WorldWideScience

Sample records for lysosomal membrane glycoproteins

  1. Stabilization of lysosomal membrane and cell membrane glycoprotein profile by Semecarpus anacardium linn. nut milk extract in experimental hepatocellular carcinoma.

    Science.gov (United States)

    Premalatha, B; Sachdanandam, P

    2000-08-01

    Semecarpus anacardium Linn. nut milk extract administered orally at a dose of 200 mg/kg/day for 14 days exerted an in vivo stabilizing effect on lysosomal membrane and glycoprotein content in rat hepatocellular carcinoma. This was demonstrated in normal rats and in animals whose biomembranes were rendered fragile by induction of hepatocellular carcinoma with aflatoxin B(1) and subsequent treatment with Semecarpus anacardium nut extract. In this condition, the discharge of lysosomal enzymes increased significantly with a subsequent increase in glycoprotein components. The nut extract administration reversed these adverse changes to near normal in treated animals. The possible reason for this reversal is discussed. Such stabilization of biomembranes by Semecarpus anacardium nut extract may have a beneficial effect in the treatment of hepatocellular carcinoma and other cancers involving abnormal fragility of lysosomes and glycoprotein content providing the extract demonstrates safety in a full toxicity study. Copyright 2000 John Wiley & Sons, Ltd.

  2. Characterization and cloning of lgp110, a lysosomal membrane glycoprotein from mouse and rat cells.

    Science.gov (United States)

    Granger, B L; Green, S A; Gabel, C A; Howe, C L; Mellman, I; Helenius, A

    1990-07-15

    lgp110 is a heavily glycosylated intrinsic protein of lysosomal membranes. Initially defined by monoclonal antibodies against mouse liver lysosomes, it consists of a 45-kilodalton core polypeptide with O-linked and 17 asparagine-linked oligosaccharide side chains in mouse cells. Sialic acid residues make the mature protein extremely acidic, with an isoelectric point of between 2 and 4 in both normal tissues and most cultured cell lines. Partial sequencing of mouse lgp110 allowed oligonucleotide probes to be constructed for the screening of several mouse cDNA libraries. A partial cDNA clone for mouse lgp110 was found and used for additional library screening, generating a cDNA clone covering all of the coding sequence of mature rat lgp110 as well as genomic clones covering most of the mouse gene. These new clones bring to seven the number of lysosomal membrane proteins whose amino acid sequences can be deduced, and two distinct but highly similar groups (designated lgp-A and lgp-B) can now be defined. Sequence comparisons suggest that differences within each group reflect species variations of the same protein and that lgp-A and lgp-B probably diverged from a common ancestor prior to the evolup4f1ary divergence of birds and mammals. Individual cells and individual lysosomes possess both lgp-A and lgp-B, suggesting that these two proteins have different functions. Mouse lgp110 is encoded by at least seven exons; intron positions suggest that the two homologous ectodomains of each lgp arose through gene duplication.

  3. Characterization of lysosomal membrane proteins of Dictyostelium discoideum. A complex population of acidic integral membrane glycoproteins, Rab GTP-binding proteins and vacuolar ATPase subunits.

    Science.gov (United States)

    Temesvari, L; Rodriguez-Paris, J; Bush, J; Steck, T L; Cardelli, J

    1994-10-14

    Highly purified lysosomes, prepared by magnetic fractionation of homogenates from Dictyostelium discoideum cells fed colloidal iron, were lysed under hypoosmotic conditions, and the membrane-associated proteins were subjected to gel electrophoresis. Thirteen major membrane polypeptides, ranging in molecular weight from 25,000 to 100,000 were identified. The isoelectric points of these proteins ranged from below 3.8 to greater than 7.0. Most of these proteins were stripped from membranes exposed to a chaotropic agent, 3,5-diodo-2-hydroxybenzoic acid lithium salt, and were therefore classified as peripheral membrane proteins. Twenty five glycoprotein species were detected by lectin blot analysis; 19 were classified as integral membrane proteins, and were, in general, larger than 45 kDa and negatively charged due in part to the presence of mannose 6-sulfate. Western blot analysis also demonstrated that a Rab 4-like GTPase, a Rab 7-like GTPase, and at least three subunits of the vacuolar ATPase were associated with the lysosomal membrane; the ATPase subunits appeared to be major proteins in lysosomal membranes. Finally, based on N-terminal sequence analysis of a major 41-kDa lysosome-associated membrane protein, we cloned a cDNA that encodes a protein (DVA41) highly homologous to a yeast and a bovine vacuolar ATPase subunit of approximately 41 kDa. The D. discoideum DVA41 gene was apparently a single copy gene, expressed at constant levels during growth and development.

  4. Vertebrate scavenger receptor class B member 2 (SCARB2: comparative studies of a major lysosomal membrane glycoprotein

    Directory of Open Access Journals (Sweden)

    Roger Stephen Holmes

    2012-06-01

    Full Text Available Scavenger receptor class B member 2 (SCARB2 (also LIMP-2, CD36L2 or LGP85 is a major lysosomal membrane glycoprotein involved in endosomal and lysosomal biogenesis and maintenance. SCARB2 acts as a receptor for the lysosomal mannose-6-phosphate independent targeting of β-glucuronidase and enterovirus 71 and influences Parkinson’s disease and epilepsy. Genetic deficiency of this protein causes deafness and peripheral neuropathy in mice as well as myoclonic epilepsy and nephrotic syndrome in humans. Comparative SCARB2 amino acid sequences and structures and SCARB2 gene locations were examined using data from several vertebrate genome projects. Vertebrate SCARB2 sequences shared 43-100% identity as compared with 30-36% sequence identities with other CD36-like superfamily members, SCARB1 and CD36. At least 10 N-glycosylation sites were conserved among most vertebrate SCARB2 proteins examined. Sequence alignments, key amino acid residues and conserved predicted secondary structures were examined, including cytoplasmic, transmembrane and external lysosomal membrane sequences: cysteine disulfide residues, thrombospondin (THP1 binding sites and 16 proline and 20 glycine conserved residues, which may contribute to short loop formation within the exomembrane SCARB2 sequences. Vertebrate SCARB2 genes contained 12 coding exons. The human SCARB2 gene contained a CpG island (CpG100, ten microRNA-binding sites and several transcription factor binding sites (including PPARA which may contribute to a higher level (2.4 times average of gene expression. Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate SCARB2 gene with vertebrate SCARB1 and CD36 genes. These suggested that SCARB2 originated from duplications of the CD36 gene in an ancestral genome forming three vertebrate CD36 gene family members: SCARB1, SCARB2 and CD36.

  5. Molecular cloning of cDNAs encoding lamp A, a human lysosomal membrane glycoprotein with apparent M sub r approx 120,000

    Energy Technology Data Exchange (ETDEWEB)

    Viitala, J.; Carlsson, S.R.; Siebert, P.D.; Fukuda, M. (La Jolla Cancer Research Foundation, CA (USA))

    1988-06-01

    Although several lysosomal membrane glycoproteins have been characterized by using specific antibodies, none of the studies so far elucidated the amino acid sequence of a lysosomal membrane glycoprotein. Here we describe cDNA clones encoding for one of the lysosome-associated membrane proteins with apparent M{sub r} {approx} 120,000, lamp A. The amino acid sequence based on the fully coded cDNA shows that as many as 18 potential N-glycosylation sites can be found in the total of 385 amino acid residues. The results obtained by endoglycosidase F digestion support the conclusion that this glycoprotein contains 18 N-glycans. These N-glycosylation sites are clustered in two domains; one contains 10 and the other contains 8 N-glycosylation sites. These domains are separated by a (proline-serine)-rich region that has a distinct homology to the IgA hinge structure. The first N-glycosylated domain is elongated to a potential leader peptide toward the NH{sub 2}-terminal end. The second N-glycosylated domain, on the other hand, is connected to a putative transmembrane portion consisting of hydrophobic amino acids. This segment, in turn, is elongated to a short cytoplasmic segment composed of 11 amino acid residues at the COOH-terminal end.

  6. Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx

    Directory of Open Access Journals (Sweden)

    Wilke Sonja

    2012-07-01

    Full Text Available Abstract Background The family of lysosome-associated membrane proteins (LAMP comprises the multifunctional, ubiquitous LAMP-1 and LAMP-2, and the cell type-specific proteins DC-LAMP (LAMP-3, BAD-LAMP (UNC-46, C20orf103 and macrosialin (CD68. LAMPs have been implicated in a multitude of cellular processes, including phagocytosis, autophagy, lipid transport and aging. LAMP-2 isoform A acts as a receptor in chaperone-mediated autophagy. LAMP-2 deficiency causes the fatal Danon disease. The abundant proteins LAMP-1 and LAMP-2 are major constituents of the glycoconjugate coat present on the inside of the lysosomal membrane, the 'lysosomal glycocalyx'. The LAMP family is characterized by a conserved domain of 150 to 200 amino acids with two disulfide bonds. Results The crystal structure of the conserved domain of human DC-LAMP was solved. It is the first high-resolution structure of a heavily glycosylated lysosomal membrane protein. The structure represents a novel β-prism fold formed by two β-sheets bent by β-bulges and connected by a disulfide bond. Flexible loops and a hydrophobic pocket represent possible sites of molecular interaction. Computational models of the glycosylated luminal regions of LAMP-1 and LAMP-2 indicate that the proteins adopt a compact conformation in close proximity to the lysosomal membrane. The models correspond to the thickness of the lysosomal glycoprotein coat of only 5 to 12 nm, according to electron microscopy. Conclusion The conserved luminal domain of lysosome-associated membrane proteins forms a previously unknown β-prism fold. Insights into the structure of the lysosomal glycoprotein coat were obtained by computational models of the LAMP-1 and LAMP-2 luminal regions.

  7. Lysosome

    National Research Council Canada - National Science Library

    Ursula Matte BSc, PhD; Gabriela Pasqualim BSc, MSc

    2016-01-01

    Since Christian de Duve first described the lysosome in the 1950s, it has been generally presented as a membrane-bound compartment containing acid hydrolases that enables the cell to degrade molecules...

  8. Lysosome

    Directory of Open Access Journals (Sweden)

    Ursula Matte BSc, PhD

    2016-12-01

    Full Text Available Since Christian de Duve first described the lysosome in the 1950s, it has been generally presented as a membrane-bound compartment containing acid hydrolases that enables the cell to degrade molecules without being digested by autolysis. For those working on the field of lysosomal storage disorders, the lack of one such hydrolase would lead to undegraded or partially degraded substrate storage inside engorged organelles disturbing cellular function by yet poorly explored mechanisms. However, in recent years, a much more complex scenario of lysosomal function has emerged, beyond and above the cellular “digestive” system. Knowledge on how the impairment of this organelle affects cell functioning may shed light on signs and symptoms of lysosomal disorders and open new roads for therapy.

  9. Turning the gun on cancer: Utilizing lysosomal P-glycoprotein as a new strategy to overcome multi-drug resistance.

    Science.gov (United States)

    Seebacher, Nicole; Lane, Darius J R; Richardson, Des R; Jansson, Patric J

    2016-07-01

    Oxidative stress plays a role in the development of drug resistance in cancer cells. Cancer cells must constantly and rapidly adapt to changes in the tumor microenvironment, due to alterations in the availability of nutrients, such as glucose, oxygen and key transition metals (e.g., iron and copper). This nutrient flux is typically a consequence of rapid growth, poor vascularization and necrosis. It has been demonstrated that stress factors, such as hypoxia and glucose deprivation up-regulate master transcription factors, namely hypoxia inducible factor-1α (HIF-1α), which transcriptionally regulate the multi-drug resistance (MDR), transmembrane drug efflux transporter, P-glycoprotein (Pgp). Interestingly, in addition to the established role of plasma membrane Pgp in MDR, a new paradigm of intracellular resistance has emerged that is premised on the ability of lysosomal Pgp to transport cytotoxic agents into this organelle. This mechanism is enabled by the topological inversion of Pgp via endocytosis resulting in the transporter actively pumping agents into the lysosome. In this way, classical Pgp substrates, such as doxorubicin (DOX), can be actively transported into this organelle. Within the lysosome, DOX becomes protonated upon acidification of the lysosomal lumen, causing its accumulation. This mechanism efficiently traps DOX, preventing its cytotoxic interaction with nuclear DNA. This review discusses these effects and highlights a novel mechanism by which redox-active and protonatable Pgp substrates can utilize lysosomal Pgp to gain access to this compartment, resulting in catastrophic lysosomal membrane permeabilization and cell death. Hence, a key MDR mechanism that utilizes Pgp (the "gun") to sequester protonatable drug substrates safely within lysosomes can be "turned on" MDR cancer cells to destroy them from within.

  10. Lysosomal enlargement and lysosomal membrane destabilisation in mussel digestive cells measured by an integrative index

    Energy Technology Data Exchange (ETDEWEB)

    Izagirre, Urtzi [Cell Biology in Environmental Toxicology Research Group, Department of Zoology and Cell Biology, School of Sciences and Technology, University of the Basque Country, P.O. box 644, E-48080 Bilbo (Spain); Marigomez, Ionan, E-mail: ionan.marigomez@ehu.e [Cell Biology in Environmental Toxicology Research Group, Department of Zoology and Cell Biology, School of Sciences and Technology, University of the Basque Country, P.O. box 644, E-48080 Bilbo (Spain)

    2009-05-15

    Lysosomal responses (enlargement and membrane destabilisation) in mussel digestive cells are well-known environmental stress biomarkers in pollution effects monitoring in marine ecosystems. Presently, in laboratory and field studies, both responses were measured separately (in terms of lysosomal volume density - Vv - and labilisation period -LP) and combined (lysosomal response index - LRI) in order to contribute to their understanding and to develop an index useful for decisions makers. LRI integrates Vv and LP, which are not necessarily dependent lysosomal responses. It is unbiased and more sensitive than Vv and LP alone and diminishes background due to confounding factors. LRI provides a simple numerical index (consensus reference = 0; critical threshold = 1) directly related to the pollution impact degree. Moreover, LRI can be represented in a way that allows the interpretation of lysosomal responses, which is useful for environmental scientists. - Lysosomal responses to pollutants measured by an integrative index.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  13. Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway.

    Science.gov (United States)

    Katayama, Kazuhiro; Kapoor, Khyati; Ohnuma, Shinobu; Patel, Atish; Swaim, William; Ambudkar, Indu S; Ambudkar, Suresh V

    2015-10-01

    P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

  14. Revealing the fate of cell surface human P-glycoprotein (ABCB1): The Lysosomal Degradation Pathway

    Science.gov (United States)

    Katayama, Kazuhiro; Kapoor, Khyati; Ohnuma, Shinobu; Patel, Atish; Swaim, William; Ambudkar, Indu S.; Ambudkar, Suresh V.

    2015-01-01

    P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7 ± 1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1± 0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp expressing cancer cells towards chemotherapeutic drugs. PMID:26057472

  15. LAMP-3 (Lysosome-Associated Membrane Protein 3) Promotes the Intracellular Proliferation of Salmonella typhimurium.

    Science.gov (United States)

    Lee, Eun-Ju; Park, Kwan-Sik; Jeon, In-Sook; Choi, Jae-Woon; Lee, Sang-Jeon; Choy, Hyun E; Song, Ki-Duk; Lee, Hak-Kyo; Choi, Joong-Kook

    2016-07-01

    Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation.

  16. Septins as modulators of endo-lysosomal membrane traffic

    Directory of Open Access Journals (Sweden)

    Kyungyeun Song

    2016-11-01

    Full Text Available Septins constitute a family of GTP-binding proteins, which assemble into non-polar filaments in a nucleotide-dependent manner. These filaments can be recruited to negatively charged membrane surfaces. When associated with membranes septin filaments can act as diffusion barriers, which confine subdomains of distinct biological functions. In addition, they serve scaffolding roles by recruiting cytosolic proteins and other cytoskeletal elements. Septins have been implicated in a large variety of membrane-dependent processes, including cytokinesis, signaling, cell migration, and membrane traffic, and several family members have been implicated in disease. However, surprisingly little is known about the molecular mechanisms underlying their biological functions. This review summarizes evidence in support of regulatory roles of septins during endo-lysosomal sorting, with a particular focus on phosphoinositides, which serve as spatial landmarks guiding septin recruitment to distinct subcellular localizations.

  17. Noxa couples lysosomal membrane permeabilization and apoptosis during oxidative stress.

    Science.gov (United States)

    Eno, Colins O; Zhao, Guoping; Venkatanarayan, Avinashnarayan; Wang, Bing; Flores, Elsa R; Li, Chi

    2013-12-01

    The exact roles of lysosomal membrane permeabilization (LMP) in oxidative stress-triggered apoptosis are not completely understood. Here, we first studied the temporal relation between LMP and mitochondrial outer membrane permeabilization (MOMP) during the initial stage of apoptosis caused by the oxidative stress inducer H2O2. Despite its essential role in mediating apoptosis, the expression of the BH3-only Bcl-2 protein Noxa was dispensable for LMP. In contrast, MOMP was dependent on Noxa expression and occurred downstream of LMP. When lysosomal membranes were stabilized by the iron-chelating agent desferrioxamine, H2O2-induced increase in DNA damage, Noxa expression, and subsequent apoptosis were abolished by the inhibition of LMP. Importantly, LMP-induced Noxa expression increase was mediated by p53 and seems to be a unique feature of apoptosis caused by oxidative stress. Finally, exogenous iron loading recapitulated the effects of H2O2 on the expression of BH3-only Bcl-2 proteins. Overall, these data reveal a Noxa-mediated signaling pathway that couples LMP with MOMP and ultimate apoptosis during oxidative stress.

  18. Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants

    NARCIS (Netherlands)

    Groux-Degroote, S.; Dijk, S.M. van; Wolthoorn, J.; Neumann, S.; Theos, A.C.; Mazière, A.M. de; Klumperman, J.; Meer, G. van; Sprong, H.

    2008-01-01

    Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal

  19. Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants

    NARCIS (Netherlands)

    Groux-Degroote, S.; Dijk, S.M. van; Wolthoorn, J.; Neumann, S.; Theos, A.C.; Mazière, A.M. de; Klumperman, J.; Meer, G. van; Sprong, H.

    2008-01-01

    Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal

  20. Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells.

    Directory of Open Access Journals (Sweden)

    Bárbara Hissa

    Full Text Available BACKGROUND: Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages and non-professional (epithelial phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. METHODOLOGY/PRINCIPAL FINDING: In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. CONCLUSION/SIGNIFICANCE: Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of

  1. Detection of glycoproteins in the Acanthamoeba plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Paatero, G.I.L. (Abo Akademi (Finland)); Gahmberg, C.G. (Univ. of Helsinki (Finland))

    1988-11-01

    In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by {sup 125}I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB{sup 3}H{sub 4} and galactose oxidase/NaB{sup 3}H{sub 4} labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M{sub r} of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with ({sup 35}S)methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.

  2. Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion.

    Directory of Open Access Journals (Sweden)

    Daniel L Glauser

    Full Text Available Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4 entry machinery--gB, gH/gL and gp150--changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion.

  3. Enantioselective effects of methamidophos on the coelomocytes lysosomal membrane stability of Eisenia fetida.

    Science.gov (United States)

    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. The Octyl Ester of Ginsenoside Rh2 Induces Lysosomal Membrane Permeabilization via Bax Translocation.

    Science.gov (United States)

    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 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.

  5. Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx

    OpenAIRE

    Wilke Sonja; Krausze Joern; Büssow Konrad

    2012-01-01

    Abstract Background The family of lysosome-associated membrane proteins (LAMP) comprises the multifunctional, ubiquitous LAMP-1 and LAMP-2, and the cell type-specific proteins DC-LAMP (LAMP-3), BAD-LAMP (UNC-46, C20orf103) and macrosialin (CD68). LAMPs have been implicated in a multitude of cellular processes, including phagocytosis, autophagy, lipid transport and aging. LAMP-2 isoform A acts as a receptor in chaperone-mediated autophagy. LAMP-2 deficiency causes the fatal Danon disease. The ...

  6. Lysosomal Membrane Permeabilization is an Early Event in Sigma-2 Receptor Ligand Mediated Cell Death in Pancreatic Cancer

    Directory of Open Access Journals (Sweden)

    Hornick John R

    2012-05-01

    Full Text Available Abstract Background Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. Results Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282 localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco. Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. Conclusions Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a

  7. Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas

    DEFF Research Database (Denmark)

    Jensen, Stine Skov; Christensen, Karina; Aaberg-Jessen, Charlotte

    Targeting lysosomes is a novel approach in cancer therapy providing a possible way of killing the otherwise apoptosis-resistant cancer cells. Recent research has thus shown that lysosome targeting compounds induce cell death in a cervix cancer cell line. Tumor stem cells in glioblastomas have...... recently been suggested to possess innate resistance mechanisms against radiation and chemotherapy possibly explaining the high level of therapeutic resistance of these tumors. Since the presence and distribution of lysosomes in tumor cells and especially in tumor stem cells in astrocytomas is unknown......, the aim of this study was to investigate the immunohistochemical expression of LAMP-1, a membrane bound protein in lysosomes, in formalin fixed paraffin embedded tumor tissue from 23 diffuse astrocytomas, 17 anaplastic astrocytomas and 72 glioblastomas. The LAMP-1 expression was scored and compared...

  8. TFEB activation promotes the recruitment of lysosomal glycohydrolases β-hexosaminidase and β-galactosidase to the plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Magini, Alessandro [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Department of Medical and Biological Sciences (DSMB), University of Udine, Udine (Italy); Polchi, Alice; Urbanelli, Lorena [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Cesselli, Daniela; Beltrami, Antonio [Department of Medical and Biological Sciences (DSMB), University of Udine, Udine (Italy); Tancini, Brunella [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Emiliani, Carla, E-mail: carla.emiliani@unipg.it [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy)

    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.

  9. Lysosomal membrane stability of the mussel, Mytilus galloprovincialis (L.), as a biomarker of tributyltin exposure.

    Science.gov (United States)

    Okoro, Hussein K; Snyman, Reinette G; Fatoki, Olalekan S; Adekola, Folahan A; Ximba, Bhekumusa J; Slabber, Michelle Y

    2015-05-01

    The effect of tributyltin (TBT) on the stability of hemocytic lysosome membranes of the mussel, Mytilus galloprovincialis, and the use thereof as a biomarker of TBT-induced stress, was investigated. Mussels were exposed to 0.1 and 1.0 µg/L tributyltin respectively for 4 weeks. Lysosomal membrane stability of hemocytes was tested weekly by means of the neutral red retention time (NRRT) assay, after which the mussel samples were analyzed for TBT content. The two exposed groups exhibited significantly increased (p galloprovincialis.

  10. Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes multidrug resistance by a novel mechanism involving the hijacking of lysosomal P-glycoprotein (Pgp).

    Science.gov (United States)

    Jansson, Patric J; Yamagishi, Tetsuo; Arvind, Akanksha; Seebacher, Nicole; Gutierrez, Elaine; Stacy, Alexandra; Maleki, Sanaz; Sharp, Danae; Sahni, Sumit; Richardson, Des R

    2015-04-10

    Multidrug resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2',7'-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport activity.

  11. [Obtaining monoclonal antibodies against outer membrane glycoproteins of Entamoeba histolytica].

    Science.gov (United States)

    Agundis, C; Isibasi, A; Ortíz, V; Reyes, J L; Paniagua, J; Ramírez, A; Kumate, J

    1990-01-01

    The goal of this paper was the production of monoclonal antibodies capable of detecting relevant antigens from the surface of Entamoeba histolytica trophozoites, with the purpose of using them as a diagnostic test. The cellular fusion for obtaining the monoclonal antibodies (mAb) was done with spleen cells from BALB/c mice, previously immunized with glycoproteins from the membrane, as well as Sp2/0 cells. The hybridoma supernatants were tested with ELISA, using glycoproteins and lipopeptide phosphoglycans (LPPG) as antigens. Seven hybridomas producing mAb against the glycoproteins were found. Among these, three recognize LPPG. The ability of reacting with the mAb against two molecules disappeared for all the LPPG positive ones when were treated with meta-periodate, and only three reacted against the glycoproteins. All of the mAb were of the Ig M isotypes. They were characterized by Dot blot and Western blot assays. From the results, one may deduce that some mAb recognize as epitopes the polysaccharide portion, and thus infer that they are directed of against the surface and therefore, in the future, could be used with a diagnostic purpose.

  12. Membrane topology analysis of HIV-1 envelope glycoprotein gp41

    Directory of Open Access Journals (Sweden)

    Xiao Dan

    2010-11-01

    Full Text Available Abstract Background The gp41 subunit of the HIV-1 envelope glycoprotein (Env has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD. An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP that is only active in the cytoplasm. The tag protein (HaloTag and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system. Results In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells, the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability. Conclusions It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.

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

    Science.gov (United States)

    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.

  14. Characterization of an estrogen-induced oviduct membrane glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Poola, I.; Lucas, J.J.

    1986-05-01

    During estrogen-induced chick oviduct differentiation a number of N-linked membrane glycoproteins are induced as judged by GDP-(/sup 14/C)Man labeling of endogenous acceptors, /sup 125/I-con A labeling as well as coomassie blue and PAS staining of SDS polyacrylamide gels. The authors have begun to characterize one of these glycoproteins having an M/sub r/ of 91 KDa. The protein has been purified via preparative SDS-PAGE and electroelution. The purified protein migrates as a single band on analytical SDS-PAGE and comigrates with an endogenous membrane glycoprotein labeled with GDP-(/sup 14/C)Man. Amino acid analysis indicates a high proportion of GLU and ASP residues (110 and 66 moles respectively). N-terminal sequence analysis by gas phase instrumentation yielded the following: X-X-VAL-ASP-VAL-ASP-ALA-THR-VAL-GLU-GLU-ASP-GLU. The protein contains about 2% neutral sugar including 6 mol Man, 2 mol Gal, 1 mol Fuc, 4 mol GlcNAc, 1 mol GalNAc and 1 mol sialic acid per mole of protein. The presence of the GalNAc residue suggests the protein contains an O-linked oligosaccharide moiety in addition to the N-linked chain(s). The detailed structure of the carbohydrate moieties is currently under investigation.

  15. Platelet membrane glycoproteins and their function: an overview.

    Science.gov (United States)

    Kunicki, T J

    1989-07-01

    The membrane glycoproteins (GP) of human platelets act as receptors that mediate two important functions, adhesion to the subendothelial matrix and platelet-platelet cohesion, or aggregation. Many of these glycoprotein receptors exist as noncovalently linked heterodimers, including those that belong to the supergene family of adhesion receptors called the integrins. Human platelets contain at least five members of this integrin family, including a collagen receptor (GP Ia-IIa; alpha 2, beta 1), a fibronectin receptor (GP Ic-IIa; alpha 5, beta 1), a laminin receptor (GP Ic'-IIa; alpha 6, beta 1), a vitronectin receptor (VnR; alpha v, beta 3), and a promiscuous, activation-dependent receptor that is thought to be the receptor most responsible for fibrinogen-dependent, platelet-platelet cohesion (GP IIb-IIIa; alpha IIb, beta 3). Some, but not all, of the integrins bind to a tripeptide sequence, arginine-glycine-aspartic acid (RGD), on the adhesive proteins. In addition to the integrins, platelets contain other membrane glyco-proteins: GP Ib-IX, a receptor for von Willebrand factor, which is thought to be the receptor most responsible for platelet adhesion to the subendothelial matrix in a flowing system; GP V, which may be associated with GP Ib-IX and whose function remains unknown; and GP IV (GP IIIb), which functions as a receptor for thrombospondin and collagen.

  16. Incorporation of Spike and Membrane Glycoproteins into Coronavirus Virions

    Directory of Open Access Journals (Sweden)

    Makoto Ujike

    2015-04-01

    Full Text Available The envelopes of coronaviruses (CoVs contain primarily three proteins; the two major glycoproteins spike (S and membrane (M, and envelope (E, a non-glycosylated protein. Unlike other enveloped viruses, CoVs bud and assemble at the endoplasmic reticulum (ER-Golgi intermediate compartment (ERGIC. For efficient virion assembly, these proteins must be targeted to the budding site and to interact with each other or the ribonucleoprotein. Thus, the efficient incorporation of viral envelope proteins into CoV virions depends on protein trafficking and protein–protein interactions near the ERGIC. The goal of this review is to summarize recent findings on the mechanism of incorporation of the M and S glycoproteins into the CoV virion, focusing on protein trafficking and protein–protein interactions.

  17. Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas.

    Science.gov (United States)

    Jensen, Stine S; Aaberg-Jessen, Charlotte; Christensen, Karina G; Kristensen, Bjarne

    2013-01-01

    Targeting of lysosomes is a novel therapeutic anti-cancer strategy for killing the otherwise apoptosis-resistant cancer cells. Such strategies are urgently needed for treatment of brain tumors, especially the glioblastoma, which is the most frequent and most malignant type. The aim of the present study was to investigate the presence of lysosomes in astrocytic brain tumors focussing also on the therapy resistant tumor stem cells. Expression of the lysosomal marker LAMP-1 (lysosomal-associated membrane protein-1) was investigated by immunohistochemistry in 112 formalin fixed paraffin embedded astrocytomas and compared with tumor grade and overall patient survival. Moreover, double immunofluorescence stainings were performed with LAMP-1 and the astrocytic marker GFAP and the putative stem cell marker CD133 on ten glioblastomas. Most tumors expressed the LAMP-1 protein in the cytoplasm of the tumor cells, while the blood vessels were positive in all tumors. The percentage of LAMP-1 positive tumor cells and staining intensities increased with tumor grade but variations in tumors of the same grade were also found. No association was found between LAMP-1 expression and patient overall survival in the individual tumor grades. LAMP-1/GFAP showed pronounced co-expression and LAMP-1/CD133 was co-expressed as well suggesting that tumor cells including the proposed tumor stem cells contain lysosomes. The results suggest that high amounts of lysosomes are present in glioblastomas and in the proposed tumor stem cells. Targeting of lysosomes may be a promising novel therapeutic strategy against this highly malignant neoplasm.

  18. The nucleotide sequence of a CpG island demonstrates the presence of the first exon of the gene encoding the human lysosomal membrane protein lamp2 and assigns the gene to Xq24.

    Science.gov (United States)

    Manoni, M; Tribioli, C; Lazzari, B; DeBellis, G; Patrosso, C; Pergolizzi, R; Pellegrini, M; Maestrini, E; Rivella, S; Vezzoni, P

    1991-03-01

    An EagI-EcoRI clone of human genomic DNA, p2-7, mapped to Xq24 has been sequenced. This analysis has confirmed the presence of a CpG island and has identified the first exon of the human LAMP2 gene, encoding a glycoprotein of the lysosomal membrane. Since the p2-7 clone corresponds to single-copy DNA, we can assign the human LAMP2 gene to Xq24.

  19. Brucella suis-Impaired Specific Recognition of Phagosomes by Lysosomes due to Phagosomal Membrane Modifications

    Science.gov (United States)

    Naroeni, Aroem; Jouy, Nicolas; Ouahrani-Bettache, Safia; Liautard, Jean-Pierre; Porte, Françoise

    2001-01-01

    Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments. PMID:11119541

  20. Cytosolic peroxidases protect the lysosome of bloodstream African trypanosomes from iron-mediated membrane damage.

    Directory of Open Access Journals (Sweden)

    Corinna Hiller

    2014-04-01

    Full Text Available African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I-II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I-II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective

  1. Phase coexistence in a triolein-phosphatidylcholine system. Implications for lysosomal membrane properties.

    Science.gov (United States)

    Pakkanen, Kirsi I; Duelund, Lars; Vuento, Matti; Ipsen, John Hjort

    2010-02-01

    The effects of tri- and monoglycerides on phospholipid (POPC) membranes were studied using spectroscopical methods. Triolein was found to form two types of POPC-rich membranes, both with POPC or as a three-component system with monopalmitin. These two membrane types were determined as co-existing phases based on their spontaneous and stable separation and named heavy and light phase according to their sedimentation behaviour. Marked differences were seen in the physical properties of these phases, even though only minor compositional variation was detected. The light, less polar phase was found to be less ordered and more fluid and seemed to allow significantly lower amount of water penetration into the membrane-water interface than pure POPC membrane. The heavy phase, apart from their slightly altered water penetration, resembled more a pure POPC membrane. As triglycerides are present in lysosomal membranes, the present results can be seen as an implication for polarity-based water permeability barrier possibly contributing to the integrity of lysosomes.

  2. COOH-terminal isoleucine of lysosome-associated membrane protein-1 is optimal for its efficient targeting to dense secondary lysosomes.

    Science.gov (United States)

    Akasaki, Kenji; Suenobu, Michihisa; Mukaida, Maki; Michihara, Akihiro; Wada, Ikuo

    2010-12-01

    Lysosome-associated membrane protein-1 (LAMP-1) consists of a highly glycosylated luminal domain, a single-transmembrane domain and a short cytoplasmic tail that possesses a lysosome-targeting signal (GYQTI(382)) at the COOH terminus. It is hypothesized that the COOH-terminal isoleucine, I(382), could be substituted with any other bulky hydrophobic amino acid residue for LAMP-1 to exclusively localize in lysosomes. In order to test this hypothesis, we compared subcellular distribution of four substitution mutants with phenylalanine, leucine, methionine and valine at the COOH-terminus (termed I382F, I382L, I382M and I382V, respectively) with that of wild-type (WT)-LAMP-1. Double-labelled immunofluorescence analyses showed that these substitution mutants were localized as significantly to late endocytic organelles as WT-LAMP-1. However, the quantitative subcellular fractionation study revealed different distribution of WT-LAMP-1 and these four COOH-terminal mutants in late endosomes and dense secondary lysosomes. WT-LAMP-1 was accumulated three to six times more in the dense lysosomal fraction than the four mutants. The level of WT-LAMP-1 in late endosomal fraction was comparable to those of I382F, I382M and I382V. Conversely, I382L in the late endosomal fraction was approximately three times more abundant than WT-LAMP-1. These findings define the presence of isoleucine residue at the COOH-terminus of LAMP-1 as critical in governing its efficient delivery to secondary lysosomes and its ratio of lysosomes to late endosomes.

  3. Urothelial endocytic vesicle recycling and lysosomal degradative pathway regulated by lipid membrane composition.

    Science.gov (United States)

    Grasso, E J; Calderón, R O

    2013-02-01

    The urothelium, a specialized epithelium that covers the mucosa cell surface of the urinary bladder, undergoes dramatic morphological changes during the micturition cycle that involve a membrane apical traffic. This traffic was first described as a lysosomal pathway, in addition to the known endocytosis/exocytosis membrane recycling. In an attempt to understand the role of membrane lipid composition in those effects, we previously described the lipid-dependent leakage of the endocytosed vesicle content. In this work, we demonstrated clear differences in the traffic of both the fluid probe and the membrane-bound probe in urothelial umbrella cells by using spectrofluorometry and/or confocal and epifluorescence microscopy. Different membrane lipid compositions were established by using three diet formulae enriched in oleic acid, linoleic acid and a commercial formula. Between three and five animals for each dietary treatment were used for each analysis. The decreased endocytosis of both fluid and membrane-bound probes (approximately 32 and 49 % lower, respectively) in oleic acid-derived umbrella cells was concomitant with an increased recycling (approximately 4.0 and 3.7 times, respectively) and diminished sorting to the lysosome (approximately 23 and 37 %, respectively) when compared with the control umbrella cells. The higher intravesicular pH and the impairment of the lysosomal pathway of oleic acid diet-derived vesicles compared to linoleic acid diet-derived vesicles and control diet-derived vesicles correlate with our findings of a lower V-ATPase activity previously reported. We integrated the results obtained in the present and previous work to determine the sorting of endocytosed material (fluid and membrane-bound probes) into the different cell compartments. Finally, the weighted average effect of the individual alterations on the intracellular distribution was evaluated. The results shown in this work add evidences for the modulatory role of the membrane

  4. Lysosomal membrane stability in laboratory- and field-exposed terrestrial isopods Porcellio scaber (Isopoda, Crustacea).

    Science.gov (United States)

    Nolde, Natasa; Drobne, Damjana; Valant, Janez; Padovan, Ingrid; Horvat, Milena

    2006-08-01

    Two established methods for assessment of the cytotoxicity of contaminants, the lysosomal latency (LL) assay and the neutral red retention (NRR) assay, were successfully applied to in toto digestive gland tubes (hepatopancreas) of the terrestrial isopod Porcellio scaber (Isopoda, Crustacea). In vitro exposure of isolated gland tubes to copper was used as a positive control to determine the performance of the two methods. Lysosomal latency and the NRR assay were then used on in vivo (via food) laboratory-exposed animals and on field populations. Arbitrarily selected criteria for determination of the fitness of P. scaber were set on the basis of lysosomal membrane stability (LMS) as assessed with in toto digestive gland tubes. Decreased LMS was detected in animals from all polluted sites, but cytotoxicity data were not in agreement with concentrations of pollutants. Lysosomal membrane stability in the digestive gland tubes of animals from an environment in Idrija, Slovenia that was highly polluted with mercury (260 microg/g dry wt food and 1,600 microg/g dry wt soil) was less affected than LMS in laboratory animals fed with 5 and 50 microg Hg/g dry weight for 3 d. This probably indicates tolerance of P. scaber to mercury in the mercury-polluted environment and/or lower bioavailability of environmental mercury. In animals from the vicinity of a thermal power plant with environmental mercury concentrations three to four orders of magnitude lower than those in Idrija, LMS was severely affected. In general, the LL assay was more sensitive than the NRR assay. The LMS assay conducted on digestive gland tubes of terrestrial isopods is highly recommended for integrated biomarker studies.

  5. Lack of the Lysosomal Membrane Protein, GLMP, in Mice Results in Metabolic Dysregulation in Liver.

    Directory of Open Access Journals (Sweden)

    Xiang Yi Kong

    Full Text Available Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1 has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmp gt/gt mice (formerly known as Ncu-g1gt/gt mice were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmp gt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmp gt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmp gt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmp gt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmp gt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmp gt/gt liver homogenates, compared to hepatocytes and liver from wild type mice. Gene expression analysis showed an increased expression of genes involved in fatty acid uptake and lipogenesis in Glmp gt/gt liver compared to wild type. Our findings are in agreement with the metabolic alterations observed in other mouse models lacking lysosomal proteins, and with alterations characteristic for advanced chronic liver injury.

  6. Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes.

    Directory of Open Access Journals (Sweden)

    Barbara Hissa

    Full Text Available In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MβCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MβCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MβCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.

  7. Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins

    OpenAIRE

    Kaariainen, L; Hashimoto, K.; Saraste, J; Virtanen, I; Penttinen, K

    1980-01-01

    Temperature-sensitive mutants of semliki forest virus (SFV) and sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chicken embryo fibroblasts. When antisera against purified glycoproteins and (125)I- labeled protein A from staphylococcus aureus were used only small amounts of virus glycoproteins were detected at the surface of SFV ts-1 and SIN Ts-10 infected cells incubated at the restrictive temperature (39 degrees C). When the muta...

  8. Effects of lysosomal membrane protein depletion on the Salmonella-containing vacuole.

    Directory of Open Access Journals (Sweden)

    Everett A Roark

    Full Text Available Salmonella is an intracellular bacterial pathogen that replicates within a membrane-bound vacuole in host cells. The major lysosomal membrane proteins 1 and 2 (LAMP-1 and LAMP-2 are recruited to the Salmonella-containing vacuole as well as Salmonella- associated filaments (Sifs that emerge from the vacuole. LAMP-1 is a dominant membrane marker for the vacuole and Sifs. Its colocalization with both is dependent on a major secreted bacterial virulence protein, SifA. Here, we show that SifA is required for the recruitment of LAMP-2 and can be used as a second independent marker for both the bacterial vacuolar membrane and Sifs. Further, RNAi studies revealed that in LAMP-1 depleted cells, the bacteria remain membrane bound as measured by their association with LAMP-2 protein. In contrast, LAMP-2 depletion increased the amount of LAMP-1 free bacteria. Together, the data suggests that despite its abundance, LAMP-1 is not essential, but LAMP-2 may be partially important for the Salmonella-containing vacuolar membrane.

  9. Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins.

    Science.gov (United States)

    Kääriäinen, L; Hashimoto, K; Saraste, J; Virtanen, I; Penttinen, K

    1980-12-01

    Temperature-sensitive mutants of semliki forest virus (SFV) and sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chicken embryo fibroblasts. When antisera against purified glycoproteins and (125)I- labeled protein A from staphylococcus aureus were used only small amounts of virus glycoproteins were detected at the surface of SFV ts-1 and SIN Ts-10 infected cells incubated at the restrictive temperature (39 degrees C). When the mutant-infected cells were shifted to the permissive temperature (28 degrees C), in the presence of cycloheximide, increasing amounts of virus glycoproteins appeared at the cell surface from 20 to 80 min after the shift. Both monensin (10muM) and carbonylcyanide-p- trifluoromethoxyphenylhydrazone (FCCP; 10-20 muM) inhibited the appearance of virus membrane glycoproteins at the cell surface. Vinblastine sulfate (10 mug/ml) inhibited the transport by approximately 50 percent, whereas cytochalasin B (1 mug/ml) had only a marginal effect. Intracellular distribution of virus glycoproteins in the mutant-infected cells was visualized in double-fluorescence studies using lectins as markers for endoplasmic reticulum and Golgi apparatus. At 39 degrees C, the virus membrane glycoproteins were located at the endoplasmic reticulum, whereas after shift to 28 degrees C, a bright juxtanuclear reticular fluorescence was seen in the location of the Golgi apparatus. In the presence of monensin, the virus glycoproteins could migrate to the Golgi apparatus, although transport to the cell surface did not take place. When the shift was carried out in the presence of FCCP, negligible fluorescence was seen in the Golgi apparatus and the glycoproteins apparently remained in the rough endoplasmic reticulum. A rapid inhibition in the accumulation of virus glycoproteins at the cell surface was obtained when FCCP was added during the active transport period, whereas with monensin there was a delay of

  10. Alternative promoter usage of the membrane glycoprotein CD36

    Directory of Open Access Journals (Sweden)

    Whatling Carl

    2006-03-01

    Full Text Available Abstract Background CD36 is a membrane glycoprotein involved in a variety of cellular processes such as lipid transport, immune regulation, hemostasis, adhesion, angiogenesis and atherosclerosis. It is expressed in many tissues and cell types, with a tissue specific expression pattern that is a result of a complex regulation for which the molecular mechanisms are not yet fully understood. There are several alternative mRNA isoforms described for the gene. We have investigated the expression patterns of five alternative first exons of the CD36 gene in several human tissues and cell types, to better understand the molecular details behind its regulation. Results We have identified one novel alternative first exon of the CD36 gene, and confirmed the expression of four previously known alternative first exons of the gene. The alternative transcripts are all expressed in more than one human tissue and their expression patterns vary highly in skeletal muscle, heart, liver, adipose tissue, placenta, spinal cord, cerebrum and monocytes. All alternative first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins. The alternative promoters lack TATA-boxes and CpG islands. The upstream region of exon 1b contains several features common for house keeping gene and monocyte specific gene promoters. Conclusion Tissue-specific expression patterns of the alternative first exons of CD36 suggest that the alternative first exons of the gene are regulated individually and tissue specifically. At the same time, the fact that all first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins may suggest that the alternative first exons are coregulated in this cell type and environmental condition. The molecular mechanisms regulating CD36 thus appear to be unusually complex, which might reflect the multifunctional role of the gene in different tissues and cellular conditions.

  11. Iron-Mediated Lysosomal Membrane Permeabilization in Ethanol-Induced Hepatic Oxidative Damage and Apoptosis: Protective Effects of Quercetin

    Science.gov (United States)

    Li, Yanyan; Chen, Man; Xu, Yanyan; Yu, Xiao; Xiong, Ting; Du, Min; Sun, Jian; Liu, Liegang; Tang, Yuhan; Yao, Ping

    2016-01-01

    Iron, in its free ferrous states, can catalyze Fenton reaction to produce OH∙, which is recognized as a crucial role in the pathogenesis of alcoholic liver diseases (ALD). As a result of continuous decomposition of iron-containing compounds, lysosomes contain a pool of redox-active iron. To investigate the important role of intralysosomal iron in alcoholic liver injury and the potential protection of quercetin, male C57BL/6J mice fed by Lieber De Carli diets containing ethanol (30% of total calories) were cotreated by quercetin or deferoxamine (DFO) for 15 weeks and ethanol-incubated mice primary hepatocytes were pretreated with FeCl3, DFO, and bafilomycin A1 at their optimal concentrations and exposure times. Chronic ethanol consumption caused an evident increase in lysosomal redox-active iron accompanying sustained oxidative damage. Iron-mediated ROS could trigger lysosomal membrane permeabilization (LMP) and subsequent mitochondria apoptosis. The hepatotoxicity was attenuated by reducing lysosomal iron while being exacerbated by escalating lysosomal iron. Quercetin substantially alleviated the alcoholic liver oxidative damage and apoptosis by decreasing lysosome iron and ameliorating iron-mediated LMP, which provided a new prospective of the use of quercetin against ALD. PMID:27057276

  12. Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells.

    Science.gov (United States)

    Wang, Zheng; Cheng, Xingan; Meng, Qianqian; Wang, Peidan; Shu, Benshui; Hu, Qiongbo; Hu, Meiying; Zhong, Guohua

    2015-07-01

    Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3.

  13. Iron-Mediated Lysosomal Membrane Permeabilization in Ethanol-Induced Hepatic Oxidative Damage and Apoptosis: Protective Effects of Quercetin.

    Science.gov (United States)

    Li, Yanyan; Chen, Man; Xu, Yanyan; Yu, Xiao; Xiong, Ting; Du, Min; Sun, Jian; Liu, Liegang; Tang, Yuhan; Yao, Ping

    2016-01-01

    Iron, in its free ferrous states, can catalyze Fenton reaction to produce OH∙, which is recognized as a crucial role in the pathogenesis of alcoholic liver diseases (ALD). As a result of continuous decomposition of iron-containing compounds, lysosomes contain a pool of redox-active iron. To investigate the important role of intralysosomal iron in alcoholic liver injury and the potential protection of quercetin, male C57BL/6J mice fed by Lieber De Carli diets containing ethanol (30% of total calories) were cotreated by quercetin or deferoxamine (DFO) for 15 weeks and ethanol-incubated mice primary hepatocytes were pretreated with FeCl3, DFO, and bafilomycin A1 at their optimal concentrations and exposure times. Chronic ethanol consumption caused an evident increase in lysosomal redox-active iron accompanying sustained oxidative damage. Iron-mediated ROS could trigger lysosomal membrane permeabilization (LMP) and subsequent mitochondria apoptosis. The hepatotoxicity was attenuated by reducing lysosomal iron while being exacerbated by escalating lysosomal iron. Quercetin substantially alleviated the alcoholic liver oxidative damage and apoptosis by decreasing lysosome iron and ameliorating iron-mediated LMP, which provided a new prospective of the use of quercetin against ALD.

  14. Iron-Mediated Lysosomal Membrane Permeabilization in Ethanol-Induced Hepatic Oxidative Damage and Apoptosis: Protective Effects of Quercetin

    Directory of Open Access Journals (Sweden)

    Yanyan Li

    2016-01-01

    Full Text Available Iron, in its free ferrous states, can catalyze Fenton reaction to produce OH∙, which is recognized as a crucial role in the pathogenesis of alcoholic liver diseases (ALD. As a result of continuous decomposition of iron-containing compounds, lysosomes contain a pool of redox-active iron. To investigate the important role of intralysosomal iron in alcoholic liver injury and the potential protection of quercetin, male C57BL/6J mice fed by Lieber De Carli diets containing ethanol (30% of total calories were cotreated by quercetin or deferoxamine (DFO for 15 weeks and ethanol-incubated mice primary hepatocytes were pretreated with FeCl3, DFO, and bafilomycin A1 at their optimal concentrations and exposure times. Chronic ethanol consumption caused an evident increase in lysosomal redox-active iron accompanying sustained oxidative damage. Iron-mediated ROS could trigger lysosomal membrane permeabilization (LMP and subsequent mitochondria apoptosis. The hepatotoxicity was attenuated by reducing lysosomal iron while being exacerbated by escalating lysosomal iron. Quercetin substantially alleviated the alcoholic liver oxidative damage and apoptosis by decreasing lysosome iron and ameliorating iron-mediated LMP, which provided a new prospective of the use of quercetin against ALD.

  15. Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas

    DEFF Research Database (Denmark)

    Jensen, Stine Skov; Christensen, Karina; Aaberg-Jessen, Charlotte

    , the aim of this study was to investigate the immunohistochemical expression of LAMP-1, a membrane bound protein in lysosomes, in formalin fixed paraffin embedded tumor tissue from 23 diffuse astrocytomas, 17 anaplastic astrocytomas and 72 glioblastomas. The LAMP-1 expression was scored and compared...... with both tumor grade and patient survival. Moreover double immunofluorescence stainings with LAMP-1 and the stem cell marker CD133 as well as the macrophage marker CD68 were performed. The results showed that LAMP-1 was expressed in the vast majority of tumors being present in the cytoplasm of single tumor...... cells, cell clusters and in blood vessel endothelial cells. The LAMP-1 expression in glioblastomas was significantly higher than in diffuse and anaplastic astrocytomas (pLAMP-1 and patient overall survival was found. Double immunofluorescence staining...

  16. Entactin: ultrastructural localization of an ubiquitous basement membrane glycoprotein in mouse skin

    DEFF Research Database (Denmark)

    Horiguchi, Y; Fine, J D; Ljubimov, A V;

    1989-01-01

    Entactin is a recently described sulfated glycoprotein component of mouse endodermal cell-derived extracellular matrix and is present in a number of basement membranes. It has been ultrastructurally localized to both lamina densa and adjacent epithelial cell membranes in rodent kidney. In the pre...

  17. Identification of the milk fat globule membrane proteins. I. Isolation and partial characterization of glycoprotein B.

    Science.gov (United States)

    Basch, J J; Farrell, H M; Greenberg, R

    1976-11-02

    The salt soluble proteins from the fat globule membrane of cow's milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.

  18. Biochemical reconstitution of hemorrhagic-fever arenavirus envelope glycoprotein-mediated membrane fusion.

    Directory of Open Access Journals (Sweden)

    Celestine J Thomas

    Full Text Available The membrane-anchored proteins of enveloped viruses form labile spikes on the virion surface, primed to undergo large-scale conformational changes culminating in virus-cell membrane fusion and viral entry. The prefusion form of these envelope glycoproteins thus represents an important molecular target for antiviral intervention. A critical roadblock to this endeavor has been our inability to produce the prefusion envelope glycoprotein trimer for biochemical and structural analysis. Through our studies of the GPC envelope glycoprotein of the hemorrhagic fever arenaviruses, we have shown that GPC is unique among class I viral fusion proteins in that the mature complex retains a stable signal peptide (SSP in addition to the conventional receptor-binding and transmembrane fusion subunits. In this report we show that the recombinant GPC precursor can be produced as a discrete native-like trimer and that its proteolytic cleavage generates the mature glycoprotein. Proteoliposomes containing the cleaved GPC mediate pH-dependent membrane fusion, a characteristic feature of arenavirus entry. This reaction is inhibited by arenavirus-specific monoclonal antibodies and small-molecule fusion inhibitors. The in vitro reconstitution of GPC-mediated membrane-fusion activity offers unprecedented opportunities for biochemical and structural studies of arenavirus entry and its inhibition. To our knowledge, this report is the first to demonstrate functional reconstitution of membrane fusion by a viral envelope glycoprotein.

  19. Mechanism of Aloe Vera extract protection against UVA: shelter of lysosomal membrane avoids photodamage.

    Science.gov (United States)

    Rodrigues, Daniela; Viotto, Ana Cláudia; Checchia, Robert; Gomide, Andreza; Severino, Divinomar; Itri, Rosangela; Baptista, Maurício S; Martins, Waleska Kerllen

    2016-03-01

    The premature aging (photoaging) of skin characterized by wrinkles, a leathery texture and mottled pigmentation is a well-documented consequence of exposure to sunlight. UVA is an important risk factor for human cancer also associated with induction of inflammation, immunosuppression, photoaging and melanogenesis. Although herbal compounds are commonly used as photoprotectants against the harmful effects of UVA, the mechanisms involved in the photodamage are not precisely known. In this study, we investigated the effects of Aloe Vera (Aloe barbadensis mil) on the protection against UVA-modulated cell killing of HaCaT keratinocytes. Aloe Vera exhibited the remarkable ability of reducing both in vitro and in vivo photodamage, even though it does not have anti-radical properties. Interestingly, the protection conferred by Aloe Vera was associated with the maintenance of membrane integrity in both mimetic membranes and intracellular organelles. The increased lysosomal stability led to a decrease in lipofuscinogenesis and cell death. This study explains why Aloe Vera extracts offer protection against photodamage at a cellular level in both the UV and visible spectra, leading to its beneficial use as a supplement in protective dermatological formulations.

  20. Increased expression of lysosome membrane protein 2 in glomeruli of patients with idiopathic membranous nephropathy

    NARCIS (Netherlands)

    Rood, I.M.; Merchant, M.L.; Wilkey, D.W.; Zhang, T.; Zabrouskov, V.; Vlag, J. van der; Dijkman, H.B.P.M.; Willemsen, B.K.; Wetzels, J.F.M.; Klein, J.B.; Deegens, J.K.J.

    2015-01-01

    Urinary microvesicles constitute a rich source of membrane-bound and intracellular proteins that may provide important clues of pathophysiological mechanisms in renal disease. In the current study, we analyzed and compared the proteome of urinary microvesicles from patients with idiopathic

  1. Lysosomal membrane stability and metallothioneins in digestive gland of mussels (Mytilus galloprovincialis Lam.) as biomarkers in a field study

    Energy Technology Data Exchange (ETDEWEB)

    Petrovic, S.; Ozretic, B.; Krajnovic-Ozretic, M. [Ruder Boskovic Inst., Center for Marine Research, Rovinj (Croatia); Bobinac, D. [Rijeka Univ., Dept. of Anatomy, Rijeka (Croatia)

    2001-07-01

    The lysosomal membrane destabilisation and the metallothionein content in the digestive gland cells of mussels (Mytilus galloprovincialis Lam.), collected along the east coast of the North Adriatic (Istrian and Kvarnerine coast, Croatia), were examined over a period of four years (1996-1999). The lysosomal membrane stability, as a biomarker of general stress, showed that the membrane labilisation period in mussels from polluted, urban- and industrial-related areas was significantly decreased (p<0.05) when compared to mussels from control, clean sea water sites. In the harbour of Rijeka, the most contaminated site, the lysosomal membrane stability was reduced by more than 70% compared to the control. This method also proved to be a useful biomarker for detection of additional stress caused by short-term hypoxia that occurred once during this study inside the polluted and periodically quite eutrophic Pula Harbour. The concentration of metallothioneins in the mussel digestive gland, as a specific biomarker of exposure to heavy metals, did not reveal significant differences (p<0.05) between sites covered by this study. (Author)

  2. Lysosomal trapping of a radiolabeled substrate of P-glycoprotein as a mechanism for signal amplification in PET

    OpenAIRE

    Kannan, Pavitra; Brimacombe, Kyle R.; Kreisl, William C.; Liow, Jeih-San; Zoghbi, Sami S.; Telu, Sanjay; Zhang, Yi; Pike, Victor W.; Halldin, Christer; Gottesman, Michael M.; Innis, Robert B.; Hall, Matthew D.

    2011-01-01

    The radiotracer [11C]N-desmethyl-loperamide (dLop) images the in vivo function of P-glycoprotein (P-gp), a transporter that blocks the entry of drugs that are substrates into brain. When P-gp is inhibited, [11C]dLop, a potent opiate agonist, enters and becomes trapped in the brain. This trapping is beneficial from an imaging perspective, because it amplifies the PET signal, essentially by accumulating radioactivity over time. As we previously demonstrated that this trapping was not caused by ...

  3. Engineered nanomaterial-induced lysosomal membrane permeabilization and anti-cathepsin agents.

    Science.gov (United States)

    Bunderson-Schelvan, Melisa; Holian, Andrij; Hamilton, Raymond F

    2017-01-01

    Engineered nanomaterials (ENMs), or small anthropogenic particles approximately < 100 nm in size and of various shapes and compositions, are increasingly incorporated into commercial products and used for industrial and medical purposes. There is an exposure risk to both the population at large and individuals in the workplace with inhalation exposures to ENMs being a primary concern. Further, there is increasing evidence to suggest that certain ENMs may represent a significant health risk, and many of these ENMs exhibit distinct similarities with other particles and fibers that are known to induce adverse health effects, such as asbestos, silica, and particulate matter (PM). Evidence regarding the importance of lysosomal membrane permeabilization (LMP) and release of cathepsins in ENM toxicity has been accumulating. The aim of this review was to describe our current understanding of the mechanisms leading to ENM-associated pathologies, including LMP and the role of cathepsins with a focus on inflammation. In addition, anti-cathepsin agents, some of which have been tested in clinical trials and may prove useful for ameliorating the harmful effects of ENM exposure, are examined.

  4. Ovine Herpesvirus 2 Glycoproteins B, H, and L Are Sufficient for, and Viral Glycoprotein Ov8 Can Enhance, Cell-Cell Membrane Fusion.

    Science.gov (United States)

    AlHajri, Salim M; Cunha, Cristina W; Nicola, Anthony V; Aguilar, Hector C; Li, Hong; Taus, Naomi S

    2017-03-15

    Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus in the genus Macavirus that is carried asymptomatically by sheep. Infection of poorly adapted animals with OvHV-2 results in sheep-associated malignant catarrhal fever, a fatal disease characterized by lymphoproliferation and vasculitis. There is no treatment or vaccine for the disease and no cell culture system to propagate the virus. The lack of cell culture has hindered studies of OvHV-2 biology, including its entry mechanism. As an alternative method to study OvHV-2 glycoproteins responsible for membrane fusion as a part of the entry mechanism, we developed a virus-free cell-to-cell membrane fusion assay to identify the minimum required OvHV-2 glycoproteins to induce membrane fusion. OvHV-2 glycoproteins B, H, and L (gB, gH, and gL) were able to induce membrane fusion together but not when expressed individually. Additionally, open reading frame Ov8, unique to OvHV-2, was found to encode a transmembrane glycoprotein that can significantly enhance membrane fusion. Thus, OvHV-2 gB, gH, and gL are sufficient to induce membrane fusion, while glycoprotein Ov8 plays an enhancing role by an unknown mechanism.IMPORTANCE Herpesviruses enter cells via attachment of the virion to the cellular surface and fusion of the viral envelope with cellular membranes. Virus-cell membrane fusion is an important step for a successful viral infection. Elucidating the roles of viral glycoproteins responsible for membrane fusion is critical toward understanding viral entry. Entry of ovine herpesvirus 2 (OvHV-2), the causative agent of sheep associated-malignant catarrhal fever, which is one of the leading causes of death in bison and other ungulates, has not been well studied due to the lack of a cell culture system to propagate the virus. The identification of OvHV-2 glycoproteins that mediate membrane fusion may help identify viral and/or cellular factors involved in OvHV-2 cell tropism and will advance investigation of cellular

  5. Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo*

    Science.gov (United States)

    Goldman, Stephen D. B.; Krise, Jeffrey P.

    2010-01-01

    The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [3H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo. PMID:20007703

  6. Niemann-Pick C1 functions independently of Niemann-Pick C2 in the initial stage of retrograde transport of membrane-impermeable lysosomal cargo.

    Science.gov (United States)

    Goldman, Stephen D B; Krise, Jeffrey P

    2010-02-12

    The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [(3)H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

  7. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) assemble via distinct modes.

    Science.gov (United States)

    Terasawa, Kazue; Tomabechi, Yuri; Ikeda, Mariko; Ehara, Haruhiko; Kukimoto-Niino, Mutsuko; Wakiyama, Motoaki; Podyma-Inoue, Katarzyna A; Rajapakshe, Anupama R; Watabe, Tetsuro; Shirouzu, Mikako; Hara-Yokoyama, Miki

    2016-10-21

    Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) have a large, heavily glycosylated luminal domain composed of two subdomains, and are the most abundant protein components in lysosome membranes. LAMP-1 and LAMP-2 have distinct functions, and the presence of both proteins together is required for the essential regulation of autophagy to avoid embryonic lethality. However, the structural aspects of LAMP-1 and LAMP-2 have not been elucidated. In the present study, we demonstrated that the subdomains of LAMP-1 and LAMP-2 adopt the unique β-prism fold, similar to the domain structure of the dendritic cell-specific-LAMP (DC-LAMP, LAMP-3), confirming the conserved aspect of this family of lysosome-associated membrane proteins. Furthermore, we evaluated the effects of the N-domain truncation of LAMP-1 or LAMP-2 on the assembly of LAMPs, based on immunoprecipitation experiments. We found that the N-domain of LAMP-1 is necessary, whereas that of LAMP-2 is repressive, for the organization of a multimeric assembly of LAMPs. Accordingly, the present study suggests for the first time that the assembly modes of LAMP-1 and LAMP-2 are different, which may underlie their distinct functions.

  8. Concanavalin A/IFN-gamma triggers autophagy-related necrotic hepatocyte death through IRGM1-mediated lysosomal membrane disruption.

    Directory of Open Access Journals (Sweden)

    Chih-Peng Chang

    Full Text Available Interferon-gamma (IFN-γ, a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1 translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/- mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.

  9. The salubrious effect of tamoxifen [correction of Tamaxifen] on serum marker enzymes, glycoproteins, and lysosomal enzymes level in breast cancer woman.

    Science.gov (United States)

    Thangaraju, M; Rameshbabu, J; Vasavi, H; Ilanchezhian, S; Vinitha, R; Sachdanandam, P

    1998-08-01

    Tumour markers correlate strongly with prognosis based on tumour burden and surgical resectability. If chemotherapy is extremely effective in certain stage of the disease, the sensitive marker may be of great use in monitoring disease response and drug treatment. Hence, this study was launched to evaluate the changes in tumour marker enzymes like lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT), alkaline phosphatase, and acid phosphatase in before and after 3 and 6 months tamoxifen treated breast cancer patients. In addition, the changes in serum glycoproteins viz., hexose, hexosamine, and sialic acid and lysosomal enzymes such as N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, and beta-D-glucuronidase were analysed in these patients. These values were compared with their age matched healthy control subjects. At 6 months evaluation, the tamoxifen treated postmenopausal breast cancer women showed a statistically significant decreased (p sialic acid and N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, and beta-D-glucuronidase were decreased significantly (p breast cancer as well as the bone demineralisation in postmenopausal breast cancer women. Thus, tamoxifen may also have its antitumour activity through its beneficial effects on tumour marker enzymes and serum proteins in breast cancer women.

  10. Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis : LAMP-2 deficient mice develop pancreatitis

    NARCIS (Netherlands)

    Mareninova, Olga A; Sendler, Matthias; Malla, Sudarshan Ravi; Yakubov, Iskandar; French, Samuel W; Tokhtaeva, Elmira; Vagin, Olga; Oorschot, Viola; Lüllmann-Rauch, Renate; Blanz, Judith; Dawson, David; Klumperman, Judith; Lerch, Markus M; Mayerle, Julia; Gukovsky, Ilya; Gukovskaya, Anna S

    2015-01-01

    BACKGROUND & AIMS: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated memb

  11. Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis : LAMP-2 deficient mice develop pancreatitis

    NARCIS (Netherlands)

    Mareninova, Olga A; Sendler, Matthias; Malla, Sudarshan Ravi; Yakubov, Iskandar; French, Samuel W; Tokhtaeva, Elmira; Vagin, Olga; Oorschot, Viola; Lüllmann-Rauch, Renate; Blanz, Judith; Dawson, David; Klumperman, Judith; Lerch, Markus M; Mayerle, Julia; Gukovsky, Ilya; Gukovskaya, Anna S

    2015-01-01

    BACKGROUND & AIMS: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated

  12. Lysosomal membrane permeabilization: Carbon nanohorn-induced reactive oxygen species generation and toxicity by this neglected mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Mei, E-mail: happy_deercn@163.com [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology 5-2, 1-1-1 Higashi, Tsukuba 305-8565 (Japan); Zhang, Minfang; Tahara, Yoshio; Chechetka, Svetlana; Miyako, Eijiro [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology 5-2, 1-1-1 Higashi, Tsukuba 305-8565 (Japan); Iijima, Sumio [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology 5-2, 1-1-1 Higashi, Tsukuba 305-8565 (Japan); Faculty of Science and Technology, Meijo University, 1-501 Shiogamaguchi, Tenpaku, Nagoya 468-8502 (Japan); Yudasaka, Masako, E-mail: m-yudasaka@aist.go.jp [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology 5-2, 1-1-1 Higashi, Tsukuba 305-8565 (Japan)

    2014-10-01

    Understanding the molecular mechanisms responsible for the cytotoxic effects of carbon nanomaterials is important for their future biomedical applications. Carbon nanotubular materials induce the generation of reactive oxygen species (ROS), which causes cell death; however, the exact details of this process are still unclear. Here, we identify a mechanism of ROS generation that is involved in the apoptosis of RAW264.7 macrophages caused by excess uptake of carbon nanohorns (CNHs), a typical type of carbon nanotubule. CNH accumulated in the lysosomes, where they induced lysosomal membrane permeabilization (LMP) and the subsequent release of lysosomal proteases, such as cathepsins, which in turn caused mitochondrial dysfunction and triggered the generation of ROS in the mitochondria. The nicotinamide adenine dinucleotide phosphate oxidase was not directly involved in CNH-related ROS production, and the ROS generation cannot be regulated by mitochondrial electron transport chain. ROS fed back to amplify the mitochondrial dysfunction, leading to the subsequent activation of caspases and cell apoptosis. Carbon nanotubules commonly accumulate in the lysosomes after internalization in cells; however, lysosomal dysfunction has not attracted much attention in toxicity studies of these materials. These results suggest that LMP, a neglected mechanism, may be the primary reason for carbon nanotubule toxicity. - Highlights: • We clarify an apoptotic mechanism of RAW264.7 cells caused by carbon nanohorns. • In the meantime, the mechanism of CNH-induced ROS generation is identified. • LMP is the initial factor of CNH-induced ROS generation and cell death. • Cathepsins work as mediators that connect LMP and mitochondrial dysfunction.

  13. Dynamic Viral Glycoprotein Machines: Approaches for Probing Transient States That Drive Membrane Fusion

    Directory of Open Access Journals (Sweden)

    Natalie K. Garcia

    2016-01-01

    Full Text Available The fusion glycoproteins that decorate the surface of enveloped viruses undergo dramatic conformational changes in the course of engaging with target cells through receptor interactions and during cell entry. These refolding events ultimately drive the fusion of viral and cellular membranes leading to delivery of the genetic cargo. While well-established methods for structure determination such as X-ray crystallography have provided detailed structures of fusion proteins in the pre- and post-fusion fusion states, to understand mechanistically how these fusion glycoproteins perform their structural calisthenics and drive membrane fusion requires new analytical approaches that enable dynamic intermediate states to be probed. Methods including structural mass spectrometry, small-angle X-ray scattering, and electron microscopy have begun to provide new insight into pathways of conformational change and fusion protein function. In combination, the approaches provide a significantly richer portrait of viral fusion glycoprotein structural variation and fusion activation as well as inhibition by neutralizing agents. Here recent studies that highlight the utility of these complementary approaches will be reviewed with a focus on the well-characterized influenza virus hemagglutinin fusion glycoprotein system.

  14. Constitutive expression of a COOH-terminal leucine mutant of lysosome-associated membrane protein-1 causes its exclusive localization in low density intracellular vesicles.

    Science.gov (United States)

    Akasaki, Kenji; Shiotsu, Keiko; Michihara, Akihiro; Ide, Norie; Wada, Ikuo

    2014-07-01

    Lysosome-associated membrane protein-1 (LAMP-1) is a type I transmembrane protein with a short cytoplasmic tail that possesses a lysosome-targeting signal of GYQTI(382)-COOH. Wild-type (WT)-LAMP-1 was exclusively localized in high density lysosomes, and efficiency of LAMP-1's transport to lysosomes depends on its COOH-terminal amino acid residue. Among many different COOH-terminal amino acid substitution mutants of LAMP-1, a leucine-substituted mutant (I382L) displays the most efficient targeting to late endosomes and lysosomes [Akasaki et al. (2010) J. Biochem. 148: , 669-679]. In this study, we generated two human hepatoma cell lines (HepG2 cell lines) that stably express WT-LAMP-1 and I382L, and compared their intracellular distributions. The subcellular fractionation study using Percoll density gradient centrifugation revealed that WT-LAMP-1 had preferential localization in the high density secondary lysosomes where endogenous human LAMP-1 was enriched. In contrast, a major portion of I382L was located in a low density fraction. The low density fraction also contained approximately 80% of endogenous human LAMP-1 and significant amounts of endogenous β-glucuronidase and LAMP-2, which probably represents occurrence of low density lysosomes in the I382L-expressing cells. Double immunofluorescence microscopic analyses distinguished I382L-containing intracellular vesicles from endogenous LAMP-1-containing lysosomes and early endosomes. Altogether, constitutive expression of I382L causes its aberrant intracellular localization and generation of low density lysosomes, indicating that the COOH-terminal isoleucine is critical for normal localization of LAMP-1 in the dense lysosomes.

  15. Morphological alteration, lysosomal membrane fragility and apoptosis of the cells of Indian freshwater sponge exposed to washing soda (sodium carbonate).

    Science.gov (United States)

    Mukherjee, Soumalya; Ray, Mitali; Dutta, Manab Kumar; Acharya, Avanti; Mukhopadhyay, Sandip Kumar; Ray, Sajal

    2015-12-01

    Washing soda is chemically known as sodium carbonate and is a component of laundry detergent. Domestic effluent, drain water and various anthropogenic activities have been identified as major routes of sodium carbonate contamination of the freshwater ecosystem. The freshwater sponge, Eunapius carteri, bears ecological and evolutionary significance and is considered as a bioresource in aquatic ecosystems. The present study involves estimation of morphological damage, lysosomal membrane integrity, activity of phosphatases and apoptosis in the cells of E. carteri under the environmentally realistic concentrations of washing soda. Exposure to washing soda resulted in severe morphological alterations and damages in cells of E. carteri. Fragility and destabilization of lysosomal membranes of E. carteri under the sublethal exposure was indicative to toxin induced physiological stress in sponge. Prolonged exposure to sodium carbonate resulted a reduction in the activity of acid and alkaline phosphatases in the cells of E. carteri. Experimental concentration of 8 mg/l of washing soda for 192 h yielded an increase in the physiological level of cellular apoptosis among the semigranulocytes and granulocytes of E. carteri, which was suggestive to possible shift in apoptosis mediated immunoprotection. The results were indicative of an undesirable shift in the immune status of sponge. Contamination of the freshwater aquifers by washing soda thus poses an alarming ecotoxicological threat to sponges.

  16. Synthesis and localization of two sulphated glycoproteins associated with basement membranes and the extracellular matrix

    DEFF Research Database (Denmark)

    Hogan, B L; Taylor, A; Kurkinen, M;

    1982-01-01

    Two sulphated glycoproteins (sgps) of apparent molecular weight (Mr) 180,000 and 150,000, are synthesized by murine PYS and PF HR9 parietal endoderm and Swiss 3T3 cells. The Mr 150,000 sgp has a similar chemical structure to the sulphated glycoprotein, C, synthesized and laid down in Reichert......'s membrane by mouse embryo parietal endoderm cells (Hogan, B. L.M., A. Taylor, and A.R. Cooper, 1982, Dev. Biol., 90:210-214). Both the Mr 180,000 and 150,000 sgps are deposited in the detergent-insoluble matrix of cultured cells, but they do not apparently undergo any disulphide-dependent intermolecular...

  17. Chromatographic finger print analysis and lysosomal membrane stabilisation activity of active fraction of Alstonia scholaris leaf extract in arthritic rats

    Directory of Open Access Journals (Sweden)

    Swapnil Goyal

    2014-01-01

    Full Text Available Object: The present study was aimed to assess the anti-arthritic activity of chloroform fraction of Alstonia scholaris leaf extract against Freund′s complete adjuvant (FCA-induced arthritis in rats. Materials and Methods: The anti-inflammatory activity of various fractions of ethanolic extract of Alstonia scholaris at concentration of 100 mg/kg was studied using the carrageenan-induced inflammatory models. The chloroform fraction shows significant anti-inflammatory activity. The chloroform fraction was further studied for anti-arthritic activity and HPTLC fingerprint analysis. For anti-arthritic activity, the active chloroform fraction was administered at the concentrations of 50 and 100 mg/kg body weight. The effect of chloroform fraction on liver ALP, ACP and LDH levels of lysosomal enzymes of FCA arthritic animals were studied. Indomethacin and prednisolone (10 mg/kg was used as standard. HPTLC studies were carried out using CAMAG HPTLC system equipped with linomat IV applicator, TLC scanner; Reprostar 3 and WIN CATS-4 software were used. Results: The chloroform fraction at 100 mg/kg, showed maximum inhibition (34.16% of inflammation induced by carrageenan. In FCA-induced arthritis, the chloroform fraction showed a highly significant reduction in paw volume (50 mg/kg-72.71%; 100 mg/kg-74.35%. The levels of lysosomal enzymes were significantly decreased in the chloroform fraction-treated groups. Conclusion: The possible mechanism of action of the chloroform fraction of Alstonia scholaris leaf extract may be through its stabilising action on lysosomal membranes. Future studies will provide new insights into the anti-arthritic activity of Alstonia scholaris and isolation of compound from it may eventually lead to development of a new class of anti-arthritic agent.

  18. The proteome of lysosomes.

    Science.gov (United States)

    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.

  19. Lysosomal cell death at a glance

    DEFF Research Database (Denmark)

    Aits, Sonja; Jaattela, Marja

    2013-01-01

    Lysosomes serve as the cellular recycling centre and are filled with numerous hydrolases that can degrade most cellular macromolecules. Lysosomal membrane permeabilization and the consequent leakage of the lysosomal content into the cytosol leads to so-called "lysosomal cell death". This form...... of cell death is mainly carried out by the lysosomal cathepsin proteases and can have necrotic, apoptotic or apoptosis-like features depending on the extent of the leakage and the cellular context. This article summarizes our current knowledge on lysosomal cell death with an emphasis on the upstream...... mechanisms that lead to lysosomal membrane permeabilization....

  20. Lysosomal membrane permeabilization is involved in oxidative stress-induced apoptotic cell death in LAMP2-deficient iPSCs-derived cerebral cortical neurons

    Directory of Open Access Journals (Sweden)

    Cheuk-Yiu Law

    2016-03-01

    Our results from cellular fractionation and inhibitor blockade experiments further revealed that oxidative stress-induced apoptosis in the LAMP2-deficient cortical neurons was caused by increased abundance of cytosolic cathepsin L. These results suggest the involvement of lysosomal membrane permeabilization in the LAMP2 deficiency associated neural injury.

  1. Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas

    DEFF Research Database (Denmark)

    Jensen, Stine S; Aaberg-Jessen, Charlotte; Christensen, Karina G

    2013-01-01

    astrocytomas and compared with tumor grade and overall patient survival. Moreover, double immunofluorescence stainings were performed with LAMP-1 and the astrocytic marker GFAP and the putative stem cell marker CD133 on ten glioblastomas. Most tumors expressed the LAMP-1 protein in the cytoplasm of the tumor...... cells, while the blood vessels were positive in all tumors. The percentage of LAMP-1 positive tumor cells and staining intensities increased with tumor grade but variations in tumors of the same grade were also found. No association was found between LAMP-1 expression and patient overall survival......Targeting of lysosomes is a novel therapeutic anti-cancer strategy for killing the otherwise apoptosis-resistant cancer cells. Such strategies are urgently needed for treatment of brain tumors, especially the glioblastoma, which is the most frequent and most malignant type. The aim of the present...

  2. Conformation of trimeric envelope glycoproteins: the CD4-dependent membrane fusion mechanism of HIV-1.

    Science.gov (United States)

    Yingliang, Wu; Hong, Yi; Zhijian, Cao; Wenxin, Li

    2007-08-01

    The HIV-1 envelope glycoproteins are assembled by the trimeric gp120s and gp41s proteins. The gp120 binds sequentially to CD4 and coreceptor for initiating virus entry. Because of noncovalent interaction and heavy glycosylation for envelope glycoproteins, it is highly difficult to determine entire envelope glycoproteins structure now. Such question extremely limits our good understanding of HIV-1 membrane fusion mechanism. Here, a novel and reasonable assembly model of trimeric gp120s and gp41s was proposed based on the conformational dynamics of trimeric gp120-gp41 complex and gp41, respectively. As for gp41, the heptad repeat sequences in the gp41 C-terminal is of enormous flexibility. On the contrary, the heptad repeat sequences in the gp41 N-terminal likely present stable three-helical bundle due to strong nonpolar interaction, and they were predicted to associate three alpha1 helixes from the non-neutralizing face of the gp120 inner domain, which is quite similar to gp41 fusion core structure. Such interaction likely leads to the formation of noncovalent gp120-gp41 complex. In the proposed assembly of trimeric gp120-gp41 complex, three gp120s present not only perfectly complementary and symmetrical distribution around the gp41, but also different flexibility degree in the different structural domains. Thus, the new model can well explain numerous experimental phenomena, present plenty of structural information, elucidate effectively HIV-1 membrane fusion mechanism, and direct to further develop vaccine and novel fusion inhibitors.

  3. TRAIL death receptor 4 signaling via lysosome fusion and membrane raft clustering in coronary arterial endothelial cells: evidence from ASM knockout mice.

    Science.gov (United States)

    Li, Xiang; Han, Wei-Qing; Boini, Krishna M; Xia, Min; Zhang, Yang; Li, Pin-Lan

    2013-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4 (DR4), have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MR) clustering and the formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and co-localized with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1 (+/+)) mice. Furthermore, TRAIL triggered ASM translocation, ceramide production, and NADPH oxidase aggregation in MR clusters in Smpd1 ( +/+ ) CAECs, whereas these observations were not found in Smpd1 (-/-) CAECs. Moreover, ASM deficiency reduced TRAIL-induced O(2) (-[Symbol: see text]) production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer, we found that Lamp-1 (lysosome membrane marker protein) and ganglioside G(M1) (MR marker) were trafficking together in Smpd1 (+/+) CAECs, which was absent in Smpd1 (-/-) CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1 (-/-) CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking, membrane fusion and formation of MR redox signaling platforms

  4. Internalization, lysosomal degradation and new synthesis of surface membrane CD4 in phorbol ester-activated T-lymphocytes and U-937 cells

    DEFF Research Database (Denmark)

    Petersen, C M; Christensen, E I; Andresen, B S

    1992-01-01

    degradation was low in resting cells. Endocytosis and/or degradation of anti-CD4 mAb was suppressed by H7, and by inhibitors of membrane traffic (Monensin) and lysosome function (methylamine, chloroquine). Immunocytochemistry localized CD4 to the surface of unstimulated T-cells. Upon PMA stimulation...... occasional labeling was seen in endosomes but whole cell CD4 decreased dramatically. However, methylamine-treated PMA blasts showed accumulation of CD4 in lysosomes and accordingly, pulse-chase experiments in biolabeled cell cultures suggested a manifest reduction of CD4 half-life in response to PMA. Despite...... in activated cells was further evidenced by metabolic labeling and Northern blot analysis demonstrating unaltered or slightly increased CD4 protein and mRNA levels resulting from PMA. Our findings demonstrate that phorbol esters downregulate the cellular CD4 pool by endocytosis and subsequent lysosomal...

  5. Membrane-Associated RING-CH proteins associate with Bap31 and target CD81 and CD44 to lysosomes.

    Directory of Open Access Journals (Sweden)

    Eric Bartee

    Full Text Available Membrane-associated RING-CH (MARCH proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER. We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins.

  6. Membrane-Associated RING-CH proteins associate with Bap31 and target CD81 and CD44 to lysosomes.

    Science.gov (United States)

    Bartee, Eric; Eyster, Craig A; Viswanathan, Kasinath; Mansouri, Mandana; Donaldson, Julie G; Früh, Klaus

    2010-12-02

    Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins.

  7. IRES-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems.

    Directory of Open Access Journals (Sweden)

    Andreas K Brödel

    Full Text Available Internal ribosome entry site (IRES elements found in the 5' untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR of the Cricket paralysis virus (CrPV genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established

  8. Structure of the Membrane Anchor of Pestivirus Glycoprotein Erns, a Long Tilted Amphipathic Helix

    Science.gov (United States)

    Aberle, Daniel; Muhle-Goll, Claudia; Bürck, Jochen; Wolf, Moritz; Reißer, Sabine; Luy, Burkhard; Wenzel, Wolfgang; Ulrich, Anne S.; Meyers, Gregor

    2014-01-01

    Erns is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the Erns membrane contact, processing and secretion. PMID:24586172

  9. Retinal glycoprotein enrichment by concanavalin a enabled identification of novel membrane autoantigen synaptotagmin-1 in equine recurrent uveitis.

    Science.gov (United States)

    Swadzba, Margarete E; Hauck, Stefanie M; Naim, Hassan Y; Amann, Barbara; Deeg, Cornelia A

    2012-01-01

    Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allowed detection of IgG reactions to low abundant proteins in sera of ERU patients. Synaptotagmin-1, a Ca2+-sensing protein in synaptic vesicles, was identified as autoantigen candidate from the pre-enriched glycoprotein fraction by mass spectrometry and was validated as a highly prevalent autoantigen by enzyme-linked immunosorbent assay. Analysis of Syt1 expression in retinas of ERU cases showed a downregulation in the majority of ERU affected retinas to 24%. Results pointed to a dysregulation of retinal neurotransmitter release in ERU. Identification of synaptotagmin-1, the first cell membrane associated autoantigen in this spontaneous autoimmune disease, demonstrated that examination of tissue fractions can lead to the discovery of previously undetected novel autoantigens. Further experiments will address its role in ERU pathology.

  10. IgG platelet antibodies in EDTA-dependent pseudothrombocytopenia bind to platelet membrane glycoprotein IIb.

    Science.gov (United States)

    Fiorin, F; Steffan, A; Pradella, P; Bizzaro, N; Potenza, R; De Angelis, V

    1998-08-01

    EDTA-dependent pseudothrombocytopenia (PTCP) consists of an inappropriate low platelet count caused by autoantibodies present in the serum samples reacting with platelets only in EDTA-anticoagulated blood. By using immunoprecipitation and Western blot techniques, we studied the immunochemical specificity of platelet agglutinating autoantibodies in the serum samples of 10 patients with PTCP. Furthermore, to evaluate a possible role of PTCP-associated IgG autoantibodies in increased platelet turnover, we assayed the plasma glycocalicin (GC) level and calculated the GC index for every patient. Our results provide direct evidence that an epitope located on platelet membrane glycoprotein IIb is recognized by PTCP-associated IgG antibodies; moreover GC levels in patients with EDTA-dependent PTCP were similar to control levels, thus excluding an increased platelet turnover. We conclude that antiplatelet antibodies directed against platelet cryptantigens are unlikely to have a major role in the increased removal of cells from circulation.

  11. Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells

    OpenAIRE

    Su Yeon Shim; Srinivasarao Karri; Sampson Law; Schatzl, Hermann M.; Sabine Gilch

    2016-01-01

    Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrPSc) of the cellular prion protein (PrPc). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrPc into PrPSc. Within neurons, PrPSc accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dy...

  12. Lysosomal membrane stability and metallothionein content in Mytilus galloprovincialis (L.), as biomarkers Combination with trace metal concentrations

    Energy Technology Data Exchange (ETDEWEB)

    Domouhtsidou, G.P.; Dailianis, S.; Kaloyianni, M.; Dimitriadis, V.K

    2004-03-01

    The simultaneous study of two biomarkers, the lysosomal membrane stability (LMS) of the digestive gland and the metallothionein (MT) content of the digestive gland, the gills and the mantle/gonad complex of the mussel Mytilus galloprovincialis was examined in an enclosed estuarine system in June and October 2001. Mussels were collected along the Gulf of Thermaikos (northern Greece) from stations displaying a pollution gradient, while Olympiada in the Gulf of Strymonikos was used as a reference station. In addition, the heavy metal (Cd, Pb, Cu and Zn) content, using atomic absorption spectrophotometry (AAS), were evaluated in the digestive gland, the gills and the mantle/gonad complex of mussels collected from the same sites and seasons. LMS values were significantly greater, and the MT content of the studied tissues were significantly less in mussels collected from the reference station compared to mussels from more polluted stations located in the Gulf of Thermaikos. Significant correlation was observed between the MT content of the gills and the mantle/gonad complex with the LMS values.

  13. Cardenolide-Induced Lysosomal Membrane Permeabilization Demonstrates Therapeutic Benefits in Experimental Human Non-Small Cell Lung Cancers

    Directory of Open Access Journals (Sweden)

    Tatjana Mijatovic

    2006-05-01

    Full Text Available Non-small cell lung cancers (NSCLCs are the leading cause of cancer deaths in most developed countries. Targeting heat shock protein 70 (Hsp70 expression and function, together with the induction of lysosomal membrane permeabilization (LMP, could overcome the multiple anti-cell death mechanisms evidenced in NSCLCs that are responsible for the failure of currently used chemotherapeutic drugs. Because cardenolides bind to the sodium pump, they affect multiple signaling pathways and thus have a number of marked effects on tumor cell behavior. The aim of the present study was to characterize in vitro and in vivo the antitumor effects of a new cardenolide (UNBS1450 on experimental human NSCLCs. UNBS1450 is a potent source of in vivo antitumor activity in the case of paclitaxeland oxaliplatin-resistant subcutaneous human NCIH727 and orthotopic A549 xenografts in nude mice. In vitro UNBS1450-mediated antitumor activity results from the induction of nonapoptotic cell death. UNBS1450 mediates the decrease of Hsp70 at both mRNA and protein levels, and this is at least partly due to UNBS1450-induced downregulation of NFAT5/ TonEBP (a factor responsible for the transcriptional control of Hsp70. These effects were paralleled by the induction of LMP, as evidenced by acridine orange staining and immunofluorescence analysis for cathepsin B accumulation.

  14. Polycyclic aromatic hydrocarbon body residues and lysosomal membrane destabilization in mussels exposed to the Dubai Star bunker fuel oil (intermediate fuel oil 380) spill in San Francisco Bay.

    Science.gov (United States)

    Hwang, Hyun-Min; Stanton, Beckye; McBride, Toby; Anderson, Michael J

    2014-05-01

    Following the spill of bunker fuel oil (intermediate fuel oil 380, approximately 1500-3000 L) into San Francisco Bay in October 2009, polycyclic aromatic hydrocarbon (PAH) concentrations in mussels from moderately oiled areas increased up to 87 554 ng/g (dry wt) and, 3 mo later, decreased to concentrations found in mussels collected prior to oiling, with a biological half-life of approximately 16 d. Lysosomal membrane destabilization increased in mussels with higher PAH body burdens.

  15. Gastrointestinal Hormone Cholecystokinin Increases P-Glycoprotein Membrane Localization and Transport Activity in Caco-2 Cells.

    Science.gov (United States)

    Yano, Kentaro; Shimizu, Saori; Tomono, Takumi; Ogihara, Takuo

    2017-09-01

    It was reported that stimulation of taste receptor type 2 member 38 by a bitter substance, phenylthiocarbamide (PTC), increased P-glycoprotein (P-gp) mRNA level and transport activity via release of the gastrointestinal hormone cholecystokinin-8 (CCK-8) at 9 h. Therefore, we hypothesized that CCK-8 and PTC might also regulate P-gp activity more rapidly via a different mechanism. As a result, we found that the pretreatment of human colon adenocarcinoma (Caco-2) cells with 10-mM PTC significantly decreased the intracellular accumulation of P-gp substrate rhodamine 123 (Rho123) compared with the control after 90-min incubation. Moreover, CCK-8 treatments significantly reduced the accumulation of Rho123 within 30 min, compared with the control. On the other hand, when Caco-2 cells were pretreated with PTC, the efflux ratio of Rho123 was significantly increased compared with control. The efflux ratio of Rho123 in CCK-8 treatment cells was also significantly increased compared with control. Furthermore, CCK-8 increased the phosphorylation of the scaffold proteins ezrin, radixin, and moesin, which regulate translocation of P-gp to the plasma membrane. Therefore, our results indicate that PTC induced release of CCK-8, which in turn induced the phosphorylation of ezrin, radixin, and moesin proteins, leading to upregulation of P-gp transport activity via increased membrane localization of P-gp. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  16. P-glycoprotein senses its substrates and the lateral membrane packing density: consequences for the catalytic cycle.

    Science.gov (United States)

    Aänismaa, Päivi; Gatlik-Landwojtowicz, Ewa; Seelig, Anna

    2008-09-23

    P-glycoprotein (ABCB1) prevents absorption (e.g., blood-brain barrier) or enhances excretion (e.g., kidney) by moving substrates from the cytosolic to the extracellular membrane leaflet at the expense of ATP hydrolysis. It translocates various drugs and functions in membranes exhibiting different lateral packing densities. To gain more functional insight, we measured the temperature dependence of the P-glycoprotein ATPase activity in NIH-MDR1-G185 cell membranes in the absence and presence of three drugs (promazine, verapamil, and PSC833), exhibiting significantly different transporter affinities. Activation enthalpies (Delta H(++)) and entropies ( TDelta S(++)) were derived from Eyring plots. In the absence of drugs, the activation enthalpy and the free energy of activation for P-glycoprotein ATPase activity was determined as Delta H(++) = 92.6 +/- 4.2 kJ/mol and Delta G(++) = 73.1 +/- 7.2 kJ/mol, respectively. Increasing the drug concentration reduced the activation enthalpy, whereby the drug with the highest transporter affinity had the strongest effect (DeltaDelta H(++) = -21%). The free energy of activation decreased for activating (DeltaDelta G(++) = approximately -3.8%) and increased for inhibitory compounds (DeltaDelta G(++) = approximately +0.7%). The drug-specific changes of the free energy of activation are thus barely above thermal energy. A comparison with literature data revealed that a decrease of the lateral membrane packing density reduces the enthalpic and the entropic contribution to the free energy of activation. Although the P-glycoprotein ATPase activity increases only slightly with decreasing lateral membrane packing density, the mode of action changes from strongly entropy-driven at high, to essentially enthalpy-driven at low packing densities. This suggests that the transporter and the membrane form a functional entity.

  17. Conglutinin binds the HIV-1 envelope glycoprotein gp 160 and inhibits its interaction with cell membrane CD4

    DEFF Research Database (Denmark)

    Andersen, Ove; Sørensen, A M; Svehag, S E

    1991-01-01

    The highly glycosylated envelope glycoprotein (gp 160) of human immunodeficiency virus (HIV) interacts with the CD4 molecule present on the membrane of CD4+ cells and is involved in the pathobiology of HIV infection. Lectins bind glycoproteins through non-covalent interactions with specific hexose...... residues. The mammalian C-type lectin bovine conglutinin was examined for its ability to interact with recombinant gp160 (rgp160) produced in vaccinia virus-infected BHK21 cells. Specific binding of conglutinin to rgp160 was demonstrated by ELISA. The interaction of bovine conglutinin with rgp160...... of the binding of rgp160 to the CD4 receptor on CEM 13 cells, as demonstrated by FACS analyses. These results indicate that conglutinin may inhibit the infection with HIV-1 through its interaction with the viral envelope glycoprotein....

  18. Structure of transmembrane domain of lysosome-associated membrane protein type 2a (LAMP-2A) reveals key features for substrate specificity in chaperone-mediated autophagy.

    Science.gov (United States)

    Rout, Ashok K; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico

    2014-12-19

    Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.

  19. Multiscale molecular dynamics simulations of lipid interactions with P-glycoprotein in a complex membrane.

    Science.gov (United States)

    Domicevica, Laura; Koldsø, Heidi; Biggin, Philip C

    2017-09-02

    P-glycoprotein (P-gp) can transport a wide range of very different hydrophobic organic molecules across the membrane. Its ability to extrude molecules from the cell creates delivery problems for drugs that target proteins in the central nervous system (CNS) and also causes drug-resistance in many forms of cancer. Whether a drug will be susceptible to export by P-gp is difficult to predict and currently this is usually assessed with empirical and/or animal models. Thus, there is a need to better understand how P-gp works at the molecular level in order to fulfil the 3Rs: Refinement, reduction and replacement of animals in research. As structural information increasingly becomes available, our understanding at the molecular level improves. Proteins like P-gp are however very dynamic entities and thus one of the most appropriate ways to study them is with molecular dynamics simulations, especially as this can capture the influence of the surrounding environment. Recent parameterization developments have meant that it is now possible to simulate lipid bilayers that more closely resemble in vivo membranes in terms of their composition. In this report we construct a complex lipid bilayer that mimics the composition of brain epithelial cells and examine the interactions of it with P-gp. We find that the negatively charged phosphatidylserine lipids in the inner leaflet of the membrane tend to form an annulus around P-gp. We also observed the interaction of cholesterol with three distinct areas of the P-gp. Potential of mean force (PMF) calculations suggest that a crevice between transmembrane helices 10 and 12 has particularly favourable interaction energy for cholesterol. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Ankyrin and band 3 differentially affect expression of membrane glycoproteins but are not required for erythroblast enucleation

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Peng, E-mail: peng-ji@fsm.northwestern.edu [Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611 (United States); Whitehead Institute for Biomedical Research, Cambridge, MA 02142 (United States); Lodish, Harvey F. [Whitehead Institute for Biomedical Research, Cambridge, MA 02142 (United States); Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer Ankyrin and band 3 are not required for erythroblasts enucleation. Black-Right-Pointing-Pointer Loss of ankyrin does not affect erythroid membrane glycoprotein expression. Black-Right-Pointing-Pointer Loss of band 3 influences erythroid membrane glycoprotein expression. -- Abstract: During late stages of mammalian erythropoiesis the nucleus undergoes chromatin condensation, migration to the plasma membrane, and extrusion from the cytoplasm surrounded by a segment of plasma membrane. Since nuclear condensation occurs in all vertebrates, mammalian erythroid membrane and cytoskeleton proteins were implicated as playing important roles in mediating the movement and extrusion of the nucleus. Here we use erythroid ankyrin deficient and band 3 knockout mouse models to show that band 3, but not ankyrin, plays an important role in regulating the level of erythroid cell membrane proteins, as evidenced by decreased cell surface expression of glycophorin A in band 3 knockout mice. However, neither band 3 nor ankyrin are required for enucleation. These results demonstrate that mammalian erythroblast enucleation does not depend on the membrane integrity generated by the ankyrin-band 3 complex.

  1. Anthrax lethal toxin induced lysosomal membrane permeabilization and cytosolic cathepsin release is Nlrp1b/Nalp1b-dependent.

    Directory of Open Access Journals (Sweden)

    Kathleen M Averette

    Full Text Available NOD-like receptors (NLRs are a group of cytoplasmic molecules that recognize microbial invasion or 'danger signals'. Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell death termed pyronecrosis. Bacillus anthracis lethal toxin (LT, is recognized by a subset of alleles of the NLR protein Nlrp1b, resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane permeabilization (LMP. The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis.

  2. Lysosomal membrane stability plays a major role in the cytotoxic activity of the anti-proliferative agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT).

    Science.gov (United States)

    Gutierrez, Elaine M; Seebacher, Nicole A; Arzuman, Laila; Kovacevic, Zaklina; Lane, Darius J R; Richardson, Vera; Merlot, Angelica M; Lok, Hiu; Kalinowski, Danuta S; Sahni, Sumit; Jansson, Patric J; Richardson, Des R

    2016-07-01

    The potent and selective anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), localizes in lysosomes and forms cytotoxic copper complexes that generate reactive oxygen species (ROS), resulting in lysosomal membrane permeabilization (LMP) and cell death. Herein, the role of lysosomal membrane stability in the anti-tumor activity of Dp44mT was investigated. Studies were performed using molecules that protect lysosomal membranes against Dp44mT-induced LMP, namely heat shock protein 70 (HSP70) and cholesterol. Up-regulation or silencing of HSP70 expression did not affect Dp44mT-induced LMP in MCF7 cells. In contrast, cholesterol accumulation in lysosomes induced by the well characterized cholesterol transport inhibitor, 3-β-[2-(diethyl-amino)ethoxy]androst-5-en-17-one (U18666A), inhibited Dp44mT-induced LMP and markedly and significantly (peffect of U18666A in increasing lysosomal cholesterol and preventing the cytotoxic activity of Dp44mT was not due to induced autophagy. Instead, U18666A was found to decrease lysosomal turnover, resulting in autophagosome accumulation. Moreover, preincubation with U18666A did not prevent the ability of Dp44mT to induce autophagosome synthesis, indicating that autophagic initiation via Dp44mT occurs independently of LMP. These studies demonstrate the significance of lysosomal membrane stability in relation to the ability of Dp44mT to execute tumor cell death and overcome pro-survival autophagy. Hence, lysosomal-dependent cell death induced by Dp44mT serves as an important anti-tumor strategy. These results are important for comprehensively understanding the mechanism of action of Dp44mT.

  3. Lysosomal cell death mechanisms in aging.

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. α-Tocopherols modify the membrane dipole potential leading to modulation of ligand binding by P-glycoprotein

    OpenAIRE

    Davis, Sterenn; Davis, Benjamin M.; Richens, Joanna L.; Vere, Kelly-Ann; Petrov, Peter G.; Winlove, C. Peter; O’Shea, Paul

    2015-01-01

    α-Tocopherol (vitamin E) has attracted considerable attention as a potential protective or palliative agent. In vitro, its free radical-scavenging antioxidant action has been widely demonstrated. In vivo, however, vitamin E treatment exhibits negligible benefits against oxidative stress. α-Tocopherol influences lipid ordering within biological membranes and its derivatives have been suggested to inhibit the multi-drug efflux pump, P-glycoprotein (P-gp). This study employs the fluorescent memb...

  5. Assembly of arenavirus envelope glycoprotein GPC in detergent-soluble membrane microdomains.

    Science.gov (United States)

    Agnihothram, Sudhakar S; Dancho, Brooke; Grant, Kenneth W; Grimes, Mark L; Lyles, Douglas S; Nunberg, Jack H

    2009-10-01

    The family Arenaviridae includes a number of highly pathogenic viruses that are responsible for acute hemorrhagic fevers in humans. Genetic diversity among arenavirus species in their respective rodent hosts supports the continued emergence of new pathogens. In the absence of available vaccines or therapeutic agents, the hemorrhagic fever arenaviruses remain a serious public health and biodefense concern. Arenaviruses are enveloped virions that assemble and bud from the plasma membrane. In this study, we have characterized the microdomain organization of the virus envelope glycoprotein (GPC) on the cell surface by using immunogold electron microscopy. We find that Junín virus (JUNV) GPC clusters into discrete microdomains of 120 to 160 nm in diameter and that this property of GPC is independent of its myristoylation and of coexpression with the virus matrix protein Z. In cells infected with the Candid#1 strain of JUNV, and in purified Candid#1 virions, these GPC microdomains are soluble in cold Triton X-100 detergent and are thus distinct from conventional lipid rafts, which are utilized by numerous other viruses for assembly. Virion morphogenesis ultimately requires colocalization of viral components, yet our dual-label immunogold staining studies failed to reveal a spatial association of Z with GPC microdomains. This observation may reflect either rapid Z-dependent budding of virus-like particles upon coassociation or a requirement for additional viral components in the assembly process. Together, these results provide new insight into the molecular basis for arenavirus morphogenesis.

  6. Methoxypolyethylene glycol-block-polycaprolactone diblock copolymers reduce P-glycoprotein efflux in the absence of a membrane fluidization effect while stimulating P-glycoprotein ATPase activity.

    Science.gov (United States)

    Zastre, Jason; Jackson, John K; Wong, Wesley; Burt, Helen M

    2007-04-01

    We have previously shown that amphiphilic diblock copolymers composed of methoxypolyethylene glycol-b-polycaprolactone (MePEG-b-PCL) increased the cellular accumulation and reduced the basolateral to apical flux of the P-glycoprotein substrate, rhodamine 123 (R-123) in caco-2 cells. The purpose of this study was to investigate membrane perturbation effects of MePEG-b-PCL diblock copolymers with erythrocyte membranes and caco-2 cells and the effect on P-gp ATPase activity. The diblock copolymer MePEG(17)-b-PCL(5) induced increasing erythrocyte hemolysis at concentrations which correlated with increasing accumulation of R-123 into caco-2 cells. However, no increase in cellular accumulation of R-123 by non-P-gp expressing cells was observed, suggesting that diblock did not enhance the transmembrane passive diffusion of R-123, but that the accumulation enhancement effect of the diblock in caco-2 cells was likely mediated primarily via P-gp inhibition. Fluorescence anisotropy measurements of membrane fluidity and P-gp ATPase activity demonstrated that MePEG(17)-b-PCL(5) decreased caco-2 membrane fluidity while stimulating ATPase activity approximately threefold at concentrations that maximally enhanced R-123 caco-2 accumulation. These results suggest that inhibition of P-gp efflux by MePEG(17)-b-PCL(5) does not appear to be related to increases in membrane fluidity or through inhibition in P-gp ATPase activities, which are two commonly reported cellular effects for P-gp inhibition mediated by surfactants.

  7. Loss of lysosomal membrane protein NCU-G1 in mice results in spontaneous liver fibrosis with accumulation of lipofuscin and iron in Kupffer cells

    Directory of Open Access Journals (Sweden)

    Xiang Y. Kong

    2014-03-01

    Full Text Available Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1gt/gt mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1gt/gt liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1gt/gt Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1gt/gt mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage.

  8. Endocytosed 2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils.

    Directory of Open Access Journals (Sweden)

    Tadakazu Okoshi

    Full Text Available Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid

  9. Biosensor based on lectin and lipid membranes for detection of serum glycoproteins in infected patients with dengue.

    Science.gov (United States)

    Luna, Débora M N; Oliveira, Maria D L; Nogueira, Maurício L; Andrade, Cesar A S

    2014-05-01

    In this work, we developed a biosystem based on Concanavalin A (ConA) and lipid membranes to recognize glycoproteins from the serum of patients contaminated with dengue serotypes 1, 2 and 3 (DENV1, DENV2 and DENV3). The modified gold electrode was characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and atomic force microscopy. Morphological analyses of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC-ConA, DPPC-ConA-DENV1, DPPC-ConA-DENV2 and DPPC-ConA-DENV3 revealed the existence of a non-uniform covering and large globules. EIS and CV measurements have shown that redox probe reactions on the modified gold electrodes were partially blocked due to the adsorption of lipid-ConA system and reveal the interaction response of the immobilized ConA to the presence of glycoproteins of dengue serum. The biosystem exhibited a wide linear response to different concentrations of sera of dengue serotypes 1, 2 and 3. A higher impedimetric response to glycoproteins present in dengue serotype 3 was observed. Our results demonstrate the applicability of lectin and lipid membranes to the development of biosensors for dengue infections.

  10. LYSOSOMAL DISRUPTION BY BACTERIAL TOXINS

    Science.gov (United States)

    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

  11. Myristoylation of the Arenavirus Envelope Glycoprotein Stable Signal Peptide Is Critical for Membrane Fusion but Dispensable for Virion Morphogenesis

    Science.gov (United States)

    York, Joanne

    2016-01-01

    ABSTRACT Arenaviruses are responsible for severe and often fatal hemorrhagic disease. In the absence of effective antiviral therapies and vaccines, these viruses pose serious threats to public health and biodefense. Arenaviruses enter the host cell by fusion of the viral and endosomal membranes, a process mediated by the virus envelope glycoprotein GPC. Unlike other class I viral fusion proteins, GPC retains its stable signal peptide (SSP) as an essential third subunit in the mature complex. SSP spans the membrane twice and is myristoylated at its cytoplasmic N terminus. Mutations that abolish SSP myristoylation have been shown to reduce pH-induced cell-cell fusion activity of ectopically expressed GPC to ∼20% of wild-type levels. In order to examine the role of SSP myristoylation in the context of the intact virus, we used reverse genetics to generate Junín viruses (Candid #1 isolate) in which the critical glycine-2 residue in SSP was either replaced by alanine (G2A) or deleted (ΔG2). These mutant viruses produced smaller foci of infection in Vero cells and showed an ∼5-fold reduction in specific infectivity, commensurate with the defect in cell-cell fusion. However, virus assembly and GPC incorporation into budded virions were unaffected. Our findings suggest that the myristate moiety is cryptically disposed in the prefusion GPC complex and may function late in the fusion process to promote merging of the viral and cellular membranes. IMPORTANCE Hemorrhagic fever arenaviruses pose significant threats to public health and biodefense. Arenavirus entry into the host cell is promoted by the virus envelope glycoprotein GPC. Unlike other viral envelope glycoproteins, GPC contains a myristoylated stable signal peptide (SSP) as an essential third subunit. Myristoylation has been shown to be important for the membrane fusion activity of recombinantly expressed GPC. Here, we use reverse genetics to study the role of SSP myristoylation in the context of the intact

  12. 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.

    Science.gov (United States)

    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.

  13. Iron-Mediated Lysosomal Membrane Permeabilization in Ethanol-Induced Hepatic Oxidative Damage and Apoptosis: Protective Effects of Quercetin

    OpenAIRE

    Yanyan Li; Man Chen; Yanyan Xu; Xiao Yu; Ting Xiong; Min Du; Jian Sun; Liegang Liu; Yuhan Tang; Ping Yao

    2016-01-01

    Iron, in its free ferrous states, can catalyze Fenton reaction to produce OH∙, which is recognized as a crucial role in the pathogenesis of alcoholic liver diseases (ALD). As a result of continuous decomposition of iron-containing compounds, lysosomes contain a pool of redox-active iron. To investigate the important role of intralysosomal iron in alcoholic liver injury and the potential protection of quercetin, male C57BL/6J mice fed by Lieber De Carli diets containing ethanol (30% of total c...

  14. Understanding the accumulation of P-glycoprotein substrates within cells: The effect of cholesterol on membrane partitioning.

    Science.gov (United States)

    Subramanian, Nandhitha; Schumann-Gillett, Alexandra; Mark, Alan E; O'Mara, Megan L

    2016-04-01

    The apparent activity of the multidrug transporter P-glycoprotein (P-gp) is enhanced by the presence of cholesterol. Whether this is due to the direct effect of cholesterol on the activity of P-gp, its effect on the local concentration of substrate in the membrane, or its effect on the rate of entry of the drug into the cell, is unknown. In this study, molecular dynamics simulation techniques coupled with potential of mean force calculations have been used to investigate the role of cholesterol in the movement of four P-gp substrates across a POPC bilayer in the presence or absence of 10% cholesterol. The simulations suggest that the presence of cholesterol lowers the free energy associated with entering the middle of the bilayer in a substrate-specific manner. These findings suggest that P-gp substrates may preferentially accumulate in cholesterol-rich regions of the membrane, which may explain its enhanced transport activity.

  15. Copper Induced Lysosomal Membrane Destabilisation in Haemolymph Cells of Mediterranean Green Crab (Carcinus aestuarii, Nardo, 1847 from the Narta Lagoon (Albania

    Directory of Open Access Journals (Sweden)

    Valbona Aliko

    2015-10-01

    Full Text Available ABSTRACTDestabilisation of blood cell lysosomes in Mediterranean green crabCarcinus aestuarii was investigated using Neutral Red Retention Assay (NRRA. Crabs collected in Narta Lagoon, Vlora (Albania during May 2014 were exposed in the laboratory to sub-lethal, environmentally realistic concentrations of copper. Neutral Red Retention Time (NRRT and glucose concentration in haemolymph of animals were measured. The mean NRRT showed a significant reduction for the animals of the treatment group compared to the control one (from 118.6 ± 28.4 to 36.4 ± 10.48 min, p<0.05, indicating damage of lysosomal membrane. Haemolymph glucose concentration was significantly higher in the treatment group (from 37.8 ± 2.7 to 137.8.4 ± 16.2 mg/dL, p<0.05 than in control group, demonstrating the presence of stress on the animals. These results showed thatC. aestuarii could be used as a successful and reliable bioindicator for evaluating the exposure to contaminants in laboratory conditions. NRRA provides a successful tool for rapid assessment of heavy metal pollution effects on marine biota.

  16. Protective Effect of Prosopis cineraria Against N-Nitrosodiethylamine Induced Liver Tumor by Modulating Membrane Bound Enzymes and Glycoproteins

    Directory of Open Access Journals (Sweden)

    Naina Mohamed Pakkir Maideen

    2012-06-01

    Full Text Available Purpose: The objective of the present study was to evaluate the protective effect of methanol extract of Prosopis cineraria (MPC against N-nitrosodiethylamine (DEN, 200mg/kg induced Phenobarbital promoted experimental liver tumors in male Wistar rats. Methods: The rats were divided into four groups, each group consisting of six animals. Group 1 served as control animals. Liver tumor was induced in group 2, 3, and 4 and Group 3 animals received MPC 200mg/kg and Group 4 animals received MPC 400mg/kg. Results: Administration of DEN has brought down the levels of membrane bound enzymes like Na+/ K+ ATPase, Mg2+ ATPase and Ca2+ATPase which were later found to be increased by the administration of Prosopis cineraria (200 and 400mg/kg in dose dependent manner. The MPC extract also suppressed the levels of glycoproteins like Hexose, Hexosamine and Sialic acid when compared to liver tumor bearing animals. Conclusions: Our study suggests that MPC may extend its protective role by modulating the levels of membrane bound enzymes and suppressing glycoprotein levels.

  17. Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP fused antigens: a potential tool to develop DNA vaccines against flaviviruses

    Directory of Open Access Journals (Sweden)

    Rafael Dhalia

    2009-12-01

    Full Text Available Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the developent of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP. The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.A vacinação é a estratégia mais prática e o melhor custo-benefício para prevenir a maioria das infecções dos flavivirus, para os quais existe vacina disponível. Entretanto, as vacinas baseadas em vírus atenuados podem potencialmente promover efeitos colaterais e, mais raramente, reações fatais. Diante deste cenário, o desenvolvimento de estratégias alternativas de vacinação, como vacinas baseadas em DNA codificando seqüências específicas dos flavivirus, está sendo considerado

  18. Sensitivity to lysosome-dependent cell death is directly regulated by lysosomal cholesterol content.

    Directory of Open Access Journals (Sweden)

    Hanna Appelqvist

    Full Text Available Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1 protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2, which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.

  19. Complex interplay between the P-glycoprotein multidrug efflux pump and the membrane: its role in modulating protein function

    Directory of Open Access Journals (Sweden)

    Frances Jane Sharom

    2014-03-01

    Full Text Available Multidrug resistance in cancer is linked to expression of the P-glycoprotein multidrug transporter (Pgp, ABCB1, which exports many structurally diverse compounds from cells. Substrates first partition into the bilayer and then interact with a large flexible binding pocket within the transporter’s transmembrane regions. Pgp has been described as a hydrophobic vacuum cleaner or an outwardly-directed drug/lipid flippase. Recent X-ray crystal structures have shed some light on the nature of the drug-binding pocket and suggested routes by which substrates can enter it from the membrane. Detergents have profound effects on Pgp function, and several appear to be substrates. Biochemical and biophysical studies in vitro, some using purified reconstituted protein, have explored the effects of the membrane environment. They have demonstrated that Pgp is involved in a complex relationship with its lipid environment, which modulates the behaviour of its substrates, as well as various functions of the protein, including ATP hydrolysis, drug binding and drug transport. Membrane lipid composition and fluidity, phospholipid headgroup and acyl chain length all influence Pgp function. Recent studies focusing on thermodynamics and kinetics have revealed some important principles governing Pgp-lipid and substrate-lipid interactions, and how these affect drug binding and transport. In some cells, Pgp is associated with cholesterol-rich microdomains which may modulate its functions. The relationship between Pgp and cholesterol remains an open question; however it clearly affects several aspects of its function in addition to substrate-membrane partitioning. The action of Pgp modulators appears to depend on their membrane permeability, and membrane fluidizers and surfactants reverse drug resistance, likely via an indirect mechanism. A detailed understanding of how the membrane affects Pgp substrates and Pgp’s catalytic cycle may lead to new strategies to combat

  20. Brief exposure to copper activates lysosomal exocytosis.

    Science.gov (United States)

    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.

  1. Dissection of the role of the stable signal peptide of the arenavirus envelope glycoprotein in membrane fusion.

    Science.gov (United States)

    Messina, Emily L; York, Joanne; Nunberg, Jack H

    2012-06-01

    The arenavirus envelope glycoprotein (GPC) retains a stable signal peptide (SSP) as an essential subunit in the mature complex. The 58-amino-acid residue SSP comprises two membrane-spanning hydrophobic regions separated by a short ectodomain loop that interacts with the G2 fusion subunit to promote pH-dependent membrane fusion. Small-molecule compounds that target this unique SSP-G2 interaction prevent arenavirus entry and infection. The interaction between SSP and G2 is sensitive to the phylogenetic distance between New World (Junín) and Old World (Lassa) arenaviruses. For example, heterotypic GPC complexes are unable to support virion entry. In this report, we demonstrate that the hybrid GPC complexes are properly assembled, proteolytically cleaved, and transported to the cell surface but are specifically defective in their membrane fusion activity. Chimeric SSP constructs reveal that this incompatibility is localized to the first transmembrane segment of SSP (TM1). Genetic changes in TM1 also affect sensitivity to small-molecule fusion inhibitors, generating resistance in some cases and inhibitor dependence in others. Our studies suggest that interactions of SSP TM1 with the transmembrane domain of G2 may be important for GPC-mediated membrane fusion and its inhibition.

  2. Biophysical characterization and membrane interaction of the two fusion loops of glycoprotein B from herpes simplex type I virus.

    Directory of Open Access Journals (Sweden)

    Annarita Falanga

    Full Text Available The molecular mechanism of entry of herpesviruses requires a multicomponent fusion system. Cell invasion by Herpes simplex virus (HSV requires four virally encoded glycoproteins: namely gD, gB and gH/gL. The role of gB has remained elusive until recently when the crystal structure of HSV-1 gB became available and the fusion potential of gB was clearly demonstrated. Although much information on gB structure/function relationship has been gathered in recent years, the elucidation of the nature of the fine interactions between gB fusion loops and the membrane bilayer may help to understand the precise molecular mechanism behind herpesvirus-host cell membrane fusion. Here, we report the first biophysical study on the two fusion peptides of gB, with a particular focus on the effects determined by both peptides on lipid bilayers of various compositions. The two fusion loops constitute a structural subdomain wherein key hydrophobic amino acids form a ridge that is supported on both sides by charged residues. When used together the two fusion loops have the ability to significantly destabilize the target membrane bilayer, notwithstanding their low bilayer penetration when used separately. These data support the model of gB fusion loops insertion into cholesterol enriched membranes.

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

    Science.gov (United States)

    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.

  4. DNA adducts, benzo(a)pyrene monooxygenase activity, and lysosomal membrane stability in Mytilus galloprovincialis from different areas in Taranto coastal waters (Italy).

    Science.gov (United States)

    Pisoni, M; Cogotzi, L; Frigeri, A; Corsi, I; Bonacci, S; Iacocca, A; Lancini, L; Mastrototaro, F; Focardi, S; Svelto, M

    2004-10-01

    The aim of this study was to investigate the impact of environmental pollution at different stations along the Taranto coastline (Ionian Sea, Puglia, Italy) using several biomarkers of exposure and the effect on mussels, Mytilus galloprovincialis, collected in October 2001 and October 2002. Five sampling sites were compared with a "cleaner" reference site in the Aeronautics Area. In this study we also investigated the differences between adduct levels in gills and digestive gland. This Taranto area is the most significant industrial settlement on the Ionian Sea known to be contaminated by polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, heavy metals, etc. Exposure to PAHs was evaluated by measuring DNA adduct levels and benzo(a)pyrene monooxygenase activity (B(a)PMO); DNA adducts were analyzed by 32P-postlabeling with nuclease P1 enhancement in both gills and digestive glands to evaluate differences between DNA adduct levels in the two tissues. B(a)PMO was assayed in the microsomal fraction of the digestive glands as a result of the high expression of P450-metabolizing enzymes in this tissue. Lysosomal membrane stability, a potential biomarker of anthropogenic stress, was also evaluated in the digestive glands of mussels, by measuring the latent activity of beta-N-acetylhexosaminidase. Induction of DNA adducts was evident in both tissues, although the results revealed large tissue differences in DNA adduct formation. In fact, gills showed higher DNA adduct levels than did digestive gland. No significant differences were found in DNA adduct levels over time, with both tissues providing similar results in both years. DNA adduct levels were correlated with B(a)PMO activity in digestive gland in both years (r = 0.60 in 2001; r = 0.73 in 2002). Increases were observed in B(a)PMO activity and DNA adduct levels at different stations; no statistical difference was observed in B(a)PMO activity over the two monitoring campaigns. The membrane labilization

  5. α-Tocopherols modify the membrane dipole potential leading to modulation of ligand binding by P-glycoprotein.

    Science.gov (United States)

    Davis, Sterenn; Davis, Benjamin M; Richens, Joanna L; Vere, Kelly-Ann; Petrov, Peter G; Winlove, C Peter; O'Shea, Paul

    2015-08-01

    α-Tocopherol (vitamin E) has attracted considerable attention as a potential protective or palliative agent. In vitro, its free radical-scavenging antioxidant action has been widely demonstrated. In vivo, however, vitamin E treatment exhibits negligible benefits against oxidative stress. α-Tocopherol influences lipid ordering within biological membranes and its derivatives have been suggested to inhibit the multi-drug efflux pump, P-glycoprotein (P-gp). This study employs the fluorescent membrane probe, 1-(3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine, to investigate whether these effects are connected via influences on the membrane dipole potential (MDP), an intrinsic property of biological membranes previously demonstrated to modulate P-gp activity. α-Tocopherol and its non-free radical-scavenging succinate analog induced similar decreases in the MDP of phosphatidylcholine vesicles. α-Tocopherol succinate also reduced the MDP of T-lymphocytes, subsequently decreasing the binding affinity of saquinavir for P-gp. Additionally, α-tocopherol succinate demonstrated a preference for cholesterol-treated (membrane microdomain enriched) cells over membrane cholesterol-depleted cells. Microdomain disruption via cholesterol depletion decreased saquinavir's affinity for P-gp, potentially implicating these structures in the influence of α-tocopherol succinate on P-gp. This study provides evidence of a microdomain dipole potential-dependent mechanism by which α-tocopherol analogs influence P-gp activity. These findings have implications for the use of α-tocopherol derivatives for drug delivery across biological barriers.

  6. A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation.

    Science.gov (United States)

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

    2016-04-01

    To mediate the degradation of biomacromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca(2+) channel TRPML1 cause lysosomal storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca(2+)-dependent centripetal movement of lysosomes towards the perinuclear region (where autophagosomes accumulate) following autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca(2+) sensor that associates physically with the minus-end-directed dynactin-dynein motor, while PtdIns(3,5)P(2), a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P(2)-TRPML1-ALG-2-dynein signalling 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 to be caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Ca(2+) release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation.

  7. BK Channels Alleviate Lysosomal Storage Diseases by Providing Positive Feedback Regulation of Lysosomal Ca2+ Release.

    Science.gov (United States)

    Cao, Qi; Zhong, Xi Zoë; Zou, Yuanjie; Zhang, Zhu; Toro, Ligia; Dong, Xian-Ping

    2015-05-26

    Promoting lysosomal trafficking represents a promising therapeutic approach for lysosome storage diseases. Efficient Ca(2+) mobilization from lysosomes is important for lysosomal trafficking. Ca(2+) release from lysosomes could generate a negative potential in the lumen to disturb subsequent Ca(2+) release in the absence of counter ion flux. Here we report that lysosomes express big-conductance Ca(2+)-activated potassium (BK) channels that form physical and functional coupling with the lysosomal Ca(2+) release channel, TRPML1. Ca(2+) release via TRPML1 causes BK activation, which in turn facilitates further lysosomal Ca(2+) release and membrane trafficking. Importantly, BK overexpression rescues the impaired TRPML1-mediated Ca(2+) release and abnormal lysosomal storage in cells from Niemann-Pick C1 patients. Therefore, we have identified a lysosomal K(+) channel that provides a positive feedback mechanism to facilitate TRPML1-mediated Ca(2+) release and membrane trafficking. Our findings suggest that upregulating BK may be a potential therapeutic strategy for certain lysosomal storage diseases and common neurodegenerative disorders.

  8. Deacylation of the transmembrane domains of Sindbis virus envelope glycoproteins E1 and E2 does not affect low-pH-induced viral membrane fusion activity

    NARCIS (Netherlands)

    Smit, JM; Bittman, R; Wilschut, J

    2001-01-01

    The envelope glycoproteins E1 and E2 of Sindbis virus are palmitoylated at cysteine residues within their transmembrane domains (E1 at position 430, and E2 at positions 388 and 390), Here, we investigated the in vitro membrane fusion activity of Sindbis virus variants (derived from the Tote 1101 inf

  9. Investigations of hemadsorption characteristics of glycoprotein HN and F antigen of parainfluenza 3 virus outer membrane in vitro

    Directory of Open Access Journals (Sweden)

    Nišavić Jakov

    2006-01-01

    Full Text Available The objective of our investigations was to examine the hemadsorption characteristics of glycoprotein HN and F antigens of the outer membrane of the parainfluenza 3 virus. Six hours following inoculation of the cell line with the Vero virus PI3, previously activated with 0.025 g/dl trypsinversen, the appearance of less intense hemadsorption was established in antigen dilutions of 1:2 and 1:4. Hemadsorption was established in the mentioned cell lines also 12 h after inoculation in virus dilutions of 1:8 and 1:16, while 24 h following inoculation, hemadsorption was present also in a virus dilution of 1:64. Inhibition of hemadsorption in inoculated cells with immune serum dilutions of 1:16 was established 24 h after inoculation of the cell line with Vero samples of the virus PI3, activated with trypsinversen and with the addition of specific immune serum. .

  10. Asparagine-linked sugar chains of glycoproteins in calf thymocyte plasma membrane. Isolation and fractionation of oligosaccharides liberated by hydrazinolysis.

    Science.gov (United States)

    Yoshima, H; Takasaki, S; Kobata, A

    1980-07-01

    The plasma membrane glycoproteins of calf thymocytes were converted to glycopeptides by exhaustive pronase digestion. Glycopeptides with asparagine-linked sugar chains were separated from those with mucine-type sugar chains by Bio-Gel P-10 column chromatography. The asparagine-linked sugar chains were released as oligosaccharides from the peptide moiety by hydrazinolysis and labeled by reduction with NaB[3H]4. The radioactive oligosaccharides were fractionated into fifteen acidic components and ten neutral components by combination of paper electrophoresis and Bio-Gel P-4 column chromatography. The acidic nature of all fifteen acidic components can be ascribed to their N-acetylneuraminic acid residues. The Bio-Gel P-4 column chromatographic patterns of the neutral oligosaccharide fraction and of the neutral fraction obtained on sialidase treatment of the pooled acidic oligosaccharide fraction were totally different, indicating that the acidic oligosaccharides are not simple sialyl derivatives of the neutral oligosaccharides.

  11. Influence of ionic strength and beta2-glycoprotein I concentration on agglutination of like-charged phospholipid membranes.

    Science.gov (United States)

    Perutková, Šárka; Frank-Bertoncelj, Mojca; Rozman, Blaž; Kralj-Iglič, Veronika; Iglič, Aleš

    2013-11-01

    The effect of ionic strength on adhesion between negatively charged giant unilamellar vesicles induced by beta2-glycoprotein I (β2-GPI) was studied experimentally and theoretically. Measuring the effective angle of contact between adhering vesicles indicated that the strength of adhesion between vesicles decreases with increasing ionic strength, and increases with concentration of β2-GPI. In the theoretical part we focused on the study of the average orientation of β2-GPI near the charged membrane and its role in mediating the attractive interactions between the vesicles. β2-GPI proteins were modelled as rods with internal distribution of electric charge. The predictions of Monte Carlo simulations show orthogonal orientation of some of the membrane attached β2-GPI in narrow gap between two vesicles. On the contrary, at larger distances between vesicles the proteins are parallelly attached to the membrane surface. A local minimum of the free energy corresponding to β2-GPI-mediated adhesion of two neighbouring vesicles was predicted. The strength of adhesion was confirmed to decrease at high ionic strength.

  12. The change of serum leptin and its relationship with platelet membrane glycoprotein Ib in patients with coronary heart disease

    Institute of Scientific and Technical Information of China (English)

    XIA Dasheng; SONG Yanqiu; LI Chao; ZHANG Feng; WEI Minxin

    2007-01-01

    The aim of this paper was to investigate the change of serum leptin and its relationship with platelet membrane glycoprotein lb(GP Ib)in patients with coronary heart disease(CHD).The enrolled included 50 patients with CHD (CHD group)and 30 patients without CHD(control group)who were diagnosed by coronary angiography.The positive percentage and the average fluorescence intensity of platelet membrane GP Ib were detected by full-blood flow cytometry.Serum leptin was detected by enzyme linked immunosorbent assay.The positive percentage and the average fluorescence intensity of platetet membrane GP Ib in the CHD group were significantly lower than those in the control group (P<0.05).After correcting the differences of systolic blood pressure,body mass index(BMI),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol (HDL-C),fasting glucose,PPBS,fasting insulin and quantita tive insulin sensitive index,serum leptin level in the CHD group was significantly higher than that in the control group (P<0.05).Single factor correlative analysis revealed that serum leptin in CHD patients was negatively correlated with the average fluorescence intensity of platelet membrane GP Ib(P<0.05).Multifactorial stepwise regression analysis showed that serum leptin in CHD patients was independently negatively correlated with the average fluorescence intensity of platelet membrane GP Ib(P<0.05).Logistic analysis demonstrated that serum leptin was independently correlated with the risk of CHD(P<0.05).Hyperleptinemia was veri fied in CHD patients.The increase of serum leptin could affect blood platelet activation.Hyperleptinemia may play an important role in the pathogenesis of CHD.

  13. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jimin, E-mail: jimin.wang@yale.edu; Li, Yue; Modis, Yorgo, E-mail: yorgo.modis@yale.edu

    2014-04-15

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed.

  14. ErbB2-associated changes in the lysosomal proteome

    DEFF Research Database (Denmark)

    Nylandsted, Jesper; Becker, Andrea C; Bunkenborg, Jakob

    2011-01-01

    Late endosomes and lysosomes (hereafter referred to as lysosomes) play an essential role in the turnover of cellular macromolecules and organelles. Their biochemical characterization has so far depended on purification methods based on either density gradient centrifugations or magnetic...... purification of iron-loaded organelles. Owing to dramatic changes in lysosomal density and stability associated with lysosomal diseases and cancer, these methods are not optimal for the comparison of normal and pathological lysosomes. Here, we introduce an efficient method for the purification of intact...... lysosomes by magnetic immunoprecipitation with antibodies against the vacuolar-type H(+) -ATPase. Quantitative MS-based proteomics analysis of the obtained lysosomal membranes identified 60 proteins, most of which have previously been associated with the lysosomal compartment. Interestingly, the lysosomal...

  15. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

    Directory of Open Access Journals (Sweden)

    Ruben R Bender

    2016-06-01

    Full Text Available Receptor-targeted lentiviral vectors (LVs can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance. Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4 was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

  16. A possible receptor for β2 glycoprotein Ⅰ on the membrane of hepatoma cell line smmc7721

    Institute of Scientific and Technical Information of China (English)

    高普均; 朴云峰; 王小丛; 曲立科; 时阳; 杨翰仪

    2003-01-01

    Objectives To study the interaction of beta-2-glycoprotein Ⅰ (β2GP Ⅰ) with the membrane of hepatocytes and determine whether β2GP Ⅰ participates in HBV infection.Methods Ligand blotting, fluorescence microscopy, and fluorescence activated cell sorter (FACS) analysis were used to detect the specific interaction of β2GP Ⅰ with the hepatoma cell line smmc7721, the gastric carcinoma cell line SGC7901, and the lymphoma cell line HL-60.Results A specific 40 kDa β2GP Ⅰ band was observed by ligand blotting in the case of smmc7721 cells. No such band was observed in SGC7901 or HL-60 cells. Fluorescence microscopy also revealed specific binding of FITC-β2GP Ⅰ to smmc7721 cells, but neither to SGC7901 nor HL-60 cells. FACS analysis demonstrated that the binding rate of FITC-β2GP Ⅰ to smmc7721 cells was significantly higher than these in SGC7901 and HL-60 cells (P<0.01). The binding rate to smmc7721 cells did not increase with increasing amounts of FITC-β2GP Ⅰ.Conclusions There is a specific β2GP Ⅰ-binding protein on the membrane of hepatoma cells in cell line smmc7721 which as the β2GP Ⅰ receptor may participate in HBV infection of hepatocytes.

  17. Identification of potential platelet alloantigens in the Equidae family by comparison of gene sequences encoding major platelet membrane glycoproteins.

    Science.gov (United States)

    Boudreaux, Mary K; Humphries, Drew M

    2013-12-01

    Platelet alloantigens in horses may play an important role in the development of neonatal alloimmune thrombocytopenia (NAIT). The objective of this study was to evaluate genes encoding major platelet glycoproteins within the Equidae family in an effort to identify potential alloantigens. DNA was isolated from blood samples obtained from Equidae family members, including a Holsteiner-Oldenburg cross, a Quarter horse, a donkey, and a Plains zebra (Equus burchelli). Gene sequences encoding equine platelet membrane glycoproteins IIb, IIIa (integrin subunits αIIb and β3), Ia (integrin subunit α2), and Ibα were determined using PCR. Gene sequences were compared to the equine genome available on GenBank. Polymorphisms that would be predicted to result in amino acid changes on platelet surfaces were documented and compared with known alloantigenic sites documented on human platelets. Amino acid differences were predicted based on nucleotide sequences for all 4 genes. Nine differences were documented for αIIb, 5 differences were documented for β3, 7 differences were documented for α2, and 16 differences were documented for Ibα outside the macroglycopeptide region. This study represents the first effort at identifying potential platelet alloantigens in members of the Equidae Family based on evaluation of gene sequences. The data obtained form the groundwork for identifying potential platelet alloantigens involved in transfusion reactions and neonatal alloimmune thrombocytopenia (NAIT). More work is required to determine whether the predicted amino acid differences documented in this study play a role in alloimmunity, and whether other polymorphisms not detected in this study are present that may result in alloimmunity. © 2013 American Society for Veterinary Clinical Pathology.

  18. Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release.

    Science.gov (United States)

    Shen, Dongbiao; Wang, Xiang; Li, Xinran; Zhang, Xiaoli; Yao, Zepeng; Dibble, Shannon; Dong, Xian-ping; Yu, Ting; Lieberman, Andrew P; Showalter, Hollis D; Xu, Haoxing

    2012-03-13

    Lysosomal lipid accumulation, defects in membrane trafficking and altered Ca(2+) homoeostasis are common features in many lysosomal storage diseases. Mucolipin transient receptor potential channel 1 (TRPML1) is the principle Ca(2+) channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca(2+) release, measured using a genetically encoded Ca(2+) indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann-Pick (NP) disease cells. Sphingomyelins (SMs) are plasma membrane lipids that undergo sphingomyelinase (SMase)-mediated hydrolysis in the lysosomes of normal cells, but accumulate distinctively in lysosomes of NP cells. Patch-clamp analyses revealed that TRPML1 channel activity is inhibited by SMs, but potentiated by SMases. In NP-type C cells, increasing TRPML1's expression or activity was sufficient to correct the trafficking defects and reduce lysosome storage and cholesterol accumulation. We propose that abnormal accumulation of luminal lipids causes secondary lysosome storage by blocking TRPML1- and Ca(2+)-dependent lysosomal trafficking.

  19. Molecular mapping of pulmonary endothelial membrane glycoproteins of the intact rabbit lung

    Energy Technology Data Exchange (ETDEWEB)

    Merker, M.P.; Carley, W.W.; Gillis, C.N. (Yale Univ. School of Medicine, New Haven, CT (USA))

    1990-09-01

    To study the biochemical characteristics of endothelium in vivo, we radioiodinated endothelial membrane proteins of the perfused rabbit lung using a water soluble analog of the Bolton-Hunter reagent, {sup 125}I-sulfosuccinimidyl (hydroxyphenyl) propionate ({sup 125}I-s-SHPP). This technique led to a 10-fold increase in specific activity of radioiodinated lung membrane protein compared with our previously reported method using lactoperoxidase and glucose oxidase-catalyzed radioiodination. Tissue autoradiography confirmed that radioiodination was largely confined to the endothelium. Perfusion pressure, wet-to-dry weight ratios, and the morphological appearance of the lungs were within normal limits, indicating that the procedure does not cause apparent lung injury. Lectin binding to a crude membrane fraction of {sup 125}I-s-SHPP labeled lung led to isolation of several putative endothelial membrane proteins. Immunoprecipitation studies with appropriate antibodies enabled the identification of radioiodinated angiotensin-converting enzyme and beta 2-microglobulin associated major histocompatibility complex class I molecules in the membrane fraction. This technique will be useful for studying biochemical responses of the endothelium in vivo to a variety of pharmacological and physiology stimuli.

  20. TRPML: transporters of metals in lysosomes essential for cell survival?

    Science.gov (United States)

    Kiselyov, Kirill; Colletti, Grace A; Terwilliger, Austen; Ketchum, Kathleen; Lyons, Christopher W P; Quinn, James; Muallem, Shmuel

    2011-09-01

    Key aspects of lysosomal function are affected by the ionic content of the lysosomal lumen and, therefore, by the ion permeability in the lysosomal membrane. Such functions include regulation of lysosomal acidification, a critical process in delivery and activation of the lysosomal enzymes, release of metals from lysosomes into the cytoplasm and the Ca(2+)-dependent component of membrane fusion events in the endocytic pathway. While the basic mechanisms of lysosomal acidification have been largely defined, the lysosomal metal transport system is not well understood. TRPML1 is a lysosomal ion channel whose malfunction is implicated in the lysosomal storage disease Mucolipidosis Type IV. Recent evidence suggests that TRPML1 is involved in Fe(2+), Ca(2+) and Zn(2+) transport across the lysosomal membrane, ascribing novel physiological roles to this ion channel, and perhaps to its relatives TRPML2 and TRPML3 and illuminating poorly understood aspects of lysosomal function. Further, alterations in metal transport by the TRPMLs due to mutations or environmental factors may contribute to their role in the disease phenotype and cell death.

  1. Identification of differential anti-neoplastic activity of copper bis(thiosemicarbazones) that is mediated by intracellular reactive oxygen species generation and lysosomal membrane permeabilization.

    Science.gov (United States)

    Stefani, Christian; Al-Eisawi, Zaynab; Jansson, Patric J; Kalinowski, Danuta S; Richardson, Des R

    2015-11-01

    Bis(thiosemicarbazones) and their copper (Cu) complexes possess unique anti-neoplastic properties. However, their mechanism of action remains unclear. We examined the structure-activity relationships of twelve bis(thiosemicarbazones) to elucidate factors regarding their anti-cancer efficacy. Importantly, the alkyl substitutions at the diimine position of the ligand backbone resulted in two distinct groups, namely, unsubstituted/monosubstituted and disubstituted bis(thiosemicarbazones). This alkyl substitution pattern governed their: (1) Cu(II/I) redox potentials; (2) ability to induce cellular (64)Cu release; (3) lipophilicity; and (4) anti-proliferative activity. The potent anti-cancer Cu complex of the unsubstituted bis(thiosemicarbazone) analog, glyoxal bis(4-methyl-3-thiosemicarbazone) (GTSM), generated intracellular reactive oxygen species (ROS), which was attenuated by Cu sequestration by a non-toxic Cu chelator, tetrathiomolybdate, and the anti-oxidant, N-acetyl-l-cysteine. Fluorescence microscopy suggested that the anti-cancer activity of Cu(GTSM) was due, in part, to lysosomal membrane permeabilization (LMP). For the first time, this investigation highlights the role of ROS and LMP in the anti-cancer activity of bis(thiosemicarbazones).

  2. Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc

    Science.gov (United States)

    Stettner, Eva; Jeffers, Scott Allen; Pérez-Vargas, Jimena; Pehau-Arnaudet, Gerard; Tortorici, M. Alejandra; Jestin, Jean-Luc; England, Patrick; Tischler, Nicole D.; Rey, Félix A.

    2016-01-01

    Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic β-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single “fusion loop”. We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal “tail” that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens. PMID:27783711

  3. Cell adhesion-dependent membrane trafficking of a binding partner for the ebolavirus glycoprotein is a determinant of viral entry.

    Science.gov (United States)

    Dube, Derek; Schornberg, Kathryn L; Shoemaker, Charles J; Delos, Sue E; Stantchev, Tzanko S; Clouse, Kathleen A; Broder, Christopher C; White, Judith M

    2010-09-21

    Ebolavirus is a hemorrhagic fever virus associated with high mortality. Although much has been learned about the viral lifecycle and pathogenesis, many questions remain about virus entry. We recently showed that binding of the receptor binding region (RBR) of the ebolavirus glycoprotein (GP) and infection by GP pseudovirions increase on cell adhesion independently of mRNA or protein synthesis. One model to explain these observations is that, on cell adhesion, an RBR binding partner translocates from an intracellular vesicle to the cell surface. Here, we provide evidence for this model by showing that suspension 293F cells contain an RBR binding site within a membrane-bound compartment associated with the trans-Golgi network and microtubule-organizing center. Consistently, trafficking of the RBR binding partner to the cell surface depends on microtubules, and the RBR binding partner is internalized when adherent cells are placed in suspension. Based on these observations, we reexamined the claim that lymphocytes, which are critical for ebolavirus pathogenesis, are refractory to infection because they lack an RBR binding partner. We found that both cultured and primary human lymphocytes (in suspension) contain an intracellular pool of an RBR binding partner. Moreover, we identified two adherent primate lymphocytic cell lines that bind RBR at their surface and strikingly, support GP-mediated entry and infection. In summary, our results reveal a mode of determining viral entry by a membrane-trafficking event that translocates an RBR binding partner to the cell surface, and they suggest that this process may be operative in cells important for ebolavirus pathogenesis (e.g., lymphocytes and macrophages).

  4. Membrane glycoprotein M6A promotes μ-opioid receptor endocytosis and facilitates receptor sorting into the recycling pathway

    Institute of Scientific and Technical Information of China (English)

    Ying-Jian Liang; Dai-Fei Wu; Ralf Stumm; Volker H(o)llt; Thomas Koch

    2008-01-01

    The interaction of μ-opioid receptor (MOPr) with the neuronal membrane glycoprotein M6a is known to facilitate MOPr endocytosis in human embryonic kidney 293 (HEK293) cells. To further study the role of M6a in the post-endocytotic sorting of MOPr, we investigated the agonist-induced co-internalization of MOPr and M6a and protein targeting after internalization in HEK293 cells that co-expressed HA-tagged MOPr and Myc-tagged M6a. We found that M6a, MOPr, and Rab 11, a marker for recycling endosomes, co-localized in endocytotic vesicles, indicating that MOPr and M6a are primarily targeted to recycling endosomes after endocytosis. Furthermore, co-expression of M6a augmented the post-endocytotic sorting of δ-opioid receptors into the recycling pathway, indicating that M6a might have a more general role in opioid receptor post-ndocytotic sorting. The enhanced post-endocytotic sorting of MOPr into the recycling pathway was accompanied by a decrease in agonist-induced receptor down-regulation of M6a in co-expressing cells. We tested the physiological relevance of these findings in primary cultures of cortical neurons and found that co-expression of M6a markedly increased the translocation of MOPrs from the plasma membrane to intracellular vesicles at steady state and significantly enhanced both constitutive and agonist-induced receptor endocytosis. In conclusion, our results strongly indicate that M6a modulates MOPr endocytosis and post-endocytotic sorting and has an important role in receptor regulation.

  5. Constitutive and functional association of the platelet collagen receptor glycoprotein VI-Fc receptor gamma-chain complex with membrane rafts.

    Science.gov (United States)

    Ezumi, Yasuharu; Kodama, Kumi; Uchiyama, Takashi; Takayama, Hiroshi

    2002-05-01

    The platelet collagen receptor glycoprotein (GP) VI-Fc receptor gamma-chain (FcRgamma) complex transduces signals in an immunoreceptorlike manner. We examined a role for the Triton X-100-insoluble membrane rafts in GPVI-FcRgamma complex signaling. Methyl-beta-cyclodextrin (MbetaCD)-induced disruption of the membrane rafts inhibited not only platelet aggregation and secretion but also tyrosine phosphorylation of signaling molecules on stimulation through the GPVI-FcRgamma complex. The GPVI-FcRgamma complex was constitutively associated with membrane rafts wherein the Src family kinases and LAT were also present. Their association was not affected by the complex engagement but was highly sensitive to MbetaCD treatment. Thus, we provide the first evidence that the GPVI-FcRgamma complex is constitutively and functionally associated with membrane rafts.

  6. D-Pinitol attenuates 7, 12 dimethylbenz [a] anthracene induced hazards through modulating protein bound carbohydrates, adenosine triphosphatases and lysosomal enzymes during experimental mammary carcinogenesis.

    Science.gov (United States)

    Rengarajan, Thamaraiselvan; Nandakumar, Natarajan; Balasubramanian, Maruthaiveeran Periyasamy

    2012-01-01

    We have reported here that the ameliorative potentials of D-Pinitol during 7, 12-Dimethylbenz [a] anthracene induced experimental breast carcinogenesis. DMBA is a potent organ specific carcinogen which is widely employed to induce mammary carcinoma in rats. D-Pinitol a natural inositol has been reported to found in soybean with many biological functions. The female sprague dawley rats were subjected to carcinogen 7, 12-DMBA and the ameliorative potentials of dietary compound D-Pinitol was investigated with reference to cell surface glycoproteins, lysosomal enzymes and adenosine triphosphatases. Interestingly, administration of D-Pinitol was found to be significantly down regulated the breast tissue glycoproteins and lysosomal enzymes and in contrast the levels of adenosine triphosphatases were remarkably up regulated. Further, the biochemical changes were well reflected and evidenced in the histology of breast and liver tissues. Thus, it can be concluded from the present study that D-Pinitol efficiently attenuates the hazardous consequences of the environmental carcinogen 7,12-DMBA through modulating cell surface glycoproteins, membrane protective role both in lysosomal and ATPase compartment via its antioxidant nature which ultimately results in the findings of future innovative remedies for genotoxin mediated hazards.

  7. Cyclic AMP-insensitive activation of c-Src and Syk protein-tyrosine kinases through platelet membrane glycoprotein VI.

    Science.gov (United States)

    Ichinohe, T; Takayama, H; Ezumi, Y; Yanagi, S; Yamamura, H; Okuma, M

    1995-11-24

    Platelet glycoprotein (GP) VI is a so-far uncharacterized 62-kDa membrane protein, whose deficiency results in selective impairment in collagen-induced platelet aggregation. Our group previously reported a human polyclonal antibody (anti-p62 IgG) that induces activation of normal, but not of GPVI-deficient, platelets in an Fc-independent manner. The F(ab')2 fragments of this antibody (F(ab')2-anti-p62) stimulated tyrosine phosphorylation of numerous proteins, which was not prevented even in the presence of cAMP-increasing agents such as prostacyclin. Pretreatment of platelets with the protein-tyrosine kinase (PTK) inhibitor tyrphostin A47 completely abolished F(ab')2-anti-p62-induced platelet aggregation in parallel with dose-dependent inhibition of protein-tyrosine phosphorylation, indicating an essential requirement of PTK activity for generating GPVI-mediated signaling. We found that two cytosolic PTKs, c-Src and Syk, became rapidly activated in response to F(ab')2-anti-p62 in a way insensitive to elevation of cAMP. In contrast, in the presence of prostacyclin, F(ab')2-anti-p62 did not stimulate tyrosine phosphorylation of the focal adhesion kinase. cAMP-insensitive activation of c-Src and Syk was also observed in collagen but not thrombin-stimulated platelets. Moreover, either F(ab')2-anti-p62 or collagen stimulated cAMP-insensitive tyrosine phosphorylation of phospholipase C-gamma 2. These results indicate that the receptor-mediated activation of several PTKs in platelets is regulated through a cAMP-sensitive or -insensitive mechanism depending on the nature of each stimulus, and also suggest that GPVI engagement is coupled to cAMP-insensitive activation of c-Src and Syk accompanied by tyrosine phosphorylation of numerous substrates including phospholipase C-gamma 2 in a manner similar to collagen stimulation.

  8. Truncation of the membrane-spanning domain of human immunodeficiency virus type 1 envelope glycoprotein defines elements required for fusion, incorporation, and infectivity.

    Science.gov (United States)

    Yue, Ling; Shang, Liang; Hunter, Eric

    2009-11-01

    The membrane-spanning domain (MSD) of the envelope (Env) glycoprotein from human (HIV) and simian immunodeficiency viruses plays a key role in anchoring the Env complex into the viral membrane but also contributes to its biological function in fusion and virus entry. In HIV type 1 (HIV-1), it has been predicted to span 27 amino acids, from lysine residue 681 to arginine 707, and encompasses an internal arginine at residue 694. By examining a series of C-terminal-truncation mutants of the HIV-1 gp41 glycoprotein that substituted termination codons for amino acids 682 to 708, we show that this entire region is required for efficient viral infection of target cells. Truncation to the arginine at residue 694 resulted in an Env complex that was secreted from the cells. In contrast, a region from residues 681 to 698, which contains highly conserved hydrophobic residues and glycine motifs and extends 4 amino acids beyond 694R, can effectively anchor the protein in the membrane, allow efficient transport to the plasma membrane, and mediate wild-type levels of cell-cell fusion. However, these fusogenic truncated Env mutants are inefficiently incorporated into budding virions. Based on the analysis of these mutants, a "snorkeling" model, in which the flanking charged amino acid residues at 681 and 694 are buried in the lipid while their side chains interact with polar head groups, is proposed for the HIV-1 MSD.

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

    Science.gov (United States)

    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.

  10. Membrane fluidization by ether, other anesthetics, and certain agents abolishes P-glycoprotein ATPase activity and modulates efflux from multidrug-resistant cells.

    Science.gov (United States)

    Regev, R; Assaraf, Y G; Eytan, G D

    1999-01-01

    The anesthetics benzyl alcohol and the nonaromatic chloroform and diethyl ether, abolish P-glycoprotein (Pgp) ATPase activity in a mode that does not fit classical competitive, noncompetitive, or uncompetitive inhibition. At concentrations similar to those required for inhibition of ATPase activity, these anesthetics fluidize membranes leading to twofold acceleration of doxorubicin flip-flop across lipid membranes and prevent photoaffinity labeling of Pgp with [125I]-iodoarylazidoprazosin. Similar concentrations of ether proved nontoxic and modulated efflux from Pgp-overexpressing cells. A similar twofold acceleration of doxorubicin flip-flop rate across membranes was observed with neutral mild detergents, including Tween 20, Nonidet P-40 and Triton X-100, and certain Pgp modulators, such as verapamil and progesterone. Concentrations of these agents, similar to those required for membrane fluidization, inhibited Pgp ATPase activity in a mode similar to that observed with the anesthetics. The mode of inhibition, i.e. lack of evidence for classical enzyme inhibition and the correlation of Pgp ATPase inhibition with membrane fluidization over a wide range of concentrations and structures of drugs favors the direct inhibition of Pgp ATPase activity by membrane fluidization. The unusual sensitivity of Pgp to membrane fluidization, as opposed to acceleration of ATPase activity of ion transporters, could fit the proposed function of Pgp as a 'flippase', which is in close contact with the membrane core.

  11. DNA encoding an HIV-1 Gag/human lysosome-associated membrane protein-1 chimera elicits a broad cellular and humoral immune response in Rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Priya Chikhlikar

    Full Text Available Previous studies of HIV-1 p55Gag immunization of mice have demonstrated the usefulness of targeting antigens to the cellular compartment containing the major histocompatibility complex type II (MHC II complex molecules by use of a DNA antigen formulation encoding Gag as a chimera with the mouse lysosome-associated membrane protein (mLAMP/gag. In the present study, we have analyzed the magnitude and breadth of Gag-specific T-lymphocyte and antibody responses elicited in Rhesus macaques after immunization with DNA encoding a human LAMP/gag (hLAMP/gag chimera. ELISPOT analyses indicated that the average Gag-specific IFN-gamma response elicited by the hLAMP/gag chimera was detectable after only two or three naked DNA immunizations in all five immunized macaques and reached an average of 1000 spot-forming cells (SFC/10(6 PBMCs. High IFN-gamma ELISPOT responses were detected in CD8(+-depleted cells, indicating that CD4(+ T-cells play a major role in these responses. The T-cell responses of four of the macaques were also tested by use of ELISPOT to 12 overlapping 15-amino acids (aa peptide pools containing ten peptides each, encompassing the complete Gag protein sequence. The two Mamu 08 immunized macaques responded to eight and twelve of the pools, the Mamu B01 to six, and the other macaque to five pools indicating that the hLAMP/gag DNA antigen formulation elicits a broad T-cell response against Gag. Additionally, there was a strong HIV-1-specific IgG response. The IgG antibody titers increased after each DNA injection, indicating a strong amnestic B-cell response, and were highly elevated in all the macaques after three immunizations. Moreover, the serum of each macaque recognized 13 of the 49 peptides of a 20-aa peptide library covering the complete Gag amino acid sequence. In addition, HIV-1-specific IgA antibodies were present in the plasma and external secretions, including nasal washes. These data support the findings of increased

  12. Methods for monitoring Ca(2+) and ion channels in the lysosome.

    Science.gov (United States)

    Zhong, Xi Zoë; Yang, Yiming; Sun, Xue; Dong, Xian-Ping

    2016-12-09

    Lysosomes and lysosome-related organelles are emerging as intracellular Ca(2+) stores and play important roles in a variety of membrane trafficking processes, including endocytosis, exocytosis, phagocytosis and autophagy. Impairment of lysosomal Ca(2+) homeostasis and membrane trafficking has been implicated in many human diseases such as lysosomal storage diseases (LSDs), neurodegeneration, myopathy and cancer. Lysosomal membrane proteins, in particular ion channels, are crucial for lysosomal Ca(2+) signaling. Compared with ion channels in the plasma membrane, lysosomal ion channels and their roles in lysosomal Ca(2+) signaling are less understood, largely due to their intracellular localization and the lack of feasible functional assays directly applied to the native environment. Recent advances in biomedical methodology have made it possible to directly investigate ion channels in the lysosomal membrane. In this review, we provide a summary of the newly developed methods for monitoring lysosomal Ca(2+) and ion channels, as well as the recent discovery of lysosomal ion channels and their significances in intracellular Ca(2+) signaling. These new techniques will expand our research scope and our understanding of the nature of lysosomes and lysosome-related diseases.

  13. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Okada, Masato, E-mail: okadam@biken.osaka-u.ac.jp [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. Black-Right-Pointing-Pointer We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. Black-Right-Pointing-Pointer The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. Black-Right-Pointing-Pointer Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. Black-Right-Pointing-Pointer The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.

  14. Intracellular interaction of human immunodeficiency virus type 1 (ARV-2) envelope glycoprotein gp160 with CD4 blocks the movement and maturation of CD4 to the plasma membrane.

    OpenAIRE

    Jabbar, M A; Nayak, D P

    1990-01-01

    The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) plays a major role in the down-regulation of its receptor, CD4. Using a transient-expression system, we investigated the interaction of the HIV-1 envelope glycoprotein with CD4 during their movement through the intracellular membrane traffic. In singly transfected cells, the envelope glyprotein gp160 was synthesized, glycosylated, and localized predominantly in the endoplasmic reticulum. Only a minor fraction of gp160 wa...

  15. The Epstein-Barr virus (EBV) glycoprotein B cytoplasmic C-terminal tail domain regulates the energy requirement for EBV-induced membrane fusion.

    Science.gov (United States)

    Chen, Jia; Zhang, Xianming; Jardetzky, Theodore S; Longnecker, Richard

    2014-10-01

    The entry of enveloped viruses into host cells is preceded by membrane fusion, which in Epstein-Barr virus (EBV) is thought to be mediated by the refolding of glycoprotein B (gB) from a prefusion to a postfusion state. In our current studies, we characterized a gB C-terminal tail domain (CTD) mutant truncated at amino acid 843 (gB843). This truncation mutant is hyperfusogenic as monitored by syncytium formation and in a quantitative fusion assay and is dependent on gH/gL for fusion activity. gB843 can rescue the fusion function of other glycoprotein mutants that have null or decreased fusion activity in epithelial and B cells. In addition, gB843 requires less gp42 and gH/gL for fusion, and can function in fusion at a lower temperature than wild-type gB, indicating a lower energy requirement for fusion activation. Since a key step in fusion is the conversion of gB from a prefusion to an active postfusion state by gH/gL, gB843 may access this activated gB state more readily. Our studies indicate that the gB CTD may participate in the fusion function by maintaining gB in an inactive prefusion form prior to activation by receptor binding. Importance: Diseases resulting from Epstein-Barr virus (EBV) infection in humans range from the fairly benign disease infectious mononucleosis to life-threatening cancer. As an enveloped virus, EBV must fuse with a host cell membrane for entry and infection by using glycoproteins gH/gL, gB, and gp42. Among these glycoproteins, gB is thought to be the protein that executes fusion. To further characterize the function of the EBV gB cytoplasmic C-terminal tail domain (CTD) in fusion, we used a previously constructed CTD truncation mutant and studied its fusion activity in the context of other EBV glycoprotein mutants. From these studies, we find that the gB CTD regulates fusion by altering the energy requirements for the triggering of fusion mediated by gH/gL or gp42. Overall, our studies may lead to a better understanding of EBV fusion

  16. TRPML and lysosomal function.

    Science.gov (United States)

    Zeevi, David A; Frumkin, Ayala; Bach, Gideon

    2007-08-01

    Mucolipin 1 (MLN1), also known as TRPML1, is a member of the mucolipin family. The mucolipins are the only lysosomal proteins within the TRP superfamily. Mutations in the gene coding for TRPML1 result in a lysosomal storage disorder (LSD). This review summarizes the current knowledge related to this protein and the rest of the mucolipin family.

  17. Physical proximity and functional association of glycoprotein 1balpha and protein-disulfide isomerase on the platelet plasma membrane

    NARCIS (Netherlands)

    Burgess, J K; Hotchkiss, K A; Suter, C; Dudman, N P; Szöllösi, J; Chesterman, C N; Chong, B H; Hogg, P J

    2000-01-01

    Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balp

  18. Release of Glycoprotein (GP1 from the Tegumental Surface of Taenia solium by Phospholipase C from Clostridium perfringens Suggests a Novel Protein-Anchor to Membranes

    Directory of Open Access Journals (Sweden)

    Abraham Landa

    2010-01-01

    Full Text Available In order to explore how molecules are linked to the membrane surface in larval Taenia solium, whole cysticerci were incubated in the presence of phospholipase C from Clostridium perfringens (PLC. Released material was collected and analyzed in polyacrylamide gels with sodium dodecyl sulfate. Two major bands with apparent molecular weights of 180 and 43 kDa were observed. Western blot of released material and localization assays in cysticerci tissue sections using antibodies against five known surface glycoproteins of T. solium cysticerci indicated that only one, previously called GP1, was released. Similar localization studies using the lectins wheat-germ-agglutinin and Concanavalin A showed that N-acetyl-D-glucosamine, N-acetylneuraminic, sialic acid, αmethyl-D-mannoside, D-manose/glucose, and N-acetyl-D-glucosamine residues are abundantly present on the surface. On the other hand, we find that treatment with PLC releases molecules from the surface; they do not reveal Cross Reacting Determinant (CRD, suggesting a novel anchor to the membrane for the glycoprotein GP1.

  19. Mechanism of pluronic effect on P-glycoprotein efflux system in blood-brain barrier: contributions of energy depletion and membrane fluidization.

    Science.gov (United States)

    Batrakova, E V; Li, S; Vinogradov, S V; Alakhov, V Y; Miller, D W; Kabanov, A V

    2001-11-01

    Pluronic block copolymer, P85, inhibits the P-glycoprotein (Pgp) drug efflux system and increases the permeability of a broad spectrum of drugs in the blood-brain barrier (BBB). This study examines the mechanisms by which P85 inhibits Pgp using bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB. The hypothesis was that simultaneous alterations in intracellular ATP levels and membrane fluidization in BBMEC monolayers by P85 results in inhibition of the drug efflux system. The methods included the use of 1) standard Pgp substrate rhodamine 123 to assay the Pgp efflux system in BBMEC, 2) luciferin/luciferase assay for ATP intracellular levels, and 3) 1,6-diphenyl-1,3,5-hexatriene for membrane microviscosity. Using 3H-labeled P85 and fluorescein-labeled P85 for confocal microscopy, this study suggests that P85 accumulates in the cells and intracellular organelles such as the mitochondria where it can interfere with metabolic processes. Following exposure of BBMEC to P85, the ATP levels were depleted, and microviscosity of the cell membranes was decreased. Furthermore, P85 treatment decreased Pgp ATPase activity in membranes expressing human Pgp. A combination of experiments examining the kinetics, concentration dependence, and directionality of P85 effects on Pgp-mediated efflux in BBMEC monolayers suggests that both energy depletion (decreasing ATP pool available for Pgp) and membrane fluidization (inhibiting Pgp ATPase activity) are critical factors contributing to the activity of the block copolymer in the BBB.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  3. Secondary Lysosomal Changes in Liver in Preclinical Drug Development

    Institute of Scientific and Technical Information of China (English)

    Vincent P. Meador; D. V. M.; Ph. D.; Diplomate ACVP

    2005-01-01

    @@ Lysosomes are intracytoplasmic membrane-bound organelles that function to degrade intracellular substances by enzymatic digestion. They occur normally in all cells, being especially prominent in phagocytic cells of the reticuloendothelial system.

  4. Lysosome Biogenesis and Autophagy

    NARCIS (Netherlands)

    Reggiori, Fulvio; Klumperman, Judith|info:eu-repo/dai/nl/075097273

    2016-01-01

    Lysosomes degrade biological components acquired by endocytosis, the major cellular pathway for internalization of extracellular material, and macroautophagy. This chapter presents an overview of these two major degradative intracellular pathways, and highlights the emerging cross talks between

  5. Endosome-lysosomes and neurodegeneration.

    Science.gov (United States)

    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.

  6. Effect of Xiaoyu Zhixue Tablet (消瘀止血片) on Expression of Platelet Membrane Glycoproteins in Patients with Hemorrhagic Thrombopathy

    Institute of Scientific and Technical Information of China (English)

    沈霖; 沈迪; 高兰; 刘仲萍; 杨艳萍; 谢晶; 周丕琪

    2004-01-01

    Objective: To observe the effect of Xiaoyu Zhixue tablet (消瘀止血片,XYZXT) on the expression of platelet membrane glycoproteins in patients with hemorrhagic thrombopathy, and to explore its possible mechanism. Methods: The total of 148 patients with hemorrhagic thrornbopathy were randomly divided into two groups, the traditional Chinese medicicne (TCM) group (n=98) treated with XYZXT and the Western medicine (WM) group (n = 50) treated with adrenosin, vitamins C, K and P, both for 6 months.The therapeutic effect and the recovery rate of platelet aggregation in the two groups were observed. And platelet membrane glycoprotein (GP) Ⅰ b/Ⅸ, GP Ⅱ b/Ⅲ a complexes, GP Ⅰ b, GP Ⅱ b, GPⅢ a and P-selectin were analyzed by flow cytometry in both groups before and after treatment and also in 34 normal healthy subjects. Results: The total effective rate of hemostasis was 89.8% in TCM group and 54.0% in the WM group (X2 =45.83, P<0.01), and the recovery rate of platelet aggregation was 72.4% and 4.0% respectively (X2 = 62.06, P<0.01). The fluorescence intensity of GP Ⅰ b/Ⅸ, GP Ⅱ b/Ⅲ a complexes, GP Ⅰ b, GP Ⅲ a and P-selectin were lower in both groups before treatment than those in the healthy subjects. Expression of above-mentioned marks was elevated in TCM group after 6 months' therapy, which was insignificantly different as compared with the healthy subjects (P>0.05) and higher than those in the WM group (P<0.05).Conclusion: One of the mechanisms in treating hemorrhagic thrombopathy with XYZXT is that it could regulate the expression of GP Ⅰ b/Ⅸ, GP Ⅱ b/Ⅲ a complexes, GP Ⅰ b, GPⅢ a and P-selectin at the level of receptor protein.

  7. A lysosome-centered view of nutrient homeostasis.

    Science.gov (United States)

    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.

  8. Mitochondrial Dysfunction in Lysosomal Storage Disorders

    Directory of Open Access Journals (Sweden)

    Mario de la Mata

    2016-10-01

    Full Text Available Lysosomal storage diseases (LSDs describe a heterogeneous group of rare inherited metabolic disorders that result from the absence or loss of function of lysosomal hydrolases or transporters, resulting in the progressive accumulation of undigested material in lysosomes. The accumulation of substances affects the function of lysosomes and other organelles, resulting in secondary alterations such as impairment of autophagy, mitochondrial dysfunction, inflammation and apoptosis. LSDs frequently involve the central nervous system (CNS, where neuronal dysfunction or loss results in progressive neurodegeneration and premature death. Many LSDs exhibit signs of mitochondrial dysfunction, which include mitochondrial morphological changes, decreased mitochondrial membrane potential (ΔΨm, diminished ATP production and increased generation of reactive oxygen species (ROS. Furthermore, reduced autophagic flux may lead to the persistence of dysfunctional mitochondria. Gaucher disease (GD, the LSD with the highest prevalence, is caused by mutations in the GBA1 gene that results in defective and insufficient activity of the enzyme β-glucocerebrosidase (GCase. Decreased catalytic activity and/or instability of GCase leads to accumulation of glucosylceramide (GlcCer and glucosylsphingosine (GlcSph in the lysosomes of macrophage cells and visceral organs. Mitochondrial dysfunction has been reported to occur in numerous cellular and mouse models of GD. The aim of this manuscript is to review the current knowledge and implications of mitochondrial dysfunction in LSDs.

  9. Ankyrin and band 3 differentially affect expression of membrane glycoproteins but are not required for erythroblast enucleation.

    Science.gov (United States)

    Ji, Peng; Lodish, Harvey F

    2012-01-27

    During late stages of mammalian erythropoiesis the nucleus undergoes chromatin condensation, migration to the plasma membrane, and extrusion from the cytoplasm surrounded by a segment of plasma membrane. Since nuclear condensation occurs in all vertebrates, mammalian erythroid membrane and cytoskeleton proteins were implicated as playing important roles in mediating the movement and extrusion of the nucleus. Here we use erythroid ankyrin deficient and band 3 knockout mouse models to show that band 3, but not ankyrin, plays an important role in regulating the level of erythroid cell membrane proteins, as evidenced by decreased cell surface expression of glycophorin A in band 3 knockout mice. However, neither band 3 nor ankyrin are required for enucleation. These results demonstrate that mammalian erythroblast enucleation does not depend on the membrane integrity generated by the ankyrin-band 3 complex.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  12. Common glycoproteins expressing polylactosamine-type glycans on matched patient primary and metastatic melanoma cells show different glycan profiles.

    Science.gov (United States)

    Kinoshita, Mitsuhiro; Mitsui, Yosuke; Kakoi, Naotaka; Yamada, Keita; Hayakawa, Takao; Kakehi, Kazuaki

    2014-02-07

    Recently, we reported comparative analysis of glycoproteins which express cancer-specific N-glycans on various cancer cells and identified 24 glycoproteins having polylactosamine (polyLacNAc)-type N-glycans that are abundantly present in malignant cells [ Mitsui et al., J. Pharm. Biomed. Anal. 2012 , 70 , 718 - 726 ]. In the present study, we applied the technique to comparative studies on common glycoproteins present in the matched patient primary and metastatic melanoma cell lines. Metastatic melanoma cells (WM266-4) contained a large amount of polyLacNAc-type N-glycans in comparison with primary melanoma cells (WM115). To identify the glycoproteins expressing these N-glycans, glycopeptides having polyLacNAc-type N-glycans were captured by a Datura stramonium agglutinin (DSA)-immobilized agarose column. The captured glycopeptides were analyzed by LC/MS after removing N-glycans, and some glycoproteins such as basigin, lysosome-associated membrane protein-1 (LAMP-1), and chondroitin sulfate proteoglycan 4 (CSPG4) were identified in both WM115 and WM266-4 cells. The expression level of polyLacNAc of CSPG4 in WM266-4 cells was significantly higher than that in WM115 cells. In addition, sulfation patterns of chondroitin sulfate (CS) chains in CSPG4 showed dramatic changes between these cell lines. These data show that characteristic glycans attached to common proteins observed in different stages of cancer cells will be useful markers for determining degree of malignancies of tumor cells.

  13. Binding of Alphaherpesvirus Glycoprotein H to Surface α4β1-Integrins Activates Calcium-Signaling Pathways and Induces Phosphatidylserine Exposure on the Plasma Membrane

    Science.gov (United States)

    Gramatica, Andrea; Herrmann, Andreas; Osterrieder, Nikolaus

    2015-01-01

    ABSTRACT Intracellular signaling connected to integrin activation is known to induce cytoplasmic Ca2+ release, which in turn mediates a number of downstream signals. The cellular entry pathways of two closely related alphaherpesviruses, equine herpesviruses 1 and 4 (EHV-1 and EHV-4), are differentially regulated with respect to the requirement of interaction of glycoprotein H (gH) with α4β1-integrins. We show here that binding of EHV-1, but not EHV-4, to target cells resulted in a rapid and significant increase in cytosolic Ca2+ levels. EHV-1 expressing EHV-4 gH (gH4) in lieu of authentic gH1 failed to induce Ca2+ release, while EHV-4 with gH1 triggered significant Ca2+ release. Blocking the interaction between gH1 and α4β1-integrins, inhibiting phospholipase C (PLC) activation, or blocking binding of inositol 1,4,5-triphosphate (IP3) to its receptor on the endoplasmic reticulum (ER) abrogated Ca2+ release. Interestingly, phosphatidylserine (PS) was exposed on the plasma membrane in response to cytosolic calcium increase after EHV-1 binding through a scramblase-dependent mechanism. Inhibition of both Ca2+ release from the ER and scramblase activation blocked PS scrambling and redirected virus entry to the endocytic pathway, indicating that PS may play a role in facilitating virus entry directly at the plasma membrane. PMID:26489864

  14. Con A-binding protein Zn-α2-glycoprotein on human sperm membrane is related to acrosome reaction and sperm fertility.

    Science.gov (United States)

    Liu, Y; Qu, F; Cao, X; Chen, G; Guo, Q; Ying, X; Guo, W; Lu, L; Ding, Z

    2012-04-01

    Fertilization, the recognition and fusion between spermatozoa and oocyte, involves various molecules on the spermatozoa and oocyte membranes. Concanavalin A (ConA)-binding proteins may be one of the molecules involved in mammal spermatozoa fertilization; however, their structure and function remain largely unknown. Here, we initially identified a ConA-binding protein, Zn-α2-glycoprotein (ZAG), involved in regulating the acrosome reaction (AR) of human spermatozoa. ZAG is localized on the pre-equatorial region covering the acrosome, neck and tail (some parts of middle piece and principal piece respectively) regions of the acrosome intact human spermatozoa, and disappears in the acrosomal region of the acrosome-reacted spermatozoa. Polyclonal antibodies against human recombinant ZAG significantly reduced the AR and sperm capability binding to human zona pellucida or penetration into zona-free hamster oocytes. Furthermore, assessment of the signaling pathways regulated by ZAG revealed that ZAG affects sperm AR through both the cAMP/PKA and PKC pathways. These results indicate that ZAG, which is present on the human sperm membrane, plays a critical role in the AR and subsequently, may be involved in sperm fertility.

  15. Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane.

    Science.gov (United States)

    Farnsworth, Aaron; Wisner, Todd W; Webb, Michael; Roller, Richard; Cohen, Gary; Eisenberg, Roselyn; Johnson, David C

    2007-06-12

    Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

  16. Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1 enhance Gag-specific immune response.

    Directory of Open Access Journals (Sweden)

    Rodrigo Maciel da Costa Godinho

    Full Text Available We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1 elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

  17. Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1) enhance Gag-specific immune response.

    Science.gov (United States)

    Godinho, Rodrigo Maciel da Costa; Matassoli, Flavio Lemos; Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J Thomas; Marques, Ernesto Torres de Azevedo; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

  18. Regulation of HIV-Gag Expression and Targeting to the Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response

    Science.gov (United States)

    Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J. Thomas; de Azevedo Marques, Ernesto Torres; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. PMID:24932692

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

    Science.gov (United States)

    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.

  20. Proton-assisted amino acid transporter PAT1 complexes with Rag GTPases and activates TORC1 on late endosomal and lysosomal membranes.

    Directory of Open Access Journals (Sweden)

    Margrét H Ögmundsdóttir

    Full Text Available Mammalian Target of Rapamycin Complex 1 (mTORC1 is activated by growth factor-regulated phosphoinositide 3-kinase (PI3K/Akt/Rheb signalling and extracellular amino acids (AAs to promote growth and proliferation. These AAs induce translocation of mTOR to late endosomes and lysosomes (LELs, subsequent activation via mechanisms involving the presence of intralumenal AAs, and interaction between mTORC1 and a multiprotein assembly containing Rag GTPases and the heterotrimeric Ragulator complex. However, the mechanisms by which AAs control these different aspects of mTORC1 activation are not well understood. We have recently shown that intracellular Proton-assisted Amino acid Transporter 1 (PAT1/SLC36A1 is an essential mediator of AA-dependent mTORC1 activation. Here we demonstrate in Human Embryonic Kidney (HEK-293 cells that PAT1 is primarily located on LELs, physically interacts with the Rag GTPases and is required for normal AA-dependent mTOR relocalisation. We also use the powerful in vivo genetic methodologies available in Drosophila to investigate the regulation of the PAT1/Rag/Ragulator complex. We show that GFP-tagged PATs reside at both the cell surface and LELs in vivo, mirroring PAT1 distribution in several normal mammalian cell types. Elevated PI3K/Akt/Rheb signalling increases intracellular levels of PATs and synergistically enhances PAT-induced growth via a mechanism requiring endocytosis. In light of the recent identification of the vacuolar H(+-ATPase as another Rag-interacting component, we propose a model in which PATs function as part of an AA-sensing engine that drives mTORC1 activation from LEL compartments.

  1. Proton-assisted amino acid transporter PAT1 complexes with Rag GTPases and activates TORC1 on late endosomal and lysosomal membranes.

    Science.gov (United States)

    Ögmundsdóttir, Margrét H; Heublein, Sabine; Kazi, Shubana; Reynolds, Bruno; Visvalingam, Shivanthy M; Shaw, Michael K; Goberdhan, Deborah C I

    2012-01-01

    Mammalian Target of Rapamycin Complex 1 (mTORC1) is activated by growth factor-regulated phosphoinositide 3-kinase (PI3K)/Akt/Rheb signalling and extracellular amino acids (AAs) to promote growth and proliferation. These AAs induce translocation of mTOR to late endosomes and lysosomes (LELs), subsequent activation via mechanisms involving the presence of intralumenal AAs, and interaction between mTORC1 and a multiprotein assembly containing Rag GTPases and the heterotrimeric Ragulator complex. However, the mechanisms by which AAs control these different aspects of mTORC1 activation are not well understood. We have recently shown that intracellular Proton-assisted Amino acid Transporter 1 (PAT1)/SLC36A1 is an essential mediator of AA-dependent mTORC1 activation. Here we demonstrate in Human Embryonic Kidney (HEK-293) cells that PAT1 is primarily located on LELs, physically interacts with the Rag GTPases and is required for normal AA-dependent mTOR relocalisation. We also use the powerful in vivo genetic methodologies available in Drosophila to investigate the regulation of the PAT1/Rag/Ragulator complex. We show that GFP-tagged PATs reside at both the cell surface and LELs in vivo, mirroring PAT1 distribution in several normal mammalian cell types. Elevated PI3K/Akt/Rheb signalling increases intracellular levels of PATs and synergistically enhances PAT-induced growth via a mechanism requiring endocytosis. In light of the recent identification of the vacuolar H(+)-ATPase as another Rag-interacting component, we propose a model in which PATs function as part of an AA-sensing engine that drives mTORC1 activation from LEL compartments.

  2. Arsenite Regulates Prolongation of Glycan Residues of Membrane Glycoprotein: A Pivotal Study via Wax Physisorption Kinetics and FTIR Imaging

    Directory of Open Access Journals (Sweden)

    Chih-Hung Lee

    2016-03-01

    Full Text Available Arsenic exposure results in several human cancers, including those of the skin, lung, and bladder. As skin cancers are the most common form, epidermal keratinocytes (KC are the main target of arsenic exposure. The mechanisms by which arsenic induces carcinogenesis remains unclear, but aberrant cell proliferation and dysregulated energy homeostasis play a significant role. Protein glycosylation is involved in many key physiological processes, including cell proliferation and differentiation. To evaluate whether arsenite exposure affected protein glycosylation, the alteration of chain length of glycan residues in arsenite treated skin cells was estimated. Herein we demonstrated that the protein glycosylation was adenosine triphosphate (ATP-dependent and regulated by arsenite exposure by using Fourier transform infrared (FTIR reflectance spectroscopy, synchrotron-radiation-based FTIR (SR-FTIR microspectroscopy, and wax physisorption kinetics coupled with focal-plane-array-based FTIR (WPK-FPA-FTIR imaging. We were able to estimate the relative length of surface protein-linked glycan residues on arsenite-treated skin cells, including primary KC and two skin cancer cell lines, HSC-1 and HaCaT cells. Differential physisorption of wax adsorbents adhered to long-chain (elongated type and short-chain (regular type glycan residues of glycoprotein of skin cell samples treated with various concentration of arsenite was measured. The physisorption ratio of beeswax remain/n-pentacosane remain for KC cells was increased during arsenite exposure. Interestingly, this increase was reversed after oligomycin (an ATP synthase inhibitor pretreatment, suggesting the chain length of protein-linked glycan residues is likely ATP-dependent. This is the first study to demonstrate the elongation and termination of surface protein-linked glycan residues using WPK-FPA-FTIR imaging in eukaryotes. Herein the result may provide a scientific basis to target surface protein

  3. Arsenite Regulates Prolongation of Glycan Residues of Membrane Glycoprotein: A Pivotal Study via Wax Physisorption Kinetics and FTIR Imaging.

    Science.gov (United States)

    Lee, Chih-Hung; Hsu, Chia-Yen; Huang, Pei-Yu; Chen, Ching-Iue; Lee, Yao-Chang; Yu, Hsin-Su

    2016-03-22

    Arsenic exposure results in several human cancers, including those of the skin, lung, and bladder. As skin cancers are the most common form, epidermal keratinocytes (KC) are the main target of arsenic exposure. The mechanisms by which arsenic induces carcinogenesis remains unclear, but aberrant cell proliferation and dysregulated energy homeostasis play a significant role. Protein glycosylation is involved in many key physiological processes, including cell proliferation and differentiation. To evaluate whether arsenite exposure affected protein glycosylation, the alteration of chain length of glycan residues in arsenite treated skin cells was estimated. Herein we demonstrated that the protein glycosylation was adenosine triphosphate (ATP)-dependent and regulated by arsenite exposure by using Fourier transform infrared (FTIR) reflectance spectroscopy, synchrotron-radiation-based FTIR (SR-FTIR) microspectroscopy, and wax physisorption kinetics coupled with focal-plane-array-based FTIR (WPK-FPA-FTIR) imaging. We were able to estimate the relative length of surface protein-linked glycan residues on arsenite-treated skin cells, including primary KC and two skin cancer cell lines, HSC-1 and HaCaT cells. Differential physisorption of wax adsorbents adhered to long-chain (elongated type) and short-chain (regular type) glycan residues of glycoprotein of skin cell samples treated with various concentration of arsenite was measured. The physisorption ratio of beeswax remain/n-pentacosane remain for KC cells was increased during arsenite exposure. Interestingly, this increase was reversed after oligomycin (an ATP synthase inhibitor) pretreatment, suggesting the chain length of protein-linked glycan residues is likely ATP-dependent. This is the first study to demonstrate the elongation and termination of surface protein-linked glycan residues using WPK-FPA-FTIR imaging in eukaryotes. Herein the result may provide a scientific basis to target surface protein-linked glycan

  4. Transformation-associated changes in sphingolipid metabolism sensitize cells to lysosomal cell death induced by inhibitors of acid sphingomyelinase

    DEFF Research Database (Denmark)

    Petersen, Nikolaj H T; Olsen, Ole D; Groth-Pedersen, Line

    2013-01-01

    Lysosomal membrane permeabilization and subsequent cell death may prove useful in cancer treatment, provided that cancer cell lysosomes can be specifically targeted. Here, we identify acid sphingomyelinase (ASM) inhibition as a selective means to destabilize cancer cell lysosomes. Lysosome......-destabilizing experimental anticancer agent siramesine inhibits ASM by interfering with the binding of ASM to its essential lysosomal cofactor, bis(monoacylglycero)phosphate. Like siramesine, several clinically relevant ASM inhibitors trigger cancer-specific lysosomal cell death, reduce tumor growth in vivo, and revert...

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

    Directory of Open Access Journals (Sweden)

    Catherine Staudt

    2016-12-01

    Full Text Available 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.

  6. The hr1 and fusion peptide regions of the subgroup B avian sarcoma and leukosis virus envelope glycoprotein influence low pH-dependent membrane fusion.

    Directory of Open Access Journals (Sweden)

    Angeline Rose Babel

    Full Text Available The avian sarcoma and leukosis virus (ASLV envelope glycoprotein (Env is activated to trigger fusion by a two-step mechanism involving receptor-priming and low pH fusion activation. In order to identify regions of ASLV Env that can regulate this process, a genetic selection method was used to identify subgroup B (ASLV-B virus-infected cells resistant to low pH-triggered fusion when incubated with cells expressing the cognate TVB receptor. The subgroup B viral Env (envB genes were then isolated from these cells and characterized by DNA sequencing. This led to identification of two frequent EnvB alterations which allowed TVB receptor-binding but altered the pH-threshold of membrane fusion activation: a 13 amino acid deletion in the host range 1 (hr1 region of the surface (SU EnvB subunit, and the A32V amino acid change within the fusion peptide of the transmembrane (TM EnvB subunit. These data indicate that these two regions of EnvB can influence the pH threshold of fusion activation.

  7. Collagen-stimulated activation of Syk but not c-Src is severely compromised in human platelets lacking membrane glycoprotein VI.

    Science.gov (United States)

    Ichinohe, T; Takayama, H; Ezumi, Y; Arai, M; Yamamoto, N; Takahashi, H; Okuma, M

    1997-01-03

    Activation of circulating platelets by subendothelial collagen is an essential event in vascular hemostasis. In human platelets, two membrane glycoprotein (GP) abnormalities, integrin alpha2 beta1 deficiency and GPVI deficiency, have been reported to result in severe hyporesponsiveness to fibrillar collagen. Although it has been well established that integrin alpha2 beta1, also known as the GPIa-IIa complex, functions as a primary platelet adhesion receptor for collagen, the mechanism by which GPVI contributes to collagen-platelet interaction has been ill defined to date. However, our recent observation that GPVI cross-linking couples to cyclic AMP-insensitive activation of c-Src and Syk tyrosine kinases suggested a potential role for GPVI in regulating protein-tyrosine phosphorylation by collagen (Ichinohe, T., Takayama, H., Ezumi, Y., Yanagi, S., Yamamura, H., and Okuma, M. (1995) J. Biol. Chem. 270, 28029-28036). To further investigate this hypothesis, here we examined the collagen-induced protein-tyrosine phosphorylation in GPVI-deficient platelets expressing normal amounts of alpha2 beta1. In response to collagen, these platelets exhibited alpha2 beta1-dependent c-Src activation accompanied by tyrosine phosphorylation of several substrates including cortactin. In contrast, severe defects were observed in collagen-stimulated Syk activation and tyrosine phosphorylation of phospholipase C-gamma2, Vav, and focal adhesion kinase, implicating a specific requirement of GPVI for recruiting these molecules to signaling cascades evoked by collagen-platelet interaction.

  8. Changes in the morphology and lability of lysosomal subpopulations in caerulein-induced acute pancreatitis.

    Science.gov (United States)

    Sarmiento, Nancy; Sánchez-Yagüe, Jesús; Juanes, Pedro P; Pérez, Nieves; Ferreira, Laura; García-Hernández, Violeta; Mangas, Arturo; Calvo, José J; Sánchez-Bernal, Carmen

    2011-02-01

    Lysosomes play an important role in acute pancreatitis (AP). Here we developed a method for the isolation of lysosome subpopulations from rat pancreas and assessed the stability of lysosomal membranes. AP was induced by four subcutaneous injections of 20 μg caerulein/kg body weight at hourly intervals. The animals were killed 9h after the first injection. Marker enzymes [N-acetyl-β-D-glucosaminidase (NAG), cathepsin B and succinate dehydrogenase (SDH)] were assayed in subcellular fractions from control pancreas and in pancreatitis. Lysosomal subpopulations were separated by Percoll density gradient centrifugation and observed by electron microscopy. NAG molecular forms were determined by DEAE-cellulose chromatography. AP was associated with: (i) increases in the specific activity of lysosomal enzymes in the soluble fraction, (ii) changes in the size and alterations in the morphology of the organelles from the lysosomal subpopulations, (iii) the appearance of large vacuoles in the primary and secondary lysosome subpopulations, (iv) the increase in the amount of the NAG form associated with the pancreatic lysosomal membrane as well as its release towards the soluble fraction. Lysosome subpopulations are separated by a combination of differential and Percoll density gradient centrifugations. Primary lysosome membrane stability decreases in AP. Copyright © 2010 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

  9. Lipid Storage Disorders Block Lysosomal Trafficking By Inhibiting TRP Channel and Calcium Release

    OpenAIRE

    2012-01-01

    Lysosomal lipid accumulation, defects in membrane trafficking, and altered Ca2+ homeostasis are common features in many lysosomal storage diseases. Mucolipin TRP channel 1 (TRPML1) is the principle Ca2+ channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca2+ release, measured using a genetically-encoded Ca2+ indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann-Pick (NP) disease cells....

  10. Glycoprotein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Schultz, Peter G. (La Jolla, CA); Wang, Lei (San Diego, CA); Zhang, Zhiwen (San Diego, CA)

    2010-11-16

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  11. Glycoprotein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Schultz, Peter G. (La Jolla, CA); Wang, Lei (San Diego, CA); Zhang, Zhiwen (San Diego, CA)

    2010-11-02

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  12. Glycoprotein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

    2009-07-14

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  13. Binding of alphaherpesvirus glycoprotein H to surface α4β1-integrins activates calcium-signaling pathways and induces phosphatidylserine exposure on the plasma membrane.

    Science.gov (United States)

    Azab, Walid; Gramatica, Andrea; Herrmann, Andreas; Osterrieder, Nikolaus

    2015-10-20

    Intracellular signaling connected to integrin activation is known to induce cytoplasmic Ca(2+) release, which in turn mediates a number of downstream signals. The cellular entry pathways of two closely related alphaherpesviruses, equine herpesviruses 1 and 4 (EHV-1 and EHV-4), are differentially regulated with respect to the requirement of interaction of glycoprotein H (gH) with α4β1-integrins. We show here that binding of EHV-1, but not EHV-4, to target cells resulted in a rapid and significant increase in cytosolic Ca(2+) levels. EHV-1 expressing EHV-4 gH (gH4) in lieu of authentic gH1 failed to induce Ca(2+) release, while EHV-4 with gH1 triggered significant Ca(2+) release. Blocking the interaction between gH1 and α4β1-integrins, inhibiting phospholipase C (PLC) activation, or blocking binding of inositol 1,4,5-triphosphate (IP3) to its receptor on the endoplasmic reticulum (ER) abrogated Ca(2+) release. Interestingly, phosphatidylserine (PS) was exposed on the plasma membrane in response to cytosolic calcium increase after EHV-1 binding through a scramblase-dependent mechanism. Inhibition of both Ca(2+) release from the ER and scramblase activation blocked PS scrambling and redirected virus entry to the endocytic pathway, indicating that PS may play a role in facilitating virus entry directly at the plasma membrane. Herpesviruses are a large family of enveloped viruses that infect a wide range of hosts, causing a variety of diseases. These viruses have developed a number of strategies for successful entry into different cell types. We and others have shown that alphaherpesviruses, including EHV-1 and herpes simplex virus 1 (HSV-1), can route their entry pathway and do so by manipulation of cell signaling cascades to ensure viral genome delivery to nuclei. We show here that the interaction between EHV-1 gH and cellular α4β1-integrins is necessary to induce emptying of ER calcium stores, which induces phosphatidylserine exposure on the plasma membrane

  14. Thiol-reactive drug substrates of human P-glycoprotein label the same sites to activate ATPase activity in membranes or dodecyl maltoside detergent micelles.

    Science.gov (United States)

    Loo, Tip W; Clarke, David M

    2017-07-08

    P-glycoprotein (P-gp, ABCB1) is an ABC drug pump that is clinically important because it is involved in multidrug resistance. Many studies have used purified P-gp in detergent (n-dodecyl-β-D-maltoside; DM) micelles to map the locations of the drug-binding sites. A potential problem is that DM could be a substrate and affect binding of drugs to P-gp. To test whether DM was a substrate of P-gp, we used an assay involving drug-rescue of the immature 150 kDa misprocessed P-gp mutant (L1260A) to show that DM is not substrate. By contrast, the detergents Triton X-100 or NP-35 were substrates because they rescued the L1260A P-gp mutant such that the major product was the mature 170 kDa protein. Cross-linking of mutant A80C/R741C in membranes can only be inhibited by the P-gp substrate tariquidar. We show that cross-linking A80C/R741C mutant was also inhibited by tariquidar in the presence of excess DM. This result suggests that the presence of DM did not affect the tariquidar-binding site. Similarly, the presence of DM did not alter the locations of other drug-binding sites since the thiol reactive forms of the substrates verapamil or rhodamine labeled the same sites in transmembrane segments 5 (I306C for verapamil) and 6 (F343C for rhodamine) whether P-gp was in native membranes or in detergent micelles. These results suggest that the presence of DM does not alter the locations of the P-gp drug-binding sites and that the detergent purified protein is suitable for mapping their locations using biochemical or structural assays. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  16. Terminal Galactosylation and Sialylation Switching on Membrane Glycoproteins upon TNF-Alpha-Induced Insulin Resistance in Adipocytes.

    Science.gov (United States)

    Parker, Benjamin L; Thaysen-Andersen, Morten; Fazakerley, Daniel J; Holliday, Mira; Packer, Nicolle H; James, David E

    2016-01-01

    Insulin resistance (IR) is a complex pathophysiological state that arises from both environmental and genetic perturbations and leads to a variety of diseases, including type-2 diabetes (T2D). Obesity is associated with enhanced adipose tissue inflammation, which may play a role in disease progression. Inflammation modulates protein glycosylation in a variety of cell types, and this has been associated with biological dysregulation. Here, we have examined the effects of an inflammatory insult on protein glycosylation in adipocytes. We performed quantitative N-glycome profiling of membrane proteins derived from mouse 3T3-L1 adipocytes that had been incubated with or without the proinflammatory cytokine TNF-alpha to induce IR. We identified the regulation of specific terminal N-glycan epitopes, including an increase in terminal di-galactose- and a decrease in biantennary alpha-2,3-sialoglycans. The altered N-glycosylation of TNF-alpha-treated adipocytes correlated with the regulation of specific glycosyltransferases, including the up-regulation of B4GalT5 and Ggta1 galactosyltransferases and down-regulation of ST3Gal6 sialyltransferase. Knockdown of B4GalT5 down-regulated the terminal di-galactose N-glycans, confirming the involvement of this enzyme in the TNF-alpha-regulated N-glycome. SILAC-based quantitative glycoproteomics of enriched N-glycopeptides with and without deglycosylation were used to identify the protein and glycosylation sites modified with these regulated N-glycans. The combined proteome and glycoproteome workflow provided a relative quantification of changes in protein abundance versus N-glycosylation occupancy versus site-specific N-glycans on a proteome-wide level. This revealed the modulation of N-glycosylation on specific proteins in IR, including those previously associated with insulin-stimulated GLUT4 trafficking to the plasma membrane. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanyan; Hu, Yahui; Feng, Yidong; Kodithuwakku, Nandani Darshika; Fang, Weirong [State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009 (China); Li, Yunman, E-mail: yunmanlicpu@hotmail.com [State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009 (China); Huang, Wenlong [Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009 (China)

    2014-01-15

    Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity and multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein. - Highlights: • The cytotoxicity and reversing effect of HZ08 was measured in Caco-2 cell line. • HZ08 inhibited the transport of Rhodamine123 across Caco-2 cell monolayer. • The efflux ratio of HZ08 was dropped when combined with P-glycoprotein

  18. The lysosome and neurodegenerative diseases

    Institute of Scientific and Technical Information of China (English)

    Lisha Zhang; Rui Sheng; Zhenghong Qin

    2009-01-01

    It has long been believed that the lysosome is an important digestive organelle. There is increasing evidence that the lysosome is also involved in pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Abnormal protein degradation and deposition induced by lysosoreal dysfunction may be the primary contributor to age-related neurodegeneration. In this review, the possible relationship between lysosome and various neurodegenerative diseases is described.

  19. Association of the Platelet Membrane Glycoprotein Ⅰa C807T Gene Polymorphism with Aspirin Resistance

    Institute of Scientific and Technical Information of China (English)

    SU Guanhua; WANG Zhaohui; DING Yanping; LIU Xiaoqian; WANG Jue

    2007-01-01

    To explore the correlation between the C807T polymorphism of platelet membrane gly- coprotein Ⅰa (GPⅠa) gene and aspirin resistance in Chinese people, 200 patients with high-risk of atherosclerosis took aspirin (100 mg/d) for 7 days. Platelet aggregation function was detected using adenosine diphosphate (ADP) and arachidonic acid (AA) before and after the administration of aspi- fin. Then the subjects were divided into three groups according to the results of platelet aggregation function: an aspirin resistant (AR) group, an aspirin semi-responder (ASR) group and an aspi- fin-sensitive (AS) group. Platelet GPⅠa gene 807CT polymorphism was examined by means of po- lymerase chain reaction-sequence specific primers (PCR-SSP). The results showed that T allelic fre- quency in AR group and ASR group were higher that of AS group (P<0.005), and the prevalence of genotypes (TT+TC) of these two groups was significantly higher than that in AS group (P<0.05). Platelet GPⅠa T allele was significantly associated with aspirin resistance as revealed by multiple logistic regression (OR=3.76, 95% CI: 2.87-9.58). The results suggest that inherited platelet GPⅠa variations may have an important impact on aspirin resistance and the presence of GPⅠa T allele may be a marker of genetic susceptibility to aspirin resistance.

  20. Syntaxin 7 and VAMP-7 are soluble N-ethylmaleimide-sensitive factor attachment protein receptors required for late endosome-lysosome and homotypic lysosome fusion in alveolar macrophages.

    Science.gov (United States)

    Ward, D M; Pevsner, J; Scullion, M A; Vaughn, M; Kaplan, J

    2000-07-01

    Endocytosis in alveolar macrophages can be reversibly inhibited, permitting the isolation of endocytic vesicles at defined stages of maturation. Using an in vitro fusion assay, we determined that each isolated endosome population was capable of homotypic fusion. All vesicle populations were also capable of heterotypic fusion in a temporally specific manner; early endosomes, isolated 4 min after internalization, could fuse with endosomes isolated 8 min after internalization but not with 12-min endosomes or lysosomes. Lysosomes fuse with 12-min endosomes but not with earlier endosomes. Using homogenous populations of endosomes, we have identified Syntaxin 7 as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome-lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late endosome and lysosome fusion as well as heterotypic late endosome-lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was a SNARE required for late endosome-lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both late endosome-lysosome fusion and homotypic lysosome fusion in vitro. These studies indicate that: 1) fusion along the endocytic pathway is a highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and human VAMP-7, are involved in fusion of vesicles in the late endocytic pathway in alveolar macrophages.

  1. A TRP Channel in the Lysosome Regulates Large Particle Phagocytosis via Focal Exocytosis

    OpenAIRE

    2013-01-01

    Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here we identified Mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers, and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML...

  2. Zinc iodide-osmium staining of membrane-coating granules in keratinized and non-keratinized mammalian oral epithelium.

    Science.gov (United States)

    Squier, C A

    1982-01-01

    Specimens of keratinized and non-keratinized oral epithelium were examined in the electron microscope after being stained with zinc iodide-osmium. In both types of tissue, reaction was seen in unmyelinated nerves, in the specific granules of epithelial Langerhans cells and within lysosome-like organelles and small vesicles associated with Golgi systems. In keratinized epithelia, the reaction was also present in the membrane-coating granules and between the deepest cells of the keratinized layer. In contrast, the membrane-coating granules of non-keratinized epithelia lacked Zn iodide-osmium staining despite the presence of reaction in adjacent Golgi systems. It is suggested that Zn iodide-osmium stains glycolipid or glycoprotein material in the cell. This material is elaborated in the Golgi systems from which lysosomes and the membrane-coating granules of keratinized tissues are probably derived.

  3. Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Russell Vassell

    Full Text Available The membrane proximal external region (MPER of the gp41 subunit of the HIV-1 envelope glycoprotein (Env contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols.

  4. Fullerene inhibits benzo(a)pyrene Efflux from Cyprinus carpio hepatocytes by affecting cell membrane fluidity and P-glycoprotein expression.

    Science.gov (United States)

    Chen, Qiqing; Hu, Xialin; Wang, Rui; Yuan, Jin; Yin, Daqiang

    2016-05-01

    P-Glycoprotein (P-gp) can protect cells by pumping out toxic compounds, and has been found widely expressed in fish tissues. Here, we illustrate the P-gp efflux ability for benzo(a)pyrene (BaP) in the hepatocytes of common carp (Cyprinus carpio) after exposing to fullerene aqueous suspension (nC60). The results revealed that nC60 increased the membrane fluidity by decreasing the ratio of saturated to unsaturated fatty acids, and increased the cholesterol contents. These findings, combined with 10-38% and 70-75% down-regulation of P-gp mRNA and protein respectively, suggested that nC60 caused inhibition on P-gp efflux transport system. Therefore, we further investigated the cellular efflux ability for BaP. Results showed unequivocally that nC60 is a potent P-gp inhibitor. The retaining BaP amounts after efflux were elevated by 1.7-2.8 fold during the 10 day exposure. Meanwhile, 5mg/L humic acid (one of the important fractions of natural organic matter, which is ubiquitous in aquatic environment) alleviated the nC60 damage to hepatocytes in terms of oxidative damage, cholesterol increment, and P-gp content reduction; and finally attenuated the suppressed P-gp efflux ability. Collectively, this study provides the first evidence of nC60 toxicity to P-gp functionality in fish and illustrates the possible mechanism of the suppressed P-gp efflux ability for BaP.

  5. Cancer-associated lysosomal changes

    DEFF Research Database (Denmark)

    Kallunki, T; Olsen, O D; Jaattela, Marja

    2013-01-01

    Rapidly dividing and invasive cancer cells are strongly dependent on effective lysosomal function. Accordingly, transformation and cancer progression are characterized by dramatic changes in lysosomal volume, composition and cellular distribution. Depending on one's point of view, the cancer-asso......:10.1038/onc.2012.292....

  6. A TRP channel in the lysosome regulates large particle phagocytosis via focal exocytosis.

    Science.gov (United States)

    Samie, Mohammad; Wang, Xiang; Zhang, Xiaoli; Goschka, Andrew; Li, Xinran; Cheng, Xiping; Gregg, Evan; Azar, Marlene; Zhuo, Yue; Garrity, Abigail G; Gao, Qiong; Slaugenhaupt, Susan; Pickel, Jim; Zolov, Sergey N; Weisman, Lois S; Lenk, Guy M; Titus, Steve; Bryant-Genevier, Marthe; Southall, Noel; Juan, Marugan; Ferrer, Marc; Xu, Haoxing

    2013-09-16

    Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here, we identified mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML1 overexpression and TRPML1 agonists facilitate both lysosomal exocytosis and particle uptake. Using time-lapse confocal imaging and direct patch clamping of phagosomal membranes, we found that particle binding induces lysosomal PI(3,5)P2 elevation to trigger TRPML1-mediated lysosomal Ca2+ release specifically at the site of uptake, rapidly delivering TRPML1-resident lysosomal membranes to nascent phagosomes via lysosomal exocytosis. Thus phagocytic ingestion of large particles activates a phosphoinositide- and Ca2+-dependent exocytosis pathway to provide membranes necessary for pseudopod extension, leading to clearance of senescent and apoptotic cells in vivo.

  7. Expression Pattern of Lysosomal Protective Protein/Cathepsin A: Implications for the analysis of hnman galactosialidosis

    NARCIS (Netherlands)

    R.J. Rottier (Robbert)

    1998-01-01

    textabstractThe lysosome represents a well characterized, membrane-contained intracellular digestive system. Iu this important organelle a battery of lysosomal hydro lases and accessory proteins work in concert on the step-wise conversion of macromolecular substrates into small biological building b

  8. Vamp-7 Mediates Vesicular Transport from Endosomes to Lysosomes

    Science.gov (United States)

    Advani, Raj J.; Yang, Bin; Prekeris, Rytis; Lee, Kelly C.; Klumperman, Judith; Scheller, Richard H.

    1999-01-01

    A more complete picture of the molecules that are critical for the organization of membrane compartments is beginning to emerge through the characterization of proteins in the vesicle-associated membrane protein (also called synaptobrevin) family of membrane trafficking proteins. To better understand the mechanisms of membrane trafficking within the endocytic pathway, we generated a series of monoclonal and polyclonal antibodies against the cytoplasmic domain of vesicle-associated membrane protein 7 (VAMP-7). The antibodies recognize a 25-kD membrane-associated protein in multiple tissues and cell lines. Immunohistochemical analysis reveals colocalization with a marker of late endosomes and lysosomes, lysosome-associated membrane protein 1 (LAMP-1), but not with other membrane markers, including p115 and transferrin receptor. Treatment with nocodozole or brefeldin A does not disrupt the colocalization of VAMP-7 and LAMP-1. Immunoelectron microscopy analysis shows that VAMP-7 is most concentrated in the trans-Golgi network region of the cell as well as late endosomes and transport vesicles that do not contain the mannose-6 phosphate receptor. In streptolysin- O–permeabilized cells, antibodies against VAMP-7 inhibit the breakdown of epidermal growth factor but not the recycling of transferrin. These data are consistent with a role for VAMP-7 in the vesicular transport of proteins from the early endosome to the lysosome. PMID:10459012

  9. Biogenesis of lysosomal enzymes in the alpha-glucosidase II-deficient modA mutant of Dictyostelium discoideum: retention of alpha-1,3-linked glucose on N-linked oligosaccharides delays intracellular transport but does not alter sorting of alpha-mannosidase or beta-glucosidase.

    Science.gov (United States)

    Ebert, D L; Bush, J M; Dimond, R L; Cardelli, J A

    1989-09-01

    The endoplasmic reticulum-localized enzyme alpha-glucosidase II is responsible for removing the two alpha-1,3-linked glucose residues from N-linked oligosaccharides of glycoproteins. This activity is missing in the modA mutant strain, M31, of Dictyostelium discoideum. Results from both radiolabeled pulse-chase and subcellular fractionation experiments indicate that this deficiency did not prevent intracellular transport and proteolytic processing of the lysosomal enzymes, alpha-mannosidase and beta-glucosidase. However, the rate at which the glucosylated precursors left the rough endoplasmic reticulum was several-fold slower than the rate at which the wild-type precursors left this compartment. Retention of glucose residues did not disrupt the binding of the precursor forms of the enzymes with intracellular membranes, indicating that the delay in movement of proteins from the ER did not result from lack of association with membranes. However, the mutant alpha-mannosidase precursor contained more trypsin-sensitive sites than did the wild-type precursor, suggesting that improper folding of precursor molecules might account for the slow rate of transport to the Golgi complex. Percoll density gradient fractionation of extracts prepared from M31 cells indicated that the proteolytically processed mature forms of alpha-mannosidase and beta-glucosidase were localized to lysosomes. Finally, the mutation in M31 may have other, more dramatic, effects on the lysosomal system since two enzymes, N-acetylglucosaminidase and acid phosphatase, were secreted much less efficiently from lysosomal compartments by the mutant strain.

  10. Lysosomal trafficking functions of mucolipin-1 in murine macrophages

    Directory of Open Access Journals (Sweden)

    Dang Hope

    2007-12-01

    Full Text Available Abstract Background Mucolipidosis Type IV is currently characterized as a lysosomal storage disorder with defects that include corneal clouding, achlorhydria and psychomotor retardation. MCOLN1, the gene responsible for this disease, encodes the protein mucolipin-1 that belongs to the "Transient Receptor Potential" family of proteins and has been shown to function as a non-selective cation channel whose activity is modulated by pH. Two cell biological defects that have been described in MLIV fibroblasts are a hyperacidification of lysosomes and a delay in the exit of lipids from lysosomes. Results We show that mucolipin-1 localizes to lysosomal compartments in RAW264.7 mouse macrophages that show subcompartmental accumulations of endocytosed molecules. Using stable RNAi clones, we show that mucolipin-1 is required for the exit of lipids from these compartments, for the transport of endocytosed molecules to terminal lysosomes, and for the transport of the Major Histocompatibility Complex II to the plasma membrane. Conclusion Mucolipin-1 functions in the efficient exit of molecules, destined for various cellular organelles, from lysosomal compartments.

  11. Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

    2016-06-30

    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 Ca(2+)-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca(2+) 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.

  13. Lysosomal acid phosphatase is internalized via clathrin-coated pits

    NARCIS (Netherlands)

    Klumperman, J.; Hille, A.; Geuze, H.J.; Peters, C.; Brodsky, F.M.; Figura, K. von

    1992-01-01

    The presence of lysosomal acid phosphatase (LAP) in coated pits at the plasma membrane was investigated by immunocytochemistry in thymidine kinase negative mouse L-cells (Ltk-) and baby hamster kidney (BHK) cells overexpressing human LAP (Ltk-LAP and BHK-LAP cells). Double immunogold labeling showed

  14. Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike.

    Directory of Open Access Journals (Sweden)

    Sai Li

    2016-02-01

    Full Text Available Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.

  15. Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike.

    Science.gov (United States)

    Li, Sai; Sun, Zhaoyang; Pryce, Rhys; Parsy, Marie-Laure; Fehling, Sarah K; Schlie, Katrin; Siebert, C Alistair; Garten, Wolfgang; Bowden, Thomas A; Strecker, Thomas; Huiskonen, Juha T

    2016-02-01

    Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.

  16. Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike.

    Directory of Open Access Journals (Sweden)

    Sai Li

    2016-02-01

    Full Text Available Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.

  17. TRPML1: an ion channel in the lysosome.

    Science.gov (United States)

    Wang, Wuyang; Zhang, Xiaoli; Gao, Qiong; Xu, Haoxing

    2014-01-01

    The first member of the mammalian mucolipin TRP channel subfamily (TRPML1) is a cation-permeable channel that is predominantly localized on the membranes of late endosomes and lysosomes (LELs) in all mammalian cell types. In response to the regulatory changes of LEL-specific phosphoinositides or other cellular cues, TRPML1 may mediate the release of Ca(2+) and heavy metal Fe(2+)/Zn(2+)ions into the cytosol from the LEL lumen, which in turn may regulate membrane trafficking events (fission and fusion), signal transduction, and ionic homeostasis in LELs. Human mutations in TRPML1 result in type IV mucolipidosis (ML-IV), a childhood neurodegenerative lysosome storage disease. At the cellular level, loss-of-function mutations of mammalian TRPML1 or its C. elegans or Drosophila homolog gene results in lysosomal trafficking defects and lysosome storage. In this chapter, we summarize recent advances in our understandings of the cell biological and channel functions of TRPML1. Studies on TRPML1's channel properties and its regulation by cellular activities may provide clues for developing new therapeutic strategies to delay neurodegeneration in ML-IV and other lysosome-related pediatric diseases.

  18. Effects of the I559P gp41 change on the conformation and function of the human immunodeficiency virus (HIV-1 membrane envelope glycoprotein trimer.

    Directory of Open Access Journals (Sweden)

    Nirmin Alsahafi

    Full Text Available The mature human immunodeficiency virus (HIV-1 envelope glycoprotein (Env trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env

  19. uPARAP/endo180 directs lysosomal delivery and degradation of collagen IV

    DEFF Research Database (Denmark)

    Kjøller, Lars; Engelholm, Lars H; Høyer-Hansen, Maria

    2004-01-01

    transmembrane glycoprotein urokinase plasminogen activator receptor-associated protein (uPARAP/endo180) directs collagen IV for lysosomal delivery and degradation. In wild-type fibroblasts, fluorescently labeled collagen IV was first internalized into vesicular structures with diffuse fluorescence eventually...... appearing uniformly within the wild-type cells after longer incubation times. In these cells, some collagen-containing vesicles were identified as lysosomes by staining for LAMP-1. In contrast, collagen IV remained extracellular and associated with fiber-like structures on uPARAP/endo180-deficient...... fibroblasts. Blocking lysosomal cysteine proteases with the inhibitor E64d resulted in strong accumulation of collagen IV in lysosomes in wild-type cells, but only very weak intracellular fluorescence accumulation in uPARAP/endo180-deficient fibroblasts. We conclude that uPARAP/endo180 is critical...

  20. Cancer-associated lysosomal changes

    DEFF Research Database (Denmark)

    Kallunki, T; Olsen, O D; Jaattela, Marja

    2013-01-01

    Rapidly dividing and invasive cancer cells are strongly dependent on effective lysosomal function. Accordingly, transformation and cancer progression are characterized by dramatic changes in lysosomal volume, composition and cellular distribution. Depending on one's point of view, the cancer-associated......-targeting anti-cancer drugs. In this review we compile our current knowledge on cancer-associated changes in lysosomal composition and discuss the consequences of these alterations to cancer progression and the possibilities they can bring to cancer therapy.Oncogene advance online publication, 9 July 2012; doi...

  1. Calpains mediate epithelial-cell death during mammary gland involution: mitochondria and lysosomal destabilization.

    Science.gov (United States)

    Arnandis, T; Ferrer-Vicens, I; García-Trevijano, E R; Miralles, V J; García, C; Torres, L; Viña, J R; Zaragozá, R

    2012-09-01

    Our aim was to elucidate the physiological role of calpains (CAPN) in mammary gland involution. Both CAPN-1 and -2 were induced after weaning and its activity increased in isolated mitochondria and lysosomes. CAPN activation within the mitochondria could trigger the release of cytochrome c and other pro-apoptotic factors, whereas in lysosomes it might be essential for tissue remodeling by releasing cathepsins into the cytosol. Immunohistochemical analysis localized CAPNs mainly at the luminal side of alveoli. During weaning, CAPNs translocate to the lysosomes processing membrane proteins. To identify these substrates, lysosomal fractions were treated with recombinant CAPN and cleaved products were identified by 2D-DIGE. The subunit b(2) of the v-type H(+) ATPase is proteolyzed and so is the lysosomal-associated membrane protein 2a (LAMP2a). Both proteins are also cleaved in vivo. Furthermore, LAMP2a cleavage was confirmed in vitro by addition of CAPNs to isolated lysosomes and several CAPN inhibitors prevented it. Finally, in vivo inhibition of CAPN1 in 72-h-weaned mice decreased LAMP2a cleavage. Indeed, calpeptin-treated mice showed a substantial delay in tissue remodeling and involution of the mammary gland. These results suggest that CAPNs are responsible for mitochondrial and lysosomal membrane permeabilization, supporting the idea that lysosomal-mediated cell death is a new hallmark of mammary gland involution.

  2. Action of low-energy monochromatic coherent light on the stability of retinal lysosomes

    Science.gov (United States)

    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.

  3. Modulatory Effect of Taurine on 7,12-Dimethylbenz(a)Anthracene-Induced Alterations in Detoxification Enzyme System, Membrane Bound Enzymes, Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue.

    Science.gov (United States)

    Vanitha, Manickam Kalappan; Baskaran, Kuppusamy; Periyasamy, Kuppusamy; Selvaraj, Sundaramoorthy; Ilakkia, Aruldoss; Saravanan, Dhiravidamani; Venkateswari, Ramachandran; Revathi Mani, Balasundaram; Anandakumar, Pandi; Sakthisekaran, Dhanapal

    2016-08-01

    The modulatory effect of taurine on 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na(+) /K(+) ATPase, Ca(2+) ATPase, and Mg(2+) ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione-S-transferase and UDP-glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA-induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA-induced breast cancer.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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/.

  6. Identification of cytoskeleton-associated proteins essential for lysosomal stability and survival of human cancer cells.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  8. Protective effect of squalene on certain lysosomal hydrolases and free amino acids in isoprenaline-induced myocardial infarction in rats

    DEFF Research Database (Denmark)

    Farvin, Sabeena; Surendraraj, A.; Anandan, R.

    2010-01-01

    This study was aimed to evaluate the preventive role of squalene on free amino acids and lysosomal alterations in experimentally induced myocardial infarction in rats. The levels of lysosomal enzymes (beta-glucuronidase, beta-galactosidase, beta-glucosidase, acid phosphatase and cathepsin D......) in plasma and lysosomal fractions, hydroxyproline content and free amino acids in heart tissue were determined. Isoprenaline administration to rats resulted in decreased stability of the membranes which was reflected by significantly (p...

  9. Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

    Energy Technology Data Exchange (ETDEWEB)

    Du, Yijun [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan (China); Pattnaik, Asit K. [School of Veterinary Medicine and Biomedical Sciences and the Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583-0900 (United States); Song, Cheng [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Yoo, Dongwan, E-mail: dyoo@illinois.edu [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Li, Gang, E-mail: dyoo@illinois.edu [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing (China)

    2012-03-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 ({omega} - 2, where {omega} is the GPI moiety at E160), P159 ({omega} - 1), and M162 ({omega} + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.

  10. Enhancing lysosomal biogenesis and autophagic flux by activating the transcription factor EB protects against cadmium-induced neurotoxicity

    Science.gov (United States)

    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

  11. Human ClC-6 is a late endosomal glycoprotein that associates with detergent-resistant lipid domains.

    Directory of Open Access Journals (Sweden)

    Sofie Ignoul

    and ClC-7 when cotransfected in COS-1 cells. CONCLUSIONS: We conclude that human ClC-6 is an endosomal glycoprotein that partitions in detergent resistant lipid domains. The differential sorting of endogenous (late endosomal versus overexpressed (early and recycling endosomal ClC-6 is reminiscent of that of other late endosomal/lysosomal membrane proteins (e.g. LIMP II, and is consistent with a rate-limiting sorting step for ClC-6 between early endosomes and its final destination in late endosomes.

  12. Interaction of mouse hepatitis virus (MHV) spike glycoprotein with receptor glycoprotein MHVR is required for infection with an MHV strain that expresses the hemagglutinin-esterase glycoprotein

    NARCIS (Netherlands)

    Gagneten, S; Gout, O; Dubois-Dalcq, M; Rottier, P; Rossen, J; Holmes, K V

    1995-01-01

    In addition to the spike (S) glycoprotein that binds to carcinoembryonic antigen-related receptors on the host cell membrane, some strains of mouse coronavirus (mouse hepatitis virus [MHV]) express a hemagglutinin esterase (HE) glycoprotein with hemagglutinating and acetylesterase activity. Virions

  13. Neuronal filopodium formation induced by the membrane glycoprotein M6a (Gpm6a) is facilitated by coronin-1a, Rac1, and p21-activated kinase 1 (Pak1).

    Science.gov (United States)

    Alvarez Juliá, Anabel; Frasch, Alberto C; Fuchsova, Beata

    2016-04-01

    Stress-responsive neuronal membrane glycoprotein M6a (Gpm6a) functions in neurite extension, filopodium and spine formation and synaptogenesis. The mechanisms of Gpm6a action in these processes are incompletely understood. Previously, we identified the actin regulator coronin-1a (Coro1a) as a putative Gpm6a interacting partner. Here, we used co-immunoprecipitation assays with the anti-Coro1a antibody to show that Coro1a associates with Gpm6a in rat hippocampal neurons. By immunofluorescence microscopy, we demonstrated that in hippocampal neurons Coro1a localizes in F-actin-enriched regions and some of Coro1a spots co-localize with Gpm6a labeling. Notably, the over-expression of a dominant-negative form of Coro1a as well as its down-regulation by siRNA interfered with Gpm6a-induced filopodium formation. Coro1a is known to regulate the plasma membrane translocation and activation of small GTPase Rac1. We show that Coro1a co-immunoprecipitates with Rac1 together with Gpm6a. Pharmacological inhibition of Rac1 resulted in a significant decrease in filopodium formation by Gpm6a. The same was observed upon the co-expression of Gpm6a with the inactive GDP-bound form of Rac1. In this case, the elevated membrane recruitment of GDP-bound Rac1 was detected as well. Moreover, the kinase activity of the p21-activated kinase 1 (Pak1), a main downstream effector of Rac1 that acts downstream of Coro1a, was required for Gpm6a-induced filopodium formation. Taken together, our results provide evidence that a signaling pathway including Coro1a, Rac1, and Pak1 facilitates Gpm6a-induced filopodium formation. Formation of filopodia by membrane glycoprotein M6a (Gpm6a) requires actin regulator coronin-1a (Coro1a), known to regulate plasma membrane localization and activation of Rac1 and its downstream effector Pak1. Coro1a associates with Gpm6a. Blockage of Coro1a, Rac1, or Pak1 interferes with Gpm6a-induced filopodium formation. Moreover, Gpm6a facilitates Rac1 membrane recruitment

  14. Syntaxin 7 and VAMP-7 are Soluble N-Ethylmaleimide–sensitive Factor Attachment Protein Receptors Required for Late Endosome–Lysosome and Homotypic Lysosome Fusion in Alveolar Macrophages

    Science.gov (United States)

    Ward, Diane McVey; Pevsner, Jonathan; Scullion, Matthew A.; Vaughn, Michael; Kaplan, Jerry

    2000-01-01

    Endocytosis in alveolar macrophages can be reversibly inhibited, permitting the isolation of endocytic vesicles at defined stages of maturation. Using an in vitro fusion assay, we determined that each isolated endosome population was capable of homotypic fusion. All vesicle populations were also capable of heterotypic fusion in a temporally specific manner; early endosomes, isolated 4 min after internalization, could fuse with endosomes isolated 8 min after internalization but not with 12-min endosomes or lysosomes. Lysosomes fuse with 12-min endosomes but not with earlier endosomes. Using homogenous populations of endosomes, we have identified Syntaxin 7 as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome–lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late endosome and lysosome fusion as well as heterotypic late endosome–lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was a SNARE required for late endosome–lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both late endosome–lysosome fusion and homotypic lysosome fusion in vitro. These studies indicate that: 1) fusion along the endocytic pathway is a highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and human VAMP-7, are involved in fusion of vesicles in the late endocytic pathway in alveolar macrophages. PMID:10888671

  15. High sphingomyelin levels induce lysosomal damage and autophagy dysfunction in Niemann Pick disease type A

    Science.gov (United States)

    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

  16. Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions.

    Science.gov (United States)

    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.

  17. Differentiation of norm and pathology during selective biochemical skreening of lysosomal storage diseases with increased excretion of oligosaccharides

    Directory of Open Access Journals (Sweden)

    N. Y. Mytsyk

    2015-06-01

    Full Text Available Oligosaccharides are a class of polymeric carbohydrates, which are constituents of a glycoside portion of glycoprotein and glycolipid molecules. The lysosomal hydrolase dysfunction due to lysosomal storage disorders results in partial or complete failure of degradation of some glycoproteins and glycolipids, causing the accumulation of specific undegraded substrates in the lysosomes of cells, the formation of the great number of oligosaccharide chains and their increased excretion with urine. Our work was aimed at detailed study of the specificities of interpreting the results of thin-layer chromatography (TLC of urine oligosaccharides in healthy persons of different age groups with the purpose of further application of these data while differentiating the norm and pathology in the course of primary selective screening of lysosomal storage disorders. The results obtained demonstrated that TLC plates for the majority of healthy persons had insignificant excretion of a number of oligosaccharides (from monosaccharides to hexasaccharides with Rlac > 0.15, which can be characterized as physiological oligosacchariduria, conditioned by the metabolism specificities in lysosomes. Therefore while interpreting the urine samples of patients with the suspected lysosomal storage disorder it is diagnostically reasonable to examine the TLC plates for the presence of both oligosaccharide groups, absent in the samples of healthy persons, and all the fractions with Rlac < 0.15.

  18. Cross-talk between TRPML1 channel, lipids and lysosomal storage diseases.

    Science.gov (United States)

    Weiss, Norbert

    2012-03-01

    Described by the Belgian cytologist Christian De Duve in 1949,(1) lysosomes (from the Greek "digestive bodies") are ubiquitous specialized intracellular organelles that ensure the degradation/recycling of macromolecules (proteins, lipids, membranes) through the activity of specific enzymes (i.e., acid hydrolases). They receive their substrates through different internalization pathways (i.e., endocytosis, phagocytosis and autophagy) and are involved in a wide range of physiological functions from cell death and signaling to cholesterol homeostasis and plasma membrane repair.(2) In Mammals, 50 soluble lysosomal hydrolases have been described, each targeting specific substrates. They are confined in the lumen of the lysosome and require an optimum pH (i.e., pH 4.5) to work. This acidic pH compared with the slightly alkaline pH of the cytosol (i.e., ~pH 7.2) is maintained by the activity of integral lysosomal membrane proteins (LMPs, that represent the second class of lysosomal proteins), including the V-type proton (H(+))-ATPase(3) and the chloride ion channel CLC7(4) that pumps protons from the cytosol across the lysosomal membrane.

  19. Biomarkers in Lysosomal Storage Diseases

    Directory of Open Access Journals (Sweden)

    Joaquin Bobillo Lobato

    2016-12-01

    Full Text Available A biomarker is generally an analyte that indicates the presence and/or extent of a biological process, which is in itself usually directly linked to the clinical manifestations and outcome of a particular disease. The biomarkers in the field of lysosomal storage diseases (LSDs have particular relevance where spectacular therapeutic initiatives have been achieved, most notably with the introduction of enzyme replacement therapy (ERT. There are two main types of biomarkers. The first group is comprised of those molecules whose accumulation is directly enhanced as a result of defective lysosomal function. These molecules represent the storage of the principal macro-molecular substrate(s of a specific enzyme or protein, whose function is deficient in the given disease. In the second group of biomarkers, the relationship between the lysosomal defect and the biomarker is indirect. In this group, the biomarker reflects the effects of the primary lysosomal defect on cell, tissue, or organ functions. There is no “gold standard” among biomarkers used to diagnosis and/or monitor LSDs, but there are a number that exist that can be used to reasonably assess and monitor the state of certain organs or functions. A number of biomarkers have been proposed for the analysis of the most important LSDs. In this review, we will summarize the most promising biomarkers in major LSDs and discuss why these are the most promising candidates for screening systems.

  20. 高尔基体驻膜糖蛋白GP73启动子克隆%Cloning of the Promoter Regions of Golgi Membrane Glycoprotein GP73

    Institute of Scientific and Technical Information of China (English)

    谢红彬; 宫钰; 彭涛

    2008-01-01

    [Objective] The aim of this study is to clone the active promoter of golgi membrane glycoprotein GP73. [Method] The sequence within the range of upstream 1 000 bp and downstream 400 bp of transcription initiation site was analyzed, the genomic DNA of hepatoma cell line Huh-7 was regarded as template for PCR amplification. The amplified fragment was cloned into recombinant construct with enhanced green fluorescent protein (EGFP) report gene. The expression of EGFP in recombinants were observed under fluorescence microscope after transfection, with the assistant of flow cytometry. [Result] The 1 310 bp ranging between upstream 980 bp and downstream 330 bp of transcription initiation site assumed promoter function. The region contains two core promoters and several conserved sequences including TATA box and many DNA binding sites such as NF-κB, AP1, GC-SP1. [Conclusion] The cloning of the promoter provides a reference for the study of the transcription mechanism of GP73.

  1. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

    Directory of Open Access Journals (Sweden)

    Christine Burkard

    2014-11-01

    Full Text Available 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.

  2. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

    Science.gov (United States)

    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.

  3. Protective effects of sinapic acid on lysosomal dysfunction in isoproterenol induced myocardial infarcted rats.

    Science.gov (United States)

    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.

  4. Artesunate Activates Mitochondrial Apoptosis in Breast Cancer Cells via Iron-catalyzed Lysosomal Reactive Oxygen Species Production*

    Science.gov (United States)

    Hamacher-Brady, Anne; Stein, Henning A.; Turschner, Simon; Toegel, Ina; Mora, Rodrigo; Jennewein, Nina; Efferth, Thomas; Eils, Roland; Brady, Nathan R.

    2011-01-01

    The antimalarial agent artesunate (ART) activates programmed cell death (PCD) in cancer cells in a manner dependent on the presence of iron and the generation of reactive oxygen species. In malaria parasites, ART cytotoxicity originates from interactions with heme-derived iron within the food vacuole. The analogous digestive compartment of mammalian cells, the lysosome, similarly contains high levels of redox-active iron and in response to specific stimuli can initiate mitochondrial apoptosis. We thus investigated the role of lysosomes in ART-induced PCD and determined that in MCF-7 breast cancer cells ART activates lysosome-dependent mitochondrial outer membrane permeabilization. ART impacted endolysosomal and autophagosomal compartments, inhibiting autophagosome turnover and causing perinuclear clustering of autophagosomes, early and late endosomes, and lysosomes. Lysosomal iron chelation blocked all measured parameters of ART-induced PCD, whereas lysosomal iron loading enhanced death, thus identifying lysosomal iron as the lethal source of reactive oxygen species upstream of mitochondrial outer membrane permeabilization. Moreover, lysosomal inhibitors chloroquine and bafilomycin A1 reduced ART-activated PCD, evidencing a requirement for lysosomal function during PCD signaling. ART killing did not involve activation of the BH3-only protein, Bid, yet ART enhanced TNF-mediated Bid cleavage. We additionally demonstrated the lysosomal PCD pathway in T47D and MDA-MB-231 breast cancer cells. Importantly, non-tumorigenic MCF-10A cells resisted ART-induced PCD. Together, our data suggest that ART triggers PCD via engagement of distinct, interconnected PCD pathways, with hierarchical signaling from lysosomes to mitochondria, suggesting a potential clinical use of ART for targeting lysosomes in cancer treatment. PMID:21149439

  5. Inhibitors of lysosomal cysteine proteases

    Directory of Open Access Journals (Sweden)

    Lyanna O. L.

    2011-04-01

    Full Text Available The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs.

  6. Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV.

    Science.gov (United States)

    Park, Soonhong; Ahuja, Malini; Kim, Min Seuk; Brailoiu, G Cristina; Jha, Archana; Zeng, Mei; Baydyuk, Maryna; Wu, Ling-Gang; Wassif, Christopher A; Porter, Forbes D; Zerfas, Patricia M; Eckhaus, Michael A; Brailoiu, Eugen; Shin, Dong Min; Muallem, Shmuel

    2016-02-01

    Mutations in TRPML1 cause the lysosomal storage disease mucolipidosis type IV (MLIV). The role of TRPML1 in cell function and how the mutations cause the disease are not well understood. Most studies focus on the role of TRPML1 in constitutive membrane trafficking to and from the lysosomes. However, this cannot explain impaired neuromuscular and secretory cells' functions that mediate regulated exocytosis. Here, we analyzed several forms of regulated exocytosis in a mouse model of MLIV and, opposite to expectations, we found enhanced exocytosis in secretory glands due to enlargement of secretory granules in part due to fusion with lysosomes. Preliminary exploration of synaptic vesicle size, spontaneous mEPSCs, and glutamate secretion in neurons provided further evidence for enhanced exocytosis that was rescued by re-expression of TRPML1 in neurons. These features were not observed in Niemann-Pick type C1. These findings suggest that TRPML1 may guard against pathological fusion of lysosomes with secretory organelles and suggest a new approach toward developing treatment for MLIV.

  7. Elevation of glycoprotein nonmetastatic melanoma protein B in type 1 Gaucher disease patients and mouse models

    NARCIS (Netherlands)

    Kramer, Gertjan; Wegdam, Wouter; Donker-Koopman, Wilma; Ottenhoff, Roelof; Gaspar, Paulo; Verhoek, Marri; Nelson, Jessica; Gabriel, Tanit; Kallemeijn, Wouter; Boot, Rolf G.; Laman, Jon D.; Vissers, Johannes P. C.; Cox, Timothy; Pavlova, Elena; Moran, Mary Teresa; Aerts, Johannes M.; van Eijk, Marco

    2016-01-01

    Gaucher disease is caused by inherited deficiency of lysosomal glucocere-brosidase. Proteome analysis of laser-dissected splenic Gaucher cells revealed increased amounts of glycoprotein nonmetastatic melanoma protein B (gpNMB). Plasma gpNMB was also elevated, correlating with chitotriosidase and CCL

  8. Elevation of glycoprotein nonmetastatic melanoma protein B in type 1 Gaucher disease patients and mouse models

    NARCIS (Netherlands)

    Kramer, G.; Wegdam, W.; Donker-Koopman, W.; Ottenhoff, R.; Gaspar, P.; Verhoek, M.; Nelson, J.; Gabriel, T.; Kallemeijn, W.W.; Boot, R.G.; Laman, J.D.; Vissers, J.P.; Cox, T.; Pavlova, E.; Moran, M.T.; Aerts, J.M.F.G.; Eijk, van M.C.

    2016-01-01

    Gaucher disease is caused by inherited deficiency of lysosomal glucocerebrosidase. Proteome analysis of laser-dissected splenic Gaucher cells revealed increased amounts of glycoprotein nonmetastatic melanoma protein B (gpNMB). Plasma gpNMB was also elevated, correlating with chitotriosidase and CCL1

  9. Structure of a trimeric variant of the Epstein–Barr virus glycoprotein B

    OpenAIRE

    2009-01-01

    Epstein–Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the str...

  10. Endosome-lysosomes, ubiquitin and neurodegeneration.

    Science.gov (United States)

    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.

  11. Membraner

    DEFF Research Database (Denmark)

    Bach, Finn

    2009-01-01

    Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...

  12. Evidence for lysosomal exocytosis and release of aggrecan-degrading hydrolases from hypertrophic chondrocytes, in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Edward R. Bastow

    2012-02-01

    The abundant proteoglycan, aggrecan, is resorbed from growth plate cartilage during endochondral bone ossification, yet mice with genetically-ablated aggrecan-degrading activity have no defects in bone formation. To account for this apparent anomaly, we propose that lysosomal hydrolases degrade extracellular, hyaluronan-bound aggrecan aggregates in growth plate cartilage, and that lysosomal hydrolases are released from hypertrophic chondrocytes into growth plate cartilage via Ca2+-dependent lysosomal exocytosis. In this study we confirm that hypertrophic chondrocytes release hydrolases via lysosomal exocytosis in vitro and we show in vivo evidence for lysosomal exocytosis in hypertrophic chondrocytes during skeletal development. We show that lysosome-associated membrane protein 1 (LAMP1 is detected at the cell surface following in vitro treatment of epiphyseal chondrocytes with the calcium ionophore, ionomycin. Furthermore, we show that in addition to the lysosomal exocytosis markers, cathepsin D and β-hexosaminidase, ionomycin induces release of aggrecan- and hyaluronan-degrading activity from cultured epiphyseal chondrocytes. We identify VAMP-8 and VAMP7 as v-SNARE proteins with potential roles in lysosomal exocytosis in hypertrophic chondrocytes, based on their colocalisation with LAMP1 at the cell surface in secondary ossification centers in mouse tibiae. We propose that resorbing growth plate cartilage involves release of destructive hydrolases from hypertrophic chondrocytes, via lysosomal exocytosis.

  13. Glucagon-like Peptide-1 Protects Pancreatic Beta-cells from Death by Increasing Autophagic Flux and Restoring Lysosomal Function.

    Science.gov (United States)

    Zummo, Francesco P; Cullen, Kirsty S; Honkanen-Scott, Minna; Shaw, James Am; Lovat, Penny E; Arden, Catherine

    2017-02-23

    Studies in animal models of type 2 diabetes have shown that glucagon-like peptide-1 (GLP-1) receptor agonists prevent β-cell loss. Whether GLP-1 mediates β-cell survival via the key lysosomal-mediated process of autophagy is unknown.Here we report that treatment of INS-1E β-cells and primary islets with glucolipotoxicity (0.5mmol/l palmitate, 25mmol/l glucose) increases LC3 II, a marker of autophagy. Further analysis indicates a blockage in autophagic flux associated with lysosomal dysfunction. Accumulation of defective lysosomes leads to lysosomal membrane permeabilisation (LMP) and release of Cathepsin D, which contributes to cell death. Our data further demonstrated defects in autophagic flux and lysosomal staining in human samples of type 2 diabetes. Co-treatment with the GLP-1 receptor agonist exendin-4 reversed the lysosomal dysfunction, relieving the impairment in autophagic flux and further stimulated autophagy. siRNA knockdown showed the restoration of autophagic flux is also essential for the protective effects of exendin-4.Collectively, our data highlights lysosomal dysfunction as a critical mediator of β-cell loss and shows that exendin-4 improves cell survival via restoration of lysosomal function and autophagic flux. Modulation of autophagy / lysosomal homeostasis may thus define a novel therapeutic strategy for type 2 diabetes, with the GLP-1 signalling pathway as a potential focus.

  14. Impact of high cholesterol in a Parkinson's disease model: Prevention of lysosomal leakage versus stimulation of α-synuclein aggregation.

    Science.gov (United States)

    Eriksson, Ida; Nath, Sangeeta; Bornefall, Per; Giraldo, Ana Maria Villamil; Öllinger, Karin

    2017-03-01

    Parkinson's disease is characterized by accumulation of intraneuronal cytoplasmic inclusions, Lewy bodies, which mainly consist of aggregated α-synuclein. Controversies exist as to whether high blood cholesterol is a risk factor for the development of the disease and whether statin treatment could have a protective effect. Using a model system of BE(2)-M17 neuroblastoma cells treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)), we found that MPP(+)-induced cell death was accompanied by cholesterol accumulation in a lysosomal-like pattern in pre-apoptotic cells. To study the effects of lysosomal cholesterol accumulation, we increased lysosomal cholesterol through pre-treatment with U18666A and found delayed leakage of lysosomal contents into the cytosol, which reduced cell death. This suggests that increased lysosomal cholesterol is a stress response mechanism to protect lysosomal membrane integrity in response to early apoptotic stress. However, high cholesterol also stimulated the accumulation of α-synuclein. Treatment with the cholesterol-lowering drug lovastatin reduced MPP(+)-induced cell death by inhibiting the production of reactive oxygen species, but did not prevent lysosomal cholesterol increase nor affect α-synuclein accumulation. Our study indicates a dual role of high cholesterol in Parkinson's disease, in which it acts both as a protector against lysosomal membrane permeabilization and as a stimulator of α-synuclein accumulation. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. IgG red blood cell autoantibodies in autoimmune hemolytic anemia bind to epitopes on red blood cell membrane band 3 glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Victoria, E.J.; Pierce, S.W.; Branks, M.J.; Masouredis, S.P. (Univ. of California, San Diego (USA))

    1990-01-01

    Red blood cell (RBC) autoantibodies from patients with IgG warm-type autoimmune hemolytic anemia were labeled with iodine 125 and their RBC binding behavior characterized. Epitope-bearing RBC membrane polypeptides were identified after autoantibody immunoprecipitation of labeled membranes and immunoblotting. Immunoaffinity isolation of labeled membrane proteins with 12 different IgG hemolytic autoantibodies with protein A-agarose revealed a major polypeptide at Mr 95 to 110 kd, which coelectrophoresed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with a membrane component isolated with sheep IgG anti-band 3. Immunoprecipitation studies with chymotrypsinized RBCs resulted in the recovery of two labeled membrane polypeptides with molecular weights characteristically resulting from the chymotryptic fragmentation of band 3. Immunoblotting with sheep IgG anti-band 3 of the immunoprecipitated polypeptides confirmed that hemolytic autoantibody binding led to recovery of band 3 or its fragments. Two 125I-labeled IgG hemolytic autoantibodies showed binding behavior consistent with epitope localization on band 3. The labeled RBC autoantibodies bound immunospecifically to all types of human RBC tested, including those of rare Rh type (Rh-null, D--) at a site density of approximately 10(6) per RBC. The 125I-IgG in two labeled autoantibodies was 84% and 92% adsorbable by human and higher nonhuman primate RBCs. Antigen-negative animal RBC bound less than 10%, consistent with immunospecific RBC binding. IgG-1 was the major subclass in five autoantibodies tested; one of six fixed complement; and autoantibody IgG appeared polyclonal by isoelectric focusing. We conclude that IgG eluted from RBCs of patients with autoimmune hemolytic anemia consists predominantly of a single totally RBC-adsorbable antibody population that binds to antigenic determinants on band 3.

  16. GNeosomes: Highly Lysosomotropic Nanoassemblies for Lysosomal Delivery.

    Science.gov (United States)

    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.

  17. Activity of lysosomal exoglycosidases in serum and synovial fluid in patients with chronic Lyme and rheumatoid arthritis.

    Science.gov (United States)

    Pancewicz, Slawomir; Popko, Janusz; Rutkowski, Ryszard; Knaś, Malgorzata; Grygorczuk, Sambor; Guszczyn, Tomasz; Bruczko, Marta; Szajda, Slawomir; Zajkowska, Joanna; Kondrusik, Maciej; Sierakowski, Stanislaw; Zwierz, Krzysztof

    2009-01-01

    Lysosomal exoglycosidases participate in the destruction of the articular cartilage by cleaving glycoside bonds in glycoproteins and proteoglycans. The aim of the study was to determine the activity of exoglycosidases: hexosaminidase, beta-glucuronidase, beta-galactosidase, alpha-mannosidase and alpha-fucosidase in serum and synovial fluid of patients with Lyme and rheumatoid arthritis. The study group consisted of 10 patients with chronic Lyme arthritis (age 18 - 74 y), 13 with rheumatoid arthritis (age 32 - 70 y) and 10 with juvenile idiopathic arthritis (age 8 - 17 y). The control group consisted of 9 healthy volunteers (age 24 - 62 y). The activity of the exoglycosidases was determined with the p-nitrophenyl derivatives of sugars as substrates. A significant increase of the activity of all the exoglycosidases in serum and in synovial fluid of the patients with different forms of arthritis was found. The ratio of synovial fluid/serum activity of exoglycosidases was above 2.0 in LA but not in JIA and RA patients. As the main source of exoglycosidases in the joint is the synovial membrane, this result supports the appropriateness of therapeutic synovectomy in chronic Lyme arthritis with knee effusion. The serum activity of hexosaminidase may be used in monitoring the course of Lyme arthritis and the efficiency of treatment.

  18. Combined effects of thermal stress and Cd on lysosomal biomarkers and transcription of genes encoding lysosomal enzymes and HSP70 in mussels, Mytilus galloprovincialis.

    Science.gov (United States)

    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.

  19. Resistance to the macrocyclic lactone moxidectin is mediated in part by membrane transporter P-glycoproteins: Implications for control of drug resistant parasitic nematodes.

    Science.gov (United States)

    Bygarski, Elizabeth E; Prichard, Roger K; Ardelli, Bernadette F

    2014-12-01

    Our objective was to determine if the resistance mechanism to moxidectin (MOX) is similar of that to ivermectin (IVM) and involves P-glycoproteins (PGPs). Several Caenorhabditis elegans strains were used: an IVM and MOX sensitive strain, 13 PGP deletion strains and the IVM-R strain which shows synthetic resistance to IVM (by creation of three point mutations in genes coding for α-subunits of glutamate gated chloride channels [GluCls]) and cross-resistance to MOX. These strains were used to compare expression of PGP genes, measure motility and pharyngeal pumping phenotypes and evaluate the ability of compounds that inhibit PGP function to potentiate sensitivity or reverse resistance to MOX. The results suggest that C. elegans may use regulation of PGPs as a response mechanism to MOX. This was indicated by the over-expression of several PGPs in both drug sensitive and IVM-R strains and the significant changes in phenotype in the IVM-R strain in the presence of PGP inhibitors. However, as the inhibitors did not completely disrupt expression of the phenotypic traits in the IVM-R strain, this suggests that there likely are multiple avenues for MOX action that may include receptors other than GluCls. If MOX resistance was mediated solely by GluCls then exposure of the IVM-R strain to PGP inhibitors should not have affected sensitivity to MOX. Targeted gene deletions showed that protection of C. elegans against MOX involves complex mechanisms and depends on the PGP gene family, particularly PGP-6. While the results presented are similar to others using IVM, there were some important differences observed with respect to PGPs which may play a role in the disparities seen in the characteristics of resistance to IVM and MOX. The similarities are of concern as parasites resistant to IVM show some degree but not complete cross-resistance to MOX; this could impact nematodes that are resistant to IVM.

  20. Resistance to the macrocyclic lactone moxidectin is mediated in part by membrane transporter P-glycoproteins: Implications for control of drug resistant parasitic nematodes

    Directory of Open Access Journals (Sweden)

    Elizabeth E. Bygarski

    2014-12-01

    Full Text Available Our objective was to determine if the resistance mechanism to moxidectin (MOX is similar of that to ivermectin (IVM and involves P-glycoproteins (PGPs. Several Caenorhabditis elegans strains were used: an IVM and MOX sensitive strain, 13 PGP deletion strains and the IVM-R strain which shows synthetic resistance to IVM (by creation of three point mutations in genes coding for α-subunits of glutamate gated chloride channels [GluCls] and cross-resistance to MOX. These strains were used to compare expression of PGP genes, measure motility and pharyngeal pumping phenotypes and evaluate the ability of compounds that inhibit PGP function to potentiate sensitivity or reverse resistance to MOX. The results suggest that C. elegans may use regulation of PGPs as a response mechanism to MOX. This was indicated by the over-expression of several PGPs in both drug sensitive and IVM-R strains and the significant changes in phenotype in the IVM-R strain in the presence of PGP inhibitors. However, as the inhibitors did not completely disrupt expression of the phenotypic traits in the IVM-R strain, this suggests that there likely are multiple avenues for MOX action that may include receptors other than GluCls. If MOX resistance was mediated solely by GluCls then exposure of the IVM-R strain to PGP inhibitors should not have affected sensitivity to MOX. Targeted gene deletions showed that protection of C. elegans against MOX involves complex mechanisms and depends on the PGP gene family, particularly PGP-6. While the results presented are similar to others using IVM, there were some important differences observed with respect to PGPs which may play a role in the disparities seen in the characteristics of resistance to IVM and MOX. The similarities are of concern as parasites resistant to IVM show some degree but not complete cross-resistance to MOX; this could impact nematodes that are resistant to IVM.

  1. Lysosomal responses to heat-shock of seasonal temperature extremes in Cd-exposed mussels.

    Science.gov (United States)

    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.

  2. Combined effects of thermal stress and Cd on lysosomal biomarkers and transcription of genes encoding lysosomal enzymes and HSP70 in mussels, Mytilus galloprovincialis

    Energy Technology Data Exchange (ETDEWEB)

    Izagirre, Urtzi; Errasti, Aitzpea; Bilbao, Eider; Múgica, María; Marigómez, Ionan, E-mail: ionan.marigomez@ehu.es

    2014-04-01

    Highlights: • Thermal stress and Cd caused lysosomal enlargement and membrane destabilisation. • hex, gusb and ctsl but not hsp70 were up-regulated at elevated temperature but down-regulated by Cd. • Thermal stress influenced lysosomal responses to Cd exposure. • The presence of Cd jeopardised responsiveness against thermal stress. - Abstract: 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 24 h 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

  3. Not nanocarbon but dispersant induced abnormality in lysosome in macrophages in vivo

    Science.gov (United States)

    Yudasaka, Masako; Zhang, Minfang; Matsumura, Sachiko; Yuge, Ryota; Ichihashi, Toshinari; Irie, Hiroshi; Shiba, Kiyotaka; Iijima, Sumio

    2015-05-01

    The properties of nanocarbons change from hydrophobic to hydrophilic as a result of coating them with dispersants, typically phospholipid polyethylene glycols, for biological studies. It has been shown that the dispersants remain attached to the nanocarbons when they are injected in mice and influence the nanocarbons’ biodistribution in vivo. We show in this report that the effects of dispersants also appear at the subcellular level in vivo. Carbon nanohorns (CNHs), a type of nanocarbon, were dispersed with ceramide polyethylene glycol (CPEG) and intravenously injected in mice. Histological observations and electron microscopy with energy dispersive x-ray analysis revealed that, in liver and spleen, the lysosome membranes were damaged, and the nanohorns formed a complex with hemosiderin in the lysosomes of the macrophages. It is inferred that the lysosomal membrane was damaged by sphigosine generated as a result of CPEG decomposition, which changed the intra lysosomal conditions, inducing the formation of the CPEG-CNH and hemosiderin complex. For comparison, when glucose was used instead of CPEG, neither the nanohorn-hemosiderin complex nor lysosomal membrane damage was found. Our results suggest that surface functionalization can control the behavior of nancarbons in cells in vivo and thereby improve their suitability for medical applications.

  4. 溶酶体膜蛋白LAMP3对肝癌转移影响的研究进展%Progress of metastasis in hepatic cell carcinoma effected by lysosome-associated mem-brane proteins(LAMP3)

    Institute of Scientific and Technical Information of China (English)

    任庆; 谭宁

    2015-01-01

    原发性肝细胞肝癌( hepatic cell carcinoma,HCC)是广西区域性高发恶性肿瘤,其预后与肝癌是否转移密切相关。目前有研究发现溶酶体膜相关蛋白LAMP3在肝癌细胞稳定过表达后,可促进肝癌细胞的转移,同时诱导细胞骨架F-actin重排并上调Cdc42活性,提示LAMP3可能是肝癌转移的促进因子。本文就溶酶体膜蛋白LAMP3对肝癌转移的意义作一综述。%Hepatic cell carcinoma( HCC)was regional and high incidence of malignant tumor in Guangxi Province, the prognosis was closely related with metastasis whether or not. At present,some studies have discovered that lyso-some-associated membrane proteins( LAMP3 )stable over expression in hepatocellular carcinoma cells,which could promote the metastasis,at the same time induced cytoskeletal F-actin rearrangement and upregulation of Cdc42 ac-tivity. Results suggest that LAMP3 is a metastasis promoting factor. The lysosomal membrane protein LAMP3 on liver metastasis were reviewed in this article.

  5. Glycoproteins: Occurrence and Significance

    Science.gov (United States)

    Wittmann, Valentin

    Protein glycosylation is regarded as the most complex form of post-translational modification leading to a heterogeneous expression of glycoproteins as mixtures of glycoforms. This chapter describes the structure and occurrence of glycoproteins with respect to their glycan chains. Discussed are different carbohydrate-peptide linkages including GPI anchors, common structures of N- and O-glycans, and the structure of glycosaminoglycans contained in proteoglycans. Also covered are the bacterial cell wall polymer peptidoglycan and the glycopeptide antibiotics of the vancomycin group. Properties and functions of the glycans contained in glycoproteins are dealt with in the next chapter of this book.

  6. Cancer cell injury by cytotoxins from cobra venom is mediated through lysosomal damage.

    Science.gov (United States)

    Feofanov, Alexei V; Sharonov, George V; Astapova, Maria V; Rodionov, Dmitriy I; Utkin, Yuriy N; Arseniev, Alexander S

    2005-08-15

    Cytotoxins from cobra venom are known to manifest cytotoxicity in various cell types. It is widely accepted that the plasma membrane is a target of cytotoxins, but the mechanism of their action remains obscure. Using the confocal spectral imaging technique, we show for the first time that cytotoxins from cobra venom penetrate readily into living cancer cells and accumulate markedly in lysosomes. Cytotoxins CT1 and CT2 from Naja oxiana, CT3 from Naja kaouthia and CT1 from Naja haje are demonstrated to possess this property with respect to human lung adenocarcinoma A549 and promyelocytic leukaemia HL60 cells. Immobilized plasma membrane binding accompanies the internalization of CT3 from Naja kaouthia in the HL60 cells, but it is very weak for other cytotoxins. Detectable membrane binding is not a property of any of the cytotoxins tested in A549 cells. The kinetics and concentration-dependence of cytotoxin accumulation in lysosomes correlate well with their cytotoxic effects. On the basis of the results obtained, we propose that lysosomes are a primary target of the lytic action of cytotoxins. Plasma membrane permeabilization seems to be a downstream event relative to lysosome rupture. Direct damage to the plasma membrane may be a complementary mechanism, but its relative contribution to the cytotoxic action depends on the cytotoxin structure and cell type.

  7. Up-regulation of lysosomal TRPML1 channels is essential for lysosomal adaptation to nutrient starvation.

    Science.gov (United States)

    Wang, Wuyang; Gao, Qiong; Yang, Meimei; Zhang, Xiaoli; Yu, Lu; Lawas, Maria; Li, Xinran; Bryant-Genevier, Marthe; Southall, Noel T; Marugan, Juan; Ferrer, Marc; Xu, Haoxing

    2015-03-17

    Upon nutrient starvation, autophagy digests unwanted cellular components to generate catabolites that are required for housekeeping biosynthesis processes. A complete execution of autophagy demands an enhancement in lysosome function and biogenesis to match the increase in autophagosome formation. Here, we report that mucolipin-1 (also known as TRPML1 or ML1), a Ca(2+) channel in the lysosome that regulates many aspects of lysosomal trafficking, plays a central role in this quality-control process. By using Ca(2+) imaging and whole-lysosome patch clamping, lysosomal Ca(2+) release and ML1 currents were detected within hours of nutrient starvation and were potently up-regulated. In contrast, lysosomal Na(+)-selective currents were not up-regulated. Inhibition of mammalian target of rapamycin (mTOR) or activation of transcription factor EB (TFEB) mimicked a starvation effect in fed cells. The starvation effect also included an increase in lysosomal proteostasis and enhanced clearance of lysosomal storage, including cholesterol accumulation in Niemann-Pick disease type C (NPC) cells. However, this effect was not observed when ML1 was pharmacologically inhibited or genetically deleted. Furthermore, overexpression of ML1 mimicked the starvation effect. Hence, lysosomal adaptation to environmental cues such as nutrient levels requires mTOR/TFEB-dependent, lysosome-to-nucleus regulation of lysosomal ML1 channels and Ca(2+) signaling.

  8. Lysosomal exoglycosidases in serum and urine of patients with pancreatic adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Anna Stypułkowska

    2010-11-01

    Full Text Available Lysosomal exoglycosidases: N-acetyl-β-D-hexosaminidase (HEX, β-D-galactosidase (GAL, ι-L-fucosidase (FUC and ι-D-mannosidase (MAN modify oligosaccharide chains of glycoconjugates in endoplasmatic reticulum and/or Golgi apparatus and degrade them in lysosomes. In acid environment of lysosome, exoglycosidases degrade oligosaccharide chains of glycoproteins, glycolipids and glycosaminoglycans by eliminating single sugars from the edges of oligosaccharide chains. Neoplasms change biochemical processes in tissues and may significantly change the activity of many enzymes including the activity of lysosomal exoglycosidasses in serum and urine of persons with neoplasmatic diseases. The aim of the present paper was evaluation the activity of HEX, GAL, FUC and MAN in serum and urine of patients with pancreatic adenocarcinoma. Serum and urine samples were collected from 15 patients with adenocarcinoma of the pancreas and 15 healthy persons. The activity of lysosomal exoglycosidases was determined by the method of Marciniak et al. adapted to serum and urine of patients with adenocarcinoma of the pancreas. Our results indicate significant decrease in activity of GAL (p=0.037 in serum of patients with pancreatic adenocarcinoma, significant increase in activity of HEX (p<0.001 and FUC (p=0.027 in serum, and HEX (p=0.003 in urine, as well as significant decrease of FUC (p=0.016 and MAN (p=0.029 in urine o patients with adenocarcinoma of the pancreas, in comparison to the control group. Increase in activity of some lysosomal enzymes in serum and urine of pancreatic adenocarcinoma patients, may indicate on destruction of pancreatic tissue by pancreatic adenocarcinoma. Determination of the HEX, GAL, FUC and MAN in serum and urine may be useful in diagnostics of pancreatic adenocarcinoma.

  9. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro.

    Science.gov (United States)

    Canfrán-Duque, Alberto; Barrio, Luis C; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A; Busto, Rebeca

    2016-03-18

    First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes' internal milieu induced by haloperidol affects lysosomal functionality.

  10. Para-toluenesulfonamide induces tongue squamous cell carcinoma cell death through disturbing lysosomal stability.

    Science.gov (United States)

    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.

  11. Streptococcus oralis Induces Lysosomal Impairment of Macrophages via Bacterial Hydrogen Peroxide.

    Science.gov (United States)

    Okahashi, Nobuo; Nakata, Masanobu; Kuwata, Hirotaka; Kawabata, Shigetada

    2016-07-01

    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.

  12. Eps8 is recruited to lysosomes and subjected to chaperone-mediated autophagy in cancer cells.

    Science.gov (United States)

    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.

  13. uPARAP/endo180 directs lysosomal delivery and degradation of collagen IV

    DEFF Research Database (Denmark)

    Kjøller, Lars; Engelholm, Lars H; Høyer-Hansen, Maria

    2004-01-01

    transmembrane glycoprotein urokinase plasminogen activator receptor-associated protein (uPARAP/endo180) directs collagen IV for lysosomal delivery and degradation. In wild-type fibroblasts, fluorescently labeled collagen IV was first internalized into vesicular structures with diffuse fluorescence eventually......Collagen turnover is crucial for tissue homeostasis and remodeling and pathological processes such as cancer invasion, but the underlying molecular mechanisms are poorly understood. A major pathway appears to be internalization and degradation by fibroblasts. We now show that the endocytic...... appearing uniformly within the wild-type cells after longer incubation times. In these cells, some collagen-containing vesicles were identified as lysosomes by staining for LAMP-1. In contrast, collagen IV remained extracellular and associated with fiber-like structures on uPARAP/endo180-deficient...

  14. Platelet Glycoprotein lb-1X and Malignancy

    Science.gov (United States)

    2011-09-01

    patient with systemic lupus erythematosus . Am J Hematol 2001; 67:262-67. 20. Arthur JF, Dunkley S and Andrews RK. Platelet glycoprotein VI-related...Moroi M. Antibody against platelet membrane glyco- protein VI in a patient with systemic lupus erythematosus . Am J Hematol 2001; 67: 262–7. 9 Arthur JF...Approved OMB No. 0704-0188 Public reporting burden for this collection of information is estimated to average 1 hour per response, including the

  15. 核黄素结合紫外光灭活血小板细菌及膜糖蛋白的变化%Effect on bacteria inactivation using riboflavin as a photosensitizer and detect membrane glycoproteins after treatment

    Institute of Scientific and Technical Information of China (English)

    卫玉芝; 许伟; 钟涛; 张循善

    2011-01-01

    Aim To explore a photochemical method using riboflavin plus UV light that can inactivate selected model bacteria and to detect its membrane glycoproteins. Methods 500 μmol · L -1 riboflavin(14 ml)was added to the apheresis platelet(125 ml)blood bag,then model bacteria(Staphylococcus epidermidis ATCC12228 and Escherichia coli ATCC25922)were added to apheresis platelet, and broadband UV light was used illuminate the blood bag, before the bacteria titer was measured. The inactivation effects of UV light plus riboflavin for bacteria was studied. The impact of a new technology for pathogen reduction based on riboflavin plus illumination at 6.2 J ·ml -1 on functional and biochemical characteristics of PLT was evaluated. Its GP CD62p and PAC-1 were detected by flow cytometry. Results With a riboflavin concentration of 50 μmol · L -1 and UV light 265 ~ 370 nm intensity of 6.2 J · ml -1, mode bacteria in apheresis platelet blood bag could be reduced to less than 1.22 logCFU/ml in 10 minutes. Conclusions Riboflavin plus ultraviolet light could significantly inactivate selected bacteria. Its membrane glycoproteins CD62p and PAC-1 was adequately maintained after treatment and during storage,this was not beyond accepted levels.%目的 探讨核黄素光化学法灭活单采血小板污染菌的效果及其膜糖蛋白表达的变化情况.方法 将14 ml浓度为500 μmol·L-1核黄素加到125 ml的单采血小板悬液中,再将一定浓度的G+菌(表皮葡萄球菌ATCC12228)和G-菌(大肠埃希氏菌ATCC25922)分别注入上述混悬液中,经(265~370 nm)广谱紫外光(6.2 J·ml-1)照射8~10 min后,测定其浓度,观察灭活效果,并用流式细胞仪检测血小板膜糖蛋白的变化情况.结果 经浓度50 μmol·L-1的核黄素结合强度为6.2 J·ml-1紫外光照射8~10 min,可将一定浓度的模型菌灭活至<1.22 logCFU/ml.结论 核黄素结合紫外光照射可以有效灭活细菌,而单采血小板的CD62p、PAC-1的阳性表达率和

  16. Effect of Xiaoyu Zhixue Tablet(消瘀止血片)on the Expression of Platelet Membrane Glycoprotein Ⅰ b/Ⅸ/Ⅴ Complex in Patients with Chronic Renal Failure

    Institute of Scientific and Technical Information of China (English)

    QIN You; SHEN Lin; LU Fu-rong; SHI Wei; LIU Jian-guo

    2008-01-01

    Objective:To investigate the effect of Xiaoyu Zhixue Tablet(消瘀止血片,XYZXT)on the expression of platelet membrane glycoprotein(GP)Ⅰ b/Ⅸ/Ⅴ complex and GP Ⅰ b α in patients with chronic renal failure(CRF)in early metaphase.Methods:Fifty-one patients with CRF in early metaphase(treated group)were treated with XYZXT,3 months as the course of treatment for 2 courses.The previous therapies remained unchanged.Flow cytometry was used to assess the expression of platelet GP Ⅰ b/Ⅸ/Ⅴ complex and GP Ⅰ b α in patients with CRF,and turbidity method was used to determine the platelet maximum aggregation rate(MAR),meanwhile the renal function was measured.The final data were compared with those before the treatment,and with those in the normal control group (3) healthy subjects).Results:Compared with the normal control group,expressions of GP Ⅰ b/Ⅸ/Ⅴ complex and GP Ⅰ b α,and platelet MAR in CRF patients were significantly lower(P=0.007,P=0.001,P=0.009)before the treatment;after the treatment with XYZXT,the above indexes in CRF patients were remarkably increased(P=0.033,P=0.026,P=0.045),but still Iower than those in the normal control group,however,it was not statistically significant.Conclusion:(1)The expression of GP Ⅰ b/Ⅸ/Ⅴ complex in CRF patients of early metaphase was decreased,which lead to platelet aggregation dysfunction.This might be one of the reasons for the hemorrhagic trend in CRF.(2)XYZXT was able to upgrade expressions of GP Ⅰ b/Ⅸ/Ⅴ complex and GP Ⅰ b α in CRF patients,improve platelet function and down-regulate platelet activation in patients with CRF.

  17. Lysosomal exoglycosidases in nasal polyps.

    Science.gov (United States)

    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

    Science.gov (United States)

    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. Glycoprotein biosynthesis in calf kidney. Glycoprotein sialyltransferase activities towards serum glycoproteins and calf Tamm-Horsfall glycoprotein.

    Science.gov (United States)

    van Dijk, W; Lasthuis, A M; van den Eijnden, D H

    1979-04-18

    CMP-AcNeu:glycoprotein sialyltransltransltransltransltransferase of calf kidney cortex was characterized using serum glycoproteins and Tamm-Horsfall glycoprotein, obtained from calf urine, as acceptors. Native calf Tamm-Horsfall glycoprotein showed the best acceptor properties, followed by desialylated calf fetuin and desialylated human alpha 1-acid glycoprotein exhibiting V values of, respectively, 114, 63 and 41 nmol/h per g wet wt. of kidney cortex and Km values of 0.12, 0.16 and 0.26 mM glycoprotein acceptor. Desialylated ovine submaxillary mucine appeared to be a very poor acceptor. Tamm-Horsfall glycoprotein sialyltransferase could be distinguished from serum glycoprotein sialyltransferase by competition studies. In addition the two glycoprotein sialyltransferase activities showed different distributions over the three regions of the calf kidney: the ratios of the Tamm-Horsfall to serum glycoprotein sialyltransferase activities decreased from 3.3 in the cortex to 0.8 and 0.4 in the medulla and the papilla, respectively. It was concluded that in calf kidney at least two different sialyltransferases exist. The high cortical Tamm-Horsfall glycoprotein sialyltransferases activity corresponds markedly to the origin of the urinary Tamm-Horsfall glycoprotein, namely the distal part of the kidney tubule. Inactivation of glycoprotein sialyltransferase activity by preincubation at various temperatures and during storage at 0 degree C, could be reduced by the addition of CMP-AcNeu. The possible relevance towards the in vivo sialylation of this finding is discussed.

  20. Sensitivity of P-glycoprotein tryptophan residues to benzodiazepines and ATP interaction.

    Science.gov (United States)

    Lima, Sofia A C; Cordeiro-da-Silva, Anabela; de Castro, Baltazar; Gameiro, Paula

    2007-01-01

    Plasma membrane P-glycoprotein is a member of the ATP-binding cassette family of membrane transporters. In the present study tryptophan intrinsic fluorescence was used to understand the P-glycoprotein response to three benzodiazepines (bromazepam, chlordiazepoxide and flurazepam) in the presence and absence of ATP. Fluorescence emission spectra showed a red shift on the maximal emission wavelength upon interaction of P-glycoprotein with all benzodiazepines. Benzodiazepine association with nucleotide-bound P-glycoprotein also showed this trend and the quenching profile was attributed to a sphere-of-action model, for static fluorescence. Furthermore, quenching data of benzodiazepine-bound P-glycoprotein with ATP were concentration dependent and saturable, indicating that nucleotide binds to P-glycoprotein whether drug is present or not. These results seems in agreement with the proposal of the ATP-switch model by Higgins and Linton, where substrate binding to the transporters initiates the transport cycle by increasing the ATP binding affinity.

  1. Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed

    Science.gov (United States)

    Serebrovskaya, Ekaterina O.; Ryumina, Alina P.; Boulina, Maria E.; Shirmanova, Marina V.; Zagaynova, Elena V.; Bogdanova, Ekaterina A.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

    2014-07-01

    KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.

  2. Application of a battery of biomarkers in mussel digestive gland to assess long-term effects of the Prestige oil spill in Galicia and the Bay of Biscay: lysosomal responses.

    Science.gov (United States)

    Garmendia, Larraitz; Izagirre, Urtzi; Cajaraville, Miren P; Marigómez, Ionan

    2011-04-01

    In order to assess the long-term lysosomal responses to the Prestige oil spill (POS), mussels, Mytilus galloprovincialis, were collected in 22 localities from Galicia and the Bay of Biscay (North Iberian peninsula) in July, and September 2003, April, July, and October 2004-2005 and April 2006. Lysosomal membrane stability (labilisation period, LP) and lysosomal structural changes (lysosomal volume density, Vv(L) and lysosomal surface-to-volume ratio, S/V(L)) were measured as general stress biomarkers. The most remarkable long-term effects after the POS were drastic changes in lysosomal size (lysosomal enlargement) and membrane stability (extremely low LP values) up to April-04. Later on, a recovery trend was envisaged all along the studied area after July-04, albeit membrane stability continued to be below 20 min throughout the studied period up to April-06, which indicates a "distress-to-moderate-stress" condition. Lysosomal Response Index (LRI) revealed that environmental stress was more marked in Galicia than in the Bay of Biscay, mainly in the first sampling year, although a "moderate-to-high-stress" condition persisted until July-05. Overall, although lysosomal size returned to reference values, membrane stability was not fully recovered indicating a stress situation throughout the studied period.

  3. Lysosomal Storage Disorders and Malignancy

    Directory of Open Access Journals (Sweden)

    Gregory M. Pastores

    2017-02-01

    Full Text Available Lysosomal storage disorders (LSDs are infrequent to rare conditions caused by mutations that lead to a disruption in the usual sequential degradation of macromolecules or their transit within the cell. Gaucher disease (GD, a lipidosis, is among the most common LSD, with an estimated incidence of 1 in 40,000 among the Caucasian, non-Jewish population. Studies have indicated an increased frequency of polyclonal and monoclonal gammopathy among patients with GD. It has been shown that two major sphingolipids that accumulate in GD, namely, β-glucosylceramide 22:0 (βGL1-22 and glucosylsphingosine (LGL1, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT cells. Investigations undertaken in an affected mouse model revealed βGL1-22- and LGL1-specific NKT cells were present and constitutively promoted the expression of a T-follicular helper (TFH phenotype; injection of these lipids led to downstream induction of germinal center B cells, hypergammaglobulinemia, and the production of antilipid antibodies. Subsequent studies have found clonal immunoglobulin in 33% of sporadic human monoclonal gammopathies is also specific for the lysolipids LGL1 and lysophosphatidylcholine (LPC. Furthermore, substrate reduction ameliorated GD-associated gammopathy in mice. It had been hypothesized that chronic antigenic stimulation by the abnormal lipid storage and associated immune dysregulation may be the underlying mechanism for the increased incidence of monoclonal and polyclonal gammopathies, as well as an increased incidence of multiple myeloma in patients with GD. Current observations support this proposition and illustrate the value of investigations into rare diseases, which as ‘experiments of nature’ may provide insights into conditions found in the general population that continue to remain incompletely understood.

  4. Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases.

    Science.gov (United States)

    Oh, Doo-Byoung

    2015-08-01

    Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy.

  5. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro

    Directory of Open Access Journals (Sweden)

    Alberto Canfrán-Duque

    2016-03-01

    Full Text Available First- and second-generation antipsychotics (FGAs and SGAs, respectively, have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanineperchlorate (DiI-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2 and LBPA (lysobisphosphatidic acid, which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1 and coatomer subunit β (β-COP were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes’ internal milieu induced by haloperidol affects lysosomal functionality.

  6. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro

    Science.gov (United States)

    Canfrán-Duque, Alberto; Barrio, Luis C.; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A.; Busto, Rebeca

    2016-01-01

    First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes’ internal milieu induced by haloperidol affects lysosomal functionality. PMID:26999125

  7. A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency

    Science.gov (United States)

    Tharkeshwar, Arun Kumar; Trekker, Jesse; Vermeire, Wendy; Pauwels, Jarne; Sannerud, Ragna; Priestman, David A.; Te Vruchte, Danielle; Vints, Katlijn; Baatsen, Pieter; Decuypere, Jean-Paul; Lu, Huiqi; Martin, Shaun; Vangheluwe, Peter; Swinnen, Johannes V.; Lagae, Liesbet; Impens, Francis; Platt, Frances M.; Gevaert, Kris; Annaert, Wim

    2017-01-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.

  8. A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency

    Science.gov (United States)

    Tharkeshwar, Arun Kumar; Trekker, Jesse; Vermeire, Wendy; Pauwels, Jarne; Sannerud, Ragna; Priestman, David A.; te Vruchte, Danielle; Vints, Katlijn; Baatsen, Pieter; Decuypere, Jean-Paul; Lu, Huiqi; Martin, Shaun; Vangheluwe, Peter; Swinnen, Johannes V.; Lagae, Liesbet; Impens, Francis; Platt, Frances M.; Gevaert, Kris; Annaert, Wim

    2017-01-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions. PMID:28134274

  9. The role of VAMP7/TI-VAMP in cell polarity and lysosomal exocytosis in vivo.

    Science.gov (United States)

    Sato, Mahito; Yoshimura, Shinichiro; Hirai, Rika; Goto, Ayako; Kunii, Masataka; Atik, Nur; Sato, Takashi; Sato, Ken; Harada, Reiko; Shimada, Junko; Hatabu, Toshimitsu; Yorifuji, Hiroshi; Harada, Akihiro

    2011-10-01

    VAMP7 or tetanus neurotoxin-insensitive vesicle- associated membrane protein (TI-VAMP) has been proposed to regulate apical transport in polarized epithelial cells, axonal transport in neurons and lysosomal exocytosis. To investigate the function of VAMP7 in vivo, we generated VAMP7 knockout mice. Here, we show that VAMP7 knockout mice are indistinguishable from control mice and display a similar localization of apical proteins in the kidney and small intestine and a similar localization of axonal proteins in the nervous system. Neurite outgrowth of cultured mutant hippocampal neurons was reduced in mutant neurons. However, lysosomal exocytosis was not affected in mutant fibroblasts. Our results show that VAMP7 is required in neurons to extend axons to the full extent. However, VAMP7 does not seem to be required for epithelial cell polarity and lysosomal exocytosis.

  10. The Phosphoinositide-Gated Lysosomal Ca(2+) Channel, TRPML1, Is Required for Phagosome Maturation.

    Science.gov (United States)

    Dayam, Roya M; Saric, Amra; Shilliday, Ryan E; Botelho, Roberto J

    2015-09-01

    Macrophages internalize and sequester pathogens into a phagosome. Phagosomes then sequentially fuse with endosomes and lysosomes, converting into degradative phagolysosomes. Phagosome maturation is a complex process that requires regulators of the endosomal pathway including the phosphoinositide lipids. Phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2 ), which respectively control early endosomes and late endolysosomes, are both required for phagosome maturation. Inhibition of PIKfyve, which synthesizes PtdIns(3,5)P2 , blocked phagosome-lysosome fusion and abated the degradative capacity of phagosomes. However, it is not known how PIKfyve and PtdIns(3,5)P2 participate in phagosome maturation. TRPML1 is a PtdIns(3,5)P2 -gated lysosomal Ca(2+) channel. Because Ca(2+) triggers membrane fusion, we postulated that TRPML1 helps mediate phagosome-lysosome fusion. Using Fcγ receptor-mediated phagocytosis as a model, we describe our research showing that silencing of TRPML1 hindered phagosome acquisition of lysosomal markers and reduced the bactericidal properties of phagosomes. Specifically, phagosomes isolated from TRPML1-silenced cells were decorated with lysosomes that docked but did not fuse. We could rescue phagosome maturation in TRPML1-silenced and PIKfyve-inhibited cells by forcible Ca(2+) release with ionomycin. We also provide evidence that cytosolic Ca(2+) concentration increases upon phagocytosis in a manner dependent on TRPML1 and PIKfyve. Overall, we propose a model where PIKfyve and PtdIns(3,5)P2 activate TRPML1 to induce phagosome-lysosome fusion.

  11. HIV-1 envelope glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Caulfield, Michael; Cupo, Albert; Dean, Hansi; Hoffenberg, Simon; King, C. Richter; Klasse, P. J.; Marozsan, Andre; Moore, John P.; Sanders, Rogier W.; Ward, Andrew; Wilson, Ian; Julien, Jean-Philippe

    2017-08-22

    The present application relates to novel HIV-1 envelope glycoproteins, which may be utilized as HIV-1 vaccine immunogens, and antigens for crystallization, electron microscopy and other biophysical, biochemical and immunological studies for the identification of broad neutralizing antibodies. The present invention encompasses the preparation and purification of immunogenic compositions, which are formulated into the vaccines of the present invention.

  12. Adaptor Protein Complexes AP-1 and AP-3 Are Required by the HHV-7 Immunoevasin U21 for Rerouting of Class I MHC Molecules to the Lysosomal Compartment

    Science.gov (United States)

    Kimpler, Lisa A.; Glosson, Nicole L.; Downs, Deanna; Gonyo, Patrick; May, Nathan A.; Hudson, Amy W.

    2014-01-01

    The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes. PMID:24901711

  13. Thrombin-induced lysosomal exocytosis in human platelets is dependent on secondary activation by ADP and regulated by endothelial-derived substances.

    Science.gov (United States)

    Södergren, Anna L; Svensson Holm, Ann-Charlotte B; Ramström, Sofia; Lindström, Eva G; Grenegård, Magnus; Öllinger, Karin

    2016-01-01

    Exocytosis of lysosomal contents from platelets has been speculated to participate in clearance of thrombi and vessel wall remodelling. The mechanisms that regulate lysosomal exocytosis in platelets are, however, still unclear. The aim of this study was to identify the pathways underlying platelet lysosomal secretion and elucidate how this process is controlled by platelet inhibitors. We found that high concentrations of thrombin induced partial lysosomal exocytosis as assessed by analysis of the activity of released N-acetyl-β-glucosaminidase (NAG) and by identifying the fraction of platelets exposing the lysosomal-associated membrane protein (LAMP)-1 on the cell surface by flow cytometry. Stimulation of thrombin receptors PAR1 or PAR4 with specific peptides was equally effective in inducing LAMP-1 surface expression. Notably, lysosomal exocytosis in response to thrombin was significantly reduced if the secondary activation by ADP was inhibited by the P2Y12 antagonist cangrelor, while inhibition of thromboxane A2 formation by treatment with acetylsalicylic acid was of minor importance in this regard. Moreover, the NO-releasing drug S-nitroso-N-acetyl penicillamine (SNAP) or the cyclic AMP-elevating eicosanoid prostaglandin I2 (PGI2) significantly suppressed lysosomal exocytosis. We conclude that platelet inhibitors that mimic functional endothelium such as PGI2 or NO efficiently counteract lysosomal exocytosis. Furthermore, we suggest that secondary release of ADP and concomitant signaling via PAR1/4- and P2Y12 receptors is important for efficient platelet lysosomal exocytosis by thrombin.

  14. Hsp70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology

    DEFF Research Database (Denmark)

    Kirkegaard, Thomas; Roth, Anke G; Petersen, Nikolaj H T

    2010-01-01

    inhibition of ASM, effectively revert the Hsp70-mediated stabilization of lysosomes. Notably, the reduced ASM activity in cells from patients with Niemann-Pick disease (NPD) A and B-severe lysosomal storage disorders caused by mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1) encoding for ASM...

  15. Lysosomal and mitochondrial permeabilization mediates zinc(II) cationic phthalocyanine phototoxicity.

    Science.gov (United States)

    Marino, Julieta; García Vior, María C; Furmento, Verónica A; Blank, Viviana C; Awruch, Josefina; Roguin, Leonor P

    2013-11-01

    In order to find a novel photosensitizer to be used in photodynamic therapy for cancer treatment, we have previously showed that the cationic zinc(II) phthalocyanine named Pc13, the sulfur-linked dye 2,9(10),16(17),23(24)-tetrakis[(2-trimethylammonium) ethylsulfanyl]phthalocyaninatozinc(II) tetraiodide, exerts a selective phototoxic effect on human nasopharynx KB carcinoma cells and induces an apoptotic response characterized by an increase in the activity of caspase-3. Since the activation of an apoptotic pathway by chemotherapeutic agents contributes to the elimination of malignant cells, in this study we investigated the molecular mechanisms underlying the antitumor action of Pc13. We found that after light exposure, Pc13 induced the production of reactive oxygen species (ROS), which are mediating the resultant cytotoxic action on KB cells. ROS led to an early permeabilization of lysosomal membranes as demonstrated by the reduction of lysosome fluorescence with acridine orange and the release of lysosomal proteases to cytosol. Treatment with antioxidants inhibited ROS generation, preserved the integrity of lysosomal membrane and increased cell proliferation in a concentration-dependent manner. Lysosome disruption was followed by mitochondrial depolarization, cytosolic release of cytochrome C and caspases activation. Although no change in the total amount of Bax was observed, the translocation of Bax from cytosol to mitochondria, the cleavage of the pro-apoptotic protein Bid, together with the decrease of the anti-apoptotic proteins Bcl-XL and Bcl-2 indicated the involvement of Bcl-2 family proteins in the induction of the mitochondrial pathway. It was also demonstrated that cathepsin D, but not caspase-8, contributed to Bid cleavage. In conclusion, Pc13-induced cell photodamage is triggered by ROS generation and activation of the mitochondrial apoptotic pathway through the release of lysosomal proteases. In addition, our results also indicated that Pc13 induced

  16. LAMP-2 is required for incorporating syntaxin-17 into autophagosomes and for their fusion with lysosomes.

    Science.gov (United States)

    Hubert, Virginie; Peschel, Andrea; Langer, Brigitte; Gröger, Marion; Rees, Andrew; Kain, Renate

    2016-10-15

    Autophagy is an evolutionarily conserved process used for removing surplus and damaged proteins and organelles from the cytoplasm. The unwanted material is incorporated into autophagosomes that eventually fuse with lysosomes, leading to the degradation of their cargo. The fusion event is mediated by the interaction between the Qa-SNARE syntaxin-17 (STX17) on autophagosomes and the R-SNARE VAMP8 on lysosomes. Cells deficient in lysosome membrane-associated protein-2 (LAMP-2) have increased numbers of autophagosomes but the underlying mechanism is poorly understood. By transfecting LAMP-2-deficient and LAMP-1/2--double-deficient mouse embryonic fibroblasts (MEFs) with a tandem fluorescent-tagged LC3 we observed a failure of fusion between the autophagosomes and the lysosomes that could be rescued by complementation with LAMP-2A. Although we observed no change in expression and localization of VAMP8, its interacting partner STX17 was absent from autophagosomes of LAMP-2-deficient cells. Thus, LAMP-2 is essential for STX17 expression by the autophagosomes and this absence is sufficient to explain their failure to fuse with lysosomes. The results have clear implications for situations associated with a reduction of LAMP-2 expression.

  17. Endo-lysosomal TRP mucolipin-1 channels trigger global ER Ca2+ release and Ca2+ influx

    Science.gov (United States)

    Kilpatrick, Bethan S.; Yates, Elizabeth; Grimm, Christian; Schapira, Anthony H.

    2016-01-01

    ABSTRACT Transient receptor potential (TRP) mucolipins (TRPMLs), encoded by the MCOLN genes, are patho-physiologically relevant endo-lysosomal ion channels crucial for membrane trafficking. Several lines of evidence suggest that TRPMLs mediate localised Ca2+ release but their role in Ca2+ signalling is not clear. Here, we show that activation of endogenous and recombinant TRPMLs with synthetic agonists evoked global Ca2+ signals in human cells. These signals were blocked by a dominant-negative TRPML1 construct and a TRPML antagonist. We further show that, despite a predominant lysosomal localisation, TRPML1 supports both Ca2+ release and Ca2+ entry. Ca2+ release required lysosomal and ER Ca2+ stores suggesting that TRPMLs, like other endo-lysosomal Ca2+ channels, are capable of ‘chatter’ with ER Ca2+ channels. Our data identify new modalities for TRPML1 action. PMID:27577094

  18. Visualization of ceramide channels in lysosomes following endogenous palmitoyl-ceramide accumulation as an initial step in the induction of necrosis

    Directory of Open Access Journals (Sweden)

    Mototeru Yamane

    2017-09-01

    Full Text Available In this study, we showed that the dual addition of glucosyl ceramide synthase and ceramidase inhibitors to A549 cell culture led to the possibility of ceramide channel formation via endogenous palmitoyl-ceramide accumulation with an increase in cholesterol contents in the lysosome membrane as an initial step prior to initiation of necrotic cell death. In addition, the dual addition led to black circular structures of 10–20 nm, interpreted as stain-filled cylindrical channels on transmission electron microscopy. The formation of palmitoyl-ceramide channels in the lysosome membrane causes the liberation of cathepsin B from lysosomes for necrotic cell death. On the other hand, necrotic cell death in the dual addition was not caused by oxidative stress or cathepsin B activity, and the cell death was free from the contribution of the translation of Bax protein to the lysosome membrane.

  19. Research progress on the location and function of P-glycoprotein in cell membrane%P糖蛋白在细胞膜的定位和功能研究进展

    Institute of Scientific and Technical Information of China (English)

    马园园; 颜天华; 程涛; 刘雯清; 曹永兵

    2014-01-01

    Objective To review the interaction between P-glycoprotein(P-gp) and plasmalemma lipids ,and to provide base for find-ing reversal agents for drug resistance .Method The literatures regarding to the relationships between plasmalemma lipids (cho-lesterol ,phospholipids and glycolipids ,etc) ,lipid raft and P-gp were retrieved .Result P-gp is one of the most sensitive trans-membrane proteins to plasmalemma lipids surroundings and has close relationships with plasmalemma lipids .Conclusion Research indicates that P-gp is one of the most important reasons caused cancer multidrug resistance ,and plasmalemma lipids can affect the activity and expression of P-gp while P-gp can influence lipids synthesis .Making their relationships clear can help us to discover reversal agents .%目的:综述P糖蛋白与细胞膜脂质的相互作用,为找到抗耐药剂提供有力的帮助。方法检索细胞膜上的脂质(胆固醇、磷脂和糖脂等)以及微区域脂筏和P糖蛋白关系的研究进展。结果 P糖蛋白属于跨膜蛋白,在所有的膜蛋白中,P糖蛋白是对所处的脂质环境最敏感的膜蛋白之一,与细胞膜脂质有密切关系。结论 P糖蛋白是引起肿瘤多药耐药的重要原因之一,细胞膜上的脂质能够影响P糖蛋白的活性和表达,同时P糖蛋白也可以影响脂质的合成,阐明二者的关系能为找到耐药逆转剂提供强有力的帮助。

  20. Structural analysis of N- and O-glycans released from glycoproteins

    DEFF Research Database (Denmark)

    Jensen, Pia Hønnerup; Karlsson, Niclas G; Kolarich, Daniel;

    2012-01-01

    This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically...

  1. AP-3 and Rabip4' coordinately regulate spatial distribution of lysosomes.

    Directory of Open Access Journals (Sweden)

    Viorica Ivan

    Full Text Available The RUN and FYVE domain proteins rabip4 and rabip4' are encoded by RUFY1 and differ in a 108 amino acid N-terminal extension in rabip4'. Their identical C terminus binds rab5 and rab4, but the function of rabip4s is incompletely understood. We here found that silencing RUFY1 gene products promoted outgrowth of plasma membrane protrusions, and polarized distribution and clustering of lysosomes at their tips. An interactor screen for proteins that function together with rabip4' yielded the adaptor protein complex AP-3, of which the hinge region in the β3 subunit bound directly to the FYVE domain of rabip4'. Rabip4' colocalized with AP-3 on a tubular subdomain of early endosomes and the extent of colocalization was increased by a dominant negative rab4 mutant. Knock-down of AP-3 had an ever more dramatic effect and caused accumulation of lysosomes in protrusions at the plasma membrane. The most peripheral lysosomes were localized beyond microtubules, within the cortical actin network. Our results uncover a novel function for AP-3 and rabip4' in regulating lysosome positioning through an interorganellar pathway.

  2. Endothelial Nlrp3 inflammasome activation associated with lysosomal destabilization during coronary arteritis.

    Science.gov (United States)

    Chen, Yang; Li, Xiang; Boini, Krishna M; Pitzer, Ashley L; Gulbins, Erich; Zhang, Yang; Li, Pin-Lan

    2015-02-01

    Inflammasomes play a critical role in the development of vascular diseases. However, the molecular mechanisms activating the inflammasome in endothelial cells and the relevance of this inflammasome activation is far from clear. Here, we investigated the mechanisms by which an Nlrp3 inflammasome is activated to result in endothelial dysfunction during coronary arteritis by Lactobacillus casei (L. casei) cell wall fragments (LCWE) in a mouse model for Kawasaki disease. Endothelial dysfunction associated with increased vascular cell adhesion protein 1 (VCAM-1) expression and endothelial-leukocyte adhesion was observed during coronary arteritis in mice treated with LCWE. Accompanied with these changes, the inflammasome activation was also shown in coronary arterial endothelium, which was characterized by a marked increase in caspase-1 activity and IL-1β production. In cultured endothelial cells, LCWE induced Nlrp3 inflammasome formation, caspase-1 activation and IL-1β production, which were blocked by Nlrp3 gene silencing or lysosome membrane stabilizing agents such as colchicine, dexamethasone, and ceramide. However, a potassium channel blocker glibenclamide or an oxygen free radical scavenger N-acetyl-l-cysteine had no effects on LCWE-induced inflammasome activation. LCWE also increased endothelial cell lysosomal membrane permeability and triggered lysosomal cathepsin B release into cytosol. Silencing cathepsin B blocked LCWE-induced Nlrp3 inflammasome formation and activation in endothelial cells. In vivo, treatment of mice with cathepsin B inhibitor also abolished LCWE-induced inflammasome activation in coronary arterial endothelium. It is concluded that LCWE enhanced lysosomal membrane permeabilization and consequent release of lysosomal cathepsin B, resulting in activation of the endothelial Nlrp3 inflammasome, which may contribute to the development of coronary arteritis.

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

    Science.gov (United States)

    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.

  4. Effects of contaminant exposure and food restriction on hepatic autophagic lysosomal parameters in Herring Gull (Larus argentatus) chicks.

    Science.gov (United States)

    Hegseth, Marit Nøst; Gorbi, Stephania; Bocchetti, Raffaella; Camus, Lionel; Gabrielsen, Geir Wing; Regoli, Francesco

    2014-08-01

    Lysosomal autophagic responses, such as lysosomal membrane stability, neutral lipids (NL), lipofuscin (LF), and malondialdehyde (MDA) levels, are valuable measures of cellular early-onset effects induced by environmental stress factors, such as contaminant exposure and fasting. In this study, these parameters were analysed and related to levels of halogenated organic contaminants (HOCs) in 40 Herring Gull (Larus argentatus) chicks. Chicks were experimentally exposed to HOCs through diet and went through a period of nutrient deprivation at the end of the experiment. HOC exposure and fasting were conducted separately and in combination. NL storages were depleted, and lysosomal membranes were destabilised after HOC exposure and nutrient deprivation. These responses were not related specifically to one type of stress or the extent of the treatment. No synergistic or additive effects from the combination of HOC exposure and fasting were observed. LF accumulated, and MDA levels increased as a result of fasting, but were unaffected by HOC exposure. LF accumulation was strongly associated with the percent weight change in the chicks. Large weight loss was associated with high LF levels, and slight weight gain was associated with low LF levels. Hence, food deprivation affected all the measured parameters, and HOC exposure decreased NL levels and lysosomal membrane stability in HG chick liver. Furthermore, autophagic lysosomal parameters have frequently been applied as biomarkers of cellular health status in previous studies of marine and terrestrial invertebrates, and this study suggests that these parameters may be good candidates for biomarkers of cellular health status in seabirds as well.

  5. Citreoviridin Induces Autophagy-Dependent Apoptosis through Lysosomal-Mitochondrial Axis in Human Liver HepG2 Cells.

    Science.gov (United States)

    Wang, Yuexia; Liu, Yanan; Liu, Xiaofang; Jiang, Liping; Yang, Guang; Sun, Xiance; Geng, Chengyan; Li, Qiujuan; Yao, Xiaofeng; Chen, Min

    2015-08-06

    Citreoviridin (CIT) is a mycotoxin derived from fungal species in moldy cereals. In our previous study, we reported that CIT stimulated autophagosome formation in human liver HepG2 cells. Here, we aimed to explore the relationship of autophagy with lysosomal membrane permeabilization and apoptosis in CIT-treated cells. Our data showed that CIT increased the expression of LC3-II, an autophagosome biomarker, from the early stage of treatment (6 h). After treatment with CIT for 12 h, lysosomal membrane permeabilization occurred, followed by the release of cathepsin D in HepG2 cells. Inhibition of autophagosome formation with siRNA against Atg5 attenuated CIT-induced lysosomal membrane permeabilization. In addition, CIT induced collapse of mitochondrial transmembrane potential as assessed by JC-1 staining. Furthermore, caspase-3 activity assay showed that CIT induced apoptosis in HepG2 cells. Inhibition of autophagosome formation attenuated CIT-induced apoptosis, indicating that CIT-induced apoptosis was autophagy-dependent. Cathepsin D inhibitor, pepstatin A, relieved CIT-induced apoptosis as well, suggesting the involvement of the lysosomal-mitochondrial axis in CIT-induced apoptosis. Taken together, our data demonstrated that CIT induced autophagy-dependent apoptosis through the lysosomal-mitochondrial axis in HepG2 cells. The study thus provides essential mechanistic insight, and suggests clues for the effective management and treatment of CIT-related diseases.

  6. Lysosomal Ca(2+) homeostasis: role in pathogenesis of lysosomal storage diseases.

    Science.gov (United States)

    Lloyd-Evans, Emyr; Platt, Frances M

    2011-08-01

    Disrupted cellular Ca(2+) signaling is believed to play a role in a number of human diseases including lysosomal storage diseases (LSD). LSDs are a group of ∼50 diseases caused predominantly by mutations in lysosomal proteins that result in accumulation of macromolecules within the lysosome. We recently reported that Niemann-Pick type C (NPC) is the first human disease to be associated with defective lysosomal Ca(2+) uptake and defective NAADP-mediated lysosomal Ca(2+) release. These defects in NPC cells leads to the disruption in endocytosis and subsequent lipid storage that is a feature of this disease. In contrast, Chediak-Higashi Syndrome cells have been reported to have enhanced lysosomal Ca(2+) uptake whilst the TRPML1 protein defective in mucolipidosis type IV is believed to function as a Ca(2+) channel. In this review we provide a summary of the current knowledge on the role of lysosomal Ca(2+) signaling in the pathogenesis of this group of diseases.

  7. Endo-lysosomal dysfunction in human proximal tubular epithelial cells deficient for lysosomal cystine transporter cystinosin.

    Directory of Open Access Journals (Sweden)

    Ekaterina A Ivanova

    Full Text Available 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.

  8. The intracellular location of lysosomal enzymes in developing Dictyostelium discoideum cells

    Energy Technology Data Exchange (ETDEWEB)

    Lenhard, J.M.

    1989-01-01

    The author has found that developing Dictyostelium cells contain two distinct acid hydrolase-containing organelles. Vesicles from cells at different stages of development were separated using Percoll density gradients. The lower density vesicles (LDVs or lysosomes) were present in nourished and starved cells. The higher density vesicles (HDVs) arose during starvation-induced differentiation. HDVs lacked two prestalk cell-specific lysosomal enzymes which were contained in LDVs. Prespore cell-specific spore coat proteins were detected in HDVs by ELISA. ({sup 35}S)sulfate labeling revealed that HDVs contained newly made glycoproteins as well as glycoproteins found in preexisting LDVs. Pulse-chase experiments using ({sup 35}S)methionine revealed that {alpha}-mannosidase from pre-existing LDVs an newly made {alpha}-mannosidase had entered HDVs. These data suggest that prespore LDVs mature to become HDVs. He has obtained evidence that HDVs are identical to prespore vesicles. Prespore vesicles are specialized secretory organelles which arise during prespore cell differentiation and which secrete their contents during terminal differentiation. As prespore vesicles secreted their contents, there was a co-incidental increase in extracellular acid hydrolase activity and a decrease in HDV-associated enzyme activity. Electron micrographs revealed that prespore cells contained two acid phosphatase-staining organelles, one of which appeared to be identical to lysosomes from nourished cells and a second which had features similar to prespore vesicles. Ricin-gold affinity electron microscopy was used to label the mucopolysaccharide component of prespore vesicles and the spore coat. Immunoelectron microscopy revealed co-localization of {alpha}-mannosidase with ricin-gold in prespore vesicles and the spore coat.

  9. Lysosomal stress: a new player in perturbed lipid metabolism

    NARCIS (Netherlands)

    Gabriel, T.L.

    2017-01-01

    Lysosomes are involved in many different essential cellular processes, among others organelle and molecule degradation, exocytosis, cell energy metabolism, cholesterol and sphingolipid level regulation. Lysosomal stress has a strong impact on the immune system, affecting specially macrophages as the

  10. Lysosomal stress: a new player in perturbed lipid metabolism

    NARCIS (Netherlands)

    Gabriel, T.L.

    2017-01-01

    Lysosomes are involved in many different essential cellular processes, among others organelle and molecule degradation, exocytosis, cell energy metabolism, cholesterol and sphingolipid level regulation. Lysosomal stress has a strong impact on the immune system, affecting specially macrophages as the

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

    Institute of Scientific and Technical Information of China (English)

    Jing Zhou; Shi-Hao Tan; Valérie Nicolas; Chantal Bauvy; Nai-Di Yang; Jianbin Zhang; Yuan Xue

    2013-01-01

    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 Torinl),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 Atg7deletion 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 antophagosome-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.

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

    Science.gov (United States)

    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.

  13. The HIV-1 envelope glycoproteins: folding, function and vaccin design

    NARCIS (Netherlands)

    Sanders, Rogier W.

    2003-01-01

    The need for a vaccine against HIV is obvious, but the development of an effective vaccine has met with frustrations. The HIV envelope glycoproteins, residing in the viral membrane, are the sole viral proteins exposed on the outside of virus particles and are therefore major targets for vaccine

  14. Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid.

    OpenAIRE

    Sturman, L S; Holmes, K V; Behnke, J.

    1980-01-01

    The two envelope glycoproteins and the viral nucleocapsid of the coronavirus A59 were isolated by solubilization of the viral membrane with Nonidet P-40 at 4 degrees C followed by sucrose density gradient sedimentation. Isolated E2 consisted of rosettes of peplomers, whereas E1, the membrane glycoprotein, was irregular and amorphous. Under certain conditions significant interactions occurred between components of Nonidet P-40-disrupted virions. Incubation of the Nonidet P-40-disrupted virus a...

  15. Characterization of Lassa virus glycoprotein oligomerization and influence of cholesterol on virus replication.

    Science.gov (United States)

    Schlie, Katrin; Maisa, Anna; Lennartz, Frank; Ströher, Ute; Garten, Wolfgang; Strecker, Thomas

    2010-01-01

    Mature glycoprotein spikes are inserted in the Lassa virus envelope and consist of the distal subunit GP-1, the transmembrane-spanning subunit GP-2, and the signal peptide, which originate from the precursor glycoprotein pre-GP-C by proteolytic processing. In this study, we analyzed the oligomeric structure of the viral surface glycoprotein. Chemical cross-linking studies of mature glycoprotein spikes from purified virus revealed the formation of trimers. Interestingly, sucrose density gradient analysis of cellularly expressed glycoprotein showed that in contrast to trimeric mature glycoprotein complexes, the noncleaved glycoprotein forms monomers and oligomers spanning a wide size range, indicating that maturation cleavage of GP by the cellular subtilase SKI-1/S1P is critical for formation of the correct oligomeric state. To shed light on a potential relation between cholesterol and GP trimer stability, we performed cholesterol depletion experiments. Although depletion of cholesterol had no effect on trimerization of the glycoprotein spike complex, our studies revealed that the cholesterol content of the viral envelope is important for the infectivity of Lassa virus. Analyses of the distribution of viral proteins in cholesterol-rich detergent-resistant membrane areas showed that Lassa virus buds from membrane areas other than those responsible for impaired infectivity due to cholesterol depletion of lipid rafts. Thus, derivation of the viral envelope from cholesterol-rich membrane areas is not a prerequisite for the impact of cholesterol on virus infectivity.

  16. Mucolipidosis type IV protein TRPML1-dependent lysosome formation.

    Science.gov (United States)

    Miller, Austin; Schafer, Jessica; Upchurch, Cameron; Spooner, Ellen; Huynh, Julie; Hernandez, Sebastian; McLaughlin, Brooke; Oden, Liam; Fares, Hanna

    2015-03-01

    Lysosomes are dynamic organelles that undergo cycles of fusion and fission with themselves and with other organelles. Following fusion with late endosomes to form hybrid organelles, lysosomes are reformed as discrete organelles. This lysosome reformation or formation is a poorly understood process that has not been systematically analyzed and that lacks known regulators. In this study, we quantitatively define the multiple steps of lysosome formation and identify the first regulator of this process.

  17. Effects of ethanol, acetaldehyde and cholesteryl esters on pancreatic lysosomes.

    OpenAIRE

    Wilson, J S; Apte, M V; Thomas, M. C.; Haber, P S; Pirola, R C

    1992-01-01

    Recent studies indicate that altered lysosomal function may be involved in the early stages of pancreatic injury. Chronic consumption of ethanol increases rat pancreatic lysosomal fragility. The aim of this study is to determine whether the lysosomal fragility observed after chronic ethanol consumption is mediated by ethanol per se, its oxidative metabolite acetaldehyde or cholesteryl esters (substances which accumulate in the pancreas after ethanol consumption). Pancreatic lysosomes from cho...

  18. Lysosomal storage disease 2 - Pompe's disease

    NARCIS (Netherlands)

    van der Ploeg, Ans T.; Reuser, Arnold J. J.

    2008-01-01

    Pompe's disease, glycogen-storage disease type II, and acid maltase deficiency are alternative names for the same metabolic disorder. It is a pan-ethnic autosomal recessive trait characterised by acid alpha-glucosidase deficiency leading to lysosomal glycogen storage. Pompe's disease is also

  19. Transport of Lysosome-Related Organelles

    NARCIS (Netherlands)

    Jordens, Ingrid

    2005-01-01

    Many intracellular compartments, including (MHC class II-containing) lysosomes, melanosomes and phagosomes, move along microtubules in a bi-directional manner due to the alternating activities of the plus-end directed kinesin motor and the minus-end directed dynein-dynactin motor. However, it is lar

  20. Transport of Lysosome-Related Organelles

    NARCIS (Netherlands)

    Jordens, Ingrid

    2005-01-01

    Many intracellular compartments, including (MHC class II-containing) lysosomes, melanosomes and phagosomes, move along microtubules in a bi-directional manner due to the alternating activities of the plus-end directed kinesin motor and the minus-end directed dynein-dynactin motor. However, it is

  1. Lysosomal proteolysis: effects of aging and insulin.

    Science.gov (United States)

    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.

  2. TPC1 has two variant isoforms, and their removal has different effects on endo-lysosomal functions compared to loss of TPC2.

    Science.gov (United States)

    Ruas, Margarida; Chuang, Kai-Ting; Davis, Lianne C; Al-Douri, Areej; Tynan, Patricia W; Tunn, Ruth; Teboul, Lydia; Galione, Antony; Parrington, John

    2014-11-01

    Organelle ion homeostasis within the endo-lysosomal system is critical for physiological functions. Two-pore channels (TPCs) are cation channels that reside in endo-lysosomal organelles, and overexpression results in endo-lysosomal trafficking defects. However, the impact of a lack of TPC expression on endo-lysosomal trafficking is unknown. Here, we characterize Tpcn1 expression in two transgenic mouse lines (Tpcn1(XG716) and Tpcn1(T159)) and show expression of a novel evolutionarily conserved Tpcn1B transcript from an alternative promoter, raising important questions regarding the status of Tpcn1 expression in mice recently described to be Tpcn1 knockouts. We show that the transgenic Tpcn1(T159) line lacks expression of both Tpcn1 isoforms in all tissues analyzed. Using mouse embryonic fibroblasts (MEFs) from Tpcn1(-/-) and Tpcn2(-/-) animals, we show that a lack of Tpcn1 or Tpcn2 expression has no significant impact on resting endo-lysosomal pH or morphology. However, differential effects in endo-lysosomal function were observed upon the loss of Tpcn1 or Tpcn2 expression; thus, while Tpcn1(-/-) MEFs have impaired trafficking of cholera toxin from the plasma membrane to the Golgi apparatus, Tpcn2(-/-) MEFs show slower kinetics of ligand-induced platelet-derived growth factor receptor β (PDGFRβ) degradation, which is dependent on trafficking to lysosomes. Our findings indicate that TPC1 and TPC2 have important but distinct roles in the endo-lysosomal pathway.

  3. Ca2+ -regulated lysosome fusion mediates angiotensin II-induced lipid raft clustering in mesenteric endothelial cells.

    Science.gov (United States)

    Han, Wei-Qing; Chen, Wen-Dong; Zhang, Ke; Liu, Jian-Jun; Wu, Yong-Jie; Gao, Ping-Jin

    2016-04-01

    It has been reported that intracellular Ca2+ is involved in lysosome fusion and membrane repair in skeletal cells. Given that angiotensin II (Ang II) elicits an increase in intracellular Ca2+ and that lysosome fusion is a crucial mediator of lipid raft (LR) clustering, we hypothesized that Ang II induces lysosome fusion and activates LR formation in rat mesenteric endothelial cells (MECs). We found that Ang II acutely increased intracellular Ca2+ content, an effect that was inhibited by the extracellular Ca2+ chelator ethylene glycol tetraacetic acid (EGTA) and the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release inhibitor 2-aminoethoxydiphenyl borate (2-APB). Further study showed that EGTA almost completely blocked Ang II-induced lysosome fusion, the translocation of acid sphingomyelinase (ASMase) to LR clusters, ASMase activation and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase activation. In contrast, 2-APB had a slight inhibitory effect. Functionally, both the lysosome inhibitor bafilomycin A1 and the ASMase inhibitor amitriptyline reversed Ang II-induced impairment of vasodilation. We conclude that Ca2+ -regulated lysosome fusion mediates the Ang II-induced regulation of the LR-redox signaling pathway and mesenteric endothelial dysfunction.

  4. ESCRT-Dependent Cell Death in a Caenorhabditis elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV.

    Science.gov (United States)

    Huynh, Julie M; Dang, Hope; Munoz-Tucker, Isabel A; O'Ketch, Marvin; Liu, Ian T; Perno, Savannah; Bhuyan, Natasha; Crain, Allison; Borbon, Ivan; Fares, Hanna

    2016-02-01

    Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The ortholog of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, USP-50, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppresses cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5.

  5. Preventive effects of p-coumaric acid on lysosomal dysfunction and myocardial infarct size in experimentally induced myocardial infarction.

    Science.gov (United States)

    Jyoti Roy, Abhro; Stanely Mainzen Prince, P

    2013-01-15

    The present study was designed to evaluate the preventive effects of p-coumaric acid on lysosomal dysfunction and myocardial infarct size in isoproterenol induced myocardial infarcted rats. Male albino Wistar rats were pretreated with p-coumaric acid (8 mg/kg body weight) daily for a period of 7 days after which isoproterenol (100mg/kg body weight) was injected subcutaneously into rats twice at an interval of 24h (8th and 9th day).The activity/levels of serum cardiac diagnostic markers, heart lysosomal lipid peroxidation products and the activities of lysosomal enzymes (β-glucuronidase, β-galactosidase, cathepsin-B and cathepsin-D) were significantly (Plysosomal fraction. The pretreatment with p-coumaric acid significantly (Plysosomal lipid peroxidation products and the activities of lysosomal enzymes. In addition, p-coumaric acid greatly reduced myocardial infarct size. p-Coumaric acid pretreatment (8 mg/kg body weight) to normal rats did not show any significant effect. Thus, this study showed that p-coumaric acid prevents lysosomal dysfunction against cardiac damage induced by isoproterenol and brings back the levels of lipid peroxidation products and activities of lysosomal enzymes to near normal levels. The in vitro study also revealed the free radical scavenging activity of p-coumaric acid. Thus, the observed effects are due to p-coumaric acid's free radical scavenging and membrane stabilizing properties.

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

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Di Malta, Chiara; Wen, Jiayu

    2014-01-01

    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...

  7. Neuroinflammatory paradigms in lysosomal storage diseases

    Directory of Open Access Journals (Sweden)

    Megan Elizabeth Bosch

    2015-10-01

    Full Text Available 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.

  8. 溶酶体相关膜蛋白2基因新突变导致Danon病的临床病理特点%Clinical and pathological features of Danon disease associated with a novel lysosome-associated membrane protein-2B mutation

    Institute of Scientific and Technical Information of China (English)

    洪道俊; 石志鸿; 张巍; 王朝霞; 袁云

    2010-01-01

    目的 报道1例Danon病患者的临床表现、病理改变特点和基因突变位点.方法 16岁男性患者表现为缓慢进展的骨骼肌无力和萎缩,以近端为主,伴随轻度脊柱强直.心电图示Ⅰ度房室传导阻滞,心脏超声示二尖瓣局限性增厚伴舒张功能损害,肌电图提示神经源性合并肌源性损害.患者左腓肠肌活体组织检查后,进行组织学、酶组织化学、电镜观察及抗肌营养不良素蛋白、层黏连蛋白α2、C5b9等免疫组织化学染色.患者及其父母进行溶酶体相关膜蛋白2(LAMP2)B基因的直接测序.结果 骨骼肌纤维内出现大小不一的自嗜空泡和镶边空泡,空泡内缺乏糖原,免疫组织化学显示多数肌纤维内的空泡边缘出现肌营养不良素蛋白、层黏连蛋白α2和C5b9的表达.电镜显示肌纤维内大量膜性空泡和溶酶体聚集.患者的LAMP2B基因9号外显子存在K402X突变,患者母亲无此突变,50名健康对照无此突变.结论 LAMP2B的9号外显子羧基末端的截断突变可以导致相对良性的Danon病,其心脏损害相对轻微.%Objectives To report the clinical and myopathological features in a case with Danon disease caused by a novel mutation in the lysosome-associated membrane protein-2 ( LAMP2 ) B gene.Methods A 16-year-old boy presenting progressive muscle weakness and atrophy, accompanied with spinal ankylosis was clinically evaluated including electrocardiogram, echocardiogram and electromyogram.Muscle biopsy was carried out in the patient.The histological staining, ultrastructural examination, and immunohistochemical staining with antibodies against dystrophin, merosin and C5b9 were performed in frozen sections.LAMP2B sequence was analyzed in the patient and his parents.Results Electrocardiogram in the patient showed Ⅰ atrioventricular block; echocardiogram revealed focal hypertrophy in mitral valve with mild cardiac diastolic dysfunction; electromyogram indicated myogenic and neurogenic

  9. Basement membrane proteoglycans and development

    DEFF Research Database (Denmark)

    Couchman, J R; Abrahamson, D R; McCarthy, K J

    1993-01-01

    Basement membranes contain distinct collagen, glycoprotein and proteoglycan species, and these exhibit considerable heterogeneity in isoform or type when different tissue types are compared. Additionally, many components are differentially expressed in organogenesis. We have considered the distri...

  10. Partial restoration of mutant enzyme homeostasis in three distinct lysosomal storage disease cell lines by altering calcium homeostasis.

    Directory of Open Access Journals (Sweden)

    Ting-Wei Mu

    2008-02-01

    Full Text Available A lysosomal storage disease (LSD results from deficient lysosomal enzyme activity, thus the substrate of the mutant enzyme accumulates in the lysosome, leading to pathology. In many but not all LSDs, the clinically most important mutations compromise the cellular folding of the enzyme, subjecting it to endoplasmic reticulum-associated degradation instead of proper folding and lysosomal trafficking. A small molecule that restores partial mutant enzyme folding, trafficking, and activity would be highly desirable, particularly if one molecule could ameliorate multiple distinct LSDs by virtue of its mechanism of action. Inhibition of L-type Ca2+ channels, using either diltiazem or verapamil-both US Food and Drug Administration-approved hypertension drugs-partially restores N370S and L444P glucocerebrosidase homeostasis in Gaucher patient-derived fibroblasts; the latter mutation is associated with refractory neuropathic disease. Diltiazem structure-activity studies suggest that it is its Ca2+ channel blocker activity that enhances the capacity of the endoplasmic reticulum to fold misfolding-prone proteins, likely by modest up-regulation of a subset of molecular chaperones, including BiP and Hsp40. Importantly, diltiazem and verapamil also partially restore mutant enzyme homeostasis in two other distinct LSDs involving enzymes essential for glycoprotein and heparan sulfate degradation, namely alpha-mannosidosis and type IIIA mucopolysaccharidosis, respectively. Manipulation of calcium homeostasis may represent a general strategy to restore protein homeostasis in multiple LSDs. However, further efforts are required to demonstrate clinical utility and safety.

  11. 血小板GPⅡb基因多态性与PCI术后血小板功能变化%Possible relationship between platelet membrane glycoprotein Ⅱ b gene polymorphism and change of platelet function after PCI

    Institute of Scientific and Technical Information of China (English)

    戚德清; 许康世

    2013-01-01

    Objective To investigate the possible relationship between platelet membrane glyco-protein (GP) IIb HPA-3gene polymorphism and changes of platelet function and the fibrinolytic activi-ty in patients with acute coronary syndrome (ACS ) ,undergoing percutaneous coronary intervention (PCI) .Methods 120 patients ,who were definitely diagnosed with ACS in the Affiliated Hospital of Guiyang Medical College ,were chosen to check platelet GP IIb HPA-3 gene polymorphism by polymer-ase chain reaction-sequence specific primers (PCR-SSP ) .Before and after PCI immediately ,and 24 hours after PCI ,the plasma GMP-140 ,vWF and PAI-1 levels were determined to analysis the change of platelet function indicators in patients with different genotype .Results The Logistic regression analysis found that GP Ⅱ bHPA -3 bb genotype is a independent risk factor for acute myocardial infarction (AMI) after correction factors .The plasma GMP-140 level in the bb genotype group was remarkably increased immediately and 24 hours after PCI among the three genotypes (P< 0 .01) .The plasma vWF level no statistical difference appeared among the three genotypes .The plasma PAI-1 level imme-diately after PCI ,there were statistical significantly differences between the bb genotype and the others (P<0 .01) .Conclusion The HPA-3 polymorphism of GP IIb is the risk factor of AMI .There is re-markable platelet activation and the fibrinolytic activity changes in patients with GP HPA-3bb geno-type than in those without it after PCI .The results are suggested that the bb genotype of platelet GP IIb HPA-3 maybe a dangerous factor in acute and sub-acute coronary thrombosis after PCI .%目的:探讨血小板膜糖蛋白(GP)Ⅱb HPA-3基因多态性与急性冠脉综合征(ACS)经皮冠状动脉介入治疗(PCI)前后血小板功能变化的关系。方法选择行 PCI的ACS患者120例测定GPⅡb HPA-3基因型,测定各基因型 PCI术前、术后即刻、术后24 h三个时间段血浆GMP-140

  12. UniProt search blastx result: AK287737 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287737 J065143M09 Q8MJJ2|LAMP3_MACMU Lysosome-associated membrane glycoprotein 3 precursor (LAMP...-3) (Lysosomal-associated membrane protein 3) (DC-lysosome-associated membrane glycoprotein) (DC LAMP) (CD208 antigen) - Macaca mulatta (Rhesus macaque) 0 ...

  13. Assembly of BODIPY-carbazole dyes with liposomes to fabricate fluorescent nanoparticles for lysosomal bioimaging in living cells.

    Science.gov (United States)

    Lv, Hai-Juan; Zhang, Xiao-Tai; Wang, Shu; Xing, Guo-Wen

    2017-01-31

    Two BODIPY-carbazole dye based fluorescent probes BCA and BCAS were designed, synthesized and encapsulated by liposomes to obtain fluorescent nanoparticles BCA-FNP and BCAS-FNP. The fluorescence imaging showed that BCA-FNP was membrane-permeable and capable of localizing lysosomes in living cells.

  14. Identification of cytoskeleton-associated proteins essential for lysosomal stability and survival of human cancer cells

    DEFF Research Database (Denmark)

    Groth-Pedersen, Line; Aits, Sonja; Corcelle-Termeau, Elisabeth

    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 si......), 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...

  15. Effects of induced breeding with various ion concentrations of cadmium on the lysosomal membrane of coelomocytes in Pheretima aspergillum%不同镉离子浓度诱导饲养对参环毛蚓体腔细胞溶酶体膜的影响

    Institute of Scientific and Technical Information of China (English)

    吴波; 李薇; 喻良文; 付玉梅

    2010-01-01

    目的 观察不同浓度镉离子诱导饲养对参环毛蚓体腔细胞溶酶体膜的毒性效应.方法 选取健康参环毛蚓70只,按照饲养土壤镉离子浓度的不同随机分为7组,每组10只.各组土壤镉离子浓度分别为0.28(对照组)、3.28、6.28、12.28、18.28、24.28和30.28 mg/kg,采用不同浓度镉离子诱导参环毛蚓染毒并饲养14 d.诱导饲养7 d和14 d时观察参环毛蚓体腔细胞的形态变化,测定其体腔细胞溶酶体中性红保留时间.结果 高浓度镉离子诱导参环毛蚓体腔细胞溶酶体的损害明显.诱导饲养7 d,对照组、3.28、6.28、12.28、18.28、24.28和30.28 mg/kg镉离子浓度组参环毛蚓体腔细胞溶酶体中性红保留时间随着镉离子浓度的增加而逐渐缩短,分别为(71.3±2.4)、(60.5±1.6)、(55.1±2.9)、(51.9±3.6)、(46.0±2.5)、(38.8±1.8)、(34.2±2.0)min.与对照组比较,诱导饲养7 d、14d时各实验组参环毛蚓溶酶体中性红保留时间明显缩短(均P<0.05).结论 体腔细胞溶酶体中性红保留时间可用于评价土壤中重金属镉对参环毛蚓的毒性效应.%Objective To investigate the toxicity effects of induced breeding with various ion concentrations of cadmium on the lysosomal membrane of coelomocytes in Pheretima aspergillum. Methods Seventy healthy Pheretima aspergilla were selected and randomized into 7 groups (n=10 each) according to different levels of cadmium ion in the soils [0.28 mg/kg (control group) , 3.28 mg/kg, 6.28 mg/kg, 12.28 mg/kg, 18.28 mg/kg, 24.28 mg/kg and 30.28 mg/kg, respectively] and were bred with the contaminating cadmium ions for 14 days. On day 7 and day 14, the morphological changes in coelomocytes were studied and neutral red retention time (NRRT) of coelomocyte lysosome was determined in Pheretima aspergilla.Results High levels of cadmium ion were associated with apparent damages to the coelomocyte lysosome in Pheretima asperillum. NRRT decreased along with increasing levels of

  16. Activity of lysosomal exoglycosidases in human gliomas.

    Science.gov (United States)

    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.

  17. Arsenic induces apoptosis by the lysosomal-mitochondrial pathway in INS-1 cells.

    Science.gov (United States)

    Pan, Xiao; Jiang, Liping; Zhong, Laifu; Geng, Chengyan; Jia, Li; Liu, Shuang; Guan, Huai; Yang, Guang; Yao, Xiaofeng; Piao, Fengyuan; Sun, Xiance

    2016-02-01

    Recently, long term arsenic exposure was considered to be associated with an increased risk of diabetes mellitus. While a relation of cause-and-effect between apoptosis of pancreatic β-cells and arsenic exposure, the precise mechanisms of these events remains unclear. The aim of this study was to explore arsenic-induced pancreatic β-cell apoptosis and the mechanisms of through the possible link between lysosomal and the mitochondrial apoptotic pathway. After exposure to 10 μM of arsenic, the reactive oxygen species (ROS) level was significantly increased at 12 h, while the mitochondrial membrane potential was reduced at 24 h and the lysosomal membrane integrity was disrupted at 48 h. A significant increase in protein expression for cytochrome c was also observed using Western blot analysis after exposure to arsenic for 48 h. To further demonstrate that arsenic reduced the lysosomal membrane integrity, cells pretreated with NH4 Cl and exposed to arsenic harbored a lower fluorescence increase than cells that were only exposed to arsenic. In addition, apoptosis was mesured using Hoechst 33342/PI dual staining by microscopy and annexin V-FITC/propidium iodide dual staining by flow cytometry. The results show an increased uptake of the arsenic dose and the cells changed from dark blue to light blue, karyopyknosis, nuclear chromatin condensation, side set or fracture, and a correlation was found between the number of apoptotic cells and arsenic dose. The result of present study suggest that arsenic may induce pancreatic β-cell apoptosis through activation of the lysosome-mitochondrial pathway.

  18. Lysosomal {beta}-mannosidase: cDNA cloning and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.; Leipprandt, J.R.; Traviss, C.E. [Michigan State Univ., East Lansing, MI (United States)] [and others

    1994-09-01

    Lysosomal {beta}-mannosidase is an exoglycosidase that cleaves the single {beta}-linked mannose residue from the non-reducing end of all N-linked glycoprotein oligosaccharides. Deficiency of this enzyme results in {beta}-mannosidosis, a severe neurodegenerative disease in goats and cattle. The human cases described have a milder, highly variable presentation. Study of the molecular pathology of this disease in ruminants and humans and development of the animal model for gene therapy studies required cloning of the gene for {beta}-mannosidase has been cloned. {beta}-Mannosidase cDNA were obtained from a bovine thyroid cDNA library by screening with mixed oligonucleotides derived from peptide sequences resulting from microsequencing of bovine {beta}-mannosidase peptides. A total of six independent positive clones were identified from 5 x 10{sup 5} plaques, covering about 80% of the C-terminal region. The missing 5{prime} region was obtained using 5{prime} RACE. The full-length construct contains 3852-bp nucleotides, encoding 879 amino acids. The initiation codon is followed by 17 amino acids containing the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrated a 4.2 kb single transcript in various tissues from both normal and affected goats and calves. The mRNA level was decreased in affected {beta}-mannosidosis animals. The gene encoding {beta}-mannosidase was localized on human chromosome 4 by Southern analysis of rodent/human somatic cell hybrids. The mutation in bovine {beta}-mannosidosis has been identified. This is the first report of cloning of the {beta}-mannosidase gene.

  19. Insight into the Mechanism of Human Herpesvirus 7 U21-mediated Diversion of Class I MHC Molecules to Lysosomes*

    Science.gov (United States)

    Glosson, Nicole L.; Gonyo, Patrick; May, Nathan A.; Schneider, Christine L.; Ristow, Laura C.; Wang, Qiuhong; Hudson, Amy W.

    2010-01-01

    The U21 open reading frame from human herpesvirus-7 encodes a membrane protein that associates with and redirects class I MHC molecules to the lysosomal compartment. The mechanism by which U21 accomplishes this trafficking excursion is unknown. Here we have examined the contribution of localization, glycosylation, domain structure, and the absence of substrate class I MHC molecules on the ability of U21 to traffic to lysosomes. Our results suggest the existence of a cellular protein necessary for U21-mediated rerouting of class I MHC molecules. PMID:20833720

  20. Retroviral Env Glycoprotein Trafficking and Incorporation into Virions

    Directory of Open Access Journals (Sweden)

    Tsutomu Murakami

    2012-01-01

    Full Text Available Together with the Gag protein, the Env glycoprotein is a major retroviral structural protein and is essential for forming infectious virus particles. Env is synthesized, processed, and transported to certain microdomains at the plasma membrane and takes advantage of the same host machinery for its trafficking as that used by cellular glycoproteins. Incorporation of Env into progeny virions is probably mediated by the interaction between Env and Gag, in some cases with the additional involvement of certain host factors. Although several general models have been proposed to explain the incorporation of retroviral Env glycoproteins into virions, the actual mechanism for this process is still unclear, partly because structural data on the Env protein cytoplasmic tail is lacking. This paper presents the current understanding of the synthesis, trafficking, and virion incorporation of retroviral Env proteins.

  1. Processing of virus-specific glycoproteins of varicella zoster virus

    Energy Technology Data Exchange (ETDEWEB)

    Namazue, J.; Campo-Vera, H.; Kitamura, K.; Okuno, T.; Yamanishi, K.

    1985-05-01

    Monoclonal antibodies to varicella zoster virus (VZV) glycoproteins were used to study the processing of three glycoproteins with molecular weights of 83K-94K (gp 2), 64K (gp 3), and 55K (gp 5). Immunoprecipitation experiments performed with VZV-infected cells, pulse labeled with (/sup 3/H)glucosamine in the presence of tunicamycin, suggest that O-linked oligosaccharide is present on the glycoprotein of gp 2. Use of the enzyme endo-beta-N-acetylglucosaminidase H revealed that the fully processed form of gp 3 had high-mannose type and that of gp 5 had only complex type of N-linked oligosaccharides. Experiments with monensin suggest that the precursor form (116K) of gp 3 is cleaved during the processing from Golgi apparatus to cell surface membrane. The extension of O-linked oligosaccharide chain and the complex type of N-linked oligosaccharide chains also occurs during this processing.

  2. Lysosomal exoglycosidases and cathepsin D in colon adenocarcinoma.

    Science.gov (United States)

    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.

  3. 乌司他丁对双瓣置换患者围术期溶酶体膜稳定性及凝血功能的影响%Effects of ulinastatin on the stability of lysosomal membrane and blood coagulation functions in patients undergoing mitral and aortic valves replacement:a double blinded randomized controlled study

    Institute of Scientific and Technical Information of China (English)

    王臻; 程亮; 曹轶; 赵璧君; 张秀静; 金振晓

    2013-01-01

    目的观察体外循环中应用乌司他丁对双瓣置换患者围术期溶酶体膜稳定性及凝血功能的影响。方法在体外循环下行二尖瓣联合主动脉瓣置换患者20例,随机分为乌司他丁组及对照组,乌司他丁组患者按20000 U/kg总量给予乌司他丁,其中半量预充,半量于主动脉开放前加入到体外循环管路中,对照组患者给予等量生理盐水。分别于术前30 min,体外循环60 min、120 min及体外循环结束后30 min采集血样,ELISA方法检测血浆中组织蛋白酶D( CTSD)及N-乙酰-β-D-氨基葡萄糖苷酶( NAG)含量;采用血栓弹力图比较手术前后凝血功能。同时,记录术后ICU滞留时间、引流量、24 h尿量及呋塞米用量,以及术后呼吸机辅助时间、死亡等情况。结果两组患者均顺利出院,无死亡,无严重并发症;术后ICU时间、机械通气时间、术后第1个24小时尿量及呋塞米用量组间无显著差异( P>0.05);与对照组比较,凝血功能各项指标在手术结束后未见明显差异( P>0.05);体外循环过程中各时间点NAG及CTSD含量组间无显著差异( P>0.05)。结论体外循环中应用乌司他丁20000 U/kg对双瓣置换患者围术期溶酶体稳定性及凝血功能的保护作用与对照组没有明显差异。%Objective To investigate the effects of ulinastatin on the stability of lysosomal membrane and blood coagulation functions in patients undergoing mitral and aortic valves replacement .Methods Twenty consecutive patients scheduled for mitral and aortic valve replacement surgery under cardiopulmonary bypass (CPB) were randomized into two groups:ulinastatin group , who received 20 000 U/kg ulinastatin during CPB with half added into the prime solution and half added to the circuit upon aortic clamping removal;control group , who received same volume of saline .Blood sample were collected at 30 min before the operation ,60 min and 120

  4. Actin-binding protein coronin 1A controls osteoclastic bone resorption by regulating lysosomal secretion of cathepsin K

    Science.gov (United States)

    Ohmae, Saori; Noma, Naruto; Toyomoto, Masayasu; Shinohara, Masahiro; Takeiri, Masatoshi; Fuji, Hiroaki; Takemoto, Kenji; Iwaisako, Keiko; Fujita, Tomoko; Takeda, Norihiko; Kawatani, Makoto; Aoyama, Mineyoshi; Hagiwara, Masatoshi; Ishihama, Yasushi; Asagiri, Masataka

    2017-01-01

    Osteoclasts degrade bone matrix proteins via the secretion of lysosomal enzymes. However, the precise mechanisms by which lysosomal components are transported and fused to the bone-apposed plasma membrane, termed ruffled border membrane, remain elusive. Here, we identified coronin 1A as a negative regulator of exocytotic release of cathepsin K, one of the most important bone-degrading enzymes in osteoclasts. The modulation of coronin 1A expression did not alter osteoclast differentiation and extracellular acidification, but strongly affected the secretion of cathepsin K and osteoclast bone-resorption activity, suggesting the coronin 1A-mediated regulation of lysosomal trafficking and protease exocytosis. Further analyses suggested that coronin 1A prevented the lipidation-mediated sorting of the autophagy-related protein LC3 to the ruffled border and attenuated lysosome–plasma membrane fusion. In this process, the interactions between coronin 1A and actin were crucial. Collectively, our findings indicate that coronin 1A is a pivotal component that regulates lysosomal fusion and the secretion pathway in osteoclast-lineage cells and may provide a novel therapeutic target for bone diseases. PMID:28300073

  5. Biphasic regulation of lysosomal exocytosis by oxidative stress.

    Science.gov (United States)

    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.

  6. A potentially dynamic lysosomal role for the endogenous TRPML proteins.

    Science.gov (United States)

    Zeevi, David A; Frumkin, Ayala; Offen-Glasner, Vered; Kogot-Levin, Aviram; Bach, Gideon

    2009-10-01

    Lysosomal storage disorders (LSDs) constitute a diverse group of inherited diseases that result from lysosomal storage of compounds occurring in direct consequence to deficiencies of proteins implicated in proper lysosomal function. Pathology in the LSD mucolipidosis type IV (MLIV), is characterized by lysosomal storage of lipids together with water-soluble materials in cells from every tissue and organ of affected patients. Mutations in the mucolipin 1 (TRPML1) protein cause MLIV and TRPML1 has also been shown to interact with two of its paralogous proteins, mucolipin 2 (TRPML2) and mucolipin 3 (TRPML3), in heterologous expression systems. Heterogeneous lysosomal storage is readily identified in electron micrographs of MLIV patient cells, suggesting that proper TRPML1 function is essential for the maintenance of lysosomal integrity. In order to investigate whether TRPML2 and TRPML3 also play a role in the maintenance of lysosomal integrity, we conducted gene-specific knockdown assays against these protein targets. Ultrastructural analysis revealed lysosomal inclusions in both TRPML2 and TRPML3 knockdown cells, suggestive of a common mechanism for these proteins, in parallel with TRPML1, in the regulation of lysosomal integrity. However, co-immunoprecipitation assays revealed that physical interactions between each of the endogenous TRPML proteins are quite limited. In addition, we found that all three endogenous proteins only partially co-localize with each other in lysosomal as well as extra-lysosomal compartments. This suggests that native TRPML2 and TRPML3 might participate with native TRPML1 in a dynamic form of lysosomal regulation. Given that depletion of TRPML2/3 led to lysosomal storage typical to an LSD, we propose that depletion of these proteins might also underlie novel LSD pathologies not described hitherto.

  7. N370S-GBA1 mutation causes lysosomal cholesterol accumulation in Parkinson's disease.

    Science.gov (United States)

    García-Sanz, Patricia; Orgaz, Lorena; Bueno-Gil, Guillermo; Espadas, Isabel; Rodríguez-Traver, Eva; Kulisevsky, Jaime; Gutierrez, Antonia; Dávila, José C; González-Polo, Rosa A; Fuentes, José M; Mir, Pablo; Vicario, Carlos; Moratalla, Rosario

    2017-08-05

    Heterozygous mutations in the GBA1 gene, which encodes the lysosomal enzyme β-glucocerebrosidase-1, increase the risk of developing Parkinson's disease, although the underlying mechanisms remain unclear. The aim of this study was to explore the impact of the N370S-GBA1 mutation on cellular homeostasis and vulnerability in a patient-specific cellular model of PD. We isolated fibroblasts from 4 PD patients carrying the N370S/wild type GBA1 mutation and 6 controls to study the autophagy-lysosome pathway, endoplasmic reticulum stress, and Golgi apparatus structure by Western blot, immunofluorescence, LysoTracker and Filipin stainings, mRNA analysis, and electron microscopy. We evaluated cell vulnerability by apoptosis, reactive oxygen species and mitochondrial membrane potential with flow cytometry. The N370S mutation produced a significant reduction in β-glucocerebrosidase-1 protein and enzyme activity and β-glucocerebrosidase-1 retention within the endoplasmic reticulum, which interrupted its traffic to the lysosome. This led to endoplasmic reticulum stress activation and triggered unfolded protein response and Golgi apparatus fragmentation. Furthermore, these alterations resulted in autophagosome and p62/SQSTM1 accumulation. This impaired autophagy was a result of dysfunctional lysosomes, indicated by multilamellar body accumulation probably caused by increased cholesterol, enlarged lysosomal mass, and reduced enzyme activity. This phenotype impaired the removal of damaged mitochondria and reactive oxygen species production and enhanced cell death. Our results support a connection between the loss of β-glucocerebrosidase-1 function, cholesterol accumulation, and the disruption of cellular homeostasis in GBA1-PD. Our work reveals new insights into the cellular pathways underlying PD pathogenesis, providing evidence that GBA1-PD shares common features with lipid-storage diseases. © 2017 International Parkinson and Movement Disorder Society. © 2017 International

  8. 采用流式细胞术分析MYH9-RD家系血小板膜糖蛋白的表达%Analysis of the expression in platelets membrane glycoprotein of the MYH9-RD family by flowcytometry

    Institute of Scientific and Technical Information of China (English)

    张淑芳; 王顺兰; 张应爱; 肖敬川; 黄邓高

    2012-01-01

    Objective To explore the pathogenic mechanism by detecting the expression of membrane glycoprotein in the platelets of nonmuscle myosin heavy chain 9 related disease (MYH9-RD)patients.Methods Periperal bloods were obtained from 11 MYH9-RD patients and 7 normal family members.Flow cytometry was used for detecting the expression of the platelet membrane glycoprotein including GP Ⅱ b/Ⅲa(CD41/61),GP Ⅰ a(CD49b),GP Ⅰ b/Ⅸ/Ⅴ (CD42a) GP Ⅰ b(CD42b) and GPⅣCD36).Results The expression fluorescence intensity of platelet membrane glycoprotein GP Ⅱ b/Ⅲ a CD41/61),GPⅠa(CD49b),GP Ⅰ b/Ⅸ/Ⅴ (CD42a) GP Ⅰ b(CD42b) and GPⅣ (CD36) are 653.7 ±192.7,420.0 ± 151.3,667.7 ± 371.3 and 236.4 ± 64.2 respectively,which are significantly higher than those in normal controls (406.7 ± 126.1,181.2 ± 29.3,271.4 ± 91.6 and 136.1 ± 23.5 ; P < 0.01) ; The expression of GP Ⅰ a(CD49b) was lower in patients with MYH9-RD (139.1 ± 54.9) than that in normal controls (192.2 ± 143.4),but there was no significant difference (P > 0.05).Conclusion In our study,the diverse clinical manifestations in patients with MYH9-RD is probably associated with the expression level of platelet membrane glycoprotein%目的 分析该非肌性肌球蛋白重链9基因相关疾病(MYH9-RD)家系患者多种血小板膜糖蛋白的表达.方法 采集MYH9-RD家系中11例患者和7名健康对照者的外周血;应用流式细胞术对血小板膜糖蛋白GPⅡb/Ⅲa(CD41/61)、GP Ⅰ a(CD49b)、GPⅠb/Ⅸ/Ⅴ(CD42a),GPⅠ b(CD42b)和GPⅣ(CD36)进行检测.结果 MYH9-RD家系GPⅡb/Ⅲa(CD41/61)、GP Ⅰ b(CD42b)、GPⅣ(CD36)和GP Ⅰ b/Ⅸ/Ⅴ(CD42a)复合物表达量分别为653.7±192.7、420.0±151.3、667.7±371.3和236.4±64.2,高于对照组(分别为406.7±126.1、181.2±29.3、271.4±91.6和136.1±23.5,P<0.01),差异具有统计学意义;MYH9-RD家系患者组GP Ⅰ a(CD49b)表达量为192.2±143.4,较对照组(139.1±54.9)稍有下降,差异无统计学意义(P>0

  9. Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury.

    Science.gov (United States)

    Godar, Rebecca J; Ma, Xiucui; Liu, Haiyan; Murphy, John T; Weinheimer, Carla J; Kovacs, Attila; Crosby, Seth D; Saftig, Paul; Diwan, Abhinav

    2015-01-01

    Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from in-vivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls. This protection was lost in mice heterozygous null for Lamp2 (coding for lysosomal-associated membrane protein 2), which demonstrate impaired autophagy in response to fasting with accumulation of autophagosomes and SQSTM1, an autophagy substrate, in the heart. In lamp2 null mice, intermittent fasting provoked progressive left ventricular dilation, systolic dysfunction and hypertrophy; worsening cardiomyocyte autophagosome accumulation and lack of protection to ischemia-reperfusion injury, suggesting that intact autophagy-lysosome machinery is essential for myocardial homeostasis during intermittent fasting and consequent ischemic cardioprotection. Fasting and refeeding cycles resulted in transcriptional induction followed by downregulation of autophagy-lysosome genes in the myocardium. This was coupled with fasting-induced nuclear translocation of TFEB (transcription factor EB), a master regulator of autophagy-lysosome machinery; followed by rapid decline in nuclear TFEB levels with refeeding. Endogenous TFEB was essential for attenuation of hypoxia-reoxygenation-induced cell death by repetitive starvation, in neonatal rat cardiomyocytes, in-vitro. Taken together, these data suggest that TFEB-mediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fasting-induced autophagy in myocardial ischemia-reperfusion injury.

  10. Lipid modification gives rise to two distinct Haloferax volcanii S-layer glycoprotein populations.

    Science.gov (United States)

    Kandiba, Lina; Guan, Ziqiang; Eichler, Jerry

    2013-03-01

    The S-layer glycoprotein is the sole component of the protein shell surrounding Haloferax volcanii cells. The deduced amino acid sequence of the S-layer glycoprotein predicts the presence of a C-terminal membrane-spanning domain. However, several earlier observations, including the ability of EDTA to selectively solubilize the protein, are inconsistent with the presence of a trans-membrane sequence. In the present report, sequential solubilization of the S-layer glycoprotein by EDTA and then with detergent revealed the existence of two distinct populations of the S-layer glycoprotein. Whereas both S-layer glycoprotein populations underwent signal peptide cleavage and N-glycosylation, base hydrolysis followed by mass spectrometry revealed that a lipid, likely archaetidic acid, modified only the EDTA-solubilized version of the protein. These observations are consistent with the S-layer glycoprotein being initially synthesized as an integral membrane protein and subsequently undergoing a processing event in which the extracellular portion of the protein is separated from the membrane-spanning domain and transferred to a waiting lipid moiety.

  11. Quantitative modeling of selective lysosomal targeting for drug design

    DEFF Research Database (Denmark)

    Trapp, Stefan; Rosania, G.; Horobin, R.W.;

    2008-01-01

    Lysosomes are acidic organelles and are involved in various diseases, the most prominent is malaria. Accumulation of molecules in the cell by diffusion from the external solution into cytosol, lysosome and mitochondrium was calculated with the Fick–Nernst–Planck equation. The cell model considers....... This demonstrates that the cell model can be a useful tool for the design of effective lysosome-targeting drugs with minimal off-target interactions....

  12. Release and uptake of lysosomal enzymes : studied in cultured cells

    OpenAIRE

    1980-01-01

    textabstractThe purpose of the experimental work described in this thesiswas to investigate some aspects of the release and uptake of lysosomal enzymes. The experiments involved the use of normal human and animal fibroblasts and some other cell types such as hepatocytes and hepatoma cells as sources of hydrolytic enzymes, and fibroblasts from patients with lysosomal storage diseases associated with a single lysosomal enzyme deficiency and with "1-cell" disease as recipient cells. In a number ...

  13. Factors and processes modulating phenotypes in neuronopathic lysosomal storage diseases

    OpenAIRE

    Jakóbkiewicz-Banecka, Joanna; Gabig-Cimińska, Magdalena; Banecka-Majkutewicz, Zyta; Banecki, Bogdan; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2013-01-01

    Lysosomal storage diseases are inherited metabolic disorders caused by genetic defects causing deficiency of various lysosomal proteins, and resultant accumulation of non-degraded compounds. They are multisystemic diseases, and in most of them (>70 %) severe brain dysfunctions are evident. However, expression of various phenotypes in particular diseases is extremely variable, from non-neuronopathic to severely neurodegenerative in the deficiency of the same enzyme. Although all lysosomal stor...

  14. A Novel High Content Imaging-Based Screen Identifies the Anti-Helminthic Niclosamide as an Inhibitor of Lysosome Anterograde Trafficking and Prostate Cancer Cell Invasion.

    Directory of Open Access Journals (Sweden)

    Magdalena L Circu

    Full Text Available Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF or acidic extracellular pH (pHe, increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 "hits" were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes

  15. Lack of Fas/CD95 surface expression in highly proliferative leukemic cell lines correlates with loss of CtBP/BARS and redirection of the protein toward giant lysosomal structures.

    Science.gov (United States)

    Monleón, Inmaculada; Iturralde, María; Martínez-Lorenzo, María José; Monteagudo, Luis; Lasierra, Pilar; Larrad, Luis; Piñeiro, Andrés; Naval, Javier; Alava, María Angeles; Anel, Alberto

    2002-07-01

    Fas/CD95 is a type-I membrane glycoprotein, which inducesapoptotic cell death when ligated by its physiological ligand. We generated previously hyperproliferative sublines derived from the human T-cell leukemia Jurkat, Jurkat-ws and Jurkat-hp, which lost Fas/CD95 surface expression. We have now observed that the total amount of Fas protein is similar in the sublines and in the parental cells, indicating that in the sublines Fas remains in an intracellular compartment. We have found that the protein is directed toward lysosomes in the sublines, where it is degraded. This defect in the secretory pathway correlates with loss of polyunsaturated fatty acids from cellular lipids, and with the lack of expression of endophilin-I and CtBP/BARS, enzymes that regulate vesicle fission by catalyzing the acylation of arachidonate into lysophosphatidic acid. In addition, great multillamer bodies, which contained acid phosphatase activity, absent in the parental Jurkat cells, were observed by transmission electron microscopy in the sublines.

  16. The Mechanism of Henipavirus Fusion: Examining the Relationships between the Attachment and Fusion Glycoproteins

    Institute of Scientific and Technical Information of China (English)

    Andrew C. Hickey; Christopher C. Broder

    2009-01-01

    The henipaviruses, represented by Nipah virus and Hendra virus, are emerging zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These viruses enter host cells via a class I viral fusion mechanism mediated by their attachment and fusion envelope glycoproteins; efficient membrane fusion requires both these glycoproteins in conjunction with specific virus receptors present on susceptible host cells. The henipavirus attachment glycoprotein interacts with a cellular B class ephrin protein receptor triggering conformational alterations leading to the activation of the viral fusion (F) glycoprotein. The analysis of monoclonal antibody (mAb) reactivity with G has revealed measurable alterations in the antigenic structure of the glycoprotein following its binding interaction with receptor. These observations only appear to occur with full-length native G glycoprotein, which is a tetrameric oligomer, and not with soluble forms of G (sG), which are disulfide-linked dimers. Single amino acid mutations in a heptad repeat-like structure within the stalk domain of G can disrupt its association with F and subsequent membrane fusion promotion activity. Notably, these mutants of G also appear to confer a postreceptor bound conformation implicating the stalk domain as an important element in the G glycoprotein's structure and functional relationship with F. Together, these observations suggest fusion is dependent on a specific interaction between the F and G glycoproteins of the henipaviruses. Further, receptor binding induces measurable changes in the G glycoprotein that appear to be greatest in respect to the interactions between the pairs of dimers comprising its native tetrameric structure. These receptor-induced conformational changes may be associated with the G glycoprotein's promotion of the fusion activity of F.

  17. Envelope glycoprotein of arenaviruses.

    Science.gov (United States)

    Burri, Dominique J; da Palma, Joel Ramos; Kunz, Stefan; Pasquato, Antonella

    2012-10-17

    Arenaviruses include lethal human pathogens which pose serious public health threats. So far, no FDA approved vaccines are available against arenavirus infections, and therapeutic options are limited, making the identification of novel drug targets for the development of efficacious therapeutics an urgent need. Arenaviruses are comprised of two RNA genome segments and four proteins, the polymerase L, the envelope glycoprotein GP, the matrix protein Z, and the nucleoprotein NP. A crucial step in the arenavirus life-cycle is the biosynthesis and maturation of the GP precursor (GPC) by cellular signal peptidases and the cellular enzyme Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P) yielding a tripartite mature GP complex formed by GP1/GP2 and a stable signal peptide (SSP). GPC cleavage by SKI-1/S1P is crucial for fusion competence and incorporation of mature GP into nascent budding virion particles. In a first part of our review, we cover basic aspects and newer developments in the biosynthesis of arenavirus GP and its molecular interaction with SKI-1/S1P. A second part will then highlight the potential of SKI-1/S1P-mediated processing of arenavirus GPC as a novel target for therapeutic intervention to combat human pathogenic arenaviruses.

  18. Envelope Glycoprotein of Arenaviruses

    Directory of Open Access Journals (Sweden)

    Antonella Pasquato

    2012-10-01

    Full Text Available Arenaviruses include lethal human pathogens which pose serious public health threats. So far, no FDA approved vaccines are available against arenavirus infections, and therapeutic options are limited, making the identification of novel drug targets for the development of efficacious therapeutics an urgent need. Arenaviruses are comprised of two RNA genome segments and four proteins, the polymerase L, the envelope glycoprotein GP, the matrix protein Z, and the nucleoprotein NP. A crucial step in the arenavirus life-cycle is the biosynthesis and maturation of the GP precursor (GPC by cellular signal peptidases and the cellular enzyme Subtilisin Kexin Isozyme-1 (SKI-1/Site-1 Protease (S1P yielding a tripartite mature GP complex formed by GP1/GP2 and a stable signal peptide (SSP. GPC cleavage by SKI-1/S1P is crucial for fusion competence and incorporation of mature GP into nascent budding virion particles. In a first part of our review, we cover basic aspects and newer developments in the biosynthesis of arenavirus GP and its molecular interaction with SKI-1/S1P. A second part will then highlight the potential of SKI-1/S1P-mediated processing of arenavirus GPC as a novel target for therapeutic intervention to combat human pathogenic arenaviruses.

  19. Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells

    DEFF Research Database (Denmark)

    Jensen, Poul Henning; Christensen, Erik Ilsø; Ebbesen, P.

    1990-01-01

    in an apparently intact form in the medium or was still cell associated. The degradation could be inhibited by inhibitors of vesicle transport and lysosomal hydrolases. By electron microscopic autoradiography, both 125I-u-PA and 125I-u-PA-inhibitor complexes were located over the cell membrane at 4 degrees C......, with the highest density of grains over the membrane at cell-cell interphases, but, after incubation at 37 degrees C, 17 and 27% of the grains for u-PA and u-PA-PAI-1 complexes, respectively, appeared over lysosomal-like bodies. These findings suggest that the u-PA receptor possesses a clearance function...

  20. Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Lee

    2015-09-01

    Full Text Available 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 Presenilin 1 knockout (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.

  1. Presenilin 1 Maintains Lysosomal Ca(2+) Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification.

    Science.gov (United States)

    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

    2015-09-01

    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 Presenilin 1 knockout (PS1KO) cells induces abnormal Ca(2+) efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca(2+). 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 Ca(2+) homeostasis, but correcting lysosomal Ca(2+) 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 Ca(2+) homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

  2. Gaucher disease: a lysosomal neurodegenerative disorder.

    Science.gov (United States)

    Huang, W J; Zhang, X; Chen, W W

    2015-04-01

    Gaucher disease is a multisystemic disorder that affects men and woman in equal numbers and occurs in all ethnic groups at any age with racial variations and an estimated worldwide incidence of 1/75,000. It is caused by a genetic deficient activity of the lysosomal enzyme glucocerebrosidase due to mutations in the β-glucocerebrosidase gene, and resulting in lack of glucocerebroside degradation. The subsequent accumulation of glucocerebroside in lysosomes of tissue macrophages primarily in the liver, bone marrow and spleen, causes damage in haematological, skeletal and nervous systems. The clinical manifestations show a high degree of variability with symptoms that varies according to organs involved. In many cases, these disorders do not correlate with mutations in the β-glucocerebrosidase gene. Although several mutations have been identified as responsible for the deficient activity of glucocerebrosidase, mechanisms by which this enzymatic defect leads to Gaucher disease remain poorly understood. Recent reports indicate the implication of complex mechanisms, including enzyme deficiency, substrate accumulation, unfolded protein response, and macrophage activation. Further elucidating these mechanisms will advance understanding of Gaucher disease and related disorders.

  3. Isolation of a surface glycoprotein from Myxococcus xanthus.

    OpenAIRE

    Maeba, P Y

    1986-01-01

    The isolation of a glycoprotein from vegetative cells of Myxococcus xanthus is reported. The protein, abbreviated VGP, was first identified during a survey of surface proteins as a major protein that could be radioiodinated in vegetative, but not developing, cells (P.Y. Maeba, J. Bacteriol. 155:1033-1041, 1983). The protein was extracted from membranes with Triton X-100 and subsequently purified by DEAE-cellulose chromatography, chromatofocusing, and gel filtration. The protein has an Mr of a...

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

    Science.gov (United States)

    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.

  5. Bile canalicular cationic dye secretion as a model for P-glycoprotein mediated transport.

    Science.gov (United States)

    Thalhammer, T; Stapf, V; Gajdzik, L; Graf, J

    1994-04-01

    This study explores properties of P-glycoprotein dependent membrane transport in rat liver with the use of acridine orange as the substrate. We studied the biliary secretion of the dye, its binding to canalicular membrane P-glycoprotein, and effects of the inhibitor cyclosporin A: acridine orange is excreted into bile together with less hydrophobic and glucuronidated metabolites. Cyclosporin A inhibited both the secretion of acridine orange and of its metabolites. In TR- animals, a rat strain that is deficient of the canalicular multi-specific organic anion transport system, non-metabolized acridine orange is the predominant species in bile and its secretion is also inhibited by cyclosporin A. Binding of acridine orange to liver P-glycoprotein was analyzed by photoaffinity labeling with azidopine, a substrate of P-glycoprotein dependent transport in multi-drug resistant tumor cells. Labeling of the immunoprecipitated P-glycoprotein was inhibited by acridine orange, verapamil, and by cyclosporin A. The results show that biliary secretion of acridine orange is highly analogous to P-glycoprotein mediated membrane drug transport in tumor cells that exhibit multi-drug resistance.

  6. Salivary agglutinin/glycoprotein-340/DMBT1

    DEFF Research Database (Denmark)

    Ligtenberg, Antoon J M; Veerman, Enno C I; Nieuw Amerongen, Arie V

    2007-01-01

    Salivary agglutinin (SAG), lung glycoprotein-340 (gp-340) and Deleted in Malignant Brain Tumours 1 (DMBT1) are three names for identical proteins encoded by the dmbt1 gene. DMBT1/SAG/gp-340 belongs to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins, a superfamily of secreted...... or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. On the one hand, DMBT1 may represent an innate defence factor acting as a pattern recognition molecule. It interacts with a broad range of pathogens, including cariogenic streptococci...... and Helicobacter pylori, influenza viruses and HIV, but also with mucosal defence proteins, such as IgA, surfactant proteins and MUC5B. Stimulation of alveolar macrophage migration, suppression of neutrophil oxidative burst and activation of the complement cascade point further to an important role...

  7. Lysosome/lipid droplet interplay in metabolic diseases.

    Science.gov (United States)

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

    Science.gov (United States)

    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.

  9. Photoaffinity labeling of the lysosomal neuraminidase from bovine testis

    NARCIS (Netherlands)

    G.T.J. van der Horst (Gijsbertus); U. Rose (Ursula); R. Brossmer (Reinhard); F.W. Verheijen (Frans)

    1990-01-01

    markdownabstractAbstract ASA-NeuAc2en, a photoreactive arylazide derivative of sialic acid, is shown to be a powerful competitive inhibitor of lysosomal neuraminidase from bovine testis (Ki ≈ 21 μM). Photoaffinity labeling and partial purification of preparations containing this lysosomal neuramin

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  12. Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6.

    Science.gov (United States)

    Poët, Mallorie; Kornak, Uwe; Schweizer, Michaela; Zdebik, Anselm A; Scheel, Olaf; Hoelter, Sabine; Wurst, Wolfgang; Schmitt, Anja; Fuhrmann, Jens C; Planells-Cases, Rosa; Mole, Sara E; Hübner, Christian A; Jentsch, Thomas J

    2006-09-12

    Mammalian CLC proteins function as Cl(-) channels or as electrogenic Cl(-)/H(+) exchangers and are present in the plasma membrane and intracellular vesicles. We now show that the ClC-6 protein is almost exclusively expressed in neurons of the central and peripheral nervous systems, with a particularly high expression in dorsal root ganglia. ClC-6 colocalized with markers for late endosomes in neuronal cell bodies. The disruption of ClC-6 in mice reduced their pain sensitivity and caused moderate behavioral abnormalities. Neuronal tissues showed autofluorescence at initial axon segments. At these sites, electron microscopy revealed electron-dense storage material that caused a pathological enlargement of proximal axons. These deposits were positive for several lysosomal proteins and other marker proteins typical for neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. However, the lysosomal pH of Clcn6(-/-) neurons appeared normal. CLCN6 is a candidate gene for mild forms of human NCL. Analysis of 75 NCL patients identified ClC-6 amino acid exchanges in two patients but failed to prove a causative role of CLCN6 in that disease.

  13. Abeta42-induced neurodegeneration via an age-dependent autophagic-lysosomal injury in Drosophila.

    Directory of Open Access Journals (Sweden)

    Daijun Ling

    Full Text Available The mechanism of widespread neuronal death occurring in Alzheimer's disease (AD remains enigmatic even after extensive investigation during the last two decades. Amyloid beta 42 peptide (Abeta(1-42 is believed to play a causative role in the development of AD. Here we expressed human Abeta(1-42 and amyloid beta 40 (Abeta(1-40 in Drosophila neurons. Abeta(1-42 but not Abeta(1-40 causes an extensive accumulation of autophagic vesicles that become increasingly dysfunctional with age. Abeta(1-42-induced impairment of the degradative function, as well as the structural integrity, of post-lysosomal autophagic vesicles triggers a neurodegenerative cascade that can be enhanced by autophagy activation or partially rescued by autophagy inhibition. Compromise and leakage from post-lysosomal vesicles result in cytosolic acidification, additional damage to membranes and organelles, and erosive destruction of cytoplasm leading to eventual neuron death. Neuronal autophagy initially appears to play a pro-survival role that changes in an age-dependent way to a pro-death role in the context of Abeta(1-42 expression. Our in vivo observations provide a mechanistic understanding for the differential neurotoxicity of Abeta(1-42 and Abeta(1-40, and reveal an Abeta(1-42-induced death execution pathway mediated by an age-dependent autophagic-lysosomal injury.

  14. Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes

    Science.gov (United States)

    Rai, Ashim; Pathak, Divya; Thakur, Shreyasi; Singh, Shampa; Dubey, Alok Kumar; Mallik, Roop

    2016-01-01

    Summary Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes. PMID:26853472

  15. Fluence- and time-dependant lysosomal and mitochondrial damage induced by LS11 PDT characterized with light scattering

    Science.gov (United States)

    Wilson, Jeremy D.; Foster, Thomas H.

    2007-02-01

    Light scattering from cells originates from sub-cellular organelles. Our measurements of angularly resolved light scattering have demonstrated that at 633 nm, the dominant scattering centers within EMT6 cells are mitochondria and lysosomes. To assess their specific contributions, we have used photodynamic therapy (PDT) to induce organelle-specific perturbations within intact cells. We have developed a coated sphere scattering model for mitochondrial swelling in response to ALA- and Pc 4-PDT, and in the case of Pc 4-PDT we have used this model to map the scattering responses into clonogenic cell survival. More recently, we demonstrated the ability to measure the size, scattering contribution, and refractive index of lysosomes within cells by exploiting the localization and high extinction of the photosensitizer LS11 and an absorbing sphere scattering model. Here we report on time- and fluence-dependant scattering measurements from cells treated with LS11-PDT. LS11-PDT causes rapid lysosomal disruption, as quantified by uptake of acridine orange, and can induce downstream effects including release of mitochondrial cytochrome c preceding the loss of mitochondrial membrane potential (Reiners et al., Cell Death Differ. 9:934, 2002). Using scattering and these various methods of analysis, we observed that the induction of lysosomal morphology changes requires a fluence significantly higher than that reported for cell killing. At lower fluences, we observe that at 1 h after irradiation there is significant mitochondrial swelling, consistent with the onset of cytochrome c-induced cell death, while the morphology of lysosomes remains unchanged. We also expand on the ideas of lysosomal staining to demonstrate the sensitivity of scattering measurements at different wavelengths to different organelle populations.

  16. Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-resistant human ovarian carcinoma cells.

    Science.gov (United States)

    Safaei, Roohangiz; Larson, Barrett J; Cheng, Timothy C; Gibson, Michael A; Otani, Shinji; Naerdemann, Wiltrud; Howell, Stephen B

    2005-10-01

    Previous work has shown that cisplatin (CDDP) becomes concentrated in lysosomes, and that acquired resistance to CDDP is associated with abnormalities of protein trafficking and secretion. The lysosomal compartment in CDDP-sensitive 2008 human ovarian carcinoma cells was compared with that in CDDP-resistant 2008/C13*5.25 subline using deconvoluting imaging and specific dyes and antibodies. The lysosomal compartment in CDDP-resistant cells was reduced to just 40% of that in the parental CDDP-sensitive cells (P<0.002). This was accompanied by a reduced expression of the lysosome-associated proteins 1 and 2 (LAMP1 and LAMP2) as determined by both microscopy and Western blot analysis. The CDDP-resistant cells released more protein as exosomes and Western blot analysis revealed that these exosomes contained substantially more LAMP1 than those released by the CDDP-sensitive cells. Following loading of the whole cell with CDDP, the exosomes released from 2008/C13*5.25 cells contained 2.6-fold more platinum than those released from sensitive cells. Enhanced exosomal export was accompanied by higher exosomal levels of the putative CDDP export transporters MRP2, ATP7A, and ATP7B. Expression profiling identified significant increases in the expression of several genes whose products function in membrane fusion and vesicle trafficking. This study shows that the lysosomal compartment of human ovarian carcinoma cells selected for stable resistance to CDDP is markedly reduced in size, and that these cells abnormally sort some lysosomal proteins and the putative CDDP transporters into an exosomal pathway that also exports CDDP.

  17. The serotonin transporter undergoes constitutive internalization and is primarily sorted to late endosomes and lysosomal degradation

    DEFF Research Database (Denmark)

    Rahbek-Clemmensen, Troels; Bay, Tina; Eriksen, Jacob

    2014-01-01

    The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of surface resident....... Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analogue JHC1-64, and by reversible and pulse chase biotinylation assays showing evidence...

  18. Contribution of mitochondria and lysosomes to photodynamic therapy-induced death in cancer cells

    Science.gov (United States)

    Nieminen, Anna-Liisa; Azizuddin, Kashif; Zhang, Ping; Kenney, Malcolm E.; Pediaditakis, Peter; Lemasters, John J.; Oleinick, Nancy L.

    2008-02-01

    In photodynamic therapy (PDT), visible light activates a photosensitizing drug added to a tissue, resulting in singlet oxygen formation and cell death. Employing confocal microscopy, we previously found that the phthalocyanine Pc 4 localized primarily to mitochondrial membranes in various cancer cell lines, resulting in mitochondrial reactive oxygen species (ROS) production, followed by inner membrane permeabilization (mitochondrial permeability transition) with mitochondrial depolarization and swelling, which in turn led to cytochrome c release and apoptotic death. Recently, derivatives of Pc 4 with OH groups added to one of the axial ligands were synthesized. These derivatives appeared to be taken up more avidly by cells and caused more cytotoxicity than the parent compound Pc 4. Using organelle-specific fluorophores, we found that one of these derivatives, Pc 181, accumulated into lysosomes and that PDT with Pc 181 caused rapid disintegration of lysosomes. We hypothesized that chelatable iron released from lysosomes during PDT contributes to mitochondrial damage and subsequent cell death. We monitored cytosolic Fe2+ concentrations after PDT with calcein. Fe2+ binds to calcein causing quenching of calcein fluorescence. After bafilomycin, an inhibitor of the vacuolar proton-translocating ATPase, calcein fluorescence became quenched, an effect prevented by starch desferal s-DFO, an iron chelator that enters cells by endocytosis. After Pc 181-PDT, cytosolic calcein fluorescence also decreased, indicating increased chelatable Fe2+ in the cytosol, and apoptosis occurred. s-DFO decreased Pc 181-PDT-induced apoptosis as measured by a decrease of caspase-3 activation. In isolated mitochondria preparations, Fe2+ induced mitochondrial swelling, which was prevented by Ru360, an inhibitor of the mitochondrial Ca2+ uniporter. The data support a hypothesis of oxidative injury in which Pc 181-PDT disintegrates lysosomes and releases constituents that synergistically promote

  19. Syntaxin 7 is localized to late endosome compartments, associates with Vamp 8, and Is required for late endosome-lysosome fusion.

    Science.gov (United States)

    Mullock, B M; Smith, C W; Ihrke, G; Bright, N A; Lindsay, M; Parkinson, E J; Brooks, D A; Parton, R G; James, D E; Luzio, J P; Piper, R C

    2000-09-01

    Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, alpha and gamma SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells.

  20. Syntaxin 7 Is Localized to Late Endosome Compartments, Associates with Vamp 8, and Is Required for Late Endosome–Lysosome Fusion

    Science.gov (United States)

    Mullock, Barbara M.; Smith, Chez W.; Ihrke, Gudrun; Bright, Nicholas A.; Lindsay, Margaret; Parkinson, Emma J.; Brooks, Doug A.; Parton, Robert G.; James, David E.; Luzio, J. Paul; Piper, Robert C.

    2000-01-01

    Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, α and γ SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells. PMID:10982406

  1. Lysosomal trafficking of {beta}-catenin induced by the tea polyphenol epigallocatechin-3-gallate

    Energy Technology Data Exchange (ETDEWEB)

    Dashwood, Wan-Mohaiza [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Carter, Orianna [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Al-Fageeh, Mohamed [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Li, Qingjie [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Dashwood, Roderick H. [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States)]. E-mail: Rod.Dashwood@oregonstate.edu

    2005-12-11

    {beta}-Catenin is a cadherin-binding protein involved in cell-cell adhesion, which also functions as a transcriptional activator when complexed in the nucleus with members of the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of proteins. There is considerable interest in mechanisms that down-regulate {beta}-catenin, since this provides an avenue for the prevention of colorectal and other cancers in which {beta}-catenin is frequently over-expressed. We show here that physiologically relevant concentrations of the tea polyphenol epigallocatechin-3-gallate (EGCG) inhibited {beta}-catenin/TCF-dependent reporter activity in human embryonic kidney 293 cells transfected with wild type or mutant {beta}-catenins, and there was a corresponding decrease in {beta}-catenin protein levels in the nuclear, cytosolic and membrane-associated fractions. However, {beta}-catenin accumulated as punctate aggregates in response to EGCG treatment, including in human colon cancer cells over-expressing {beta}-catenin endogenously. Confocal microscopy studies revealed that the aggregated {beta}-catenin in HEK293 cells was extra-nuclear and co-localized with lysosomes, suggesting that EGCG activated a pathway involving lysosomal trafficking of {beta}-catenin. Lysosomal inhibitors leupeptin and transepoxysuccinyl-L-leucylamido(4-guanido)butane produced an increase in {beta}-catenin protein in total cell lysates, without a concomitant increase in {beta}-catenin transcriptional activity. These data provide the first evidence that EGCG facilitates the trafficking of {beta}-catenin into lysosomes, presumably as a mechanism for sequestering {beta}-catenin and circumventing further nuclear transport and activation of {beta}-catenin/TCF/LEF signaling.

  2. The lysosomal stability as a biomarker for the determination of pollution in aquatic environments

    Directory of Open Access Journals (Sweden)

    Maria Loreto Nazar

    2008-10-01

    Full Text Available This work studied the effects caused by five different formulae of gasoline on the stability of the lysosomes isolated from the liver of the tilapia fish (Oreochromis niloticus. The functional integrity of the lysosomal membranes was evaluated via the acid phosphatase activity. The results showed that there were significant changes in the stability of the lysosomes exposed to the presence of the hydrocarbons in the environment. Therefore, considering the method's simplicity, the sensitivity of the responses and its low cost the assessment of the lysosomal activity could be an important tool for the study of the effects of pollution in the aquatic environments.A procura de biomarcadores de agentes poluidores, mais simples e menos custosos, tem levado ao estudo dos lisossomos, isolados de animais componentes da biota nos ambientes contaminados, principalmente por poluentes com características lipofílicas, a exemplo dos hidrocarbonetos policíclicos e seus derivados. Este trabalho estudou os efeitos provocados por 05 diferentes formulações de gasolina sobre a estabilidade de lisossomos, isolados de fígado de tilápia (Oreochromis niloticus. A integridade funcional das membranas lisossômicas foi avaliada através da atividade da fosfatase ácida, expressa em mU/mg de proteínas totais. Os resultados obtidos mostraram que existem alterações significativas na estabilidade dos lisossomos isolados de fígado de tilápias submetidas aos efeitos de hidrocarbonetos presentes no meio ambiente. Portanto, levando em conta a simplicidade, a sensibilidade de resposta e o baixo custo, os autores recomendam a avaliação da atividade lisossômica, como uma importante ferramenta para o estudo dos efeitos da poluição dos meios aquáticos.

  3. A study on platelet indices and platelet membrane glycoprotein in children with primary immune thrombocytope-nia%儿童原发性免疫性血小板减少症血小板参数及功能变化

    Institute of Scientific and Technical Information of China (English)

    白静; 刘文君

    2015-01-01

    目的:探讨儿童原发性免疫性血小板减少症(ITP)治疗前后血小板参数及功能的变化。方法应用全自动血细胞分析仪和FCM微量全血法检测新诊断ITP患儿18例治疗前(ITP组)及治疗后达完全反应组(ITP-CR组)以及儿外科择期手术患儿17例(对照组)的血小板参数、IPF%、IPC、及膜糖蛋白(CD62p、PAC-1、CD42b)。结果与对照组比较,ITP组MPV、PDW、P-LCR、IPF%明显升高(P<0.05),而PLT、PCT、IPC降低(P<0.05);且ADP激活前、后三种膜糖蛋白(PAC-1、CD62P、CD42b)的表达也明显降低(P<0.05);ITP-CR组与ITP组比较,MPV、PDW、P-LCR、IPF%降低,PLT、PCT、IPC及三种膜糖蛋白(PAC-1、CD62p、CD42b)的表达升高(P<0.05)。结论 ITP患儿体内外血小板均处于低活化状态,血小板功能降低;血小板相关参数为ITP的诊断及疗效判定提供依据。%ObjectiveTo investigate the change of the platelet function in children with primary immune thrombocy-topenia (ITP) before and after treatment.MethodsThe platelet indices, immature platelet fraction (IPF%), immature platelet counts (IPC), granule membrane glycoproteins of platelet (CD62p, PAC-1, CD42b) were tested by automatic blood cell analyzer and lfow cytometry (FCM) in 18 children with ITP before treatment and after complete response and in 17 children undergoing elective surgery (control gourp).ResultsCompared with control group, MPV, PDW, P-LCR and IPF% were signiifcantly higher and PLT, PCT, IPC were signiifcantly lower in children with ITP. The expressions of three platelet membrane glycoproteins (PAC-1, CD62p, CD42b) were signiifcantly lower in children with ITP. After treatment, children with ITP achieved completely response. Compared with before treatment, MPV, PDW, P-LCR and IPF% were signiifcantly lower, and PLT, PCT, IPC, and the expressions of three platelet membrane glycoproteins (PAC-1, CD62p and CD42b) were significantly

  4. Changes of Platelet Parameters in Patients with Sepsis and Its Relationship with Platelet Membrane Glycoprotein%脓毒血症患者血小板参数变化及其与血小板膜糖蛋白的关系

    Institute of Scientific and Technical Information of China (English)

    陈玉时

    2014-01-01

    目的:探讨脓毒血症患者血小板参数变化及其与血小板膜糖蛋白的关系,以寻求精确判断血小板功能状态的办法。方法:选取2013年2月-2014年1月于本院住院治疗的脓毒症患者48例为病例组,并选取同期健康体检者32例作为对照组,应用全自动血球分析仪检测血小板参数PC、MPV、PDW,采用流式细胞仪检测血小板膜糖蛋白PAC-1、CD62P的水平。结果:与对照组比较,病例组PC显著降低,MPV、PDW均显著增大,PAC-1、CD62P均表达上调,差异均有统计学意义(P0.05)。结论:脓毒症患者体内存在血小板明显活化,血小板参数与血小板膜糖蛋白PAC-1、CD62P共同参与血小板活化过程。%Objective:To investigate the changes of platelet parameters in patients with sepsis and its relationship with Platelet membrane glycoprotein, in order to estimate accurately the function of platelet.Method:48 patients with sepsis treated in our hospital from February 2013 to January 2014 were selected as sepsis group, and 32 healthy subjects were selected as control group, the platelet parameters PC, MPV, PDW were detected with automatic blood cell analyzer, and the platelet membrane glycoprotein PAC-1, CD62P were detected with flow cytometry.Result:Compared with the control group, PC of the sepsis group was significantly decreased, MPV, PDW were significantly increased, the expression of PAC-1, CD62P were upregulated,there were statistically significant difference between the two groups (P<0.05). Pearson correlation analysis showed that, there were a significant positive correlation between MPV and the expression of PAC-1, CD62P(r=0.377,0.306,P<0.05),and non significant correlation between PDW, PV and the expression of PAC-1, CD62P(P<0.05).Conclusion:Platelet was activated in patient with sepsis, and platelet parameters and platelet membrane glycoprotein PAC-1, CD62P pay a role in the pathological process.

  5. PURIFICATION OF THE INTEGRAL MEMBRANE-GLYCOPROTEINS-D OF HERPES-SIMPLEX VIRUS TYPE-1 AND TYPE-2, PRODUCED IN THE RECOMBINANT BACULOVIRUS EXPRESSION SYSTEM, BY ION-EXCHANGE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

    NARCIS (Netherlands)

    DAMHOF, RA; FEIJLBRIEF, M; WELLINGWESTER, S; WELLING, GW

    1994-01-01

    Selective elution of Sendai virus integral membrane proteins by ion-exchange high-performance liquid chromatography (HPIEC) using different detergent concentrations was reported before [S. Welling-Wester, M. Feijlbrief, D.G.A.M. Koedijk, M.A. Braaksma, B.R.K. Douma and G.W. Welling, J. Chromatogr.,

  6. PURIFICATION OF THE INTEGRAL MEMBRANE-GLYCOPROTEINS-D OF HERPES-SIMPLEX VIRUS TYPE-1 AND TYPE-2, PRODUCED IN THE RECOMBINANT BACULOVIRUS EXPRESSION SYSTEM, BY ION-EXCHANGE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

    NARCIS (Netherlands)

    DAMHOF, RA; FEIJLBRIEF, M; WELLINGWESTER, S; WELLING, GW

    1994-01-01

    Selective elution of Sendai virus integral membrane proteins by ion-exchange high-performance liquid chromatography (HPIEC) using different detergent concentrations was reported before [S. Welling-Wester, M. Feijlbrief, D.G.A.M. Koedijk, M.A. Braaksma, B.R.K. Douma and G.W. Welling, J. Chromatogr.,

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

    Directory of Open Access Journals (Sweden)

    Stern Stephan T

    2012-06-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

    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.

  9. Development of oligoclonal nanobodies for targeting the tumor-associated glycoprotein 72 antigen

    DEFF Research Database (Denmark)

    Sharifzadeh, Zahra; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad Ali;

    2013-01-01

    The tumor-associated glycoprotein 72 (TAG-72) is a membrane mucin whose over-expression is correlated with advanced tumor stage and increased invasion and metastasis. In this study, we identified a panel of four nanobodies, single variable domains of dromedary heavy-chain antibodies that specific......The tumor-associated glycoprotein 72 (TAG-72) is a membrane mucin whose over-expression is correlated with advanced tumor stage and increased invasion and metastasis. In this study, we identified a panel of four nanobodies, single variable domains of dromedary heavy-chain antibodies...

  10. Cyclosporin a, but not FK506, induces osmotic lysis of pancreas zymogen granules, intra-acinar enzyme release, and lysosome instability by activating K+ channel.

    Science.gov (United States)

    Lee, Wing-Kee; Braun, Matthias; Langelüddecke, Christian; Thévenod, Frank

    2012-05-01

    The immunosuppressant tacrolimus (FK506) has improved pancreas allograft survival compared with cyclosporin A (CsA), possibly because of reduced acute pancreatitis following ischemia-reperfusion injury. Ion permeabilities in zymogen granule (ZG) membranes, including a KCNQ1 K channel, promote hormone-stimulated enzyme secretion. We investigated whether a differential modulation of ZG and lysosomal ion permeabilities and enzyme secretion by CsA/FK506 contributes to pancreatitis. Rat ZGs and lysosomes were isolated by gradient centrifugation, ion permeabilities assayed by osmotic lysis, and single-channel currents recorded in a planar lipid bilayer. Amylase release was measured in permeabilized acini and lysosomal cathepsin B release detected by immunoblotting. CsA (1-10 μM), but not FK506, enhanced ZGs osmotic lysis by selectively increasing K permeability up to 5-fold. Zymogen granule membrane K channels showed ∼2-fold increased single-channel open probability with CsA only. Cyclosporin A selectively increased basal (∼2-fold), but not cholecystokinin-octapeptide (1 nM)-induced amylase secretion in K medium only. Cyclosporin A (5 μM), but not FK506, increased cathepsin B release from lysosomes. Cyclosporin A selectively opens the ZG K channel and induces cathepsin B release from lysosomes, which cause increased in situ lysis of ZGs and may aggravate or fuel acute allograft pancreatitis following hypoxia-reperfusion injury.

  11. Critical role of CAV1/caveolin-1 in cell stress responses in human breast cancer cells via modulation of lysosomal function and autophagy.

    Science.gov (United States)

    Shi, Yin; Tan, Shi-Hao; Ng, Shukie; Zhou, Jing; Yang, Na-Di; Koo, Gi-Bang; McMahon, Kerrie-Ann; Parton, Robert G; Hill, Michelle M; Del Pozo, Miguel A; Kim, You-Sun; Shen, Han-Ming

    2015-01-01

    CAV1 (caveolin 1, caveolae protein, 22kDa) is well known as a principal scaffolding protein of caveolae, a specialized plasma membrane structure. Relatively, the caveolae-independent function of CAV1 is less studied. Autophagy is a process known to involve various membrane structures, including autophagosomes, lysosomes, and autolysosomes for degradation of intracellular proteins and organelles. Currently, the function of CAV1 in autophagy remains largely elusive. In this study, we demonstrate for the first time that CAV1 deficiency promotes both basal and inducible autophagy. Interestingly, the promoting effect was found mainly in the late stage of autophagy via enhancing lysosomal function and autophagosome-lysosome fusion. Notably, the regulatory function of CAV1 in lysosome and autophagy was found to be caveolae-independent, and acts through lipid rafts. Furthermore, the elevated autophagy level induced by CAV1 deficiency serves as a cell survival mechanism under starvation. Importantly, downregulation of CAV1 and enhanced autophagy level were observed in human breast cancer cells and tissues. Taken together, our data reveal a novel function of CAV1 and lipid rafts in breast cancer development via modulation of lysosomal function and autophagy.

  12. Early involvement of lysosome dysfunction in the degeneration of cerebral cortical neurons caused by the lipid peroxidation product 4-hydroxynonenal.

    Science.gov (United States)

    Zhang, Shi; Eitan, Erez; Mattson, Mark P

    2017-03-01

    Free radical-mediated oxidative damage to proteins, lipids, and DNA occurs in neurons during acute brain injuries and in neurodegenerative disorders. Membrane lipid peroxidation contributes to neuronal dysfunction and death, in part by disrupting neuronal ion homeostasis and cellular bioenergetics. Emerging findings suggest that 4-hydroxynonenal (HNE), an aldehyde produced during lipid peroxidation, impairs the function of various proteins involved in neuronal homeostasis. Here we tested the hypothesis that HNE impairs the cellular system that removes damaged proteins and organelles, the autophagy-lysosome pathway in rat primary cortical neurons. We found that HNE, at a concentration that causes apoptosis over a 48-72 h period, increases protein levels of LC3 II and p62 and within 1 and 4 h of exposure, respectively; LC3 II and p62 immunoreactive puncta were observed in the cytoplasm of HNE-treated neurons at 6 h. The extent of up-regulation of p62 and LC3 II in response to HNE was not affected by co-treatment with the lysosome inhibitor bafilomycin A1, suggesting that the effects of HNE on autophagy were secondary to lysosome inhibition. Indeed, we found that neurons exposed to HNE exhibit elevated pH levels, and decreased protein substrate hydrolysis and cathepsin B activity. Neurons exposed to HNE also exhibited the accumulation of K63-linked polyubiquitinated proteins, which are substrates targeted for lysosomal degradation. Moreover, we found that the levels of LAMP2a and constitutively active heat-shock protein 70, and numbers of LAMP2a-positive lysosomes, are decreased in neurons exposed to HNE. Our findings demonstrate that the lipid peroxidation product HNE causes early impairment of lysosomes which may contribute to the accumulation of damaged and dysfunctional proteins and organelles and consequent neuronal death. Because impaired lysosome function is increasingly recognized as an early event in the neuronal death that occurs in neurodegenerative

  13. From biological membranes to biomimetic model membranes

    Directory of Open Access Journals (Sweden)

    Eeman, M.

    2010-01-01

    Full Text Available Biological membranes play an essential role in the cellular protection as well as in the control and the transport of nutrients. Many mechanisms such as molecular recognition, enzymatic catalysis, cellular adhesion and membrane fusion take place into the biological membranes. In 1972, Singer et al. provided a membrane model, called fluid mosaic model, in which each leaflet of the bilayer is formed by a homogeneous environment of lipids in a fluid state including globular assembling of proteins and glycoproteins. Since its conception in 1972, many developments were brought to this model in terms of composition and molecular organization. The main development of the fluid mosaic model was made by Simons et al. (1997 and Brown et al. (1997 who suggested that membrane lipids are organized into lateral microdomains (or lipid rafts with a specific composition and a molecular dynamic that are different to the composition and the dynamic of the surrounding liquid crystalline phase. The discovery of a phase separation in the plane of the membrane has induced an explosion in the research efforts related to the biology of cell membranes but also in the development of new technologies for the study of these biological systems. Due to the high complexity of biological membranes and in order to investigate the biological processes that occur on the membrane surface or within the membrane lipid bilayer, a large number of studies are performed using biomimicking model membranes. This paper aims at revisiting the fundamental properties of biological membranes in terms of membrane composition, membrane dynamic and molecular organization, as well as at describing the most common biomimicking models that are frequently used for investigating biological processes such as membrane fusion, membrane trafficking, pore formation as well as membrane interactions at a molecular level.

  14. Glycoproteins of the carcinoembryonic antigen (CEA) family are expressed in sweat and sebaceous glands of human fetal and adult skin.

    Science.gov (United States)

    Metze, D; Bhardwaj, R; Amann, U; Eades-Perner, A M; Neumaier, M; Wagener, C; Jantscheff, P; Grunert, F; Luger, T A

    1996-01-01

    The carcinoembryonic antigen (CEA) family comprises a group of glycoproteins including the classical CEA, nonspecific cross-reacting antigens (NCA), and biliary glycoprotein (BGP). CEA glycoproteins have been identified in many glandular and mucosal tissues. In view of their putative role in cell adhesion, protein sorting, and signal transduction, CEA glycoproteins are thought to be involved in embryogenesis, architectual integrity, and secretory mechanisms of glandular epithelia. Since there are few data available on the expression of CEA-like proteins in human skin, the aim of this study was to immunohistochemically specify and localize the CEA glycoproteins in cutaneous adult and fetal glands using a panel of well-characterized antibodies. The secretory parts of eccrine sweat glands expressed CEA, NCA-90, and BGP, whereas apocrine glands remained unreactive for CEA glycoproteins. The ductal epithelia of both eccrine and apocrine glands contained CEA and NCA-90. Sebaceous glands were stained for BGP only. Electron microscopy of sweat glands showed CEA glycoprotein expression in cytoplasmic organelles and on microvilli lining the ductal surface. In sebaceous glands, BGP were demonstrated in small vesicles and along the cell membranes of differentiating sebocytes. Fetal development of cutaneous glands was associated with early expression of CEA glycoproteins. Additionally, mice transgenic for human CEA were shown to express CEA in sweat glands. The overall distribution of CEA glycoproteins in cutaneous glands was consistent with that in epithelia of other glandular tissues.

  15. In vitro generation of human cytotoxic lymphocytes by virus. Viral glycoproteins induce nonspecific cell-mediated cytotoxicity without release of interferon.

    Science.gov (United States)

    Casali, P; Sissons, J G; Buchmeier, M J; Oldstone, M B

    1981-09-01

    Purified hemagglutinin and fusion glycoproteins of measles virus either in soluble form or inserted in artifical membranes bind to human peripheral blood lymphocytes and induce cell-mediated cytotoxicity (CMC) in a dose-response fashion. Both autologous and heterologous noninfected target cells are lysed in vitro. The expression of CMC is not inhibited by anti-measles virus antibody added to lymphocytes previously exposed to viral glycoproteins. THe killer lymphocytes are Fc receptor positive, both erythrocyte-rosetting and non-erythrocyte-rosetting, as assessed by both positive and negative selection experiments. The induction of nonspecific CMC by viral glycoproteins either in the soluble state or inserted into artificial membranes could be segregated from the CMC associated with whole virions. First, on kinetics studies, purified viral glycoproteins induced CMC more rapidly than did whole virus. Second, viral glycoprotein-produced response occurred in the absence of detectable release of interferon into the culture medium, whereas CMC activity due to whole virions was associated with interferon release. The fact that purified measles virus glycoproteins integrated into artificial membrane bilayers were as efficient as their soluble counterparts in inducing CMC suggests that the hydrophobic portion of the glycoproteins was not involved in the induction and expression of the lytic activity. Purified glycoproteins from lymphocytic choriomeningitis virus behave similarly, although this virus is unrelated to measles virus. It is inferred that interferon-independent CMC induced by viral glycoproteins might account for some of the biological reactions occurring early in the control of a viral infection.

  16. Lysosomal-associated protein multispanning transmembrane 5 gene (LAPTM5 is associated with spontaneous regression of neuroblastomas.

    Directory of Open Access Journals (Sweden)

    Jun Inoue

    Full Text Available BACKGROUND: Neuroblastoma (NB is the most frequently occurring solid tumor in children, and shows heterogeneous clinical behavior. Favorable tumors, which are usually detected by mass screening based on increased levels of catecholamines in urine, regress spontaneously via programmed cell death (PCD or mature through differentiation into benign ganglioneuroma (GN. In contrast, advanced-type NB tumors often grow aggressively, despite intensive chemotherapy. Understanding the molecular mechanisms of PCD during spontaneous regression in favorable NB tumors, as well as identifying genes with a pro-death role, is a matter of urgency for developing novel approaches to the treatment of advanced-type NB tumors. PRINCIPAL FINDINGS: We found that the expression of lysosomal associated protein multispanning transmembrane 5 (LAPTM5 was usually down-regulated due to DNA methylation in an NB cell-specific manner, but up-regulated in degenerating NB cells within locally regressing areas of favorable tumors detected by mass-screening. Experiments in vitro showed that not only a restoration of its expression but also the accumulation of LAPTM5 protein, was required to induce non-apoptotic cell death with autophagic vacuoles and lysosomal destabilization with lysosomal-membrane permeabilization (LMP in a caspase-independent manner. While autophagy is a membrane-trafficking pathway to degrade the proteins in lysosomes, the LAPTM5-mediated lysosomal destabilization with LMP leads to an interruption of autophagic flux, resulting in the accumulation of immature autophagic vacuoles, p62/SQSTM1, and ubiqitinated proteins as substrates of autophagic degradation. In addition, ubiquitin-positive inclusion bodies appeared in degenerating NB cells. CONCLUSIONS: We propose a novel molecular mechanism for PCD with the accumulation of autophagic vacuoles due to LAPTM5-mediated lysosomal destabilization. LAPTM5-induced cell death is lysosomal cell death with impaired autophagy

  17. Generation of H9 T-cells stably expressing a membrane-bound form of the cytoplasmic tail of the Env-glycoprotein: lack of transcomplementation of defective HIV-1 virions encoding C-terminally truncated Env

    Directory of Open Access Journals (Sweden)

    Bosch Valerie

    2006-05-01

    Full Text Available Abstract H9-T-cells do not support the replication of mutant HIV-1 encoding Env protein lacking its long cytoplasmic C-terminal domain (Env-CT. Here we describe the generation of a H9-T-cell population constitutively expressing the HIV-1 Env-CT protein domain anchored in the cellular membrane by it homologous membrane-spanning domain (TMD. We confirmed that the Env-TMD-CT protein was associated with cellular membranes, that its expression did not have any obvious cytotoxic effects on the cells and that it did not affect wild-type HIV-1 replication. However, as measured in both a single-round assay as well as in spreading infections, replication competence of mutant pNL-Tr712, lacking the Env-CT, was not restored in this H9 T-cell population. This means that the Env-CT per se cannot transcomplement the replication block of HIV-1 virions encoding C-terminally truncated Env proteins and suggests that the Env-CT likely exerts its function only in the context of the complete Env protein.

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

    Science.gov (United States)

    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.

  19. Evaluation of the expression of P-glycoprotein in propoxur-resistant Caco-2 cells.

    Directory of Open Access Journals (Sweden)

    Shabnam Yazdian

    2014-10-01

    Full Text Available There is a great concern about the effect of propoxur, as one of the more common N-methyl carbamate pesticides, on human health due to its extensive use in agricultural and non-agricultural applications. Caco-2 cells became resistant to propoxur, and the resistance was confirmed through MTT assay. Then the cell membrane integrity and P-glycoprotein expression were measured by LDH assay and western blot analysis, respectively and compared to the parent cells.  Contrary to what was expected, the expression of P-glycoprotein in propoxur resistant cells was lower than parent cells.This study indicates that the resistance to propoxur may not be related to P-glycoprotein expression directly, since P-glycoprotein expression has decreased in these cells.

  20. Distribution of surface glycoproteins on influenza A virus determined by electron cryotomography.

    Science.gov (United States)

    Wasilewski, Sebastian; Calder, Lesley J; Grant, Tim; Rosenthal, Peter B

    2012-12-07

    We use electron cryotomography to reconstruct virions of two influenza A H3N2 virus strains. The maps reveal the structure of the viral envelope containing hemagglutinin (HA) and neuraminidase (NA) glycoproteins and the virus interior containing a matrix layer and an assembly of ribonucleoprotein particles (RNPs) that package the genome. We build a structural model for the viral surface by locating copies of the X-ray structure of the HA ectodomain into density peaks on the virus surface. We calculate inter-glycoprotein distances and the fractional volume occupied by glycoproteins. The models suggest that for typical HA densities on virus, Fabs can bind to epitopes on the HA stem domain. The models also show how membrane curvature may influence the number of glycoproteins that can simultaneously interact with a target surface of receptors.

  1. 抗血小板膜糖蛋白Ib单克隆抗体的制备、鉴定及其功能的初步研究%Development,identification and function assay of monoclonal antibody against platelet membrane glycoprotein Ib

    Institute of Scientific and Technical Information of China (English)

    谷颖; 季顺东; 赵益明; 沈飞; 阮长耿

    2011-01-01

    AIM: To prepare and identify a monoclonal antibody (mAb) against human platelet glycoprotein Ib and make its application. METHODS: BALB/c mice were immunized with human platelets washed, and the spleen cells of them were fused with myloma cells. A hybridoma cell was screened by indirect ELISA and cloned, and the mAb were purified from the ascites of mice. Ig subclass was analysed by double immunodiffusion. The antigen recognized by monoclonal antibody was identified by flow cytometry and radioimmunoassy, respectively. The inhibition of mAb on plasma von Willebrand factor ristocetin cofactor activity (vWF: Rcof) was investigated by enzyme linked immunosorbent assay (ELISA). RESULTS: A mudne mAb against human platelet membrane glycoprotein (GP) lb was developed and denominated as SZ-151. SZ-151 belonged to IgG1 subclass and its titer in ascites was 1:20 000. Flow cytometry and radioimmunoassy showed that the antigen recognized by monoclonal antibody SZ-151 was platelet membrane GPIb. ELISA showed that SZ-151 did not inhibit plasma von Willebrand factor ristocetin cofactor activity. CONCLUSION: A mAb, SZ-151 against platelet glycoprotein Ib was developed, which could be useful in assays of plasma von Willebrand factor ristocetin cofactor activity(vWF: Rcof)and can be used for diagnose patients with vWD.%目的:制备抗人血小板膜糖蛋白Ib(GPIb)单克隆抗体(mAb),并进行生化鉴定与初步探讨其临床应用.方法:采用血小板免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞融合.经间接ELISA法筛选和克隆化培养,获得1株抗人GPIb mAb的杂交瘤细胞,纯化其腹水获得mAb;免疫双扩散鉴定其抗体亚类;流式细胞术和免疫放射法鉴定其识别的抗原;ELISA法检测该抗体对血浆血管性血友病因子(vWF)的瑞斯托霉素辅因子活性的影响.结果:成功制备了1株抗人GPIb mAb,命名为SZ-151.ELISA法测定其腹水效价为1:20 000,免疫双扩散鉴定其为IgG1亚类.免疫放射法和流式

  2. Recent Progress in Electrochemical Biosensors for Glycoproteins.

    Science.gov (United States)

    Akiba, Uichi; Anzai, Jun-Ichi

    2016-12-01

    This review provides an overview of recent progress in the development of electrochemical biosensors for glycoproteins. Electrochemical glycoprotein sensors are constructed by combining metal and carbon electrodes with glycoprotein-selective binding elements including antibodies, lectin, phenylboronic acid and molecularly imprinted polymers. A recent trend in the preparation of glycoprotein sensors is the successful use of nanomaterials such as graphene, carbon nanotube, and metal nanoparticles. These nanomaterials are extremely useful for improving the sensitivity of glycoprotein sensors. This review focuses mainly on the protocols for the preparation of glycoprotein sensors and the materials used. Recent improvements in glycoprotein sensors are discussed by grouping the sensors into several categories based on the materials used as recognition elements.

  3. Recent Progress in Electrochemical Biosensors for Glycoproteins

    Directory of Open Access Journals (Sweden)

    Uichi Akiba

    2016-12-01

    Full Text Available This review provides an overview of recent progress in the development of electrochemical biosensors for glycoproteins. Electrochemical glycoprotein sensors are constructed by combining metal and carbon electrodes with glycoprotein-selective binding elements including antibodies, lectin, phenylboronic acid and molecularly imprinted polymers. A recent trend in the preparation of glycoprotein sensors is the successful use of nanomaterials such as graphene, carbon nanotube, and metal nanoparticles. These nanomaterials are extremely useful for improving the sensitivity of glycoprotein sensors. This review focuses mainly on the protocols for the preparation of glycoprotein sensors and the materials used. Recent improvements in glycoprotein sensors are discussed by grouping the sensors into several categories based on the materials used as recognition elements.

  4. HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.

    Science.gov (United States)

    Albecka, Anna; Laine, Romain F; Janssen, Anne F J; Kaminski, Clemens F; Crump, Colin M

    2016-01-01

    Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment.

  5. Cationic lipids delay the transfer of plasmid DNA to lysosomes.

    Science.gov (United States)

    Wattiaux, R; Jadot, M; Laurent, N; Dubois, F; Wattiaux-De Coninck, S

    1996-10-14

    Plasmid 35S DNA, naked or associated with different cationic lipid preparations was injected to rats. Subcellular distribution of radioactivity in the liver one hour after injection, was established by centrifugation methods. Results show that at that time, 35S DNA has reached lysosomes. On the contrary, when 35S DNA was complexed with lipids, radioactivity remains located in organelles whose distribution after differential and isopycnic centrifugation, is clearly distinct from that of arylsulfatase, lysosome marker enzyme. Injection of Triton WR 1339, a specific density perturbant of lysosomes, four days before 35S DNA injection causes a density decrease of radioactivity bearing structures, apparent one hour after naked 35S DNA injection but visible only after more than five hours, when 35S DNA associated with a cationic lipid is injected. These observations show that cationic lipids delay the transfer to lysosomes, of plasmid DNA taken up by the liver.

  6. LINGO-1 promotes lysosomal degradation of amyloid-β protein precursor.

    Science.gov (United States)

    de Laat, Rian; Meabon, James S; Wiley, Jesse C; Hudson, Mark P; Montine, Thomas J; Bothwell, Mark

    2015-01-01

    Sequential proteolytic cleavages of amyloid-β protein precursor (AβPP) by β-secretase and γ-secretase generate amyloid β (Aβ) peptides, which are thought to contribute to Alzheimer's disease (AD). Much of this processing occurs in endosomes following endocytosis of AβPP from the plasma membrane. However, this pathogenic mode of processing AβPP may occur in competition with lysosomal degradation of AβPP, a common fate of membrane proteins trafficking through the endosomal system. Following up on published reports that LINGO-1 binds and promotes the amyloidogenic processing of AβPP we have examined the consequences of LINGO-1/AβPP interactions. We report that LINGO-1 and its paralogs, LINGO-2 and LINGO-3, decrease processing of AβPP in the amyloidogenic pathway by promoting lysosomal degradation of AβPP. We also report that LINGO-1 levels are reduced in AD brain, representing a possible pathogenic mechanism stimulating the generation of Aβ peptides in AD.

  7. LINGO-1 promotes lysosomal degradation of amyloid-β protein precursor

    Directory of Open Access Journals (Sweden)

    Rian de Laat

    2015-03-01

    Full Text Available Sequential proteolytic cleavages of amyloid-β protein precursor (AβPP by β-secretase and γ-secretase generate amyloid β (Aβ peptides, which are thought to contribute to Alzheimer's disease (AD. Much of this processing occurs in endosomes following endocytosis of AβPP from the plasma membrane. However, this pathogenic mode of processing AβPP may occur in competition with lysosomal degradation of AβPP, a common fate of membrane proteins trafficking through the endosomal system. Following up on published reports that LINGO-1 binds and promotes the amyloidogenic processing of AβPP we have examined the consequences of LINGO-1/AβPP interactions. We report that LINGO-1 and its paralogs, LINGO-2 and LINGO-3, decrease processing of AβPP in the amyloidogenic pathway by promoting lysosomal degradation of AβPP. We also report that LINGO-1 levels are reduced in AD brain, representing a possible pathogenic mechanism stimulating the generation of Aβ peptides in AD.

  8. WO3/Pt nanoparticles promote light-induced lipid peroxidation and lysosomal instability within tumor cells

    Science.gov (United States)

    Clark, Andrea J.; Petty, Howard R.

    2016-02-01

    Although metal-metal oxide nanoparticles have attracted considerable interest as catalysts, they have attracted little interest in nanomedicine. This is likely due to the fact that metal oxide semiconductors generally require biologically harmful ultraviolet excitation. In contrast, this study focuses upon WO3/Pt nanoparticles, which can be excited by visible light. To optimize the nanoparticles’ catalytic performance, platinization was performed at alkaline pH. These nanoparticles destroyed organic dyes, consumed dissolved oxygen and produced hydroxyl radicals. 4T1 breast cancer cells internalized WO3/Pt nanoparticles within the membrane-bound endo-lysosomal compartment as shown by electron and fluorescence microscopy. During visible light exposure, but not in darkness, WO3/Pt nanoparticles manufacture reactive oxygen species, promote lipid peroxidation, and trigger lysosomal membrane disruption. As cells of the immune system degrade organic molecules, produce reactive oxygen species, and activate the lipid peroxidation pathway within target cells, these nanoparticles mimic the chemical attributes of immune effector cells. These biomimetic nanoparticles should become useful in managing certain cancers, especially ocular cancer.

  9. Inhibitory effect of mTOR activator MHY1485 on autophagy: suppression of lysosomal fusion.

    Directory of Open Access Journals (Sweden)

    Yeon Ja Choi

    Full Text Available Autophagy is a major degradative process responsible for the disposal of cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During the autophagic process, cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm and finally fuse with lysosomes. In the present study, we investigated the inhibition of autophagy by a synthesized compound, MHY1485, in a culture system by using Ac2F rat hepatocytes. Autophagic flux was measured to evaluate the autophagic activity. Autophagosomes were visualized in Ac2F cells transfected with AdGFP-LC3 by live-cell confocal microscopy. In addition, activity of mTOR, a major regulatory protein of autophagy, was assessed by western blot and docking simulation using AutoDock 4.2. In the result, treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy. The levels of p62 and beclin-1 did not show significant change after treatment with MHY1485. Decreased co-localization of autophagosomes and lysosomes in confocal microscopic images revealed the inhibitory effect of MHY1485 on lysosomal fusion during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time-dependent manner. Furthermore, MHY1485 induced mTOR activation and correspondingly showed a higher docking score than PP242, a well-known ATP-competitive mTOR inhibitor, in docking simulation. In conclusion, MHY1485 has an inhibitory effect on the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. MHY1485 also induces mTOR activity, providing a possibility for another regulatory mechanism of autophagy by the MHY compound. The significance of this study is the finding of a novel

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

    Science.gov (United States)

    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.

  11. 川崎病患儿血小板膜糖蛋白Ibα基因多态性分析%Investigation of platelet membrane glycoprotein Ibα gene polymorphisms in children with Kawasaki disease

    Institute of Scientific and Technical Information of China (English)

    李卓颖; 黄麟; 杨作成

    2013-01-01

    Objective To investigate the association between platelet glycoprotein Ibα (GPIbα) gene HPA-2a/b polymorphisms and the risk of Kawasaki disease (KD) and that complicated with coronary artery lesion (CAL).Methods A total of 30 patients with KD and 60 healthy controls were genotyped by polymerase chain reaction-restriction fragment length polymorphism and agarose gel electrophoresis for the HPA-2a/b polymorphism in GPIbα gene.Results For HPA-2a/b polymorphism in GPIbα gene,there was only genotype TC and CC of HPA-2a/b polymorphism in GPIbα gene in children with KD and healthy controls,and genotype TF was not found in both groups.There were no significant differences between KD patients and the controls in genotype frequencies of CC,TC and TT and allele frequencies of C and T (x2 =0.052,0.048,all P > 0.05) ; also there was no significant difference between KD patients with CAL and that without CAL in genotype and allele frequencies (x2 =2.672,2.481,all P > 0.05).Conclusion No association is found between HPA-2a/b polymorphism in GPIbα gene and the risk of KD or its complication of CAL in this study.%目的 研究血小板膜糖蛋白Ibα(GPIbα)基因HPA-2a/b多态性与KD发病及并发冠状动脉损伤(CAL)之间的关系.方法 应用聚合酶链反应-限制性内切酶片断长度多态性分析技术结合琼脂糖凝胶电泳技术,检测30例KD患儿和60例健康对照组儿童GPIbα基因HPA-2a/b多态性位点的基因型和等位基因分布.结果 本组KD病例和健康对照组儿童GPIbα基因HPA-2a/b的基因型均只有TC型和CC型,而未发现TT型.KD组GPIbα基因HPA-2a/b多态性的CC、TC、TT基因型分布频率和C、T等位基因频率与健康对照组比较差异均无统计学意义(x2=0.052、0.048,P均>0.05),KD组中合并CAL组与无CAL组基因型分布频率和等位基因频率比较差异亦无统计学意义(x2=2.672、2.481,P均>0.05).结论 GPIbα基因的HPA-2a/b多态性与KD及其CAL的发生均无明显相关性.

  12. Lysosome-associated protein 1 (LAMP-1) and lysosome-associated protein 2 (LAMP-2) in a larger family carrier of Fabry disease.

    Science.gov (United States)

    Pereira, Ester M; do Monte, Semiramis J H; do Nascimento, Fernando F; de Castro, Jose A F; Sousa, Jackeline L M; Filho, Henrique C S A L C; da Silva, Raimundo N; Labilloy, Anatália; Monte Neto, José T; da Silva, Adalberto S

    2014-02-15

    This study investigated the potential relationship between the expression levels of lysosome-associated membrane proteins (LAMP) 1 and 2 and responses to enzyme replacement therapy (ERT) in the members of a single family with Fabry disease (FD). LAMP levels were assessed by flow cytometry in leukocytes from 17 FD patients who received an eight-month course of ERT course and 101 healthy individuals. We found that phagocytic cells from the FD patients had higher expression levels of both LAMP-1 and LAMP-2, relative to the levels in phagocytes from the healthy controls (p=0.001). Furthermore, the LAMP-1 and LAMP-2 levels in phagocytes from the FD carriers continuously decreased with ERT administration to reach levels similar to those in healthy controls. We suggest that LAMP-1 and LAMP-2 could be used as additional markers with which to assess ERT effectiveness in FD.

  13. Pathogenesis of mucosal injury in the blind loop syndrome. Brush border enzyme activity and glycoprotein degradation.

    Science.gov (United States)

    Jonas, A; Flanagan, P R; Forstner, G G

    1977-12-01

    The effect of intestinal bacterial over-growth on brush border hydrolases and brush border glycoproteins was studied in nonoperated control rats, control rats with surgically introduced jejunal self-emptying blind loops, and rats with surgically introduced jejunal self-filling blind loops. Data were analyzed from blind loop segments, segments above and below the blind loops, and three corresponding segments in the nonoperated controls. Rats with self-filling blind loops had significantly greater fat excretion than controls and exhibited significantly lower conjugated:free bile salt ratios in all three segments. Maltase, sucrase, and lactase activities were significantly reduced in homogenates and isolated brush borders from the self-filling blind loop, but alkaline phosphatase was not affected. The relative degradation rate of homogenate and brush border glycoproteins was assessed by a double-isotope technique involving the injection of d-[6-(3)H]glucosamine 3 h and d-[U-(14)C]glucosamine 19 h before sacrifice, and recorded as a (3)H:(14)C ratio. The relative degradation rate in both homogenate and brush border fractions was significantly greater in most segments from rats with self-filling blind loops. In the upper and blind loop segments from rats with self-filling blind loops, the (3)H:(14)C ratios were higher in the brush border membrane than in the corresponding homogenates, indicating that the increased rates of degradation primarily involve membrane glycoproteins. Incorporation of d-[6-(3)H]glucosamine by brush border glycoproteins was not reduced in rats with self-filling blind loops, suggesting that glycoprotein synthesis was not affected. Polyacrylamide gel electrophoresis of brush border glycoproteins from the contaminated segments indicated that the large molecular weight glycoproteins, which include many of the surface hydrolases, were degraded most rapidly. Brush border maltase, isolated by immunoprecipitation, had (3)H:(14)C ratios characteristic of

  14. Expression of platelet membrane glycoproteins and their clinical significance in patients infected by severe fever with thrombocytopenia syndrome virus%新型布尼亚病毒感染患者血小板膜糖蛋白的表达及临床意义

    Institute of Scientific and Technical Information of China (English)

    杨海波; 李世波; 杨序春; 庄晓玲; 黄燕燕; 周吉航; 杨志强; 周世权; 邓杰

    2013-01-01

    目的 探讨新型布尼亚病毒感染患者血小板膜糖蛋白表达的意义.方法 收集2011年5月至2013年5月浙江省舟山医院收治的并发多器官功能障碍综合征(MODS)(8例)和未并发MODS(20例)新型布尼亚病毒感染患者.对两组患者血小板进行常规检测并比较,检测两组患者二磷酸腺苷(ADP)诱导前后的血小板膜糖蛋白(CD41a、CD42a、CD61、CD62p)的阳性表达率和平均荧光强度(MFI),采用独立样本t检验比较两组间的差异.结果 MODS组与非MODS组ADP诱导前比较,血小板的计数差异无统计学意义(t=0.139,P>0.05),但平均体积偏大(t=6.417,P<0.01),血小板分布宽度大(t =9.664,P<0.01);MODS组患者血小板膜糖蛋白阳性表达率和MFI较非MODS组患者显著增高(CD41a:t=14.486和49.419,P<0.01;CD42a:t =41.693和58.160,P<0.01;CD61:t=15.452和19.251,P<0.01;CD62p:t 18.230和24.258,P<0.05);经ADP诱导活化处理后MODS组患者血小板膜糖蛋白阳性表达率和MFI未出现明显变化(CD41a:t=1.364和1.237,P>0.05;CD42a:t=1.247和1.448,P>0.05; CD61:t=1.258和1.354,P> 0.05;CD62p:t=1.213和1.147,P>0.05),而非MODS组患者经ADP诱导活化处理后血小板膜糖蛋白阳性表达率和MFI均明显升高(CD41a:t=15.891和18.417,P<0.01;CD42a:t=54.367和14.217,P<0.01;CD61:t=16.257和21.348,P<0.01;CD62p:t =58.268和18.145,P<0.01).结论 新型布尼亚病毒感染并发MODS患者血小板膜糖蛋白阳性表达率和MFI较轻症患者高,而且持续活化表达能力极低,血小板膜糖蛋白的检测可以作为判断新布尼亚病毒感染患者疾病严重程度的重要指标之一.%Objective To investigate the expression of platelet membrane glycoproteins in patients infected with severe fever with thrombocytopenia syndrome virus (SFTSV).Methods Twenty-eight SFTSV infected patients,including 8 cases with multiple organ dysfunction syndrome (MODS) and 20 without MODS,were collected from Zhoushan Hospital during May 2011 and

  15. Localization of the glycoprotein Cb42 in larvae of the screwworm fly Chrysomya bezziana (Diptera: Calliphoridae

    Directory of Open Access Journals (Sweden)

    Creig Eisemanni

    2000-10-01

    Full Text Available The glycoprotein Cb-peritrophin-42 was localized in third instar larvae of Chrysomya bezziana using primary antibodies raised against a non-glycosylated bacterial recombinant form of this protein. Both immunofluorescent antibody techniques on unfixed whole mounts of gut tissues and immunogold electron microscopy techniques on ultra-thin sections of fixed and embedded tissues were employed. The protein was shown to be exposed over the whole of both surfaces of the peritrophic membrane and to occur throughout its thickness. Immunogold labelling indicated that Cb-peritrophin-42 was expressed in the peritrophic membrane-secreting cells of the cardia, a specialized peritrophic membrane-forming organ situated at the junction of the foregut and midgut. The accessibility of Cb-peritrophin-42 present in intact peritrophic membrane to the primary antibodies used in the immunofluorescent antibody localization indicates that this glycoprotein is a potential molecular target for vaccination of host animals against larvae of Chrysomya bezziana.

  16. Redistribution of cathepsin B activity from the endosomal-lysosomal pathway in chick intestine within 3 min of calcium absorption.

    Science.gov (United States)

    Nemere, I; Norman, A W

    1991-06-01

    Earlier work has suggested that calcium-containing lysosomes are involved in 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-stimulated intestinal absorption of the divalent cation. In the present report immunofluorescent labelling studies on fixed frozen sections of chick intestine were undertaken to determine whether lysosomes could respond to calcium transport conditions in less than 5 min. Tissue prepared from vitamin D-deficient chicks dosed with vehicle or 1.3 nmol of 1,25(OH)2D3 15 h prior to use was immunofluorescently labelled for cathepsin B, a lysosomal protease. In the absence of calcium absorption, punctate staining was found in the region below the terminal web, and more diffusely in the cytoplasm. The intensity of staining was noticeably greater in sections from 1,25(OH)2D3-treated than control chicks. In sections prepared after 3 min of calcium absorption, cathepsin B staining was localized near the basal and lateral membranes of the epithelial cells. After 30 min of transport, the protease was found in the villus core regardless of vitamin D status; however, immunoreactivity within the epithelial cells of 1,25(OH)2D3-treated chick intestine had returned to pretransport intensity, whereas that of controls had not. To further investigate the specificity of the cathepsin B antibody, the intracellular compartmentalization of the protease was determined by biochemical methods. Using dosing procedures and calcium transport times equivalent to those for the immunofluorescent studies mucosae were collected by scraping, homogenized, and subcellular fractions prepared by a combination of differential and Percoll gradient centrifugation. In the absence of calcium transport, cathepsin B-specific activity was enhanced in whole homogenates, endocytic vesicles, and a lysosomal fraction prepared from intestinal epithelium of 1,25(OH)2D3-treated chicks, relative to vitamin D-deficient controls. After 3 min of calcium absorption, a profound (approximately 4-fold) decrease in

  17. Stabilization of HIV-1 envelope glycoprotein trimers to induce neutralizing antibodies

    NARCIS (Netherlands)

    de Taeye, S.W.

    2017-01-01

    HIV-1 has evolved various tricks to prevent the development of a potent humoral immune response. The only target for neutralizing antibodies (NAbs) is the HIV-1 envelope glycoprotein (Env), which is the sole viral protein embedded in the viral membrane. It consists of three gp41 subunits and three g

  18. Effect of luteolin on glycoproteins metabolism in 1, 2-dimethylhydrazine induced experimental colon carcinogenesis

    Directory of Open Access Journals (Sweden)

    Manju Vaiyapuri

    2008-12-01

    carcinogenesis. Thus, the present study indicates that luteolin has protected the cell surface and maintained the structural integrity of the cell membranes during DMH induced colon carcinogenesis. Keywords: Colon cancer, 1, 2-dimethylhydrazine, luteolin, glycoproteins Received: 23 January 2009 / Received in revised form: 17 February 2009, Accepted: 28 February 2009, Published online: 3 March 2009

  19. Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer

    NARCIS (Netherlands)

    Walls, Alexandra C; Tortorici, M Alejandra; Bosch, Berend-Jan; Frenz, Brandon; Rottier, Peter J M; DiMaio, Frank; Rey, Félix A; Veesler, David

    2016-01-01

    The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion f

  20. Structure of Acidic pH Dengue Virus Showing the Fusogenic Glycoprotein Trimers

    NARCIS (Netherlands)

    Zhang, Xinzheng; Sheng, Ju; Austin, S. Kyle; Hoornweg, Tabitha E.; Smit, Jolanda M.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G.

    2015-01-01

    Flaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions, the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid membrane in host cells to initiate viral fusion and nucleocapsid penetration into the cytoplasm. However,

  1. Calpain-controlled detachment of major glycoproteins from the cytoskeleton regulates adhesive properties of activated phosphatidylserine-positive platelets.

    Science.gov (United States)

    Artemenko, Elena O; Yakimenko, Alena O; Pichugin, Alexey V; Ataullakhanov, Fazly I; Panteleev, Mikhail A

    2016-02-15

    In resting platelets, adhesive membrane glycoproteins are attached to the cytoskeleton. On strong activation, phosphatidylserine(PS)-positive and -negative platelet subpopulations are formed. Platelet activation is accompanied by cytoskeletal rearrangement, although the glycoprotein attachment status in these two subpopulations is not clear. We developed a new, flow cytometry-based, single-cell approach to investigate attachment of membrane glycoproteins to the cytoskeleton in cell subpopulations. In PS-negative platelets, adhesive glycoproteins integrin αIIbβ3, glycoprotein Ib and, as shown for the first time, P-selectin were associated with the cytoskeleton. In contrast, this attachment was disrupted in PS-positive platelets; it was retained to some extent only in the small convex regions or 'caps'. It correlated with the degradation of talin and filamin observed only in PS-positive platelets. Calpain inhibitors essentially prevented the disruption of membrane glycoprotein attachment in PS-positive platelets, as well as talin and filamin degradation. With the suggestion that detachment of glycoproteins from the cytoskeleton may affect platelet adhesive properties, we investigated the ability of PS-positive platelets to resist shear-induced breakaway from the immobilized fibrinogen. Shear rates of 500/s caused PS-positive platelet breakaway, but their adhesion stability increased more than 10-fold after pretreatment of the platelets with calpain inhibitor. In contrast, the ability of PS-positive platelets to adhere to immobilized von Willebrand's factor at 100/s was low, but this was not affected by the preincubation of platelets with a calpain inhibitor. Our data suggest that calpain-controlled detachment of membrane glycoproteins is a new mechanism that is responsible for the loss of ability of the procoagulant platelets to resist detachment from thrombi by high shear stress.

  2. Lysosomal storage disorders: Molecular basis and laboratory testing

    Directory of Open Access Journals (Sweden)

    Filocamo Mirella

    2011-03-01

    Full Text Available Abstract Lysosomal storage disorders (LSDs are a large group of more than 50 different inherited metabolic diseases which, in the great majority of cases, result from the defective function of specific lysosomal enzymes and, in cases, of non-enzymatic lysosomal proteins or non-lysosomal proteins involved in lysosomal biogenesis. The progressive lysosomal accumulation of undegraded metabolites results in generalised cell and tissue dysfunction, and, therefore, multi-systemic pathology. Storage may begin during early embryonic development, and the clinical presentation for LSDs can vary from an early and severe phenotype to late-onset mild disease. The diagnosis of most LSDs--after accurate clinical/paraclinical evaluation, including the analysis of some urinary metabolites--is based mainly on the detection of a specific enzymatic deficiency. In these cases, molecular genetic testing (MGT can refine the enzymatic diagnosis. Once the genotype of an individual LSD patient has been ascertained, genetic counselling should include prediction of the possible phenotype and the identification of carriers in the family at risk. MGT is essential for the identification of genetic disorders resulting from non-enzymatic lysosomal protein defects and is complementary to biochemical genetic testing (BGT in complex situations, such as in cases of enzymatic pseudodeficiencies. Prenatal diagnosis is performed on the most appropriate samples, which include fresh or cultured chorionic villus sampling or cultured amniotic fluid. The choice of the test--enzymatic and/or molecular--is based on the characteristics of the defect to be investigated. For prenatal MGT, the genotype of the family index case must be known. The availability of both tests, enzymatic and molecular, enormously increases the reliability of the entire prenatal diagnostic procedure. To conclude, BGT and MGT are mostly complementary for post- and prenatal diagnosis of LSDs. Whenever genotype

  3. Involvement of oxidative stress and mitochondrial/lysosomal cross-talk in olanzapine cytotoxicity in freshly isolated rat hepatocytes.

    Science.gov (United States)

    Eftekhari, Aziz; Azarmi, Yadollah; Parvizpur, Alireza; Eghbal, Mohammad Ali

    2016-01-01

    1. Olanzapine (OLZ) is a widely used atypical antipsychotic agent for the treatment of schizophrenia and other disorders. Serious hepatotoxicity and elevated liver enzymes have been reported in patients receiving OLZ. However, the cellular and molecular mechanisms of the OLZ hepatotoxicity are unknown. 2. In this study, the cytotoxic effect of OLZ on freshly isolated rat hepatocytes was assessed. Our results showed that the cytotoxicity of OLZ in hepatocytes is mediated by overproduction of reactive oxygen species (ROS), mitochondrial potential collapse, lysosomal membrane leakiness, GSH depletion and lipid peroxidation preceding cell lysis. All the aforementioned OLZ-induced cellular events were significantly (p lysosomal involvement following the initiation of oxidative stress in hepatocytes.

  4. Inspired by nonenveloped viruses escaping from endo-lysosomes: a pH-sensitive polyurethane micelle for effective intracellular trafficking

    Science.gov (United States)

    Song, Nijia; Zhou, Lijuan; Li, Jiehua; Pan, Zhicheng; He, Xueling; Tan, Hong; Wan, Xinyuan; Li, Jianshu; Ran, Rong; Fu, Qiang

    2016-03-01

    A multifunctional drug delivery system (DDS) for cancer therapy still faces great challenges due to multiple physiological barriers encountered in vivo. To increase the efficacy of current cancer treatment a new anticancer DDS mimicking the response of nonenveloped viruses, triggered by acidic pH to escape endo-lysosomes, is developed. Such a smart DDS is self-assembled from biodegradable pH-sensitive polyurethane containing hydrazone bonds in the backbone, named pHPM. The pHPM exhibits excellent micellization characteristics and high loading capacity for hydrophobic chemotherapeutic drugs. The responses of the pHPM in acidic media, undergoing charge conversion and hydrophobic core exposure, resulting from the detachment of the hydrophilic polyethylene glycol (PEG) shell, are similar to the behavior of a nonenveloped virus when trapped in acidic endo-lysosomes. Moreover, the degradation mechanism was verified by gel permeation chromatography (GPC). The endo-lysosomal membrane rupture induced by these transformed micelles is clearly observed by transmission electron microscopy. Consequently, excellent antitumor activity is confirmed both in vitro and in vivo. The results verify that the pHPM could be a promising new drug delivery tool for the treatment of cancer and other diseases.A multifunctional drug delivery system (DDS) for cancer therapy still faces great challenges due to multiple physiological barriers encountered in vivo. To increase the efficacy of current cancer treatment a new anticancer DDS mimicking the response of nonenveloped viruses, triggered by acidic pH to escape endo-lysosomes, is developed. Such a smart DDS is self-assembled from biodegradable pH-sensitive polyurethane containing hydrazone bonds in the backbone, named pHPM. The pHPM exhibits excellent micellization characteristics and high loading capacity for hydrophobic chemotherapeutic drugs. The responses of the pHPM in acidic media, undergoing charge conversion and hydrophobic core

  5. CUP-5, the C. elegans ortholog of the mammalian lysosomal channel protein MLN1/TRPML1, is required for proteolytic degradation in autolysosomes.

    Science.gov (United States)

    Sun, Tao; Wang, Xingwei; Lu, Qun; Ren, Haiyan; Zhang, Hong

    2011-11-01

    The process of macroautophagy (herein referred to as autophagy) involves the formation of a closed double-membrane structure, called the autophagosome, and its subsequent fusion with lysosomes to form an autolysosome. Lysosomes are regenerated from autolysosomes after degradation of the sequestrated materials. In this study, we showed that mutations in cup-5, encoding the C. elegans Mucolipin 1 homolog, cause defects in the autophagy pathway. In cup-5 mutants, a variety of autophagy substrates accumulate in enlarged vacuoles that display characteristics of late endosomes and lysosomes, indicating defective proteolytic degradation in autolysosomes. We further revealed that lysosomes in coelomocytes (scavenger cells located in the body cavity) are smaller in size and more numerous in mutants with loss of autophagy activity. Furthermore, the enlarged vacuole accumulation abnormality and embryonic lethality of cup-5 mutants are partially suppressed by reduced autophagy activity. Our results indicate that the basal constitutive level of autophagy activity regulates the size and number of lysosomes and provides insights into the molecular mechanisms underlying mucolipidosis type IV disease.

  6. The importance of drug-transporting P-glycoproteins in toxicology.

    Science.gov (United States)

    van Tellingen, O

    2001-03-31

    The importance of specific transport in toxicology is becoming increasingly clear and the work on P-glycoprotein has certainly been a major contribution to these growing insights. P-Glycoproteins were discovered by their ability to confer multidrug resistance in mammalian tumour cells. They are localised in the cell membrane where they actively extrude a wide range of compounds including many anti-cancer drugs from the cell. Besides in tumour cells, drug-transporting P-glycoproteins are also expressed in a polarised fashion in normal tissues that perform an excretory or barrier function, such as the liver, kidneys, intestines, brain endothelial cells. Based on this expression profile, it has been proposed that P-glycoproteins are important in protecting the host by reducing exposure to xenobiotics. Further studies with P-glycoprotein knockout mice have clearly established this protective function. In general, the clearance of substrate drugs is lower in knockout mice due to a diminished hepatobiliary excretion, direct intestinal excretion and/or increased enterohepatic cycling. Moreover, their uptake in sanctuary sites, such as the brain or the foetus, was profoundly higher in P-glycoprotein knockout mice, as was the uptake of drugs from the gastro-intestinal tract into the systemic circulation following oral ingestion. These results clearly highlight the impact that transport proteins can play in toxicology.

  7. Molecular docking studies with rabies virus glycoprotein to design viral therapeutics

    Directory of Open Access Journals (Sweden)

    Tomar N

    2010-01-01

    Full Text Available The genome of rabies virus encodes five proteins; the nucleoprotein, the phosphoprotein, the matrix protein, the glycoprotein, and the RNA-dependent RNA polymerase. Among these, the glycoprotein is the most important as it is the major contributor to pathogenicity and virus neutralizing antibody response. Keeping in mind that glycoprotein is the only protein exposed on the surface of virus and is thought to be responsible for the interaction with the cell membrane, it was attempted to target glycoprotein by a ligand polyethylene glycol 4000, which blocks its active site, as seen by molecular operating environment software, so that it may be possible to prevent the spread of virus into the host. The ligand polyethylene glycol 4000 was retrieved from Research Collaboratory for Structural Bioinformatics protein data bank by providing the glycoprotein sequence to the databank. In this study it was observed that the ligand was successfully docked on a major portion of antigenic site II of glycoprotein by mimicking the virus neutralizing antibodies. This knowledge may be important for the development of novel therapies for the treatment of rabies and other viral diseases in the future.

  8. Swainsonine-induced lysosomal storage disease in goats caused by the ingestion of Turbina cordata in Northeastern Brazil.

    Science.gov (United States)

    Dantas, A F M; Riet-Correa, F; Gardner, D R; Medeiros, R M T; Barros, S S; Anjos, B L; Lucena, R B

    2007-01-01

    A disease of the central nervous system in goats was observed in the municipalities of Juazeiro, Casa Nova and Curaça, state of Bahia, and Petrolina, state of Pernambuco, Northeastern Brazil. The disease was produced experimentally in two goats by the administration of dry Turbina cordata mixed with grain. Clinical signs were observed after the ingestion of 62 and 106 g/kg body weight in 28 and 54 days, respectively. The concentration of swainsonine in the plant varied from less than 0.001% to 0.14% (dry weight). Clinical signs of natural and experimental cases included difficulties in standing, ataxia, hypermetria, wide-based stance, intention tremors, spastic paresis mainly in the hind legs, nystagmus, abnormal postural reactions, head tilting, and falling. Diffuse vacuolation of neurons, epithelial cells of pancreas, thyroids, and renal tubules were observed on the histology. From the electron microscopy of Purkinje cells the vacuoles represented dilated lysosomes. These findings demonstrated that T. cordata causes an acquired glycoprotein lysosomal storage disease. The intoxication occurs at least in an area of 27,000 km2 causing severe losses in goats, and some farmers report the disease also in cattle.

  9. Glycoprotein fucosylation is increased in seminal plasma of subfertile men

    Directory of Open Access Journals (Sweden)

    Beata Olejnik

    2015-04-01

    Full Text Available Fucose, the monosaccharide frequent in N- and O-glycans, is a part of Lewis-type antigens that are known to mediate direct sperm binding to the zona pellucida. Such interaction was found to be inhibited in vitroby fucose-containing oligo- and polysaccharides, as well as neoglycoproteins. The objective of this study was to screen seminal plasma proteins of infertile/subfertile men for the content and density of fucosylated glycoepitopes, and compare them to samples of fertile normozoospermic subjects. Seminal proteins were separated in polyacrylamide gel electrophoresis and blotted onto nitrocellulose membrane and probed with fucose-specific Aleuria aurantia lectin (AAL. Twelve electrophoretic bands were selected for quantitative densitometric analysis. It was found that the content, and especially the density of fucosylated glycans, were higher in glycoproteins present in seminal plasma of subfertile men. No profound differences in fucosylation density were found among the groups of normozoospermic, oligozoospermic, asthenozoospermic, and oligoasthenozoospermic subfertile men. According to the antibody probing, AAL-reactive bands can be attributed to male reproductive tract glycoproteins, including prostate-specific antigen, prostatic acid phosphatase, glycodelin and chorionic gonadotropin. Fibronectin, α1 -acid glycoprotein, α1 -antitrypsin, immunoglobulin G and antithrombin III may also contribute to this high fucosylation. It is suggested that the abundant fucosylated glycans in the sperm environment could interfere with the sperm surface and disturb the normal course of the fertilization cascade.

  10. Lysosomal iron mobilization and induction of the mitochondrial permeability transition in acetaminophen-induced toxicity to mouse hepatocytes.

    Science.gov (United States)

    Kon, Kazuyoshi; Kim, Jae-Sung; Uchiyama, Akira; Jaeschke, Hartmut; Lemasters, John J

    2010-09-01

    Acetaminophen induces the mitochondrial permeability transition (MPT) in hepatocytes. Reactive oxygen species (ROS) trigger the MPT and play an important role in AAP-induced hepatocellular injury. Because iron is a catalyst for ROS formation, our aim was to investigate the role of chelatable iron in MPT-dependent acetaminophen toxicity to mouse hepatocytes. Hepatocytes were isolated from fasted male C3Heb/FeJ mice. Necrotic cell killing was determined by propidium iodide fluorometry. Mitochondrial membrane potential was visualized by confocal microscopy of tetramethylrhodamine methylester. Chelatable ferrous ion was monitored by calcein quenching, and 70 kDa rhodamine-dextran was used to visualize lysosomes. Cell killing after acetaminophen (10mM) was delayed and decreased by more than half after 6 h by 1mM desferal or 1mM starch-desferal. In a cell-free system, ferrous but not ferric iron quenched calcein fluorescence, an effect reversed by dipyridyl, a membrane-permeable iron chelator. In hepatocytes loaded with calcein, intracellular calcein fluorescence decreased progressively beginning about 4 h after acetaminophen. Mitochondria then depolarized after about 6 h. Dipyridyl (20mM) dequenched calcein fluorescence. Desferal and starch-desferal conjugate prevented acetaminophen-induced calcein quenching and mitochondrial depolarization. As calcein fluorescence became quenched, lysosomes disappeared, consistent with release of iron from ruptured lysosomes. In conclusion, an increase of cytosolic chelatable ferrous iron occurs during acetaminophen hepatotoxicity, which triggers the MPT and cell killing. Disrupted lysosomes are the likely source of iron, and chelation of this iron decreases acetaminophen toxicity to hepatocytes.

  11. Triazoles inhibit cholesterol export from lysosomes by binding to NPC1.

    Science.gov (United States)

    Trinh, Michael N; Lu, Feiran; Li, Xiaochun; Das, Akash; Liang, Qiren; De Brabander, Jef K; Brown, Michael S; Goldstein, Joseph L

    2017-01-03

    Niemann-Pick C1 (NPC1), a membrane protein of lysosomes, is required for the export of cholesterol derived from receptor-mediated endocytosis of LDL. Lysosomal cholesterol export is reportedly inhibited by itraconazole, a triazole that is used as an antifungal drug [Xu et al. (2010) Proc Natl Acad Sci USA 107:4764-4769]. Here we show that posaconazole, another triazole, also blocks cholesterol export from lysosomes. We prepared P-X, a photoactivatable cross-linking derivative of posaconazole. P-X cross-linked to NPC1 when added to intact cells. Cross-linking was inhibited by itraconazole but not by ketoconazole, an imidazole that does not block cholesterol export. Cross-linking of P-X was also blocked by U18666A, a compound that has been shown to bind to NPC1 and inhibit cholesterol export. P-X also cross-linked to purified NPC1 that was incorporated into lipid bilayer nanodiscs. In this in vitro system, cross-linking of P-X was inhibited by itraconazole, but not by U18666A. P-X cross-linking was not prevented by deletion of the N-terminal domain of NPC1, which contains the initial binding site for cholesterol. In contrast, P-X cross-linking was reduced when NPC1 contained a point mutation (P691S) in its putative sterol-sensing domain. We hypothesize that the sterol-sensing domain has a binding site that can accommodate structurally different ligands.

  12. Crystal Structure of Glycoprotein C from a Hantavirus in the Post-fusion Conformation

    Science.gov (United States)

    Willensky, Shmuel; Bignon, Eduardo A.; Tischler, Nicole D.; Dessau, Moshe

    2016-01-01

    Hantaviruses are important emerging human pathogens and are the causative agents of serious diseases in humans with high mortality rates. Like other members in the Bunyaviridae family their M segment encodes two glycoproteins, GN and GC, which are responsible for the early events of infection. Hantaviruses deliver their tripartite genome into the cytoplasm by fusion of the viral and endosomal membranes in response to the reduced pH of the endosome. Unlike phleboviruses (e.g. Rift valley fever virus), that have an icosahedral glycoprotein envelope, hantaviruses display a pleomorphic virion morphology as GN and GC assemble into spikes with apparent four-fold symmetry organized in a grid-like pattern on the viral membrane. Here we present the crystal structure of glycoprotein C (GC) from Puumala virus (PUUV), a representative member of the Hantavirus genus. The crystal structure shows GC as the membrane fusion effector of PUUV and it presents a class II membrane fusion protein fold. Furthermore, GC was crystallized in its post-fusion trimeric conformation that until now had been observed only in Flavi- and Togaviridae family members. The PUUV GC structure together with our functional data provides intriguing evolutionary and mechanistic insights into class II membrane fusion proteins and reveals new targets for membrane fusion inhibitors against these important pathogens. PMID:27783673

  13. The Link Between Lysosomal Storage Disorders and More Common Diseases

    Directory of Open Access Journals (Sweden)

    Michael Beck MD

    2016-12-01

    Full Text Available In the last decades, it has become more and more evident that lysosomal storage disorders and common neurodegenerative diseases such as Alzheimer and Parkinson diseases have clinical, neuropathological, and genetic features in common, including lysosomal dysfunction and impaired autophagy. Patients with Gaucher and even carriers of Gaucher disease have an increased risk to develop Parkinson disease. Likewise, individuals who are heterozygous for a mutation of a gene that causes an adult form of neuronal ceroid lipofuscinosis are more likely to be affected by a form of frontotemporal dementia in their later life. A further example is the gene NAGLU encoding the enzyme α- N -acetylglucosaminidase, which is deficient in patients with mucopolysaccharidosis type IIIB. Mutations of the NAGLU gene have been observed in patients affected by an axonal neuropathy. An interesting unexpected finding was the link between stuttering and genes that are essential for the function of all lysosomal enzymes. This review will present some example of the association of lysosomal storage disorders and neurodegenerative disease and discuss possible pathogenic mechanisms that are common to both conditions. The understanding of the pathophysiology of the endosomal–lysosomal–autophagic system may help to develop drugs, which might provide benefit not only for patients with rare lysosomal storage disorders but also for individuals affected by more common diseases.

  14. The Link Between Lysosomal Storage Disorders and More Common Diseases

    Directory of Open Access Journals (Sweden)

    Michael Beck MD

    2016-12-01

    Full Text Available In the last decades, it has become more and more evident that lysosomal storage disorders and common neurodegenerative diseases such as Alzheimer and Parkinson diseases have clinical, neuropathological, and genetic features in common, including lysosomal dysfunction and impaired autophagy. Patients with Gaucher and even carriers of Gaucher disease have an increased risk to develop Parkinson disease. Likewise, individuals who are heterozygous for a mutation of a gene that causes an adult form of neuronal ceroid lipofuscinosis are more likely to be affected by a form of frontotemporal dementia in their later life. A further example is the gene NAGLU encoding the enzyme α-N-acetylglucosaminidase, which is deficient in patients with mucopolysaccharidosis type IIIB. Mutations of the NAGLU gene have been observed in patients affected by an axonal neuropathy. An interesting unexpected finding was the link between stuttering and genes that are essential for the function of all lysosomal enzymes. This review will present some example of the association of lysosomal storage disorders and neurodegenerative disease and discuss possible pathogenic mechanisms that are common to both conditions. The understanding of the pathophysiology of the endosomal–lysosomal–autophagic system may help to develop drugs, which might provide benefit not only for patients with rare lysosomal storage disorders but also for individuals affected by more common diseases.

  15. Rab2 promotes autophagic and endocytic lysosomal degradation.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  17. A biotin-avidin enzyme-linked immunosorbent assay for studying platelet membrane glycoprotein Ⅱb/Ⅲa, CD62p expression and its clinical application%BA-ELISA定量检测人血小板膜糖蛋白Ⅱb/Ⅲa、CD62p的表达及其临床应用

    Institute of Scientific and Technical Information of China (English)

    张有涛; 赵益明; 朱明清; 蒋敏; 程寅峰; 史进方; 顾国浩; 阮长耿

    2012-01-01

    Objective To develop a biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) method for detecting platelet membrane glycoprotein function, and evaluate its clinical application. Methods With the monoclonal antibodies (mAb) recognizing GPⅡb/Ⅲa (7E3) , CD62p (SZ51) and biotin-avidin system, to develop a biotin-avidin-ELISA ( BA-ELISA). The levels of GP Ⅱ b/ Ⅲ a and CD62p were measured in patients with acute myocardial infarction ( AMI) , acute cerebral infarction (ACI) , diabetes mellitus ( DM) or in the healthy people. This BA-ELISA was compared with flow cytometry ( FCM). Inhibition of membrane glycoprotein expression was evaluated with inhibitory mAb, SZ21 and aspirin (acetylsalicylic acid,ASA) respectively. Results The BA-ELISA detected platelet count were as low as 3. 13 × 109/L or 6. 25 × 109/L in the platelet-rich plasma (PRP) of the specimens. Both of the interassay and intraassay coefficient variation ( CV) were less than 10%. Adenosine diphosphate ( ADP) -induced, or non-ADP-induced GP Ⅱ b/ Ⅲ a and CD62p expression in AMI, ACI, DM were significantly higher than those in the controls ( all P < 0.01). Either SZ21 or aspirin inhibited the expression of GP H b/ M a and CD62p induced by ADP. A high correlation was showed between BA-ELISA and FCM methods. Conclusion These observations indicate that BA-ELISA is a sensitive and high-throughput assay for evaluating platelet membrane glycoprotein expression and activation extent. This method is suitable to screen inhibitors/activators of platelet activation and has a potential in use for diagnostic purposes.%目的 建立测定血小板膜糖蛋白功能的生物素-亲和素-酶联免疫吸附(BA-ELISA)方法,并探讨其临床应用价值.方法 采用抗血小板膜糖蛋白Ⅱb/Ⅲa( GPⅡb/Ⅲa)单抗7E3和抗血小板CD62p单抗SZ51及生物素-亲和素系统建立BA-ELISA方法,并应用该方法检测50名健康志愿者、30例急性心肌梗死(AMI)、30例急性脑梗死(ACI)和30例

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

    Science.gov (United States)

    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.

  19. Crystal structure of the Epstein-Barr virus (EBV) glycoprotein H/glycoprotein L (gH/gL) complex.

    Science.gov (United States)

    Matsuura, Hisae; Kirschner, Austin N; Longnecker, Richard; Jardetzky, Theodore S

    2010-12-28

    The Epstein-Barr virus (EBV) is a γ-herpesvirus that infects B cells and epithelial cells and that has been linked to malignancies in both cell types in vivo. EBV, like other herpesviruses, has three glycoproteins, glycoprotein B (gB), gH, and gL, that form the core membrane fusion machinery mediating viral penetration into the cell. The gH and gL proteins associate to form a heterodimeric complex, which is necessary for efficient membrane fusion and also implicated in direct binding to epithelial cell receptors required for viral entry. To gain insight into the mechanistic role of gH/gL, we determined the crystal structure of the EBV gH/gL complex. The structure is comprised of four domains organized along the longest axis of the molecule. Comparisons with homologous HSV-2 gH/gL and partial pseudorabies virus gH structures support the domain boundaries determined for the EBV gH/gL structure and illustrate significant differences in interdomain packing angles. The gL subunit and N-terminal residues of gH form a globular domain at one end of the structure, implicated in interactions with gB and activation of membrane fusion. The C-terminal domain of gH, proximal to the viral membrane, is also implicated in membrane fusion. The gH/gL structure locates an integrin binding motif, implicated in epithelial cell entry, on a prominent loop in the central region of the structure. Multiple regions of gH/gL, including its two extreme ends, are functionally important, consistent with the multiple roles of gH/gL in EBV entry.

  20. The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Changkeun; Guo, Jiangtao; Zeng, Weizhong; Kim, Sunghoon; She, Ji; Cang, Chunlei; Ren, Dejian; Jiang, Youxing

    2017-07-19

    TMEM175 is a lysosomal K+ channel that is important for maintaining the membrane potential and pH stability in lysosomes1. It contains two homologous copies of a six-transmembrane-helix (6-TM) domain, which has no sequence homology to the canonical tetrameric K+ channels and lacks the TVGYG selectivity filter motif found in these channels2, 3, 4. The prokaryotic TMEM175 channel, which is present in a subset of bacteria and archaea, contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 channel from Chamaesiphon minutus, CmTMEM175, the architecture of which represents a completely different fold from that of canonical K+ channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swapping. The highly conserved TM1 helix acts as the pore-lining inner helix, creating an hourglass-shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the carboxy-terminal half of the TM1 helices form a bottleneck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter is primarily responsible for channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears to be distinct from that of the classical K+ channel family.

  1. The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture.

    Science.gov (United States)

    Lee, Changkeun; Guo, Jiangtao; Zeng, Weizhong; Kim, Sunghoon; She, Ji; Cang, Chunlei; Ren, Dejian; Jiang, Youxing

    2017-07-27

    TMEM175 is a lysosomal K(+) channel that is important for maintaining the membrane potential and pH stability in lysosomes. It contains two homologous copies of a six-transmembrane-helix (6-TM) domain, which has no sequence homology to the canonical tetrameric K(+) channels and lacks the TVGYG selectivity filter motif found in these channels. The prokaryotic TMEM175 channel, which is present in a subset of bacteria and archaea, contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 channel from Chamaesiphon minutus, CmTMEM175, the architecture of which represents a completely different fold from that of canonical K(+) channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swapping. The highly conserved TM1 helix acts as the pore-lining inner helix, creating an hourglass-shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the carboxy-terminal half of the TM1 helices form a bottleneck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter is primarily responsible for channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears to be distinct from that of the classical K(+) channel family.

  2. Nanoparticle size and combined toxicity of TiO2 and DSLS (surfactant) contribute to lysosomal responses in digestive cells of mussels exposed to TiO2 nanoparticles.

    Science.gov (United States)

    Jimeno-Romero, A; Oron, M; Cajaraville, M P; Soto, M; Marigómez, I

    2016-10-01

    The aim of this investigation was to understand the bioaccumulation, cell and tissue distribution and biological effects of disodium laureth sulfosuccinate (DSLS)-stabilised TiO2 nanoparticles (NPs) in marine mussels, Mytilus galloprovincialis. Mussels were exposed in vivo to 0.1, 1 and 10 mg Ti/L either as TiO2 NPs (60 and 180 nm) or bulk TiO2, as well as to DSLS alone. A significant Ti accumulation was observed in mussels exposed to TiO2 NPs, which were localised in endosomes, lysosomes and residual bodies of digestive cells, and in the lumen of digestive tubules, as demonstrated by ultrastructural observations and electron probe X-ray microanalysis. TiO2 NPs of 60 nm were internalised within digestive cell lysosomes to a higher extent than TiO2 NPs of 180 nm, as confirmed by the quantification of black silver deposits after autometallography. The latter were localised mainly forming large aggregates in the lumen of the gut. Consequently, lysosomal membrane stability (LMS) was significantly reduced upon exposure to both TiO2 NPs although more markedly after exposure to TiO2-60 NPs. Exposure to bulk TiO2 and to DSLS also affected the stability of the lysosomal membrane. Thus, effects on the lysosomal membrane depended on the nanoparticle size and on the combined biological effects of TiO2 and DSLS.

  3. Importance of lysosomal cysteine proteases in lung disease

    Directory of Open Access Journals (Sweden)

    Chapman Harold A

    2000-11-01

    Full Text Available Abstract The human lysosomal cysteine proteases are a family of 11 proteases whose members include cathepsins B, C, H, L, and S. The biology of these proteases was largely ignored for decades because of their lysosomal location and the belief that their function was limited to the terminal degradation of proteins. In the past 10 years, this view has changed as these proteases have been found to have specific functions within cells. This review highlights some of these functions, specifically their roles in matrix remodeling and in regulating the immune response, and their relationship to lung diseases.

  4. PDT: loss of autophagic cytoprotection after lysosomal photodamage

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  6. Analyzing Lysosome-Related Organelles by Electron Microscopy

    KAUST Repository

    Hurbain, Ilse

    2017-04-29

    Intracellular organelles have a particular morphological signature that can only be appreciated by ultrastructural analysis at the electron microscopy level. Optical imaging and associated methodologies allow to explore organelle localization and their dynamics at the cellular level. Deciphering the biogenesis and functions of lysosomes and lysosome-related organelles (LROs) and their dysfunctions requires their visualization and detailed characterization at high resolution by electron microscopy. Here, we provide detailed protocols for studying LROs by transmission electron microscopy. While conventional electron microscopy and its recent improvements is the method of choice to investigate organelle morphology, immunoelectron microscopy allows to localize organelle components and description of their molecular make up qualitatively and quantitatively.

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

    Science.gov (United States)

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

    2016-05-23

    Impaired homeostasis of lysosomal Ca(2+) causes lysosome dysfunction and lysosomal storage diseases (LSDs), but the mechanisms by which lysosomes acquire and refill Ca(2+) are not known. We developed a physiological assay to monitor lysosomal Ca(2+) store refilling using specific activators of lysosomal Ca(2+) channels to repeatedly induce lysosomal Ca(2+) release. In contrast to the prevailing view that lysosomal acidification drives Ca(2+) into the lysosome, inhibiting the V-ATPase H(+) pump did not prevent Ca(2+) refilling. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca(2+) prevented lysosomal Ca(2+) stores from refilling. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca(2+) refilling of lysosomes, but not in cells lacking IP3Rs. Furthermore, reducing ER Ca(2+) 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 Ca(2+)for the lysosome.

  8. Synthetic High-Density Lipoprotein-Like Nanocarrier Improved Cellular Transport of Lysosomal Cholesterol in Human Sterol Carrier Protein-Deficient Fibroblasts.

    Science.gov (United States)

    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.

  9. Myelin-associated glycoprotein and its axonal receptors.

    Science.gov (United States)

    Schnaar, Ronald L; Lopez, Pablo H H

    2009-11-15

    Myelin-associated glycoprotein (MAG) is expressed on the innermost myelin membrane wrap, directly apposed to the axon surface. Although it is not required for myelination, MAG enhances long-term axon-myelin stability, helps to structure nodes of Ranvier, and regulates the axon cytoskeleton. In addition to its role in axon-myelin stabilization, MAG inhibits axon regeneration after injury; MAG and a discrete set of other molecules on residual myelin membranes at injury sites actively signal axons to halt elongation. Both the stabilizing and the axon outgrowth inhibitory effects of MAG are mediated by complementary MAG receptors on the axon surface. Two MAG receptor families have been described, sialoglycans (specifically gangliosides GD1a and GT1b) and Nogo receptors (NgRs). Controversies remain about which receptor(s) mediates which of MAG's biological effects. Here we review the findings and challenges in associating MAG's biological effects with specific receptors.

  10. Genomic expression analyses reveal lysosomal, innate immunity proteins, as disease correlates in murine models of a lysosomal storage disorder.

    Directory of Open Access Journals (Sweden)

    Md Suhail Alam

    Full Text Available Niemann-Pick Type C (NPC disease is a rare, genetic, lysosomal disorder with progressive neurodegeneration. Poor understanding of the pathophysiology and a lack of blood-based diagnostic markers are major hurdles in the treatment and management of NPC and several additional, neurological lysosomal disorders. To identify disease severity correlates, we undertook whole genome expression profiling of sentinel organs, brain, liver, and spleen of Balb/c Npc1(-/- mice relative to Npc1(+/- at an asymptomatic stage, as well as early- and late-symptomatic stages. Unexpectedly, we found prominent up regulation of innate immunity genes with age-dependent change in their expression, in all three organs. We shortlisted a set of 12 secretory genes whose expression steadily increased with age in both brain and liver, as potential plasma correlates of neurological and/or liver disease. Ten were innate immune genes with eight ascribed to lysosomes. Several are known to be elevated in diseased organs of murine models of other lysosomal diseases including Gaucher's disease, Sandhoff disease and MPSIIIB. We validated the top candidate lysozyme, in the plasma of Npc1(-/- as well as Balb/c Npc1(nmf164 mice (bearing a point mutation closer to human disease mutants and show its reduction in response to an emerging therapeutic. We further established elevation of innate immunity in Npc1(-/- mice through multiple functional assays including inhibition of bacterial infection as well as cellular analysis and immunohistochemistry. These data revealed neutrophil elevation in the Npc1(-/- spleen and liver (where large foci were detected proximal to damaged tissue. Together our results yield a set of lysosomal, secretory innate immunity genes that have potential to be developed as pan or specific plasma markers for neurological diseases associated with lysosomal storage and where diagnosis is a major problem. Further, the accumulation of neutrophils in diseased organs

  11. Synthesis and translocation of gangliosides and glycoproteins during urethane anesthesia

    Energy Technology Data Exchange (ETDEWEB)

    Domowicz, M.S.; Kivatinitz, S.C.; Caputto, B.L.; Caputto, R.

    1988-05-01

    In this work, we have studied (a) the contents of gangliosides, glycoproteins, and phospholipids of the vesicle and plasma membrane fractions from brains of anesthetized and control rats and chickens and (b) the labeling of gangliosides and glycoproteins in the retina ganglion cell layer and optic tectum of urethane-anesthetized and control chickens after intraocular injection of a labeled N-acetylneuraminic acid precursor and the distribution of the label after subcellular fractionation. We found an increase in the content of gangliosides relative to protein in the vesicle fraction of both anesthetized rats and chickens relative to their controls. Other values were not affected by anesthesia. These results do not reflect a faster synthesis of gangliosides stimulated by urethane, because their rate of labeling was diminished in anesthetized animals. During the 4-h period after the animals were injected intraocularly with the radioactive precursor, the highest values of ganglioside-specific radioactivity were found in the vesicle fraction of control and anesthetized animals; at longer intervals, the specific radioactivity of the vesicle and plasma membrane fractions became rather similar. These data are in accordance with previous studies from this laboratory suggesting that the synthesis of the carbohydrate chain of gangliosides is regulated by the physiological demands made by the neurotransmitting system.

  12. 1,25-Dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysosomes containing calcium and calcium-binding protein (calbindin-D28K).

    Science.gov (United States)

    Nemere, I; Leathers, V; Norman, A W

    1986-12-05

    A variety of intestinal cell organelles and proteins have been proposed to mediate 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated calcium absorption. In the present study biochemical analyses were undertaken to determine the subcellular localization of 45Ca after calcium transport in vivo in ligated duodenal loops of vitamin D-deficient chicks injected with 1.3 nmol of 1,25-(OH)2D3 or vehicle 15 h prior to experimentation. Separation of Golgi, mitochondria, basal lateral membrane, and lysosome fractions in the epithelial homogenates was achieved by differential sedimentation followed by centrifugation in Percoll gradients and evaluation of appropriate marker enzyme activities. Both vitamin D-deficient and 1,25-(OH)2D3-treated chicks had the highest levels of 45Ca-specific activity in lysosomal fractions. The lysosomes were also the only organelles to exhibit a 1,25-(OH)2D3-mediated difference in calcium content, increasing to 138% of controls. Lysosomes prepared from 1,25-(OH)2D3-treated chicks also contained the greatest levels of immunoreactive calbindin-D28k (calcium-binding protein). Chloroquine, a drug known to interfere with lysosomal function, was tested and found to inhibit 1,25-(OH)2D3-stimulated intestinal calcium absorption. Neither 1,25-(OH)2D3 nor chloroquine affected [3H]2O transport. In additional experiments, microsomal membranes (105,000 X g pellets) were subjected to gradient centrifugation. The highest levels of 45Ca-specific activity and calcium-binding protein in material from 1,25-(OH)2D3-treated chicks were found in fractions denser than endoplasmic reticulum and may represent endocytic vesicles. In studies on intestinal mucosa of 1,25-(OH)2D3-treated birds fractionated after 30 min of exposure to lumenal Ca2+ or Ca2+ plus chloroquine, 45Ca was found to accumulate in lysosomes and putative endocytic vesicles, relative to controls. A mechanism involving vesicular flow is proposed for 1,25-(OH)2D3-mediated intestinal calcium transport

  13. Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase

    Science.gov (United States)

    Glukhova, Alisa; Hinkovska-Galcheva, Vania; Kelly, Robert; Abe, Akira; Shayman, James A.; Tesmer, John J. G.

    2015-03-01

    Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid-metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. Here we describe several high-resolution crystal structures of human LPLA2 and a low-resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/β hydrolase core of LPLA2 form domains that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The LCAT structure suggests the molecular basis underlying human disease for most of the known LCAT missense mutations, and paves the way for rational development of new therapeutics to treat LCAT deficiency, atherosclerosis and acute coronary syndrome.

  14. A role for the deep orange and carnation eye color genes in lysosomal delivery in Drosophila.

    Science.gov (United States)

    Sevrioukov, E A; He, J P; Moghrabi, N; Sunio, A; Krämer, H

    1999-10-01

    Deep orange and carnation are two of the classic eye color genes in Drosophila. Here, we demonstrate that Deep orange is part of a protein complex that localizes to endosomal compartments. A second component of this complex is Carnation, a homolog of Sec1p-like regulators of membrane fusion. Because complete loss of deep orange function is lethal, the role of this complex in intracellular trafficking was analyzed in deep orange mutant clones. Retinal cells devoid of deep orange function completely lacked pigmentation and exhibited exaggerated multivesicular structures. Furthermore, a defect in endocytic trafficking was visualized in developing photoreceptor cells. These results provide direct evidence that eye color mutations of the granule group also disrupt vesicular trafficking to lysosomes.

  15. Recent advances in gene therapy for lysosomal storage disorders

    Directory of Open Access Journals (Sweden)

    Rastall DP

    2015-06-01

    Full Text Available David PW Rastall,1 Andrea Amalfitano1,2 1Department of Microbiology and Molecular Genetics, 2Department of Pediatrics, College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA Abstract: 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. Keywords: human trials, clinical trials, gene therapy, lysosomal storage disease, blood-brain barrier, adeno-associated virus, lentivirus, adenovirus 

  16. The frequency of lysosomal storage diseases in The Netherlands

    NARCIS (Netherlands)

    Poorthuis, BJHM; Wevers, RA; Kleijer, WJ; Groener, JEM; de Jong, JGN; van Weely, S; Niezen-Koning, KE; van Diggelen, OP

    1999-01-01

    We have calculated the relative frequency and the birth prevalence of lysosomal storage diseases (LSDs) in The Netherlands based on all 963 enzymatically confirmed cases diagnosed during the period 1970-1996. The combined birth prevalence for all LSDs is 14 per 100,000 live births. Glycogenosis type

  17. Clinical, biochemical and genetic heterogeneity in lysosomal storage diseases

    NARCIS (Netherlands)

    A.J.J. Reuser (Arnold)

    1977-01-01

    textabstractThe history of lysosomal storage diseases dates back to the end of the last century when the first clinical reports appeared of patients suffering from these genetic, metabolic disorders (Tay, 1881; Gaucher, 1882; Sachs, 1887; Fabry, 1898). About seventy years wouid pass before the term

  18. The frequency of lysosomal storage diseases in The Netherlands

    NARCIS (Netherlands)

    Poorthuis, BJHM; Wevers, RA; Kleijer, WJ; Groener, JEM; de Jong, JGN; van Weely, S; Niezen-Koning, KE; van Diggelen, OP

    1999-01-01

    We have calculated the relative frequency and the birth prevalence of lysosomal storage diseases (LSDs) in The Netherlands based on all 963 enzymatically confirmed cases diagnosed during the period 1970-1996. The combined birth prevalence for all LSDs is 14 per 100,000 live births. Glycogenosis type

  19. Release and uptake of lysosomal enzymes : studied in cultured cells

    NARCIS (Netherlands)

    D.J.J. Halley (Dicky)

    1980-01-01

    textabstractThe purpose of the experimental work described in this thesiswas to investigate some aspects of the release and uptake of lysosomal enzymes. The experiments involved the use of normal human and animal fibroblasts and some other cell types such as hepatocytes and hepatoma cells as sources

  20. Neuronopathic Lysosomal Storage Diseases: Clinical and Pathologic Findings

    Science.gov (United States)

    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,…

  1. Spore surface glycoproteins of Colletotrichum lindemuthianum are recognized by a monoclonal antibody which inhibits adhesion to polystyrene.

    Science.gov (United States)

    Hughes, H B; Carzaniga, R; Rawlings, S L; Green, J R; O'Connell, R J

    1999-08-01

    Conidia (spores) of Colletotrichum lindemuthianum, a fungal plant pathogen causing bean anthracnose, adhere to the aerial parts of host plants to initiate the infection process. These spores possess a fibrillar 'spore coat' as well as a cell wall. In a previous study a mAb, UB20, was raised that recognized glycoproteins on the spore surface. In this study UB20 was used to localize and characterize these glycoproteins and to investigate their possible role in adhesion. Glycoproteins recognized by UB20 were concentrated on the outer surface of the spore coat and, to a lesser extent, at the plasma membrane/cell wall interface. Extraction of spores with hot water or 0.2% SDS resulted in removal of the spore coat. Western blotting with UB20 showed that a relatively small number of glycoproteins were extracted by these procedures, including a major component at 110 kDa. Biotinylation of carbohydrate moieties, together with cell fractionation, confirmed that these glycoproteins were exposed at the surface of the spores. In adhesion assays, > 90% of ungerminated conidia attached to polystyrene Petri dishes within 30 min. UB20 IgG at low concentrations inhibited attachment in an antigen-specific manner. This suggests that the glycoproteins recognized by this mAb may function in the initial rapid attachment of conidia to hydrophobic substrata. Polystyrene microspheres bound selectively to the 110 kDa glycoprotein in Western blots, providing further evidence that this component could mediate interactions with hydrophobic substrata.

  2. Boronic acid functionalized peptidyl synthetic lectins: Combinatorial library design, peptide sequencing, and selective glycoprotein recognition

    Science.gov (United States)

    Bicker, Kevin L.; Sun, Jing; Lavigne, John J.; Thompson, Paul R.

    2011-01-01

    Aberrant glycosylation of cell membrane and secreted glycoproteins is a hallmark of various disease states, including cancer. The natural lectins currently used in the recognition of these glycoproteins are costly, difficult to produce, and unstable towards rigorous use. Herein we describe the design and synthesis of several boronic acid functionalized peptide-based synthetic lectin (SL) libraries, as well as the optimized methodology for obtaining peptide sequences of these SLs. SL libraries were subsequently used to identify SLs with as high as 5-fold selectivity for various glycoproteins. SLs will inevitably find a role in cancer diagnositics, given that they do not suffer from the drawbacks of natural lectins and that the combinatorial nature of these libraries allows for the identification of an SL for nearly any glycosylated biomolecule. PMID:21405093

  3. Olaquindox induces DNA damage via the lysosomal and mitochondrial pathway involving ROS production and p53 activation in HEK293 cells.

    Science.gov (United States)

    Yang, Yang; Jiang, Liping; She, Yan; Chen, Min; Li, Qiujuan; Yang, Guang; Geng, Chengyan; Tang, Liyun; Zhong, Laifu; Jiang, Lijie; Liu, Xiaofang

    2015-11-01

    Olaquindox (OLA) is a potent antibacterial agent used as a feed additive and growth promoter. In this study, the genotoxic potential of OLA was investigated in the human embryonic kidney cell line 293 (HEK293). Results showed that OLA caused significant increases of DNA migration. Lysosomal membrane permeability and mitochondrial membrane potential were reduced after treatment with OLA. OLA was shown to induce ROS production and GSH depletion. The expression of p53 protein is increased in cells incubated with OLA. The activation of p53 and ATM gene was assessed by exposure to OLA. Furthermore, NAC reduced DNA migration, ROS formation, GSH depletion and the expression of the p53 protein and gene. And desipramine significantly decreased AO fluorescence intensity and the expression of the p53 protein and gene. These results support the assumption that OLA exerted genotoxic effects and induced DNA strand breaks in HEK293 cells, possibly through lysosomal-mitochondrial pathway involving ROS production and p53 activation.

  4. The research of neurospecific proteins and lysosomal peptidehydrolases in frontal neocortex during forming conditioned reaction of active avoiding of rats

    Directory of Open Access Journals (Sweden)

    Vyatkin O. K.

    2009-04-01

    Full Text Available Dynamics of the activity of neuronal cell adhesion molecule (NCAM and lysosomal cysteine cathepsins B, L, H was researched in frontal neocortex of rat brain during forming a conditioned reaction of active avoiding. The quantitative estimation of NCAM content in the neocortex membrane fraction was carried on by ELISA in 3, 7, 14 and 21 days after starting animals’ training. The dynamics correlation between the NCAM content increasing and cysteine cathepsins activity was obtained, especially for aminopeptidase cathepsin H during the process of memory engram forming in frontal neocortex of rat brain.

  5. Elevation of glycoprotein nonmetastatic melanoma protein B in type 1 Gaucher disease patients and mouse models.

    Science.gov (United States)

    Kramer, Gertjan; Wegdam, Wouter; Donker-Koopman, Wilma; Ottenhoff, Roelof; Gaspar, Paulo; Verhoek, Marri; Nelson, Jessica; Gabriel, Tanit; Kallemeijn, Wouter; Boot, Rolf G; Laman, Jon D; Vissers, Johannes P C; Cox, Timothy; Pavlova, Elena;