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:3.4.21.69 (APC)
16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human protein C (HPC) undergoes several post-translational modifications, including gamma-carboxylation, N-linked glycosylation, and internal proteolytic processing. We have utilized a recombinant human kidney cell line (293) secreting correctly modified HPC (rHPC) to study the processing reactions for the modification of this complex protein. gamma-Carboxylation was shown to proceed via a vitamin K-dependent pathway and was required for both efficient secretion and anticoagulant activity. rHPC was rapidly secreted following the addition of vitamin K to depleted cells, and secretion was not inhibited by cyclohexamide indicating that non gamma-carboxylated rHPC accumulates as an intracellular releasable pool. However, in cells grown in the presence of vitamin K, the majority of intracellular rHPC was gamma-carboxylated, suggesting that this post-translational modification is not rate limiting for secretion under conditions optimal for vitamin K-dependent carboxylation. Nonglycosylated rHPC was found to be secreted inefficiently, and processing of the N-linked core in the endoplasmic reticulum, but not in the Golgi, was required for secretion. Further, the intracellular rHPC present in vitamin K-supplemented cells was core glycosylated, but not processed past the high mannose step. gamma-Carboxylation occurred after core glycosylation, indicating that this modification is not cotranslational. Further, glycosylation and gamma-carboxylation were not coupled and did not need to proceed sequentially. Proteolytic processing of the internal KR dipeptide was found to occur late in the secretion pathway, and the cleavage was calcium-dependent. The secretion rate of rHPC was also calcium-dependent but was independent of the calcium effect on internal KR dipeptide removal, indicating that cleavage is not required for efficient secretion. Our results define the sequence of processing events, the subcellular localization of the processing reactions, and the rate-limiting steps in the secretion pathway for this complex protein.
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PMID:Post-translational processing events in the secretion pathway of human protein C, a complex vitamin K-dependent antithrombotic factor. 152 91

Surfactant protein C (SP-C) is a 3.7 kDa, hydrophobic protein that enhances the adsorption of phospholipids in pulmonary surfactant. SP-C is generated by proteolytic processing of a 21 kDa precursor. Murine fetal lung explant cultures and a Chinese hamster ovary cell line expressing recombinant human SP-C gene (CHO/SPC) were used to determine the subcellular location and post-translational modification(s) of proSP-C. After in vitro translation, proSP-C of Mr = 21,000 was generated. ProSP-C was associated with canine pancreatic microsomes during co-translation and was partially protected from digestion with proteinase K, supporting the concept that proSP-C enters but does not completely traverse the membrane of the endoplasmic reticulum (ER). Association of proSP-C isoforms of 21 and 26 kDa with intracellular membranes was demonstrated by subcellular fractionation of CHO/SPC cells. Pulse/chase experiments demonstrated that the 21 kDa SP-C proprotein was synthesized first and after 15 min was modified to produce a 26 kDa isoform in CHO/SPC cells or a 24 kDa isoform in murine fetal lung. Both the 21 and 26 kDa proSP-C isoforms were detected after labelling CHO/SPC cells with [3H]palmitic acid. The formation of the 26 kDa proSP-C isoform in CHO/SPC cells and the 24 kDa proSP-C isoform in murine fetal lung was blocked by cerulenin, an inhibitor of fatty acid synthesis. In conclusion, proSP-C is associated with subcellular membranes. ProSP-C is palmitoylated and undergoes additional post-translational modification that is blocked by an inhibitor of fatty acid synthesis.
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PMID:Surfactant protein C precursor is palmitoylated and associates with subcellular membranes. 156 93

The presence of nineteen blood coagulation factors and fibrinolysis factors was immunohistochemically evaluated in human lymph node germinal centers (GCs). Twelve of these factors were detected within lymphoid GCs. The predominant pattern was dendritic with occasional crescent-shaped, ring-shaped or 'moth-eaten' appearance. Immunostains of factor VIII-related antigen, factor I, protein C, tetranectin, antithrombin III, type 2-plasminogen activator inhibitor, and alpha 2-plasmin inhibitor were almost entirely absent from GCs, although they reacted in vascular wall and lumen, respectively. The immunostaining to high molecular weight kininogen, kallikrein, factors XII, X, V, II, XIIIa, XIIIs, plasminogen, tissue-plasminogen activator, and type 1-plasminogen activator inhibitor more frequently revealed a positive dendritic pattern. Immuno-electron microscopy demonstrated factor X and factor XIIIa attached to the cell surfaces of lymphocytes, macrophages, and follicular dendritic cells (FDCs); and in the intercellular space within GCs, especially attached to the labyrinthine-like structure of FDCs. No reaction products were observed in the perinuclear cisternae and rough endoplasmic reticulum in either lymphocytes or FDCs. Our data demonstrate that human lymphoid GCs really contain some of the proteins related to the blood coagulation and fibrinolysis cascades.
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PMID:Localization of blood coagulation factors and fibrinolysis factors within lymphoid germinal centers in human lymph nodes. 168 Aug 35

We have shown previously that specific Ag presentation is prevented by the inhibition of protein synthesis but nonspecific presentation is not. In the present paper, Ag presentation by Ag-specific B cells was examined for sensitivity to brefeldin A (BFA), which blocks protein export from the endoplasmic reticulum. A20-HL B lymphoma expressing surface receptors specific for TNP was used as a B cell, and TNP-OVA was used as a specific Ag. The presence of BFA during pulsing of A20-HL cells with TNP-OVA inhibited the ability of the pulsed cells to stimulate 42-6A T cell clone, specific for OVA323-339 and Iad. The inhibition was not due to nonspecific toxicity of BFA, because the presence of BFA during pulsing of A20-HL cells with OVA323-339 did not affect their APC function. Ag binding to the receptor on A20-HL cells and internalization by the cells were observed in the presence of BFA. Thus, BFA might inhibit intracellular processing of specific Ag or intracellular complex formation of antigenic peptide from specific Ag with MHC class II molecules. Nonspecific Ag presentation by A20-HL cells, however, was resistant to BFA. A20-HL cells pulsed with OVA in the presence of BFA, even after fixation, could stimulate 42-6A cells to produce IL-2, although the IL-2 production was lower than that induced by A20-HL cells pulsed in the absence of BFA. These results suggest that the processing pathways for specific Ag and nonspecific Ag are different from each other, at least partly, in A20-HL cells.
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PMID:Inhibition by brefeldin A of the specific B cell antigen presentation to MHC class II-restricted T cells. 194 Mar 37

The protein C-protein S anticoagulant pathway is closely linked to the endothelium. In this paper the synthesis and release of the vitamin K-dependent coagulation factor protein S is demonstrated. Western blotting, after SDS PAGE of Triton X-100 extracts of bovine aortic endothelial cells grown in serum-free medium, demonstrated the presence of protein S. A single major band was observed at Mr approximately 75,000, closely migrating with protein S purified from plasma absent from cells treated with cycloheximide. Metabolic labeling of endothelial cells with [35S]methionine confirmed de novo synthesis of protein S. Using a radioimmunoassay, endothelium was found to release 180 fmol/10(5) cells per 24 h and contain 44 fmol/10(5) cells of protein S antigen. Protein S released from endothelium was functionally active and could promote activated protein C-mediated factor Va inactivation on the endothelial cell surface. Warfarin decreased secretion of protein S antigen by greater than 90% and increased intracellular accumulation by almost twofold. Morphological studies demonstrated intracellular protein S was in the Golgi complex, concentrated at the trans face, rough endoplasmic reticulum, lysosomes, and in vesicles at the periphery. In contrast, protein S was not found in vascular fibroblasts or smooth muscle cells. A pool of intracellular protein S could be released rapidly by the calcium ionophore A23187 (5 microM). This effect was dependent on the presence of calcium in the culture medium and could be blocked by LaCl3, which suggests that cytosolic calcium flux may be responsible for protein S release. These results demonstrate that endothelial cells, but not the subendothelial cells of the vessel wall, can synthesize and release protein S, which indicates a new mechanism by which the inner lining of the vessel wall can contribute to the prevention of thrombotic events.
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PMID:Participation of endothelial cells in the protein C-protein S anticoagulant pathway: the synthesis and release of protein S. 293 94

Virus particles, lacking the spike G-glycoproteins, are produced during infection of Vero cells with the vesicular stomatitis virus mutant ts045 at the restrictive temperature 39.5 degrees C. At this temperature the mutated G proteins are blocked in their intracellular transport in the endoplasmic reticulum. We have studied the role of the G proteins in the formation of these spikeless virus particles. The results showed that the spikeless particles contain a full complement of membrane anchors, derived from the carboxy-terminal end of the G protein. Our observations suggest that virus particles are formed at the restrictive temperature with G protein which is later cleaved to produce spikeless particles. We suggest that this is due to a leak of G protein to the cell surface at 39.5 degrees C where budding then takes place, presumably driven by a G protein C-terminal tail--nucleocapsid interaction.
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PMID:The budding mechanism of spikeless vesicular stomatitis virus particles. 301 69

Warfarin, an antagonist of vitamin K, is known to disrupt the microsomal vitamin K cycle, which results in a decrease in the plasma level of protein C, an anticoagulant factor, as well as some other vitamin K-dependent coagulation factors. Here, we examined the effect of warfarin on the secretion of recombinant protein C expressed in human kidney 293 or BHK cells. In transient expression, warfarin caused a 2-4-fold decrease in the quantity of protein C secreted, compared to findings with vitamin K-treated cells. Pulse-chase experiments using stable cells showed that, although recombinant protein C was secreted in the presence of vitamin K, the decrease in the total amount of radioactivity in the warfarin-treated cells suggested intracellular degradation. This degradation depended on the concentration of warfarin and was not inhibited by an endoplasmic reticulum (ER)-Golgi transport inhibitor (brefeldin A) or by lysosomotropic inhibitors (chloroquine and NH4Cl). Thus, protein C synthesized in the presence of warfarin is probably selectively degraded, and this degradation occurs in a pre-Golgi, nonlysosomal compartment. Among the protease inhibitors tested, N-alpha-acetyl-Leu-Leu-methioninal and N-alpha-acetyl-Leu-Leu-norleucinal blocked the degradation of protein C precursor synthesized in the presence of warfarin, and the precursor accumulated intracellularly, in a dose-dependent manner. Both inhibitors, however, did not disturb the secretion of protein C precursor in the vitamin K-treated cells. Thus, a cysteine protease(s) appeared to be responsible for the degradation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Warfarin causes the degradation of protein C precursor in the endoplasmic reticulum. 782 66

Among the self antigens, immunoglobulins, and in particular idiotypes, are of special interest because of their extreme sequence heterogeneity and their postulated involvement in regulatory interactions in the immune system. We have therefore studied antigen processing and presentation of variable region peptides, processed idiotypes, to MHC class II molecule-restricted T cells. The immunoglobulin used has been the lambda 2(315) light chain produced by the BALB/c MOPC 315 plasmacytoma (alpha, lambda 2). The minimum length of a stimulatory synthetic idiotypic peptide comprises residues 91-101 of lambda 2(315) and is presented by the I-E(d) molecule to CD4+ T cells. T cell clones with specificity for the 91-101(lambda 2(315))/I-E(d) complex utilize a limited TCR repertoire and are of both Th1 and Th2 type. For presentation, extracellular lambda 2(315) requires endocytosis and processing, as previously described for conventional exogenous antigens. In addition, a B lymphoma cell can process and present its own endogenous lambda 2(315). This was shown by transfecting manipulated lambda 2(315) gene variants into B lymphoma cells, followed by evaluation of the APC function of the transfectants. These studies demonstrated that surface expression or secretion of lambda 2(315) is not necessary for presentation and suggested that the endoplasmic reticulum may be a processing compartment. To extend our findings to naive Id+ B cells and anti-Id T cells, we have generated lambda 2(315)-transgenic as well as TCR-transgenic mice. A model is presented for a T-B cell interaction based on presentation of processed idiotypes.
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PMID:Processing and presentation of idiotypes to MHC-restricted T cells. 829 47

T lymphocytes are activated upon binding of their Ag receptors to a complex of Ag-derived peptides and MHC class I or class II molecules expressed on the surface of APC. It is now well established that APC degrade exogenous Ag in acidic endosomal compartments, and that Ag fragments bind to class II molecules moving through these compartments on their way to the surface of the APC. Although peptides derived from some endogenous Ag can also bind to class II molecules and subsequently be recognized by class II-restricted T cells, the intracellular trafficking pathways that enable endogenous proteins to be processed for association with class II molecules remain controversial. We have analyzed the mechanism by which the envelope (env) protein of the HIV-1 is processed in infected cells for recognition by class II-restricted T cells. A large number of env-specific class II-restricted human CTL clones were shown to lyse B-lymphoblastoid cell lines expressing the env. A novel dilutional assay proved that A novel dilutional assay proved that recognition of endogenous env protein was not a consequence of release and re-uptake of the env protein and subsequent processing by the standard class II-restricted pathway. Processing of endogenous env protein required that the protein be co-translationally translocated into the endoplasmic reticulum (ER) and then exit the ER, since the class II-restricted CTL did not recognize env protein localized to the cytosol or retained in the ER of target cells. Under these conditions, however, class I-restricted recognition was readily demonstrated. Finally, class II-restricted recognition was strikingly dependent upon the steady state level of surface env protein, since extracellular reagents that removed intact env protein from the surface of target cells inhibited recognition. This inhibition operated at the Ag-processing level rather than at the level of subsequent Ag recognition. These results provide the first direct evidence that endogenously synthesized membrane proteins enter the class II-restricted Ag-processing pathway after expression on the cell surface in an intact form.
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PMID:HIV-1 envelope protein is expressed on the surface of infected cells before its processing and presentation to class II-restricted T lymphocytes. 837 62

A cDNA encoding a form of hen egg lysozyme (HEL) lacking a leader sequence and predicted to be localized in the cytoplasm, was transfected into MHC class II-positive B lymphoma cells. Cytoplasmically expressed HEL (cytHEL) had a half-life of less than 5 min and did not react with HEL specific mAb suggesting non-native conformation. Cells expressing cytoplasmic HEL, as well as cells previously reported to express a low level of HEL retained in the endoplasmic reticulum (ERHEL), constitutively presented the HEL determinant encoded by residues 46-61 to a sensitive class II-restricted T hybridoma (3A9). Constitutive presentation of HEL determinants was not detectable in cytHEL or ERHEL transfectants using T hybridomas with lower sensitivity to exogenous Ag. Constitutive presentation of HEL46-61 derived from cytoplasmic HEL was demonstrable in multiple transfected clones and was most obvious when a CMV rather than SV40 promoter was used to express the cytHEL gene. The presentation of HEL46-61 by cytHEL transfectants was not due to HEL reuptake by bystander cells because there was no biochemical evidence of cytHEL shedding and cytHEL supernatants added to indicator APC did not result in HEL46-61 presentation. Constitutive presentation of endogenous HEL46-61 by the cytHEL and ERHEL transfectants was inhibited by chloroquine, and recovery of presentation of endogenous HEL was slower in cytHEL compared with ERHEL transfectants. The findings indicate that class II-restricted presentation of Ag retained in the cytoplasm or endoplasmic reticulum does take place but probably requires abundant levels of intracellular Ag and is easily disrupted by lysosomotropic agents. These pathways of presentation may be important when high levels of foreign endoplasmic reticulum-retained or cytoplasmic Ag are present (e.g., viral infection), and during the acquisition of self-tolerance by highly sensitive developing T cells.
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PMID:Class II-restricted presentation of a hen egg lysozyme determinant derived from endogenous antigen sequestered in the cytoplasm or endoplasmic reticulum of the antigen presenting cells. 847 26


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