Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have utilized a mouse mammary tumor virus (MMTV) glycoprotein gene containing a mutation in the endoproteolytic cleavage site to investigate the biological significance of processing, the structural requirements for and the events involved in the proteolytic maturation of MMTV. Using oligonucleotide-directed mutagenesis, the endoproteolytic cleavage site within the MMTV glycoprotein was mutated from Arg-Ala-Lys-Arg to Arg-Ala-Asn-Gln and both the wild-type and mutated genes were transfected and expressed in HTC rat hepatoma cells. Indirect immunofluorescence, steady state radiolabeling and pulse-chase kinetic experiments demonstrated that this mutated glycoprotein was transported to the cell surface with the same efficiency as the wild-type maturation products; however, proteolytic cleavage and fusogenic activity were almost completely abolished. Consistent with the lack of cleavage, the endoglycosidase H-resistant precursor, gp78, accumulated on the cell surface and in the extracellular environment. When HTC cells expressing the wild-type MMTV glycoprotein were treated with the Golgi mannosidase I and II inhibitors, deoxymannojirimycin and swainsonine respectively, the resulting endogycosidase H-sensitive glycoproteins were processed efficiently. Taken together, these results suggest that proteolytic processing of the MMTV glycoprotein most likely occurs in the trans Golgi or at a later step in the exocytic pathway and occurs after the formation of an endoglycosidase H-resistant, terminally sialylated intermediate. Moreover, the acquisition of endoglycosidase H-resistant oligosaccharides is not a prerequisite for recognition by the cellular proteases to generate the viral maturation products. Our evidence also suggests that the processing of the MMTV envelope glycoprotein is required for the functional exposure of the fusion domain which is involved in viral infectivity.
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PMID:Trafficking of wild-type and an endoproteolytic-site mutant of the mouse mammary tumor virus glycoprotein. 838 4

Primary rat hepatocytes and two hepatoma cell lines have been used to study whether high mannose-type N-glycans of plasma membrane glycoproteins may be modified by the removal of mannose residues even after transport to the cell surface. To examine glycan remodeling of cell surface glycoproteins, high mannose-type glycoforms were generated by adding the reversible mannosidase I inhibitor deoxymannojirimycin during metabolic labeling with [3H]mannose, thereby preventing further processing of high mannose-type N-glycans to complex structures. Upon transport to the cell surface, glycoproteins were additionally labeled with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. This strategy allowed us to follow selectively the fate of cell surface glycoproteins. Postbiosynthetic demannosylation was monitored by determining the conversion of Man8-9GlcNAc2 to smaller structures during reculture of cells in the absence of deoxymannojirimycin. The results show that high mannose-type N-glycans of selected cell surface glycoproteins are trimmed from Man8-9GlcNAc2 to Man5GlcNAc2 with Man7GlcNAc2 and Man6GlcNAc2 formed as intermediates. It could be clearly shown in MH 7777 as well as in HepG2 cells that demannosylation affects plasma membrane glycoproteins after they are routed to the cell surface. As was determined for total cell surface glycoproteins in HepG2 cells, this process occurs with a half-time of 6.7 h. By analyzing the size of high mannose-type glycans of glycoproteins isolated from the cell surface at the end of the reculture period, i.e. after trimming had occurred, we were able to demonstrate that glycoproteins carrying trimmed high mannose glycans become exposed at the cell surface. From these data we conclude that cell surface glycoproteins can be trimmed by mannosidases at sites peripheral to N-acetylglucosaminyltransferase I without further processing of their glycans to the complex form. This glycan remodeling may occur at the cell surface or during endocytosis and recycling back to the cell surface.
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PMID:Cell surface glycoproteins undergo postbiosynthetic modification of their N-glycans by stepwise demannosylation. 942 72

The role of conformation-based quality control in the early secretory pathway is to eliminate misfolded polypeptides and unassembled multimeric protein complexes from the endoplasmic reticulum, ensuring the deployment of only functional molecules to distal sites. The intracellular fate of terminally misfolded human alpha1-antitrypsin was examined in hepatoma cells to identify the functional role of asparagine-linked oligosaccharide modification in the selection of glycoproteins for degradation by the cytosolic proteasome. Proteasomal degradation required physical interaction with the molecular chaperone calnexin. Altered sedimentation of intracellular complexes following treatment with the specific proteasome inhibitor lactacystin, and in combination with mannosidase inhibition, revealed that the removal of mannose from attached oligosaccharides abrogates the release of misfolded alpha1-antitrypsin from calnexin prior to proteasomal degradation. Intracellular turnover was arrested with kifunensine, implicating the participation of endoplasmic reticulum mannosidase I in the disposal process. Accelerated degradation occurred in a mannosidase-independent manner and was arrested by lactacystin, in response to the posttranslational inhibition of glucosidase II, demonstrating that the attenuated removal of glucose from attached oligosaccharides functions as the underlying rate-limiting step in the proteasome-mediated pathway. A model is proposed in which the removal of mannose from multiple attached oligosaccharides directs calnexin in the selection of misfolded alpha1-antitrypsin for degradation by the proteasome.
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PMID:Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome. 1002 9

In the early secretory pathway, a distinct set of processing enzymes and family of lectins facilitate the folding and quality control of newly synthesized glycoproteins. In this regard, we recently identified a mechanism in which processing by endoplasmic reticulum mannosidase I, which attenuates the removal of glucose from asparagine-linked oligosaccharides, sorts terminally misfolded alpha(1)-antitrypsin for proteasome-mediated degradation in response to its abrogated physical dissociation from calnexin (Liu, Y., Choudhury, P., Cabral, C., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861-5867). In the present study, we examined the quality control of genetic variant PI Z, which undergoes inappropriate polymerization following biosynthesis. Here we show that in stably transfected hepatoma cells the additional processing of asparagine-linked oligosaccharides by endoplasmic reticulum mannosidase II partitions variant PI Z away from the conventional disposal mechanism in response to an arrested posttranslational interaction with calnexin. Intracellular disposal is accomplished by a nonproteasomal system that functions independently of cytosolic components but is sensitive to tyrosine phosphatase inhibition. The functional role of ER mannosidase II in glycoprotein quality control is discussed.
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PMID:Processing by endoplasmic reticulum mannosidases partitions a secretion-impaired glycoprotein into distinct disposal pathways. 1082 1

The exocytic pathway provides a physical route through which newly synthesized secretory and membrane proteins are deployed to the eukaryote cell surface. For newly synthesized alpha1-antitrypsin (AAT), the modification of its asparagine-linked oligosaccharides by a slow-acting mannosidase partitions the misfolded monomer into the proteasomal degradation pathway. Herein, we asked whether, and how, modification by endoplasmic reticulum mannosidase I (ERManI) contributes to the preferential selection of the misfolded AAT monomer for proteasomal degradation. Transiently expressed mutant and WT AAT variants underwent rapid destabilization in response to an artificially elevated ERManI concentration in the murine hepatoma cell line, Hepa1a. Based on the mannosidase- and lactacystin-sensitive properties of intracellular turnover, a stochastic model is proposed in which the delayed onset of the glycan modification, relative to the duration of nonnative protein structure, coordinates the preferential degradation of the misfolded monomer and spares the native molecule from destruction. Newly synthesized endogenous transferrin underwent degradation in response to an elevated concentration of ERManI, whereas the nonglycosylated secretory glycoprotein albumin was not affected. Taken together, these findings indicate that efficient conformational maturation might function as the initial quality control standard for a broad population of glycoproteins.
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PMID:Elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation. 1281 1

In the early secretory pathway, opportunistic cleavage of asparagine-linked oligosaccharides by endoplasmic reticulum (ER) mannosidase I targets misfolded glycoproteins for dislocation into the cytosol and destruction by 26 S proteasomes. The low basal concentration of the glycosidase is believed to coordinate the glycan cleavage with prolonged conformation-based ER retention, ensuring that terminally misfolded glycoproteins are preferentially targeted for destruction. Herein the intracellular fate of human ER mannosidase I was monitored to determine whether a post-translational process might contribute to the regulation of its intracellular concentration. The transiently expressed recombinant human glycosidase was subject to rapid intracellular turnover in mouse hepatoma cells, as was the endogenous mouse ortholog. Incubation with either chloroquine or leupeptin, but not lactacystin, led to intracellular stabilization, implicating the involvement of lysosomal acid hydrolases. Inhibition of protein synthesis with cycloheximide led to intracellular depletion of the glycosidase and concomitant ablation of asparagine-linked glycoprotein degradation, confirming the physiologic relevance of the destabilization process. Metabolic incorporation of radiolabeled phosphate, detection by anti-phosphoserine antiserum, and the stabilizing effect of general serine kinase inhibition implied that ER mannosidase I is subjected to regulated proteolysis. Stabilization in response to genetically engineered removal of the amino-terminal cytoplasmic tail, a postulated regulatory domain, and colocalization of green fluorescent protein fusion proteins with Lamp1 provided two additional lines of evidence to support the hypothesis. A model is proposed in which proteolytically driven checkpoint control of ER mannosidase I contributes to the establishment of an equitable glycoprotein quality control standard by which the efficiency of asparagine-linked glycoprotein conformational maturation is measured.
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PMID:Human endoplasmic reticulum mannosidase I is subject to regulated proteolysis. 1716 54