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)

Afipia felis is a Gram-negative bacterium that causes some cases of human Cat Scratch Disease. A. felis can survive and multiply in several mammalian cell types, including macrophages, but the precise intracellular compartmentalization of A. felis-containing phagosomes is unknown. Here, we demonstrate that, in murine macrophages, most A. felis-containing phagosomes exclude lysosomal tracer loaded into macrophage lysosomes before, as well as endocytic tracer loaded after, establishment of an infection. Established Afipia-containing phagosomes possess neither early endosomal marker proteins [early endosome antigen 1 (EEA1), Rab5, transferrin receptor, trytophane aspartate containing coat protein (TACO)] nor late endosomal or lysosomal proteins [cathepsin D, beta-glucuronidase, vacuolar proton-pumping ATPase, rab7, mannose-6-phosphate receptor, vesicle-associated membrane protein 8, lysosome-associated membrane proteins LAMP-1 and LAMP-2]. Those bacteria that will be found in a nonendosomal compartment enter the macrophage via an EEA1-negative compartment, which remains negative for LAMP-1. The smaller subpopulation of afipiae whose phagosomes will be part of the endocytic system enters into an EEA1-positive compartment, which also subsequently acquires LAMP-1. Killing of Afipia or opsonization with immune antibodies leads to a strong increase in the percentage of A. felis-containing phagosomes that interact with the endocytic system. We conclude that most phagosomes containing A. felis are disconnected from the endosome-lysosome continuum, that their unusual compartmentalization is decided at uptake, and that this compartmentalization requires bacterial viability.
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PMID:Afipia felis induces uptake by macrophages directly into a nonendocytic compartment. 1140 61

Salmonella typhimurium is a facultative intracellular pathogen that colonizes host cells throughout the course of infection. A unique feature of this pathogen is its ability to enter into (invade) epithelial cells and elongate the vacuole within which it resides into tubular structures called Salmonella-induced filaments (Sifs). In this study we sought to characterize the mechanism of Sif formation by immunofluorescence analysis using subcellular markers. The late endosomal lipid lysobisphosphatidic acid associated in a punctate pattern with the Salmonella-containing vacuole, starting 90 min after infection and increasing thereafter. Lysobisphosphatidic acid-rich vesicles were also found to interact with Sifs, at numerous sites along the tubules. Similarly, cholesterol-rich vesicles were also found in association with intracellular bacteria and Sifs. The lysosomal hydrolase cathepsin D was present in Sifs, both in a punctate pattern and, at later times, predominantly in an uninterrupted linear pattern. Rab7 associated with Sifs and expression of the N125I dominant negative mutant of this GTPase inhibited Sif formation. Transfection of HeLa cells with a vector encoding SifA fused to the green fluorescent protein caused swelling and aggregation of lysobisphosphatidic acid-containing compartments, suggesting that this virulence factor directs membrane fusion events involving late endosomes. Our findings demonstrate that Sif formation involves fusion of late endocytic compartments with the Salmonella-containing vacuole, and suggest that SifA modulates this event.
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PMID:Characterization of Salmonella-induced filaments (Sifs) reveals a delayed interaction between Salmonella-containing vacuoles and late endocytic compartments. 1155 18

Modifications occurring during the transformation of phagosomes into mature phagolysosomes were investigated in osteoclast-like cells (OCLs) and macrophages using latex beads as markers for the isolation of phagosomal compartments (LBC) at different time points after phagocytosis. In OCLs, newly formed LBC acquired cathepsin K, tartarate-resistant phosphatase (TRAP), lysosome-associated membrane protein-1 (Lamp-1), and cathepsin D, and rapidly lost annexin II in a time-dependent manner. The levels of Rab7 and c-Src in OCLs initially increased and then gradually decreased during the transformation from early to late endosomal LBC or phagolysosomes. Receptor activator of NF-kappaB (RANKL) significantly increased the LBC levels of cathepsin K, TRAP, and c-Src, whereas calcitonin decreased the LBC levels of cathepsin K, TRAP, and Rab7, indicating that the transformation of early to late endosomal elements and lysosomes in OCLs is also regulated by osteoclastogenesis regulatory factors. On the other hand, changes in the LBC levels of Lamp-1, cathepsin D, and annexin II in macrophages were comparable to those in OCLs. However, contrary to osteoclastic LBC, Rab7 levels of macrophage LBC decreased in a time-dependent manner. Macrophage LBC were devoid of cathepsin K, TRAP, and c-Src in all transformation stages. These observations suggest that OCLs and macrophages have different phagosome maturation mechanisms that involve the specific and regulated acquisition of markers from endocytic organelles. The results also demonstrate that the use of LBC is a useful system in which to identify and characterize molecules involved in these different endocytic pathways.
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PMID:Characterization of phagosomal subpopulations along endocytic routes in osteoclasts and macrophages. 1172 83

Previously, we showed that live Salmonella-containing phagosomes (LSP) recruit early acting Rab5 and promote fusion with early endosomes, thus avoiding transport to the lysosomes. Therefore, live Salmonella survive in a specialized compartment. Here we show that scavenger-receptor-mediated intracellular delivery of muramyl dipeptide (MDP) to macrophages leads to efficient killing of Salmonella both in vitro and in vivo. To understand the intracellular trafficking modulation of Salmonella by delivery of MDP, we investigated the levels of endocytic Rab proteins, which are the major regulators of vesicular transport. Western blot analysis reveals reduced Rab5 and enhanced Rab7 content in the maleylated bovine serum albumin-MDP (MBSA-MDP)-treated cells. The reduced content of Rab5 in the treated cells and on phagosomes inhibits the fusion of Salmonella-containing phagosomes with early endosomes, and the enhanced Rab7 content in these cells facilitated targeting of LSP to lysosomes, which contain cathepsin D and vacuolar ATPase, for killing. In vitro reconstitution of lysosomal transport demonstrated that a reduced content of Rab5 and an enhanced level of Rab7 in MBSA-MDP-treated cells is primarily responsible for targeting Salmonella to lysosomes. Intracellular delivery of MDP thus offers a general strategy against macrophage-associated infections caused by intracellular pathogens that survive in the host cell by resisting transport to lysosomes.
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PMID:Diverting intracellular trafficking of Salmonella to the lysosome through activation of the late endocytic Rab7 by intracellular delivery of muramyl dipeptide. 1218 55

Nramp2 (DMT1) is a pH-dependent divalent cation transporter that acts as the transferrin-independent iron uptake system at the intestinal brush border and also transports iron released from transferrin across the membrane of acidified endosomes. In this study, RAW264.7 macrophages and 2 independently derived murine Sertoli cells lines, TM4 and 15P-1, were used to further study the subcellular localization of Nramp2/DMT1 in phagocytic cells, including possible recruitment to the phagosomal membrane. Nramp2/DMT1 was localized primarily to the EEA1-positive recycling endosome compartment, with some overlapping staining with Lamp1-positive late endosomes. After phagocytosis, immunofluorescence analysis and in vitro biochemical studies using purified latex bead-containing phagosomes indicated Nramp2/DMT1 recruitment to the membrane of Lamp1, cathepsin D, and rab7-positive phagosomes. Nramp2/DMT1 was also found associated with erythrocyte-containing phagosomes in RAW macrophages and with the periphery of sperm-containing phagosomes in Sertoli cells. These results suggest that, as for the macrophage-specific Nramp1 protein, Nramp2/DMT1 may transport divalent metals from the phagosomal space.
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PMID:Iron transporter Nramp2/DMT-1 is associated with the membrane of phagosomes in macrophages and Sertoli cells. 1223 76

Molecular mechanisms of endocytosis in the genetically and biochemically tractable professional phagocyte Dictyostelium discoideum reveal a striking degree of similarity to higher eukaryotic cells. Pulse-chase feeding with latex beads allowed purification of phagosomes at different stages of maturation. Gentle ATP stripping of an actin meshwork entrapping contaminating organelles resulted in a 10-fold increase in yield and purity, as confirmed by electron microscopy. Temporal profiling of signaling, cytoskeletal, and trafficking proteins resulted in a complex molecular fingerprint of phagosome biogenesis and maturation. First, nascent phagosomes were associated with coronin and rapidly received a lysosomal glycoprotein, LmpB. Second, at least two phases of delivery of lysosomal hydrolases (cathepsin D [CatD] and cysteine protease [CPp34]) were accompanied by removal of plasma membrane components (PM4C4 and biotinylated surface proteins). Third, a phase of late maturation, preparing for final exocytosis of undigested material, included quantitative recycling of hydrolases and association with vacuolin. Also, lysosomal glycoproteins of the Lmp family showed distinct trafficking kinetics. The delivery and recycling of CatD was directly visualized by confocal microscopy. This heavy membrane traffic of cargos was precisely accompanied by regulatory proteins such as the Rab7 GTPases and the endosomal SNAREs Vti1 and VAMP7. This initial molecular description of phagocytosis demonstrates the feasibility of a comprehensive analysis of phagosomal lipids and proteins in genetically modified strains.
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PMID:High-resolution dissection of phagosome maturation reveals distinct membrane trafficking phases. 1238 53

Aspergillus fumigatus is the most prevalent airborne fungal pathogen responsible for fatal invasive aspergillosis in immunocompromised patients. Upon arrival in the lung alveolus, conidia of A. fumigatus are phagocytosed by alveolar macrophages, the major phagocytic cells of the lung. Engulfment and intracellular trafficking of A. fumigatus conidia in alveolar macrophages of two different origins, the murine cell line MH-S and human pulmonary alveolar macrophages, were analyzed by electron microscopy and immunofluorescence. Phagocytosis of A. fumigatus conidia required actin polymerization and phosphatidylinositol 3-kinase activity. Fusion of A. fumigatus phagosomes with early and late endosomes was shown by immunolabeling with specific markers for the transferrin receptor, early endosome antigen, and Rab7. Maturation of A. fumigatus phagolysosomes was monitored by using a fixable acidotropic probe, LysoTracker Red DND-99, and an anti-cathepsin D antibody. Bafilomycin A-induced inhibition of lysosomal acidification abolished the conidial killing by the macrophages. These data suggest that the maturation of A. fumigatus phagosomes results from fusion with the compartments of the endocytic pathway and that the killing of conidia depends on phagolysosome acidification. A model for the phagocytosis of A. fumigatus conidia by alveolar macrophages is proposed on the basis of these results.
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PMID:Phagocytosis and intracellular fate of Aspergillus fumigatus conidia in alveolar macrophages. 1254 May 71

Escherichia coli K1 has been shown to invade human brain microvascular endothelial cells (HBMEC) in vitro and translocate the blood-brain barrier in vivo, but it is unclear how E. coli K1 traverses HBMEC. We have previously shown that internalized E. coli K1 is localized within membrane-bound vacuole in HBMEC. The present study was carried out to understand intracellular trafficking of E. coli K1 containing vacuoles (ECVs) in HBMEC. ECVs initially acquired two early endosomal marker proteins, EEA1 and transferrin receptor. Rab7 and Lamp-1, markers for late endosome and late endosome/lysosome, respectively, were subsequently recruited on the ECVs, which was confirmed with flow cytometry analysis of ECVs. However, ECVs did not obtain cathepsin D, a lysosomal enzyme, even after 120 min incubation, suggesting that E. coli K1 avoids lysosomal fusion. In contrast, isogenic K1 capsule-deletion mutant obtained early and late endosomal markers on vacuolar membranes and allowed lysosomal fusion with subsequent degradation inside vacuoles. This observation was consistent with the decreased intracellular survival of K1 capsule-deletion mutant, even though the binding and internalization rates of the mutant were higher than those of the parent E. coli K1 strain. This is the first demonstration that E. coli K1, via the K1 capsule on the bacterial surface, modulates the maturation process of ECVs and prevents fusion with lysosomes, which is an event necessary for traversal of the blood-brain barrier as live bacteria.
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PMID:The K1 capsule modulates trafficking of E. coli-containing vacuoles and enhances intracellular bacterial survival in human brain microvascular endothelial cells. 1267 82

Mice double deficient in LAMP-1 and -2 were generated. The embryos died between embryonic days 14.5 and 16.5. An accumulation of autophagic vacuoles was detected in many tissues including endothelial cells and Schwann cells. Fibroblast cell lines derived from the double-deficient embryos accumulated autophagic vacuoles and the autophagy protein LC3II after amino acid starvation. Lysosomal vesicles were larger and more peripherally distributed and showed a lower specific density in Percoll gradients in double deficient when compared with control cells. Lysosomal enzyme activities, cathepsin D processing and mannose-6-phosphate receptor expression levels were not affected by the deficiency of both LAMPs. Surprisingly, LAMP-1 and -2 deficiencies did not affect long-lived protein degradation rates, including proteolysis due to chaperone-mediated autophagy. The LAMP-1/2 double-deficient cells and, to a lesser extent, LAMP-2 single-deficient cells showed an accumulation of unesterified cholesterol in endo/lysosomal, rab7, and NPC1 positive compartments as well as reduced amounts of lipid droplets. The cholesterol accumulation in LAMP-1/2 double-deficient cells could be rescued by overexpression of murine LAMP-2a, but not by LAMP-1, highlighting the more prominent role of LAMP-2. Taken together these findings indicate partially overlapping functions for LAMP-1 and -2 in lysosome biogenesis, autophagy, and cholesterol homeostasis.
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PMID:Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/LAMP-2 double-deficient fibroblasts. 1512 81

Acquisition of microbicidal properties by phagosomes requires the action of molecules which regulate the interactions between phagosomes and endocytic organelles. Members of the protein kinase C (PKC) superfamily of serine/threonine kinases are recruited to phagosomes with various kinetics during phagolysosome biogenesis. To study the role of PKC-alpha in this process, we compared the composition of latex bead-containing phagosomes isolated from control and dominant-negative (DN) PKC-alpha-overexpressing RAW 264.7 macrophages. Western blot analysis indicated that the levels of both lysosomal-associated membrane protein-1 and flotillin-1, which are acquired through interactions with late endosomes and lysosomes, are reduced in phagosomes from DN PKC-alpha-overexpressing macrophages. Proteomic characterization of latex bead-containing phagosomes revealed that recruitment of the small GTPase Rab7, cathepsin D, and cathepsin S is inhibited by DN PKC-alpha. Collectively, these data provide evidence that PKC-alpha plays a role in phagolysosome biogenesis, a critical process of the innate immune response against infections.
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PMID:Proteomic analysis reveals a role for protein kinase C-alpha in phagosome maturation. 1518 55


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