Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Ad2 E3-10.4K protein is required together with the E3-14.5K protein to down-regulate the epidermal growth factor receptor in adenovirus-infected cells. Both proteins are also required to prevent tumor necrosis factor cytolysis under certain conditions. 10.4K is a 91 amino acid membrane-associated protein that migrates as two bands, upper and lower, on SDS-PAGE. We show here that the upper band is the primary translation product which initiates at AUG2173 in the E3 transcription unit of Ad2. The upper band is processed slowly (greater than 4 hr to complete) into the lower band by proteolytic cleavage between residues Ala22 and Ala23 by a microsome-associated protease. The upper and lower bands become equal in abundance, after which they are very stable. The N-terminus of the in vivo-derived upper band is not blocked to sequencing and it retains its initiating Met. 10.4K has a hydrophobic domain (H1) near its N-terminus that is probably a signal sequence for membrane insertion; cleavage of this signal is atypical because it was not cotranslational in vivo and it was not complete. 10.4K has a second hydrophobic domain (H2) located within residues 35-60. H2 appears to be a transmembrane (stop transfer) domain because both the upper and the lower 10.4K bands remained associated with membranes after extraction at pH 11.5, because both bands were extracted into the detergent phase with Triton X-114, and because both bands were only partially reduced in size when 10.4K-containing microsomes were digested with proteinase K. These proteinase K-digested bands were immunoprecipitated with an antipeptide antiserum against residues 19-34 but not with an antiserum against residues 68-80 or 77-91, indicating that both 10.4K bands are orientated in the membrane with the C-terminus in the cytoplasm. We conclude that the lower band of 10.4K is a type I bitopic membrane protein and suggest that the upper band is a polytopic membrane protein with both the H1 and the H2 hydrophobic domains spanning the membrane.
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PMID:The E3-10.4K protein of adenovirus is an integral membrane protein that is partially cleaved between Ala22 and Ala23 and has a Ccyt orientation. 153 Dec 78

We previously reported that the adenovirus type 5 E3 14.5-kilodalton protein (14.5K) forms a complex with E3 10.4K and that both proteins are required to down-regulate the epidermal growth factor receptor in adenovirus-infected human cells. Both proteins are also required to prevent cytolysis by tumor necrosis factor of most mouse cell lines infected by adenovirus mutants that lack E3 14.7K. The E3 14.5K amino acid sequence suggests that 14.5K is an integral membrane protein with an N-terminal signal sequence for membrane insertion. Here we show that 14.5K was found exclusively in cytoplasmic membrane fractions. Radiochemical sequencing of 14.5K indicated that the N-terminal signal sequence is cleaved predominantly between Cys-18 and Ser-19. With a mutant that does not express 10.4K, cleavage occurs predominantly between Phe-17 and Cys-18, indicating that the presence or absence of 10.4K affects the signal cleavage site. 14.5K was extracted into the detergent phase with Triton X-114, it remained associated with membranes after extraction with Na2CO3 at pH 11.5, and it was partially protected by membranes from proteinase K digestion; these observations indicate that 14.5K is an integral membrane protein. Proteinase K digestion followed by immunoprecipitation with antipeptide antisera directed against the N or C terminus of mature 14.5K indicated that 14.5K is oriented in the membrane with its N terminus in the lumen and its C terminus in the cytoplasm. Thus, 14.5K is a type I bitopic membrane protein. Previous studies indicated that 10.4K is also an integral membrane protein oriented with its C terminus in the cytoplasm. Altogether, these findings suggest that cytoplasmic membranes are the site of action when 10.4K and 14.5K down-regulate the epidermal growth factor receptor and prevent tumor necrosis factor cytolysis.
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PMID:The adenovirus E3 14.5-kilodalton protein, which is required for down-regulation of the epidermal growth factor receptor and prevention of tumor necrosis factor cytolysis, is an integral membrane protein oriented with its C terminus in the cytoplasm. 153 70

Monocytes lysing a variety of tumor cells were isolated by adhesion to autologous serum-coated plastic surfaces. When the blood monocytes were co-cultured with K562 cells for 3-24 h, the supernatants contained soluble factors, termed monocyte cytotoxic factors (MCF), capable of lysing K562 and other tumor cells in a 48-h microcytotoxicity assay. The production of MCF was mediated by typical monocytes expressing a surface phenotype of CD11 (+), CD16 (-), LeuM1 (+). When target cells were pretreated with actinomycin D, they showed an increase in their susceptibility to lysis by MCF. Addition of the drug to MCF assays also resulted in an enhancement of MCF-mediated lysis. Thus, the lytic activity of MCF was detectable in an 18-h assay. The presence of interferon (IFN)-alpha or -gamma augmented the biological activity of MCF, while pretreatment of target cells with IFN did not enhance MCF activity. The absorption of MCF activity was not elevated by actinomycin D or IFN. MCF lysed target cells that were resistant to tumor necrosis factor (TNF). One result of importance is that MCF lysed autologous and allogeneic freshly isolated human tumor cells. The lysis of fresh human tumor cells by MCF was not inhibited by monoclonal antibodies directed against TNF, lymphotoxin (LT), IFN-alpha, IFN-gamma, or interleukin 1 (IL-1). Furthermore, TNF, LT, IFNs, and IL-1 did not kill fresh human tumor cells. MCF activity was stable at low temperatures but was destroyed by heating. The biological activity of MCF was reduced or abolished by serum, trypsin, chymotrypsin and proteinase K, indicating the proteinaceous nature of MCF. The lytic activity was resistant to protease inhibitors. These data indicate that MCF is a noble cytokine that acts on human fresh tumor cells.
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PMID:[Production and function of the monocyte cytotoxic factor (MCF)]. 244 Mar 86

Marked tumor-regressing activity was induced in the serum of S180 tumor-bearing mice by injection of an antitumor polysaccharide, CM-TAK [carboxymethylated beta(1-3)glucan]. Maximal activity was induced 7-14 days after the tumor transplantation and 10-12 h after CM-TAK treatment. A quantitative assay for the activity was established on the basis of the initial decrease in the number of the tumor cells within 24 h. The factor with tumor-regressing activity was purified 10,000-fold by the series of hydroxylapatite chromatography, ammonium sulfate precipitation, anion-exchange chromatography, gel filtration, and boronate-mediated affinity chromatography. The molecular weight was estimated to be 250,000 by gel filtration. The activity was proteinase K sensitive, but relatively resistant to trypsin. Neuraminidase did not affect the activity. It is believed that the tumor-regressing factor is different from the tumor necrosis factor.
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PMID:Antitumor polysaccharide-induced tumor-regressing factor in the serum of tumor-bearing mice: purification and characterization. 372 39

Mycoplasma fermentans is one of several Mycoplasma species that have been reported to stimulate tumor necrosis factor (TNF) secretion from monocytes. This activity has been associated primarily with the mycoplasma membrane fraction. In this article, we have characterized a membrane protein that stimulates TNF and interleukin 1 beta secretion. The TNF-releasing activity partitioned into the Triton X-114 detergent phase, suggesting that the molecules is hydrophobic. The secretion of TNF is elevated in the presence of serum, which suggests that a serum component may play a role in the interaction between this mycoplasma protein and monocytes. Treatment of monocytes with monoclonal anti-CD14 antibody had no effect on the levels of TNF-releasing activity. By using the monocyte Western blot (immunoblot) technique, we have determined the molecular mass of the active molecule to be 48 kDa. This molecule appears to be distinct from the recently described family of variable lipoproteins of M. fermentans. Mycoplasma particulate material treated with proteinase K lost all inducing activity, whereas lipoprotein lipase-treated samples retained some level of activity.
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PMID:A 48-kilodalton Mycoplasma fermentans membrane protein induces cytokine secretion by human monocytes. 752 Apr 21

Mycoplasmal products may exert a number of diverse in vitro effects on cells of the immune system. A macrophage-activating substance from Mycoplasma fermentans was described in this laboratory and named mycoplasma-derived high-molecular-weight material (MDHM). Using synthesis of nitric oxide by peritoneal cells from endotoxin low-responder mice as an assay system, MDHM was purified as follows. After freeze-thawing of M. fermentans, MDHM activity was sedimented with the membrane fraction. Membranes were delipidated with chloroform-methanol, and MDHM activity was extracted with octyl glucoside. Coextracted proteins were degraded by proteinase K. MDHM was further purified by reversed-phase high-pressure liquid chromatography and eluted in one major and one minor peak of activity. Neither carbohydrates nor amino acids were found as constituents. MDHM had the following properties: it partitioned into the phenol phase upon phenol-water extraction and into the Triton phase after extraction with Triton X-114. MDHM was not inactivated by either phospholipase A2 or triglyceride lipases. However, mild periodate treatment led to a > 95% loss of activity. Also, alkaline hydrolysis at 25 degrees C completely abolished MDHM activity with a half-life of 2 min. MDHM activity was spread out over a wide molecular weight range upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes, whereas after proteinase treatment MDHM activity migrated close to the front. These features of MDHM, taken together, speak in favor of an amphiphilic molecule with a lipid moiety carrying fatty acids in ester linkage and a polyol moiety of unknown character. MDHM was active in the nanogram-per-milliliter range, activating macrophages to release nitric oxide, interleukin-6, and tumor necrosis factor.
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PMID:Purification and partial biochemical characterization of a Mycoplasma fermentans-derived substance that activates macrophages to release nitric oxide, tumor necrosis factor, and interleukin-6. 806 96

Blood lymphocytes of cancer patients lysed autologous, freshly isolated tumor cells. The autologous tumor-killing (ATK) activity is strongly associated with postoperative clinical course, indicating that ATK is a meaningful prognostic indicator and provides evidence for immunological control of tumor growth and metastasis. Large granular lymphocytes (LGL) with ATK activity released a soluble cytotoxic factor(s), termed LGL-CF (LGL-derived cytotoxic factor) during interaction with autologous tumor cells. The cytotoxic factor lysed autologous and allogeneic freshly isolated human tumor cells, while they were resistant to any of recombinant cytokines, including tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN) alpha, IFN gamma, interleukin (IL)-1 alpha and IL-2. Biological activity of LGL-CF was not abrogated by monoclonal and polyclonal antibodies against these cytokines. LGL-CF also exhibited lysis of a variety of tumor cell lines, but not of nonmalignant cells. Actinomycin D augmented the lysis of LGL-CF. LGL-CF was stable at 56 degrees C, but was destroyed at 100 degrees C. Treatment of LGL-CF with trypsin or proteinase K reduced or abrogated the lytic effect, respectively, while it was resistant to papain, catalase, and superoxide dismutase. These results indicate that LGL produce a novel cytotoxic factor in response to autologous tumor cells that mediates lysis of fresh human tumor cells.
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PMID:Role of NK cell cytotoxic factor against fresh human tumors. 825 31

To identify the mediators that stimulate periapical bone resorption following infection, a rat model system was used in which active (rapid) and chronic (slow) phases of bone destruction can be distinguished. Extracts of inflammatory tissues from active lesions contained high levels of bone-resorbing activity, which was destroyed by proteinase K and heat (70 degrees C), but was unaffected by polymyxin B, indicating the presence of protein mediator(s) rather than lipopolysaccharide. Fast-performance liquid chromatography gel filtration of extracts of active lesions demonstrated that most activity was associated with macromolecules of MW 30-60 kDa and 15-20 kDa, consistent with bone resorptive cytokines, including interleukin 1 (IL-1) and tumor necrosis factor (TNF). Inhibition with cytokine-specific antisera demonstrated that resorbing activity in active lesions was significantly neutralized by anti-IL-1 alpha, whereas anti-IL-1 beta, anti-TNF alpha and anti-TNF beta had only slight effect. A lower amount of resorbing activity was present in extracts of chronic lesions, which was also neutralized only by anti-IL-1 alpha. Inflammatory tissue explants produced more IL-1 alpha than IL-1 beta in vitro, confirming findings with extracts, and high levels of IL-1 alpha were present in active lesions by radioimmunoassay. These data indicate that bone resorption stimulated by bacterial infection is primarily mediated by IL-1 alpha in this model. The similarity of cytokines in active and chronic lesions suggests that quantitative rather than qualitative differences in these mediators may account for lesion progression.
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PMID:The role of interleukin-1 alpha in the pathogenesis of periapical bone destruction in a rat model system. 851 Sep 85

We have developed enhanced immunohistochemical protocols for detecting autonomic nerve fibers and splenocyte-associated proteins in rat spleen. This includes norepinephrine-synthesizing enzymes (dopamine-beta hydroxylase (DBH) and tyrosine hydroxylase (TH)), neuropeptide Y (NPY), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), c-fos protein, inducible nitric oxide synthase (iNOS), and the macrophage cell marker ED1. Animals were divided into sham-operated and splenic nerve-sectioned groups for detection of DBH, TH, and NPY. For immunodetection of TNF-alpha, iNOS, IFN-gamma and c-fos, animals were injected IV with saline or 100 microg of lipopolysaccharide (LPS) and were sacrificed at various time intervals post injection. Rats were perfused with 4% paraformaldehyde, spleens removed and cryoprotected, and 50-microm floating sections were cut on a freezing microtome. Immunodetection was performed with various detection systems and substrate/chromogen solutions, and in some cases using pretreatment with proteinase K (PK) for antigen unmasking. PK pretreatment increased immunostaining for DBH, TH, NPY, IFN-gamma, iNOS, and ED1, and the improvement was concentration-dependent. Using NPY immunostaining to index the signal-to-noise ratio for various substrates and detection systems, we found that an alkaline phosphatase detection system with NBT/BCIP as a substrate was the best procedure for light microscopy, whereas the CY3-labeled secondary antibody technique proved optimal for fluorescent microscopy. Surgical transection of the splenic nerve eliminated all nerve fiber staining for DBH, TH, and NPY. TNF-alpha, IFN-gamma, c-fos, and iNOS proteins were observed in the spleen in a time-dependent manner after LPS stimulation. Fluorescent double labeling, visualized with fluorescent confocal scanning laser microscopy, revealed many NPY fibers distributed among the ED1-labeled macrophages. These results demonstrate that immunohistochemistry can be used to index the activational effects of an immune challenge on splenocytes in situ and verifies that splenic immune cells are innervated by the sympathetic nervous system.
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PMID:Enhanced immunohistochemical detection of autonomic nerve fibers, cytokines and inducible nitric oxide synthase by light and fluorescent microscopy in rat spleen. 911 Dec 38

LeIF, a gene homologue of the eukaryotic initiation factor 4A was first described as a leishmanial antigen that induced a Th1-type T cell response in peripheral blood mononuclear cells (PBMC) from leishmaniasis patients. Moreover, the interferon (IFN)-gamma production by PBMC was found to be interleukin (IL)-12 dependent. Herein, we characterize the effects of LeIF on cytokine production and expression of surface molecules by normal human monocytes as well as by monocyte-derived macrophages and dendritic cells (MoDC). LeIF was a strong inducer of IL-12 and, to a lesser extent, of IL-10 and tumor necrosis factor (TNF)-alpha in macrophages and MoDC. IL-12 production did not require CD40 triggering, confirming that the ability of LeIF to induce IL-12 was not mediated through an effect on T cells. However, addition of soluble CD40 ligand (L) synergistically augmented IL-12 production in macrophages and MoDC. The cytokine-inducing activity of LeIF is located in the N-terminal portion of the molecule and was both proteinase K sensitive and polymyxin B resistant. LeIF, lipopolysaccharide and fixed Staphylococcus aureus all induced comparable amounts of IL-12, validating the potent cytokine-inducing effects of LeIF. Moreover, of these stimuli, LeIF had the highest IL-12/IL-10 and IL-12/TNF-alpha ratio demonstrating the preference of LeIF for IL-12 induction. Studies investigating the expression of surface molecules showed that LeIF up-regulated B7-1 and CD54 (ICAM-1) on macrophages and MoDC. To our knowledge this is the first report describing IL-12 production, up-regulation of co-stimulatory and intercellular adhesion molecules by monocytic antigen-presenting cells in response to a protein from a pathogenic microorganism. These immunomodulatory characteristics of LeIF might be excellent properties for a Th1-type adjuvant.
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PMID:A Leishmania protein that modulates interleukin (IL)-12, IL-10 and tumor necrosis factor-alpha production and expression of B7-1 in human monocyte-derived antigen-presenting cells. 936 20


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