Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.23.5 (cathepsin D)
4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We show that insulin-dependent signals regulate azurophil granule-selective macroautophagy in human myeloid cells. Depletion of insulin from an insulin-transferrin-supplemented serum-free medium caused growth retardation of myeloblastic HL-60 cells, in which sequestration of electronic-dense cytoplasmic materials by autophagosomes was observed. Positive immunoreactivity with anti-CD68, anti-cathepsin D, and anti-myeloperoxidase antibodies indicated that the sequestrated materials were azurophil granules, the granulocyte/macrophage lineage-specific lysosome-like particles. By contrast, other organelles, including the mitochondria, endoplasmic reticulum, and Golgi apparatus remained intact, indicating that the macroautophagy selectively targeted azurophil granules. The addition of insulin induced rapid activations of p70S6K and Akt, and the cells were rescued from macroautophagy. Rapamycin, an inhibitor of mammalian target of rapamycin, did not block the insulin-mediated rescue from macroautophagy, although it nullified the activation of p70S6K and cell growth. Low doses of LY294002, a phosphatidyl-inositol-3-kinase inhibitor, which abolished cell growth and p70S6K activity but did not influence Akt activity, did not block the insulin-mediated rescue either. By contrast, low doses of Akt-specific inhibitors, which inhibited neither cell growth nor p70S6K activity, completely blocked the insulin-mediated rescue from macroautophagy. Thus, insulin-dependent signals are responsible for the control of azurophil granule-selective macroautophagy via Akt-dependent pathways, while p70S6K-dependent pathways promote cell growth.
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PMID:Insulin-dependent signaling regulates azurophil granule-selective macroautophagy in human myeloblastic cells. 1296 Feb 28

Variant late infantile neuronal ceroid lipofuscinosis, a lysosomal storage disorder characterized by progressive mental deterioration and blindness, is caused by mutations in a polytopic membrane protein (CLN6) with unknown intracellular localization and function. In this study, transient transfection of BHK21 cells with CLN6 cDNA and immunoblot analysis using peptide-specific CLN6 antibodies demonstrated the expression of a approximately 27-kDa protein that does not undergo proteolytic processing. Cross-linking experiments revealed the presence of CLN6 dimers. Using double immunofluorescence microscopy, epitope-tagged CLN6 was shown to be retained in the endoplasmic reticulum (ER) with no colocalization with the cis-Golgi or lysosomal markers. The translocation into the ER and proper folding were confirmed by the N-linked glycosylation of a mutant CLN6 polypeptide. Pulse-chase labeling of fibroblasts from CLN6 patients and from sheep (OCL6) and mouse (nclf) models of the disease followed by immunoprecipitation of cathepsin D indicated that neither the synthesis, sorting nor the proteolytic processing of this lysosomal enzyme was affected in CLN6-defective cells. However, the degradation of the endocytosed index protein arylsulfatase A was strongly reduced in all of the mutant CLN6 cell lines compared with controls. These data suggest that defects in the ER-resident CLN6 protein lead to lysosomal dysfunctions, which may result in lysosomal accumulation of storage material.
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PMID:Defective endoplasmic reticulum-resident membrane protein CLN6 affects lysosomal degradation of endocytosed arylsulfatase A. 1501 Apr 53

Brucella is responsible for one of the major worldwide zoonoses. Over the last century, several vaccines have been used against brucellosis. Among these, the rough vaccine Brucella abortus RB51 was introduced with the idea that it would not interfere with the diagnosis of brucellosis. Recently, RB51 has been isolated from milk and vaginal exudates from vaccinated cows, thus raising the possibility of extensive bacterial replication in these animals. We hypothesized that shedding of RB51 might be related to a change in its intracellular cell cycle. Therefore, we have compared the intracellular trafficking in CHO cells of the virulent B. abortus 2308 and two RB51 strains, the vaccinal strain and the one isolated from cow milk. Both RB51 strains were transiently observed in phagosomes characterized by the presence of the early endosomal marker EEA1 and then were found in cathepsin D-enriched lysosomal compartments, in which they eventually underwent degradation at later post-infection times. In contrast, the virulent 2308 strain replicated within the endoplasmic reticulum. These results suggest that a change in intracellular trafficking cannot account for Brucella shedding in adult vaccinated cows.
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PMID:Intracellular trafficking study of a RB51 B. abortus vaccinal strain isolated from cow milk. 1503 39

In transgenic mice expressing human mutant beta-amyloid precursor protein (APP) and mutant presenilin-1 (PS1), Abeta antibodies labeled granules, about 1 microm in diameter, in the perikaryon of neurons clustered in the isocortex, hippocampus, amygdala, thalamus, and brainstem. The granules were present before the onset of Abeta deposits; their number increased up to 9 months and decreased in 15-month-old animals. They were immunostained by antibodies against Abeta 40, Abeta 42, and APP C-terminal region. In double immunofluorescence experiments, the intracellular Abeta co-localized with lysosome markers and less frequently with MG160, a Golgi marker. Abeta accumulation correlated with an increased volume of lysosomes and Golgi apparatus, while the volume of endoplasmic reticulum and early endosomes did not change. Some granules were immunolabeled with an antibody against flotillin-1, a raft marker. At electron microscopy, Abeta, APP-C terminal, cathepsin D, and flotillin-1 epitopes were found in the lumen of multivesicular bodies. This study shows that Abeta peptide and APP C-terminal region accumulate in multivesicular bodies containing lysosomal enzymes, while APP N-terminus is excluded from them. Multivesicular bodies could secondarily liberate their content in the extracellular space as suggested by the association of cathepsin D with Abeta peptide in the extracellular space.
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PMID:Subcellular topography of neuronal Abeta peptide in APPxPS1 transgenic mice. 1550 18

Cathepsin E is an aspartic proteinase that has been implicated in Ag processing within the class II MHC pathway. In this study, we document the presence of cathepsin E message and protein in human myeloid dendritic cells, the preeminent APCs of the immune system. Cathepsin E is found in a perinuclear compartment, which is likely to form part of the endoplasmic reticulum, and also a peripheral compartment just beneath the cell membrane, with a similar distribution to that of Texas Red-dextran within 2 min of endocytosis. To investigate the function of cathepsin E in processing, a new soluble targeted inhibitor was synthesized by linking the microbial aspartic proteinase inhibitor pepstatin to mannosylated BSA via a cleavable disulfide linker. This inhibitor was shown to block cathepsin D/E activity in cell-free assays and within dendritic cells. The inhibitor blocked the ability of dendritic cells from wild-type as well as cathepsin D-deficient mice to present intact OVA, but not an OVA-derived peptide, to cognate T cells. The data therefore support the hypothesis that cathepsin E has an important nonredundant role in the class II MHC Ag processing pathway within dendritic cells.
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PMID:The expression and function of cathepsin E in dendritic cells. 1569 5

Legionella pneumophila is an intracellular pathogen that modulates the biogenesis of its phagosome to evade endocytic vesicle traffic. The Legionella-containing phagosome (LCP) does not acquire any endocytic markers and is remodeled by the endoplasmic reticulum during early stages. Here we show that intracellular replication of L. pneumophila is inhibited in gamma interferon (IFN-gamma)-activated, bone marrow-derived mouse macrophages and IFN-gamma-activated, human monocyte-derived macrophages in a dose-dependent manner. This inhibition of intracellular replication is associated with the maturation of the LCP into a phagolysosome, as documented by the acquisition of LAMP-2, cathepsin D, and lysosomal tracer Texas Red ovalbumin, and with the failure of the LCP to be remodeled by the rough endoplasmic reticulum. We conclude that IFN-gamma-activated macrophages override the ability of L. pneumophila to evade endocytic fusion and that the LCP is processed through the "default" endosomal-lysosomal degradation pathway.
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PMID:Maturation of the Legionella pneumophila-containing phagosome into a phagolysosome within gamma interferon-activated macrophages. 1584 27

Cells in the Brucella spp. are intracellular pathogens that survive and replicate within host monocytes. Brucella maintains persistent infections in animals despite the production of high levels of anti-Brucella-specific antibodies. To determine the effect of antibody opsonization on the ability of Brucella to establish itself within monocytes, the intracellular trafficking of virulent Brucella abortus 2308 and attenuated hfq and bacA mutants was followed in the human monocytic cell line THP-1. Early trafficking events of B. abortus 2308-containing phagosomes (BCP) were indistinguishable from those seen for control particles (heat-killed B. abortus 2308, live Escherichia coli HB101, or latex beads). All phagosomes transiently communicated the early-endosomal compartment and rapidly matured into LAMP-1(+), cathepsin D(+), and acidic phagosomes. By 2 h postinfection, however, the number of cathepsin D(+) BCP was significantly lower for live B. abortus 2308-infected cells than for either Brucella mutant strains or control particles. B. abortus 2308 persisted within these cathepsin D(-), LAMP-1(+), and acidic vesicles; however, at the onset of intracellular replication, the numbers of acidic B. abortus 2308 BCP decreased while remaining cathepsin D(-) and LAMP-1(+). In contrast to B. abortus 2308, the isogenic hfq and bacA mutants remained in acidic, LAMP-1(+) phagosomes and failed to initiate intracellular replication. Notably, markers specific for the host endoplasmic reticulum were absent from the BCPs throughout the course of the infection. Thus, opsonized B. abortus in human monocytes survives within phagosomes that remain in the endosomal pathway and replication of virulent B. abortus 2308 within these vesicles corresponds with an increase in intraphagosomal pH.
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PMID:Opsonized virulent Brucella abortus replicates within nonacidic, endoplasmic reticulum-negative, LAMP-1-positive phagosomes in human monocytes. 1590

The cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to alpha-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomal-lysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes.
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PMID:Involvement of a novel Q-SNARE, D12, in quality control of the endomembrane system. 1635 70

We investigated the trafficking of Burkholderia cenocepacia, an opportunistic respiratory pathogen of persons with cystic fibrosis (CF), in immortalized CF airway epithelial cells in vitro. Our results indicate that bacteria enter cells in a process involving actin rearrangement. Whereas both live and heat-killed bacteria reside transiently in early endosomes, only live bacteria escape from late endosomes to colocalize in vesicles positive for lysosomal membrane marker LAMP1, endoplasmic reticulum (ER) membrane marker calnexin, and autophagosome marker monodansylcadavarine (MDC). Twenty-four hours after infection, microcolonies of live bacteria were observed in the perinuclear area colocalizing with calnexin. In contrast, after ingestion, dead bacteria colocalized with late endosome marker Rab7, and lysosome markers LAMP1 and cathepsin D, but not with calnexin or MDC. Six to eight hours after ingestion of dead bacteria, degraded bacterial particles were observed in the cytoplasm and in vesicles positive for cathepsin D. These results indicate that live B. cenocepacia gain entry into human CF airway cells by endocytosis, escape from late endosomes to enter autophagosomes that fail to fuse with mature lysosomes, and undergo replication in the ER. This survival and replication strategy may contribute to the capacity of B. cenocepacia to persist in the lungs of infected CF patients.
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PMID:Intracellular trafficking and replication of Burkholderia cenocepacia in human cystic fibrosis airway epithelial cells. 1692 64

Legionella survives intracellularly by preventing fusion with lysosomes, due to phagosome escape from the endocytic pathway at an early stage of phagosome maturation, and by creating a replicative organelle that acquires endoplasmic reticulum (ER) characteristics through sustained interactions and fusion with the ER. Intracellular replication of Legionella pneumophila in mouse macrophages is controlled by the Lgn1 locus. Functional complementation in vivo has identified the Birc1e/Naip5 gene as being responsible for the Lgn1 effect. To understand the function and temporal site of action of Birc1e/Naip5 in susceptibility to L. pneumophila, we examined the biogenesis of Legionella-containing vacuoles (LCVs) formed in permissive A/J macrophages and in their Birc1e/Naip5 transgenic non-permissive counterpart. Birc1e/Naip5 effects on acquisition of lysosomal and ER markers were evident within 1-2 h following infection. A significantly higher proportion of LCVs formed in Birc1e/Naip5 transgenic macrophages had acquired the lysosomal markers cathepsin D and Lamp1 by 2 h post infection, whereas a significantly higher proportion of LCVs formed in permissive macrophages were positively stained for the ER markers BAP31 and calnexin, 6 h post infection. Likewise, studies by electron microscopy showed acquisition of lysosomal contents (horseradish peroxidase), within the first hour following phagocytic uptake, by LCVs formed in Birc1e/Naip5 transgenic macrophages and delivery of the ER marker glucose 6-phosphatase (G6Pase) only to the lumen of LCVs formed in A/J macrophages. Finally, a larger proportion of LCVs formed in A/J macrophages were studded with ribosomes 24 h post infection, compared with LCVs formed in Birc1e/Naip5 transgenic macrophages. These results suggest that sensing of L. pneumophila products by Birc1e/Naip5 in macrophages occurs rapidly following phagocytosis, a process that antagonizes the ability of L. pneumophila to remodel its phagosome into a specialized vacuole with ER characteristics.
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PMID:Birc1e/Naip5 rapidly antagonizes modulation of phagosome maturation by Legionella pneumophila. 1708 31


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