Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.23.5 (cathepsin D)
 4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies have indicated that acid-optimal cysteine proteinase(s) in the endosomal-lysosomal compartments, cathepsins, play a critical role in the proteolytic processing of endocytosed proteins to generate the antigenic peptides presented to the immune system on major histocompatibility complex (MHC) class II molecules. The presentation of these peptides and the expression of MHC class II molecules by macrophages and lymphocytes are stimulated by gamma-interferon (gamma-IFN). We found that treatment of human U-937 monocytes with gamma-IFN increased the activities and the content of the two major lysosomal cysteine proteinases, cathepsins B and L. Assays of protease activity, enzyme-linked immunosorbant assays (ELISA) and immunoblotting showed that this cytokine increased the amount of cathepsin B 5-fold and cathepsin L 3-fold in the lysosomal fraction. By contrast, the aspartic proteinase, cathepsin D, in this fraction was not significantly altered by gamma-IFN treatment. An induction of cathepsins B and L was also observed in mouse macrophages, but not in HeLa cells. These results suggest coordinate regulation in monocytes of the expression of cathepsins B and L and MHC class II molecules. Presumably, this induction of cysteine proteases contributes to the enhancement of antigen presentation by gamma-IFN.
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PMID:Gamma-interferon causes a selective induction of the lysosomal proteases, cathepsins B and L, in macrophages. 772 59

A functional approach of gene cloning was applied to HeLa cells in an attempt to isolate positive mediators of programmed cell death. The approach was based on random inactivation of genes by transfections with antisense cDNA expression libraries, followed by the selection of cells that survived in the presence of the external apoptotic stimulus. An antisense cDNA fragment identical to human cathepsin D aspartic protease was rescued by this positive selection. The high cathepsin D antisense RNA levels protected the HeLa cells from interferon-gamma- and Fas/APO-1-induced death. Pepstatin A, an inhibitor of cathepsin D, suppressed cell death in these systems and interfered with the TNF-alpha-induced programmed cell death of U937 cells as well. During cell death, expression of cathepsin D was elevated and processing of the protein was affected, which resulted in high steady-state levels of an intermediate, proteolytically active, single chain form of this protease. Overexpression of cathepsin D by ectopic expression induced cell death in the absence of any external stimulus. Altogether, these results suggest that this well-known endoprotease plays an active role in cytokine-induced programmed cell death, thus adding cathepsin D to the growing list of proteases that function as positive mediators of apoptosis.
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PMID:Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha. 867 Aug 91

Interleukin-6 (IL-6), a four-helix bundle protein, is a multifunctional cytokine which plays an important role in the regulation of the immune system, hematopoiesis, and inflammatory response, as well as in the pathogenesis of multiple myeloma. We have previously shown that a single-disulfide variant of human IL-6, lacking 22 N-terminal amino acids and the disulfide bond connecting Cys-45 and Cys-51 in the 185-residue chain of the wild-type protein, fully retains the conformational, stability, and functional properties of the full-length human IL-6 [Breton et al. (1995) Eur. J. Biochem. 227, 573-581]. In this study, we have investigated the conformational and stability properties of mutant IL-6 at acidic pH (A-state). Using far- and near-ultraviolet (UV) circular dichroism (CD), fluorescence emission, and second-derivative absorption spectroscopy, we have established that mutant IL-6 at pH 2.0 fully retains the helical secondary structure of the native protein at pH 7.5, while the tertiary interactions are much weaker. At variance from the native species, mutant IL-6 in the A-state binds 1-anilinonaphthalene-8-sulfonic acid (ANS), a property considered most typical of a protein in the molten globule state. The pH-induced conformational change from the native to the A-state, monitored either by near-UV CD or by ANS-binding measurements, shows a transition midpoint at pH approximately 4.5, thus indicating that the partial unfolding of the protein is mediated by the titration of glutamic and/or aspartic acid residues. At pH 2.0, the thermal denaturation of mutant IL-6 occurs as a broad process of low cooperativity with a transition at 50-60 degrees C, whereas at pH 7.5 the thermal unfolding is cooperative and characterized by a transition midpoint at 65 degrees C. Of interest, the unfolding of the A-state is not complete even up to approximately 85 degrees C. The urea-mediated unfolding profile of mutant IL-6, measured by far-UV CD, is essentially identical at both pH 7.5 and 2.0, with a midpoint of the cooperative unfolding transition at 5.5 +/- 0.1 M denaturant. Both thermal and urea denaturations of the A-state are complex and cannot fit to a two-state model for unfolding. The unusual stability of mutant IL-6 in acid is also reflected by the resistance to proteolysis at pH 3.6-4.0 by Staphylococcus aureus V8 protease or cathepsin D, an acid protease released by machrophages upon inflammatory stimulation. It is suggested that the molten globule state of IL-6 at acidic pH can play a role in the biological activity of this cytokine, which can exert its activity also at mildly acidic pH, as in inflammation sites.
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PMID:Acid-induced molten globule state of a fully active mutant of human interleukin-6. 878 6

Gallium arsenide (GaAs) is a semiconductor utilized in the electronics industry. Chemical exposure of animals causes a local inflammatory reaction, but systemic immunosuppression. Mice were administered i.p. 200 mg/kg GaAs crystals or latex beads, or vehicle. Five days after exposure, splenic macrophages were defective, whereas thioglycolate-elicited peritoneal macrophages (PEC) were more efficient in processing the Ag, pigeon cytochrome c, than vehicle control macrophages. Various aspects of the MHC class II Ag-processing pathway were examined. Both macrophage populations normally presented a peptide fragment to the CD4+ T cells. Surface MHC class II expression on the PEC was up-regulated, but splenic cells had normal MHC class II expression. PEC had elevated levels of glutathione and cysteine, major physiologic reducing thiols. However, the cysteine content of splenic macrophages was diminished. Proteolytic activities of aspartyl cathepsin D, and thiol cathepsins B and L were decreased significantly in splenic macrophages. On the other hand, thiol cathepsin activities were increased selectively in PEC. Latex bead-exposed PEC were not more potent APC, and their thiol cathepsin activities were unchanged, indicating that phagocytosis and nonspecific irritation were not responsible. The phenotype of PEC directly exposed to GaAs mirrored cytokine-activated macrophages, in contrast to splenic macrophages from a distant site. Therefore, GaAs exposure differentially modulated cathepsin activities in splenic macrophages and PEC, which correlated with their Ag-processing efficiency. Perhaps such distinct alterations may contribute to the local inflammation and systemic immunotoxicity caused by chemical exposure.
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PMID:Gallium arsenide modulates proteolytic cathepsin activities and antigen processing by macrophages. 972 6

The process of apoptosis (programmed cell death) has become the subject of intensive and extensive research over the past few years. Various approaches are being used to identify and study genes which function as positive mediators of apoptosis. Here, we address a novel approach of gene cloning aimed at isolating intracellular death promoting genes by utilizing a functional screen. This method, called TKO, was based on transfection of cells with an anti-sense cDNA library, followed by the selection of transfectants which survived in the continuous presence of a killing cytokine-interferon-gamma. It led to the identification of five novel apoptotic genes and to the finding that a known protease-cathepsin D, is actively recruited to the death process. The five novel apoptotic genes (named DAP genes for: Death Associated Proteins) code for proteins which display a diverse spectrum of biochemical activities. The list comprises a novel type of calcium/calmodulin-regulated kinase which carries ankyrin repeats and a death domain (DAP-kinase), a nucleotide-binding protein (DAP-3), a small proline-rich cytoplasmic protein (DAP-1), and a novel homolog of the eIF4G translation initiation factor (DAP-5). Extensive studies proved that these genes are critical for mediating cell death initiated by interferon-gamma, and in some of the tested cases also cell death induced by Fas/APO-1, TNF-alpha, and a detachment from extracellular matrix. Moreover, one of these genes, DAP-kinase, was recently found to display strong tumor suppressive activities, coupling the control of apoptosis to metastasis. The advantage of functional approaches of gene cloning is that they select the relevant rate limiting genes along the death pathways in a complete unbiased manner. As a consequence, novel targets and unpredicted mechanisms emerged. A few examples illustrating this important point will be discussed. One relates to the calcium/calmodulin-dependent DAP-kinase, which is localized to the actin microfilaments. It was found that the correct localization of DAP-kinase to the microfilament network was critical for the execution of the apoptotic process, and more specifically for the disruption of the stress fibers--a typical hallmark of apoptosis. Another important breakthrough step in our understanding of apoptotic processes relates to the identification and analysis of the DAP-5 gene. The structure/ function features of this novel translation regulator resemble the proteolytically cleaved eIF4G which appears in cells upon infection with some RNA viruses and which directs cap-independent translation. Thus, the rescue of DAP-5 highlighted the importance of regulation of protein translation in certain apoptotic systems. Finally, the isolation of cathespin D by our method suggests that lysosomal proteases are recruited during apoptosis, in addition to the well known caspase family of proteases, and that a unique pattern of regulation affecting the processing of this protease takes place. The major challenge now is to analyse how these diverse DAP gene activities constitute biochemical pathway(s) leading to programmed cell death, and what is their functional position with respect to other known positive mediators and suppressors of apoptosis such as the Bcl2 and caspase family members.
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PMID:Death associated proteins (DAPs): from gene identification to the analysis of their apoptotic and tumor suppressive functions. 991 95

Production of alpha-1-antitrypsin (AAT) by human monocytes is an important factor in controlling tissue damage by proteases in the microenvironment of inflammation. Increases, of four- to eightfold, in numbers of macrophages and levels of AAT and its cleavage fragments have been found in various inflammatory loci. We have found that the C-terminal peptide (C-36) of AAT, produced by specific proteinase cleavage when added in its fibrillar form at concentrations >/=5 microM to monocytes in culture for 24 h, significantly increases low density lipoprotein (LDL) binding and uptake, up-regulates levels of LDL receptors and also induces proinflammatory cytokine (interleukin-1, interleukin-6 and tumour necrosis factor alpha) production and glutathione reductase activity. Because it is known that various cells selectively internalize surface receptors and their ligands through receptor-mediated endocytosis via clathrin-coated pits, we tested whether antibodies raised against the clathrin heavy chain would block the effects of the fibrillar form of C-36 on human monocytes in culture. Addition of excess anti-(clathrin HC) with 10 microM fibrillar C-36 diminished the stimulatory effects of the latter on LDL binding, uptake and LDL receptor levels. In contrast, however, in the presence of anti-(clathrin HC), the potentially cytotoxic effects of fibrils, such as induction of cytokines, free radicals and cytosolic activity of cathepsin D, were much greater than those observed when cells were treated with fibrils alone. These results suggest that endocytosis is the pathway by which C-36 fibrils upregulate LDL receptors, and may be the natural mechanism for fibril clearance. We infer that human monocytes clear C-36 fibrils by a clathrin-dependent pathway, presumably endocytotic, and that loss of this pathway amplifies the cytotoxic effects of the fibrils by increasing their availability to other specific or nonspecific sites through which they exert their cytotoxic effects.
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PMID:Human monocyte activation by cleaved form of alpha-1-antitrypsin involvement of the phagocytic pathway. 1051 80

Lymphocyte proliferation and cytokine production were measured in groups of mice vaccinated (but not subsequently challenge infected) with recombinant forms of Schistosoma japonicum cathepsin D aspartic protease, rSjASP1 (expressed in bacteria; enzymatically inactive) and rSjASP2 (expressed in insect cells; enzymatically active). Both forms of the schistosome enzyme induced significant proliferation of splenocytes recovered from vaccinated mice, and expression of interferon (IFN)-gamma, interleukin (IL)-4 and IL-10 mRNA in these cells was detected using reverse transcriptase-polymerase chain reaction. Secretion of IFN-gamma, IL-4 and IL-10 by splenocytes from vaccinated mice was confirmed and quantified using enzyme-linked immunosorbent assay. IFN-gamma was the most abundant cytokine produced, followed by IL-4 and IL-10 in rank order. These findings indicated that vaccination of mice with the schistosome protease induces a mixed Th1/Th2 cytokine response, which may explain the modest level of protection after challenge infection in cathepsin D-vaccinated mice, reported previously.
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PMID:Cellular responses to Schistosoma japonicum cathepsin D aspartic protease. 1216 22

Differential techniques have revealed several novel genes and peptides involved in trophoblast development including PL74/gdf15/MIC-1, a TGFbeta family cytokine that controls apoptosis and differentiation, PL48, a new serine-threonine protein kinase, serum and glucocorticoid-induced kinase, PBK-1, a tunicamycin-responsive gene, a cathepsin D-like gene (DAP-1) and hypoxia- regulated genes HRF-1,2,6,8 and HIF-1alpha, HIF-1beta, and hEPAS-1. Syncytin, a cell fusion- inducing gene, has been cloned from placenta where it regulates cell fusion. ERV-3 has also been demonstrated to promote cell fusion. These two genes represent the first demonstrated functions of endogenous retroviral sequences in human tissues. Endoglin, PlGF, TGFbeta3, IGF-II, IGFBP-1, and a placental IGFBP protease have found new roles in regulating cytotrophoblast proliferation and invasiveness. A specific placental p105 rasGAP protein has been identified. The homeobox genes DLX4, HB24, MSX2 and MOX2 also likely play a role in development at the epithelial-mesenchymal boundary. Transcription factors such as TEF-5, Hand1, HEB, HASH-2 and two genes represented by ESTs may have regulatory roles in placental development. Evidence suggests that the placenta has an unusual two-cell system for apoptosis regulation in which the cytotrophoblast may direct later apoptotic events in the syncytium, and with syncytialization possibly triggered by the "phosphatidylserine flip". Thus, the placenta is both a rich source of new growth-regulatory substances, and a model system for originating new paradigms of developmental biology.
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PMID:Life and death in the placenta: new peptides and genes regulating human syncytiotrophoblast and extravillous cytotrophoblast lineage formation and renewal. 1236 35

In several 'in vitro' models of apoptosis, lysosomal proteolysis has been shown to play an active role in mediating the death signal by cytokines or antiblastic drugs. Depending on the experimental cell model and the cytotoxic stimulus applied, an increased expression and the cytosolic translocation of either cathepsin D or B have been reported in apoptotic cells. We have analysed the involvement of these lysosomal proteases in a canonical apoptotic cell model, namely L929 fibroblasts, in which apoptosis was induced by cytotoxic agents acting through different mechanisms: (i) the cytokine TNFalpha, which triggers the cell suicide via interaction with its membrane receptor, and (ii) the topoisomerase II-inhibitor etoposide (VP16), which directly causes DNA damage. In both cases the activity of cathepsins B and D increased in apoptosing cultures. CA074-Me, a specific inhibitor of cathepsin B, and Leupeptin, a broad inhibitor of serine and cysteine proteases (among which is cathepsin B), did not exert any protection from TNFalpha. In contrast, pre-loading the cells with pepstatin A, a specific inhibitor of cathepsin D, protected L929 cells from TNFalpha cytotoxicity by more than 50%. However, no protection was observed if pepstatin A was added concomitantly with the cytokine. Inhibition of either cathepsin B or D did not impede apoptosis induced by etoposide. Lysosomal integrity was preserved and cathepsin D remained still confined in vesicular structures in apoptotic cells treated with either TNFalpha or etoposide. It follows that proteolysis by cathepsin D is likely to represent an early event in the death pathway triggered by TNFalpha and occurs within the endosomal-lysosomal compartment.
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PMID:Endosomal-lysosomal proteolysis mediates death signalling by TNFalpha, not by etoposide, in L929 fibrosarcoma cells: evidence for an active role of cathepsin D. 1243 11

The effects of grepafloxacin on the release of cytokines, chemical mediators, hydrolytic enzyme activities, and lipoxygenation in zymogen A- or Staphylococcus aureus-stimulated human THP-1 monocytes were evaluated. Initially, consistent with stimulation of phagocytic mechanisms of the monocytes, increases in cyclic adenosine monophosphate (cAMP) release, nitric oxide [NO] release, and hydrogen peroxide [H(2)O(2)] release, with a small decrease in cellular pH, occurred within 2 h. Enzymatic activities associated with oxygen burst of phagocytic cells (e.g., protein kinase C and nicotinamide adenine dinucleotide phosphate, reduced (NADPH) oxidase) were elevated, suggesting that monocytes attempted to destroy the invading organism through an innate phagocytic cidal immunologic mechanism. After 1-2 h of exposure to grepafloxacin, the oxygen burst and the release of proinflammatory cytokines and chemical mediators were suppressed. After 4 h, suppression of n-acetyl glucosaminidase (NAG) and cathepsin D activities and lipid peroxidation occurred, suppressing the pathogen-induced spread of infection and inflammation. Release of tumor necrosis factor (TNFalpha), interleukin (IL)-1, IL-6, and IL-8 was inhibited by grepafloxacin in a concentration-dependent manner, suggesting a reduction in the acute-phase inflammatory responses initiated by cytokine release from monocytes. Later, S. aureus were killed through inhibition of DNA synthesis, consistent with a bacteriostatic effect. Drug action against invading organisms appears to occur through multiple processes. Modulation of the innate immune system occurs within the first hour, causing the activation of cytokines, chemical mediators, and hydrolytic enzymes. A second phase between 2-4 h appears to involve the suppression of cellular components involved in inflammation and the spread of the infection. The third response, an apparent bacteriostatic inhibition of DNA synthesis, causes bacterial death.
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PMID:In-vitro anti-inflammatory and immunomodulatory effects of grepafloxacin in zymogen A- or Staphylococcus aureus-stimulated human THP-1 monocytes. 1282 12


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