Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain inovalbumin-immunized mice. (41/912)

We previously reported that CA074, a specific inhibitor of cathepsin B, modulates specific immune responses from the T helper 2 (Th2) type to Th1 type in BALB/c mice infected with Leishmania major. In the present study, we found that a similar type of immune deviation was also induced in mice immunized with ovalbumin (OVA). However, treatment of mice with pepstatin A, a specific cathepsin D inhibitor, suppressed the OVA-specific proliferation of lymphocytes and blocked the development of both Th1 and Th2 cellular responses. These inhibitors did not appear to have any direct influence in vitro on functions of naive lymphocytes. OVA antigen (47 000 MW) was digested mainly into 40 000 MW protein in vitro by lysosomal proteases from naive BALB/c mice, and its digestion was markedly inhibited by the addition of CA074, but not by addition of pepstatin A, during incubation. However, pepstatin A strongly suppressed the degradation of the major histocompatibility complex class II-associated invariant chain (Ii) molecule in vivo and in vitro. Thus, cathepsin B appears to process antigens directed to preferential activation of Th2 cells, while cathepsin D may be responsible for the degradation of Ii, the processing of which is essential in initiating the antigen-specific activation of Th1 and Th2 CD4+ T cells. These lysosomal proteases may have different functions in regulating immune responses.  (+info)

Stallion epididymal fluid proteome: qualitative and quantitative characterization; secretion and dynamic changes of major proteins. (42/912)

Proteins present in and secreted into the lumen of various regions of the stallion epididymis were characterized qualitatively and quantitatively by two-dimensional electrophoresis. Using this proteomic approach, 201 proteins were found in the lumen and 117 were found that were secreted by the epithelium in various parts of the organ. Eighteen proteins made up 92.6% of the total epididymal secretory activity, lactoferrin (41.2%) and clusterin (24.8%) being the most abundant. Procathepsin D, HE1/CTP (cholesterol transfer protein), GPX (glutathione peroxidase), beta-N-acetyl-hexosaminidase, and PGDS (prostaglandin D2 synthase) were the other major compounds secreted. The most abundant proteins found in the luminal fluid were albumin and the secreted proteins: lactoferrin, PGDS, GPX, HE1/CTP, and hexosaminidase. Three main secretory epididymal regions were identified from the protein pattern, i.e., regions E0-E2, E3-E5, and E6-E9. Region E0-E2 was characterized by the secretion of clusterin (53%), PGDS (44%), and GPX (6%). Region E3-E5 had the highest number of secreted proteins, the highest protein concentrations (60-80 mg/ml), and the highest spermatocrit value (85%). Lactoferrin (60% in E4), clusterin (29% in E3), hexosaminidase (10% in E3), and procathepsin D (6.9% in E4) were the most abundant proteins in this region. Region E6-E9, in which few region-specific secreted compounds were found, was characterized by a high quantity of lactoferrin in the luminal fluid (2-14 mg/ml). Comparison between the secretion of the major proteins and their concentrations in the lumen throughout the organ showed that the behavior of each protein is specific, in particular for the three isoforms of clusterin.  (+info)

Live Salmonella modulate expression of Rab proteins to persist in a specialized compartment and escape transport to lysosomes. (43/912)

We investigated the intracellular route of Salmonella in macrophages to determine a plausible mechanism for their survival in phagocytes. Western blot analysis of isolated phagosomes using specific antibodies revealed that by 5 min after internalization dead Salmonella-containing phagosomes acquire transferrin receptors (a marker for early endosomes), whereas by 30 min the dead bacteria are found in vesicles carrying the late endosomal markers cation-dependent mannose 6-phosphate receptors, Rab7 and Rab9. In contrast, live Salmonella-containing phagosomes (LSP) retain a significant amount of Rab5 and transferrin receptor until 30 min, selectively deplete Rab7 and Rab9, and never acquire mannose 6-phosphate receptors even 90 min after internalization. Retention of Rab5 and Rab18 and selective depletion of Rab7 and Rab9 presumably enable the LSP to avoid transport to lysosomes through late endosomes. The presence of immature cathepsin D (48 kDa) and selective depletion of the vacuolar ATPase in LSP presumably contributes to the less acidic pH of LSP. In contrast, proteolytically processed cathepsin D (M(r) 17,000) was detected by 30 min on the dead Salmonella-containing phagosomes. Morphological analysis also revealed that after uptake by macrophages, the dead Salmonella are transported to lysosomes, whereas the live bacteria persist in compartments that avoid fusion with lysosomes, indicating that live Salmonella bypass the normal endocytic route targeted to lysosomes and mature in a specialized compartment.  (+info)

Phenotypic modulation by fibronectin enhances the angiotensin II-generating system in cultured vascular smooth muscle cells. (44/912)

We previously demonstrated that homogeneous cultures of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats produce angiotensin II (Ang II) in response to increases in the levels of angiotensinogen, cathepsin D, and angiotensin-converting enzyme (ACE). The change of VSMCs from the contractile to the synthetic phenotype increased the amount of synthetic organelles, resulting in the production of proteases and growth factors. To evaluate the contribution of the synthetic phenotype to the generation of Ang II, we examined the effect of fibronectin (FN), which reportedly induces the synthetic phenotype, on the Ang II-generating system in VSMCs. Cultured VSMCs from Wistar-Kyoto rats were incubated with an active fragment of FN, Arg-Gly-Asp-Ser, for 24, 48, or 72 hours after synchronization of the cell cycle with 0. 2% calf serum for 48 hours. Immunofluorescence and protein levels of alpha-smooth muscle (SM) actin and expression of SM22alpha mRNA, apparent in the contractile phenotype, were suppressed by FN, whereas expression of matrix Gla mRNA and osteopontin mRNA and protein, apparent in the synthetic phenotype, was increased. FN (1 to 1000 microg/mL) dose-dependently increased DNA synthesis in the VSMCs, which was inhibited by the Ang II type 1 receptor antagonist CV-11974. Ang II-like immunoreactivity as determined by radioimmunoassay was significantly increased in conditioned medium from the VSMCs. In addition, mRNA for the Ang II-generating proteases cathepsin D and ACE was increased by FN. Expression of transforming growth factor-beta1, platelet-derived growth factor A-chain, and basic fibroblast growth factor mRNAs was also increased by FN. These results indicate that the changes accompanying the alteration to the synthetic phenotype in homogeneous cultures of VSMCs increase expression of proteases such as cathepsin D and ACE, which then produce Ang II, and that these changes increase expression of growth factors that then induce growth of VSMCs.  (+info)

Aspartic proteases from the nematode Caenorhabditis elegans. Structural organization and developmental and cell-specific expression of asp-1. (45/912)

A Caenorhabditis elegans gene (asp-1) and cDNA that encode a homologue of cathepsin D aspartic protease were cloned and characterized. The asp-1 mRNA is transcribed from a single exon, and it begins with the SL1 trans-splice leader sequence. The protein (ASP-1) is expressed as a 396-amino acid, 42.7-kDa pre-pro-peptide that is post-translationally processed into a approximately 40-kDa lysosomal protein. ASP-1 shares approximately 60% sequence identity with the aspartic protease precursor from the nematode Strongyloides stercoralis. The amino acid sequences adjacent to the two active site aspartic acid residues in ASP-1 are 100% identical to those in other eukaryotic aspartic proteases. In addition, ASP-1 contains conserved, potential disulfide bond-forming cysteine residues and N-glycosylation sites. The asp-1 gene is exclusively transcribed in the intestinal cells, with the highest levels of expression observed at late embryonic and early larval stages of development. asp-1 transcription is not observed in adult nematodes or mature larvae. Furthermore, transcription predominantly occurs in eight anterior cells of the intestine (int6-int8). Analyses of ASP-1 nucleotide and amino acid sequences revealed the presence of five additional C. elegans aspartic proteases.  (+info)

A mutation in the ovine cathepsin D gene causes a congenital lysosomal storage disease with profound neurodegeneration. (46/912)

The neuronal ceroid lipofuscinoses (NCLs) constitute a group of neurodegenerative storage diseases characterized by progressive psychomotor retardation, blindness and premature death. Pathologically, there is accumulation of autofluorescent material in lysosome-derived organelles in a variety of cell types, but neurons in the central nervous system appear to be selectively affected and undergo progressive death. In this report we show that a novel form of NCL, congenital ovine NCL, is caused by a deficiency in the lysosomal aspartyl proteinase cathepsin D. A single nucleotide mutation in the cathepsin D gene results in conversion of an active site aspartate to asparagine, leading to production of an enzymatically inactive but stable protein. This results in severe cerebrocortical atrophy and early death, providing strong evidence for an important role of cathepsin D in neuronal development and/or homeostasis.  (+info)

Proteolysis of microtubule associated protein 2 and sensitivity of pancreatic tumours to docetaxel. (47/912)

We have studied the state of microtubule associated protein 2 (MAP2) in the pancreatic ductal adenocarcinomas P03 and P02 (sensitive and refractory to docetaxel respectively) since they express the corresponding mRNA and MAP2-related peptides. Immunohistochemical localization showed that in tumour P03 the MAP2-related peptides are highly expressed and confined to the epithelial malignant cells while in P02 the Intensity of the immunostaining is lower. However, anti alpha-tubulin staining followed a similar pattern suggesting that the net amount of macromolecular structures in the sensitive tumour is higher than in the refractory one. This may explain its higher sensitivity to docetaxel, because tubulin assembled into microtubules is the target of the drug. We found that protein extracts from both tumours differed in their proteolytic activity on rat brain MAP2. Since the proteolysis pattern obtained was similar to the one produced by Cathepsin D, we studied the effect of MAP2 proteolysed by this enzyme on microtubule formation in vitro. Proteolysis was found to increase the tendency of tubulin to assemble into macromolecular structures (microtubules and aggregates) in the presence of docetaxel. This suggests that in vivo proteolysis of MAP2 might increase microtubule alterations and potentiate the antitumour effect of docetaxel.  (+info)

Invasive properties of murine squamous carcinoma cells: secretion of matrix-degrading cathepsins is attributable to a deficiency in the mannose 6-phosphate/insulin-like growth factor II receptor. (48/912)

Penetration of basement membrane layers is a hallmark feature of metastatic tumor cells. The invasive propensity of murine SCC-VII squamous carcinoma cells is in part attributable to the extracellular action of the lysosomal cysteine proteinase cathepsin B. Although most noncancerous cells store this enzyme in the lysosomes, we found that SCC-VII cells release a large fraction (42%) of their newly synthesized procathepsin B into the culture medium. Procathepsins D and L, the precursors of other major lysosomal proteinases, are also secreted in significant amounts (24 and 75%, respectively). In contrast, normal murine 3T3-L1 fibroblasts exocytose only minor amounts of their newly synthesized procathepsins B (10%), D (<1%), and L (16%). Western blotting analysis revealed that SCC-VII cells are deficient in the 300 kDa mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R), a tumor suppressor with a central role in the intracellular transport of lysosomal enzymes. Consistent with the absence of M6P/IGF2R, SCC-VII cells lack dense lysosomes, with the bulk of intracellular acid hydrolases residing in late endosomes/ prelysosomes. On the other hand, the synthesis of the M6P recognition marker on lysosomal enzymes is not impaired in SCC-VII cells, because [33P]Pi is incorporated into the carbohydrate moieties of procathepsins B, D, and L. Furthermore, 69% of the phosphorylated N-linked oligosaccharides synthesized by SCC-VII cells carry phosphomonoester groups and as such constitute high-affinity ligands for M6P receptors. SCC-VII cells express the 46 kDa cation-dependent M6P receptor (MPR46), but intracellular retention of procathepsins B, D, and L is not affected by ammonium chloride and chloroquine, agents known to perturb the M6P receptor system. Our results indicate that failure to express the 300 kDa M6P/IGF2R may enhance the metastatic capacity of tumor cells by inducing the secretion of procathepsins B, D, and L.  (+info)