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: UMLS:C0038362 (stomatitis)
8,852 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Endogenous processing of viral glycoproteins for presentation to CD4+T cells is a poorly investigated aspect of antigen processing and presentation. This pathway may involve not only pathogens, but also self proteins, and may thus be involved in self-tolerance. We have characterized the processing of the endoplasmic reticulum-restricted glycoprotein (G) of vesicular stomatitis virus, termed poison tail (Gpt), biochemically and enzymatically, and by T cell recognition assays. Expressed with a vaccinia vector, Gpt remains endoglycosidase H-sensitive and does not mature to endoglycosidase D sensitivity. The protein is degraded in the ER with a T1/2 of 4 h. Gpt peptides are not secreted since Gpt-infected cells are unable to sensitize uninfected antigen-presenting cells in an innocent bystander assay. Using flow cytometry, Gpt is undetectable on the plasma membrane; in contrast, wild-type G is readily found on the surface or secreted into the milieu as soluble G following infection of A20 cells with a vaccinia recombinant expressing G. The degradation of Gpt is sensitive to the thiol reagent diamide and occurs optimally at physiological pH. A series of proteolytic inhibitors were tested: 3,4-dichloroisocoumarin and 1-chloro-3-tosylamido-7-amino-2-heptanone inhibited degradation, which suggests the involvement of a serine protease. The degradation does not require transport to the Golgi complex, and is not sensitive to a variety of lysosomotropic agents. We show that the degradation products include the immunogenic epitopes recognized by a panel of T cell clones and hybridomas.
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PMID:Processing of a viral glycoprotein in the endoplasmic reticulum for class II presentation. 766 84

Rubella virus (RV) envelope glycoproteins, E2 and E1, form a heterodimeric complex that is targeted to medial/trans-Golgi cisternae. To identify the Golgi targeting signal(s) for the E2/E1 spike complex, we constructed chimeric proteins consisting of domains from RV glycoproteins and vesicular stomatitis virus (VSV) G protein. The location of the chimeric proteins in stably transfected Chinese hamster ovary cells was determined by immunofluorescence, immunoelectron microscopy, and by the extent of processing of their N-linked glycans. A trans-dominant Golgi retention signal was identified within the C-terminal region of E2. When the transmembrane (TM) and cytoplasmic (CT) domains of VSV G were replaced with those of RV E2, the hybrid protein (G-E2TMCT+) was retained in the Golgi. Transport of G-E2TMCT+ to the Golgi was rapid (t1/2 = 10-20 min). The G-E2TMCT+ protein was determined to be distal to or within the medial Golgi based on acquisition of endo H resistance but proximal to the trans-Golgi network since it lacked sialic acid. Deletion analysis revealed that only the TM domain of E2 was required for Golgi targeting. Although the cytoplasmic domain of E2 was not necessary for Golgi retention, it was required for efficient transport of VSV G-RV chimeras out of the endoplasmic reticulum. When assayed in sucrose velocity sedimentations gradients, the Golgi-retained G-E2TMCT+ protein behaved as a dimer. Unlike virtually all other Golgi targeting signals, the E2 TM domain does not contain any polar amino acids. The TM and CT domains of E1 were not required for targeting of E2 and E1 to the Golgi indicating that a heterodimer of two integral membrane proteins can be retained in the Golgi by a single retention signal.
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PMID:Targeting of a heterodimeric membrane protein complex to the Golgi: rubella virus E2 glycoprotein contains a transmembrane Golgi retention signal. 774 96

Many soluble resident proteins of the endoplasmic reticulum share a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. Current opinion favours a model in which these proteins can escape from the endoplasmic reticulum (ER) by bulk flow and are recognized and sorted in the Golgi apparatus by binding to a specific KDEL-receptor, which returns them to the ER. Through biochemical, morphological and mutational analysis we have studied the mechanisms that determine the localization of calreticulin, a soluble 60 kDa KDEL-protein of the ER. Immunogold labelling established the ER localization of calreticulin in transfected and nontransfected COS cells. Although the ER cisternae in transfected cells were enormously dilated and heavily labelled by gold particles we found no significant label in any other compartment. In vivo pulse chase experiments with [35S]methionine followed by biochemical fractionation of calreticulin overexpressing COS cells (50- to 100-fold) revealed that only a minor part of labelled calreticulin leaves the ER. Retrieval from the Golgi was confirmed by a partial redistribution of the endogenous KDEL-receptor as shown by double immunofluorescence. These data suggest a KDEL-independent retention of calreticulin in the ER. Further supporting evidence has come from morphological in vivo studies using calreticulin-transfected and vesicular stomatitis virus (ts045)-infected COS cells. Stimulation of vesicular transport from the ER by releasing the temperature-dependent transport block for the viral G-protein resulted in a small but significant appearance of calreticulin in a post-ER compartment. In contrast a calreticulin mutant, which lacked the Ca(2+)-binding domain but included the KDEL sequence, could escape from the ER to a much higher extent. Secretion of the nonmutated calreticulin was very low (1-2% of total calreticulin in 3 hours) compared to the mutated form (18% in 3 hours). Deletion of the KDEL sequence led to an increase in secretion to 29% over a 3 hour period, which is much less than expected for a secretory protein. Taken together these results strongly support the hypothesis of two independently operating retention/retrieval mechanisms for calreticulin: one providing for direct retention in the ER with a very high capacity and having Ca(2+)-dependent properties; the other a KDEL-based retrieval system for escaped calreticulin present in the Golgi apparatus.
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PMID:Retention and retrieval: both mechanisms cooperate to maintain calreticulin in the endoplasmic reticulum. 787 39

Fluorescence recovery after photobleaching (FRAP) has been a powerful tool for characterizing the mobility of cell surface membrane proteins. However, the application of FRAP to the study of intracellular membrane proteins has been hampered by the lack of specific probes and their physical inaccessibility in the cytoplasm. We have measured the mobility of a model transmembrane protein, the temperature-sensitive vesicular stomatitis viral membrane glycoprotein (ts-O45-G), in transit from the endoplasmic reticulum (ER) to the Golgi complex. ts-O45-G accumulates in the ER at nonpermissive temperature (39.5 degrees C) and is transported via the Golgi complex to the surface upon shifting cells to the permissive temperature (31 degrees C). Rhodamine-labeled Fab fragments against a cytoplasmic epitope of ts-O45-G (rh-P5D4-Fabs) were microinjected into cells to visualize the intracellular viral membrane protein and to determine its mobility by FRAP with a confocal microscope. Moreover, we have measured the effects of microinjected antibodies against beta-COP on the mobility of ts-O45-G following release of the temperature block. FRAP was essentially complete when rh-P5D4-Fab-injected cells were bleached either following release of labeled ts-O45-G from the ER or upon its accumulation at 20 degrees C in the trans-Golgi network (TGN). In contrast, recovery was reduced by about one third when infected cells had been injected with antibodies that bind to beta-COP in vivo. The diffusion constant of mobile ts-O45-G under all conditions was approximately 10 x 10(-10) cm2/s. These results validate the feasibility of FRAP for the study of an intracellular transmembrane protein and provide the first evidence that such a protein is highly mobile.
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PMID:The intracellular mobility of a viral membrane glycoprotein measured by confocal microscope fluorescence recovery after photobleaching. 792 37

Cytotoxic T lymphocytes (CTL) recognize antigenic peptides bound to major histocompatibility complex class I antigens on the cell surface of virus-infected cells. It is believed that the majority of peptides originate from cytoplasmic degradation of proteins assumed to be mediated by the "20S" proteasome. Cytosolic peptides are then translocated, presumably by transporters associated with antigen processing (TAP-1 and -2), into the lumen of the endoplasmic reticulum (ER) where binding and formation of the ternary complex between heavy chain, beta2-microglobulin (beta 2m) and peptide occurs. In this study, we have analyzed and compared the phenotype of two mutant cell lines, the thymoma cell line RMA-S and a small lung carcinoma cell line CMT.64, in order to address the mechanism that underlies the antigen processing deficiency of CMT.64 cells. Unlike RMA-S cells, vesicular stomatitis virus (VSV)-infected CMT.64 cells are not recognized by specific CTL. Interferon gamma (IFN-gamma) treatment of CMT.64 cells restores the ability of these cells to process and present VSV in the context of Kb. We show that although CMT.64 cells express a low level of beta 2m, the recognition of VSV-specific CTL is not restored by increasing the amount of beta 2m synthesized in CMT.64 cells. In addition, we find that CMT.64 cells express moderate levels of Kb heavy chain molecules, but most of it is unstable and rapidly degraded in the absence of IFN-gamma treatment. We infer that the antigen processing deficiency does not lie at the level of beta 2m or Kb production. We find also that the mRNAs for both TAP-1 and -2 are present in RMA and RMA-S cells but are absent in uninduced CMT.64 cells. Upon IFN-gamma induction, both mRNAs are highly expressed in CMT-64 cells. In addition, we find that the low molecular mass polypeptides 2 and 7, and additional components of the proteasome are induced by IFN-gamma in CMT-64 cells. Finally, introduction of the rat TAP-1 gene in CMT.64 cells restores CTL recognition of VSV-infected cells. These results indicate that a TAP-1 homodimer may translocate peptides in the ER and explain partially the CMT.64 defect and the RMA-S phenotype. These findings link a dysfunction in the transport and/or generation of antigenic peptides to the capacity of tumor cells to evade immunosurveillance and provide a unique model system to dissect this phenomenon.
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PMID:Comparison of cell lines deficient in antigen presentation reveals a functional role for TAP-1 alone in antigen processing. 793 Oct 74

The endoplasmic reticulum (ER) contains molecular chaperones that facilitate the folding of proteins in mammalian cells. Biosynthetic labeling was used to study the interactions of two chaperones, BiP and calnexin, with vesicular stomatitis virus (VSV) glycoprotein (G protein). Coimmunoprecipitation of G protein with the chaperones showed that BiP bound maximally to early folding intermediates of G protein, whereas calnexin bound after a short lag to more folded molecules. Castanospermine, an inhibitor of ER glucosidases, blocked the binding of proteins to calnexin and inhibited G protein folding. Interaction with calnexin was necessary for efficient folding of G protein and for retention of partially folded forms.
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PMID:Folding of VSV G protein: sequential interaction with BiP and calnexin. 793 87

Syntaxins are a family of vesicular transport receptors that are involved in membrane traffic through both the constitutive and regulated secretory pathways. Syntaxins 1A/B,2,3, and 4 are principally associated with the plasma membrane. Two of the syntaxins, 1A and 1B, have been suggested to be the docking receptors for synaptic vesicles with the presynaptic membrane. The most distant member of the family, syntaxin 5, has been found in the Golgi region and has significant homology (35% identity) with Sed5p, an essential protein in yeast which is required for vesicular transport from the endoplasmic reticulum (ER) to the Golgi stack. Here we present evidence that syntaxin 5 performs an analogous function in ER to Golgi transport in mammalian cells. Transient expression of an hemagglutinin-tagged full-length clone of syntaxin 5 and a truncated mutant lacking the transmembrane domain inhibited the transport of vesicular stomatitis virus glycoprotein to the Golgi stack. Under these conditions, vesicular stomatitis virus glycoprotein accumulated in pre-Golgi intermediates, which were strongly enriched in syntaxin 5. Our results suggest that syntaxin 5 is the functional mammalian homologue of Sed5p and provides evidence for its role in regulating the potential targeting and/or fusion of carrier vesicles following export from the ER.
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PMID:Syntaxin 5 regulates endoplasmic reticulum to Golgi transport. 796 11

In polarized Madin-Darby canine kidney cells the newly synthesized plasma membrane proteins, on the exocytic pathway, are sorted in the trans-Golgi network (TGN) and delivered directly to the apical or basolateral surface. Forskolin, isobutylmethylxanthine, and dibutyryl cAMP, all known to activate protein kinase A, stimulated transport of influenza hemagglutinin (HA) from the TGN to the apical surface. The same reagents, however, did not affect the transport of HA from the endoplasmic reticulum to the Goli complex nor did they affect transport of vesicular stomatitis virus G protein from the TGN to the basolateral surface. The addition of staurosporin, a general protein kinase inhibitor, did not affect the transport of HA in nontreated cells but blocked the stimulation caused by the above reagents. Apical transport of HA was also stimulated by phorbol ester, an activator of protein kinase C. Activation of apical transport by phorbol ester as well as aluminum fluoride (Pimplikar, S. W., and Simons, K. (1993) Nature 362, 456-458) was also negated by staurosporin. These results show that in polarized Madin-Darby canine kidney cells, protein kinase A and protein kinase C selectively stimulate the apical transport.
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PMID:Activators of protein kinase A stimulate apical but not basolateral transport in epithelial Madin-Darby canine kidney cells. 803 64

In the class II region of the major histocompatibility complex (MHC(, four genes implicated in MHC class I-mediated antigen processing have been described. Two genes (TAP1 and TAP2) code for multimembrane-spanning ATP-binding transporter proteins and two genes (LMP2 and LMP7) code for subunits of the proteasome. While TAP1 and TAP2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP1/2 and LMP2/7 genes, it was recently shown that expression of TAP1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA-2 by HLA-A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP2/7, we transfected T2 cells with TAP1, TAP2 and either of the H-2Kb, Db or Kd genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus-specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP1/2 gene products, presented all tested viral antigens restricted through either the H-2Kb, Db or Kd class I molecules. We conclude that the proteasome subunits LMP2/7 as well as other gene products in the MHC class II region, except from TAP1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.
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PMID:Presentation of viral antigens restricted by H-2Kb, Db or Kd in proteasome subunit LMP2- and LMP7-deficient cells. 805 44

Fluorescence-activated cell sorting was used to isolate 19 independent, temperature-sensitive, low density lipoprotein (LDL) receptor-deficient Chinese hamster ovary cell mutants that define three recessive complementation groups, ldlE, ldlF, ldlG. LDL receptor activity, essentially normal at the permissive temperature (34 degrees C), was virtually abolished in the mutants after incubation for 8-12 h at the nonpermissive temperature (39-40.5 degrees C). The mutants died after incubation for > 24 h at 39.5 degrees C. These mutants exhibited two striking and unexpected abnormalities that suggest that they define three genes important for general vesicular membrane traffic. First, LDL receptors were degraded abnormally rapidly at the nonpermissive temperature (chloroquine inhibited this degradation in ldlE and ldlG, but not in ldlF). In ldlE cells, the rapid degradation did not require efficient receptor clustering into coated pits and was not observed for all cell surface proteins. This selective degradation may be due to endocytic missorting. Second, the mutants exhibited temperature-sensitive defects in the posttranslational processing and intracellular transport of many membrane-associated and secreted proteins, including the LDL, mannose 6-phosphate/insulin-like growth factor II, and scavenger receptors, the vesicular stomatitis virus G protein and decay accelerating factor. Although the initial synthesis, folding, and processing of precursor forms of these proteins in the endoplasmic reticulum were apparently normal at the nonpermissive temperature, there was either a delay or a block in oligosaccharide processing associated with endoplasmic reticulum to medial Golgi transport at the nonpermissive temperature. This was accompanied by a dramatic inhibition of total soluble protein secretion. The posttranslational processing defects, the instability of cell surface LDL receptors, and the defective protein secretion exhibited by these mutants suggest that the ldlE-G gene products regulate or participate in reactions that are vital for a variety of secretory and endocytic membrane transport processes. This suggestion is strongly supported by our recent observation that a cDNA encoding a component of the coatomer, epsilon-COP, corrects the mutant phenotypes of ldlF cells (Guo, Q., Vasile, E., and Krieger, M. (1994) J. Cell Biol. 125, 1213-1224). Thus, these mutant cells should prove useful for further genetic and biochemical analysis of the mechanisms underlying intracellular membrane traffic.
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PMID:Isolation of three classes of conditional lethal Chinese hamster ovary cell mutants with temperature-dependent defects in low density lipoprotein receptor stability and intracellular membrane transport. 806 14


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