-coated carriers that transport their cargo from the trans-Golgi network and plasma membrane to the endosomes. However, increasing evidence points to additional mechanisms participating in the biogenesis of lysosomes. In some cell types, for example, there are alternatives to the Man-6-P receptors for the transport of some acid hydrolases. In addition, several “non-consensus” sorting motifs have been identified, and atypical transport routes to endolysosomes have been brought to light. These “unconventional” or “less known” transport mechanisms are the focus of this review. PMID:28036022

  19. Passive diffusion of non-electrolytes across the lysosome membrane.

    PubMed Central

    Iveson, G P; Bird, S J; Lloyd, J B

    1989-01-01

    An osmotic-protection method has been used to study the permeability of rat liver lysosomes to 43 organic non-electrolytes of formula weights ranging from 62 to 1000. A lysosome-rich centrifugal fraction of rat liver homogenate was resuspended in an unbuffered 0.25 M solution of test solute, pH 7.0, and incubated at 25 degrees C for 60 min. The free and total activities of 4-methylumbelliferyl N-acetyl-beta-D-glucosaminidase were measured after incubation for 0, 30 and 60 min. Three patterns of results were seen. In pattern A the percentage free activity remained low throughout the 60 min incubation, indicating little or no solute entry into the lysosomes. In pattern B, the percentage free activity was initially low, but rose substantially during the incubation, indicating solute entry. In pattern C there was not even initial osmotic protection, indicating very rapid solute entry. The rapidity of solute entry into the lysosomes showed no correlation with the formula weight, but a perfect inverse correlation with the hydrogen-bonding capacity of the solutes. The results, which can be used to predict the ability of further compounds to cross the lysosome membrane by unassisted diffusion, are discussed in the context of metabolite and drug release from lysosomes in vivo. PMID:2775227

  20. IN VITRO INDUCTION OF LYSOSOMAL ENZYMES BY PHAGOCYTOSIS

    PubMed Central

    Axline, Stanton G.; Cohn, Zanvil A.

    1970-01-01

    The in vitro induction of lysosomal enzymes by phagocytosis was demonstrated in cultivated mouse peritoneal macrophages. The contribution of each of several steps in the endocytic process to enzyme induction was examined. The enzymatic response after the uptake of equal numbers of erythrocytes (RBC) and nondigestible particles were compared. Phagocytosis of RBC produced a marked increase in the levels of acid phosphatase, β-glucuronidase, and cathepsin D. Puromycin (1 µg/ml) inhibited the enzyme response. In contrast, phagocytosis of polyvinyl toluene, polystyrene, and insoluble starch particles produced no increase in macrophage lysosomal enzymes, although fusion of phagosomes with preexisting lysosomes occurred normally. The endocytic stimulus to synthesis of inducible lysosomal enzymes, therefore, occurred at or beyond the stage of digestion. Purified protein (bovine gamma globulin) aggregates and homopolymer coacervates of poly-l-glutamic acid: poly-l-lysine were effective inducers of lysosomal acid phosphatase, β-glucuronidase, and cathepsin D, whereas homopolymers of the same D-amino acids were ineffective as inducers. Both the quantity of phagocytized substrate and its rate of enzymatic hydrolysis appear to control the level and persistance of lysosomal hydrolases. PMID:4911552

  1. Lysosomal photodamage induces cell death via mitochondrial apoptotic pathway

    NASA Astrophysics Data System (ADS)

    Liu, Lei; Wang, Xian-wang; Li, Hui

    2009-11-01

    Lysosomal photosensitizers have been used in photodynamic therapy (PDT). Combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. The lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, we selected N-aspartyl chlorin e6 (NPe6), an effective photosensitizer which preferentially accumulates in lysosomes, to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells treated by NPe6-PDT was studied using real-time single-cell analysis. In this study, the fluorescence probes Cyto c-GFP and DsRed-Mit were used to detect the spatial and temporal changes of cytochrome c in real-time in sub-cell level; the Rhodamine 123 dyes were used to monitor the changes of mitochondrial membrane potential. The results showed that, after PDT treatment,the mitochondrial membrane potential decreased, and cytochrome c released from mitochondria; The caspase-3 was activated obviously. These results suggested that lysosomal photodamage activates mitochondrial apoptotic pathway to induce cell death.

  2. Studies on the involvement of lysosomes in estrogen action, I. Isolation and enzymatic properties of pig endometrial lysosomes.

    PubMed

    Sierralta, W; Truitt, A J; Jungblut, P W

    1978-04-01

    Pig endometrium cells, collected by curettage and homogenized in an all-glass Potter Elvehjem homogenizer, gave a considerably higher yield of intact mitochondria and lysosomes than homogenates of whole uterus obtained with the Ultraturrax or the Parr bomb. After homogenization of the cells and subfractionation in the presence of Mg2, mitochondria and lysosomes equilibrated at the same modal density in isopycnic centrifugation. Homogenization and subfractionation in buffers devoid of divalent cations and containing EDTA resulted in a decrease in the buoyant density of mitochondria, allowing for a separation from lysosomes. The pH optima and the specific activities of two mitochondrial enzymes and eight hydrolyases used as marker enzymes were determined. The morphological characteristics of fractions were established by electron microscopy. Preliminary results indicate an involvement of lysosomes in steroid metabolism rather than in steroid and receptor translocation into the nucleus.

  3. Enzymatic reduction of disulfide bonds in lysosomes: Characterization of a Gamma-interferon-inducible lysosomal thiol reductase (GILT)

    NASA Astrophysics Data System (ADS)

    Arunachalam, Balasubramanian; Phan, Uyen T.; Geuze, Hans J.; Cresswell, Peter

    2000-01-01

    Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter- and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin, but the mechanism of lysosomal reduction is unknown. We describe here a lysosomal thiol reductase optimally active at low pH and capable of catalyzing disulfide bond reduction both in vivo and in vitro. The active site, determined by mutagenesis, consists of a pair of cysteine residues separated by two amino acids, similar to other enzymes of the thioredoxin family. The enzyme is a soluble glycoprotein that is synthesized as a precursor. After delivery into the endosomal/lysosomal system by the mannose 6-phosphate receptor, N- and C-terminal prosequences are removed. The enzyme is expressed constitutively in antigen-presenting cells and induced by IFN-γ in other cell types, suggesting a potentially important role in antigen processing.

  4. Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis

    PubMed Central

    Napolitano, Gennaro; Johnson, Jennifer L; He, Jing; Rocca, Celine J; Monfregola, Jlenia; Pestonjamasp, Kersi; Cherqui, Stephanie; Catz, Sergio D

    2015-01-01

    Metabolite accumulation in lysosomal storage disorders (LSDs) results in impaired cell function and multi-systemic disease. Although substrate reduction and lysosomal overload-decreasing therapies can ameliorate disease progression, the significance of lysosomal overload-independent mechanisms in the development of cellular dysfunction is unknown for most LSDs. Here, we identify a mechanism of impaired chaperone-mediated autophagy (CMA) in cystinosis, a LSD caused by defects in the cystine transporter cystinosin (CTNS) and characterized by cystine lysosomal accumulation. We show that, different from other LSDs, autophagosome number is increased, but macroautophagic flux is not impaired in cystinosis while mTOR activity is not affected. Conversely, the expression and localization of the CMA receptor LAMP2A are abnormal in CTNS-deficient cells and degradation of the CMA substrate GAPDH is defective in Ctns−/− mice. Importantly, cysteamine treatment, despite decreasing lysosomal overload, did not correct defective CMA in Ctns−/− mice or LAMP2A mislocalization in cystinotic cells, which was rescued by CTNS expression instead, suggesting that cystinosin is important for CMA activity. In conclusion, CMA impairment contributes to cell malfunction in cystinosis, highlighting the need for treatments complementary to current therapies that are based on decreasing lysosomal overload. PMID:25586965

  5. Clinical Features of Lysosomal Acid Lipase Deficiency

    PubMed Central

    Burton, Barbara K.; Deegan, Patrick B.; Enns, Gregory M.; Guardamagna, Ornella; Horslen, Simon; Hovingh, Gerard K.; Lobritto, Steve J.; Malinova, Vera; McLin, Valerie A.; Raiman, Julian; Di Rocco, Maja; Santra, Saikat; Sharma, Reena; Sykut-Cegielska, Jolanta; Whitley, Chester B.; Eckert, Stephen; Valayannopoulos, Vassili; Quinn, Anthony G.

    2015-01-01

    Abstract Objective: The aim of this study was to characterize key clinical manifestations of lysosomal acid lipase deficiency (LAL D) in children and adults. Methods: Investigators reviewed medical records of LAL D patients ages ≥5 years, extracted historical data, and obtained prospective laboratory and imaging data on living patients to develop a longitudinal dataset. Results: A total of 49 patients were enrolled; 48 had confirmed LAL D. Mean age at first disease-related abnormality was 9.0 years (range 0–42); mean age at diagnosis was 15.2 years (range 1–46). Twenty-nine (60%) were male patients, and 27 (56%) were <20 years of age at the time of consent/assent. Serum transaminases were elevated in most patients with 458 of 499 (92%) of alanine aminotransferase values and 265 of 448 (59%) of aspartate aminotransferase values above the upper limit of normal. Most patients had elevated low-density lipoprotein (64% patients) and total cholesterol (63%) at baseline despite most being on lipid-lowering therapies, and 44% had high-density lipoprotein levels below the lower limit of normal. More than half of the patients with liver biopsies (n = 31, mean age 13 years) had documented evidence of steatosis (87%) and/or fibrosis (52%). Imaging assessments revealed that the median liver volume was ∼1.15 multiples of normal (MN) and median spleen volume was ∼2.2 MN. Six (13%) patients had undergone a liver transplant (ages 9–43.5 years). Conclusion: This study provides the largest longitudinal case review of patients with LAL D and confirms that LAL D is predominantly a pediatric disease causing early and progressive hepatic dysfunction associated with dyslipidemia that often leads to liver failure and transplantation. PMID:26252914

  6. Clinical Features of Lysosomal Acid Lipase Deficiency.

    PubMed

    Burton, Barbara K; Deegan, Patrick B; Enns, Gregory M; Guardamagna, Ornella; Horslen, Simon; Hovingh, Gerard K; Lobritto, Steve J; Malinova, Vera; McLin, Valerie A; Raiman, Julian; Di Rocco, Maja; Santra, Saikat; Sharma, Reena; Sykut-Cegielska, Jolanta; Whitley, Chester B; Eckert, Stephen; Valayannopoulos, Vassili; Quinn, Anthony G

    2015-12-01

    The aim of this study was to characterize key clinical manifestations of lysosomal acid lipase deficiency (LAL D) in children and adults. Investigators reviewed medical records of LAL D patients ages ≥5 years, extracted historical data, and obtained prospective laboratory and imaging data on living patients to develop a longitudinal dataset. A total of 49 patients were enrolled; 48 had confirmed LAL D. Mean age at first disease-related abnormality was 9.0 years (range 0-42); mean age at diagnosis was 15.2 years (range 1-46). Twenty-nine (60%) were male patients, and 27 (56%) were <20 years of age at the time of consent/assent. Serum transaminases were elevated in most patients with 458 of 499 (92%) of alanine aminotransferase values and 265 of 448 (59%) of aspartate aminotransferase values above the upper limit of normal. Most patients had elevated low-density lipoprotein (64% patients) and total cholesterol (63%) at baseline despite most being on lipid-lowering therapies, and 44% had high-density lipoprotein levels below the lower limit of normal. More than half of the patients with liver biopsies (n = 31, mean age 13 years) had documented evidence of steatosis (87%) and/or fibrosis (52%). Imaging assessments revealed that the median liver volume was ∼1.15 multiples of normal (MN) and median spleen volume was ∼2.2 MN. Six (13%) patients had undergone a liver transplant (ages 9-43.5 years). This study provides the largest longitudinal case review of patients with LAL D and confirms that LAL D is predominantly a pediatric disease causing early and progressive hepatic dysfunction associated with dyslipidemia that often leads to liver failure and transplantation.

  7. Caenorhabditis elegans HOPS and CCZ-1 mediate trafficking to lysosome-related organelles independently of RAB-7 and SAND-1

    PubMed Central

    Delahaye, Jared L.; Foster, Olivia K.; Vine, Annalise; Saxton, Daniel S.; Curtin, Thomas P.; Somhegyi, Hannah; Salesky, Rebecca; Hermann, Greg J.

    2014-01-01

    As early endosomes mature, the SAND-1/CCZ-1 complex acts as a guanine nucleotide exchange factor (GEF) for RAB-7 to promote the activity of its effector, HOPS, which facilitates late endosome–lysosome fusion and the consumption of AP-3–containing vesicles. We show that CCZ-1 and the HOPS complex are essential for the biogenesis of gut granules, cell type–specific, lysosome-related organelles (LROs) that coexist with conventional lysosomes in Caenorhabditis elegans intestinal cells. The HOPS subunit VPS-18 promotes the trafficking of gut granule proteins away from lysosomes and functions downstream of or in parallel to the AP-3 adaptor. CCZ-1 also acts independently of AP-3, and ccz-1 mutants mistraffic gut granule proteins. Our results indicate that SAND-1 does not participate in the formation of gut granules. In the absence of RAB-7 activity, gut granules are generated; however, their size and protein composition are subtly altered. These observations suggest that CCZ-1 acts in partnership with a protein other than SAND-1 as a GEF for an alternate Rab to promote gut granule biogenesis. Point mutations in GLO-1, a Rab32/38-related protein, predicted to increase spontaneous guanine nucleotide exchange, specifically suppress the loss of gut granules by ccz-1 and glo-3 mutants. GLO-3 is known to be required for gut granule formation and has homology to SAND-1/Mon1–related proteins, suggesting that CCZ-1 functions with GLO-3 upstream of the GLO-1 Rab, possibly as a GLO-1 GEF. These results support LRO formation occurring via processes similar to conventional lysosome biogenesis, albeit with key molecular differences. PMID:24501423

  8. Caenorhabditis elegans HOPS and CCZ-1 mediate trafficking to lysosome-related organelles independently of RAB-7 and SAND-1.

    PubMed

    Delahaye, Jared L; Foster, Olivia K; Vine, Annalise; Saxton, Daniel S; Curtin, Thomas P; Somhegyi, Hannah; Salesky, Rebecca; Hermann, Greg J

    2014-04-01

    As early endosomes mature, the SAND-1/CCZ-1 complex acts as a guanine nucleotide exchange factor (GEF) for RAB-7 to promote the activity of its effector, HOPS, which facilitates late endosome-lysosome fusion and the consumption of AP-3-containing vesicles. We show that CCZ-1 and the HOPS complex are essential for the biogenesis of gut granules, cell type-specific, lysosome-related organelles (LROs) that coexist with conventional lysosomes in Caenorhabditis elegans intestinal cells. The HOPS subunit VPS-18 promotes the trafficking of gut granule proteins away from lysosomes and functions downstream of or in parallel to the AP-3 adaptor. CCZ-1 also acts independently of AP-3, and ccz-1 mutants mistraffic gut granule proteins. Our results indicate that SAND-1 does not participate in the formation of gut granules. In the absence of RAB-7 activity, gut granules are generated; however, their size and protein composition are subtly altered. These observations suggest that CCZ-1 acts in partnership with a protein other than SAND-1 as a GEF for an alternate Rab to promote gut granule biogenesis. Point mutations in GLO-1, a Rab32/38-related protein, predicted to increase spontaneous guanine nucleotide exchange, specifically suppress the loss of gut granules by ccz-1 and glo-3 mutants. GLO-3 is known to be required for gut granule formation and has homology to SAND-1/Mon1-related proteins, suggesting that CCZ-1 functions with GLO-3 upstream of the GLO-1 Rab, possibly as a GLO-1 GEF. These results support LRO formation occurring via processes similar to conventional lysosome biogenesis, albeit with key molecular differences.

  9. Lysosomal Degradation of α-Synuclein in Vivo*

    PubMed Central

    Mak, Sally K.; McCormack, Alison L.; Manning-Boğ, Amy B.; Cuervo, Ana Maria; Di Monte, Donato A.

    2010-01-01

    Pathologic accumulation of α-synuclein is a feature of human parkinsonism and other neurodegenerative diseases. This accumulation may be counteracted by mechanisms of protein degradation that have been investigated in vitro but remain to be elucidated in animal models. In this study, lysosomal clearance of α-synuclein in vivo was indicated by the detection of α-synuclein in the lumen of lysosomes isolated from the mouse midbrain. When neuronal α-synuclein expression was enhanced as a result of toxic injury (i.e. treatment of mice with the herbicide paraquat) or transgenic protein overexpression, the intralysosomal content of α-synuclein was also significantly increased. This effect was paralleled by a marked elevation of the lysosome-associated membrane protein type 2A (LAMP-2A) and the lysosomal heat shock cognate protein of 70 kDa (hsc70), two essential components of chaperone-mediated autophagy (CMA). Immunofluorescence microscopy revealed an increase in punctate (lysosomal) LAMP-2A staining that co-localized with α-synuclein within nigral dopaminergic neurons of paraquat-treated and α-synuclein-overexpressing animals. The data provide in vivo evidence of lysosomal degradation of α-synuclein under normal conditions and, quite importantly, under conditions of enhanced protein burden. In the latter, increased lysosomal clearance of α-synuclein was mediated, at least in part, by CMA induction. It is conceivable that these neuronal mechanisms of protein clearance play an important role in neurodegenerative processes characterized by abnormal α-synuclein buildup. PMID:20200163

  10. Endolyn-78, a membrane glycoprotein present in morphologically diverse components of the endosomal and lysosomal compartments: implications for lysosome biogenesis

    PubMed Central

    1989-01-01

    A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn- 78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with

  11. Lysosomal Multienzyme Complex: Pros and Cons of Working Together

    PubMed Central

    Bonten, Erik J.; Annunziata, Ida; d’Azzo, Alessandra

    2014-01-01

    The ubiquitous distribution of lysosomes and their heterogeneous protein composition reflects the versatility of these organelles in maintaining cell homeostasis and their importance in tissue differentiation and remodeling. In lysosomes, the degradation of complex, macromolecular substrates requires the synergistic action of multiple hydrolases that usually work in a stepwise fashion. This catalytic machinery explains the existence of lysosomal enzyme complexes that can be dynamically assembled and disassembled to efficiently and quickly adapt to the pool of substrates to be processed or degraded, adding extra tiers to the regulation of the individual protein components. An example of such a complex is the one composed of three hydrolases that are ubiquitously but differentially expressed: the serine carboxypeptidase, Protective Protein/Cathepsin A (PPCA), the sialidase, Neuraminidase-1 (NEU1), and the glycosidase β-Galactosidase (β-GAL). Next to this ‘core’ complex, the existence of sub-complexes, that may contain additional components, and function at the cell surface or extracellularly, suggests as yet unexplored functions of these enzymes. Here we review how studies of basic biological processes in the mouse models of three lysosomal storage disorders, galactosialidosis, sialidosis, and GM1-gangliosidosis, revealed new and unexpected roles for the three respective affected enzymes, Ppca, Neu1 and β-Gal, that go beyond their canonical degradative activities. These findings have broadened our perspective on their functions and may pave the way for the development of new therapies for these lysosomal storage disorders. PMID:24337808

  12. Oxidative damage and redox in Lysosomal Storage Disorders: Biochemical markers.

    PubMed

    Donida, Bruna; Jacques, Carlos Eduardo Diaz; Mescka, Caroline Paula; Rodrigues, Daiane Grigolo Bardemaker; Marchetti, Desirèe Padilha; Ribas, Graziela; Giugliani, Roberto; Vargas, Carmen Regla

    2017-03-01

    Lysosomal Storage Disorders (LSD) comprise a heterogeneous group of >50 genetic disorders caused by mutations in genes that encode lysosomal enzymes, transport proteins or other gene products essential for a functional lysosomal system. As a result, abnormal accumulation of substrates within the lysosome leads to a progressive cellular impairment and dysfunction of numerous organs and systems. The exact mechanisms underlying the pathophysiology of LSD remain obscure. Previous studies proposed a relationship between oxidative stress and the pathogenesis of several inborn errors of metabolism, including LSD. Considering these points, in this paper it was reviewed oxidative stress and emerging antioxidant therapy in LSD, emphasizing studies with biological samples from patients affected by this group of conditions. These studies allow presuming that metabolites accumulated in LSD cause an increase of lysosomes' number and size, which may induce excessive production of reactive species and/or deplete the tissue antioxidant capacity, leading to damage in biomolecules. In vitro and in vivo evidence showed that cell oxidative process occurs in LSD and probably contributes to the pathophysiology of these disorders. In this context, it is possible to suggest that, in the future, antioxidants could come to be used as adjuvant therapy for LSD patients.

  13. Membrane proteins of dense lysosomes from Chinese hamster ovary cells

    SciTech Connect

    Chance, S.C.

    1987-01-01

    In this work membrane proteins from lysosomes were studied in order to gain more information on the biogenesis and intracellular sorting of this class of membrane proteins. Membrane proteins were isolated from a purified population of lysosomes. These proteins were then examined for various co- and post-translational modifications which could serve as potential intracellular sorting signals. Biochemical analysis using marker enzymatic activities detected no plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, mitochondria, or cytosol. Analysis after incorporation of ({sup 3}H)thymidine or ({sup 3}H)uridine detected no nuclei or ribosomes. A fraction containing integral membrane proteins was obtained from the dense lysosomes by extraction with Triton X-114. Twenty-three polypeptides which incorporated both ({sup 35}S)methionine and ({sup 3}H)leucine were detected by SDS PAGE in this membrane fraction, and ranged in molecular weight from 30-130 kDa. After incorporation by cells of various radioactive metabolic precursors, the membrane fraction from dense lysosomes was examined and was found to be enriched in mannose, galactose, fucose, palmitate, myristate, and sulfate, but was depleted in phosphate. The membrane fraction from dense lysosomes was then analyzed by SDS PAGE to determine the apparent molecular weights of modified polypepties.

  14. Proteasomal and lysosomal protein degradation and heart disease.

    PubMed

    Wang, Xuejun; Robbins, Jeffrey

    2014-06-01

    In the cell, the proteasome and lysosomes represent the most important proteolytic machineries, responsible for the protein degradation in the ubiquitin-proteasome system (UPS) and autophagy, respectively. Both the UPS and autophagy are essential to protein quality and quantity control. Alterations in cardiac proteasomal and lysosomal degradation are remarkably associated with most heart disease in humans and are implicated in the pathogenesis of congestive heart failure. Studies carried out in animal models and in cell culture have begun to establish both sufficiency and, in some cases, the necessity of proteasomal functional insufficiency or lysosomal insufficiency as a major pathogenic factor in the heart. This review article highlights some recent advances in the research into proteasome and lysosome protein degradation in relation to cardiac pathology and examines the emerging evidence for enhancing degradative capacities of the proteasome and/or lysosome as a new therapeutic strategy for heart disease. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy". Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. BACE2 degradation mediated by the macroautophagy-lysosome pathway.

    PubMed

    Liu, Xi; Wang, Zhe; Wu, Yili; Wang, Jianping; Song, Weihong

    2013-06-01

    Neuritic plaque is the pathological hallmark in Alzheimer's disease (AD). Amyloid-β protein (Aβ), the central component of neuritic plaques, is generated from amyloid-β precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. β-site APP cleaving enzyme 2 (BACE2), a homolog of BACE1, functions differently from BACE1 in APP processing. BACE1 is the β-secretase essential for Aβ production, and BACE2, a θ-secretase, cleaves APP within the Aβ domain, preventing Aβ production. Elucidation of the mechanism underlying BACE2 degradation is important for defining its biological features and its potential role in Alzheimer's disease drug development. In this report we first showed that the half-life of BACE2 is approximately 20 h. Lysosomal inhibition increased BACE2 protein levels whereas proteasomal inhibition had no effect on BACE2 protein expression. Furthermore, we identified that macroautophagy mediated BACE2 degradation. Finally, we showed that lysosomal inhibition increased BACE2 cleavage of APP. Taken together, our in vitro study showed that BACE2 is degraded through the macrophagy-lysosome pathway and that lysosomal inhibition affects BACE2 processing of APP. Modulation of BACE2 degradation via the lysosomal pathway could be a new target for AD drug development.

  16. Lysosome-mediated processing of chromatin in senescence.

    PubMed

    Ivanov, Andre; Pawlikowski, Jeff; Manoharan, Indrani; van Tuyn, John; Nelson, David M; Rai, Taranjit Singh; Shah, Parisha P; Hewitt, Graeme; Korolchuk, Viktor I; Passos, Joao F; Wu, Hong; Berger, Shelley L; Adams, Peter D

    2013-07-08

    Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C-negative, but strongly γ-H2AX-positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression.

  17. Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress

    PubMed Central

    Pascua-Maestro, Raquel

    2017-01-01

    Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular

  18. Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress.

    PubMed

    Pascua-Maestro, Raquel; Diez-Hermano, Sergio; Lillo, Concepción; Ganfornina, Maria D; Sanchez, Diego

    2017-02-01

    Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular

  19. GFP-like proteins stably accumulate in lysosomes.

    PubMed

    Katayama, Hiroyuki; Yamamoto, Akitsugu; Mizushima, Noboru; Yoshimori, Tamotsu; Miyawaki, Atsushi

    2008-01-01

    Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its GFP variants (Aequorea GFPs), and more recently the novel GFP-like proteins from Anthozoa have greatly advanced our technologies for fluorescently labeling cells, organelles, and proteins. It has been shown, however, that some GFP-like proteins have a tendency to oligomerize and aggregate. Transfection of GFP-like proteins into cultured mammalian cells results in bright punctate structures, which are thought to be cytosolic protein aggregates. In this study, we demonstrate that these structures are not cytosolic aggregates but lysosomes that have accumulated the GFP-like proteins. Our biochemical and immunocytochemical experiments have revealed that certain GFP-like proteins expressed in the cytosol enter lysosomes possibly by an autophagy-related mechanism, but retain their fluorescence because of resistance not only to acidity but also to lysosomal proteases.

  20. Cellular proteostasis: degradation of misfolded proteins by lysosomes

    PubMed Central

    Jackson, Matthew P.

    2016-01-01

    Proteostasis refers to the regulation of the cellular concentration, folding, interactions and localization of each of the proteins that comprise the proteome. One essential element of proteostasis is the disposal of misfolded proteins by the cellular pathways of protein degradation. Lysosomes are an important site for the degradation of misfolded proteins, which are trafficked to this organelle by the pathways of macroautophagy, chaperone-mediated autophagy and endocytosis. Conversely, amyloid diseases represent a failure in proteostasis, in which proteins misfold, forming amyloid deposits that are not degraded effectively by cells. Amyloid may then exacerbate this failure by disrupting autophagy and lysosomal proteolysis. However, targeting the pathways that regulate autophagy and the biogenesis of lysosomes may present approaches that can rescue cells from the deleterious effects of amyloidogenic proteins. PMID:27744333

  1. Salinomycin kills cancer stem cells by sequestering iron in lysosomes

    NASA Astrophysics Data System (ADS)

    Mai, Trang Thi; Hamaï, Ahmed; Hienzsch, Antje; Cañeque, Tatiana; Müller, Sebastian; Wicinski, Julien; Cabaud, Olivier; Leroy, Christine; David, Amandine; Acevedo, Verónica; Ryo, Akihide; Ginestier, Christophe; Birnbaum, Daniel; Charafe-Jauffret, Emmanuelle; Codogno, Patrice; Mehrpour, Maryam; Rodriguez, Raphaël

    2017-10-01

    Cancer stem cells (CSCs) represent a subset of cells within tumours that exhibit self-renewal properties and the capacity to seed tumours. CSCs are typically refractory to conventional treatments and have been associated to metastasis and relapse. Salinomycin operates as a selective agent against CSCs through mechanisms that remain elusive. Here, we provide evidence that a synthetic derivative of salinomycin, which we named ironomycin (AM5), exhibits a more potent and selective activity against breast CSCs in vitro and in vivo, by accumulating and sequestering iron in lysosomes. In response to the ensuing cytoplasmic depletion of iron, cells triggered the degradation of ferritin in lysosomes, leading to further iron loading in this organelle. Iron-mediated production of reactive oxygen species promoted lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis. These findings reveal the prevalence of iron homeostasis in breast CSCs, pointing towards iron and iron-mediated processes as potential targets against these cells.

  2. Parkinson's Disease Shares the Lysosome with Gaucher's Disease

    PubMed Central

    Dawson, Ted M.; Dawson, Valina L.

    2015-01-01

    Summary The second most common neurodegenerative disorder, Parkinson's disease (PD) is an age dependent progressive neurodegenerative disorder that affects movement. While many of the causes of PD remain unclear, a consistent finding in PD is the abnormal accumulation of α-synuclein that has lead to the widely held notion that PD is a synucleinopathy. In a recent Cell manuscript Mazzuli et al., provide a potential mechanistic link between Gaucher's disease, a glycolipid lysosomal storage disorder due to Glucocerebrocidase (GBA) deficiency and PD. The authors reveal a reciprocal connection between the loss of GBA activity and accumulation of α-synuclein in the lysosome establishing a bidirectional positive feed back loop with pathologic consequences. These findings should stimulate further work on role of the lysosome in PD pathogenesis and the identification of new treatment strategies for PD. PMID:21753118

  3. Aneuploidy triggers a TFEB-mediated lysosomal stress response

    PubMed Central

    Santaguida, Stefano; Amon, Angelika

    2015-01-01

    Aneuploidy, defined as an alteration in chromosome number that is not a multiple of the haploid complement, severely affects cellular physiology. Changes in chromosome number lead to imbalances in cellular protein composition, thus disrupting cellular processes and causing proteins to misfold and aggregate. We recently reported that in mammalian cells protein aggregates are readily encapsulated within autophagosomes but are not degraded by lysosomes. This leads to a lysosomal stress response in which the transcription factor TFEB induces expression of factors needed for macroautophagy-mediated protein degradation. Our studies uncover lysosomal degradation defects as a feature of the aneuploid state, and a role for the transcription factor TFEB in the response thereto. PMID:26571033

  4. Rab2 promotes autophagic and endocytic lysosomal degradation.

    PubMed

    Lőrincz, Péter; Tóth, Sarolta; Benkő, Péter; Lakatos, Zsolt; Boda, Attila; Glatz, Gábor; Zobel, Martina; Bisi, Sara; Hegedűs, Krisztina; Takáts, Szabolcs; Scita, Giorgio; Juhász, Gábor

    2017-07-03

    Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes. © 2017 Lőrincz et al.

  5. Rotenone impairs autophagic flux and lysosomal functions in Parkinson's disease.

    PubMed

    Wu, F; Xu, H-D; Guan, J-J; Hou, Y-S; Gu, J-H; Zhen, X-C; Qin, Z-H

    2015-01-22

    Rotenone is an environmental neurotoxin that induces accumulation of α-synuclein and degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc), but the molecular mechanisms are not fully understood. We investigated whether rotenone induced impairment of autophagic flux and lysosomal functions. Autophagy flux, accumulation of α-synuclein, lysosomal membrane integrity and neurodegeneration were assessed in the rotenone-treated rat model and PC12 cells, and the effects of the autophagy inducer trehalose on rotenone's cytotoxicity were also studied. Rotenone administration significantly reduced motor activity and caused a loss of tyrosine hydroxylase in SNpc of Lewis rats. The degeneration of nigral dopaminergic neurons was accompanied by the deposition of α-synuclein aggregates, autophagosomes and redistribution of cathepsin D from lysosomes to the cytosol. In cultured PC12 cells, rotenone also induced increases in protein levels of α-synuclein, microtubule-associated protein 1 light chain 3-II, Beclin 1, and p62. Rotenone increased lysosomal membrane permeability as evidenced by leakage of N-acetyl-beta-d-glucosaminidase and cathepsin D, the effects were blocked by reactive oxygen species scavenger tiron. Autophagy inducer trehalose enhanced the nuclear translocation of transcription factor EB, accelerated the clearance of autophagosomes and α-synuclein and attenuated rotenone-induced cell death of PC12 cells. Meanwhile, administration of trehalose to rats in drinking water (2%) decreased rotenone-induced dopaminergic neurons loss in SNpc. These studies indicate that the lysosomal dysfunction contributes to rotenone's neurotoxicity and restoration of lysosomal function could be a new therapeutic strategy for Parkinson's disease. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Specific lysosomal transport of small neutral amino acids

    SciTech Connect

    Pisoni, R.L.; Flickinger, K.S.; Thoene, J.G.; Christensen, H.N.

    1986-05-01

    Studies of amino acid exodus from lysosomes have allowed us previously to describe transport systems specific for cystine and another for cationic amino acids in fibroblast lysosomes. They are now able to study amino acid uptake into highly purified fibroblast lysosomes obtained by separating crude granular fraction on gradients formed by centrifugation in 35% isoosmotic Percoll solutions. Analog inhibition and saturation studies indicate that L-(/sup 14/C)proline (50 ..mu..M) uptake by fibroblast lysosomes at 37/sup 0/C in 50 mM citrate/tris pH 7.0 buffer containing 0.25 M sucrose is mediated by two transport systems, one largely specific for L-proline and the other for which transport is shared with small neutral amino acids such as alanine, serine and threonine. At 7 mM, L-proline inhibits L-(/sup 14/C)proline uptake almost completely, whereas ala, ser, val, thr, gly, N-methylalanine and sarcosine inhibit proline uptake by 50-65%. The system shared by alanine, serine and threonine is further characterized by these amino acids strongly inhibiting the uptakes of each other. Lysosomal proline transport is selective for the L-isomer of the amino acid, and is scarcely inhibited by 7 mM arg, glu, asp, leu, phe, his, met, (methylamino) isobutyrate, betaine or N,N-dimethylglycine. Cis or trans-4-hydroxy-L-proline inhibit proline uptake only slightly. In sharp contrast to the fibroblast plasma membrane in which Na/sup +/ is required for most proline and alanine transport, lysosomal uptake of these amino acids occurs independently of Na/sup +/.

  7. Targeting Androgen Receptor by Lysosomal Degradation in Prostate Cancer

    DTIC Science & Technology

    2014-09-01

    mTOR allosteric inhibitors as therapy for Ewing sarcoma . ■ ASSOCIATED CONTENT * S Supporting Information Table S1: Preliminary list of FLAG-His- EWS -Fli...course of this project to demonstrate the role of the TFEB – lysosome pathway in the turnover of EWS -Fli-1, a fusion oncoprotein of Ewing sarcoma (Note...degradation by TFEB (Figure 2A) and a lysosomal protease, cathepsin D, can proteolyze EWS -Fli-1 both in 293 cells (Figure 2B) and in A673 Ewing sarcoma

  8. PDT: loss of autophagic cytoprotection after lysosomal photodamage

    NASA Astrophysics Data System (ADS)

    Kessel, David; Price, Michael

    2012-02-01

    Photodynamic therapy is known to evoke both autophagy and apoptosis. Apoptosis is an irreversible death pathway while autophagy can serve a cytoprotective function. In this study, we examined two photosensitizing agents that target lysosomes, although they differ in the reactive oxygen species (ROS) formed during irradiation. With both agents, the 'shoulder' on the PDT dose-response curve was substantially attenuated, consistent with loss of a cytoprotective pathway. In contrast, this 'shoulder' is commonly observed when PDT targets mitochondria or the ER. We propose that lysosomal targets may offer the possibility of promoting PDT efficacy by eliminating a potentially protective pathway.

  9. Genomic organization of the human lysosomal acid lipase gene (LIPA)

    SciTech Connect

    Aslandis, C.; Klima, H.; Lackner, K.J.; Schmitz, G. )

    1994-03-15

    Defects in the human lysosomal acid lipase gene are responsible for cholesteryl ester storage disease (CESD) and Wolman disease. Exon skipping as the cause for CESD has been demonstrated. The authors present here a summary of the exon structure of the entire human lysosomal acid lipase gene consisting of 10 exons, together with the sizes of genomic EcoRI and SacI fragments hybridizing to each exon. In addition, the DNA sequence of the putative promoter region is presented. The EMBL accession numbers for adjacent intron sequences are given. 7 refs., 2 figs., 1 tab.

  10. Lysosome stability during lytic infection by simian virus 40.

    PubMed

    Einck, K H; Norkin, L C

    1979-01-01

    By 48 h postinfection, 40--80% of SV40-infected CV-1 cells have undergone irreversible injury as indicated by trypan blue staining. Nevertheless, at this time the lysosomes of these cells appear as discrete structures after vital staining with either acridine orange or neutral red. Lysosomes, vitally stained with neutral red at 24 h postinfection, were still intact in cells stained with trypan blue at 48 h. Acid phosphatase activity is localized in discrete cytoplasmic particles at 48 h, as indicated by histochemical staining of both fixed and unfixed cells.

  11. Protein kinase C-δ isoform mediates lysosome labilization in DNA damage-induced apoptosis

    PubMed Central

    PARENT, NICOLAS; SCHERER, MAX; LIEBISCH, GERHARD; SCHMITZ, GERD; BERTRAND, RICHARD

    2013-01-01

    A lysosomal pathway, characterized by the partial rupture or labilization of lysosomal membranes (LLM) and cathepsin release into the cytosol, is evoked during the early events of 20-S-camptothecin lactone (CPT)-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. Recently, in a comparative proteomics analysis performed on highly-enriched lysosomal extracts, we identified proteins whose translocation to lysosomes correlated with LLM induction after CPT treatment, including protein kinase C-δ (PKC-δ). In this study, we show that the PKC-δ translocation to lysosomes is required for LLM, as silencing its expression with RNA interference or suppressing its activity with the inhibitor, rottlerin, prevents CPT-induced LLM. PKC-δ translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase in ceramide (CER) content in lysosomes. The accumulation of endogenous CER in lysosomes is a critical event for CPT-induced LLM as suppressing PKC-δ or ASM activity reduces both the CPT-mediated CER generation in lysosomes and CPT-induced LLM. These findings reveal a novel mechanism by which PKC-δ mediates ASM phosphorylation/activation and CER accumulation in lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment. PMID:21174057

  12. The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes

    PubMed Central

    Garrity, Abigail G; Wang, Wuyang; Collier, Crystal MD; Levey, Sara A; Gao, Qiong; Xu, Haoxing

    2016-01-01

    Impaired homeostasis of lysosomal Ca2+ causes lysosome dysfunction and lysosomal storage diseases (LSDs), but the mechanisms by which lysosomes acquire and refill Ca2+ are not known. We developed a physiological assay to monitor lysosomal Ca2+ store refilling using specific activators of lysosomal Ca2+ channels to repeatedly induce lysosomal Ca2+ release. In contrast to the prevailing view that lysosomal acidification drives Ca2+ into the lysosome, inhibiting the V-ATPase H+ pump did not prevent Ca2+ refilling. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca2+ prevented lysosomal Ca2+ stores from refilling. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca2+ refilling of lysosomes, but not in cells lacking IP3Rs. Furthermore, reducing ER Ca2+ or blocking IP3Rs caused a dramatic LSD-like lysosome storage phenotype. By closely apposing each other, the ER may serve as a direct and primary source of Ca2+for the lysosome. DOI: http://dx.doi.org/10.7554/eLife.15887.001 PMID:27213518

  13. Properties of the membrane proteins of rat liver lysosomes. The majority of lysosomal membrane proteins are exposed to the cytoplasm.

    PubMed Central

    Schneider, D L; Burnside, J; Gorga, F R; Nettleton, C J

    1978-01-01

    Rat liver lysosomes were lysed and subfractionated by differential centrifugation through 0.2M-NaCl to yield a membranous pellet. This membrane fraction contains less than 20% of the lysosomal protein, adenosine triphosphatase activity of about 1.2mumol/min per mg of protein, 120nmol of thiol groups/mg of protein and at least 16 protein and glycoprotein bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The gel patterns of membranes isolated from lysosomes after treatment with (1) [125I]iodidehydrogen peroxide-lactoperoxidase, (2) toluene 2,4-di-isocyanate-activated bovine serum albumin, (3) trypsin and (4) subtilisin indicate that most of the membrane proteins are exposed to the cytoplasm. These exposed proteins are candidates for intracellular receptors which recognize either substances that are to be degraded or vesicles containing those substances. PMID:153136

  14. Ferritin-stimulated lipid peroxidation, lysosomal leak, and macroautophagy promote lysosomal "metastability" in primary hepatocytes determining in vitro cell survival.

    PubMed

    Krenn, Margit A; Schürz, Melanie; Teufl, Bernhard; Uchida, Koji; Eckl, Peter M; Bresgen, Nikolaus

    2015-03-01

    Several pathologies are associated with elevated levels of serum ferritin, for which growth inhibitory properties have been reported; however, the underlying mechanisms are still poorly defined. Previously we have described cytotoxic properties of isoferritins released from primary hepatocytes in vitro, which induce apoptosis in an iron and oxidative stress-dependent mode. Here we show that this ferritin species stimulates endosome clustering and giant endosome formation in primary hepatocytes accompanied by enhanced lysosomal membrane permeability (LMP). In parallel, protein modification by lipid peroxidation-derived 4-hydroxynonenal (HNE) is strongly promoted by ferritin, the HNE-modified proteins (HNE-P) showing remarkable aggregation. Emphasizing the prooxidant context, GSH is rapidly depleted and the GSH/GSSG ratio is substantially declining in ferritin-treated cells. Furthermore, ferritin triggers a transient upregulation of macroautophagy which is abolished by iron chelation and apparently supports HNE-P clearance. Macroautophagy inhibition by 3-methyladenine strongly amplifies ferritin cytotoxicity in a time- and concentration-dependent mode, suggesting an important role of macroautophagy on cellular responses to ferritin endocytosis. Moreover, pointing at an involvement of lysosomal proteolysis, ferritin cytotoxicity and lysosome fragility are aggravated by the protease inhibitor leupeptin. In contrast, EGF which suppresses ferritin-induced cell death attenuates ferritin-mediated LMP. In conclusion, we propose that HNE-P accumulation, lysosome dysfunction, and macroautophagy stimulated by ferritin endocytosis provoke lysosomal "metastability" in primary hepatocytes which permits cell survival as long as in- and extrinsic determinants (e.g., antioxidant availability, damage repair, EGF signaling) keep the degree of lysosomal destabilization below cell death-inducing thresholds.

  15. A Rab3a-dependent complex essential for lysosome positioning and plasma membrane repair

    PubMed Central

    Encarnação, Marisa; Mateus, Denisa; Michelet, Xavier; Santarino, Inês; Hsu, Victor W.; Brenner, Michael B.

    2016-01-01

    Lysosome exocytosis plays a major role in resealing plasma membrane (PM) disruptions. This process involves two sequential steps. First, lysosomes are recruited to the periphery of the cell and then fuse with the damaged PM. However, the trafficking molecular machinery involved in lysosome exocytosis and PM repair (PMR) is poorly understood. We performed a systematic screen of the human Rab family to identify Rabs required for lysosome exocytosis and PMR. Rab3a, which partially localizes to peripheral lysosomes, was one of the most robust hits. Silencing of Rab3a or its effector, synaptotagmin-like protein 4a (Slp4-a), leads to the collapse of lysosomes to the perinuclear region and inhibition of PMR. Importantly, we have also identified a new Rab3 effector, nonmuscle myosin heavy chain IIA, as part of the complex formed by Rab3a and Slp4-a that is responsible for lysosome positioning at the cell periphery and lysosome exocytosis. PMID:27325790

  16. The relationship between Cd-induced autophagy and lysosomal activation in WRL-68 cells.

    PubMed

    Meng, Su-Fang; Mao, Wei-Ping; Wang, Fang; Liu, Xiao-Qian; Shao, Luan-Luan

    2015-11-01

    This study shows that Cd induces autophagy in the human's embryonic normal liver cell line (WRL-68). The expression of LC3B-II and the mature cathepsin L were analyzed by Western blotting. The autophagosomes and lysosomes were directly visualized by electron microscopy and confocal microscopy analysis in Cd-exposed WRL-68 cells. In this study, we first found that autophagy induced the activation of lysosomal function in WRL-68 cells. The lysosomal activation was markedly decreased when the cells were co-treated with 3-MA (an inhibitor of autophagy). Secondly, we provided the evidence that the activation of lysosomal function depended on autophagosome-lysosome fusion. The colocalization of lysosome-associated membrane protein-2 (LAMP2) and GFP-LC3 was significantly reduced, when they were treated with thapsigargin (an inhibitor of autophagosome-lysosome fusion). We demonstrated that deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, which suggests that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Thirdly, we provided evidence that the activation of lysosomal function was associated with lysosomal acid. We investigated the relationship between autophagosome-lysosome fusion and pH in acidic compartments by visualizing fusion process in WRL-68 cells. This suggests that increasing pH in acidic compartments in WRL-68 cells inhibits the autophagosome-lysosome fusion. Finally, we found that the activation of lysosomal function was associated with Ca(2+) stores and the intracellular Ca(2+) channels or pumps were possibly pH-dependent.

  17. PIG7 promotes leukemia cell chemosensitivity via lysosomal membrane permeabilization.

    PubMed

    Liu, Jiazhuo; Peng, Leiwen; Niu, Ting; Wu, Yu; Li, Jianjun; Wang, Fangfang; Zheng, Yuhuan; Liu, Ting

    2016-01-26

    PIG7 localizes to lysosomal membrane in leukemia cells. Our previous work has shown that transduction of pig7 into a series of leukemia cell lines did not result in either apoptosis or differentiation of most tested cell lines. Interestingly, it did significantly sensitize these cell lines to chemotherapeutic drugs. Here, we further investigated the mechanism underlying pig7-induced improved sensitivity of acute leukemia cells to chemotherapy. Our results demonstrated that the sensitization effect driven by exogenous pig7 was more effective in drug-resistant leukemia cell lines which had lower endogenous pig7 expression. Overexpression of pig7 did not directly activate the caspase apoptotic pathway, but decreased the lysosomal stability. The expression of pig7 resulted in lysosomal membrane permeabilization (LMP) and lysosomal protease (e.g. cathepsin B, D, L) release. Moreover, we also observed increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) induced by pig7. Some autophagy markers such as LC3I/II, ATG5 and Beclin-1, and necroptosis maker MLKL were also stimulated. However, intrinsic antagonism such as serine/cysteine protease inhibitors Spi2A and Cystatin C prevented downstream effectors from triggering leukemia cells, which were only on the "verge of apoptosis". When combined with chemotherapy, LMP increased and more proteases were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways.

  18. Lysosomal Cholesterol Accumulation Sensitizes To Acetaminophen Hepatotoxicity by Impairing Mitophagy.

    PubMed

    Baulies, Anna; Ribas, Vicent; Núñez, Susana; Torres, Sandra; Alarcón-Vila, Cristina; Martínez, Laura; Suda, Jo; Ybanez, Maria D; Kaplowitz, Neil; García-Ruiz, Carmen; Fernández-Checa, Jose C

    2015-12-11

    The role of lysosomes in acetaminophen (APAP) hepatotoxicity is poorly understood. Here, we investigated the impact of genetic and drug-induced lysosomal cholesterol (LC) accumulation in APAP hepatotoxicity. Acid sphingomyelinase (ASMase)(-/-) mice exhibit LC accumulation and higher mortality after APAP overdose compared to ASMase(+/+) littermates. ASMase(-/-) hepatocytes display lower threshold for APAP-induced cell death and defective fusion of mitochondria-containing autophagosomes with lysosomes, which decreased mitochondrial quality control. LC accumulation in ASMase(+/+) hepatocytes caused by U18666A reproduces the susceptibility of ASMase(-/-) hepatocytes to APAP and the impairment in the formation of mitochondria-containing autolysosomes. LC extraction by 25-hydroxycholesterol increased APAP-mediated mitophagy and protected ASMase(-/-) mice and hepatocytes against APAP hepatotoxicity, effects that were reversed by chloroquine to disrupt autophagy. The regulation of LC by U18666A or 25-hydroxycholesterol did not affect total cellular sphingomyelin content or its lysosomal distribution. Of relevance, amitriptyline-induced ASMase inhibition in human hepatocytes caused LC accumulation, impaired mitophagy and increased susceptibility to APAP. Similar results were observed upon glucocerebrosidase inhibition by conduritol β-epoxide, a cellular model of Gaucher disease. These findings indicate that LC accumulation determines susceptibility to APAP hepatotoxicity by modulating mitophagy, and imply that genetic or drug-mediated ASMase disruption sensitizes to APAP-induced liver injury.

  19. Lysosomal Cholesterol Accumulation Sensitizes To Acetaminophen Hepatotoxicity by Impairing Mitophagy

    PubMed Central

    Baulies, Anna; Ribas, Vicent; Núñez, Susana; Torres, Sandra; Alarcón-Vila, Cristina; Martínez, Laura; Suda, Jo; Ybanez, Maria D.; Kaplowitz, Neil; García-Ruiz, Carmen; Fernández-Checa, Jose C.

    2015-01-01

    The role of lysosomes in acetaminophen (APAP) hepatotoxicity is poorly understood. Here, we investigated the impact of genetic and drug-induced lysosomal cholesterol (LC) accumulation in APAP hepatotoxicity. Acid sphingomyelinase (ASMase)−/− mice exhibit LC accumulation and higher mortality after APAP overdose compared to ASMase+/+ littermates. ASMase−/− hepatocytes display lower threshold for APAP-induced cell death and defective fusion of mitochondria-containing autophagosomes with lysosomes, which decreased mitochondrial quality control. LC accumulation in ASMase+/+ hepatocytes caused by U18666A reproduces the susceptibility of ASMase−/− hepatocytes to APAP and the impairment in the formation of mitochondria-containing autolysosomes. LC extraction by 25-hydroxycholesterol increased APAP-mediated mitophagy and protected ASMase−/− mice and hepatocytes against APAP hepatotoxicity, effects that were reversed by chloroquine to disrupt autophagy. The regulation of LC by U18666A or 25-hydroxycholesterol did not affect total cellular sphingomyelin content or its lysosomal distribution. Of relevance, amitriptyline-induced ASMase inhibition in human hepatocytes caused LC accumulation, impaired mitophagy and increased susceptibility to APAP. Similar results were observed upon glucocerebrosidase inhibition by conduritol β-epoxide, a cellular model of Gaucher disease. These findings indicate that LC accumulation determines susceptibility to APAP hepatotoxicity by modulating mitophagy, and imply that genetic or drug-mediated ASMase disruption sensitizes to APAP-induced liver injury. PMID:26657973

  20. Genetic Regulation of Caenorhabditis elegans Lysosome Related Organelle Function

    PubMed Central

    Soukas, Alexander A.; Carr, Christopher E.; Ruvkun, Gary

    2013-01-01

    Lysosomes are membrane-bound organelles that contain acid hydrolases that degrade cellular proteins, lipids, nucleic acids, and oligosaccharides, and are important for cellular maintenance and protection against age-related decline. Lysosome related organelles (LROs) are specialized lysosomes found in organisms from humans to worms, and share many of the features of classic lysosomes. Defective LROs are associated with human immune disorders and neurological disease. Caenorhabditis elegans LROs are the site of concentration of vital dyes such as Nile red as well as age-associated autofluorescence. Even though certain short-lived mutants have high LRO Nile red and high autofluorescence, and other long-lived mutants have low LRO Nile red and low autofluorescence, these two biologies are distinct. We identified a genetic pathway that modulates aging-related LRO phenotypes via serotonin signaling and the gene kat-1, which encodes a mitochondrial ketothiolase. Regulation of LRO phenotypes by serotonin and kat-1 in turn depend on the proton-coupled, transmembrane transporter SKAT-1. skat-1 loss of function mutations strongly suppress the high LRO Nile red accumulation phenotype of kat-1 mutation. Using a systems approach, we further analyzed the role of 571 genes in LRO biology. These results highlight a gene network that modulates LRO biology in a manner dependent upon the conserved protein kinase TOR complex 2. The results implicate new genetic pathways involved in LRO biology, aging related physiology, and potentially human diseases of the LRO. PMID:24204312

  1. Selective Lysosomal Transporter Degradation by Organelle Membrane Fusion.

    PubMed

    McNally, Erin Kate; Karim, Mahmoud Abdul; Brett, Christopher Leonard

    2017-01-23

    Lysosomes rely on their resident transporter proteins to return products of catabolism to the cell for reuse and for cellular signaling, metal storage, and maintaining the lumenal environment. Despite their importance, little is known about the lifetime of these transporters or how they are regulated. Using Saccharomyces cerevisiae as a model, we discovered a new pathway intrinsic to homotypic lysosome membrane fusion that is responsible for their degradation. Transporter proteins are selectively sorted by the docking machinery into an area between apposing lysosome membranes, which is internalized and degraded by lumenal hydrolases upon organelle fusion. These proteins have diverse lifetimes that are regulated in response to protein misfolding, changing substrate levels, or TOR activation. Analogous to endocytosis for controlling surface protein levels, the "intralumenal fragment pathway" is critical for lysosome membrane remodeling required for organelle function in the context of cellular protein quality control, ion homeostasis, and metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Structure of human saposin A at lysosomal pH

    SciTech Connect

    Hill, Chris H.; Read, Randy J.; Deane, Janet E.

    2015-06-27

    A 1.8 Å resolution structure of the sphingolipid activator protein saposin A has been determined at pH 4.8, the physiologically relevant lysosomal pH for hydrolase enzyme activation and lipid-transfer activity. The saposins are essential cofactors for the normal lysosomal degradation of complex glycosphingolipids by acid hydrolase enzymes; defects in either saposin or hydrolase function lead to severe metabolic diseases. Saposin A (SapA) activates the enzyme β-galactocerebrosidase (GALC), which catalyzes the breakdown of β-d-galactocerebroside, the principal lipid component of myelin. SapA is known to bind lipids and detergents in a pH-dependent manner; this is accompanied by a striking transition from a ‘closed’ to an ‘open’ conformation. However, previous structures were determined at non-lysosomal pH. This work describes a 1.8 Å resolution X-ray crystal structure determined at the physiologically relevant lysosomal pH 4.8. In the absence of lipid or detergent at pH 4.8, SapA is observeed to adopt a conformation closely resembling the previously determined ‘closed’ conformation, showing that pH alone is not sufficient for the transition to the ‘open’ conformation. Structural alignments reveal small conformational changes, highlighting regions of flexibility.

  3. Lysosomal β-glucuronidase regulates Lyme and rheumatoid arthritis severity.

    PubMed

    Bramwell, Kenneth K C; Ma, Ying; Weis, John H; Chen, Xinjian; Zachary, James F; Teuscher, Cory; Weis, Janis J

    2014-01-01

    Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most prevalent arthropod-borne illness in the United States and remains a clinical and social challenge. The spectrum of disease severity among infected patients suggests that host genetics contribute to pathogenic outcomes, particularly in patients who develop arthritis. Using a forward genetics approach, we identified the lysosomal enzyme β-glucuronidase (GUSB), a member of a large family of coregulated lysosomal enzymes, as a key regulator of Lyme-associated arthritis severity. Severely arthritic C3H mice possessed a naturally occurring hypomorphic allele, Gusbh. C57BL/6 mice congenic for the C3H Gusb allele were prone to increased Lyme-associated arthritis severity. Radiation chimera experiments revealed that resident joint cells drive arthritis susceptibility. C3H mice expressing WT Gusb as a transgene were protected from severe Lyme arthritis. Importantly, the Gusbh allele also exacerbated disease in a serum transfer model of rheumatoid arthritis. A known GUSB function is the prevention of lysosomal accumulation of glycosaminoglycans (GAGs). Development of Lyme and rheumatoid arthritis in Gusbh-expressing mice was associated with heightened accumulation of GAGs in joint tissue. We propose that GUSB modulates arthritis pathogenesis by preventing accumulation of proinflammatory GAGs within inflamed joint tissue, a trait that may be shared by other lysosomal exoglycosidases.

  4. Lysosomal Dysfunction Promotes Cleavage and Neurotoxicity of Tau In Vivo

    PubMed Central

    Sharp, Katherine A.; Loewen, Carin A.; Mulkearns, Erin; Tyynelä, Jaana; Scherzer, Clemens R.; Feany, Mel B.

    2010-01-01

    Expansion of the lysosomal system, including cathepsin D upregulation, is an early and prominent finding in Alzheimer's disease brain. Cell culture studies, however, have provided differing perspectives on the lysosomal connection to Alzheimer's disease, including both protective and detrimental influences. We sought to clarify and molecularly define the connection in vivo in a genetically tractable model organism. Cathepsin D is upregulated with age in a Drosophila model of Alzheimer's disease and related tauopathies. Genetic analysis reveals that cathepsin D plays a neuroprotective role because genetic ablation of cathepsin D markedly potentiates tau-induced neurotoxicity. Further, generation of a C-terminally truncated form of tau found in Alzheimer's disease patients is significantly increased in the absence of cathepsin D. We show that truncated tau has markedly increased neurotoxicity, while solubility of truncated tau is decreased. Importantly, the toxicity of truncated tau is not affected by removal of cathepsin D, providing genetic evidence that modulation of neurotoxicity by cathepsin D is mediated through C-terminal cleavage of tau. We demonstrate that removing cathepsin D in adult postmitotic neurons leads to aberrant lysosomal expansion and caspase activation in vivo, suggesting a mechanism for C-terminal truncation of tau. We also demonstrate that both cathepsin D knockout mice and cathepsin D–deficient sheep show abnormal C-terminal truncation of tau and accompanying caspase activation. Thus, caspase cleavage of tau may be a molecular mechanism through which lysosomal dysfunction and neurodegeneration are causally linked in Alzheimer's disease. PMID:20664788

  5. Sphingosine-induced apoptosis is dependent on lysosomal proteases.

    PubMed Central

    Kågedal, K; Zhao, M; Svensson, I; Brunk, U T

    2001-01-01

    We propose a new mechanism for sphingosine-induced apoptosis, involving relocation of lysosomal hydrolases to the cytosol. Owing to its lysosomotropic properties, sphingosine, which is also a detergent, especially when protonated, accumulates by proton trapping within the acidic vacuolar apparatus, where most of its action as a detergent would be exerted. When sphingosine was added in low-to-moderate concentrations to Jurkat and J774 cells, partial lysosomal rupture occurred dose-dependently, starting within a few minutes. This phenomenon preceded caspase activation, as well as changes of mitochondrial membrane potential. High sphingosine doses rapidly caused extensive lysosomal rupture and ensuing necrosis, without antecedent apoptosis or caspase activation. The sphingosine effect was prevented by pre-treatment with another, non-toxic, lysosomotropic base, ammonium chloride, at 10 mM. The lysosomal protease inhibitors, pepstatin A and epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester ('E-64d'), inhibited markedly sphingosine-induced caspase activity to almost the same degree as the general caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone ('Z-VAD-FMK'), although they did not by themselves inhibit caspases. We conclude that cathepsin D and one or more cysteine proteases, such as cathepsins B or L, are important mediators of sphingosine-induced apoptosis, working upstream of the caspase cascade and mitochondrial membrane-potential changes. PMID:11583579

  6. Neuronopathic Lysosomal Storage Diseases: Clinical and Pathologic Findings

    ERIC Educational Resources Information Center

    Prada, Carlos E.; Grabowski, Gregory A.

    2013-01-01

    Background: The lysosomal--autophagocytic system diseases (LASDs) affect multiple body systems including the central nervous system (CNS). The progressive CNS pathology has its onset at different ages, leading to neurodegeneration and early death. Methods: Literature review provided insight into the current clinical neurological findings,…

  7. Lysosomal β-glucuronidase regulates Lyme and rheumatoid arthritis severity

    PubMed Central

    Bramwell, Kenneth K.C.; Ma, Ying; Weis, John H.; Chen, Xinjian; Zachary, James F.; Teuscher, Cory; Weis, Janis J.

    2013-01-01

    Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most prevalent arthropod-borne illness in the United States and remains a clinical and social challenge. The spectrum of disease severity among infected patients suggests that host genetics contribute to pathogenic outcomes, particularly in patients who develop arthritis. Using a forward genetics approach, we identified the lysosomal enzyme β-glucuronidase (GUSB), a member of a large family of coregulated lysosomal enzymes, as a key regulator of Lyme-associated arthritis severity. Severely arthritic C3H mice possessed a naturally occurring hypomorphic allele, Gusbh. C57BL/6 mice congenic for the C3H Gusb allele were prone to increased Lyme-associated arthritis severity. Radiation chimera experiments revealed that resident joint cells drive arthritis susceptibility. C3H mice expressing WT Gusb as a transgene were protected from severe Lyme arthritis. Importantly, the Gusbh allele also exacerbated disease in a serum transfer model of rheumatoid arthritis. A known GUSB function is the prevention of lysosomal accumulation of glycosaminoglycans (GAGs). Development of Lyme and rheumatoid arthritis in Gusbh-expressing mice was associated with heightened accumulation of GAGs in joint tissue. We propose that GUSB modulates arthritis pathogenesis by preventing accumulation of proinflammatory GAGs within inflamed joint tissue, a trait that may be shared by other lysosomal exoglycosidases. PMID:24334460

  8. PIG7 promotes leukemia cell chemosensitivity via lysosomal membrane permeabilization

    PubMed Central

    Niu, Ting; Wu, Yu; Li, Jianjun; Wang, Fangfang; Zheng, Yuhuan; Liu, Ting

    2016-01-01

    PIG7 localizes to lysosomal membrane in leukemia cells. Our previous work has shown that transduction of pig7 into a series of leukemia cell lines did not result in either apoptosis or differentiation of most tested cell lines. Interestingly, it did significantly sensitize these cell lines to chemotherapeutic drugs. Here, we further investigated the mechanism underlying pig7-induced improved sensitivity of acute leukemia cells to chemotherapy. Our results demonstrated that the sensitization effect driven by exogenous pig7 was more effective in drug-resistant leukemia cell lines which had lower endogenous pig7 expression. Overexpression of pig7 did not directly activate the caspase apoptotic pathway, but decreased the lysosomal stability. The expression of pig7 resulted in lysosomal membrane permeabilization (LMP) and lysosomal protease (e.g. cathepsin B, D, L) release. Moreover, we also observed increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) induced by pig7. Some autophagy markers such as LC3I/II, ATG5 and Beclin-1, and necroptosis maker MLKL were also stimulated. However, intrinsic antagonism such as serine/cysteine protease inhibitors Spi2A and Cystatin C prevented downstream effectors from triggering leukemia cells, which were only on the “verge of apoptosis”. When combined with chemotherapy, LMP increased and more proteases were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways. PMID:26716897

  9. Lysosomal cathepsins and their regulation in aging and neurodegeneration.

    PubMed

    Stoka, Veronika; Turk, Vito; Turk, Boris

    2016-12-01

    Lysosomes and lysosomal hydrolases, including the cathepsins, have been shown to change their properties with aging brain a long time ago, although their function was not really understood. The first biochemical and clinical studies were followed by a major expansion in the last 20 years with the development of animal disease models and new approaches leading to a major advancement of understanding of the role of physiological and degenerative processes in the brain at the molecular level. This includes the understanding of the major role of autophagy and the cathepsins in a number of diseases, including its critical role in the neuronal ceroid lipofuscinosis. Similarly, cathepsins and some other lysosomal proteases were shown to have important roles in processing and/or degradation of several important neuronal proteins, thereby having either neuroprotective or harmful roles. In this review, we discuss lysosomal cathepsins and their regulation with the focus on cysteine cathepsins and their endogenous inhibitors, as well as their role in several neurodegenerative diseases. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Neuronopathic Lysosomal Storage Diseases: Clinical and Pathologic Findings

    ERIC Educational Resources Information Center

    Prada, Carlos E.; Grabowski, Gregory A.

    2013-01-01

    Background: The lysosomal--autophagocytic system diseases (LASDs) affect multiple body systems including the central nervous system (CNS). The progressive CNS pathology has its onset at different ages, leading to neurodegeneration and early death. Methods: Literature review provided insight into the current clinical neurological findings,…

  11. Cholesterol transport through lysosome-peroxisome membrane contacts.

    PubMed

    Chu, Bei-Bei; Liao, Ya-Cheng; Qi, Wei; Xie, Chang; Du, Ximing; Wang, Jiang; Yang, Hongyuan; Miao, Hong-Hua; Li, Bo-Liang; Song, Bao-Liang

    2015-04-09

    Cholesterol is dynamically transported among organelles, which is essential for multiple cellular functions. However, the mechanism underlying intracellular cholesterol transport has remained largely unknown. We established an amphotericin B-based assay enabling a genome-wide shRNA screen for delayed LDL-cholesterol transport and identified 341 hits with particular enrichment of peroxisome genes, suggesting a previously unappreciated pathway for cholesterol transport. We show dynamic membrane contacts between peroxisome and lysosome, which are mediated by lysosomal Synaptotagmin VII binding to the lipid PI(4,5)P2 on peroxisomal membrane. LDL-cholesterol enhances such contacts, and cholesterol is transported from lysosome to peroxisome. Disruption of critical peroxisome genes leads to cholesterol accumulation in lysosome. Together, these findings reveal an unexpected role of peroxisome in intracellular cholesterol transport. We further demonstrate massive cholesterol accumulation in human patient cells and mouse model of peroxisomal disorders, suggesting a contribution of abnormal cholesterol accumulation to these diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Lysosome-Related Organelles in Intestinal Cells Are a Zinc Storage Site in C. elegans

    PubMed Central

    Roh, Hyun Cheol; Collier, Sara; Guthrie, James; Robertson, J. David; Kornfeld, Kerry

    2014-01-01

    SUMMARY Zinc is an essential trace element involved in many biological processes and human diseases. Because zinc deficiency and excess are deleterious, animals require homeostatic mechanisms to maintain zinc levels in response to dietary fluctuations. Here we demonstrate that lysosome-related organelles in intestinal cells of C. elegans, called gut granules, function as the major site of zinc storage. Zinc storage in gut granules promotes detoxification and subsequent mobilization, linking cellular and organismal zinc metabolism. The cation diffusion facilitator protein CDF-2 plays a critical role in this process by transporting zinc into gut granules. In response to high dietary zinc, gut granules displayed structural changes characterized by a bilobed morphology with asymmetric distributions of zinc and molecular markers. We defined a genetic pathway that mediates the formation of bilobed morphology. These findings elucidate mechanisms of zinc storage, detoxification and mobilization in C. elegans and may be relevant to other animals. PMID:22225878

  13. Toxicity detection using lysosomal enzymes, glycoamylase and thioredoxin fused with fluorescent protein in Saccharomyces cerevisiae.

    PubMed

    Nguyen, Ngoc-Tu; Shin, Hwa-Yoon; Kim, Yang-Hoon; Min, Jiho

    2015-11-20

    Saccharomyces cerevisiae is the simplest and a favorite eukaryotic system that contains lysosome and thus, is a suitable organism for monitoring some toxic effects in environmental pollution. In this study, S. cerevisiae was transformed with two recombinant plasmids. Sporulation-specific glycoamylase (SGA1), which was upregulated in response to arsenic, was fused with the blue fluorescent protein (BFP) for the construction of an oxidative stress-causing chemicals sensor. Additionally, thioredoxin (TRX2), a protein overexpressed exclusively under tetracycline's influence, fused with the cyan fluorescent protein (CFP) to create a detector for this kind of chemical. In summary, we developed two recombinant S. cerevisiae that facilitate the detection of both kinds of toxic chemicals, specifically visualized by different color indicators.

  14. Lysosome size, motility and stress response regulated by fronto-temporal dementia modifier TMEM106B.

    PubMed

    Stagi, Massimiliano; Klein, Zoe A; Gould, Travis J; Bewersdorf, Joerg; Strittmatter, Stephen M

    2014-07-01

    Fronto-temporal lobar degeneration with TDP-43 (FTLD-TDP) is a fatal neurodegeneration. TMEM106B variants are linked to FTLD-TDP risk, and TMEM106B is lysosomal. Here, we focus on neuronal TMEM106B, and demonstrate co-localization and traffic with lysosomal LAMP-1. pH-sensitive reporters demonstrate that the TMEM106B C-terminus is lumenal. The TMEM106B N-terminus interacts with endosomal adaptors and other TMEM106 proteins. TMEM106B knockdown reduces neuronal lysosomal number and diameter by STED microscopy, and overexpression enlarges LAMP-positive structures. Reduction of TMEM106B increases axonally transported lysosomes, while TMEM106B elevation inhibits transport and yields large lysosomes in the soma. TMEM106B overexpression alters lysosomal stress signaling, causing a translocation of the mTOR-sensitive transcription factor, TFEB, to neuronal nuclei. TMEM106B loss-of-function delays TFEB translocation after Torin-1-induced stress. Enlarged TMEM106B-overexpressing lysosomes maintain organelle integrity longer after lysosomal photodamage than do control lysosomes, while small TMEM106B-knockdown lysosomes are more sensitive to illumination. Thus, neuronal TMEM106B plays a central role in regulating lysosomal size, motility and responsiveness to stress, highlighting the possible role of lysosomal biology in FTLD-TDP.

  15. Lysosome size, motility and stress response regulated by Fronto-Temporal Dementia modifier TMEM106B

    PubMed Central

    Stagi, Massimiliano; Klein, Zoe A.; Gould, Travis J.; Bewersdorf, Joerg; Strittmatter, Stephen M.

    2014-01-01

    Fronto-Temporal Lobar Degeneration with TDP-43 (FTLD-TDP) is a fatal neurodegeneration. TMEM106B variants are linked to FTLD-TDP risk, and TMEM106B is lysosomal. Here, we focus on neuronal TMEM106B, and demonstrate co-localization and traffic with lysosomal LAMP-1. pH-sensitive reporters demonstrate that the TMEM106B C-terminus is lumenal. The TMEM106B N-terminus interacts with endosomal adaptors and other TMEM106 proteins. TMEM106B knockdown reduces neuronal lysosomal number and diameter by STED microscopy, and overexpression enlarges LAMP-positive structures. Reduction of TMEM106B increases axonally transported lysosomes, while TMEM106B elevation inhibits transport and yields large lysosomes in the soma. TMEM106B overexpression alters lysosomal stress signaling, causing a translocation of the mTOR-sensitive transcription factor, TFEB, to neuronal nuclei. TMEM106B loss-of-function delays TFEB translocation after Torin-1-induced stress. Enlarged TMEM106B-overexpressing lysosomes maintain organelle integrity longer after lysosomal photodamage than do control lysosomes, while small TMEM106B-knockdown lysosomes are more sensitive to illumination. Thus, neuronal TMEM106B plays a central role in regulating lysosomal size, motility and responsiveness to stress, highlighting the possible role of lysosomal biology in FTLD-TDP. PMID:25066864

  16. The influence of oxidation of membrane thiol groups on lysosomal proton permeability.

    PubMed Central

    Wan, F Y; Wang, Y N; Zhang, G J

    2001-01-01

    The influence of oxidation of membrane thiol groups on lysosomal proton permeability was studied by measuring lysosomal pH with FITC-conjugated dextran, determining the membrane potential with 3,3'-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Residual membrane thiol groups were measured with 5,5'-dithiobis-(2-nitrobenzoic acid). The lysosomal membrane thiol groups were modified by treatment with diamide and dithiothreitol. SDS/PAGE revealed aggregations of the membrane proteins induced by the treatment of lysosomes with diamide. The cross-linkage of proteins could be abolished by subsequent treatment with dithiothreitol, indicating that the proteins were linked via disulphide bonds. Treating the lysosomes with diamide decreased their membrane thiol groups and caused increases in lysosomal pH, membrane potential and proton leakage, which could be reversed by treatment of the lysosomes with dithiothreitol. This indicates that the lysosomal proton permeability can be increased by oxidation of the membrane thiol groups and restored to the normal level by reduction of the groups. Treatment of the lysosomes with N-ethylmaleimide reduced their membrane thiol groups but did not change the lysosomal pH or their degree of proton leakage. It suggests that protein aggregation may be an important mechanism for the increase in lysosomal proton permeability. The results raise the possibility that the proton permeability of lysosomes in vivo may be affected by the redox states of their membrane thiol groups. PMID:11716763

  17. P-selectin targeting to secretory lysosomes of Rbl-2H3 cells.

    PubMed

    Kaur, Jasber; Cutler, Daniel F

    2002-03-22

    The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes.

  18. LYSOSOMAL INSTABILITY AND CATHEPSIN B RELEASE DURING ACETAMINOPHEN HEPATOTOXICITY

    PubMed Central

    Woolbright, Benjamin L.; Ramachandran, Anup; McGill, Mitchell R.; Yan, Hui-min; Bajt, Mary Lynn; Sharpe, Matthew R.; Lemasters, John J.; Jaeschke, Hartmut

    2012-01-01

    Acetaminophen (APAP) overdose is currently the most frequent cause of drug-induced liver failure in the United States. Recently, it was shown that lysosomal iron translocates to mitochondria where it contributes to collapse of the mitochondrial membrane potential. Therefore, the purpose of this study was to investigate if cathepsin B, a lysosomal protease, is involved in APAP-induced hepatotoxicity. Cathepsin B activity was measured in subcellular liver fractions of C57Bl/6 mice 3 hr after 300 mg/kg APAP treatment. There was a significant increase in cytoplasmic cathepsin activity, concurrent with a decrease in microsomal activity, indicative of lysosomal cathepsin B release. To investigate the effect of cathepsin B on hepatotoxicity, the cathepsin inhibitor AC-LVK-CHO was given 1 hr prior to 300 mg/kg APAP treatment along with vehicle control. There was no difference between groups in serum ALT values, or by histological evaluation of necrosis, although cathepsin B activity was inhibited by 70–80% compared to controls. These findings were confirmed with a different inhibitor (z-FA-fmk) in vivo and in vitro. Hepatocytes were exposed to 5 mM acetaminophen. Lysotracker staining confirmed lysosomal instability, and cathepsin B release, but there was no reduction in cell death after treatment with cathepsin B inhibitors. Finally, cathepsin B release was measured in clinical samples from patients with APAP-induced liver injury. Low levels of cathepsin B were released into plasma from overdose patients. Conclusion APAP overdose causes lysosomal instability and release of cathepsin B into the cytosol but does not contribute to liver injury under these conditions. PMID:22900545

  19. Characterization of the complex formed by β-glucocerebrosidase and the lysosomal integral membrane protein type-2.

    PubMed

    Zunke, Friederike; Andresen, Lisa; Wesseler, Sophia; Groth, Johann; Arnold, Philipp; Rothaug, Michelle; Mazzulli, Joseph R; Krainc, Dimitri; Blanz, Judith; Saftig, Paul; Schwake, Michael

    2016-04-05

    The lysosomal integral membrane protein type-2 (LIMP-2) plays a pivotal role in the delivery of β-glucocerebrosidase (GC) to lysosomes. Mutations in GC result in Gaucher's disease (GD) and are the major genetic risk factor for the development of Parkinson's disease (PD). Variants in the LIMP-2 gene cause action myoclonus-renal failure syndrome and also have been linked to PD. Given the importance of GC and LIMP-2 in disease pathogenesis, we studied their interaction sites in more detail. Our previous data demonstrated that the crystal structure of LIMP-2 displays a hydrophobic three-helix bundle composed of helices 4, 5, and 7, of which helix 5 and 7 are important for ligand binding. Here, we identified a similar helical motif in GC through surface potential analysis. Coimmunoprecipitation and immunofluorescence studies revealed a triple-helical interface region within GC as critical for LIMP-2 binding and lysosomal transport. Based on these findings, we generated a LIMP-2 helix 5-derived peptide that precipitated and activated recombinant wild-type and GD-associated N370S mutant GC in vitro. The helix 5 peptide fused to a cell-penetrating peptide also activated endogenous lysosomal GC and reduced α-synuclein levels, suggesting that LIMP-2-derived peptides can be used to activate endogenous as well as recombinant wild-type or mutant GC efficiently. Our data also provide a structural model of the LIMP-2/GC complex that will facilitate the development of GC chaperones and activators as potential therapeutics for GD, PD, and related synucleinopathies.

  20. Characterization of the complex formed by β-glucocerebrosidase and the lysosomal integral membrane protein type-2

    PubMed Central

    Zunke, Friederike; Andresen, Lisa; Wesseler, Sophia; Groth, Johann; Arnold, Philipp; Rothaug, Michelle; Mazzulli, Joseph R.; Krainc, Dimitri; Blanz, Judith; Saftig, Paul; Schwake, Michael

    2016-01-01

    The lysosomal integral membrane protein type-2 (LIMP-2) plays a pivotal role in the delivery of β-glucocerebrosidase (GC) to lysosomes. Mutations in GC result in Gaucher's disease (GD) and are the major genetic risk factor for the development of Parkinson's disease (PD). Variants in the LIMP-2 gene cause action myoclonus-renal failure syndrome and also have been linked to PD. Given the importance of GC and LIMP-2 in disease pathogenesis, we studied their interaction sites in more detail. Our previous data demonstrated that the crystal structure of LIMP-2 displays a hydrophobic three-helix bundle composed of helices 4, 5, and 7, of which helix 5 and 7 are important for ligand binding. Here, we identified a similar helical motif in GC through surface potential analysis. Coimmunoprecipitation and immunofluorescence studies revealed a triple-helical interface region within GC as critical for LIMP-2 binding and lysosomal transport. Based on these findings, we generated a LIMP-2 helix 5-derived peptide that precipitated and activated recombinant wild-type and GD-associated N370S mutant GC in vitro. The helix 5 peptide fused to a cell-penetrating peptide also activated endogenous lysosomal GC and reduced α-synuclein levels, suggesting that LIMP-2–derived peptides can be used to activate endogenous as well as recombinant wild-type or mutant GC efficiently. Our data also provide a structural model of the LIMP-2/GC complex that will facilitate the development of GC chaperones and activators as potential therapeutics for GD, PD, and related synucleinopathies. PMID:27001828

  1. Marked enhancement of lysosomal targeting and efficacy of ErbB2-targeted drug delivery by HSP90 inhibition

    PubMed Central

    Mohapatra, Bhopal; Luan, Haitao; Soni, Kruti; Zhang, Jinjin; Storck, Matthew A.; Feng, Dan; Bielecki, Timothy A.; Band, Vimla; Cohen, Samuel M.; Bronich, Tatiana K.; Band, Hamid

    2016-01-01

    Targeted delivery of anticancer drugs to tumor cells using monoclonal antibodies against oncogenic cell surface receptors is an emerging therapeutic strategy. These strategies include drugs directly conjugated to monoclonal antibodies through chemical linkers (Antibody-Drug Conjugates, ADCs) or those encapsulated within nanoparticles that in turn are conjugated to targeting antibodies (Antibody-Nanoparticle Conjugates, ANPs). The recent FDA approval of the ADC Trastuzumab-TDM1 (Kadcyla®; Genentech; San Francisco) for the treatment of ErbB2-overexpressing metastatic breast cancer patients has validated the strong potential of these strategies. Even though the activity of ANPs and ADCs is dependent on lysosomal traffic, the roles of the endocytic route traversed by the targeted receptor and of cancer cell-specific alterations in receptor dynamics on the efficiency of drug delivery have not been considered in these new targeted therapies. For example, constitutive association with the molecular chaperone HSP90 is thought to either retard ErbB2 endocytosis or to promote its recycling, traits undesirable for targeted therapy with ANPs and ADCs. HSP90 inhibitors are known to promote ErbB2 ubiquitination, targeting to lysosome and degradation. We therefore hypothesized that ErbB2-targeted drug delivery using Trastuzumab-conjugated nanoparticles could be significantly improved by HSP90 inhibitor-promoted lysosomal traffic of ErbB2. Studies reported here validate this hypothesis and demonstrate, both in vitro and in vivo, that HSP90 inhibition facilitates the intracellular delivery of Trastuzumab-conjugated ANPs carrying a model chemotherapeutic agent, Doxorubicin, specifically into ErbB2-overexpressing breast cancer cells, resulting in improved antitumor activity. These novel findings highlight the need to consider oncogene-specific alterations in receptor traffic in the design of targeted drug delivery strategies. We suggest that combination of agents that enhance

  2. Marked enhancement of lysosomal targeting and efficacy of ErbB2-targeted drug delivery by HSP90 inhibition.

    PubMed

    Raja, Srikumar M; Desale, Swapnil S; Mohapatra, Bhopal; Luan, Haitao; Soni, Kruti; Zhang, Jinjin; Storck, Matthew A; Feng, Dan; Bielecki, Timothy A; Band, Vimla; Cohen, Samuel M; Bronich, Tatiana K; Band, Hamid

    2016-03-01

    Targeted delivery of anticancer drugs to tumor cells using monoclonal antibodies against oncogenic cell surface receptors is an emerging therapeutic strategy. These strategies include drugs directly conjugated to monoclonal antibodies through chemical linkers (Antibody-Drug Conjugates, ADCs) or those encapsulated within nanoparticles that in turn are conjugated to targeting antibodies (Antibody-Nanoparticle Conjugates, ANPs). The recent FDA approval of the ADC Trastuzumab-TDM1 (Kadcyla; Genentech; San Francisco) for the treatment of ErbB2-overexpressing metastatic breast cancer patients has validated the strong potential of these strategies. Even though the activity of ANPs and ADCs is dependent on lysosomal traffic, the roles of the endocytic route traversed by the targeted receptor and of cancer cell-specific alterations in receptor dynamics on the efficiency of drug delivery have not been considered in these new targeted therapies. For example, constitutive association with the molecular chaperone HSP90 is thought to either retard ErbB2 endocytosis or to promote its recycling, traits undesirable for targeted therapy with ANPs and ADCs. HSP90 inhibitors are known to promote ErbB2 ubiquitination, targeting to lysosome and degradation. We therefore hypothesized that ErbB2-targeted drug delivery using Trastuzumab-conjugated nanoparticles could be significantly improved by HSP90 inhibitor-promoted lysosomal traffic of ErbB2. Studies reported here validate this hypothesis and demonstrate, both in vitro and in vivo, that HSP90 inhibition facilitates the intracellular delivery of Trastuzumab-conjugated ANPs carrying a model chemotherapeutic agent, Doxorubicin, specifically into ErbB2-overexpressing breast cancer cells, resulting in improved antitumor activity. These novel findings highlight the need to consider oncogene-specific alterations in receptor traffic in the design of targeted drug delivery strategies. We suggest that combination of agents that enhance receptor

  3. The SM protein Car/Vps33A regulates SNARE-mediated trafficking to lysosomes and lysosome-related organelles.

    PubMed

    Akbar, Mohammed A; Ray, Sanchali; Krämer, Helmut

    2009-03-01

    The SM proteins Vps33A and Vps33B are believed to act in membrane fusions in endosomal pathways, but their specific roles are controversial. In Drosophila, Vps33A is the product of the carnation (car) gene. We generated a null allele of car to test its requirement for trafficking to different organelles. Complete loss of car function is lethal during larval development. Eye-specific loss of Car causes late, light-independent degeneration of photoreceptor cells. Earlier in these cells, two distinct phenotypes were detected. In young adults, autophagosomes amassed indicating that their fusion with lysosomes requires Car. In eye discs, endocytosed receptors and ligands accumulate in Rab7-positive prelysosomal compartments. The requirement of Car for late endosome-to-lysosome fusion in imaginal discs is specific as early endosomes are unaffected. Furthermore, lysosomal delivery is not restored by expression of dVps33B. This specificity reflects the distinct pattern of binding to different Syntaxins in vitro: dVps33B predominantly binds the early endosomal Avl and Car to dSyntaxin16. Consistent with a role in Car-mediated fusion, dSyntaxin16 is not restricted to Golgi membranes but also present on lysosomes.

  4. The SM Protein Car/Vps33A Regulates SNARE-mediated Trafficking to Lysosomes and Lysosome-related Organelles

    PubMed Central

    Akbar, Mohammed A.; Ray, Sanchali

    2009-01-01

    The SM proteins Vps33A and Vps33B are believed to act in membrane fusions in endosomal pathways, but their specific roles are controversial. In Drosophila, Vps33A is the product of the carnation (car) gene. We generated a null allele of car to test its requirement for trafficking to different organelles. Complete loss of car function is lethal during larval development. Eye-specific loss of Car causes late, light-independent degeneration of photoreceptor cells. Earlier in these cells, two distinct phenotypes were detected. In young adults, autophagosomes amassed indicating that their fusion with lysosomes requires Car. In eye discs, endocytosed receptors and ligands accumulate in Rab7-positive prelysosomal compartments. The requirement of Car for late endosome-to-lysosome fusion in imaginal discs is specific as early endosomes are unaffected. Furthermore, lysosomal delivery is not restored by expression of dVps33B. This specificity reflects the distinct pattern of binding to different Syntaxins in vitro: dVps33B predominantly binds the early endosomal Avl and Car to dSyntaxin16. Consistent with a role in Car-mediated fusion, dSyntaxin16 is not restricted to Golgi membranes but also present on lysosomes. PMID:19158398

  5. β2-microglobulin amyloid fibrils are nanoparticles that disrupt lysosomal membrane protein trafficking and inhibit protein degradation by lysosomes.

    PubMed

    Jakhria, Toral; Hellewell, Andrew L; Porter, Morwenna Y; Jackson, Matthew P; Tipping, Kevin W; Xue, Wei-Feng; Radford, Sheena E; Hewitt, Eric W

    2014-12-26

    Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β2-microglobulin (β2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β2m fibrils did not, however, cause cell death. Instead, fragmented β2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Autophagic flux promotes cisplatin resistance in human ovarian carcinoma cells through ATP-mediated lysosomal function.

    PubMed

    Ma, Liwei; Xu, Ye; Su, Jing; Yu, Huimei; Kang, Jinsong; Li, Hongyan; Li, Xiaoning; Xie, Qi; Yu, Chunyan; Sun, Liankun; Li, Yang

    2015-11-01

    Lysosomes are involved in promoting resistance of cancer cells to chemotherapeutic agents. However, the mechanisms underlying lysosomal influence of cisplatin resistance in ovarian cancer remain incompletely understood. We report that, compared with cisplatin-sensitive SKOV3 cells, autophagy increases in cisplatin-resistant SKOV3/DDP cells treated with cisplatin. Inhibition of early-stage autophagy enhanced cisplatin-mediated cytotoxicity in SKOV3/DDP cells, but autophagy inhibition at a later stage by disturbing autophagosome-lysosome fusion is more effective. Notably, SKOV3/DDP cells contained more lysosomes than cisplatin-sensitive SKOV3 cells. Abundant lysosomes and lysosomal cathepsin D activity were required for continued autolysosomal degradation and maintenance of autophagic flux in SKOV3/DDP cells. Furthermore, SKOV3/DDP cells contain abundant lysosomal ATP required for lysosomal function, and inhibition of lysosomal ATP accumulation impaired lysosomal function and blocked autophagic flux. Therefore, our findings suggest that lysosomes at least partially contribute to cisplatin resistance in ovarian cancer cells through their role in cisplatin-induced autophagic processes, and provide insight into the mechanism of cisplatin resistance in tumors.

  7. TFEB-mediated increase in peripheral lysosomes regulates store-operated calcium entry

    PubMed Central

    Sbano, Luigi; Bonora, Massimo; Marchi, Saverio; Baldassari, Federica; Medina, Diego L.; Ballabio, Andrea; Giorgi, Carlotta; Pinton, Paolo

    2017-01-01

    Lysosomes are membrane-bound organelles mainly involved in catabolic processes. In addition, lysosomes can expel their contents outside of the cell via lysosomal exocytosis. Some of the key steps involved in these important cellular processes, such as vesicular fusion and trafficking, require calcium (Ca2+) signaling. Recent data show that lysosomal functions are transcriptionally regulated by transcription factor EB (TFEB) through the induction of genes involved in lysosomal biogenesis and exocytosis. Given these observations, we investigated the roles of TFEB and lysosomes in intracellular Ca2+ homeostasis. We studied the effect of transient modulation of TFEB expression in HeLa cells by measuring the cytosolic Ca2+ response after capacitative Ca2+ entry activation and Ca2+ dynamics in the endoplasmic reticulum (ER) and directly in lysosomes. Our observations show that transient TFEB overexpression significantly reduces cytosolic Ca2+ levels under a capacitative influx model and ER re-uptake of calcium, increasing the lysosomal Ca2+ buffering capacity. Moreover, lysosomal destruction or damage abolishes these TFEB-dependent effects in both the cytosol and ER. These results suggest a possible Ca2+ buffering role for lysosomes and shed new light on lysosomal functions during intracellular Ca2+ homeostasis. PMID:28084445

  8. TM7SF1 (GPR137B): a novel lysosome integral membrane protein.

    PubMed

    Gao, Jialin; Xia, Libin; Lu, Meiqing; Zhang, Binhua; Chen, Yueping; Xu, Rang; Wang, Lizhuo

    2012-09-01

    In the previous proteomic study of human placenta, transmembrane 7 superfamily member 1 (TM7SF1) was found enriched in lysosome compartments. TM7SF1 encodes a 399-amino acid protein with a calculated molecular mass of 45 kDa. Bioinformatic analysis of its amino acid sequence showed that it is a multipass transmembrane protein containing a potential dileucine-based lysosomal targeting signal and four putative N-glycosylation sites. By percoll-gradient centrifugation and further subfraction ways, the lysosomal solute and membrane compartments were isolated respectively. Immunoblotting analysis indicated that TM7SF1 was co-fractioned with lysosome associated membrane protein 2 (LAMP2), which was only detected in lysosomal membrane compartments whereas not detected in the solute compartments. Using specific anti-TM7SF1 antibody and double-immunofluorescence with lysosome membrane protein LAMP1 and Lyso-Tracker Red, the colocalisations of endogenous TM7SF1 with lysosome and late endosome markers were demonstrated. All of this indicated that TM7SF1 is an integral lysosome membrane protein. Rat ortholog of TM7SF1 was found to be strongly expressed in heart, liver, kidney and brain while not or low detected in other tissues. In summary, TM7SF1 was a lysosomal integral membrane protein that shows tissue-specific expression. As a G-protein-coupled receptor in lysosome membrane, TM7SF1 was predicted function as signal transduction across lysosome membrane.

  9. The Role of Oxidized Cholesterol in Diabetes-Induced Lysosomal Dysfunction in the Brain.

    PubMed

    Sims-Robinson, Catrina; Bakeman, Anna; Rosko, Andrew; Glasser, Rebecca; Feldman, Eva L

    2016-05-01

    Abnormalities in lysosomal function have been reported in diabetes, aging, and age-related degenerative diseases. These lysosomal abnormalities are an early manifestation of neurodegenerative diseases and often precede the onset of clinical symptoms such as learning and memory deficits; however, the mechanism underlying lysosomal dysfunction is not known. In the current study, we investigated the mechanism underlying lysosomal dysfunction in the cortex and hippocampi, key structures involved in learning and memory, of a type 2 diabetes (T2D) mouse model, the leptin receptor deficient db/db mouse. We demonstrate for the first time that diabetes leads to destabilization of lysosomes as well as alterations in the protein expression, activity, and/or trafficking of two lysosomal enzymes, hexosaminidase A and cathepsin D, in the hippocampus of db/db mice. Pioglitazone, a thiazolidinedione (TZD) commonly used in the treatment of diabetes due to its ability to improve insulin sensitivity and reverse hyperglycemia, was ineffective in reversing the diabetes-induced changes on lysosomal enzymes. Our previous work revealed that pioglitazone does not reverse hypercholesterolemia; thus, we investigated whether cholesterol plays a role in diabetes-induced lysosomal changes. In vitro, cholesterol promoted the destabilization of lysosomes, suggesting that lysosomal-related changes associated with diabetes are due to elevated levels of cholesterol. Since lysosome dysfunction precedes neurodegeneration, cognitive deficits, and Alzheimer's disease neuropathology, our results may provide a potential mechanism that links diabetes with complications of the central nervous system.

  10. The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis.

    PubMed

    Roczniak-Ferguson, Agnes; Petit, Constance S; Froehlich, Florian; Qian, Sharon; Ky, Jennifer; Angarola, Brittany; Walther, Tobias C; Ferguson, Shawn M

    2012-06-12

    Lysosomes are the major cellular site for clearance of defective organelles and digestion of internalized material. Demand on lysosomal capacity can vary greatly, and lysosomal function must be adjusted to maintain cellular homeostasis. Here, we identified an interaction between the lysosome-localized mechanistic target of rapamycin complex 1 (mTORC1) and the transcription factor TFEB (transcription factor EB), which promotes lysosome biogenesis. When lysosomal activity was adequate, mTOR-dependent phosphorylation of TFEB on Ser(211) triggered the binding of 14-3-3 proteins to TFEB, resulting in retention of the transcription factor in the cytoplasm. Inhibition of lysosomal function reduced the mTOR-dependent phosphorylation of TFEB, resulting in diminished interactions between TFEB and 14-3-3 proteins and the translocation of TFEB into the nucleus, where it could stimulate genes involved in lysosomal biogenesis. These results identify TFEB as a target of mTOR and suggest a mechanism for matching the transcriptional regulation of genes encoding proteins of autophagosomes and lysosomes to cellular need. The closely related transcription factors MITF (microphthalmia transcription factor) and TFE3 (transcription factor E3) also localized to lysosomes and accumulated in the nucleus when lysosome function was inhibited, thus broadening the range of physiological contexts under which this regulatory mechanism may prove important.

  11. Effect of pH on the formation of lysosome-alginate beads for antimicrobial activity.

    PubMed

    Park, Hyun Jung; Min, Jiho; Ahn, Joo-Myung; Cho, Sung-Jin; Ahn, Ji-Young; Kim, Yang-Hoon

    2015-02-01

    In this study, we developed lysosome-alginate beads for application as an oral drug delivery system (ODDS). The beads harboring lysosomes, which have antimicrobial activity, and various concentrations of alginate were characterized and optimized. For application as an ODDS, pH-dependent lysosome-alginate beads were generated, and the level of lysosome release was investigated by using antimicrobial tests. At low pH, lysosomes were not released from the lysosome-alginate beads; however, at neutral pH, similar to the pH in the intestine, lysosome release was confirmed, as determined by a high antimicrobial activity. This study shows the potential of such an ODDS for the in vivo treatment of infection with pathogens.

  12. Resolution of lysosomes in living cells with a ratiometric molecular pH-meter.

    PubMed

    Li, Zhu; Wu, Shuqi; Han, Jiahuai; Yang, Liu; Han, Shoufa

    2013-09-30

    Intracellular acidic vesicles, constituted mostly by lysosomes, mediated a variety of biological events ranging from endocytosis, apoptosis, to cancer metastasis, etc. A chimeric molecular pH-meter (Lyso-DR), comprised of a dansyl fluorophore and proton activatable rhodamine-lactam, was prepared for ratiometric reporting of intralysosomal acidity. Exclusively confined in lysosomes, Lyso-DR exhibited pH dependent dual fluorescence emission bands which enable resolution of individual lysosomes in terms of acidity and quantitation of the overall intracellular lysosomal acidity, e.g. the lysosomal pH of HeLa cells is around pH 5.0 whereas that of L929 cells is around pH 6.2. Lyso-DR effectively differentiated the lysosomal pH changes of cells undergoing apoptosis vs necrosis, suggesting its utility in investigations on lysosome involved biomedical processes. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Biogenesis of lysosomes in marshall cells and in cells of the male reproductive system.

    PubMed

    Morales, C R; Fuska, J; Zhao, Q; Lefrancois, S

    2001-05-01

    The mechanism of plasma membrane trafficking and degradation is still poorly understood. This investigation deals with the biogenesis of lysosomes during endocytic flow in Marshall cells and in various cell types of the male reproductive system. Marshall cells were exposed to ammonium chloride (NH4Cl) and leupeptin after labeling with cationic ferritin. In some experiments, the treated cells were immunogold labeled with anti-prosaposin antibody. NH4Cl and leupeptin are lysosomotropic agents that affect the endosomal-lysosomal progression. Testes, efferent ducts and epididymis from mouse mutants with defects affecting plasma membrane degradation were also used to analyze this process. NH4Cl produced a retention of cationic ferritin in endosomes and hindered the endosomal/lysosomal progression. Leupeptin did not affect this process. NH4Cl decreased the labeling of prosaposin in endosomes and lysosomes, while leupeptin increased the labeling of prosaposin in lysosomes. The number of lysosomes per cytoplasmic area was higher in treated cells than in controls. These findings suggest that leupeptin affected lysosomes whereas NH4Cl affected both endosomes and lysosomes. The endosomal and lysosomal accumulation of prosaposin induced by the treatment with NH4Cl and leupeptin indicated that the site of entry of prosaposinwas both the lysosome and endosome. Electron microscopy (EM) of tissues from mouse mutants with defects affecting plasma membrane degradation substantiated these observations. The EM analysis revealed a selective accumulation of multivesicular bodies (MVBs) and the disappearance of lysosomes, in testicular fibroblasts, nonciliated cells of the efferent ducts and principal cells of the epididymis, suggesting that MVBs are precursors of lysosomes. (1) endosomes and MVBs are a required steps for degradation of membranes; (2) endosomes and MVBs are precursors of lysosomes; and (3) endosomes, MVBs, and lysosomes appear to be transient organelles. Copyright 2001

  14. Phosphoinositides in the mammalian endo-lysosomal network

    PubMed Central

    Cullen, Peter J.; Carlton, Jeremy G.

    2014-01-01

    The endo-lysosomal system is an interconnected tubulo-vesicular network that acts as a sorting station to process and distribute internalised cargo. This network accepts cargoes from both the plasma membrane and the biosynthetic pathway, and directs these cargos either towards the lysosome for degradation, the peri-nuclear recycling endosome for return to the cell surface, or to the trans-Golgi network. These intracellular membranes are variously enriched in different phosphoinositides that help to shape compartmental identity. These lipids act to localise a number of phosphoinositide-binding proteins that function as sorting machineries to regulate endosomal cargo sorting. Herein we discuss regulation of these machineries by phosphoinositides and explore how phosphoinositide-switching contributes toward sorting decisions made at this platform. PMID:22374088

  15. Role of Endosomes and Lysosomes in Human Disease

    PubMed Central

    Maxfield, Frederick R.

    2014-01-01

    In addition to their roles in normal cell physiology, endocytic processes play a key role in many diseases. In this review, three diseases are discussed as examples of the role of endocytic processes in disease. The uptake of cholesterol via LDL is central to our understanding of atherosclerosis, and the study of this disease led to many of the key breakthroughs in understanding receptor-mediated endocytosis. Alzheimer’s disease is a growing burden as the population ages. Endosomes and lysosomes play important but only partially understood roles in both the formation and the degradation of the amyloid fibrils that are associated with Alzheimer’s disease. Inherited lysosomal storage diseases are individually rare, but collectively they affect many individuals. Recent advances are leading to improved enzyme replacement therapy and are also leading to small-molecule drugs to treat some of these diseases. PMID:24789821

  16. Zinc Chelation Mediates the Lysosomal Disruption without Intracellular ROS Generation

    PubMed Central

    Matias, Andreza Cândido; Manieri, Tânia Maria; Cerchiaro, Giselle

    2016-01-01

    We report the molecular mechanism for zinc depletion caused by TPEN (N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine) in neuroblastoma cells. The activation of p38 MAP kinase and subsequently caspase 3 is not due to or followed by redox imbalance or ROS generation, though these are commonly observed in literature. We found that TPEN is not responsible for ROS generation and the mechanism involves essentially lysosomal disruption caused by intracellular zinc depletion. We also observed a modest activation of Bax and no changes in the Bcl-2 proteins. As a result, we suggest that TPEN causes intracellular zinc depletion which can influence the breakdown of lysosomes and cell death without ROS generation. PMID:27123155

  17. Immune response hinders therapy for lysosomal storage diseases

    PubMed Central

    Ponder, Katherine P.

    2008-01-01

    Enzyme replacement therapy (ERT) for the lysosomal storage disease mucopolysaccharidosis I (MPS I) involves i.v. injection of α-l-iduronidase, which can be taken up by cells throughout the body. While a significant immune response to ERT has been shown in patients with MPS I, little is known about what effect anti-enzyme antibodies have on treatment efficacy. In this issue of the JCI, Dickson et al. demonstrate that anti-enzyme antibodies inhibit enzyme uptake and substantially limit the therapeutic efficacy of ERT in canines with MPS I (see the related article beginning on page 2868). Furthermore, the induction of immune tolerance — via oral delivery of cyclosporine A and azathioprine for two months at the time of initiation of ERT with recombinant human α-l-iduronidase — improved enzyme uptake in organs. Therefore, transient immunosuppression may enhance ERT for lysosomal storage diseases. PMID:18654672

  18. Immune response hinders therapy for lysosomal storage diseases.

    PubMed

    Ponder, Katherine P

    2008-08-01

    Enzyme replacement therapy (ERT) for the lysosomal storage disease mucopolysaccharidosis I (MPS I) involves i.v. injection of alpha-l-iduronidase, which can be taken up by cells throughout the body. While a significant immune response to ERT has been shown in patients with MPS I, little is known about what effect anti-enzyme antibodies have on treatment efficacy. In this issue of the JCI, Dickson et al. demonstrate that anti-enzyme antibodies inhibit enzyme uptake and substantially limit the therapeutic efficacy of ERT in canines with MPS I (see the related article beginning on page 2868). Furthermore, the induction of immune tolerance--via oral delivery of cyclosporine A and azathioprine for two months at the time of initiation of ERT with recombinant human alpha-L-iduronidase--improved enzyme uptake in organs. Therefore, transient immunosuppression may enhance ERT for lysosomal storage diseases.

  19. PLEKHM1: Adapting to life at the lysosome.

    PubMed

    McEwan, David G; Dikic, Ivan

    2015-04-03

    The endosomal system and autophagy are 2 intertwined pathways that share a number of common protein factors as well as a final destination, the lysosome. Identification of adaptor platforms that can link both pathways are of particular importance, as they serve as common nodes that can coordinate the different trafficking arms of the endolysosomal system. Using a mass spectrometry approach to identify interaction partners of active (GTP-bound) RAB7, the late endosome/lysosome GTPase, and yeast 2-hybrid screening to identify LC3/GABARAP interaction partners we discovered the multivalent adaptor protein PLEKHM1. We discovered a highly conserved LC3-interaction region (LIR) between 2 PH domains of PLEKHM1 that mediated direct binding to all LC3/GABARAP family members. Subsequent mass spectrometry analysis of PLEKHM1 precipitated from cells revealed the HOPS (homotypic fusion and protein sorting) complex as a prominent interaction partner. Functionally, depletion of PLEKHM1, HOPS, or RAB7 results in decreased autophagosome-lysosome fusion. In Plekhm1 knockout (KO) mouse embryonic fibroblasts (MEFs) we observed increased lipidated LC3B, decreased colocalization between LC3B and LAMP1 under amino acid starvation conditions and decreased autolysosome formation. Finally, PLEKHM1 binding to LC3-positive autophagosomes was also essential for selective autophagy pathways, as shown by clearance of puromycin-aggregates, in a PLEKHM1-LIR-dependent manner. Overall, we have identified PLEKHM1 as an endolysosomal adaptor platform that acts as a central hub to integrate endocytic and autophagic pathways at the lysosome.

  20. Doxorubicin Blocks Cardiomyocyte Autophagic Flux by Inhibiting Lysosome Acidification

    PubMed Central

    Li, Dan L.; Wang, Zhao V.; Ding, Guanqiao; Tan, Wei; Luo, Xiang; Criollo, Alfredo; Xie, Min; Jiang, Nan; May, Herman; Kyrychenko, Viktoriia; Schneider, Jay W.; Gillette, Thomas G.; Hill, Joseph A.

    2016-01-01

    Background The clinical use of doxorubicin is limited by cardiotoxicity. Histopathologic changes include interstitial myocardial fibrosis and appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Methods and Results Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, we studied animals with diminished autophagic activity due to haploinsufficiency for Beclin 1. Beclin 1+/− mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely, animals over-expressing Beclin 1 manifested an amplified cardiotoxic response. Conclusions Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity. PMID:26984939

  1. Doxorubicin Blocks Cardiomyocyte Autophagic Flux by Inhibiting Lysosome Acidification.

    PubMed

    Li, Dan L; Wang, Zhao V; Ding, Guanqiao; Tan, Wei; Luo, Xiang; Criollo, Alfredo; Xie, Min; Jiang, Nan; May, Herman; Kyrychenko, Viktoriia; Schneider, Jay W; Gillette, Thomas G; Hill, Joseph A

    2016-04-26

    The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, we studied animals with diminished autophagic activity resulting from haploinsufficiency for Beclin 1. Beclin 1(+/-) mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely, animals overexpressing Beclin 1 manifested an amplified cardiotoxic response. Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity. © 2016 American Heart Association, Inc.

  2. Lysosomal storage diseases: diagnostic confirmation and management of presymptomatic individuals.

    PubMed

    Wang, Raymond Y; Bodamer, Olaf A; Watson, Michael S; Wilcox, William R

    2011-05-01

    To develop educational guidelines for the diagnostic confirmation and management of individuals identified by newborn screening, family-based testing after proband identification, or carrier testing in at-risk populations, and subsequent prenatal or postnatal testing of those who are presymptomatic for a lysosomal storage disease. Review of English language literature and discussions in a consensus development panel comprised an international group of experts in the clinical and laboratory diagnosis, treatment and management, newborn screening, and genetic aspects of lysosomal storage diseases. Although clinical trial and longitudinal data were used when available, the evidence in the literature is limited and consequently the recommendations must be considered as expert opinion. Guidelines were developed for Fabry, Gaucher, and Niemann-Pick A/B diseases, glycogen storage type II (Pompe disease), globoid cell leukodystrophy (Krabbe disease), metachromatic leukodystrophy, and mucopolysaccharidoses types I, II, and VI. These guidelines serve as an educational resource for confirmatory testing and subsequent clinical management of presymptomatic individuals suspected to have a lysosomal storage disease; they also help to define a research agenda for longitudinal studies such as the American College of Medical Genetics/National Institutes of Health Newborn Screening Translational Research Network.

  3. Discriminating lysosomal membrane protein types using dynamic neural network.

    PubMed

    Tripathi, Vijay; Gupta, Dwijendra Kumar

    2014-01-01

    This work presents a dynamic artificial neural network methodology, which classifies the proteins into their classes from their sequences alone: the lysosomal membrane protein classes and the various other membranes protein classes. In this paper, neural networks-based lysosomal-associated membrane protein type prediction system is proposed. Different protein sequence representations are fused to extract the features of a protein sequence, which includes seven feature sets; amino acid (AA) composition, sequence length, hydrophobic group, electronic group, sum of hydrophobicity, R-group, and dipeptide composition. To reduce the dimensionality of the large feature vector, we applied the principal component analysis. The probabilistic neural network, generalized regression neural network, and Elman regression neural network (RNN) are used as classifiers and compared with layer recurrent network (LRN), a dynamic network. The dynamic networks have memory, i.e. its output depends not only on the input but the previous outputs also. Thus, the accuracy of LRN classifier among all other artificial neural networks comes out to be the highest. The overall accuracy of jackknife cross-validation is 93.2% for the data-set. These predicted results suggest that the method can be effectively applied to discriminate lysosomal associated membrane proteins from other membrane proteins (Type-I, Outer membrane proteins, GPI-Anchored) and Globular proteins, and it also indicates that the protein sequence representation can better reflect the core feature of membrane proteins than the classical AA composition.

  4. Enzymatic Screening and Diagnosis of Lysosomal Storage Diseases

    PubMed Central

    Yu, Chunli; Sun, Qin; Zhou, Hui

    2016-01-01

    Lysosomal storage diseases (LSDs) are a group of more than 50 genetic disorders. Clinical symptoms are caused by the deficiency of specific enzyme (enzymes) function and resultant substrate accumulation in the lysosomes, which leads to impaired cellular function and progressive tissue and organ dysfunction. Measurement of lysosomal enzyme activity plays an important role in the clinical diagnosis of LSDs. The major enzymatic testing methods include fluorometric assays using artificial 4-methylumbelliferyl (4-MU) substrates, spectrophotometric assays and radioactive assays with radiolabeled natural substrates. As many effective treatment options have become available, presymptomatic diagnosis and early intervention are imperative. Many methods were developed in the past decade for newborn screening (NBS) of selective LSDs in dried blood spot (DBS) specimens. Modified fluorometric assays with 4-MU substrates, MS/MS or LC-MS/MS multiplex enzyme assays, digital microfluidic fluorometric assays, and immune-quantification assays for enzyme contents have been reported in NBS of LSDs, each with its own advantages and limitations. Active technical validation studies and pilot screening studies have been conducted or are ongoing. These studies have provided insight in the efficacy of various methodologies. In this review, technical aspects of the enzyme assays used in clinical diagnosis and NBS are summarized. The important findings from pilot NBS studies are also reviewed. PMID:27293520

  5. Enzyme therapy for lysosomal acid lipase deficiency in the mouse.

    PubMed

    Du, H; Schiavi, S; Levine, M; Mishra, J; Heur, M; Grabowski, G A

    2001-08-01

    Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of the triglycerides (TG) and cholesteryl esters (CE) delivered to lysosomes. Its deficiency produces two human phenotypes, Wolman disease (WD) and cholesteryl ester storage disease (CESD). A targeted disruption of the LAL locus produced a null (lal( -/-)) mouse model that mimics human WD/CESD. The potential for enzyme therapy was tested using mannose terminated human LAL expressed in Pichia pastoris (phLAL), purified, and administered by tail vein injections to lal( -/-) mice. Mannose receptor (MR)-dependent uptake and lysosomal targeting of phLAL were evidenced ex vivo using competitive assays with MR-positive J774E cells, a murine monocyte/macrophage line, immunofluorescence and western blots. Following (bolus) IV injection, phLAL was detected in Kupffer cells, lung macrophages and intestinal macrophages in lal( -/-) mice. Two-month-old lal( -/-) mice received phLAL (1.5 U/dose) or saline injections once every 3 days for 30 days (10 doses). The treated lal( -/-) mice showed nearly complete resolution of hepatic yellow coloration; hepatic weight decreased by approximately 36% compared to PBS-treated lal( -/-) mice. Histologic analyses of numerous tissues from phLAL-treated mice showed reductions in macrophage lipid storage. TG and cholesterol levels decreased by approximately 50% in liver, 69% in spleen and 50% in small intestine. These studies provide feasibility for LAL enzyme therapy in human WD and CESD.

  6. Structure of human saposin A at lysosomal pH.

    PubMed

    Hill, Chris H; Read, Randy J; Deane, Janet E

    2015-07-01

    The saposins are essential cofactors for the normal lysosomal degradation of complex glycosphingolipids by acid hydrolase enzymes; defects in either saposin or hydrolase function lead to severe metabolic diseases. Saposin A (SapA) activates the enzyme β-galactocerebrosidase (GALC), which catalyzes the breakdown of β-D-galactocerebroside, the principal lipid component of myelin. SapA is known to bind lipids and detergents in a pH-dependent manner; this is accompanied by a striking transition from a `closed' to an `open' conformation. However, previous structures were determined at non-lysosomal pH. This work describes a 1.8 Å resolution X-ray crystal structure determined at the physiologically relevant lysosomal pH 4.8. In the absence of lipid or detergent at pH 4.8, SapA is observeed to adopt a conformation closely resembling the previously determined `closed' conformation, showing that pH alone is not sufficient for the transition to the `open' conformation. Structural alignments reveal small conformational changes, highlighting regions of flexibility.

  7. Astrocyte dysfunction triggers neurodegeneration in a lysosomal storage disorder.

    PubMed

    Di Malta, Chiara; Fryer, John D; Settembre, Carmine; Ballabio, Andrea

    2012-08-28

    The role of astrocytes in neurodegenerative processes is increasingly appreciated. Here we investigated the contribution of astrocytes to neurodegeneration in multiple sulfatase deficiency (MSD), a severe lysosomal storage disorder caused by mutations in the sulfatase modifying factor 1 (SUMF1) gene. Using Cre/Lox mouse models, we found that astrocyte-specific deletion of Sumf1 in vivo induced severe lysosomal storage and autophagy dysfunction with consequential cytoplasmic accumulation of autophagic substrates. Lysosomal storage in astrocytes was sufficient to induce degeneration of cortical neurons in vivo. Furthermore, in an ex vivo coculture assay, we observed that Sumf1(-/-) astrocytes failed to support the survival and function of wild-type cortical neurons, suggesting a non-cell autonomous mechanism for neurodegeneration. Compared with the astrocyte-specific deletion of Sumf1, the concomitant removal of Sumf1 in both neurons and glia in vivo induced a widespread neuronal loss and robust neuroinflammation. Finally, behavioral analysis of mice with astrocyte-specific deletion of Sumf1 compared with mice with Sumf1 deletion in both astrocytes and neurons allowed us to link a subset of neurological manifestations of MSD to astrocyte dysfunction. This study indicates that astrocytes are integral components of the neuropathology in MSD and that modulation of astrocyte function may impact disease course.

  8. Alterations in membrane trafficking and pathophysiological implications in lysosomal storage disorders.

    PubMed

    Kuech, Eva-Maria; Brogden, Graham; Naim, Hassan Y

    2016-11-01

    Lysosomal storage disorders are a heterogeneous group of more than 50 distinct inborn metabolic diseases affecting about 1 in 5000 to 7000 live births. The diseases often result from mutations followed by functional deficiencies of enzymes or transporters within the acidic environment of the lysosome, which mediate the degradation of a wide subset of substrates, including glycosphingolipids, glycosaminoglycans, cholesterol, glycogen, oligosaccharides, peptides and glycoproteins, or the export of the respective degradation products from the lysosomes. The progressive accumulation of uncleaved substrates occurs in multiple organs and finally causes a broad spectrum of different pathologies including visceral, neurological, skeletal and hematologic manifestations. Besides deficient lysosomal enzymes and transporters other defects may lead to lysosomal storage disorders, including activator defects, membrane defects or defects in modifier proteins. In this review we concentrate on four different lysosomal storage disorders: Niemann-Pick type C, Fabry disease, Gaucher disease and Pompe disease. While the last three are caused by defective lysosomal hydrolases, Niemann-Pick type C is caused by the inability to export LDL-derived cholesterol out of the lysosome. We want to emphasise potential implications of membrane trafficking defects on the pathology of these diseases, as many mutations interfere with correct lysosomal protein trafficking and alter cellular lipid homeostasis. Current therapeutic strategies are summarised, including substrate reduction therapy as well as pharmacological chaperone therapy which directly aim to improve folding and lysosomal transport of misfolded mutant proteins.

  9. Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins

    SciTech Connect

    Storrie, B.; Sachdeva, M.; Viers, V.S.

    1984-02-01

    We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.

  10. MCOLN1 is a ROS sensor in lysosomes that regulates autophagy

    PubMed Central

    Zhang, Xiaoli; Cheng, Xiping; Yu, Lu; Yang, Junsheng; Calvo, Raul; Patnaik, Samarjit; Hu, Xin; Gao, Qiong; Yang, Meimei; Lawas, Maria; Delling, Markus; Marugan, Juan; Ferrer, Marc; Xu, Haoxing

    2016-01-01

    Cellular stresses trigger autophagy to remove damaged macromolecules and organelles. Lysosomes ‘host' multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and lysosomes. For example, transcription factor (TF)EB, which regulates autophagy and lysosome biogenesis, is activated following the inhibition of mTOR, a lysosome-localized nutrient sensor. Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca2+-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca2+ release. This activation triggers calcineurin-dependent TFEB-nuclear translocation, autophagy induction and lysosome biogenesis. When TRPML1 is genetically inactivated or pharmacologically inhibited, clearance of damaged mitochondria and removal of excess ROS are blocked. Furthermore, TRPML1's ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage. Hence, TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell. PMID:27357649

  11. Molecular pathogenesis and in vitro correction of the only known human lysosomal amidase deficiency, aspartylglucosaminuria

    SciTech Connect

    Jalanko, A.; Enomaa, N.; Isoniemi, A.

    1994-09-01

    Aspartylglucosaminuria (AGU) is an infantile-onset lysosomal storage disease with a severe neurodegenerative course. This disease is caused by mutations in the aspartylglucosaminidase (AGA) gene and it is strongly enriched, with the carrier frequency of 1:40, in the genetically isolated population of Finland, where one mutation, AGU{sub Fin} accounts for 98% of disease alleles. We have isolated the AGA cDNA and characterized in detail the individual steps in the intracellular biosynthesis and processing of this lysosomal amidase. Analysis of cellular consequences of the AGU{sub Fin} and twelve other mutations have revealed that naturally occurring AGU-mutations cause a rather uniform clinical phenotype and typically affect the folding and intracellular stability of the enzyme. In vitro expression of mutagenized AGA polypeptides have exposed regions important for the activity. This data combined with the preliminary data from crystallization studies have given insight to the three-dimensional structure of the enzyme, including the active site. This information is essential considering the therapy trials, which we have initiated using AGU patients fibroblasts and murine neural cell lines as targets. Complete correction of the enzyme deficiency was obtained in vitro with recombinant AGA enzyme purified from CHO-cells and with retrovirus-mediated transfer of AGA. To elucidate tissue consequences in the CNS, and to facilitate further therapy studies, we have cloned the genomic AGA DNA of mouse and constructed ES cell lines carrying targeted mutations in the AGA gene. Subsequent creation of the murine models for AGU will allow the molecular studies of phenotypes of the knock-out mice and those homozygous for less dramatic mutations mimicking the disease in man, and will give insight to the pathogenesis of the CNS symptoms of AGU.

  12. A non-conserved miRNA regulates lysosomal function and impacts on a human lysosomal storage disorder.

    PubMed

    Frankel, Lisa B; Di Malta, Chiara; Wen, Jiayu; Eskelinen, Eeva-Liisa; Ballabio, Andrea; Lund, Anders H

    2014-12-19

    Sulfatases are key enzymatic regulators of sulfate homeostasis with several biological functions including degradation of glycosaminoglycans (GAGs) and other macromolecules in lysosomes. In a severe lysosomal storage disorder, multiple sulfatase deficiency (MSD), global sulfatase activity is deficient due to mutations in the sulfatase-modifying factor 1 (SUMF1) gene, encoding the essential activator of all sulfatases. We identify a novel regulatory layer of sulfate metabolism mediated by a microRNA. miR-95 depletes SUMF1 protein levels and suppresses sulfatase activity, causing the disruption of proteoglycan catabolism and lysosomal function. This blocks autophagy-mediated degradation, causing cytoplasmic accumulation of autophagosomes and autophagic substrates. By targeting miR-95 in cells from MSD patients, we can effectively increase residual SUMF1 expression, allowing for reactivation of sulfatase activity and increased clearance of sulfated GAGs. The identification of this regulatory mechanism opens the opportunity for a unique therapeutic approach in MSD patients where the need for exogenous enzyme replacement is circumvented.

  13. The Ankrd13 Family of Ubiquitin-interacting Motif-bearing Proteins Regulates Valosin-containing Protein/p97 Protein-mediated Lysosomal Trafficking of Caveolin 1*

    PubMed Central

    Burana, Daocharad; Yoshihara, Hidehito; Tanno, Hidetaka; Yamamoto, Akitsugu; Saeki, Yasushi; Tanaka, Keiji; Komada, Masayuki

    2016-01-01

    Caveolin 1 (Cav-1) is an oligomeric protein that forms flask-shaped, lipid-rich pits, termed caveolae, on the plasma membrane. Cav-1 is targeted for lysosomal degradation in ubiquitination- and valosin-containing protein (VCP)-dependent manners. VCP, an ATPase associated with diverse cellular activities that remodels or segregates ubiquitinated protein complexes, has been proposed to disassemble Cav-1 oligomers on the endosomal membrane, facilitating the trafficking of Cav-1 to the lysosome. Genetic mutations in VCP compromise the lysosomal trafficking of Cav-1, leading to a disease called inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia (IBMPFD). Here we identified the Ankrd13 family of ubiquitin-interacting motif (UIM)-containing proteins as novel VCP-interacting molecules on the endosome. Ankrd13 proteins formed a ternary complex with VCP and Cav-1 and exhibited high binding affinity for ubiquitinated Cav-1 oligomers in an UIM-dependent manner. Mass spectrometric analyses revealed that Cav-1 undergoes Lys-63-linked polyubiquitination, which serves as a lysosomal trafficking signal, and that the UIMs of Ankrd13 proteins bind preferentially to this ubiquitin chain type. The overexpression of Ankrd13 caused enlarged hollow late endosomes, which was reminiscent of the phenotype of the VCP mutations in IBMPFD. Overexpression of Ankrd13 proteins also stabilized ubiquitinated Cav-1 oligomers on the limiting membrane of enlarged endosomes. The interaction with Ankrd13 was abrogated in IMBPFD-associated VCP mutants. Collectively, our results suggest that Ankrd13 proteins cooperate with VCP to regulate the lysosomal trafficking of ubiquitinated Cav-1. PMID:26797118

  14. The Ankrd13 Family of Ubiquitin-interacting Motif-bearing Proteins Regulates Valosin-containing Protein/p97 Protein-mediated Lysosomal Trafficking of Caveolin 1.

    PubMed

    Burana, Daocharad; Yoshihara, Hidehito; Tanno, Hidetaka; Yamamoto, Akitsugu; Saeki, Yasushi; Tanaka, Keiji; Komada, Masayuki

    2016-03-18

    Caveolin 1 (Cav-1) is an oligomeric protein that forms flask-shaped, lipid-rich pits, termed caveolae, on the plasma membrane. Cav-1 is targeted for lysosomal degradation in ubiquitination- and valosin-containing protein (VCP)-dependent manners. VCP, an ATPase associated with diverse cellular activities that remodels or segregates ubiquitinated protein complexes, has been proposed to disassemble Cav-1 oligomers on the endosomal membrane, facilitating the trafficking of Cav-1 to the lysosome. Genetic mutations in VCP compromise the lysosomal trafficking of Cav-1, leading to a disease called inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia (IBMPFD). Here we identified the Ankrd13 family of ubiquitin-interacting motif (UIM)-containing proteins as novel VCP-interacting molecules on the endosome. Ankrd13 proteins formed a ternary complex with VCP and Cav-1 and exhibited high binding affinity for ubiquitinated Cav-1 oligomers in an UIM-dependent manner. Mass spectrometric analyses revealed that Cav-1 undergoes Lys-63-linked polyubiquitination, which serves as a lysosomal trafficking signal, and that the UIMs of Ankrd13 proteins bind preferentially to this ubiquitin chain type. The overexpression of Ankrd13 caused enlarged hollow late endosomes, which was reminiscent of the phenotype of the VCP mutations in IBMPFD. Overexpression of Ankrd13 proteins also stabilized ubiquitinated Cav-1 oligomers on the limiting membrane of enlarged endosomes. The interaction with Ankrd13 was abrogated in IMBPFD-associated VCP mutants. Collectively, our results suggest that Ankrd13 proteins cooperate with VCP to regulate the lysosomal trafficking of ubiquitinated Cav-1. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Signals for the lysosome: a control center for cellular clearance and energy metabolism

    PubMed Central

    Settembre, Carmine; Fraldi, Alessandro; Medina, Diego L.

    2015-01-01

    Preface For a long time lysosomes were considered merely to be cellular “incinerators” involved in the degradation and recycling of cellular waste. However, there is now compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signaling and energy metabolism. Furthermore, the essential role of lysosomes in the autophagic pathway puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master gene, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy, has revealed how the lysosome adapts to environmental cues, such as starvation, and suggests novel therapeutic strategies for modulating lysosomal function in human disease. PMID:23609508

  16. Identification of a Lysosomal Pathway That Modulates Glucocorticoid Signaling and the Inflammatory Response

    PubMed Central

    He, Yuanzheng; Xu, Yong; Zhang, Chenghai; Gao, Xiang; Dykema, Karl J.; Martin, Katie R.; Ke, Jiyuan; Hudson, Eric A.; Khoo, Sok Kean; Resau, James H.; Alberts, Arthur S.; MacKeigan, Jeffrey P.; Furge, Kyle A.; Xu, H. Eric

    2013-01-01

    The antimalaria drug chloroquine has been used as an anti-inflammatory agent for treating systemic lupus erythematosus and rheumatoid arthritis. We report that chloroquine promoted the transrepression of proinflammatory cytokines by the glucocorticoid receptor (GR). In a mouse collagen-induced arthritis model, chloroquine enhanced the therapeutic effects of glucocorticoid treatment. By inhibiting lysosome function, chloroquine synergistically activated glucocorticoid signaling. Lysosomal inhibition by either bafilomycin A1 (an inhibitor of the vacuolar adenosine triphosphatase) or knockdown of transcription factor EB (TFEB, a master activator of lysosomal biogenesis) mimicked the effects of chloroquine. The abundance of the GR, as well as that of the androgen receptor and estrogen receptor, correlated with changes in lysosomal biogenesis. Thus, we showed that glucocorticoid signaling is regulated by lysosomes, which provides a mechanistic basis for treating inflammation and autoimmune diseases with a combination of glucocorticoids and lysosomal inhibitors. PMID:21730326

  17. Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection.

    PubMed

    Qi, Xiaopeng; Man, Si Ming; Malireddi, R K Subbarao; Karki, Rajendra; Lupfer, Christopher; Gurung, Prajwal; Neale, Geoffrey; Guy, Clifford S; Lamkanfi, Mohamed; Kanneganti, Thirumala-Devi

    2016-09-19

    Lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic, and degenerative diseases in humans. In this study, we identified a key cell-intrinsic role for cathepsin B as a negative feedback regulator of lysosomal biogenesis and autophagy. Mice and macrophages lacking cathepsin B activity had increased resistance to the cytosolic bacterial pathogen Francisella novicida Genetic deletion or pharmacological inhibition of cathepsin B down-regulated mechanistic target of rapamycin activity and prevented cleavage of the lysosomal calcium channel TRPML1. These events drove transcription of lysosomal and autophagy genes via transcription factor EB, which increased lysosomal biogenesis and activation of autophagy initiation kinase ULK1 for clearance of the bacteria. Our results identified a fundamental biological function of cathepsin B in providing a checkpoint for homeostatic maintenance of lysosome populations and basic recycling functions in the cell. © 2016 Qi et al.

  18. Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection

    PubMed Central

    Malireddi, R.K. Subbarao; Karki, Rajendra; Lupfer, Christopher; Gurung, Prajwal; Lamkanfi, Mohamed

    2016-01-01

    Lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic, and degenerative diseases in humans. In this study, we identified a key cell-intrinsic role for cathepsin B as a negative feedback regulator of lysosomal biogenesis and autophagy. Mice and macrophages lacking cathepsin B activity had increased resistance to the cytosolic bacterial pathogen Francisella novicida. Genetic deletion or pharmacological inhibition of cathepsin B down-regulated mechanistic target of rapamycin activity and prevented cleavage of the lysosomal calcium channel TRPML1. These events drove transcription of lysosomal and autophagy genes via transcription factor EB, which increased lysosomal biogenesis and activation of autophagy initiation kinase ULK1 for clearance of the bacteria. Our results identified a fundamental biological function of cathepsin B in providing a checkpoint for homeostatic maintenance of lysosome populations and basic recycling functions in the cell. PMID:27551156

  19. In vivo and in vitro analysis of lysosomes and acid phosphatase activity in human chagasic placentas.

    PubMed

    Fretes, R E; De Fabro, S P

    1995-12-01

    A structural, cytochemical, stereological, and biochemical study of lysosomes and a lysosome marker, the enzyme acid phosphatase, was performed, both in placentas at term from chagasic pregnant women without fetal infection and in normal placentas at term cocultured in vitro with Trypanosoma cruzi. It was found that in placentas from chagasic women lysosomes were normally distributed in the trophoblast. Stereological analysis showed that lysosomes and cytochemical acid phosphatase (AcP) activity were increased in the trophoblast of chagasic placentas. AcP activity increased in subcellular fractions of the isolated trophoblast from chagasic placentas, and the lysosomal fraction of those placentas exhibited the highest value of enzymatic activity in comparison to controls (P < 0.05). No differences in AcP activity were observed between homogenates of normal placentas cocultured with T. cruzi and controls. These data suggest that the placental lysosome population might be involved in the process of placental infection by T. cruzi.

  20. Biliary copper excretion by hepatocyte lysosomes in the rat. Major excretory pathway in experimental copper overload

    SciTech Connect

    Gross, J.B. Jr.; Myers, B.M.; Kost, L.J.; Kuntz, S.M.; LaRusso, N.F.

    1989-01-01

    We investigated the hypothesis that lysosomes are the main source of biliary copper in conditions of hepatic copper overload. We used a rat model of oral copper loading and studied the relationship between the biliary output of copper and lysosomal hydrolases. Male Sprague-Dawley rats were given tap water with or without 0.125% copper acetate for up to 36 wk. Copper loading produced a 23-fold increase in the hepatic copper concentration and a 30-65% increase in hepatic lysosomal enzyme activity. Acid phosphatase histochemistry showed that copper-loaded livers contained an increased number of hepatocyte lysosomes; increased copper concentration of these organelles was confirmed directly by both x ray microanalysis and tissue fractionation. The copper-loaded rats showed a 16-fold increase in biliary copper output and a 50-300% increase in biliary lysosomal enzyme output. In the basal state, excretory profiles over time were similar for biliary outputs of lysosomal enzymes and copper in the copper-loaded animals but not in controls. After pharmacologic stimulation of lysosomal exocytosis, biliary outputs of copper and lysosomal hydrolases in the copper-loaded animals remained coupled: injection of colchicine or vinblastine produced an acute rise in the biliary output of both lysosomal enzymes and copper to 150-250% of baseline rates. After these same drugs, control animals showed only the expected increase in lysosomal enzyme output without a corresponding increase in copper output. We conclude that the hepatocyte responds to an increased copper load by sequestering excess copper in an increased number of lysosomes that then empty their contents directly into bile. The results provide direct evidence that exocytosis of lysosomal contents into biliary canaliculi is the major mechanism for biliary copper excretion in hepatic copper overload.

  1. Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments.

    PubMed

    Pizarro-Cerdá, J; Moreno, E; Sanguedolce, V; Mege, J L; Gorvel, J P

    1998-05-01

    Virulent and attenuated Brucella abortus strains attach to and penetrate nonprofessional phagocytic HeLa cells. Compared to pathogenic Brucella, the attenuated strain 19 hardly replicates within cells. The majority of the strain 19 bacteria colocalized with the lysosome marker cathepsin D, suggesting that Brucella-containing phagosomes had fused with lysosomes, in which they may have degraded. The virulent bacteria prevented lysosome-phagosome fusion and were found distributed in the perinuclear region within compartments resembling autophagosomes.

  2. Musings on genome medicine: enzyme-replacement therapy of the lysosomal storage diseases

    PubMed Central

    2009-01-01

    The lysosomal storage diseases, such as Gaucher's disease, mucopolysaccharidosis I, II and IV, Fabry's disease, and Pompe's disease, are rare inherited disorders whose symptoms result from enzyme deficiency causing lysosomal accumulation. Until effective gene-replacement therapy is developed, expensive, and at best incomplete, enzyme-replacement therapy is the only hope for sufferers of rare lysosomal storage diseases. Preventive strategies involving carrier detection should be a priority toward the successful management of these conditions. PMID:20017892

  3. Role of lysosome rupture in controlling Nlrp3 signaling and necrotic cell death

    PubMed Central

    Lima, Jr., Heriberto; Jacobson, Lee S.; Goldberg, Michael F.; Chandran, Kartik; Diaz-Griffero, Felipe; Lisanti, Michael P.; Brojatsch, Jürgen

    2013-01-01

    The Nod-like receptor, Nlrp3, has been linked to inflammatory diseases and adjuvant-mediated immune responses. A wide array of structurally diverse agents does not interact directly with Nlrp3, but is thought to activate the Nlrp3 inflammasome by inducing a common upstream signal, such as lysosome rupture. To test the connection between lysosome integrity and Nlrp3 signaling, we analyzed inflammasome activation following stimulation of murine macrophages with lysosome-destabilizing agents and pyroptosis inducers. Here we provide evidence that lysosomal rupture and the corresponding release of lysosomal hydrolases is an early event in macrophages exposed to the lysosome-destabilizing adjuvants LLOMe and alum. Lysosome rupture preceded cell death induction mediated by these agents and was associated with the degradation of low-molecular weight proteins, including the inflammasome component caspase-1. Proteolysis of caspase-1 was controlled by specific cathepsins, but was independent of autocatalytic processes and Nlrp3 signaling. Consistent with these findings, lysosome-disrupting agents triggered only minimal caspase-1 activation and failed to cause caspase-1-dependent cell death (pyroptosis), generally associated with Nlrp3 signaling. In contrast, lysosome rupture was a late event in macrophages exposed to prototypical pyroptosis inducers. These agents triggered extensive Nlrp3 signaling prior to lysosome rupture with only minimal impact on the cellular proteome. Taken together, our findings suggest that lysosome impairment triggers a cascade of events culminating in cell death but is not crucial for Nlrp3 signaling. The significant differences observed between lysosome-disrupting agents and pyroptosis inducers might explain the distinct immunologic responses associated with these compounds. PMID:23708522

  4. Lysosomal glycogen storage induced by Acarbose, a 1,4-alpha-glucosidase inhibitor.

    PubMed Central

    Geddes, R; Taylor, J A

    1985-01-01

    The 1,4-alpha-glucosidase inhibitor. Acarbose, when injected intraperitoneally disturbs liver lysosome metabolism, causing distinct and persistent inhibition of the enzymes and acute disturbances of lysosomal glycogen metabolism. A feedback control mechanism appears to operate, affecting cytosolic carbohydrate metabolism. A model is suggested for the adult form of lysosomal storage disease. The biochemical effects closely resemble those occurring in glycogenosis type II (Pompe's disease), and these have been confirmed by electron microscopy. Images Fig. 3. PMID:3893420

  5. Phorbol ester induces elevated oxidative activity and alkalization in a subset of lysosomes

    SciTech Connect

    Chen, Chii-Shiarng )

    2002-01-01

    Background: Lysosomes are acidic organelles that play multiple roles in various cellular oxidative activities such as the oxidative burst during cytotoxic killing. It remains to be determined how lysosomal lumen oxidative activity and pH interact and are regulated. Here, I report the use of fluorescent probes to measure oxidative activity and pH of lysosomes in live macrophages upon treatment with the tumor promotor phorbol 12-myristate 13-acetate (PMA), and provide novel insight regarding the regulation of lysosomal oxidative activity and pH. Results: The substrate used to measure oxidative activity was bovine serum albumin covalently coupled to dihydro-2?, 4,5,6,7,7?-hexafluorofluorescein (OxyBURST Green H2HFF BSA). During pulse-chase procedures with live macrophages, this reduced dye was internalized through an endocytic pathway and accumulated in the lysosomes. Oxidation of this compound results in a dramatic increase of fluorescence intensity. By using low-light level fluorescence microscopy, I determined that phorbol ester treatment results in increased oxidative activity and pH elevation in different subsets of lysosomes. Furthermore, lysosomes with stronger oxidative activity tended to exclude the acidotropic lysosomal indicator, and thus exhibit higher alkalinity. Conclusions: Results indicate that there is a regulatory mechanism between lysosomal oxidative activity and pH. Activation of lysosomal Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase by phorbol ester may result in increase of intralysosomal O2?- and H2O2, concurrent with pH elevation due to consumption of H+ and generation of OH-. Furthermore, effect of phorbol ester on elevated oxidative activity and pH is heterogeneous among total lysosomal population. Higher oxidative activity and/or pH are only observed in subsets of lysosomes.

  6. A new lysosomal storage disorder resembling Morquio syndrome in sibs.

    PubMed

    Perrin, Laurence; Fenneteau, Odile; Ilharreborde, Brice; Capri, Yline; Gérard, Marion; Quoc, Emmanuel Bui; Passemard, Sandrine; Ghoumid, Jamal; Caillaud, Catherine; Froissart, Roseline; Tabet, Anne-Claude; Lebon, Sophie; El Ghouzzi, Vincent; Mazda, Keyvan; Verloes, Alain

    2012-03-01

    We report two male sibs, born from unrelated French Caribbean parents, presenting with an unclassifiable storage disorder. Pregnancy and delivery were uneventful. Stunted growth was noted during the first year of life. Both children have short stature (below - 4SD) with short trunk, barrel chest, micromelia with rhizomelic shortening, severe kyphoscoliosis, pectus carinatum, short hands and feet with metatarsus adductus, and excessive joint laxity of the small joints. Learning difficulties with borderline intelligence quotient (IQ) were noted in one of them. They had no hepatomegaly, no splenomegaly, and no dysmorphism. Skeletal X-rays survey demonstrated generalized platyspondyly with tongue-like deformity of the anterior part of the vertebral bodies, hypoplasia of the odontoid process, generalized epiphyseal dysplasia and abnormally shaped metaphyses. The acetabular roofs had a trident aspect. Ophthalmologic and cardiac examinations were normal. Spine deformity required surgical correction in one of the patient at age 4 years. Lysosomal enzymes assays including N-acetylgalactosamine-6-sulfate sulfatase and β-galactosidase were normal, excluding mucopolysaccharidoses type IV A and IV B (Morquio syndrome), respectively. Qualitative analysis found traces of dermatan and chondroitin-sulfates in urine, but quantitative glycosaminoglycan excretion fell within normal limits. They were no vacuolated lymphocytes. Abnormal coarse inclusions were present in eosinophils. Mild Alder anomaly was observed in polymorphonuclears. Granulations were discretely metachromatic with toluidine blue. Those morphological anomalies are in favor of a lysosomal storage disease. No inclusions were found in skin fibroblasts. We hypothesize that these two boys have a distinct autosomal recessive or X-linked lysosomal storage disorder of unknown origin that shares clinical and radiological features with Morquio disease.

  7. Facilitating Facilitators: Enhancing PBL through a Structured Facilitator Development Program

    ERIC Educational Resources Information Center

    Salinitri, Francine D.; Wilhelm, Sheila M.; Crabtree, Brian L.

    2015-01-01

    With increasing adoption of the problem-based learning (PBL) model, creative approaches to enhancing facilitator training and optimizing resources to maintain effective learning in small groups is essential. We describe a theoretical framework for the development of a PBL facilitator training program that uses the constructivist approach as the…

  8. Facilitating Facilitators: Enhancing PBL through a Structured Facilitator Development Program

    ERIC Educational Resources Information Center

    Salinitri, Francine D.; Wilhelm, Sheila M.; Crabtree, Brian L.

    2015-01-01

    With increasing adoption of the problem-based learning (PBL) model, creative approaches to enhancing facilitator training and optimizing resources to maintain effective learning in small groups is essential. We describe a theoretical framework for the development of a PBL facilitator training program that uses the constructivist approach as the…

  9. Sub-lethal oxidative stress induces lysosome biogenesis via a lysosomal membrane permeabilization-cathepsin-caspase 3-transcription factor EB-dependent pathway.

    PubMed

    Leow, San Min; Chua, Shu Xian Serene; Venkatachalam, Gireedhar; Shen, Liang; Luo, Le; Clement, Marie-Veronique

    2016-12-18

    Here we provide evidence to link sub-lethal oxidative stress to lysosomal biogenesis. Exposure of cells to sub-lethal concentrations of exogenously added hydrogen peroxide resulted in cytosol to nuclear translocation of the Transcription Factor EB (TFEB), the master controller of lysosome biogenesis and function. Nuclear translocation of TFEB was dependent upon the activation of a cathepsin-caspase 3 signaling pathway, downstream of a lysosomal membrane permeabilization and accompanied by a significant increase in lysosome numbers as well as induction of TFEB dependent lysosome-associated genes expression such as Ctsl, Lamp2 and its spliced variant Lamp2a, Neu1and Ctsb and Sqstm1 and Atg9b. The effects of sub-lethal oxidative stress on lysosomal gene expression and biogenesis were rescued upon gene silencing of caspase 3 and TFEB. Notably, caspase 3 activation was not associated with phenotypic hallmarks of apoptosis, evidenced by the absence of caspase 3 substrate cleavage, such as PARP, Lamin A/C or gelsolin. Taken together, these data demonstrate for the first time an unexpected and non-canonical role of a cathepsin-caspase 3 axis in the nuclear translocation of TFEB leading to lysosomes biogenesis under conditions of sub-lethal oxidative stress.

  10. Cathepsin inhibition-induced lysosomal dysfunction enhances pancreatic beta-cell apoptosis in high glucose.

    PubMed

    Jung, Minjeong; Lee, Jaemeun; Seo, Hye-Young; Lim, Ji Sun; Kim, Eun-Kyoung

    2015-01-01

    Autophagy is a lysosomal degradative pathway that plays an important role in maintaining cellular homeostasis. We previously showed that the inhibition of autophagy causes pancreatic β-cell apoptosis, suggesting that autophagy is a protective mechanism for the survival of pancreatic β-cells. The current study demonstrates that treatment with inhibitors and knockdown of the lysosomal cysteine proteases such as cathepsins B and L impair autophagy, enhancing the caspase-dependent apoptosis of INS-1 cells and islets upon exposure to high concentration of glucose. Interestingly, treatment with cathepsin B and L inhibitors prevented the proteolytic processing of cathepsins B, D and L, as evidenced by gradual accumulation of the respective pro-forms. Of note, inhibition of aspartic cathepsins had no effect on autophagy and cell viability, suggesting the selective role of cathepsins B and L in the regulation of β-cell autophagy and apoptosis. Lysosomal localization of accumulated pro-cathepsins in the presence of cathepsin B and L inhibitors was verified via immunocytochemistry and lysosomal fractionation. Lysotracker staining indicated that cathepsin B and L inhibitors led to the formation of severely enlarged lysosomes in a time-dependent manner. The abnormal accumulation of pro-cathepsins following treatment with inhibitors of cathepsins B and L suppressed normal lysosomal degradation and the processing of lysosomal enzymes, leading to lysosomal dysfunction. Collectively, our findings suggest that cathepsin defects following the inhibition of cathepsin B and L result in lysosomal dysfunction and consequent cell death in pancreatic β-cells.

  11. Deficiency of ATP13A2 leads to lysosomal dysfunction, α-synuclein accumulation, and neurotoxicity.

    PubMed

    Usenovic, Marija; Tresse, Emilie; Mazzulli, Joseph R; Taylor, J Paul; Krainc, Dimitri

    2012-03-21

    The autophagy-lysosomal pathway plays an important role in the clearance of long-lived proteins and dysfunctional organelles. Lysosomal dysfunction has been implicated in several neurodegenerative disorders including Parkinson's disease and related synucleinopathies that are characterized by accumulations of α-synuclein in Lewy bodies. Recent identification of mutations in genes linked to lysosomal function and neurodegeneration has offered a unique opportunity to directly examine the role of lysosomes in disease pathogenesis. Mutations in lysosomal membrane protein ATP13A2 (PARK9) cause familial Kufor-Rakeb syndrome characterized by early-onset parkinsonism, pyramidal degeneration and dementia. While previous data suggested a role of ATP13A2 in α-synuclein misfolding and toxicity, the mechanistic link has not been established. Here we report that loss of ATP13A2 in human fibroblasts from patients with Kufor-Rakeb syndrome or in mouse primary neurons leads to impaired lysosomal degradation capacity. This lysosomal dysfunction results in accumulation of α-synuclein and toxicity in primary cortical neurons. Importantly, silencing of endogenous α-synuclein attenuated the toxicity in ATP13A2-depleted neurons, suggesting that loss of ATP13A2 mediates neurotoxicity at least in part via the accumulation of α-synuclein. Our findings implicate lysosomal dysfunction in the pathogenesis of Kufor-Rakeb syndrome and suggest that upregulation of lysosomal function and downregulation of α-synuclein represent important therapeutic strategies for this disorder.

  12. Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

    PubMed Central

    Lu, Yingying; Dong, Shichen; Hao, Baixia; Li, Chang; Zhu, Kaiyuan; Guo, Wenjing; Wang, Qian; Cheung, King-Ho; Wong, Connie WM; Wu, Wu-Tian; Markus, Huss; Yue, Jianbo

    2014-01-01

    Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca2+ content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy. PMID:25483964

  13. Autophagy failure in Alzheimer’s disease and the role of defective lysosomal acidification

    PubMed Central

    Wolfe, Devin M.; Lee, Ju-hyun; Kumar, Asok; Lee, Sooyeon; Orenstein, Samantha J.; Nixon, Ralph A.

    2013-01-01

    Autophagy is a lysosomal degradative process to recycle cellular waste and eliminate potentially toxic damaged organelles and protein aggregates. The important cytoprotective functions of autophagy are evidenced by the diverse pathogenic consequences that may stem from autophagy dysregulation in a growing number of neurodegenerative disorders. In many of the diseases associated with autophagy anomalies, it is the final stage of autophagy-lysosomal degradation that is disrupted. In several disorders, including AD, defective lysosomal acidification contributes to this proteolytic failure. The complex regulation of lysosomal pH makes this process vulnerable to disruption by many factors and reliable lysosomal pH measurements have become increasingly important in investigations of disease mechanisms. Although various reagents for pH quantification have been developed over several decades, they are not all equally well-suited for measuring the pH of lysosomes. Here, we evaluate the most commonly used pH probes for sensitivity and localization and identify Lysosensor Yellow/Blue-Dextran, among currently used probes, as having the most optimal profile of properties for measuring lysosomal pH. In addition, we review evidence that lysosomal acidification is defective in Alzheimer’s disease (AD) and extend our original findings of elevated lysosomal pH in presenilin 1 (PS1)-deficient blastocysts and neurons to additional cell models of PS1- and PS1/2-deficiency, to fibroblasts from AD patients with PS1 mutations, and to neurons in the PS/APP mouse model of AD. PMID:23773064

  14. Lysosomal responses in the digestive gland of the freshwater mussel, Dreissena polymorpha, experimentally exposed to cadmium

    SciTech Connect

    Giamberini, Laure . E-mail: giamb@sciences.univ-metz.fr; Cajaraville, Miren P.

    2005-06-01

    In order to examine the possible use of lysosomal response as a biomarker of freshwater quality, structural changes of lysosomes were measured by image analysis in the digestive gland of the zebra mussel, Dreissena polymorpha, exposed in laboratory conditions to cadmium. Mussels were exposed to the metal (10 and 200 {mu}g/L) for 3 weeks and randomly collected after 7 and 21 days. At each treatment day, digestive tissues were excised and {beta}-glucuronidase activity was revealed in cryotome sections. Four stereological parameters were calculated: lysosomal volume density, lysosomal surface density, lysosomal surface to volume ratio, and lysosomal numerical density. The changes observed in this study reflected a general activation of the lysosomal system, including an increase in both the number and the size of lysosomes in the digestive gland cells of mussels exposed to cadmium. The digestive lysosomal response in zebra mussels was related to exposure time and to metal concentration, demonstrating the potential of this biomarker in freshwater biomonitoring.

  15. Lysosomes serve as a platform for hepatitis A virus particle maturation and nonlytic release.

    PubMed

    Seggewiß, Nicole; Paulmann, Dajana; Dotzauer, Andreas

    2016-01-01

    Early studies on hepatitis A virus (HAV) in cell culture demonstrated the inclusion of several viral particles in an intracellular lipid-bilayer membrane. However, the origin of these virus-associated membranes and the mechanism for the non-lytic release of HAV into bile are still unknown. Analyzing the association of this virus with cell organelles, we found that newly synthesized HAV particles accumulate in lysosomal organelles and that lysosomal enzymes are involved in the maturation cleavage of the virion. Furthermore, by inhibiting the processes of fusion of lysosomes with the plasma membrane, we found that the nonlytic release of HAV from infected cells occurs via lysosome-related organelles.

  16. Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of LysosomesV⃞

    PubMed Central

    Cordonnier, Marie-Neige; Dauzonne, Daniel; Louvard, Daniel; Coudrier, Evelyne

    2001-01-01

    An earlier report suggested that actin and myosin I alpha (MMIα), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIα were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIα. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIα impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIα contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors. PMID:11739797

  17. Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence.

    PubMed

    Davis, Michael J; Eastman, Alison J; Qiu, Yafeng; Gregorka, Brian; Kozel, Thomas R; Osterholzer, John J; Curtis, Jeffrey L; Swanson, Joel A; Olszewski, Michal A

    2015-03-01

    Upon ingestion by macrophages, Cryptococcus neoformans can survive and replicate intracellularly unless the macrophages become classically activated. The mechanism enabling intracellular replication is not fully understood; neither are the mechanisms that allow classical activation to counteract replication. C. neoformans-induced lysosome damage was observed in infected murine bone marrow-derived macrophages, increased with time, and required yeast viability. To demonstrate lysosome damage in the infected host, we developed a novel flow cytometric method for measuring lysosome damage. Increased lysosome damage was found in C. neoformans-containing lung cells compared with C. neoformans-free cells. Among C. neoformans-containing myeloid cells, recently recruited cells displayed lower damage than resident cells, consistent with the protective role of recruited macrophages. The magnitude of lysosome damage correlated with increased C. neoformans replication. Experimental induction of lysosome damage increased C. neoformans replication. Activation of macrophages with IFN-γ abolished macrophage lysosome damage and enabled increased killing of C. neoformans. We conclude that induction of lysosome damage is an important C. neoformans survival strategy and that classical activation of host macrophages counters replication by preventing damage. Thus, therapeutic strategies that decrease lysosomal damage, or increase resistance to such damage, could be valuable in treating cryptococcal infections.

  18. hLGDB: a database of human lysosomal genes and their regulation.

    PubMed

    Brozzi, Alessandro; Urbanelli, Lorena; Germain, Pierre Luc; Magini, Alessandro; Emiliani, Carla

    2013-01-01

    Lysosomes are cytoplasmic organelles present in almost all eukaryotic cells, which play a fundamental role in key aspects of cellular homeostasis such as membrane repair, autophagy, endocitosis and protein metabolism. The characterization of the genes and enzymes constituting the lysosome represents a central issue to be addressed toward a better understanding of the biology of this organelle. In humans, mutations that cause lysosomal enzyme deficiencies result in >50 different disorders and severe pathologies. So far, many experimental efforts using different methodologies have been carried out to identity lysosomal genes. The Human Lysosome Gene Database (hLGDB) is the first resource that provides a comprehensive and accessible census of the human genes belonging to the lysosomal system. This database was developed by collecting and annotating gene lists from many different sources. References to the studies that have identified each gene are provided together with cross databases gene related information. Special attention has been given to the regulation of the genes through microRNAs and the transcription factor EB. The hLGDB can be easily queried to retrieve, combine and analyze information on different lists of lysosomal genes and their regulation by microRNA (binding sites predicted by five different algorithms). The hLGDB is an open access dynamic project that will permit in the future to collapse in a unique publicly accessible resource all the available biological information about lysosome genes and their regulation. Database URL: http://lysosome.unipg.it/.

  19. hLGDB: a database of human lysosomal genes and their regulation

    PubMed Central

    Brozzi, Alessandro; Urbanelli, Lorena; Luc Germain, Pierre; Magini, Alessandro; Emiliani, Carla

    2013-01-01

    Lysosomes are cytoplasmic organelles present in almost all eukaryotic cells, which play a fundamental role in key aspects of cellular homeostasis such as membrane repair, autophagy, endocitosis and protein metabolism. The characterization of the genes and enzymes constituting the lysosome represents a central issue to be addressed toward a better understanding of the biology of this organelle. In humans, mutations that cause lysosomal enzyme deficiencies result in >50 different disorders and severe pathologies. So far, many experimental efforts using different methodologies have been carried out to identity lysosomal genes. The Human Lysosome Gene Database (hLGDB) is the first resource that provides a comprehensive and accessible census of the human genes belonging to the lysosomal system. This database was developed by collecting and annotating gene lists from many different sources. References to the studies that have identified each gene are provided together with cross databases gene related information. Special attention has been given to the regulation of the genes through microRNAs and the transcription factor EB. The hLGDB can be easily queried to retrieve, combine and analyze information on different lists of lysosomal genes and their regulation by microRNA (binding sites predicted by five different algorithms). The hLGDB is an open access dynamic project that will permit in the future to collapse in a unique publicly accessible resource all the available biological information about lysosome genes and their regulation. Database URL: http://lysosome.unipg.it/ PMID:23584836

  20. Lysosomal cysteine proteases: structure, function and inhibition of cathepsins.

    PubMed

    Roberts, Rebecca

    2005-12-01

    Lysosomal cysteine proteases, a subgroup of the cathepsin family, are critical for normal cellular functions such as general protein turnover, antigen processing and bone remodeling. In the past decade, the number of identified human cathepsins has more than doubled and their known role in several pathologies has expanded rapidly. Increased understanding of the structure and mechanism of this class of enzymes has brought on a new fervor in the design of small molecule inhibitors with the hope of producing specific, therapeutic drugs for diseases such as arthritis, allergy, multiple sclerosis, atherosclerosis, Alzheimer's disease and cancer.

  1. Lysosomal storage diseases: natural history and ethical and economic aspects.

    PubMed

    Beutler, Ernest

    2006-07-01

    Potential treatment for lysosomal diseases now includes enzyme replacement therapy, substrate reduction therapy, and chaperone therapy. The first two of these have been implemented commercially, and the spectrum of diseases that are now treatable has expanded from Gaucher disease to include several other disorders. Treatment of these diseases is extremely costly. We explore some of the reasons for the high cost and discuss how, by proper selection of patients and appropriate dosing, the economic burden on society of treating these disease may be ameliorated, at least in part. However, the cost of treating rare diseases is a growing problem that society needs to address.

  2. Leaving the lysosome behind: novel developments in autophagy inhibition

    PubMed Central

    Solitro, Abigail R; MacKeigan, Jeffrey P

    2016-01-01

    The search for a single silver bullet for the treatment of cancer has now been overshadowed by the identification of multiple therapeutic targets unique to each malignancy and even to each patient. In recent years, autophagy has emerged as one such therapeutic target. In response to both therapeutic and oncogenic stress, cancer cells upregulate and demonstrate an increased dependence upon this intracellular recycling process. Particularly in malignancies that currently lack targeted therapeutic options, autophagy inhibitors are the next hopeful prospects for the treatment of this disease. In this review, we discuss the rapid evolution of autophagy inhibitors from early lysosomotropic agents to next-generation lysosome-targeted drugs and beyond. PMID:26689099

  3. Molecular basis of multiple sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease - Lysosomal storage disorders caused by defects of non-lysosomal proteins.

    PubMed

    Dierks, Thomas; Schlotawa, Lars; Frese, Marc-André; Radhakrishnan, Karthikeyan; von Figura, Kurt; Schmidt, Bernhard

    2009-04-01

    Multiple sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been

  4. Lysosomal Acid Lipase Hydrolyzes Retinyl Ester and Affects Retinoid Turnover.

    PubMed

    Grumet, Lukas; Eichmann, Thomas O; Taschler, Ulrike; Zierler, Kathrin A; Leopold, Christina; Moustafa, Tarek; Radovic, Branislav; Romauch, Matthias; Yan, Cong; Du, Hong; Haemmerle, Guenter; Zechner, Rudolf; Fickert, Peter; Kratky, Dagmar; Zimmermann, Robert; Lass, Achim

    2016-08-19

    Lysosomal acid lipase (LAL) is essential for the clearance of endocytosed cholesteryl ester and triglyceride-rich chylomicron remnants. Humans and mice with defective or absent LAL activity accumulate large amounts of cholesteryl esters and triglycerides in multiple tissues. Although chylomicrons also contain retinyl esters (REs), a role of LAL in the clearance of endocytosed REs has not been reported. In this study, we found that murine LAL exhibits RE hydrolase activity. Pharmacological inhibition of LAL in the human hepatocyte cell line HepG2, incubated with chylomicrons, led to increased accumulation of REs in endosomal/lysosomal fractions. Furthermore, pharmacological inhibition or genetic ablation of LAL in murine liver largely reduced in vitro acid RE hydrolase activity. Interestingly, LAL-deficient mice exhibited increased RE content in the duodenum and jejunum but decreased RE content in the liver. Furthermore, LAL-deficient mice challenged with RE gavage exhibited largely reduced post-prandial circulating RE content, indicating that LAL is required for efficient nutritional vitamin A availability. In summary, our results indicate that LAL is the major acid RE hydrolase and required for functional retinoid homeostasis.

  5. Lysosomal Acid Lipase Hydrolyzes Retinyl Ester and Affects Retinoid Turnover*

    PubMed Central

    Grumet, Lukas; Eichmann, Thomas O.; Zierler, Kathrin A.; Leopold, Christina; Moustafa, Tarek; Radovic, Branislav; Romauch, Matthias; Yan, Cong; Haemmerle, Guenter; Zechner, Rudolf; Fickert, Peter; Lass, Achim

    2016-01-01

    Lysosomal acid lipase (LAL) is essential for the clearance of endocytosed cholesteryl ester and triglyceride-rich chylomicron remnants. Humans and mice with defective or absent LAL activity accumulate large amounts of cholesteryl esters and triglycerides in multiple tissues. Although chylomicrons also contain retinyl esters (REs), a role of LAL in the clearance of endocytosed REs has not been reported. In this study, we found that murine LAL exhibits RE hydrolase activity. Pharmacological inhibition of LAL in the human hepatocyte cell line HepG2, incubated with chylomicrons, led to increased accumulation of REs in endosomal/lysosomal fractions. Furthermore, pharmacological inhibition or genetic ablation of LAL in murine liver largely reduced in vitro acid RE hydrolase activity. Interestingly, LAL-deficient mice exhibited increased RE content in the duodenum and jejunum but decreased RE content in the liver. Furthermore, LAL-deficient mice challenged with RE gavage exhibited largely reduced post-prandial circulating RE content, indicating that LAL is required for efficient nutritional vitamin A availability. In summary, our results indicate that LAL is the major acid RE hydrolase and required for functional retinoid homeostasis. PMID:27354281

  6. From Lysosomal Storage Diseases to NKT Cell Activation and Back

    PubMed Central

    Pereira, Cátia S.; Ribeiro, Helena; Macedo, M. Fatima

    2017-01-01

    Lysosomal storage diseases (LSDs) are inherited metabolic disorders characterized by the accumulation of different types of substrates in the lysosome. With a multisystemic involvement, LSDs often present a very broad clinical spectrum. In many LSDs, alterations of the immune system were described. Special emphasis was given to Natural Killer T (NKT) cells, a population of lipid-specific T cells that is activated by lipid antigens bound to CD1d (cluster of differentiation 1 d) molecules at the surface of antigen-presenting cells. These cells have important functions in cancer, infection, and autoimmunity and were altered in a variety of LSDs’ mouse models. In some cases, the observed decrease was attributed to defects in either lipid antigen availability, trafficking, processing, or loading in CD1d. Here, we review the current knowledge about NKT cells in the context of LSDs, including the alterations detected, the proposed mechanisms to explain these defects, and the relevance of these findings for disease pathology. Furthermore, the effect of enzyme replacement therapy on NKT cells is also discussed. PMID:28245613

  7. [Therapy of lysosomal storage diseases: update and perspectives].

    PubMed

    Lara-Aguilar, Ricardo Alejandro; Juárez-Vázquez, Clara Ibet; Medina-Lozano, Claudina

    2011-01-01

    Lysosomal storage diseases (LSD) are caused by monogenic mutations in genes coding for multiple aberrant proteins involved in the catabolism of complex lipids, glycosaminoglycans, oligosaccharides, or nucleic acids. The pathophysiology of the LSD is due to abnormal accumulation of non-hydrolyzed substrate in the lysosomes, affecting the architecture and function of cells, tissues and organs. Due to their genic and allelic heterogeneity the LSD present a wide clinical spectrum in severity of symptoms, evolution and age of onset. The therapeutic strategy has two goals: 1) Palliative management of symptoms (splenectomy, surgery to improve or restore joints or bones, drugs for CNS symptoms, etc.), and 2) The correction of activity of the mutant protein, the former has two approaches: A) Replacing deficient protein (bone marrow transplantation, hematopoietic stem cells or umbilical cord blood cells; replacement with recombinant enzyme and gene therapy) and B) Activate or enhanced the functionality of the mutant enzyme with therapeutic small molecules. Neither of the known treatments is able to address all aspects of these multisystemic disorders, nor cure the patients. Currently, the combination of corrective therapy (CT) with paliative therapy (PT) is the most promising strategy to solve most of the multisystem manifestations. The multidisciplinary medical care is fundamental for diagnosis, treatment and control of disease. Nanotechnology opens a promising new era in the treatment of LSD. Finally, the LSD that has CT must be included in newborn screening programs in order to implement timely treatment and prevent irreversible damage.

  8. Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes.

    PubMed

    Cano, Victoria; March, Catalina; Insua, Jose Luis; Aguiló, Nacho; Llobet, Enrique; Moranta, David; Regueiro, Verónica; Brennan, Gerard P; Millán-Lou, Maria Isabel; Martín, Carlos; Garmendia, Junkal; Bengoechea, José A

    2015-11-01

    Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of phosphoinositide 3-kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella-containing vacuole (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not co-localize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the down-regulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation that may contribute to Klebsiella pathogenesis.

  9. Group XV phospholipase A2, a lysosomal phospholipase A2

    PubMed Central

    Shayman, James A.; Kelly, Robert; Kollmeyer, Jessica; He, Yongqun; Abe, Akira

    2010-01-01

    A phospholipase A2 was identified from MDCK cell homogenates with broad specificity toward glycerophospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. The phospholipase has the unique ability to transacylate short chain ceramides. This phospholipase is calcium-independent, localized to lysosomes, and has an acidic pH optimum. The enzyme was purified from bovine brain and found to be a water-soluble glycoprotein consisting of a single peptide chain with a molecular weight of 45 kDa. The primary structure deduced from the DNA sequences is highly conserved between chordates. The enzyme was named lysosomal phospholipase A2 (LPLA2) and subsequently designated group XV phospholipase A2. LPLA2 has 49 percent of amino acid sequence identity to lecithin cholesterol acyltransferase and is a member of the αβ-hydrolase superfamily. LPLA2 is highly expressed in alveolar macrophages. A marked accumulation of glycerophospholipids and extensive lamellar inclusion bodies, a hallmark of cellular phospholipidosis, is observed in alveolar macrophages in LPLA2−/− mice. This defect can also be reproduced in macrophages that are exposed to cationic amphiphilic drugs such as amiodarone. In addition, older LPLA2−/− mice develop a phenotype similar to human autoimmune disease. These observations indicate that LPLA2 may play a primary role in phospholipid homeostasis, drug toxicity, and host defense. PMID:21074554

  10. Nonsense Suppression as an Approach to Treat Lysosomal Storage Diseases

    PubMed Central

    Keeling, Kim M.

    2016-01-01

    In-frame premature termination codons (PTCs) (also referred to as nonsense mutations) comprise ~10% of all disease-associated gene lesions. PTCs reduce gene expression in two ways. First, PTCs prematurely terminate translation of an mRNA, leading to the production of a truncated polypeptide that often lacks normal function and/or is unstable. Second, PTCs trigger degradation of an mRNA by activating nonsense-mediated mRNA decay (NMD), a cellular pathway that recognizes and degrades mRNAs containing a PTC. Thus, translation termination and NMD are putative therapeutic targets for the development of treatments for genetic diseases caused by PTCs. Over the past decade, significant progress has been made in the identification of compounds with the ability to suppress translation termination of PTCs (also referred to as readthrough). More recently, NMD inhibitors have also been explored as a way to enhance the efficiency of PTC suppression. Due to their relatively low threshold for correction, lysosomal storage diseases are a particularly relevant group of diseases to investigate the feasibility of nonsense suppression as a therapeutic approach. In this review, the current status of PTC suppression and NMD inhibition as potential treatments for lysosomal storage diseases will be discussed. PMID:28367323

  11. New therapeutic prospects for the glycosphingolipid lysosomal storage diseases.

    PubMed

    Platt, F M; Butters, T D

    1998-08-15

    The glycosphingolipid (GSL) lysosomal storage diseases result from mutations in the genes that encode the enzymes required for glycosphingolipid catabolism within lysosomes. They are relatively rare diseases, but are frequently severe in terms of their pathology. Many involve progressive neurodegeneration, and in the most severe forms result in death in early infancy. The therapeutic options for treating these diseases are limited, and for the majority of these disorders there are currently no therapies available. To date, most research has focused on correcting the genetic lesion by gene therapy or by augmenting the enzyme activity deficient in these patients by introducing fully functional enzyme. This can be achieved by bone marrow transplantation or intravenous infusion of purified or recombinant enzyme (enzyme replacement). Gene therapy and enzyme replacement therapy are disease specific, and pharmacological approaches for the treatment of these disorders have not been fully explored. In this commentary, the problems associated with disease therapy are discussed, and a pharmacological agent (N-butyldeoxynojirimycin) is presented for the potential generic treatment of this family of disorders. Successful prevention of glycosphingolipid storage in a mouse model of Tay-Sachs disease suggests that this strategy merits clinical evaluation.

  12. G Protein–Coupled Receptor Sorting to Endosomes and Lysosomes

    PubMed Central

    Marchese, Adriano; Paing, May M.; Temple, Brenda R.S.; Trejo, JoAnn

    2010-01-01

    The heptahelical G protein–coupled receptors (GPCRs) belong to the largest family of cell surface signaling receptors encoded in the human genome. GPCRs signal to diverse extracellular stimuli and control a vast number of physiological responses, making this receptor class the target of nearly half the drugs currently in use. In addition to rapid desensitization, receptor trafficking is crucial for the temporal and spatial control of GPCR signaling. Sorting signals present in the intracytosolic domains of GPCRs regulate trafficking through the endosomal-lysosomal system. GPCR internalization is mediated by serine and threonine phosphorylation and arrestin binding. Short, linear peptide sequences including tyrosine- and dileucine-based motifs, and PDZ ligands that are recognized by distinct endocytic adaptor proteins also mediate internalization and endosomal sorting of GPCRs. We present new data from bioinformatic searches that reveal the presence of these types of sorting signals in the cytoplasmic tails of many known GPCRs. Several recent studies also indicate that the covalent modification of GPCRs with ubiquitin serves as a signal for internalization and lysosomal sorting, expanding the diversity of mechanisms that control trafficking of mammalian GPCRs. PMID:17995450

  13. USP8 controls the trafficking and sorting of lysosomal enzymes.

    PubMed

    MacDonald, Ewan; Urbé, Sylvie; Clague, Michael J

    2014-08-01

    The endosomal deubiquitylase USP8 has profound effects on endosomal morphology and organisation. Previous reports have proposed both positive (EGFR, MET) and negative roles in the down-regulation of receptors (Frizzled, Smoothened). Here we report an additional influence of USP8 on the retromer-dependent shuttling of ci-M6PR between the sorting endosome and biosynthetic pathway. Depletion of USP8 leads to a steady state redistribution of ci-M6PR from the Trans-Golgi Network (TGN) to endosomal compartments. Consequently we observe a defect in sorting of lysosomal enzymes, evidenced by increased levels of unprocessed Cathepsin D, which is secreted into the medium. The normal distribution of receptor can be restored by expression of siRNA-resistant USP8 but not by a catalytically inactive mutant or a truncated form, lacking a MIT domain required for endosomal localisation. We suggest that effects of USP8 depletion may reflect the loss of ESCRT-0 components which associate with retromer components Vps35 and SNX1, whilst failure to efficiently deliver lysosomal enzymes may also contribute to the observed block in receptor tyrosine kinase degradation.

  14. Inherited metabolic disorders: prenatal diagnosis of lysosomal storage disorders.

    PubMed

    Verma, Jyotsna; Thomas, Divya C; Sharma, Sandeepika; Jhingan, Geetu; Saxena, Renu; Kohli, Sudha; Puri, Ratna D; Bijarnia, Sunita; Verma, Ishwar C

    2015-11-01

    To offer accurate prenatal diagnosis of lysosomal storage disorders in early pregnancy. Prenatal enzymatic diagnoses of Gaucher, Fabry, Pompe, Niemann Pick A/B, Tay Sach, Sandoff, GM1, mucoplysaccharidoses, Wolman, Krabbe, Metachromatic leukodystrophy and Batten diseases were made in uncultured chorionic villi samples by fluorometric/spectrophotometric methods. Of 331 prenatal enzymatic diagnosis, 207 fetuses (67%) were normal and 124 (37%) were affected. The interpretation of affected, normal and carrier fetuses was done using their respective reference ranges as well as % enzyme activity of normal mean. The prenatal molecular confirmation was feasible in 43 biochemically diagnosed fetuses. Of the 207 normal reported fetuses, post natal enzymatic confirmation was done in 23 babies, clinical status of another 165 babies was assessed as unaffected via questionnaire on telephone and 19 were lost to follow-up. In affected pregnancies, 123 opted for termination of which 44 were confirmed enzymatically after abortion. A single false positive was determined to be a carrier by prenatal mutation analysis and carried to term. We recommend uncultured chorionic villi for reliable prenatal enzymatic diagnosis of various lysosomal storage disorders on account of the low rate of false positive (0.5%) and false negative (2.2%) results. © 2015 John Wiley & Sons, Ltd.

  15. Gene therapy for the neurological manifestations in lysosomal storage disorders.

    PubMed

    Cheng, Seng H

    2014-09-01

    Over the past several years, considerable progress has been made in the development of gene therapy as a therapeutic strategy for a variety of inherited metabolic diseases, including neuropathic lysosomal storage disorders (LSDs). The premise of gene therapy for this group of diseases is borne of findings that genetic modification of a subset of cells can provide a more global benefit by virtue of the ability of the secreted lysosomal enzymes to effect cross-correction of adjacent and distal cells. Preclinical studies in small and large animal models of these disorders support the application of either a direct in vivo approach using recombinant adeno-associated viral vectors or an ex vivo strategy using lentiviral vector-modified hematopoietic stem cells to correct the neurological component of these diseases. Early clinical studies utilizing both approaches have begun or are in late-stage planning for a small number of neuropathic LSDs. Although initial indications from these studies are encouraging, it is evident that second-generation vectors that exhibit a greater safety profile and transduction activity may be required before this optimism can be fully realized. Here, I review recent progress and the remaining challenges to treat the neurological aspects of various LSDs using this therapeutic paradigm.

  16. Classification of Subcellular Location by Comparative Proteomic Analysis of Native and Density-shifted Lysosomes*

    PubMed Central

    Della Valle, Maria Cecilia; Sleat, David E.; Zheng, Haiyan; Moore, Dirk F.; Jadot, Michel; Lobel, Peter

    2011-01-01

    One approach to the functional characterization of the lysosome lies in the use of proteomic methods to identify proteins in subcellular fractions enriched for this organelle. However, distinguishing between true lysosomal residents and proteins from other cofractionating organelles is challenging. To this end, we implemented a quantitative mass spectrometry approach based on the selective decrease in the buoyant density of liver lysosomes that occurs when animals are treated with Triton-WR1339. Liver lysosome-enriched preparations from control and treated rats were fractionated by isopycnic sucrose density gradient centrifugation. Tryptic peptides derived from gradient fractions were reacted with isobaric tag for relative and absolute quantitation eight-plex labeling reagents and analyzed by two-dimensional liquid chromatography matrix-assisted laser desorption ionization time-of-flight MS. Reporter ion intensities were used to generate relative protein distribution profiles across both types of gradients. A distribution index was calculated for each identified protein and used to determine a probability of lysosomal residence by quadratic discriminant analysis. This analysis suggests that several proteins assigned to the lysosome in other proteomics studies are not true lysosomal residents. Conversely, results support lysosomal residency for other proteins that are either not or only tentatively assigned to this location. The density shift for two proteins, Cu/Zn superoxide dismutase and ATP-binding cassette subfamily B (MDR/TAP) member 6, was corroborated by quantitative Western blotting. Additional balance sheet analyses on differential centrifugation fractions revealed that Cu/Zn superoxide dismutase is predominantly cytosolic with a secondary lysosomal localization whereas ATP-binding cassette subfamily B (MDR/TAP) member 6 is predominantly lysosomal. These results establish a quantitative mass spectrometric/subcellular fractionation approach for

  17. Beneficial effects of lysosome-modulating and other pharmacological and nanocarrier agents on amyloid-beta-treated cells.

    PubMed

    Kanazirska, Marie V; Fuchs, Philipp M; Chen, Liping; Lal, Sumit; Verma, Jyoti; Vassilev, Peter M

    2012-12-01

    The progression of Alzheimer's disease (AD) is accompanied by disturbances of the endosome/lysosome (EL) system and there is accumulation of peptides of the AD-associated amyloid beta (Abeta) type in EL vesicles of affected neurons. EL modulating agents partially ameliorate the Abeta-mediated cell abnormalities. However, no extensive studies on the potential pharmaceutical applications of combinations of such agents and their synergistic effects have been performed. This study shows the beneficial anti-amyloid effects of several combinations of lysosomal modulators and other pharmacological and new nanobiotechnological agents. Some agents potentiated each other's action and some of them facilitated the anti-amyloid actions of memantine, a modifier of Ca2+-permeable channels involved in AD and one of the few drugs used for treatment of AD. Another compound used in nanobiotechnology ameliorated as a nanocarrier the beneficial effects of some of these potential pharmaceutical agents. They may be considered as additional drugs to improve the efficacy of the therapeutic approaches for AD and related neurodegenerative disorders.

  18. Camalexin-Induced Apoptosis in Prostate Cancer Cells Involves Alterations of Expression and Activity of Lysosomal Protease Cathepsin D

    PubMed Central

    Smith, Basil; Randle, Diandra; Mezencev, Roman; Thomas, LeeShawn; Hinton, Cimona; Marah, Valerie

    2015-01-01

    Camalexin, the phytoalexin produced in the model plant Arabidopsis thaliana, possesses antiproliferative and cancer chemopreventive effects. We have demonstrated that the cytostatic/cytotoxic effects of camalexin on several prostate cancer (PCa) cells are due to oxidative stress. Lysosomes are vulnerable organelles to Reactive Oxygen Species (ROS)-induced injuries, with the potential to initiate and or facilitate apoptosis subsequent to release of proteases such as cathepsin D (CD) into the cytosol. We therefore hypothesized that camalexin reduces cell viability in PCa cells via alterations in expression and activity of CD. Cell viability was evaluated by MTS cell proliferation assay in LNCaP and ARCaP Epithelial (E) cells, and their respective aggressive sublines C4-2 and ARCaP Mesenchymal (M) cells, whereby the more aggressive PCa cells (C4-2 and ARCaPM) displayed greater sensitivity to camalexin treatments than the lesser aggressive cells (LNCaP and ARCaPE). Immunocytochemical analysis revealed CD relocalization from the lysosome to the cytosol subsequent to camalexin treatments, which was associated with increased protein expression of mature CD; p53, a transcriptional activator of CD; BAX, a downstream effector of CD, and cleaved PARP, a hallmark for apoptosis. Therefore, camalexin reduces cell viability via CD and may present as a novel therapeutic agent for treatment of metastatic prostate cancer cells. PMID:24699144

  19. Protective effects of sinapic acid on lysosomal dysfunction in isoproterenol induced myocardial infarcted rats.

    PubMed

    Roy, Subhro Jyoti; Stanely Mainzen Prince, Ponnian

    2012-11-01

    In the pathology of myocardial infarction, lysosomal lipid peroxidation and resulting enzyme release play an important role. We evaluated the protective effects of sinapic acid on lysosomal dysfunction in isoproterenol induced myocardial infarcted rats. Male Wistar rats were treated with sinapic acid (12 mg/kg body weight) orally daily for 10 days and isoproterenol (100 mg/kg body weight) was injected twice at an interval of 24 h (9th and 10th day). Then, lysosomal lipid peroxidation, lysosomal enzymes in serum, heart homogenate, lysosomal fraction and myocardial infarct size were measured. Isoproterenol induced myocardial infarcted rats showed a significant increase in serum creatine kinase-MB and lysosomal lipid peroxidation. The activities of β-glucuronidase, β-galactosidase, cathepsin-B and D were significantly increased in serum, heart and the activities of β-glucuronidase and cathepsin-D were significantly decreased in lysosomal fraction of myocardial infarcted rats. Pre-and-co-treatment with sinapic acid normalized all the biochemical parameters and reduced myocardial infarct size in myocardial infarcted rats. In vitro studies confirmed the free radical scavenging effects of sinapic acid. The possible mechanisms for the observed effects are attributed to sinapic acid's free radical scavenging and membrane stabilizing properties. Thus, sinapic acid has protective effects on lysosomal dysfunction in isoproterenol induced myocardial infarcted rats.

  20. The phytoestrogen genistein modulates lysosomal metabolism and transcription factor EB (TFEB) activation.

    PubMed

    Moskot, Marta; Montefusco, Sandro; Jakóbkiewicz-Banecka, Joanna; Mozolewski, Paweł; Węgrzyn, Alicja; Di Bernardo, Diego; Węgrzyn, Grzegorz; Medina, Diego L; Ballabio, Andrea; Gabig-Cimińska, Magdalena

    2014-06-13

    Genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) has been previously proposed as a potential drug for use in substrate reduction therapy for mucopolysaccharidoses, a group of inherited metabolic diseases caused by mutations leading to inefficient degradation of glycosaminoglycans (GAGs) in lysosomes. It was demonstrated that this isoflavone can cross the blood-brain barrier, making it an especially desirable potential drug for the treatment of neurological symptoms present in most lysosomal storage diseases. So far, no comprehensive genomic analyses have been performed to elucidate the molecular mechanisms underlying the effect elicited by genistein. Therefore, the aim of this work was to identify the genistein-modulated gene network regulating GAG biosynthesis and degradation, taking into consideration the entire lysosomal metabolism. Our analyses identified over 60 genes with known roles in lysosomal biogenesis and/or function whose expression was enhanced by genistein. Moreover, 19 genes whose products are involved in both GAG synthesis and degradation pathways were found to be remarkably differentially regulated by genistein treatment. We found a regulatory network linking genistein-mediated control of transcription factor EB (TFEB) gene expression, TFEB nuclear translocation, and activation of TFEB-dependent lysosome biogenesis to lysosomal metabolism. Our data indicate that the molecular mechanism of genistein action involves not only impairment of GAG synthesis but more importantly lysosomal enhancement via TFEB. These findings contribute to explaining the beneficial effects of genistein in lysosomal storage diseases as well as envisage new therapeutic approaches to treat these devastating diseases.

  1. Autophagy-lysosomal pathway is involved in lipid degradation in rat liver.

    PubMed

    Skop, V; Cahová, M; Papáčková, Z; Páleníčková, E; Daňková, H; Baranowski, M; Zabielski, P; Zdychová, J; Zídková, J; Kazdová, L

    2012-01-01

    We present data supporting the hypothesis that the lysosomal-autophagy pathway is involved in the degradation of intracellular triacylglycerols in the liver. In primary hepatocytes cultivated in the absence of exogenous fatty acids (FFA), both inhibition of autophagy flux (asparagine) or lysosomal activity (chloroquine) decreased secretion of VLDL (very low density lipoproteins) and formation of FFA oxidative products while the stimulation of autophagy by rapamycine increased some of these parameters. Effect of rapamycine was completely abolished by inactivation of lysosomes. Similarly, when autophagic activity was influenced by cultivating the hepatocytes in "starving" (amino-acid poor medium) or "fed" (serum-supplemented medium) conditions, VLDL secretion and FFA oxidation mirrored the changes in autophagy being higher in starvation and lower in fed state. Autophagy inhibition as well as lysosomal inactivation depressed FFA and DAG (diacylglycerol) formation in liver slices in vitro. In vivo, intensity of lysosomal lipid degradation depends on the formation of autophagolysosomes, i.e. structures bringing the substrate for degradation and lysosomal enzymes into contact. We demonstrated that lysosomal lipase (LAL) activity in liver autophagolysosomal fraction was up-regulated in fasting and down-regulated in fed state together with the increased translocation of LAL and LAMP2 proteins from lysosomal pool to this fraction. Changes in autophagy intensity (LC3-II/LC3-I ratio) followed a similar pattern.

  2. The Octyl Ester of Ginsenoside Rh2 Induces Lysosomal Membrane Permeabilization via Bax Translocation.

    PubMed

    Chen, Fang; Zhang, Bing; Sun, Yong; Xiong, Zeng-Xing; Peng, Han; Deng, Ze-Yuan; Hu, Jiang-Ning

    2016-04-25

    Ginsenoside Rh2 is a potential pharmacologically active metabolite of ginseng. Previously, we have reported that an octyl ester derivative of ginsenoside Rh2 (Rh2-O), has been confirmed to possess higher bioavailability and anticancer effect than Rh2 in vitro. In order to better assess the possibility that Rh2-O could be used as an anticancer compound, the underlying mechanism was investigated in this study. The present results revealed that lysosomal destabilization was involved in the early stage of cell apoptosis in HepG2 cells induced by Rh2-O. Rh2-O could induce an early lysosomal membrane permeabilization with the release of lysosomal protease cathepsins to the cytosol in HepG2 cells. The Cat B inhibitor (leu) and Cat D inhibitor (pepA) inhibited Rh2-O-induced HepG2 apoptosis as well as tBid production and Δφm depolarization, indicating that lysosomal permeabilization occurred upstream of mitochondrial dysfunction. In addition, Rh2-O induced a significant increase in the protein levels of DRAM1 and Bax (p < 0.05) in lysosomes of HepG2 cells. Knockdown of Bax partially inhibited Rh2-O-induced Cat D release from lysosomes. Thus it was concluded that Rh2-O induced apoptosis of HepG2 cells through activation of the lysosomal-mitochondrial apoptotic pathway involving the translocation of Bax to the lysosome.

  3. Imidazoacridinone-dependent lysosomal photodestruction: a pharmacological Trojan horse approach to eradicate multidrug-resistant cancers

    PubMed Central

    Adar, Y; Stark, M; Bram, E E; Nowak-Sliwinska, P; van den Bergh, H; Szewczyk, G; Sarna, T; Skladanowski, A; Griffioen, A W; Assaraf, Y G

    2012-01-01

    Multidrug resistance (MDR) remains a primary hindrance to curative cancer therapy. Thus, introduction of novel strategies to overcome MDR is of paramount therapeutic significance. Sequestration of chemotherapeutics in lysosomes is an established mechanism of drug resistance. Here, we show that MDR cells display a marked increase in lysosome number. We further demonstrate that imidazoacridinones (IAs), which are cytotoxic fluorochromes, undergo a dramatic compartmentalization in lysosomes because of their hydrophobic weak base nature. We hence developed a novel photoactivation-based pharmacological Trojan horse approach to target and eradicate MDR cancer cells based on photo-rupture of IA-loaded lysosomes and tumor cell lysis via formation of reactive oxygen species. Illumination of IA-loaded cells resulted in lysosomal photodestruction and restoration of parental cell drug sensitivity. Lysosomal photodestruction of MDR cells overexpressing the key MDR efflux transporters ABCG2, ABCB1 or ABCC1 resulted in 10- to 52-fold lower IC50 values of various IAs, thereby restoring parental cell sensitivity. Finally, in vivo application of this photodynamic therapy strategy after i.v. injection of IAs in human ovarian tumor xenografts in the chorioallantoic membrane model revealed selective destruction of tumors and their associated vasculature. These findings identify lysosomal sequestration of IAs as an Achilles heel of MDR cells that can be harnessed to eradicate MDR tumor cells via lysosomal photodestruction. PMID:22476101

  4. Characterization of storage material in cultured fibroblasts by specific lectin binding in lysosomal storage diseases.

    PubMed

    Virtanen, I; Ekblom, P; Laurila, P; Nordling, S; Raivio, K O; Aula, P

    1980-11-01

    The lysosomal storage material in cultured fibroblasts from patients with various lysosomal storage diseases was characterized by fluorescence microscopy using lectins specific for different saccharide moieties. In normal fibroblasts and cultured amniotic fluid cells lectins specific for mannosyl and glucosyl moieties, Con A and LcA gave a bright perinuclear cytoplasmic staining corresponding to the localization of endoplasmic reticulum in the cells. All other lectins stained the Golgi apparatus as a juxtanuclear reticular structure. In fucosidosis fibroblasts, only lectins specific for fucosyl groups LTA and UEA, distinctly stained the lysosomal inclusions. The lysosomes in mannosidosis fibroblasts did not react with Con A and LcA, both specific for mannosyl moieties of glycoconjugates, but were brightly labeled with WGA, a lectin specific for N-acetyl glucosaminyl moieties. In I-cell fibroblasts, the numerous perinuclear phase-dense granules, representing abnormal lysosomes, were labeled with every lectin used. In fibroblasts from patients with Salla disease, a newly discovered lysosomal storage disorder, the lysosomes were brightly stained only with LPA, indicating the presence of increased amounts of sialic acid residues in the lysosomal inclusions.

  5. Lipophilic cationic drugs increase the permeability of lysosomal membranes in a cell culture system.

    PubMed

    Kornhuber, Johannes; Henkel, Andreas W; Groemer, Teja W; Städtler, Sven; Welzel, Oliver; Tripal, Philipp; Rotter, Andrea; Bleich, Stefan; Trapp, Stefan

    2010-07-01

    Lysosomes accumulate many drugs several fold higher compared to their extracellular concentration. This mechanism is believed to be responsible for many pharmacological effects. So far, uptake and release kinetics are largely unknown and interactions between concomitantly administered drugs often provoke mutual interference. In this study, we addressed these questions in a cell culture model. The molecular mechanism for lysosomal uptake kinetics was analyzed by live cell fluorescence microscopy in SY5Y cells using four drugs (amantadine, amitriptyline, cinnarizine, flavoxate) with different physicochemical properties. Drugs with higher lipophilicity accumulated more extensively within lysosomes, whereas a higher pK(a) value was associated with a more rapid uptake. The drug-induced displacement of LysoTracker was neither caused by elevation of intra-lysosomal pH, nor by increased lysosomal volume. We extended our previously developed numerical single cell model by introducing a dynamic feedback mechanism. The empirical data were in good agreement with the results obtained from the numerical model. The experimental data and results from the numerical model lead to the conclusion that intra-lysosomal accumulation of lipophilic xenobiotics enhances lysosomal membrane permeability. Manipulation of lysosomal membrane permeability might be useful to overcome, for example, multi-drug resistance by altering subcellular drug distribution.

  6. Autophagic lysosomal reformation depends on mTOR reactivation in H2O2-induced autophagy.

    PubMed

    Zhang, Jiqian; Zhou, Wei; Lin, Jun; Wei, Pengfei; Zhang, Yunjiao; Jin, Peipei; Chen, Ming; Man, Na; Wen, Longping

    2016-01-01

    Autophagic lysosomal reformation, a key cellular process for maintaining lysosome homeostasis in elevated autophagy, so far has only been reported for cells under certain forms of starvation. For this reason, it is controversial that whether this phenomenon is starvation-specific and its importance in lysosomal regeneration at the late stage of autophagy is often challenged. Here we show that exogenous hydrogen peroxide (H2O2) induced lysosome depletion and recovery characteristic of autophagic lysosomal reformation, and we confirmed the occurrence of autophagic lysosomal reformation after H2O2 treatment by demonstrating Rab7 dissociation from autolysosomes, recruitment of Phosphatidylinositol 4-phosphate (PI4P) and clathrin to the surface of autolysosomes, and the existence of tubular "pro-lysosome" structures extending from autolysosomes. Similar to starvation, H2O2 caused an initial deactivation and a subsequent reactivation for mTOR, and mTOR reactivation was essential for ALR. Our results provided a first non-starvation example of autophagic lysosomal reformation and provide evidence for its importance for some autophagic processes other than that of starvation.

  7. Failure of lysosome clustering and positioning in the juxtanuclear region in cells deficient in rapsyn

    PubMed Central

    Aittaleb, Mohamed; Chen, Po-Ju; Akaaboune, Mohammed

    2015-01-01

    ABSTRACT Rapsyn, a scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at contacts between motor neurons and differentiating muscle cells. Rapsyn is also expressed in cells that do not express AChRs. However, its function in these cells remains unknown. Here, we show that rapsyn plays an AChR-independent role in organizing the distribution and mobility of lysosomes. In cells devoid of AChRs, rapsyn selectively induces the clustering of lysosomes at high density in the juxtanuclear region without affecting the distribution of other intracellular organelles. However, when the same cells overexpress AChRs, rapsyn is recruited away from lysosomes to colocalize with AChR clusters on the cell surface. In rapsyn-deficient (Rapsn−/−) myoblasts or cells overexpressing rapsyn mutants, lysosomes are scattered within the cell and highly dynamic. The increased mobility of lysosomes in Rapsn−/− cells is associated with a significant increase in lysosomal exocytosis, as evidenced by increased release of lysosomal enzymes and plasma membrane damage when cells were challenged with the bacterial pore-forming toxin streptolysin-O. These findings uncover a new link between rapsyn, lysosome positioning, exocytosis and plasma membrane integrity. PMID:26330529

  8. Activation of Peroxisome Proliferator-activated Receptor α Induces Lysosomal Biogenesis in Brain Cells

    PubMed Central

    Ghosh, Arunava; Jana, Malabendu; Modi, Khushbu; Gonzalez, Frank J.; Sims, Katherine B.; Berry-Kravis, Elizabeth; Pahan, Kalipada

    2015-01-01

    Lysosomes are ubiquitous membrane-enclosed organelles filled with an acidic interior and are central to the autophagic, endocytic, or phagocytic pathway. In contrast to its classical function as the waste management machinery, lysosomes are now considered to be an integral part of various cellular signaling processes. The diverse functionality of this single organelle requires a very complex and coordinated regulation of its activity with transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, at its core. However, mechanisms by which TFEB is regulated are poorly understood. This study demonstrates that gemfibrozil, an agonist of peroxisome proliferator-activated receptor (PPAR) α, alone and in conjunction with all-trans-retinoic acid is capable of enhancing TFEB in brain cells. We also observed that PPARα, but not PPARβ and PPARγ, is involved in gemfibrozil-mediated up-regulation of TFEB. Reporter assay and chromatin immunoprecipitation studies confirmed the recruitment of retinoid X receptor α, PPARα, and PGC1α on the PPAR-binding site on the Tfeb promoter as well. Subsequently, the drug-mediated induction of TFEB caused an increase in lysosomal protein and the lysosomal abundance in cell. Collectively, this study reinforces the link between lysosomal biogenesis and lipid metabolism with TFEB at the crossroads. Furthermore, gemfibrozil may be of therapeutic value in the treatment of lysosomal storage disorders in which autophagy-lysosome pathway plays an important role. PMID:25750174

  9. Signaling from lysosomes to mitochondria sensitizes cancer cells to photodynamic treatment

    NASA Astrophysics Data System (ADS)

    Hung, Hsin-I.; Quiogue, Geraldine; Lemasters, John J.; Nieminen, Anna-Liisa

    2011-02-01

    Previously, we showed that photosensitizers that localize to lysosomes are more effective in killing cancer cells than ones directed to mitochondria after photodynamic treatment (PDT). The photosensitizer, phthalocyanine 4 (Pc 4), localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. However, analogues of Pc 4 (e.g., Pc 181) that primarily target lysosomes still produce mitochondria-mediated cell death, although the time course is slower compared to Pc 4-PDT. In A431 epidermoid carcinoma cells, these new analogues preferentially localized in lysosomes were highly efficient in inducing apoptotic cell death. To assess further how lysosomes contribute to PDT, we monitored cell killing of A431 cells after Pc 4-PDT in the presence and absence of bafilomycin, an inhibitor of the acidic vacuolar proton pump that collapses the pH gradient of the lysosomal/endosomal compartment. Bafilomycin by itself was not toxic but greatly enhanced Pc 4-PDT-induced mitochondrial depolarization and cell killing. Both depolarization and cell killing were substantially prevented by iron chelators. The fact that Pc 4-PDT plus bafilomycin treatment did not induce lysosomal membrane damage prior to mitochondrial depolarization suggests that bafilomycin instead induced release of redox active iron from lysosomes into the cytosol that further translocated into mitochondria, where iron-mediated free radical formation occurred. In conclusion, agents that disturb lysosomal function could potentially be used as adjuvants with mitochondrion-targeted photosensitizers to enhance phototoxicity.

  10. The role of intraorganellar Ca(2+) in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles.

    PubMed

    Pryor, P R; Mullock, B M; Bright, N A; Gray, S R; Luzio, J P

    2000-05-29

    We have investigated the requirement for Ca(2+) in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca(2+). Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca(2+) reduced the amount of fusion. The requirement for Ca(2+) for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome-lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca(2+) plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.

  11. Lysosomal cathepsin B participates in the podosome-mediated extracellular matrix degradation and invasion via secreted lysosomes in v-Src fibroblasts

    PubMed Central

    Tu, Chun; Ortega-Cava, Cesar F.; Chen, Gengsheng; Fernandes, Norvin D.; Cavallo-Medved, Dora; Sloane, Bonnie F.; Band, Vimla; Band, Hamid

    2009-01-01

    Podosomes mediate cell migration and invasion by coordinating the reorganization of actin cytoskeleton and focal matrix degradation. Matrix metalloproteases and serine proteases have been found to function at podosomes. The lysosomal cysteine cathepsins, a third major class of matrix-degrading enzymes involved in tumor invasion and tissue remodeling, have yet to be linked to podosome with the exception of cathepsin K in osteoclasts. Using inhibitors and shRNA-mediated depletion, we show that cathepsin B participate in podosomes-mediated focal matrix degradation and invasion in v-Src transformed fibroblasts., We observed that lysosomal marker LAMP-1 localized at the center of podosome rosettes protruding into extracellular matrix using confocal microscopy. Time-lapse live-cell imaging revealed that lysosomal vesicles moved to and fused with podosomes. Disruption of lysosomal pH gradient with Bafilomycin A1, chloroquine or ammonium chloride greatly enhanced the formation of podosomes and increased the matrix degradation. Live cell imaging showed that actin-structures, induced shortly after Bafilomycin A1 treatment, were closely associated with lysosomes. Overall, our results suggest that cathepsin B, delivered by lysosomal vesicles, are involved in the matrix degradtion of podosomes. PMID:19010886

  12. The Role of Intraorganellar Ca2+In Late Endosome–Lysosome Heterotypic Fusion and in the Reformation of Lysosomes from Hybrid Organelles

    PubMed Central

    Pryor, Paul R.; Mullock, Barbara M.; Bright, Nicholas A.; Gray, Sally R.; Luzio, J. Paul

    2000-01-01

    We have investigated the requirement for Ca2+ in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca2+. Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca2+ reduced the amount of fusion. The requirement for Ca2+ for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome–lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca2+ plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway. PMID:10831609

  13. Quantification of Lysosomal Membrane Permeabilization by Cytosolic Cathepsin and β-N-Acetyl-Glucosaminidase Activity Measurements.

    PubMed

    Jäättelä, Marja; Nylandsted, Jesper

    2015-11-02

    Programmed cell death involving lysosomal membrane permeabilization (LMP) is an alternative cell death pathway induced under various cellular conditions and by numerous cytotoxic stimuli. The method presented here to quantify LMP takes advantage of the detergent digitonin, which creates pores in cellular membranes by replacing cholesterol. The difference in cholesterol content between the plasma membrane (high) and lysosomal membrane (low) allows titration of digitonin to a concentration that permeabilizes the plasma membrane but leaves lysosomal membranes intact. The extent of LMP is determined by measuring the cytosolic activity of lysosomal hydrolases (e.g., cysteine cathepsins) and/or β-N-acetyl-glucosaminidase in the digitonin-extracted cytoplasm and comparing it to the total cellular enzyme activity. Digitonin extraction of the cytosol can be combined with precipitation of protein and/or western blot analysis for detection of lysosomal proteins (e.g., cathepsins).

  14. The lysosome as a command-and-control center for cellular metabolism

    PubMed Central

    2016-01-01

    Lysosomes are membrane-bound organelles found in every eukaryotic cell. They are widely known as terminal catabolic stations that rid cells of waste products and scavenge metabolic building blocks that sustain essential biosynthetic reactions during starvation. In recent years, this classical view has been dramatically expanded by the discovery of new roles of the lysosome in nutrient sensing, transcriptional regulation, and metabolic homeostasis. These discoveries have elevated the lysosome to a decision-making center involved in the control of cellular growth and survival. Here we review these recently discovered properties of the lysosome, with a focus on how lysosomal signaling pathways respond to external and internal cues and how they ultimately enable metabolic homeostasis and cellular adaptation. PMID:27621362

  15. Automated measurement of lysosomal structure alterations in oocytes of mussels exposed to petroleum hydrocarbons.

    PubMed

    Cajaraville, M P; Marigómez, J A; Angulo, E

    1991-09-01

    The present study examines the structure of the lysosomal system of mature oocytes in mussels, Mytilus galloprovincialis, after a 21 day exposure to the water accommodated fraction (WAF) of two crude oils (types Ural and Maya) and of a commercial lubricant oil. The automated image analysis indicates that lysosomes, showing cytochemically demonstrable beta-glucuronidase activity, are smaller and much more numerous in oocytes of mussels treated with a 40% dose of Ural- and Lubricant-WAF when compared to controls. It is suggested that the structure of the lysosomal system of oocytes is different from that of somatic cells (i.e., digestive cells) and that budding or "fission" into smaller bodies occurs in oocyte lysosomes under certain petroleum hydrocarbon-exposure conditions. These changes in the lysosomal compartment appear to be associated to the process of gamete release or spawning.

  16. High sphingomyelin levels induce lysosomal damage and autophagy dysfunction in Niemann Pick disease type A

    PubMed Central

    Gabandé-Rodríguez, E; Boya, P; Labrador, V; Dotti, C G; Ledesma, M D

    2014-01-01

    Niemann Pick disease type A (NPA), which is caused by loss of function mutations in the acid sphingomyelinase (ASM) gene, is a lysosomal storage disorder leading to neurodegeneration. Yet, lysosomal dysfunction and its consequences in the disease are poorly characterized. Here we show that undegraded molecules build up in neurons of acid sphingomyelinase knockout mice and in fibroblasts from NPA patients in which autophagolysosomes accumulate. The latter is not due to alterations in autophagy initiation or autophagosome–lysosome fusion but because of inefficient autophago–lysosomal clearance. This, in turn, can be explained by lysosomal membrane permeabilization leading to cytosolic release of Cathepsin B. High sphingomyelin (SM) levels account for these effects as they can be induced in control cells on addition of the lipid and reverted on SM-lowering strategies in ASM-deficient cells. These results unveil a relevant role for SM in autophagy modulation and characterize autophagy anomalies in NPA, opening new perspectives for therapeutic interventions. PMID:24488099

  17. Ubiquitin trafficking to the lysosome: keeping the house tidy and getting rid of unwanted guests.

    PubMed

    Purdy, Georgiana E; Russell, David G

    2007-01-01

    Bacterial killing by autophagic delivery to the lysosomal compartment has been shown for Mycobacteria, Streptococcus, Shigella, Legionella and Salmonella, indicating an important role for this conserved trafficking pathway for the control of intracellular bacterial pathogens.(1-5) In a recent study we found that solubilized lysosomes isolated from bone marrow-derived macrophages had potent antibacterial properties against M. tuberculosis and M. smegmatis that were associated with ubiquitin and ubiquitin-derived peptides. We propose that ubiquitinated proteins are delivered to the lysosomal compartment, where degradation by lysosomal proteinases generates ubiquitin-derived peptides with antimycobacterial properties. This surprising finding provokes a number of questions regarding the nature and trafficking of ubiquitin and ubiquitin-modified proteins in mammalian cells. We discuss the possible role(s) that the multivesicular body (MVB), the late endosome and the autophagosome may play in trafficking of ubiquitinated proteins to the lysosome.

  18. Alteration of epithelial cell lysosomal integrity induced by bacterial cholesterol‐dependent cytolysins

    PubMed Central

    Malet, Julien Karim

    2016-01-01

    Abstract Bacterial pathogens can interfere during infection with host cell organelles, such as mitochondria, the endoplasmic reticulum‐Golgi system or nuclei. As important cellular functions are often compartmentalized in these organelles, their targeting allows pathogens to manipulate key host functions during infection. Here, we identify lysosomes as a new class of organelles targeted by the pathogenic bacterium Listeria monocytogenes. We demonstrate that extracellular Listeria, via secretion of the pore‐forming toxin listeriolysin O, alters lysosomal integrity in epithelial cells but not in macrophages. Listeriolysin O induces lysosomal membrane permeabilization and release of lysosomal content, such as cathepsins proteases, which remain transiently active in the host cytosol. We furthermore show that other bacterial pore‐forming toxins, such as perfringolysin O and pneumolysin, also induce lysosomes alteration. Together, our data unveil a novel activity of bacterial cholesterol‐dependent cytolysins. PMID:27739224

  19. The effects of hydrocortisone and glycyrrhizine on the enzyme releases of arylsulfatase and hyaluronidase from lysosomes of liver.

    PubMed

    Ozeki, T; Tokawa, Y; Ogasawara, T; Sato, K; Kan, M

    1978-03-15

    Hydrocortisone and glycyrrhizine act as both stabilizers and labilizers of the lysosomes of liver. The effect of both agents on the lysosomes is changeable according to the duration of their administration.

  20. The Coxiella burnetii Dot/Icm system creates a comfortable home through lysosomal renovation.

    PubMed

    Newton, Hayley J; Roy, Craig R

    2011-01-01

    Understanding the molecular pathogenesis of Coxiella burnetii, the causative agent of human Q fever, has historically been hindered by the technical difficulties of genetically manipulating obligate intracellular bacteria. The recent development of culture conditions suitable for axenic propagation of C. burnetii has paved the way for the application of a range of genetic techniques to address key questions within the field. Recent studies using mutational analysis have revealed that the C. burnetii Dot/Icm type 4 secretion system (T4SS) is an important virulence determinant that is essential for renovation of a lysosome into a mature Coxiella-containing vacuole (CCV) permissive of intracellular replication. Interestingly, a mutant of C. burnetii deficient in Dot/Icm function was found to be capable of replicating within the parasitophorous vacuole created by Leishmania amazonensis, which indicates that C. burnetii replication is not dependent on the cohort of Dot/Icm effector proteins per se but rather that the collective actions of effectors are required to create the specialized niche supportive of replication. Thus, a role for the Dot/Icm T4SS during the intracellular life cycle of C. burnetii has been more clearly defined by these studies, which demonstrate that advances in genetic analysis should allow future studies to focus on the intricacies of Dot/Icm effector functions that facilitate development of the unique CCV.

  1. The Coxiella burnetii Dot/Icm System Creates a Comfortable Home through Lysosomal Renovation

    PubMed Central

    Newton, Hayley J.; Roy, Craig R.

    2011-01-01

    ABSTRACT Understanding the molecular pathogenesis of Coxiella burnetii, the causative agent of human Q fever, has historically been hindered by the technical difficulties of genetically manipulating obligate intracellular bacteria. The recent development of culture conditions suitable for axenic propagation of C. burnetii has paved the way for the application of a range of genetic techniques to address key questions within the field. Recent studies using mutational analysis have revealed that the C. burnetii Dot/Icm type 4 secretion system (T4SS) is an important virulence determinant that is essential for renovation of a lysosome into a mature Coxiella-containing vacuole (CCV) permissive of intracellular replication. Interestingly, a mutant of C. burnetii deficient in Dot/Icm function was found to be capable of replicating within the parasitophorous vacuole created by Leishmania amazonensis, which indicates that C. burnetii replication is not dependent on the cohort of Dot/Icm effector proteins per se but rather that the collective actions of effectors are required to create the specialized niche supportive of replication. Thus, a role for the Dot/Icm T4SS during the intracellular life cycle of C. burnetii has been more clearly defined by these studies, which demonstrate that advances in genetic analysis should allow future studies to focus on the intricacies of Dot/Icm effector functions that facilitate development of the unique CCV. PMID:22010216

  2. The phylogeny and evolution of deoxyribonuclease II: An enzyme essential for lysosomal DNA degradation

    PubMed Central

    Shpak, Max; Kugelman, Jeffrey R.; Varela-Ramirez, Armando; Aguilera, Renato J.

    2008-01-01

    Deoxyribonuclease II (DNase II) is an endonuclease with optimal activity at low pH, localized within the lysosomes of higher eukaryotes. The origin of this enzyme remains in dispute, and its phylogenetic distribution leaves many questions about its subsequent evolutionary history open. Earlier studies have documented its presence in various metazoans, as well as in Dictyostelium, Trichomonas and, anomalously, a single genus of bacteria (Burkholderia). This study makes use of searches of the genomes of various organisms against known DNase II query sequences, in order to determine the likely point of origin of this enzyme among cellular life forms. Its complete absence from any other bacteria makes prokaryotic origin unlikely. Convincing evidence exists for DNase II homologs in Alveolates such as Paramecium, Heterokonts such as diatoms and water molds, and even tentative matches in green algae. Apparent absences include red algae, plants, fungi, and a number of parasitic organisms. Based on this phylogenetic distribution and hypotheses of eukaryotic relationships, the most probable explanation is that DNase II has been subject to multiple losses. The point of origin is debatable, though its presence in Trichomonas and perhaps in other evolutionarily basal “Excavate” protists such as Reclinomonas, strongly support the hypothesis that DNase II arose as a plesiomorphic trait in eukaryotes. It probably evolved together with phagocytosis, specifically to facilitate DNA degradation and bacteriotrophy. The various absences in many eukaryotic lineages are accounted for by loss of phagotrophic function in intracellular parasites, in obligate autotrophs, and in saprophytes. PMID:18226927

  3. Endosomal type Iγ PIP 5-kinase controls EGF receptor lysosomal sorting.

    PubMed

    Sun, Yue; Hedman, Andrew C; Tan, Xiaojun; Schill, Nicholas J; Anderson, Richard A

    2013-04-29

    Endosomal trafficking and degradation of epidermal growth factor receptor (EGFR) play an essential role in the control of its signaling. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)) is an established regulator of endocytosis, whereas PtdIns3P modulates endosomal trafficking. However, we demonstrate here that type I gamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme that synthesizes PtdIns4,5P(2), controls endosome-to-lysosome sorting of EGFR. In this pathway, PIPKIγi5 interacts with sorting nexin 5 (SNX5), a protein that binds PtdIns4,5P(2) and other phosphoinositides. PIPKIγi5 and SNX5 localize to endosomes, and loss of either protein blocks EGFR sorting into intraluminal vesicles (ILVs) of the multivesicular body. Loss of ILV sorting greatly enhances and prolongs EGFR signaling. PIPKIγi5 and SNX5 prevent Hrs ubiquitination, and this facilitates the Hrs association with EGFR that is required for ILV sorting. These findings reveal that PIPKIγi5 and SNX5 form a signaling nexus that controls EGFR endosomal sorting, degradation, and signaling.

  4. Identification of cytoskeleton-associated proteins essential for lysosomal stability and survival of human cancer cells.

    PubMed

    Groth-Pedersen, Line; Aits, Sonja; Corcelle-Termeau, Elisabeth; Petersen, Nikolaj H T; Nylandsted, Jesper; Jäättelä, Marja

    2012-01-01

    Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 siRNAs was preceded by lysosomal membrane permeabilization, and all identified siRNAs induced several changes in the endo-lysosomal compartment, i.e. increased lysosomal volume (KIF11, KIF20A, KIF25, MYO1G, MYH1), increased cysteine cathepsin activity (KIF20A, KIF25), altered lysosomal localization (KIF25, MYH1, TPM2), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide or cisplatin. Similarly to KIF11 siRNA, the KIF11 inhibitor monastrol induced lysosomal membrane permeabilization and sensitized several cancer cell lines to siramesine. While KIF11 inhibitors are under clinical development as mitotic blockers, our data reveal a new function for KIF11 in controlling lysosomal stability and introduce six other molecular motors as putative cancer drug targets.

  5. Mild MPP(+) exposure impairs autophagic degradation through a novel lysosomal acidity-independent mechanism.

    PubMed

    Miyara, Masatsugu; Kotake, Yaichiro; Tokunaga, Wataru; Sanoh, Seigo; Ohta, Shigeru

    2016-10-01

    Parkinson's disease (PD) is the second most common neurodegenerative disorder, but its underlying cause remains unknown. Although recent studies using PD-related neurotoxin MPP(+) suggest autophagy involvement in the pathogenesis of PD, the effect of MPP(+) on autophagic processes under mild exposure, which mimics the slow progressive nature of PD, remains largely unclear. We examined the effect of mild MPP(+) exposure (10 and 200 μM for 48 h), which induces a more slowly developing cell death, on autophagic processes and the mechanistic differences with acute MPP(+) toxicity (2.5 and 5 mM for 24 h). In SH-SY5Y cells, mild MPP(+) exposure predominantly inhibited autophagosome degradation, whereas acute MPP(+) exposure inhibited both autophagosome degradation and basal autophagy. Mild MPP(+) exposure reduced lysosomal hydrolase cathepsin D activity without changing lysosomal acidity, whereas acute exposure decreased lysosomal density. Lysosome biogenesis enhancers trehalose and rapamycin partially alleviated mild MPP(+) exposure induced impaired autophagosome degradation and cell death, but did not prevent the pathogenic response to acute MPP(+) exposure, suggesting irreversible lysosomal damage. We demonstrated impaired autophagic degradation by MPP(+) exposure and mechanistic differences between mild and acute MPP(+) toxicities. Mild MPP(+) toxicity impaired autophagosome degradation through novel lysosomal acidity-independent mechanisms. Sustained mild lysosomal damage may contribute to PD. We examined the effects of MPP(+) on autophagic processes under mild exposure, which mimics the slow progressive nature of Parkinson's disease, in SH-SY5Y cells. This study demonstrated impaired autophagic degradation through a reduction in lysosomal cathepsin D activity without altering lysosomal acidity by mild MPP(+) exposure. Mechanistic differences between acute and mild MPP(+) toxicity were also observed. Sustained mild damage of lysosome may be an underlying cause

  6. Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions.

    PubMed

    Lee, Sook-Jeong; Park, Mi-Ha; Kim, Hyun-Jae; Koh, Jae-Young

    2010-08-01

    Cellular zinc plays a key role in lysosomal change and cell death in neurons and astrocytes under oxidative stress. Here, using astrocytes lacking metallothionein-3 (MT3), a potential source of labile zinc in the brain, we studied the role of MT3 in oxidative stress responses. H(2)O(2) induced a large increase in labile zinc in wild-type (WT) astrocytes, but stimulated only a modest rise in MT3-null astrocytes. In addition, H(2)O(2)-induced lysosomal membrane permeabilization (LMP) and cell death were comparably attenuated in MT3-null astrocytes. Expression and glycosylation of Lamp1 (lysosome-associated membrane protein 1) and Lamp2 were increased in MT3-null astrocytes, and the activities of several lysosomal enzymes were significantly reduced, indicating an effect of MT3 on lysosomal components. Consistent with lysosomal dysfunction in MT3-null cells, the level of LC3-II (microtubule-associated protein 1 light chain 3), a marker of early autophagy, was increased by oxidative stress in WT astrocytes, but not in MT3-null cells. Similar changes in Lamp1, LC3, and cathepsin-D were induced by the lysosomal inhibitors bafilomycin A1, chloroquine, and monensin, indicating that lysosomal dysfunction may lie upstream of changes observed in MT3-null astrocytes. Consistent with this idea, lysosomal accumulation of cholesterol and lipofuscin were augmented in MT3-null astrocytes. Similar to the results seen in MT3-null cells, MT3 knockdown by siRNA inhibited oxidative stress-induced increases in zinc and LMP. These results indicate that MT3 may play a key role in normal lysosomal function in cultured astrocytes.

  7. Lysosomal dysfunction causes neurodegeneration in mucolipidosis II 'knock-in' mice.

    PubMed

    Kollmann, K; Damme, M; Markmann, S; Morelle, W; Schweizer, M; Hermans-Borgmeyer, I; Röchert, A K; Pohl, S; Lübke, T; Michalski, J-C; Käkelä, R; Walkley, S U; Braulke, T

    2012-09-01

    Mucolipidosis II is a neurometabolic lysosomal trafficking disorder of infancy caused by loss of mannose 6-phosphate targeting signals on lysosomal proteins, leading to lysosomal dysfunction and accumulation of non-degraded material. However, the identity of storage material and mechanisms of neurodegeneration in mucolipidosis II are unknown. We have generated 'knock-in' mice with a common mucolipidosis II patient mutation that show growth retardation, progressive brain atrophy, skeletal abnormalities, elevated lysosomal enzyme activities in serum, lysosomal storage in fibroblasts and brain and premature death, closely mimicking the mucolipidosis II disease in humans. The examination of affected mouse brains at different ages by immunohistochemistry, ultrastructural analysis, immunoblotting and mass spectrometric analyses of glycans and anionic lipids revealed that the expression and proteolytic processing of distinct lysosomal proteins such as α-l-fucosidase, β-hexosaminidase, α-mannosidase or Niemann-Pick C2 protein are more significantly impacted by the loss of mannose 6-phosphate residues than enzymes reaching lysosomes independently of this targeting mechanism. As a consequence, fucosylated N-glycans, GM2 and GM3 gangliosides, cholesterol and bis(monoacylglycero)phosphate accumulate progressively in the brain of mucolipidosis II mice. Prominent astrogliosis and the accumulation of organelles and storage material in focally swollen axons were observed in the cerebellum and were accompanied by a loss of Purkinje cells. Moreover, an increased neuronal level of the microtubule-associated protein 1 light chain 3 and the formation of p62-positive neuronal aggregates indicate an impairment of constitutive autophagy in the mucolipidosis II brain. Our findings demonstrate the essential role of mannose 6-phosphate for selected lysosomal proteins to maintain the capability for degradation of sequestered components in lysosomes and autophagolysosomes and prevent

  8. Oxidant-induced autophagy and ferritin degradation contribute to epithelial–mesenchymal transition through lysosomal iron

    PubMed Central

    Sioutas, Apostolos; Vainikka, Linda K; Kentson, Magnus; Dam-Larsen, Sören; Wennerström, Urban; Jacobson, Petra; Persson, Hans Lennart

    2017-01-01

    Purpose Transforming growth factor (TGF)-β1 triggers epithelial–mesenchymal transition (EMT) through autophagy, which is partly driven by reactive oxygen species (ROS). The aim of this study was to determine whether leaking lysosomes and enhanced degradation of H-ferritin could be involved in EMT and whether it could be possible to prevent EMT by iron chelation targeting of the lysosome. Materials and methods EMT, H-ferritin, and autophagy were evaluated in TGF-β1-stimulated A549 human lung epithelial cells cultured in vitro using Western blotting, with the additional morphological assessment of EMT. By using immunofluorescence and flow cytometry, lysosomes and ROS were assessed by acridine orange and 6-carboxy-2′,7′-dichlorodihydrofluorescein acetate assays, respectively. Results TGF-β1-stimulated cells demonstrated a loss of H-ferritin, which was prevented by the antioxidant N-acetyl-L-cysteine (NAC) and inhibitors of lysosomal degradation. TGF-β1 stimulation generated ROS and autophagosome formation and led to EMT, which was further promoted by the additional ROS-generating cytokine, tumor necrosis factor-α. Lysosomes of TGF-β1-stimulated cells were sensitized to oxidants but also completely protected by lysosomal loading with dextran-bound deferoxamine (DFO). Autophagy and EMT were prevented by NAC, DFO, and inhibitors of autophagy and lysosomal degradation. Conclusion The findings of this study support the role of enhanced autophagic degradation of H-ferritin as a mechanism for increasing the vulnerability of lysosomes to iron-driven oxidant injury that triggers further autophagy during EMT. This study proposes that lysosomal leakage is a novel pathway of TGF-β1-induced EMT that may be prevented by iron-chelating drugs that target the lysosome. PMID:28405169

  9. Cloning and expression of mouse legumain, a lysosomal endopeptidase.

    PubMed

    Chen, J M; Dando, P M; Stevens, R A; Fortunato, M; Barrett, A J

    1998-10-01

    Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain.

  10. Cloning and expression of mouse legumain, a lysosomal endopeptidase.

    PubMed Central

    Chen, J M; Dando, P M; Stevens, R A; Fortunato, M; Barrett, A J

    1998-01-01

    Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain. PMID:9742219

  11. Lysine fatty acylation promotes lysosomal targeting of TNF-α.

    PubMed

    Jiang, Hong; Zhang, Xiaoyu; Lin, Hening

    2016-04-15

    Tumor necrosis factor-α (TNF-α) is a proinflammation cytokine secreted by various cells. Understanding its secretive pathway is important to understand the biological functions of TNF-α and diseases associated with TNF-α. TNF-α is one of the first proteins known be modified by lysine fatty acylation (e.g. myristoylation). We previously demonstrated that SIRT6, a member of the mammalian sirtuin family of enzymes, can remove the fatty acyl modification on TNF-α and promote its secretion. However, the mechanistic details about how lysine fatty acylation regulates TNF-α secretion have been unknown. Here we present experimental data supporting that lysine fatty acylation promotes lysosomal targeting of TNF-α. The result is an important first step toward understanding the biological functions of lysine fatty acylation.

  12. Ten plus one challenges in diseases of the lysosomal system.

    PubMed

    Grabowski, Gregory A; Whitley, Chester

    The advent of the first effective specific therapy for a lysosomal storage disease (LSDs), Gaucher disease type 1, by Roscoe O. Brady was foundational for development of additional treatments for this group of rare diseases. The past 26years, since the approval of enzyme therapy for Gaucher disease type 1, have witnessed a burgeoning understanding of LSDs at genetic, molecular, biochemical, cell biologic, and clinical levels. Simultaneously, this expansion of knowledge has exposed our incomplete understanding of the individual pathophysiologies of LSDs as well as difficult challenges for improvement in therapy and therapeutic outcomes for afflicted individuals. Here, 10 such challenges/problems representing major impediments, which need to be overcome, to move forward toward the goals of more effective and complete therapies for these devastating diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Molecular structure and functional characterization of the gamma-interferon-inducible lysosomal thiol reductase (GILT) gene in largemouth bass (Microptenus salmoides).

    PubMed

    Yang, Qian; Zhang, Jiaxin; Hu, Lingling; Lu, Jia; Sang, Ming; Zhang, Shuangquan

    2015-12-01

    The enzyme gamma-interferon-inducible lysosomal thiol reductase (GILT) plays a role in facilitating the processing and presentation of major histocompatibility complex (MHC) class II-restricted antigens and is also involved in MHC I-restricted antigens in adaptive immunity catalyzing disulfide bond reduction in mammals. In this study, we cloned a GILT gene homolog from largemouth bass (designated 'lbGILT'), a freshwater fish belonging to Perciformes and known for its nutritive value. We obtained the full-length cDNA of lbGILT by reverse transcription PCR and rapid amplification of cDNA ends. This cDNA is comprised of a 5'-untranslated region (UTR) of 87 bp, a 3'-UTR of 189 bp, and an open reading frame of 771 bp. It encodes a protein of 256 amino acids with a deduced molecular weight of 28.548 kDa and a predicted isoelectric point of 5.62. The deduced protein possesses the typical structural features of known GILTs, including an active site motif, two potential N-linked glycosylation sites, a GILT signature sequence, and six conserved cysteines. Tissue-specific expression of lbGILT was shown by real-time quantitative PCR. The expression of lbGILT mRNA was obviously up regulated in spleen and kidney after induction with lipopolysaccharide. Recombinant lbGILT was produced as an inclusion body with a His6 tag in ArcticExpress (DE3), and the protein was then washed, solubilized, and refolded. The refolded lbGILT showed reduction activity against an IgG substrate. These results suggest that lbGILT plays a role in innate immunity.

  14. Synergistic Anticancer Action of Lysosomal Membrane Permeabilization and Glycolysis Inhibition.

    PubMed

    Kosic, Milica; Arsikin-Csordas, Katarina; Paunovic, Verica; Firestone, Raymond A; Ristic, Biljana; Mircic, Aleksandar; Petricevic, Sasa; Bosnjak, Mihajlo; Zogovic, Nevena; Mandic, Milos; Bumbasirevic, Vladimir; Trajkovic, Vladimir; Harhaji-Trajkovic, Ljubica

    2016-10-28

    We investigated the in vitro and in vivo anticancer effect of combining lysosomal membrane permeabilization (LMP)-inducing agent N-dodecylimidazole (NDI) with glycolytic inhibitor 2-deoxy-d-glucose (2DG). NDI-triggered LMP and 2DG-mediated glycolysis block synergized in inducing rapid ATP depletion, mitochondrial damage, and reactive oxygen species production, eventually leading to necrotic death of U251 glioma cells but not primary astrocytes. NDI/2DG-induced death of glioma cells was partly prevented by lysosomal cathepsin inhibitor E64 and antioxidant α-tocopherol, suggesting the involvement of LMP and oxidative stress in the observed cytotoxicity. LMP-inducing agent chloroquine also displayed a synergistic anticancer effect with 2DG, whereas glucose deprivation or glycolytic inhibitors iodoacetate and sodium fluoride synergistically cooperated with NDI, thus further indicating that the anticancer effect of NDI/2DG combination was indeed due to LMP and glycolysis block. The two agents synergistically induced ATP depletion, mitochondrial depolarization, oxidative stress, and necrotic death also in B16 mouse melanoma cells. Moreover, the combined oral administration of NDI and 2DG reduced in vivo melanoma growth in C57BL/6 mice by inducing necrotic death of tumor cells, without causing liver, spleen, or kidney toxicity. Based on these results, we propose that NDI-triggered LMP causes initial mitochondrial damage that is further increased by 2DG due to the lack of glycolytic ATP required to maintain mitochondrial health. This leads to a positive feedback cycle of mitochondrial dysfunction, ATP loss, and reactive oxygen species production, culminating in necrotic cell death. Therefore, the combination of LMP-inducing agents and glycolysis inhibitors seems worthy of further exploration as an anticancer strategy. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Lysosomal exoglycosidases and cathepsin D in colon adenocarcinoma.

    PubMed

    Waszkiewicz, Napoleon; Zalewska-Szajda, Beata; Szajda, Sławomir D; Kępka, Alina; Waszkiewicz, Magdalena; Roszkowska-Jakimiec, Wiesława; Wojewódzka-Żeleźniakowicz, Marzena; Milewska, Anna J; Dadan, Jacek; Szulc, Agata; Zwierz, Krzysztof; Ladny, Jerzy R

    2012-01-01

    Changes in the structure of membrane glycoconjugates and activity of glycosidases and proteases are important in tumor formation. The aim of the study was to compare the specific activity of lysosomal exoglycosidases: N-acetyl-β-D-hexosaminidase (HEX), its isoenzymes A (HEX A) and B (HEX B), β-D-galactosidase (GAL), α-fucosidase (FUC), and α-mannosidase (MAN) with the activity of cathepsin D (CD) in serum, urine, and carcinoma tissue of patients with colon adenocarcinoma. The specific activity of HEX, HEX A, HEX B, GAL, FUC, MAN, and CD was assayed in serum, urine, and carcinoma tissue of 12 patients with colon adenocarcinoma. Lysosomal exoglycosidases and CD have similar specific activity in colon adenocarcinoma tissue and urine, which is higher than their activity in serum (with the exception of the highest specific activity of CD in urine). A positive correlation was observed between the specific activity of CD and that of HEX, HEX A, FUC, and MAN in the carcinoma tissue and urine as well as between CD and GAL in the urine of patients with colon adenocarcinoma. Negative correlations were observed between protein levels and the specific activity of HEX, HEX A, FUC, MAN, and CD in the carcinoma tissue and urine, and between protein levels and GAL in urine. Increased degradation and remodeling of glycoconjugates in the colon adenocarcinoma tissue is reflected by increased specific activity of exoglycosidases and CD. The results suggest a strong effect of exoglycosidase action on tissue degradation and a potential role of exoglycosidases in the initiation of proteolysis.

  16. Critical Roles of Lysosomal Acid Lipase in Myelopoiesis

    PubMed Central

    Qu, Peng; Shelley, William C.; Yoder, Mervin C.; Wu, Lingyan; Du, Hong; Yan, Cong

    2010-01-01

    Lysosomal acid lipase (LAL) is a key enzyme that cleaves cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. Genetic ablation of the lal gene (lal−/−) in mice has resulted in a systemic increase of macrophages and neutrophils, causing severe inflammation and pathogenesis in multiple organs. We hypothesized that aberrant growth and differentiation of myeloid cells in lal−/− mice arises from dysregulated production of progenitor cells in the bone marrow. Indeed, lal−/− mice displayed increased numbers of primitive lin−Sca-1+c-Kit+ (LSK) cells and granulocyte-macrophage precursors (GMP). Increased high proliferative potential colony-forming cells (HPP-CFC) were enumerated from cultured lal−/− bone marrow cells, as were significantly more CFU-GM, CFU-G, and CFU-M colonies. As a consequence, lal−/− mice developed significant myeloid infiltration, particularly with CD11b+/Gr-1+ myeloid-derived suppressive cells in multiple organs. Both decreased apoptosis and increased proliferation contribute to the systemic increase of myeloid cells in lal−/− myeloid cells. These lal−/− CD11b+/Gr-1+ cells displayed suppressive activity on T cell proliferation and function in vitro. Bone marrow chimeras confirmed that the myeloproliferative disorder in lal−/− mice was primarily attributable to autonomous defects in myeloid progenitor cells, although the hematopoietic microenvironment in the lal−/− mice did not support hematopoiesis normally. These results provide evidence that LAL is an important regulator of myelopoiesis during hematopoietic development, differentiation, and homeostasis. PMID:20348241

  17. Processing and activation of latent heparanase occurs in lysosomes.

    PubMed

    Zetser, Anna; Levy-Adam, Flonia; Kaplan, Victoria; Gingis-Velitski, Svetlana; Bashenko, Yulia; Schubert, Shay; Flugelman, Moshe Y; Vlodavsky, Israel; Ilan, Neta

    2004-05-01

    Heparanase is a heparan sulfate degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Heparanase is synthesized as a 65 kDa non-active precursor that subsequently undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. The protease responsible for heparanase processing is currently unknown, as is the sub-cellular processing site. In this study, we characterize an antibody (733) that preferentially recognizes the active 50 kDa heparanase form as compared to the non-active 65 kDa heparanase precursor. We have utilized this and other anti-heparanase antibodies to study the cellular localization of the latent 65 kDa and active 50 kDa heparanase forms during uptake and processing of exogenously added heparanase. Interestingly, not only the processed 50 kDa, but also the 65 kDa heparanase precursor was localized to perinuclear vesicles, suggesting that heparanase processing occurs in lysosomes. Indeed, heparanase processing was completely inhibited by chloroquine and bafilomycin A1, inhibitors of lysosome proteases. Similarly, processing of membrane-targeted heparanase was also chloroquine-sensitive, further ruling out the plasma membrane as the heparanase processing site. Finally, we provide evidence that antibody 733 partially neutralizes the enzymatic activity of heparanase, suggesting that the N-terminal region of the molecule is involved in assuming an active conformation. Monoclonal antibodies directed to this region are likely to provide specific heparanase inhibitors and hence assist in resolving heparanase functions under normal and pathological conditions.

  18. Synthetic High-Density Lipoprotein-Like Nanocarrier Improved Cellular Transport of Lysosomal Cholesterol in Human Sterol Carrier Protein-Deficient Fibroblasts.

    PubMed

    Nam, Da-Eun; Kim, Ok-Kyung; Park, Yoo Kyoung; Lee, Jeongmin

    2016-01-01

    Sterol carrier protein-2 (SCP-2), which is not found in tissues of people with Zellweger syndrome, facilitates the movement of cholesterol within cells, resulting in abnormal accumulation of cholesterol in SCP-2-deficient cells. This study investigated whether synthetic high-density lipoprotein-like nanocarrier (sHDL-NC) improves the cellular transport of lysosomal cholesterol to plasma membrane in SCP-2-deficient fibroblasts. Human SCP-2-deficient fibroblasts were incubated with [(3)H-cholesterol]LDL as a source of cholesterol and sHDL-NC. The cells were fractionated by centrifugation permit tracking of [(3)H]-cholesterol from lysosome into plasma membrane. Furthermore, cellular content of cholesteryl ester as a storage form and mRNA expression of low-density lipoprotein (LDL) receptor were measured to support the cholesterol transport to plasma membrane. Incubation with sHDL-NC for 8 h significantly increased uptake of [(3)H]-cholesterol to lysosome by 53% and further enhanced the transport of [(3)H]-cholesterol to plasma membrane by 32%. Treatment with sHDL-NC significantly reduced cellular content of cholesteryl ester and increased mRNA expression of LDL receptor (LDL-R). In conclusion, sHDL-NC enables increased transport of lysosomal cholesterol to plasma membrane. In addition, these data were indirectly supported by decreased cellular content of cholesteryl ester and increased gene expression of LDL-R. Therefore, sHDL-NC may be a useful vehicle for transporting cholesterol, which may help to prevent accumulation of cholesterol in SCP-2-deficient fibroblasts.

  19. Antioxidant, genotoxic and lysosomal biomarkers in the freshwater bivalve (Unio pictorum) transplanted in a metal polluted river basin.

    PubMed

    Guidi, Patrizia; Frenzilli, Giada; Benedetti, Maura; Bernardeschi, Margherita; Falleni, Alessandra; Fattorini, Daniele; Regoli, Francesco; Scarcelli, Vittoria; Nigro, Marco

    2010-10-01

    The freshwater painter's mussel (Unio pictorum) was used as sentinel species to assess the chemical disturbance in an Italian river (the river Cecina) characterized by elevated levels of trace metals of both natural and anthropogenic origin. Organisms were transplanted for 4 weeks in different locations of the river basin and the bioaccumulation of metals was integrated with a wide battery of biomarkers consisting of oxidative, genotoxic and lysosomal responses. Such parameters included the levels of individual antioxidants (catalase, glutathione-S-transferases, glutathione reductase, Se-dependent and Se-independent glutathione peroxidases, total glutathione), the total oxyradical scavenging capacity (TOSC), metallothionein-like proteins, the assessment of DNA integrity, chromosomal damages and lysosomal membrane stability. Elevated levels of several metals were measured in sediments, but the relatively low tissue concentrations suggested a moderate bioaccumulation, possibly due to a high excretion efficiency, of U. pictorum and/or to a limited bioavailability of these elements, partly deriving from erosion of bedrocks. Among antioxidant responses, those based on glutathione metabolism and the activity of catalase were mostly affected in bivalves showing a significant accumulation of arsenic, mercury and/or nickel. In these specimens, the content of glutathione and the activities of glutathione reductase and glutathione peroxidases (H2O2) were respectively 9-, 6- and 4-fold lower than in controls, while a 3-fold increase was observed for catalase. Despite some differences in the response of individual antioxidants, a significant reduction of the capability to neutralize peroxyl radicals was observed in bivalves caged in all the impacted sites of the river basin; these organisms also exhibited a significant impairment at the DNA, chromosomal and lysosomal levels. Considering the mild contamination gradient in the investigated area, the overall results suggested that

  20. Combined effects of thermal stress and Cd on lysosomal biomarkers and transcription of genes encoding lysosomal enzymes and HSP70 in mussels, Mytilus galloprovincialis.

    PubMed

    Izagirre, Urtzi; Errasti, Aitzpea; Bilbao, Eider; Múgica, María; Marigómez, Ionan

    2014-04-01

    In estuaries and coastal areas, intertidal organisms may be subject to thermal stress resulting from global warming, together with pollution. In the present study, the combined effects of thermal stress and exposure to Cd were investigated in the endo-lysosomal system of digestive cells in mussels, Mytilus galloprovincialis. Mussels were maintained for 24h at 18°C and 26°C seawater temperature in absence and presence of 50 μg Cd/L seawater. Cadmium accumulation in digestive gland tissue, lysosomal structural changes and membrane stability were determined. Semi-quantitative PCR was applied to reveal the changes elicited by the different experimental conditions in hexosaminidase (hex), β-glucuronidase (gusb), cathepsin L (ctsl) and heat shock protein 70 (hsp70) gene transcription levels. Thermal stress provoked lysosomal enlargement whilst Cd-exposure led to fusion of lysosomes. Both thermal stress and Cd-exposure caused lysosomal membrane destabilisation. hex, gusb and ctsl genes but not hsp70 gene were transcriptionally up-regulated as a result of thermal stress. In contrast, all the studied genes were transcriptionally down-regulated in response to Cd-exposure. Cd bioaccumulation was comparable at 18°C and 26°C seawater temperatures but interactions between thermal stress and Cd-exposure were remarkable both in lysosomal biomarkers and in gene transcription. hex, gusb and ctsl genes, reacted to elevated temperature in absence of Cd but not in Cd-exposed mussels. Therefore, thermal stress resulting from global warming might influence the use and interpretation of lysosomal biomarkers in marine pollution monitoring programmes and, vice versa, the presence of pollutants may condition the capacity of mussels to respond against thermal stress in a climate change scenario.

  1. Eucommia ulmoides cortex, geniposide and aucubin regulate lipotoxicity through the inhibition of lysosomal BAX.

    PubMed

    Lee, Geum-Hwa; Lee, Mi-Rin; Lee, Hwa-Young; Kim, Seung Hyun; Kim, Hye-Kyung; Kim, Hyung-Ryong; Chae, Han-Jung

    2014-01-01

    In this study we examined the inhibition of hepatic dyslipidemia by Eucommia ulmoides extract (EUE). Using a screening assay for BAX inhibition we determined that EUE regulates BAX-induced cell death. Among various cell death stimuli tested EUE regulated palmitate-induced cell death, which involves lysosomal BAX translocation. EUE rescued palmitate-induced inhibition of lysosomal V-ATPase, α-galactosidase, α-mannosidase, and acid phosphatase, and this effect was reversed by bafilomycin, a lysosomal V-ATPase inhibitor. The active components of EUE, aucubin and geniposide, showed similar inhibition of palmitate-induced cell death to that of EUE through enhancement of lysosome activity. Consistent with these in vitro findings, EUE inhibited the dyslipidemic condition in a high-fat diet animal model by regulating the lysosomal localization of BAX. This study demonstrates that EUE regulates lipotoxicity through a novel mechanism of enhanced lysosomal activity leading to the regulation of lysosomal BAX activation and cell death. Our findings further indicate that geniposide and aucubin, active components of EUE, may be therapeutic candidates for non-alcoholic fatty liver disease.

  2. Acute and chronic mitochondrial respiratory chain deficiency differentially regulate lysosomal biogenesis

    PubMed Central

    Fernández-Mosquera, Lorena; Diogo, Cátia V.; Yambire, King Faisal; Santos, Gabriela L.; Luna Sánchez, Marta; Bénit, Paule; Rustin, Pierre; Lopez, Luis Carlos; Milosevic, Ira; Raimundo, Nuno

    2017-01-01

    Mitochondria are key cellular signaling platforms, affecting fundamental processes such as cell proliferation, differentiation and death. However, it remains unclear how mitochondrial signaling affects other organelles, particularly lysosomes. Here, we demonstrate that mitochondrial respiratory chain (RC) impairments elicit a stress signaling pathway that regulates lysosomal biogenesis via the microphtalmia transcription factor family. Interestingly, the effect of mitochondrial stress over lysosomal biogenesis depends on the timeframe of the stress elicited: while RC inhibition with rotenone or uncoupling with CCCP initially triggers lysosomal biogenesis, the effect peaks after few hours and returns to baseline. Long-term RC inhibition by long-term treatment with rotenone, or patient mutations in fibroblasts and in a mouse model result in repression of lysosomal biogenesis. The induction of lysosomal biogenesis by short-term mitochondrial stress is dependent on TFEB and MITF, requires AMPK signaling and is independent of calcineurin signaling. These results reveal an integrated view of how mitochondrial signaling affects lysosomes, which is essential to fully comprehend the consequences of mitochondrial malfunction, particularly in the context of mitochondrial diseases. PMID:28345620

  3. Activity-dependent trafficking of lysosomes in dendrites and dendritic spines.

    PubMed

    Goo, Marisa S; Sancho, Laura; Slepak, Natalia; Boassa, Daniela; Deerinck, Thomas J; Ellisman, Mark H; Bloodgood, Brenda L; Patrick, Gentry N

    2017-08-07

    In neurons, lysosomes, which degrade membrane and cytoplasmic components, are thought to primarily reside in somatic and axonal compartments, but there is little understanding of their distribution and function in dendrites. Here, we used conventional and two-photon imaging and electron microscopy to show that lysosomes traffic bidirectionally in dendrites and are present in dendritic spines. We find that lysosome inhibition alters their mobility and also decreases dendritic spine number. Furthermore, perturbing microtubule and actin cytoskeletal dynamics has an inverse relationship on the distribution and motility of lysosomes in dendrites. We also find trafficking of lysosomes is correlated with synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. Strikingly, lysosomes traffic to dendritic spines in an activity-dependent manner and can be recruited to individual spines in response to local activation. These data indicate the position of lysosomes is regulated by synaptic activity and thus plays an instructive role in the turnover of synaptic membrane proteins. © 2017 Goo et al.

  4. Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes

    PubMed Central

    1996-01-01

    We have followed the transfer of EGF-EGF receptor (EGFR) complexes from endosomal vacuoles that contain transferrin receptors (TfR) to lysosome vacuoles identified by their content of HRP loaded as a 15-min pulse 4 h previously. We show that the HRP-loaded lysosomes are lysosomal- associated membrane protein-1 (LAMP-1) positive, mannose-6-phosphate receptor (M6PR) negative. and contain active acid hydrolase. EGF-EGFR complexes are delivered to these lysosomes intact and are then rapidly degraded. Preactivating the HRP contained within the preloaded lysosomes inhibits the delivery of EGFR and degradation of EGF, and results in the accumulation of EGFR-containing multivesicular bodies (MVB). With time these accumulating MVB undergo a series of maturation changes that include the loss of TfR, the continued recruitment of EGFR, and the accumulation of internal vesicles, but they remain LAMP-1 and M6PR negative. The mature MVB are often seen to make direct contact with lysosomes containing preactivated HRP, but their perimeter membranes remain intact. Together our observations suggest that the transfer of EGF-EGFR complexes from the TfR-containing endosome compartment to the lysosomes that degrade them employs a single vacuolar intermediate, the maturing MVB, and can be achieved by a single heterotypic fusion step. PMID:8601581

  5. The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes

    PubMed Central

    Khatter, Divya; Raina, Vivek B.; Dwivedi, Devashish; Sindhwani, Aastha; Bahl, Surbhi; Sharma, Mahak

    2015-01-01

    The homotypic fusion and protein sorting (HOPS) complex is a multi-subunit complex conserved from yeast to mammals that regulates late endosome and lysosome fusion. However, little is known about how the HOPS complex is recruited to lysosomes in mammalian cells. Here, we report that the small GTPase Arl8b, but not Rab7 (also known as RAB7A), is essential for membrane localization of the human (h)Vps41 subunit of the HOPS complex. Assembly of the core HOPS subunits to Arl8b- and hVps41-positive lysosomes is guided by their subunit–subunit interactions. RNA interference (RNAi)-mediated depletion of hVps41 resulted in the impaired degradation of EGFR that was rescued upon expression of wild-type but not an Arl8b-binding-defective mutant of hVps41, suggesting that Arl8b-dependent lysosomal localization of hVps41 is required for its endocytic function. Furthermore, we have also identified that the Arl8b effector SKIP (also known as PLEKHM2) interacts with and recruits HOPS subunits to Arl8b and kinesin-positive peripheral lysosomes. Accordingly, RNAi-mediated depletion of SKIP impaired lysosomal trafficking and degradation of EGFR. These findings reveal that Arl8b regulates the association of the human HOPS complex with lysosomal membranes, which is crucial for the function of this tethering complex in endocytic degradation. PMID:25908847

  6. Lysosomal cholesterol accumulation: driver on the road to inflammation during atherosclerosis and non-alcoholic steatohepatitis.

    PubMed

    Hendrikx, T; Walenbergh, S M A; Hofker, M H; Shiri-Sverdlov, R

    2014-05-01

    Many studies show an association between the accumulation of cholesterol inside lysosomes and the progression towards inflammatory disease states that are closely related to obesity. While in the past, the knowledge regarding lysosomal cholesterol accumulation was limited to its association with plaque severity during atherosclerosis, recently, a growing body of evidence indicates a causal link between lysosomal cholesterol accumulation and inflammation. These findings make lysosomal cholesterol accumulation an important target for intervention in metabolic diseases that are characterized by the presence of an inflammatory response. In this review, we aim to show the importance of cholesterol trapping inside lysosomes to the development of inflammation by focusing upon cardiovascular disease and non-alcoholic steatohepatitis (NASH) in particular. We summarize current data supporting the hypothesis that lysosomal cholesterol accumulation plays a key role in the development of inflammation during atherosclerosis and NASH. In addition, potential mechanisms by which disturbed lysosomal function can trigger the inflammatory response, the challenges in improving cholesterol trafficking in macrophages and recent successful research directions will be discussed.

  7. Autophagosome–lysosome fusion is independent of V-ATPase-mediated acidification

    PubMed Central

    Mauvezin, Caroline; Nagy, Péter; Juhász, Gábor; Neufeld, Thomas P.

    2015-01-01

    The ATP-dependent proton pump V-ATPase ensures low intralysosomal pH, which is essential for lysosomal hydrolase activity. Based on studies with the V-ATPase inhibitor BafilomycinA1, lysosomal acidification is also thought to be required for fusion with incoming vesicles from the autophagic and endocytic pathways. Here we show that loss of V-ATPase subunits in the Drosophila fat body causes an accumulation of non-functional lysosomes, leading to a block in autophagic flux. However, V-ATPase-deficient lysosomes remain competent to fuse with autophagosomes and endosomes, resulting in a time-dependent formation of giant autolysosomes. In contrast, BafilomycinA1 prevents autophagosome–lysosome fusion in these cells, and this defect is phenocopied by depletion of the Ca2+ pump SERCA, a secondary target of this drug. Moreover, activation of SERCA promotes fusion in a BafilomycinA1-sensitive manner. Collectively, our results indicate that lysosomal acidification is not a prerequisite for fusion, and that BafilomycinA1 inhibits fusion independent of its effect on lysosomal pH. PMID:25959678

  8. Action of low-energy monochromatic coherent light on the stability of retinal lysosomes

    NASA Astrophysics Data System (ADS)

    Metelitsina, Irina P.; Leus, N. F.

    1995-05-01

    The data had been obtained during the experiment in vitro by irradiation of solubilized lysosomal enzymes, retinal homogenates and native lysosomes enabled us to conclude that the laser beam ((lambda) equals 632.8 nm, power density from 0.1 to 15.0 mWt/cm2) acts on the level of membranous structures of lysosomes. During irradiation of rabbits eyes in vitro with an unfocused laser beam (power density on the cornea aur face from 0.01 to 15.0 mWt/cm2 was shown, that low-energy, ranged from 0.01 to 1.0 mWt/cm2 promotes stabilization of lysosomal membranes. Irradiation with laser beam of 8.0 mWt/cm2 and more power induces destabilization of lysosomal membranes. We have also shown that vitamins A and E effecting membranotropic on lysosomes may be corrected by low-energy radiation of helium-neon laser. It is substantiated experimentally that the stabilizing effect of vitamin E may be intensified in case of the combined action of laser radiation on lysosomes. The labilizing effect of vitamin A on membranes of organelles, as was studied, may be weakened by application of laser radiation of low intensities.

  9. An unusual lysosome compartment involved in vitellogenin endocytosis by Xenopus oocytes

    PubMed Central

    1985-01-01

    We have investigated the lysosomal compartment of Xenopus oocytes to determine the possible role of this organelle in the endocytic pathway of the yolk protein precursor, vitellogenin. Oocytes have lysosome-like organelles of unusual enzymatic composition at all stages of their development, and the amount of hydrolase activity increases steadily throughout oogenesis. These unusual lysosomes appear to be located primarily in a peripheral zone of oocyte cytoplasm. At least two distinct populations of lysosomal organelles can be identified after sucrose density gradient fractionation of vitellogenic oocytes. Most enzyme activity resides in a compartment of large size and high density that appears to be a subpopulation of yolk platelets that are less dense than most platelets within the cell. The appearance of this high density peak of lysosomal enzyme activity coincides with the time of onset of vitellogenin endocytosis during oocyte development. The data suggest that endocytic vesicles that contain vitellogenin fuse with modified lysosomes shortly after their internalization by the oocyte. Pulse-chase experiments with radiolabeled vitellogenin suggest that the ligand passes through the low density platelet compartment en route to the heavy platelets. The accumulation of yolk proteins apparently results from a failure of these molecules to undergo complete digestion after their entry into an unusual lysosomal compartment. The yolk platelets that these proteins finally enter for prolonged storage appear to be a postlysosomal organelle. PMID:4055890

  10. Iowa Mutant Apolipoprotein A-I (ApoA-IIowa) Fibrils Target Lysosomes

    PubMed Central

    Kameyama, Hirokazu; Nakajima, Hiroyuki; Nishitsuji, Kazuchika; Mikawa, Shiho; Uchimura, Kenji; Kobayashi, Norihiro; Okuhira, Keiichiro; Saito, Hiroyuki; Sakashita, Naomi

    2016-01-01

    The single amino acid mutation G26R in human apolipoprotein A-I (apoA-IIowa) is the first mutation that was associated with familial AApoA1 amyloidosis. The N-terminal fragments (amino acid residues 1–83) of apoA-I containing this mutation deposit as amyloid fibrils in patients’ tissues and organs, but the mechanisms of cellular degradation and cytotoxicity have not yet been clarified. In this study, we demonstrated degradation of apoA-IIowa fibrils via the autophagy-lysosomal pathway in human embryonic kidney 293 cells. ApoA-IIowa fibrils induced an increase in lysosomal pH and the cytosolic release of the toxic lysosomal protease cathepsin B. The mitochondrial dysfunction caused by apoA-IIowa fibrils depended on cathepsin B and was ameliorated by increasing the degradation of apoA-IIowa fibrils. Thus, although apoA-IIowa fibril transport to lysosomes and fibril degradation in lysosomes may have occurred, the presence of an excess number of apoA-IIowa fibrils, more than the lysosomes could degrade, may be detrimental to cells. Our results thus provide evidence that the target of apoA-IIowa fibrils is lysosomes, and we thereby gained a novel insight into the mechanism of AApoA1 amyloidosis. PMID:27464946

  11. Intracellular target for alpha-terthienyl photosensitization: involvement of lysosomal membrane damage.

    PubMed

    Sasaki, M; Koyama, S; Tokiwa, K; Fujita, H

    1993-05-01

    Intracellular targets for the photosensitizer alpha-terthienyl (alpha T) were examined by fluorescence microscopy and microfluorospectrometry using human nonkeratinized buccal cells. Intracellular distribution of alpha T was observed as fluorescent patches widely dispersed in the cytoplasm. The distribution of the fluorescent patches was compared with that of acid phosphatase activity visualized as an azo dye produced by the fast garnet 2-methyl-4-[(2-methyl-phenyl)azo]benzenediasonium sulfate reaction. Because both the distribution sites coincided, lysosomes were the likely sites of intracellular affinity of alpha T. However, because acid phosphatase is not a specific lysosomal marker, we tried to detect another lysosomal enzyme, beta-galactosidase, to confirm if the fluorescent patches were lysosomes, using fluorescein-di-(beta-D-galactopyranoside) (FDG) as a fluorogenic substrate. Without UV-A (320-400 nm) irradiation of the cells after uptake of alpha T and FDG, no significant fluorescence was observed. In contrast, with prior UV-A irradiation in the presence of alpha T and FDG, the bright yellow fluorescence of fluorescein, which is the digested product of FDG, was clearly detected in the cells by fluorescence microscopy. This observation implied that inflow of external FDG into the lysosomes is caused by lysosomal membrane damage on alpha T photosensitization. The present results indicated that lysosomes are the primary photosensitization site of alpha T.

  12. Antimicrobial Properties of Lysosomal Enzymes Immobilized on NH₂Functionalized Silica-Encapsulated Magnetite Nanoparticles.

    PubMed

    Bang, Seung Hyuck; Sekhon, Simranjeet Singh; Cho, Sung-Jin; Kim, So Jeong; Le, Thai-Hoang; Kim, Pil; Ahn, Ji-Young; Kim, Yang-Hoon; Min, Jiho

    2016-01-01

    The immobilization efficiency, antimicrobial activity and recovery of lysosomal enzymes on NH2 functionalized magnetite nanoparticles have been studied under various conditions. The immobi- lization efficiency depends upon the ratio of the amount of enzyme and magnetite and it shows an increase with magnetite concentration which is due to the presence of amine group at the magnetite surface that leads to a strong attraction. The optimized reaction time to immobilize the lysosomal enzymes on magnetite was determined by using a rolling method. The immobilization efficiency increases with reaction time and reached a plateau after 5 minutes and then remained constant for 10 minutes. However, after 30 minutes the immobilization efficiency decreased to 85%, which is due to the weaker electrostatic interactions between magnetite and detached lysosomal enzymes. The recovery and stability of immobilized lysosomal enzymes has also been studied. The antimicrobial activity was almost 100% but it decreased upon reuse and no activity was observed after its reuse for seven times. The storage stability of lysosomal enzymes as an antimicrobial agent was about 88%, which decreased to 53% after one day and all activity of immobilized lysosomal enzymes was maintained after five days. Thus, the lysosomal enzymes immobilized on magnetite nanoparticles could potentially be used as antimicrobial agents to remove bacteria.

  13. Drosophila Mitf regulates the V-ATPase and the lysosomal-autophagic pathway

    PubMed Central

    Bouché, Valentina; Espinosa, Alma Perez; Leone, Luigi; Sardiello, Marco; Ballabio, Andrea; Botas, Juan

    2016-01-01

    ABSTRACT An evolutionarily conserved gene network regulates the expression of genes involved in lysosome biogenesis, autophagy, and lipid metabolism. In mammals, TFEB and other members of the MiTF-TFE family of transcription factors control this network. Here we report that the lysosomal-autophagy pathway is controlled by Mitf gene in Drosophila melanogaster. Mitf is the single MiTF-TFE family member in Drosophila and prior to this work was known only for its function in eye development. We show that Mitf regulates the expression of genes encoding V-ATPase subunits as well as many additional genes involved in the lysosomal-autophagy pathway. Reduction of Mitf function leads to abnormal lysosomes and impairs autophagosome fusion and lipid breakdown during the response to starvation. In contrast, elevated Mitf levels increase the number of lysosomes, autophagosomes and autolysosomes, and decrease the size of lipid droplets. Inhibition of Drosophila MTORC1 induces Mitf translocation to the nucleus, underscoring conserved regulatory mechanisms between Drosophila and mammalian systems. Furthermore, we show Mitf-mediated clearance of cytosolic and nuclear expanded ATXN1 (ataxin 1) in a cellular model of spinocerebellar ataxia type 1 (SCA1). This remarkable observation illustrates the potential of the lysosomal-autophagy system to prevent toxic protein aggregation in both the cytoplasmic and nuclear compartments. We anticipate that the genetics of the Drosophila model and the absence of redundant MIT transcription factors will be exploited to investigate the regulation and function of the lysosomal-autophagy gene network. PMID:26761346

  14. Positive Lysosomal Modulation As a Unique Strategy to Treat Age-Related Protein Accumulation Diseases

    PubMed Central

    Wisniewski, Meagan L.; Butler, David

    2012-01-01

    Abstract Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and α-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator Z-Phe-Ala-diazomethylketone (PADK). Such positive modulators of the lysosomal system have been studied in the well-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Active cathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulation reduced the pre-existing PHF deposits, restored tubulin structure and transport, and recovered synaptic components. Further proof-of-principle studies used Alzheimer disease mouse models. It was recently reported that systemic PADK administration caused dramatic increases in cathepsin B protein and activity levels, whereas neprilysin, insulin-degrading enzyme, α-secretase, and β-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Aβ) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Aβ1–38 peptide corresponded with decreased levels of Aβ1–42, supporting the lysosome's antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomal system, identifying lysosomal modulation as an avenue for disease-modifying therapies. From the in vitro and in vivo findings, unique lysosomal modulators represent a minimally invasive, pharmacologically controlled strategy against protein accumulation disorders

  15. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

    PubMed

    Burkard, Christine; Verheije, Monique H; Wicht, Oliver; van Kasteren, Sander I; van Kuppeveld, Frank J; Haagmans, Bart L; Pelkmans, Lucas; Rottier, Peter J M; Bosch, Berend Jan; de Haan, Cornelis A M

    2014-11-01

    Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.

  16. Autophagic lysosome reformation dysfunction in glucocerebrosidase deficient cells: relevance to Parkinson disease

    PubMed Central

    Magalhaes, Joana; Gegg, Matthew E.; Migdalska-Richards, Anna; Doherty, Mary K.; Whitfield, Phillip D.; Schapira, Anthony H.V.

    2016-01-01

    Glucocerebrosidase (GBA1) gene mutations increase the risk of Parkinson disease (PD). While the cellular mechanisms associating GBA1 mutations and PD are unknown, loss of the glucocerebrosidase enzyme (GCase) activity, inhibition of autophagy and increased α-synuclein levels have been implicated. Here we show that autophagy lysosomal reformation (ALR) is compromised in cells lacking functional GCase. ALR is a cellular process controlled by mTOR which regenerates functional lysosomes from autolysosomes formed during macroautophagy. A decrease in phopho-S6K levels, a marker of mTOR activity, was observed in models of GCase deficiency, including primary mouse neurons and the PD patient derived fibroblasts with GBA1 mutations, suggesting that ALR is compromised. Importantly Rab7, a GTPase crucial for endosome-lysosome trafficking and ALR, accumulated in GCase deficient cells, supporting the notion that lysosomal recycling is impaired. Recombinant GCase treatment reversed ALR inhibition and lysosomal dysfunction. Moreover, ALR dysfunction was accompanied by impairment of macroautophagy and chaperone-mediated autophagy, increased levels of total and phosphorylated (S129) monomeric α-synuclein, evidence of amyloid oligomers and increased α-synuclein release. Concurrently, we found increased cholesterol and altered glucosylceramide homeostasis which could compromise ALR. We propose that GCase deficiency in PD inhibits lysosomal recycling. Consequently neurons are unable to maintain the pool of mature and functional lysosomes required for the autophagic clearance of α-synuclein, leading to the accumulation and spread of pathogenic α-synuclein species in the brain. Since GCase deficiency and lysosomal dysfunction occur with ageing and sporadic PD pathology, the decrease in lysosomal reformation may be a common feature in PD. PMID:27378698

  17. Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner

    PubMed Central

    Burkard, Christine; Verheije, Monique H.; Wicht, Oliver; van Kasteren, Sander I.; van Kuppeveld, Frank J.; Haagmans, Bart L.; Pelkmans, Lucas; Rottier, Peter J. M.; Bosch, Berend Jan; de Haan, Cornelis A. M.

    2014-01-01

    Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion. PMID:25375324

  18. Electron probe X-ray analysis on human hepatocellular lysosomes with copper deposits: copper binding to a thiol-protein in lysosomes

    SciTech Connect

    Hanaichi, T.; Kidokoro, R.; Hayashi, H.; Sakamoto, N.

    1984-11-01

    Livers of eight patients with chronic liver diseases were investigated by energy dispersive x-ray analysis. First, three kinds of preparations (osmium-Epon sections, glutaraldehyde-frozen sections, and unfixed-frozen sections) were compared for element detectability at a subcellular level. The glutaraldehyde-frozen sections were satisfactory as far as copper, sulfur, and phosphorus were concerned. Five patients (one patient with Wilson's disease, one chronic cholestasis, one chronic hepatitis, and two asymptomatic primary biliary cirrhosis) yielded x-ray images of copper and sulfur consistent with hepatocellular lysosomes. Second, the glutaraldehyde-frozen sections were utilized for a study of copper deposits in the patients' livers. There was a significant correlation between copper and sulfur contents in the lysosomes of all patients studied but no correlation in the remainder of the cytoplasm. Zinc was not detected in the lysosomes. Whatever the content of copper in the lysosomes, the ratio of delta copper to phosphorus (weight/weight) to delta sulfur to phosphorus was 0.60. These data indicate that most lysosomal copper binds to a thiol protein, probably metallothionein, in the liver.

  19. A Two-Photon Fluorescent Probe for Lysosomal Thiols in Live Cells and Tissues

    PubMed Central

    Fan, Jiangli; Han, Zhichao; Kang, Yao; Peng, Xiaojun

    2016-01-01

    Lysosome-specific fluorescent probes are exclusive to elucidate the functions of lysosomal thiols. Moreover, two-photon microscopy offers advantages of less phototoxicity, better three dimensional spatial localization, deeper penetration depth and lower self-absorption. However, such fluorescent probes for thiols are still rare. In this work, an efficient two-photon fluorophore 1,8-naphthalimide-based probe conjugating a 2,4-dinitrobenzenesulfonyl chloride and morpholine was designed and synthesized, which exhibited high selectivity and sensitivity towards lysosomal thiols by turn-on fluorescence method quantitatively and was successfully applied to the imaging of thiols in live cells and tissues by two-photon microscopy. PMID:26794434

  20. Self-eating in skeletal development: implications for lysosomal storage disorders.

    PubMed

    Settembre, Carmine; Arteaga-Solis, Emilio; Ballabio, Andrea; Karsenty, Gerard

    2009-02-01

    Macroautophagy (a.k.a. autophagy) is a cellular process aimed at the recycling of proteins and organelles that is achieved when autophagosomes fuse with lysosomes. Accordingly, lysosomal dysfunctions are often associated with impaired autophagy. We demonstrated that inactivation of the sulfatase modifying factor 1 gene (Sumf1), a gene mutated in multiple sulfatase deficiency (MSD), causes glycosaminoglycans (GAGs) to accumulate in lysosomes, which in turn disrupts autophagy. We utilized a murine model of MSD to study how impairment of this process affects chondrocyte viability and thus skeletal development.

  1. A quantitative model of traffic between plasma membrane and secondary lysosomes: evaluation of inflow, lateral diffusion, and degradation

    PubMed Central

    1988-01-01

    We present here a mathematical model that accounts for the various proportions of plasma membrane constituents occurring in the lysosomal membrane of rat fibroblasts (Draye, J.-P., J. Quintart, P. J. Courtoy, and P. Baudhuin. 1987. Eur. J. Biochem. 170: 395-403; Draye, J.-P., P. J. Courtoy, J. Quintart, and P. Baudhuin. 1987. Eur. J. Biochem. 170:405-411). It is based on contents of plasma membrane markers in purified lysosomal preparations, evaluations of their half-life in lysosomes and measurements of areas of lysosomal and plasma membranes by morphometry. In rat fibroblasts, structures labeled by a 2-h uptake of horseradish peroxidase followed by a 16-h chase (i.e., lysosomes) occupy 3% of the cellular volume and their total membrane area corresponds to 30% of the pericellular membrane area. Based on the latter values, the model predicts the rate of inflow and outflow of plasma membrane constituents into lysosomal membrane, provided their rate of degradation is known. Of the bulk of polypeptides iodinated at the cell surface, only 4% reach the lysosomes every hour, where the major part (integral of 83%) is degraded with a half-life in lysosomes of integral to 0.8 h. For specific plasma membrane constituents, this model can further account for differences in the association to the lysosomal membrane by variations in the rate either of lysosomal degradation, of inflow along the pathway from the pericellular membrane to the lysosomes, or of lateral diffusion. PMID:2848849

  2. Theranostic iridium(III) complexes as one- and two-photon phosphorescent trackers to monitor autophagic lysosomes.

    PubMed

    He, Liang; Tan, Cai-Ping; Ye, Rui-Rong; Zhao, Yi-Zhi; Liu, Ya-Hong; Zhao, Qiang; Ji, Liang-Nian; Mao, Zong-Wan

    2014-11-03

    During autophagy, the intracellular components are captured in autophagosomes and delivered to lysosomes for degradation and recycling. Changes in lysosomal trafficking and contents are key events in the regulation of autophagy, which has been implicated in many physiological and pathological processes. In this work, two iridium(III) complexes (LysoIr1 and LysoIr2) are developed as theranostic agents to monitor autophagic lysosomes. These complexes display lysosome-activated phosphorescence and can specifically label lysosomes with high photostability. Simultaneously, they can induce autophagy potently without initiating an apoptosis response. We demonstrate that LysoIr2 can effectively implement two functions, namely autophagy induction and lysosomal tracking, in the visualization of autophagosomal-lysosomal fusion. More importantly, they display strong two-photon excited fluorescence (TPEF), which is favorable for live cell imaging and in vivo applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Enantioselective effects of methamidophos on the coelomocytes lysosomal membrane stability of Eisenia fetida.

    PubMed

    Chen, Linhua; Lu, Xianting; Ma, Yun

    2012-12-01

    Many of organophosphorous insecticides are chiral compounds. In this study, the enantioselective effects of organophosphate insecticide methamidophos on the coelomocytes lysosomal membrane stability of earthworm Eisenia fetida were studied: (1) The enantiomers of methamidophos were absolutely separated by high-performance liquid chromatography with a commercial chiral column; (2) The neutral red retention assay was used to judge the lysosomal membrane stability. The results showed that with the concentration increasing, lysosomal membranes have been significantly destroyed by individual stereoisomers and racemate of methamidophos. The neutral red retention times were significantly descended from 76.88 to 29.78 min. Both (+)- and (-)-methamidophos showed more prone to destroy the integrity of the lysosomal membrane than the racemate. However, the different effect between stereoisomers is slight.

  4. Lysosomal acid lipase: at the crossroads of normal and atherogenic cholesterol metabolism

    PubMed Central

    Dubland, Joshua A.; Francis, Gordon A.

    2015-01-01

    Unregulated cellular uptake of apolipoprotein B-containing lipoproteins in the arterial intima leads to the formation of foam cells in atherosclerosis. Lysosomal acid lipase (LAL) plays a crucial role in both lipoprotein lipid catabolism and excess lipid accumulation as it is the primary enzyme that hydrolyzes cholesteryl esters derived from both low density lipoprotein (LDL) and modified forms of LDL. Evidence suggests that as atherosclerosis progresses, accumulation of excess free cholesterol in lysosomes leads to impairment of LAL activity, resulting in accumulation of cholesteryl esters in the lysosome as well as the cytosol in foam cells. Impaired metabolism and release of cholesterol from lysosomes can lead to downstream defects in ATP-binding cassette transporter A1 regulation, needed to offload excess cholesterol from plaque foam cells. This review focuses on the role LAL plays in normal cholesterol metabolism and how the associated changes in its enzymatic activity may ultimately contribute to atherosclerosis progression. PMID:25699256

  5. Increased endocytotic and lysosomal activities in denervated type I and type II muscle fibres.

    PubMed

    Lawoko, G; Tågerud, S; Libelius, R

    1992-01-01

    Previous work has shown that increased endocytotic and lysosomal activities occur in the endplate region of denervated skeletal muscle fibres. This, however, does not engage all fibres of a muscle at a given time after denervation. The present study was carried out in order to determine if both type I (slow) and type II (fast) muscle fibres can react to denervation by increased endocytotic and lysosomal activities. Uptake of horseradish peroxidase as a marker for endocytosis was studied in conjunction with acid phosphatase staining for lysosomal activity in type I and type II fibres of the denervated mouse hemidiaphragm. Fibre typing was performed using a monoclonal antibody against fast skeletal myosin and by adenosine triphosphatase staining. The results show that increased endocytosis and lysosomal activation occur in both type I and type II fibres after denervation.

  6. Lysosome biogenesis mediated by vps-18 affects apoptotic cell degradation in Caenorhabditis elegans.

    PubMed

    Xiao, Hui; Chen, Didi; Fang, Zhou; Xu, Jing; Sun, Xiaojuan; Song, Song; Liu, Jiajia; Yang, Chonglin

    2009-01-01

    Appropriate clearance of apoptotic cells (cell corpses) is an important step of programmed cell death. Although genetic and biochemical studies have identified several genes that regulate the engulfment of cell corpses, how these are degraded after being internalized in engulfing cell remains elusive. Here, we show that VPS-18, the Caenorhabditis elegans homologue of yeast Vps18p, is critical to cell corpse degradation. VPS-18 is expressed and functions in engulfing cells. Deletion of vps-18 leads to significant accumulation of cell corpses that are not degraded properly. Furthermore, vps-18 mutation causes strong defects in the biogenesis of endosomes and lysosomes, thus affecting endosomal/lysosomal protein degradation. Importantly, we demonstrate that phagosomes containing internalized cell corpses are unable to fuse with lysosomes in vps-18 mutants. Our findings thus provide direct evidence for the important role of endosomal/lysosomal degradation in proper clearance of apoptotic cells during programmed cell death.

  7. Emerging therapies for neurodegenerative lysosomal storage disorders - from concept to reality.

    PubMed

    Hemsley, Kim M; Hopwood, John J

    2011-10-01

    Lysosomal storage disorders are inherited metabolic diseases in which a mutation in a gene encoding a lysosomal enzyme or lysosome-related protein results in the intra-cellular accumulation of substrate and reduced cell/tissue function. Few patients with neurodegenerative lysosomal storage disorders have access to safe and effective treatments although many therapeutic strategies have been or are presently being studied in vivo thanks to the availability of a large number of animal models. This review will describe the comparative advancement of a variety of therapeutic strategies through the 'research pipeline'. Our goal is to provide information for clinicians, researchers and patients/families alike on the leading therapeutic candidates at this point in time, and also to provide information on emerging approaches that may provide a safe and effective treatment in the future. The length of the pipeline represents the significant and sustained effort required to move a novel concept from the laboratory into the clinic.

  8. Autophagy Regulates Cholesterol Efflux from Macrophage Foam Cells via Lysosomal Acid Lipase

    PubMed Central

    Ouimet, Mireille; Franklin, Vivian; Mak, Esther; Liao, Xianghai; Tabas, Ira; Marcel, Yves L.

    2012-01-01

    SUMMARY The lipid droplet (LD) is the major site of cholesterol storage in macrophage foam cells and is a potential therapeutic target for the treatment of atherosclerosis. Cholesterol, stored as cholesteryl esters (CEs), is liberated from this organelle and delivered to cholesterol acceptors. The current paradigm attributes all cytoplasmic CE hydrolysis to the action of neutral CE hydrolases. Here, we demonstrate an important role for lysosomes in LD CE hydrolysis in cholesterol-loaded macrophages, in addition to that mediated by neutral hydrolases. Furthermore, we demonstrate that LDs are delivered to lysosomes via autophagy, where lysosomal acid lipase (LAL) acts to hydrolyze LD CE to generate free cholesterol mainly for ABCA1-dependent efflux; this process is specifically induced upon macrophage cholesterol loading. We conclude that, in macrophage foam cells, lysosomal hydrolysis contributes to the mobilization of LD-associated cholesterol for reverse cholesterol transport. PMID:21641547

  9. Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space.

    PubMed

    Coppens, Isabelle; Dunn, Joe Dan; Romano, Julia D; Pypaert, Marc; Zhang, Hui; Boothroyd, John C; Joiner, Keith A

    2006-04-21

    The intracellular compartment harboring Toxoplasma gondii satisfies the parasite's nutritional needs for rapid growth in mammalian cells. We demonstrate that the parasitophorous vacuole (PV) of T. gondii accumulates material coming from the host mammalian cell via the exploitation of the host endo-lysosomal system. The parasite actively recruits host microtubules, resulting in selective attraction of endo-lysosomes to the PV. Microtubule-based invaginations of the PV membrane serve as conduits for the delivery of host endo-lysosomes within the PV. These tubular conduits are decorated by a parasite coat, including the tubulogenic protein GRA7, which acts like a garrote that sequesters host endocytic organelles in the vacuolar space. These data define an unanticipated process allowing the parasite intimate and concentrated access to a diverse range of low molecular weight components produced by the endo-lysosomal system. More generally, they identify a unique mechanism for unidirectional transport and sequestration of host organelles.

  10. Analysis of lysosomal membrane proteins exposed to melanin in HeLa cells

    PubMed Central

    2016-01-01

    Objectives There have been developed to use targeting ability for antimicrobial, anticancerous, gene therapy and cosmetics through analysis of various membrane proteins isolated from cell organelles. Methods It was examined about the lysosomal membrane protein extracted from lysosome isolated from HeLa cell treated by 100 ppm melanin for 24 hours in order to find associated with targeting ability to melanin using by 2-dimensional electrophoresis. Results The result showed 14 up-regulated (1.5-fold) and 13 down-regulated (2.0-fold) spots in relation to melanin exposure. Conclusions It has been found that lysosomal membrane proteins are associated with melanin to decolorize and quantity through cellular activation of lysosome. PMID:27158002

  11. Non-neuronopathic lysosomal storage disorders: Disease spectrum and treatments.

    PubMed

    Pastores, Gregory M; Hughes, Derralynn A

    2015-03-01

    Distinctive facial features, hepatosplenomegaly or cardiomyopathy with or without associated skeletal dysplasia are clinical manifestations that may be suggestive of an underlying lysosomal storage disorder (LSD), However, these features may not be evident in certain subtypes associated primarily with central nervous system involvement. Age at onset can be broad, ranging from infancy to adulthood. Diagnosis may be delayed, as manifestations may be slow to evolve (taking months to years), particularly in those with later (adult-)onset, and in isolated cases (i.e., those without a prior family history). Diagnosis of individual subtypes can be confirmed using a combination of biochemical and molecular assays. In a few LSDs, treatment with hematopoietic stem cell transplantation, enzyme replacement or substrate reduction therapy is available. Symptomatic and palliative measure may enhance quality of life for both treatable and currently untreatable cases. Genetic counseling is important, so patients and their families can be informed of reproductive risks, disease prognosis and therapeutic options. Investigations of underlying disease mechanisms are enhancing knowledge about rare diseases, but also other more common medical conditions, on account of potential convergent disease pathways.

  12. IRE1 directs proteasomal and lysosomal degradation of misfolded rhodopsin

    PubMed Central

    Chiang, Wei-Chieh; Messah, Carissa; Lin, Jonathan H.

    2012-01-01

    Endoplasmic reticulum (ER) is responsible for folding of secreted and membrane proteins in eukaryotic cells. Disruption of ER protein folding leads to ER stress. Chronic ER stress can cause cell death and is proposed to underlie the pathogenesis of many human diseases. Inositol-requiring enzyme 1 (IRE1) directs a key unfolded protein response signaling pathway that controls the fidelity of ER protein folding. IRE1 signaling may be particularly helpful in preventing chronic ER stress and cell injury by alleviating protein misfolding in the ER. To examine this, we used a chemical-genetic approach to selectively activate IRE1 in mammalian cells and tested how artificial IRE1 signaling affected the fate of misfolded P23H rhodopsin linked to photoreceptor cell death. We found that IRE1 signaling robustly promoted the degradation of misfolded P23H rhodopsin without affecting its wild-type counterpart. We also found that IRE1 used both proteasomal and lysosomal degradation pathways to remove P23H rhodopsin. Surprisingly, when one degradation pathway was compromised, IRE1 signaling could still promote misfolded rhodopsin degradation using the remaining pathway. Last, we showed that IRE1 signaling also reduced levels of several other misfolded rhodopsins with lesser effects on misfolded cystic fibrosis transmembrane conductance regulator. Our findings reveal the diversity of proteolytic mechanisms used by IRE1 to eliminate misfolded rhodopsin. PMID:22219383

  13. Pulmonary and generalized lysosomal storage induced by amphiphilic drugs.

    PubMed Central

    Hruban, Z

    1984-01-01

    Administration of amphiphilic drugs to experimental animals causes formation of myelinoid bodies in many cell types, accumulation of foamy macrophages in pulmonary alveoli and pulmonary alveolar proteinosis. These changes are the result of an interaction between the drugs and phospholipids which leads to an alteration in physicochemical properties of the phospholipids. Impairment of the digestion of altered pulmonary secretions in phagosomes of macrophages results in accumulation of foam cells in pulmonary alveoli. Impairment of the metabolism of altered phospholipids removed by autophagy induces an accumulation of myelinoid bodies. The administration of amphiphilic compounds thus causes pulmonary intra-alveolar histiocytosis which is a part of a drug-induced lysosomal storage or generalized lipidosis. The accumulation of drug-lipid complexes in myelinoid bodies and in pulmonary foam cells may lead to alteration of cellular functioning and to clinical disease. Currently over 50 amphiphilic drugs are known. Unique pharmacological properties necessitate clinical use of some of these drugs. The occurrence and severity of potential clinical side effects depend on the nature of each drug, dosage and duration of treatment, simultaneous administration of other drugs and foods, individual metabolic pattern of the patient and other factors. Further studies on factors preventing and potentiating adverse effects of amphiphilic drugs are indicated. Images FIGURE 1. FIGURE 2. FIGURE 3. FIGURE 4. FIGURE 5. FIGURE 6. FIGURE 7. FIGURE 8. FIGURE 9. FIGURE 10. PMID:6376111

  14. Oxidized phagosomal NOX2 is replenished from lysosomes.

    PubMed

    Dingjan, Ilse; Linders, Peter T A; van den Bekerom, Luuk; Baranov, Maksim V; Halder, Partho; Ter Beest, Martin; van den Bogaart, Geert

    2017-02-15

    In dendritic cells, the NADPH oxidase 2 (NOX2) is recruited to the phagosomal membrane during antigen uptake. NOX2 produces reactive oxygen species (ROS) in the lumen of the phagosome which kill ingested pathogens, delay antigen breakdown and alter the peptide repertoire for presentation to T cells. How the integral membrane component of NOX2, cytochrome b558, traffics to phagosomes is incompletely understood. In this study, we show in dendritic cells derived from human blood-isolated monocytes that cytochrome b558 is initially recruited to the phagosome from the plasma membrane during phagosome formation. Cytochrome b558 also traffics from a lysosomal pool to phagosomes and this is required to replenish oxidatively damaged NOX2. We identified syntaxin-7, SNAP23 and VAMP8 as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediating this process. Our data describe a key mechanism of how dendritic cells sustain ROS production after antigen uptake required to initiate T cell responses.

  15. Magnesium Modulates Doxorubicin Activity through Drug Lysosomal Sequestration and Trafficking.

    PubMed

    Trapani, Valentina; Luongo, Francesca; Arduini, Daniela; Wolf, Federica I

    2016-03-21

    Magnesium is directly involved in the control of cell growth and survival, but its role in cancer biology and therapy is multifaceted; in particular, it is highly controversial whether magnesium levels can affect therapy outcomes. Here we investigated whether magnesium availability can modulate cellular responses to the widely used chemotherapeutic doxorubicin. We used an in vitro model consisting of mammary epithelial HC11 cells and found that high magnesium availability was correlated with diminished sensitivity both in cells chronically adapted to high magnesium concentrations and in acutely magnesium-supplemented cells. This decrease in sensitivity resulted from reduced intracellular doxorubicin accumulation in the face of a similar drug uptake rate. We observed that high-magnesium conditions caused a decrease in intracellular drug retention by altering drug lysosomal sequestration and trafficking. In our model, magnesium supplementation correspondingly modulated expression of the TRPM7 channel, which is known to control cytoskeletal organization and dynamics and may be involved in the proposed mechanism. Our findings suggest that magnesium supplementation in hypomagnesemic cancer patients may hinder response to therapy.

  16. The Lysosomal Protein Saposin B Binds Chloroquine.

    PubMed

    Huta, Brian P; Mehlenbacher, Matthew R; Nie, Yan; Lai, Xuelei; Zubieta, Chloe; Bou-Abdallah, Fadi; Doyle, Robert P

    2016-02-04

    Chloroquine (CQ) has been widely used in the treatment of malaria since the 1950s, though toxicity and resistance is increasingly limiting its use in the clinic. More recently, CQ is also becoming recognized as an important therapeutic compound for the treatment of autoimmune disorders and has shown activity as an anticancer agent. However, the full extent of CQ pharmacology in humans is still unclear. Herein, we demonstrate that the lysosomal protein saposin B (sapB), critical for select lipid degradation, binds CQ with implications for both CQ function and toxicity. Using isothermal titration calorimetry (ITC) and fluorescence quenching experiments, CQ was shown to bind to the dimeric form of sapB at both pH 5.5 and pH 7.4 with an average binding affinity of 2.3×10(4)  m(-1). X-ray crystallography confirmed this, and the first complete crystal structure of sapB with a bound small molecule (CQ) is reported. The results suggest that sapB might play a role in mitigating CQ-based toxicity and that sapB might itself be overwhelmed by CQ causing impaired lipid degradation.

  17. Lysosomal trafficking of TGFBIp via caveolae-mediated endocytosis.

    PubMed

    Choi, Seung-Il; Maeng, Yong-Sun; Kim, Tae-Im; Lee, Yangsin; Kim, Yong-Sun; Kim, Eung Kweon

    2015-01-01

    Transforming growth factor-beta-induced protein (TGFBIp) is ubiquitously expressed in the extracellular matrix (ECM) of various tissues and cell lines. Progressive accumulation of mutant TGFBIp is directly involved in the pathogenesis of TGFBI-linked corneal dystrophy. Recent studies reported that mutant TGFBIp accumulates in cells; however, the trafficking of TGFBIp is poorly understood. Therefore, we investigated TGFBIp trafficking to determine the route of its internalization and secretion and to elucidate its roles in the pathogenesis of granular corneal dystrophy type 2 (GCD2). Our data indicate that newly synthesized TGFBIp was secreted via the endoplasmic reticulum/Golgi-dependent secretory pathway, and this secretion was delayed in the corneal fibroblasts of patients with GCD2. We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation. In addition, the proteasome inhibitor MG132 inhibits the endocytosis of TGFBIp. Co-immunoprecipitation revealed that TGFBIp interacted with integrin αVβ3. Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp. These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy.

  18. Less Is More: Substrate Reduction Therapy for Lysosomal Storage Disorders

    PubMed Central

    Coutinho, Maria Francisca; Santos, Juliana Inês; Alves, Sandra

    2016-01-01

    Lysosomal storage diseases (LSDs) are a group of rare, life-threatening genetic disorders, usually caused by a dysfunction in one of the many enzymes responsible for intralysosomal digestion. Even though no cure is available for any LSD, a few treatment strategies do exist. Traditionally, efforts have been mainly targeting the functional loss of the enzyme, by injection of a recombinant formulation, in a process called enzyme replacement therapy (ERT), with no impact on neuropathology. This ineffectiveness, together with its high cost and lifelong dependence is amongst the main reasons why additional therapeutic approaches are being (and have to be) investigated: chaperone therapy; gene enhancement; gene therapy; and, alternatively, substrate reduction therapy (SRT), whose aim is to prevent storage not by correcting the original enzymatic defect but, instead, by decreasing the levels of biosynthesis of the accumulating substrate(s). Here we review the concept of substrate reduction, highlighting the major breakthroughs in the field and discussing the future of SRT, not only as a monotherapy but also, especially, as complementary approach for LSDs. PMID:27384562

  19. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

    PubMed

    Kook, Seunghyi; Wang, Ping; Young, Lisa R; Schwake, Michael; Saftig, Paul; Weng, Xialian; Meng, Ying; Neculai, Dante; Marks, Michael S; Gonzales, Linda; Beers, Michael F; Guttentag, Susan

    2016-04-15

    The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients.

  20. Neuronal-Targeted TFEB Accelerates Lysosomal Degradation of APP, Reducing Aβ Generation and Amyloid Plaque Pathogenesis.

    PubMed

    Xiao, Qingli; Yan, Ping; Ma, Xiucui; Liu, Haiyan; Perez, Ronaldo; Zhu, Alec; Gonzales, Ernesto; Tripoli, Danielle L; Czerniewski, Leah; Ballabio, Andrea; Cirrito, John R; Diwan, Abhinav; Lee, Jin-Moo

    2015-09-02

    In AD, an imbalance between Aβ production and removal drives elevated brain Aβ levels and eventual amyloid plaque deposition. APP undergoes nonamyloidogenic processing via α-cleavage at the plasma membrane, amyloidogenic β- and γ-cleavage within endosomes to generate Aβ, or lysosomal degradation in neurons. Considering multiple reports implicating impaired lysosome function as a driver of increased amyloidogenic processing of APP, we explored the efficacy of targeting transcription factor EB (TFEB), a master regulator of lysosomal pathways, to reduce Aβ levels. CMV promoter-driven TFEB, transduced via stereotactic hippocampal injections of adeno-associated virus particles in APP/PS1 mice, localized primarily to neuronal nuclei and upregulated lysosome biogenesis. This resulted in reduction of APP protein, the α and β C-terminal APP fragments (CTFs), and in the steady-state Aβ levels in the brain interstitial fluid. In aged mice, total Aβ levels and amyloid plaque load were selectively reduced in the TFEB-transduced hippocampi. TFEB transfection in N2a cells stably expressing APP695, stimulated lysosome biogenesis, reduced steady-state levels of APP and α- and β-CTFs, and attenuated Aβ generation by accelerating flux through the endosome-lysosome pathway. Cycloheximide chase assays revealed a shortening of APP half-life with exogenous TFEB expression, which was prevented by concomitant inhibition of lysosomal acidification. These data indicate that TFEB enhances flux through lysosomal degradative pathways to induce APP degradation and reduce Aβ generation. Activation of TFEB in neurons is an effective strategy to attenuate Aβ generation and attenuate amyloid plaque deposition in AD. A key driver for AD pathogenesis is the net balance between production and clearance of Aβ, the major component of amyloid plaques. Here we demonstrate that lysosomal degradation of holo-APP influences Aβ production by limiting the availability of APP for amyloidogenic

  1. Neuronal-Targeted TFEB Accelerates Lysosomal Degradation of APP, Reducing Aβ Generation and Amyloid Plaque Pathogenesis

    PubMed Central

    Xiao, Qingli; Yan, Ping; Ma, Xiucui; Liu, Haiyan; Perez, Ronaldo; Zhu, Alec; Gonzales, Ernesto; Tripoli, Danielle L.; Czerniewski, Leah; Ballabio, Andrea; Cirrito, John R.

    2015-01-01

    In AD, an imbalance between Aβ production and removal drives elevated brain Aβ levels and eventual amyloid plaque deposition. APP undergoes nonamyloidogenic processing via α-cleavage at the plasma membrane, amyloidogenic β- and γ-cleavage within endosomes to generate Aβ, or lysosomal degradation in neurons. Considering multiple reports implicating impaired lysosome function as a driver of increased amyloidogenic processing of APP, we explored the efficacy of targeting transcription factor EB (TFEB), a master regulator of lysosomal pathways, to reduce Aβ levels. CMV promoter-driven TFEB, transduced via stereotactic hippocampal injections of adeno-associated virus particles in APP/PS1 mice, localized primarily to neuronal nuclei and upregulated lysosome biogenesis. This resulted in reduction of APP protein, the α and β C-terminal APP fragments (CTFs), and in the steady-state Aβ levels in the brain interstitial fluid. In aged mice, total Aβ levels and amyloid plaque load were selectively reduced in the TFEB-transduced hippocampi. TFEB transfection in N2a cells stably expressing APP695, stimulated lysosome biogenesis, reduced steady-state levels of APP and α- and β-CTFs, and attenuated Aβ generation by accelerating flux through the endosome-lysosome pathway. Cycloheximide chase assays revealed a shortening of APP half-life with exogenous TFEB expression, which was prevented by concomitant inhibition of lysosomal acidification. These data indicate that TFEB enhances flux through lysosomal degradative pathways to induce APP degradation and reduce Aβ generation. Activation of TFEB in neurons is an effective strategy to attenuate Aβ generation and attenuate amyloid plaque deposition in AD. SIGNIFICANCE STATEMENT A key driver for AD pathogenesis is the net balance between production and clearance of Aβ, the major component of amyloid plaques. Here we demonstrate that lysosomal degradation of holo-APP influences Aβ production by limiting the availability of

  2. Lysosomal responses to heat-shock of seasonal temperature extremes in Cd-exposed mussels.

    PubMed

    Múgica, M; Izagirre, U; Marigómez, I

    2015-07-01

    The present study was aimed at determining the effect of temperature extremes on lysosomal biomarkers in mussels exposed to a model toxic pollutant (Cd) at different seasons. For this purpose, temperature was elevated 10°C (from 12°C to 22°C in winter and from 18°C to 28°C in summer) for a period of 6h (heat-shock) in control and Cd-exposed mussels, and then returned back to initial one. Lysosomal membrane stability and lysosomal structural changes in digestive gland were investigated. In winter, heat-shock reduced the labilisation period (LP) of the lysosomal membrane, especially in Cd-exposed mussels, and provoked transient lysosomal enlargement. LP values recovered after the heat-shock cessation but lysosomal enlargement prevailed in both experimental groups. In summer, heat-shock induced remarkable reduction in LP and lysosomal enlargement (more markedly in Cd-exposed mussels), which recovered within 3 days. Besides, whilst heat-shock effects on LP were practically identical for Cd-exposed mussels in winter and summer, the effects were longer-lasting in summer than in winter for control mussels. Thus, lysosomal responsiveness after heat-shock was higher in summer than in winter but recovery was faster as well, and therefore the consequences of the heat shock seem to be more decisive in winter. In contrast, inter-season differences were attenuated in the presence of Cd. Consequently, mussels seem to be better prepared in summer than in winter to stand short periods of abrupt temperature change; this is, however, compromised when mussels are exposed to pollutants such as Cd.

  3. Role of Lysosomal Enzyme Release in Circulatory Shock and Critical Illness.

    DTIC Science & Technology

    1978-06-01

    association of lysosomal enzyme release and the severity of shock in the human system in states of circulatory shocl4 and conditions of critical...antereo) 6-iI SUMMARY To determine the association of lysosomal enzyme release and the severity of shock in the human system in states of circulatory...The results, however, do not show any adverse effects from the introduction of exogenous cathepsin D into the systemic circulation during hemorrhagic

  4. Observation of intracellular interactions between DNA origami and lysosomes by the fluorescence localization method.

    PubMed

    Fu, Meifang; Dai, Luru; Jiang, Qiao; Tang, Yunqing; Zhang, Xiaoming; Ding, Baoquan; Li, Junbai

    2016-07-28

    We obtained the fluorescence localization images of tube DNA origami nanostructures in NIH 3T3 cells for the first time. The fluorescence localization images of tube DNA origami nanostructures and TIRF images of lysosomes were combined and they revealed the detailed interactions between the two structures. Quantitative analysis illustrated that the tube origami can be captured as well as degraded by lysosomes with time.

  5. The BH3 Mimetic Obatoclax Accumulates in Lysosomes and Causes Their Alkalinization.

    PubMed

    Stamelos, Vasileios A; Fisher, Natalie; Bamrah, Harnoor; Voisey, Carolyn; Price, Joshua C; Farrell, William E; Redman, Charles W; Richardson, Alan

    2016-01-01

    Obatoclax belongs to a class of compounds known as BH3 mimetics which function as antagonists of Bcl-2 family apoptosis regulators. It has undergone extensive preclinical and clinical evaluation as a cancer therapeutic. Despite this, it is clear that obatoclax has additional pharmacological effects that contribute to its cytotoxic activity. It has been claimed that obatoclax, either alone or in combination with other molecularly targeted therapeutics, induces an autophagic form of cell death. In addition, obatoclax has been shown to inhibit lysosomal function, but the mechanism of this has not been elucidated. We have evaluated the mechanism of action of obatoclax in eight ovarian cancer cell lines. Consistent with its function as a BH3 mimetic, obatoclax induced apoptosis in three cell lines. However, in the remaining cell lines another form of cell death was evident because caspase activation and PARP cleavage were not observed. Obatoclax also failed to show synergy with carboplatin and paclitaxel, chemotherapeutic agents which we have previously shown to be synergistic with authentic Bcl-2 family antagonists. Obatoclax induced a profound accumulation of LC-3 but knockdown of Atg-5 or beclin had only minor effects on the activity of obatoclax in cell growth assays suggesting that the inhibition of lysosomal function rather than stimulation of autophagy may play a more prominent role in these cells. To evaluate how obatoclax inhibits lysosomal function, confocal microscopy studies were conducted which demonstrated that obatoclax, which contains two basic pyrrole groups, accumulates in lysosomes. Studies using pH sensitive dyes demonstrated that obatoclax induced lysosomal alkalinization. Furthermore, obatoclax was synergistic in cell growth/survival assays with bafilomycin and chloroquine, two other drugs which cause lysosomal alkalinization. These studies explain, for the first time, how obatoclax inhibits lysosomal function and suggest that lysosomal

  6. Lysosomal exocytosis in response to subtle membrane damage following nanosecond pulse exposure

    NASA Astrophysics Data System (ADS)

    Dalzell, Danielle R.; Roth, Caleb C.; Bernhard, Joshua A.; Payne, Jason A.; Wilmink, Gerald J.; Ibey, Bennett L.

    2011-03-01

    The cellular response to subtle membrane damage following exposure to nanosecond electric pulses (nsEP) is not well understood. Recent work has shown that when cells are exposed to nsEP, ion permeable nanopores (< 2nm) are created in the plasma membrane in contrast to larger diameter pores (> 2nm) created by longer micro and millisecond duration pulses. Macroscopic damage to a plasma membrane by a micropipette has been shown to cause internal vesicles (lysosomes) to undergo exocytosis to repair membrane damage, a calcium mediated process called lysosomal exocytosis. Formation of large pores in the plasma membrane by electrical pulses has been shown to elicit lysosomal exocytosis in a variety of cell types. Our research objective is to determine whether lysosomal exocytosis will occur in response to nanopores formed by exposure to nsEP. In this paper we used propidium iodide (PI) and Calcium Green-1 AM ester (CaGr) to differentiate between large and small pores formed in CHO-K1 cells following exposure to either 1 or 20, 600-ns duration electrical pulses at 16.2 kV/cm. This information was compared to changes in membrane organization observed by increases in FM1-43 fluorescence, both in the presence and absence of calcium ions in the outside buffer. In addition, we monitored the real time migration of lysosomes within the cell using Cellular Lights assay to tag LAMP-1, a lysosomal membrane protein. Both 1 and 20 pulses elicited a large influx of extracellular calcium, while little PI uptake was observed following a single pulse exposure. Statistically significant increases in FM1-43 fluorescence were seen in samples containing calcium suggesting that calcium-triggered membrane repair may be occurring. Lastly, density of lysosomes within cells, specifically around the nucleus, appeared to change rapidly upon nsEP stimulation suggesting lysosomal migration.

  7. Streptococcus oralis Induces Lysosomal Impairment of Macrophages via Bacterial Hydrogen Peroxide

    PubMed Central

    Okahashi, Nobuo; Kuwata, Hirotaka; Kawabata, Shigetada

    2016-01-01

    Streptococcus oralis, an oral commensal, belongs to the mitis group of streptococci and occasionally causes opportunistic infections, such as bacterial endocarditis and bacteremia. Recently, we found that the hydrogen peroxide (H2O2) produced by S. oralis is sufficient to kill human monocytes and epithelial cells, implying that streptococcal H2O2 is a cytotoxin. In the present study, we investigated whether streptococcal H2O2 impacts lysosomes, organelles of the intracellular digestive system, in relation to cell death. S. oralis infection induced the death of RAW 264 macrophages in an H2O2-dependent manner, which was exemplified by the fact that exogenous H2O2 also induced cell death. Infection with either a mutant lacking spxB, which encodes pyruvate oxidase responsible for H2O2 production, or Streptococcus mutans, which does not produce H2O2, showed less cytotoxicity. Visualization of lysosomes with LysoTracker revealed lysosome deacidification after infection with S. oralis or exposure to H2O2, which was corroborated by acridine orange staining. Similarly, fluorescent labeling of lysosome-associated membrane protein-1 gradually disappeared during infection with S. oralis or exposure to H2O2. The deacidification and the following induction of cell death were inhibited by chelating iron in lysosomes. Moreover, fluorescent staining of cathepsin B indicated lysosomal destruction. However, treatment of infected cells with a specific inhibitor of cathepsin B had negligible effects on cell death; instead, it suppressed the detachment of dead cells from the culture plates. These results suggest that streptococcal H2O2 induces cell death with lysosomal destruction and then the released lysosomal cathepsins contribute to the detachment of the dead cells. PMID:27113357

  8. [Experimental models of lysosomal phase reactivity in blood leukocytes exposed to low doses of potassium cyanide].

    PubMed

    Dolgushin, M V; Khomuev, G D

    2013-01-01

    Cytochemical analysis of acid phosphatase was used to evaluate lysosomal membranes stability under oral intake of potassium cyanide by rats over one month in daily doses of 1.30 mg/kg (1/10 LD50) and 0.65 mg/kg (1/20 LD50). The authors demonstrated phase-related dose-dependent changes in the lysosomal state, and the main response feature was associated with functional activation that usually followed the membrane alteration.

  9. Eps8 is recruited to lysosomes and subjected to chaperone-mediated autophagy in cancer cells.

    PubMed

    Welsch, Thilo; Younsi, Alexander; Disanza, Andrea; Rodriguez, Jose Antonio; Cuervo, Ana Maria; Scita, Giorgio; Schmidt, Jan

    2010-07-15

    Eps8 controls actin dynamics directly through its barbed end capping and actin-bundling activity, and indirectly by regulating Rac-activation when engaged into a trimeric complex with Eps8-Abi1-Sos1. Recently, Eps8 has been associated with promotion of various solid malignancies, but neither its mechanisms of action nor its regulation in cancer cells have been elucidated. Here, we report a novel association of Eps8 with the late endosomal/lysosomal compartment, which is independent from actin polymerization and specifically occurs in cancer cells. Endogenous Eps8 localized to large vesicular lysosomal structures in metastatic pancreatic cancer cell lines, such as AsPC-1 and Capan-1 that display high Eps8 levels. Additionally, ectopic expression of Eps8 increased the size of lysosomes. Structure-function analysis revealed that the region encompassing the amino acids 184-535 of Eps8 was sufficient to mediate lysosomal recruitment. Notably, this fragment harbors two KFERQ-like motifs required for chaperone-mediated autophagy (CMA). Furthermore, Eps8 co-immunoprecipitated with Hsc70 and LAMP-2, which are key elements for the CMA degradative pathway. Consistently, in vitro, a significant fraction of Eps8 bound to (11.9+/-5.1%) and was incorporated into (5.3+/-6.5%) lysosomes. Additionally, Eps8 binding to lysosomes was competed by other known CMA-substrates. Fluorescence recovery after photobleaching revealed that Eps8 recruitment to the lysosomal membrane was highly dynamic. Collectively, these results indicate that Eps8 in certain human cancer cells specifically localizes to lysosomes, and is directed to CMA. These results open a new field for the investigation of how Eps8 is regulated and contributes to tumor promotion in human cancers.

  10. Eps8 is recruited to lysosomes and subjected to chaperone-mediated autophagy in cancer cells

    PubMed Central

    Welsch, Thilo; Younsi, Alexander; Disanza, Andrea; Rodriguez, Jose Antonio; Cuervo, Ana Maria; Scita, Giorgio; Schmidt, Jan

    2015-01-01

    Eps8 controls actin dynamics directly through its barbed end capping and actin-bundling activity, and indirectly by regulating Rac-activation when engaged into a trimeric complex with Eps8- Abi1-Sos1. Recently, Eps8 has been associated with promotion of various solid malignancies, but neither its mechanisms of action nor its regulation in cancer cells have been elucidated. Here, we report a novel association of Eps8 with the late endosomal/lysosomal compartment, which is independent from actin polymerization and specifically occurs in cancer cells. Endogenous Eps8 localized to large vesicular lysosomal structures in metastatic pancreatic cancer cell lines, such as AsPC-1 and Capan-1 that display high Eps8 levels. Additionally, ectopic expression of Eps8 increased the size of lysosomes. Structure–function analysis revealed that the region encompassing the amino acids 184–535 of Eps8 was sufficient to mediate lysosomal recruitment. Notably, this fragment harbors two KFERQ-like motifs required for chaperone-mediated autophagy (CMA). Furthermore, Eps8 co-immunoprecipitated with Hsc70 and LAMP-2, which are key elements for the CMA degradative pathway. Consistently, in vitro, a significant fraction of Eps8 bound to (11.9± 5.1%) and was incorporated into (5.3± 6.5%) lysosomes. Additionally, Eps8 binding to lysosomes was competed by other known CMA-substrates. Fluorescence recovery after photobleaching revealed that Eps8 recruitment to the lysosomal membrane was highly dynamic. Collectively, these results indicate that Eps8 in certain human cancer cells specifically localizes to lysosomes, and is directed to CMA. These results open a new field for the investigation of how Eps8 is regulated and contributes to tumor promotion in human cancers PMID:20184880

  11. Impulse control disorder, lysosomal malfunction and ATP13A2 insufficiency in Parkinsonism.

    PubMed

    Liu, Jun-Ping; Li, Jianfeng; Lu, Yanhua; Wang, Lihui; Chen, Gang

    2017-02-01

    Lysosomal transport of cargos in neurons is essential for neuronal proteostasis, transmission and functional motors and behaviours. Lysosomal malfunction including storage disorders is involved in the pathogenesis of Parkinson's disease (PD). Given the unclear molecular mechanisms of diverse defects in PD phenotypes, especially behavioural deficits, this mini review explores the cellular contexts of PD impulse control disorders and the molecular aspects of lysosomal cross-membrane transports. Focuses are paid to trace metal involvements in α-synuclein assembly in Lewy bodies, the functions and molecular interactions of ATP13A2 as ATPase transporters in lysosomal membranes for cross-membrane trafficking and lysosomal homeostasis, and our current understandings of the neural circuits in ICD. Erroneously polarized distributions of cargos such as metals and lipids on each side of lysosomal membranes triggered by gene mutations and deregulated expression of ATP13A2 may thus instigate sensing protein structural changes such as aggregations, organelle degeneration, and specific neuronal ageing and death in Parkinsonism. © 2016 John Wiley & Sons Australia, Ltd.

  12. The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells.

    PubMed

    Padman, Benjamin S; Bach, Markus; Lucarelli, Giuseppe; Prescott, Mark; Ramm, Georg

    2013-11-01

    Mitophagy is a selective pathway, which targets and delivers mitochondria to the lysosomes for degradation. Depolarization of mitochondria by the protonophore CCCP is a strategy increasingly used to experimentally trigger not only mitophagy, but also bulk autophagy. Using live-cell fluorescence microscopy we found that treatment of HeLa cells with CCCP caused redistribution of mitochondrially targeted dyes, including DiOC6, TMRM, MTR, and MTG, from mitochondria to the cytosol, and subsequently to lysosomal compartments. Localization of mitochondrial dyes to lysosomal compartments was caused by retargeting of the dye, rather than delivery of mitochondrial components to the lysosome. We showed that CCCP interfered with lysosomal function and autophagosomal degradation in both yeast and mammalian cells, inhibited starvation-induced mitophagy in mammalian cells, and blocked the induction of mitophagy in yeast cells. PARK2/Parkin-expressing mammalian cells treated with CCCP have been reported to undergo high levels of mitophagy and clearance of all mitochondria during extensive treatment with CCCP. Using correlative light and electron microscopy in PARK2-expressing HeLa cells, we showed that mitochondrial remnants remained present in the cell after 24 h of CCCP treatment, although they were no longer easily identifiable as such due to morphological alterations. Our results showed that CCCP inhibits autophagy at both the initiation and lysosomal degradation stages. In addition, our data demonstrated that caution should be taken when using organelle-specific dyes in conjunction with strategies affecting membrane potential.

  13. Para-toluenesulfonamide induces tongue squamous cell carcinoma cell death through disturbing lysosomal stability.

    PubMed

    Liu, Zhe; Liang, Chenyuan; Zhang, Zhuoyuan; Pan, Jian; Xia, Hui; Zhong, Nanshan; Li, Longjiang

    2015-11-01

    Para-toluenesulfonamide (PTS) has been implicated with anticancer effects against a variety of tumors. In the present study, we investigated the inhibitory effects of PTS on tongue squamous cell carcinoma (Tca-8113) and explored the lysosomal and mitochondrial changes after PTS treatment in vitro. High-performance liquid chromatography showed that PTS selectively accumulated in Tca-8113 cells with a relatively low concentration in normal fibroblasts. Next, the effects of PTS on cell viability, invasion, and cell death were determined. PTS significantly inhibited Tca-8113 cells' viability and invasive ability with increased cancer cell death. Flow cytometric analysis and the lactate dehydrogenase release assay showed that PTS induced cancer cell death by activating apoptosis and necrosis simultaneously. Morphological changes, such as cellular shrinkage, nuclear condensation as well as formation of apoptotic body and secondary lysosomes, were observed, indicating that PTS might induce cell death through disturbing lysosomal stability. Lysosomal integrity assay and western blot showed that PTS increased lysosomal membrane permeabilization associated with activation of lysosomal cathepsin B. Finally, PTS was shown to inhibit ATP biosynthesis and induce the release of mitochondrial cytochrome c. Therefore, our findings provide a novel insight into the use of PTS in cancer therapy.

  14. The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes

    PubMed Central

    Marwaha, Rituraj; Arya, Subhash B.; Jagga, Divya; Kaur, Harmeet

    2017-01-01

    Endocytic, autophagic, and phagocytic vesicles move on microtubule tracks to fuse with lysosomes. Small GTPases, such as Rab7 and Arl8b, recruit their downstream effectors to mediate this transport and fusion. However, the potential cross talk between these two GTPases is unclear. Here, we show that the Rab7 effector PLEKHM1 simultaneously binds Rab7 and Arl8b, bringing about clustering and fusion of late endosomes and lysosomes. We show that the N-terminal RUN domain of PLEKHM1 is necessary and sufficient for interaction with Arl8b and its subsequent localization to lysosomes. Notably, we also demonstrate that Arl8b mediates recruitment of HOPS complex to PLEKHM1-positive vesicle contact sites. Consequently, Arl8b binding to PLEKHM1 is required for its function in delivery and, therefore, degradation of endocytic and autophagic cargo in lysosomes. Finally, we also show that PLEKHM1 competes with SKIP for Arl8b binding, which dictates lysosome positioning. These findings suggest that Arl8b, along with its effectors, orchestrates lysosomal transport and fusion. PMID:28325809

  15. Interaction of arylsulfatase A with UDP-N-acetylglucosamine:Lysosomal enzyme-N-acetylglucosamine-1-phosphotransferase.

    PubMed

    Schierau, A; Dietz, F; Lange, H; Schestag, F; Parastar, A; Gieselmann, V

    1999-02-05

    The critical step in lysosomal targeting of soluble lysosomal enzymes is the recognition by an UDP-N-acetylglucosamine:lysosomal enzyme-N-acetylglucosamine-1-phosphotransferase. The structure of the determinant common to all lysosomal enzymes for proper recognition by the phosphotransferase is not completely understood. Our current knowledge is largely based on the introduction of targeted amino acid substitutions into lysosomal enzymes and analysis of their effects on phosphotransferase recognition. We have investigated the effect of eight anti-arylsulfatase A monoclonal antibodies on the interaction of arylsulfatase A with the lysosomal enzyme phosphotransferase in vitro. We also show that a lysine-rich surface area of arylsulfatases A and B is essential for proper recognition by the phosphotransferase. Monoclonal antibodies bind to at least six different epitopes at different locations on the surface of arylsulfatase A. All antibodies bind outside the lysine-rich recognition area, but nevertheless Fab fragments of these antibodies prevent interaction of arylsulfatase A with the phosphotransferase. Our data support a model in which binding of arylsulfatase A to the phosphotransferase is not restricted to a limited surface area but involves the simultaneous recognition of large parts of arylsulfatase A.

  16. Uptake and degradation of cytoplasmic RNA by lysosomes in the perfused rat liver

    SciTech Connect

    Heydrick, S.J.; Lardeux, B.; Mortimore, G.E.

    1987-05-01

    The release of (/sup 14/C)cytidine has been shown previously to be a valid marker for RNA degradation in rat hepatocytes. The breakdown of RNA measured with this marker in perfused livers prelabeled in vivo with (6-/sup 14/C)orotic acid was found to be regulated acutely by perfusate amino acids over a wide range, from 0.29 to 3.48%/h. This regulation paralleled that of lysosomal proteolysis. Chloroquine inhibited RNA degradation 60-70%. In subsequent cell fractionation studies labelled cytidine was released; the distribution of this release paralleled that of a lysosomal marker enzyme. The release plateaued after two hours, defining a distinct lysosomal pool of RNA. The lysosomal location of the RNA pool was confirmed in experiments where a 22% increase in the apparent pool size was obtained by lowering the homogenate pH from 7.0 to 5.5. The pool size correlated linearly with the rate of RNA degradation measured during perfusion, giving a turnover constant in reasonable agreement with values reported for autophagy. These results indicate that cytoplasmic RNA degradation occurs primarily in the lysosome and is regulated under these conditions by the amino acid control of lysosomal sequestration of cytoplasm.

  17. Regulation of mTORC1 by lysosomal calcium and calmodulin

    PubMed Central

    Li, Ruo-Jing; Xu, Jing; Fu, Chenglai; Zhang, Jing; Zheng, Yujun George; Jia, Hao; Liu, Jun O

    2016-01-01

    Blockade of lysosomal calcium release due to lysosomal lipid accumulation has been shown to inhibit mTORC1 signaling. However, the mechanism by which lysosomal calcium regulates mTORC1 has remained undefined. Herein we report that proper lysosomal calcium release through the calcium channel TRPML1 is required for mTORC1 activation. TRPML1 depletion inhibits mTORC1 activity, while overexpression or pharmacologic activation of TRPML1 has the opposite effect. Lysosomal calcium activates mTORC1 by inducing association of calmodulin (CaM) with mTOR. Blocking the interaction between mTOR and CaM by antagonists of CaM significantly inhibits mTORC1 activity. Moreover, CaM is capable of stimulating the kinase activity of mTORC1 in a calcium-dependent manner in vitro. These results reveal that mTOR is a new type of CaM-dependent kinase, and TRPML1, lysosomal calcium and CaM play essential regulatory roles in the mTORC1 signaling pathway. DOI: http://dx.doi.org/10.7554/eLife.19360.001 PMID:27787197

  18. Distinct Protein Sorting and Localization to Premelanosomes, Melanosomes, and Lysosomes in Pigmented Melanocytic Cells✪

    PubMed Central

    Raposo, Graça; Tenza, Danielle; Murphy, Diane M.; Berson, Joanne F.; Marks, Michael S.

    2001-01-01

    Melanosomes and premelanosomes are lysosome-related organelles with a unique structure and cohort of resident proteins. We have positioned these organelles relative to endosomes and lysosomes in pigmented melanoma cells and melanocytes. Melanosome resident proteins Pmel17 and TRP1 localized to separate vesicular structures that were distinct from those enriched in lysosomal proteins. In immunogold-labeled ultrathin cryosections, Pmel17 was most enriched along the intralumenal striations of premelanosomes. Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane. Both proteins were largely excluded from lysosomal compartments enriched in LAMP1 and cathepsin D. By kinetic analysis of fluid phase uptake and immunogold labeling, premelanosomal proteins segregated from endocytic markers within an unusual endosomal compartment. This compartment contained Pmel17, was accessed by BSA–gold after 15 min, was acidic, and displayed a cytoplasmic planar coat that contained clathrin. Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells. Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted. PMID:11266471

  19. Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.

    PubMed

    Raposo, G; Tenza, D; Murphy, D M; Berson, J F; Marks, M S

    2001-02-19

    Melanosomes and premelanosomes are lysosome-related organelles with a unique structure and cohort of resident proteins. We have positioned these organelles relative to endosomes and lysosomes in pigmented melanoma cells and melanocytes. Melanosome resident proteins Pmel17 and TRP1 localized to separate vesicular structures that were distinct from those enriched in lysosomal proteins. In immunogold-labeled ultrathin cryosections, Pmel17 was most enriched along the intralumenal striations of premelanosomes. Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane. Both proteins were largely excluded from lysosomal compartments enriched in LAMP1 and cathepsin D. By kinetic analysis of fluid phase uptake and immunogold labeling, premelanosomal proteins segregated from endocytic markers within an unusual endosomal compartment. This compartment contained Pmel17, was accessed by BSA-gold after 15 min, was acidic, and displayed a cytoplasmic planar coat that contained clathrin. Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells. Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted.

  20. FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1

    PubMed Central

    Chen, Zhi; Ji, Xin; Qiao, Xianfeng; Jin, Yaping; Liu, Wei

    2016-01-01

    The intracellular amino acid pool within lysosome is a signal that stimulates the nutrient-sensing mTORC1 signalling pathway. The signal transduction cascade has garnered much attention, but little is known about the sequestration of the signalling molecules within the lysosome. Using human HEK293 cells as a model, we found that suppression of the BHD syndrome gene FLCN reduced the leucine level in lysosome, which correlated with decreased mTORC1 activity. Both consequences could be reversed by supplementation with high levels of leucine, but not other tested amino acids. Conversely, overexpressed FLCN could sequester lysosomal leucine and stimulate mTORC1 in an amino acid limitation environment. These results identify a novel function of FLCN: it controls mTORC1 by modulating the leucine signal in lysosome. Furthermore, we provided evidence that FLCN exerted this role by inhibiting the accumulation of the amino acid transporter PAT1 on the lysosome surface, thereby maintaining the signal level within the organelle. PMID:27280402

  1. Lysosome abnormalities and lipofucsin content of nerve cells of oedematous human cerebral cortex.

    PubMed

    Castejón, O J

    2004-01-01

    Lysosome alterations and lipofucsin content of nerve cells, capillary endothelial cells and pericytes were examined in the anoxic-ischaemic brain parenchyma of thirty two patients with congenital hydrocephalus, complicated brain traumatic injuries, brain tumours and vascular anomalies. Cortical biopsies of frontal, parietal and temporal cortex were processed for transmission electron microscopy. In oedematous non pyramidal and pyramidal nerve cells, lysosomes showed fragmentation of their limiting membranes and an associated dense granulation. Areas of cytoplasmic focal necrosis were observed surrounding the lysosomes. Lipofucsin granules were also observed in neonate and infant patients with congenital hydrocephalus, suggesting that lipofucsin formation is a life span process. Lysosomes coexisting with an increased amount of lipofucsin granules were observed in young and adult patients with brain trauma, tumours and vascular anomalies. Phagocytic astrocytes and activated oligodendroglial cells showed the overall spectrum of an altered endosomal/lysosomal system. Lipofucsin granules and multivesicular bodies also were distinguished in endothelial and pericyte cells. The role of released and activated lysosomal enzymes is discussed in relation with the cytoplasmatic focal necrosis of nerve cells and the genesis of moderate and severe oedema.

  2. Lysosomal Membrane Permeabilization Induces Cell Death in a Mitochondrion-dependent Fashion

    PubMed Central

    Boya, Patricia; Andreau, Karine; Poncet, Delphine; Zamzami, Naoufal; Perfettini, Jean-Luc; Metivier, Didier; Ojcius, David M.; Jäättelä, Marja; Kroemer, Guido

    2003-01-01

    A number of diseases are due to lysosomal destabilization, which results in damaging cell loss. To investigate the mechanisms of lysosomal cell death, we characterized the cytotoxic action of two widely used quinolone antibiotics: ciprofloxacin (CPX) or norfloxacin (NFX). CPX or NFX plus UV light (NFX*) induce lysosomal membrane permeabilization (LMP), as detected by the release of cathepsins from lysosomes. Inhibition of the lysosomal accumulation of CPX or NFX suppresses their capacity to induce LMP and to kill cells. CPX- or NFX-triggered LMP results in caspase-independent cell death, with hallmarks of apoptosis such as chromatin condensation and phosphatidylserine exposure on the plasma membrane. LMP triggers mitochondrial membrane permeabilization (MMP), as detected by the release of cytochrome c. Both CPX and NFX* cause Bax and Bak to adopt their apoptotic conformation and to insert into mitochondrial membranes. Bax−/− Bak−/− double knockout cells fail to undergo MMP and cell death in response to CPX- or NFX-induced LMP. The single knockout of Bax or Bak (but not Bid) or the transfection-enforced expression of mitochondrion-targeted (but not endoplasmic reticulum–targeted) Bcl-2 conferred protection against CPX (but not NFX*)-induced MMP and death. Altogether, our data indicate that mitochondria are indispensable for cell death initiated by lysosomal destabilization. PMID:12756268

  3. Endolysosomal transport of newly-synthesized cathepsin D in a sucrose model of lysosomal storage.

    PubMed

    Hamer, Isabelle; Jadot, Michel

    2005-10-01

    Newly-synthesized soluble lysosomal enzymes are transported from the trans-Golgi network to lysosomes by a mannose 6-phosphate receptor-mediated pathway. Lysosomal storage of indigestible material has been reported to perturb the biosynthesis and the fate of lysosomal hydrolases. In this study, we have focused our attention on the last steps in the transport of newly-synthesized cathepsin D to lysosomes in sucrose-treated WI-38 fibroblasts. Pulse-chase experiments indicate that, in sucrose-treated cells, cathepsin D maturation is delayed by 2 to 4 h. By subcellular fractionation, we show that newly-synthesized cathepsin D precursors transit through organelles endowed with a high sedimentation coefficient. These organelles are recovered in the dense region of a self-forming Percoll density gradient while the bulk of hydrolytic activities is redistributed to the low density region. Only later, are the precursors delivered to organelles containing the bulk of active hydrolases. There, procathepsin D is proteolytically processed into its 31 kDa-mature form. Our results suggest that when sucrose is present, the delayed maturation of procathepsin D is related to the delivery of the polypeptides into an organelle behaving in centrifugation like lysosomes but which is poorly efficient in proteolytic processing of procathepsin D. This low proteolytic activity of this organelle could be due to its poor ability to interact with hydrolase-containing structures.

  4. Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A.

    PubMed

    Bohl, Cornelius; Pomorski, Adam; Seemann, Susanne; Knospe, Anne-Marie; Zheng, Chaonan; Krężel, Artur; Rolfs, Arndt; Lukas, Jan

    2017-08-15

    Fluorescence-based live-cell imaging (LCI) of lysosomal glycosidases is often hampered by unfavorable pH and redox conditions that reduce fluorescence output. Moreover, most lysosomal glycosidases are low-mass soluble proteins that do not allow for bulky fluorescent protein fusions. We selected α-galactosidase A (GALA) as a model lysosomal glycosidase involved in Anderson-Fabry disease (AFD) for the current LCI approach. Examination of the subcellular localization of AFD-causing mutants can reveal the mechanism underlying cellular trafficking deficits. To minimize genetic GALA modification, we employed a biarsenical labeling protocol with tetracysteine (TC-tag) detection. We tested the efficiency of halogen substituted biarsenical probes to interact with C-terminally TC-tagged GALA peptide at pH 4.5 in vitro and identified F2FlAsH-EDT2 as a superior detection reagent for GALA. This probe provides improved signal/noise ratio in labeled COS-7 cells transiently expressing TC-tagged GALA. The investigated fluorescence-based LCI technology of TC-tagged lysosomal protein using an improved biarsenical probe can be used to identify novel compounds that promote proper trafficking of mutant GALA to lysosomal compartments and rescue the mutant phenotype.-Bohl, C., Pomorski, A., Seemann, S., Knospe, A.-M., Zheng, C., Krężel, A., Rolfs, A., Lukas, J. Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A. © FASEB.

  5. Activation of the transcription factor EB rescues lysosomal abnormalities in cystinotic kidney cells.

    PubMed

    Rega, Laura R; Polishchuk, Elena; Montefusco, Sandro; Napolitano, Gennaro; Tozzi, Giulia; Zhang, Jinzhong; Bellomo, Francesco; Taranta, Anna; Pastore, Anna; Polishchuk, Roman; Piemonte, Fiorella; Medina, Diego L; Catz, Sergio D; Ballabio, Andrea; Emma, Francesco

    2016-04-01

    Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disease characterized by accumulation of cystine into lysosomes secondary to mutations in the cystine lysosomal transporter, cystinosin. The defect initially causes proximal tubular dysfunction (Fanconi syndrome) which in time progresses to end-stage renal disease. Cystinotic patients treated with the cystine-depleting agent, cysteamine, have improved life expectancy, delayed progression to chronic renal failure, but persistence of Fanconi syndrome. Here, we have investigated the role of the transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in conditionally immortalized proximal tubular epithelial cells derived from the urine of a healthy volunteer or a cystinotic patient. Lack of cystinosin reduced TFEB expression and induced TFEB nuclear translocation. Stimulation of endogenous TFEB activity by genistein, or overexpression of exogenous TFEB lowered cystine levels within 24 hours in cystinotic cells. Overexpression of TFEB also stimulated delayed endocytic cargo processing within 24 hours. Rescue of other abnormalities of the lysosomal compartment was observed but required prolonged expression of TFEB. These abnormalities could not be corrected with cysteamine. Thus, these data show that the consequences of cystinosin deficiency are not restricted to cystine accumulation and support the role of TFEB as a therapeutic target for the treatment of lysosomal storage diseases, in particular of cystinosis. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  6. ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes

    PubMed Central

    Liu, Jun; Liu, Wenjing; Yang, Jun

    2016-01-01

    We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1–3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca2+-dependent lysosomal exocytosis. PMID:26864824

  7. PLEKHM1/DEF8/RAB7 complex regulates lysosome positioning and bone homeostasis

    PubMed Central

    Fujiwara, Toshifumi; Ye, Shiqiao; Winchell, Caylin G.; Andrews, Norma W.; Voth, Daniel E.; Varughese, Kottayil I.; Mackintosh, Samuel G.; Feng, Yunfeng; Nakamura, Takashi; Manolagas, Stavros C.

    2016-01-01

    Mutations of the Plekhm1 gene in humans and rats cause osteopetrosis, an inherited bone disease characterized by diminished bone resorption by osteoclasts. PLEKHM1 binds to RAB7 and is critical for lysosome trafficking. However, the molecular mechanisms by which PLEKHM1 regulates lysosomal pathways remain unknown. Here, we generated germline and conditional Plekhm1-deficient mice. These mice displayed no overt abnormalities in major organs, except for an increase in trabecular bone mass. Furthermore, loss of PLEKHM1 abrogated the peripheral distribution of lysosomes and bone resorption in osteoclasts. Mechanistically, we indicated that DEF8 interacts with PLEKHM1 and promotes its binding to RAB7, whereas the binding of FAM98A and NDEL1 with PLEKHM1 connects lysosomes to microtubules. Importantly, suppression of these proteins results in lysosome positioning and bone resorption defects similar to those of Plekhm1-null osteoclasts. Thus, PLHKEM1, DEF8, FAM98A, and NDEL1 constitute a molecular complex that regulates lysosome positioning and secretion through RAB7. PMID:27777970

  8. TMEM175 deficiency impairs lysosomal and mitochondrial function and increases α-synuclein aggregation

    PubMed Central

    Jinn, Sarah; Drolet, Robert E.; Cramer, Paige E.; Wong, Andus Hon-Kit; Toolan, Dawn M.; Gretzula, Cheryl A.; Voleti, Bhavya; Vassileva, Galya; Disa, Jyoti; Tadin-Strapps, Marija; Stone, David J.

    2017-01-01

    Parkinson disease (PD) is a neurodegenerative disorder pathologically characterized by nigrostriatal dopamine neuron loss and the postmortem presence of Lewy bodies, depositions of insoluble α-synuclein, and other proteins that likely contribute to cellular toxicity and death during the disease. Genetic and biochemical studies have implicated impaired lysosomal and mitochondrial function in the pathogenesis of PD. Transmembrane protein 175 (TMEM175), the lysosomal K+ channel, is centered under a major genome-wide association studies peak for PD, making it a potential candidate risk factor for the disease. To address the possibility that variation in TMEM175 could play a role in PD pathogenesis, TMEM175 function was investigated in a neuronal model system. Studies confirmed that TMEM175 deficiency results in unstable lysosomal pH, which led to decreased lysosomal catalytic activity, decreased glucocerebrosidase activity, impaired autophagosome clearance by the lysosome, and decreased mitochondrial respiration. Moreover, TMEM175 deficiency in rat primary neurons resulted in increased susceptibility to exogenous α-synuclein fibrils. Following α-synuclein fibril treatment, neurons deficient in TMEM175 were found to have increased phosphorylated and detergent-insoluble α-synuclein deposits. Taken together, data from these studies suggest that TMEM175 plays a direct and critical role in lysosomal and mitochondrial function and PD pathogenesis and highlight this ion channel as a potential therapeutic target for treating PD. PMID:28193887

  9. Involvement of BimL activation in apoptosis induced by lysosomal photodamage

    NASA Astrophysics Data System (ADS)

    Liu, Lei; Wang, Xianwang; Li, Hui

    2008-12-01

    Lysosomal photosensitizers have been used in photodynamic therapy (PDT). Combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. The lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, we selected N-aspartyl chlorin e6 (NPe6), an effective photosensitizer which preferentially accumulates in lysosomes, to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells treated by NPe6-PDT was studied using real-time single-cell analysis. Confocal imaging of cells transfected with BimL-GFP demonstrated that BimL translocated to mitochondria after NPe6-PDT treatment for about 150 min, and then sequestered into clusters associated with the mitochondira within 30 min. The activation of BimL proved to be an important event in the apoptotic machinery, as demonstrated by the significant protection of cell death in samples suppressed the expression level of endogenous BimL. This study demonstrates that BimL activation was involved in the cell death induced by PDT with lysosomal photosensitizer.

  10. "Facilitated consensus," "ethics facilitation," and unsettled cases.

    PubMed

    Aulisio, Mark R

    2011-01-01

    In "Consensus, Clinical Decision Making, and Unsettled Cases:' David M. Adams and William J.Winslade' make multiple references to both editions of the American Society of Bioethics and Humanities (ASBH) Core Competencies for Healthcare Ethics Consultation in their discussion of two assumptions that are supposed to be at the heart of the facilitated consensus model's inability to handle unsettled cases; that is, that: 1. Consultants "should maintain a kind of moral impartiality or neutrality throughout the process," "explicitly condemn[ing] anything resembling a substantive 'ethics' recommendation, and 2. "What counts as the proper set of allowable options among which the parties are to deliberate will itself always be clearly discernible' Herein, I argue that neither of these assumptions is required by ASBH's ethics facilitation approach. I then conclude by suggesting that, despite their fundamentally mistaken interpretation of the ASBH approach-perhaps even because of it-Adams and Winslade have made two important contributions to the ethics consultation literature.

  11. The Novel Neuronal Ceroid Lipofuscinosis Gene MFSD8 Encodes a Putative Lysosomal Transporter

    PubMed Central

    Siintola, Eija ; Topcu, Meral ; Aula, Nina ; Lohi, Hannes ; Minassian, Berge A. ; Paterson, Andrew D. ; Liu, Xiao-Qing ; Wilson, Callum ; Lahtinen, Ulla ; Anttonen, Anna-Kaisa ; Lehesjoki, Anna-Elina 

    2007-01-01

    The late-infantile–onset forms are the most genetically heterogeneous group among the autosomal recessively inherited neurodegenerative disorders, the neuronal ceroid lipofuscinoses (NCLs). The Turkish variant was initially considered to be a distinct genetic entity, with clinical presentation similar to that of other forms of late-infantile–onset NCL (LINCL), including age at onset from 2 to 7 years, epileptic seizures, psychomotor deterioration, myoclonus, loss of vision, and premature death. However, Turkish variant LINCL was recently found to be genetically heterogeneous, because mutations in two genes, CLN6 and CLN8, were identified to underlie the disease phenotype in a subset of patients. After a genomewide scan with single-nucleotide–polymorphism markers and homozygosity mapping in nine Turkish families and one Indian family, not linked to any of the known NCL loci, we mapped a novel variant LINCL locus to chromosome 4q28.1-q28.2 in five families. We identified six different mutations in the MFSD8 gene (previously denoted “MGC33302”), which encodes a novel polytopic 518–amino acid membrane protein that belongs to the major facilitator superfamily of transporter proteins. MFSD8 is expressed ubiquitously, with several alternatively spliced variants. Like the majority of the previously identified NCL proteins, MFSD8 localizes mainly to the lysosomal compartment. However, the function of MFSD8 remains to be elucidated. Analysis of the genome-scan data suggests the existence of at least three more genes in the remaining five families, further corroborating the great genetic heterogeneity of LINCLs. PMID:17564970

  12. Lysosomal acid lipase in mesenchymal stem cell stimulation of tumor growth and metastasis

    PubMed Central

    Zhao, Ting; Yan, Cong; Du, Hong

    2016-01-01

    Bone marrow mesenchymal stem cells (MSCs) are an important participant in the tumor microenvironment, in which they promote tumor growth and progression. Here we report for the first time that depletion of lysosomal acid lipase (LAL) in MSCs impairs their abilities to stimulate tumor growth and metastasis both in allogeneic and syngeneic mouse models. Reduced cell viability was observed in LAL-deficient (lal−/−) MSCs, which was a result of both increased apoptosis and decreased proliferation due to cell cycle arrest. The synthesis and secretion of cytokines and chemokines that are known to mediate MSCs' tumor-stimulating and immunosuppressive effects, i.e., IL-6, MCP-1 and IL-10, were down-regulated in lal−/− MSCs. When tumor cells were treated with the conditioned medium from lal−/− MSCs, decreased proliferation was observed, accompanied by reduced activation of oncogenic intracellular signaling molecules in tumor cells. Co-injection of lal−/− MSCs and B16 melanoma cells into wild type mice not only induced CD8+ cytotoxic T cells, but also decreased accumulation of tumor-promoting Ly6G+CD11b+ myeloid-derived suppressor cells (MDSCs), which may synergistically contribute to the impairment of tumor progression. Furthermore, lal−/− MSCs showed impaired differentiation towards tumor-associated fibroblasts. In addition, MDSCs facilitated MSC proliferation, which was mediated by MDSC-secreted cytokines and chemokines. Our results indicate that LAL plays a critical role in regulating MSCs' ability to stimulate tumor growth and metastasis, which provides a mechanistic basis for targeting LAL in MSCs to reduce the risk of cancer metastasis. PMID:27531897

  13. Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts.

    PubMed

    Kelly, B M; Yu, C Z; Chang, P L

    1989-02-01

    Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.

  14. TFEB activation promotes the recruitment of lysosomal glycohydrolases β-hexosaminidase and β-galactosidase to the plasma membrane

    SciTech Connect

    Magini, Alessandro; Polchi, Alice; Urbanelli, Lorena; Cesselli, Daniela; Beltrami, Antonio; Tancini, Brunella; Emiliani, Carla

    2013-10-18

    Highlights: •TFEB activation promotes the increase of Hex and Gal activities. •The increase of Hex and Gal activities is related to transcriptional regulation. •TFEB promotes the recruitment of mature Hex and Gal on cell surface. -- Abstract: Lysosomes are membrane-enclosed organelles containing acid hydrolases. They mediate a variety of physiological processes, such as cellular clearance, lipid homeostasis, energy metabolism and pathogen defence. Lysosomes can secrete their content through a process called lysosome exocytosis in which lysosomes fuse with the plasma membrane realising their content into the extracellular milieu. Lysosomal exocytosis is not only responsible for the secretion of lysosomal enzymes, but it also has a crucial role in the plasma membrane repair. Recently, it has been demonstrated that lysosome response to the physiologic signals is regulated by the transcription factor EB (TFEB). In particular, lysosomal secretion is transcriptionally regulated by TFEB which induces both the docking and fusion of lysosomes with the plasma membrane. In this work we demonstrated that TFEB nuclear translocation is accompanied by an increase of mature glycohydrolases β-hexosaminidase and β-galactosidase on cell surface. This evidence contributes to elucidate an unknown TFEB biological function leading the lysosomal glycohydrolases on plasma membrane.

  15. Enhancing lysosomal biogenesis and autophagic flux by activating the transcription factor EB protects against cadmium-induced neurotoxicity

    PubMed Central

    Pi, Huifeng; Li, Min; Tian, Li; Yang, Zhiqi; Yu, Zhengping; Zhou, Zhou

    2017-01-01

    Cadmium (Cd), a highly ubiquitous heavy metal, is a well-known inducer of neurotoxicity. However, the mechanism underlying cadmium-induced neurotoxicity remains unclear. In this study, we found that Cd inhibits autophagosome-lysosome fusion and impairs lysosomal function by reducing the levels of lysosomal-associated membrane proteins, inhibiting lysosomal proteolysis and altering lysosomal pH, contributing to defects in autophagic clearance and subsequently leading to nerve cell death. In addition, Cd decreases transcription factor EB (TFEB) expression at both the mRNA and protein levels. Furthermore, Cd induces the nuclear translocation of TFEB and TFEB target-gene expression, associated with compromised lysosomal function or a compensatory effect after the impairment of the autophagic flux. Notably, restoration of the levels of lysosomal-associated membrane protein, lysosomal proteolysis, lysosomal pH and autophagic flux through Tfeb overexpression protects against Cd-induced neurotoxicity, and this protective effect is incompletely dependent on TFEB nuclear translocation. Moreover, gene transfer of the master autophagy regulator TFEB results in the clearance of toxic proteins and the correction of Cd-induced neurotoxicity in vivo. Our study is the first to demonstrate that Cd disrupts lysosomal function and autophagic flux and manipulation of TFEB signalling may be a therapeutic approach for antagonizing Cd-induced neurotoxicity. PMID:28240313

  16. Facilitating Organizational Change.

    ERIC Educational Resources Information Center

    1999

    The first of the three papers in this symposium, "Conflicts that Arise in Small Group Facilitation: A Descriptive Study of Accounts, Actions, Outcomes, and Assessments" (Judith A. Kolb, William J. Rothwell), contains self-report verbatim accounts contributed by facilitators and the results of a literature review on small group conflict.…

  17. Evaluator or Facilitator.

    ERIC Educational Resources Information Center

    Bolding, James T.

    1978-01-01

    In American schools, the classroom teacher must act in two conflicting capacities: as a facilitator of learning and as an evaluator of his own facilitating activities. To avoid problems inherent in this, the evaluator role could be assigned elsewhere, as in the Boy Scouts' merit badge system. (SJL)

  18. STRUCTURAL BASIS OF STEROL BINDING BY NPC2, A LYSOSOMAL PROTEIN DEFICIENT IN NIEMANN-PICK TYPE C2 DISEASE*

    PubMed Central

    Xu, Sujuan; Benoff, Brian; Liou, Heng-Ling; Lobel, Peter; Stock, Ann M.

    2013-01-01

    NPC2 is a small lysosomal glycoprotein that binds cholesterol with submicromolar affinity. Deficiency in NPC2 is the cause of Niemann Pick type C2 disease, a fatal neurovisceral disorder characterized by accumulation of cholesterol in lysosomes. Here we report the crystal structure of bovine NPC2 bound to cholesterol-3-O-sulfate, an analog that binds with greater apparent affinity than cholesterol. Structures of both apo- and sterol-bound NPC2 were observed within the same crystal lattice, with an asymmetric unit containing one molecule of apoNPC2 and two molecules of sterol-bound NPC2. As predicted from a previously determined structure of apoNPC2, the sterol binds in a deep hydrophobic pocket sandwiched between the two β sheets of NPC2, with only the sulfate substituent of the ligand exposed to solvent. In the two available structures of apoNPC2, the incipient ligand-binding pocket, which ranges from a loosely packed hydrophobic core to a small tunnel, is too small to accommodate cholesterol. In the presence of sterol, the pocket expands, facilitated by a slight separation of the β strands and substantial reorientation of some side chains, resulting in a perfect molding of the pocket around the hydrocarbon portion of cholesterol. A notable feature is the repositioning of two aromatic residues at the tunnel entrance that are essential for NPC2 function. The NPC2 structures provide evidence of a malleable binding site, consistent with the previously documented broad range of sterol ligand specificity. PMID:17573352

  19. Loss of AP-5 results in accumulation of aberrant endolysosomes: defining a new type of lysosomal storage disease.

    PubMed

    Hirst, Jennifer; Edgar, James R; Esteves, Typhaine; Darios, Frédéric; Madeo, Marianna; Chang, Jaerak; Roda, Ricardo H; Dürr, Alexandra; Anheim, Mathieu; Gellera, Cinzia; Li, Jun; Züchner, Stephan; Mariotti, Caterina; Stevanin, Giovanni; Blackstone, Craig; Kruer, Michael C; Robinson, Margaret S

    2015-09-01

    Adaptor proteins (AP 1-5) are heterotetrameric complexes that facilitate specialized cargo sorting in vesicular-mediated trafficking. Mutations in AP5Z1, encoding a subunit of the AP-5 complex, have been reported to cause hereditary spastic paraplegia (HSP), although their impact at the cellular level has not been assessed. Here we characterize three independent fibroblast lines derived from skin biopsies of patients harbouring nonsense mutations in AP5Z1 and presenting with spastic paraplegia accompanied by neuropathy, parkinsonism and/or cognitive impairment. In all three patient-derived lines, we show that there is complete loss of AP-5 ζ protein and a reduction in the associated AP-5 µ5 protein. Using ultrastructural analysis, we show that these patient-derived lines consistently exhibit abundant multilamellar structures that are positive for markers of endolysosomes and are filled with aberrant storage material organized as exaggerated multilamellar whorls, striated belts and 'fingerprint bodies'. This phenotype can be replicated in a HeLa cell culture model by siRNA knockdown of AP-5 ζ. The cellular phenotype bears striking resemblance to features described in a number of lysosomal storage diseases (LSDs). Collectively, these findings reveal an emerging picture of the role of AP-5 in endosomal and lysosomal homeostasis, illuminates a potential pathomechanism that is relevant to the role of AP-5 in neurons and expands the understanding of recessive HSPs. Moreover, the resulting accumulation of storage material in endolysosomes leads us to propose that AP-5 deficiency represents a new type of LSDs.

  20. Structural Basis of Sterol Binding by NPC2, a Lysosomal Protein Deficient in Niemann-Pick Type C2 Disease

    SciTech Connect

    Xu,S.; Benoff, B.; Liou, H.; Lobel, P.; Stock, A.

    2007-01-01

    NPC2 is a small lysosomal glycoprotein that binds cholesterol with submicromolar affinity. Deficiency in NPC2 is the cause of Niemann-Pick type C2 disease, a fatal neurovisceral disorder characterized by accumulation of cholesterol in lysosomes. Here we report the crystal structure of bovine NPC2 bound to cholesterol-3-O-sulfate, an analog that binds with greater apparent affinity than cholesterol. Structures of both apo-bound and sterol-bound NPC2 were observed within the same crystal lattice, with an asymmetric unit containing one molecule of apoNPC2 and two molecules of sterol-bound NPC2. As predicted from a previously determined structure of apoNPC2, the sterol binds in a deep hydrophobic pocket sandwiched between the two {beta}-sheets of NPC2, with only the sulfate substituent of the ligand exposed to solvent. In the two available structures of apoNPC2, the incipient ligand-binding pocket, which ranges from a loosely packed hydrophobic core to a small tunnel, is too small to accommodate cholesterol. In the presence of sterol, the pocket expands, facilitated by a slight separation of the {beta}-strands and substantial reorientation of some side chains, resulting in a perfect molding of the pocket around the hydrocarbon portion of cholesterol. A notable feature is the repositioning of two aromatic residues at the tunnel entrance that are essential for NPC2 function. The NPC2 structures provide evidence of a malleable binding site, consistent with the previously documented broad range of sterol ligand specificity.