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:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Most cases of cystic fibrosis are caused by mutations that interfere with the biosynthetic folding of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to the rapid degradation of CFTR molecules that have not matured beyond the endoplasmic reticulum (ER). The mechanism by which integral membrane proteins including CFTR are recognized and targeted for ER degradation and the proteolytic machinery involved in this process are not well understood. We show here that the degradation of both wild-type and mutant CFTR is inhibited by two potent proteasome inhibitors that induce the accumulation of polyubiquitinated forms of immature CFTR. CFTR degradation was also inhibited by coexpression of a dominant negative ubiquitin mutant and in cells bearing a temperature-sensitive mutation in the ubiquitin-activating enzyme, confirming that ubiquitination is required for rapid CFTR degradation.
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PMID:Degradation of CFTR by the ubiquitin-proteasome pathway. 755 63

The molecular components of the quality control system that rapidly degrades abnormal membrane and secretory proteins have not been identified. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane protein to which this quality control is stringently applied; approximately 75% of the wild-type precursor and 100% of the delta F508 CFTR variant found in most CF patients are rapidly degraded before exiting from the ER. We now show that this ER degradation is sensitive to inhibitors of the cytosolic proteasome, including lactacystin and certain peptide aldehydes. One of the latter compounds, MG-132, also completely blocks the ATP-dependent conversion of the wild-type precursor to the native folded form that enables escape from degradation. Hence, CFTR and presumably other intrinsic membrane proteins are substrates for proteasomal degradation during their maturation within the ER.
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PMID:Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. 755 64

Ubiquitination is a covalent protein modification that can target proteins in eukaryotic cells for degradation by the 26 S proteasome. Substrates for this degradation pathway include abnormal proteins that arise from misfolding and/or mutation. How and when the ubiquitination machinery recognizes misfolded proteins and targets them for degradation remains largely unknown. We have previously shown that cystic fibrosis transmembrane conductance regulator (CFTR), is rapidly degraded in a ubiquitin-dependent fashion, without any detectable lag following its synthesis (Ward, C. L., and Kopito, R. R. (1994) J. Biol. Chem. 269, 25710-25718), suggesting that ubiquitination and protein synthesis may be temporally linked. In the present study, we have investigated the timing of CFTR ubiquitination relative to its translation in reticulocyte lysates containing 125I-ubiquitin. In synchronized, proteasome-inhibited lysates, translation of full-length CFTR chains was completed in approximately 30 min, whereas modification of CFTR with [125I]ubiquitin was evident by 20 min, indicating that ubiquitination precedes the completion of full-length polypeptide chains. Moreover, ubiquitin was also found to be transferred to nascent CFTR chains while attached to ribosomes. Together, these data establish that ubiquitination, which is widely assumed to be a post-translational event, can occur cotranslationally and suggest a role for ubiquitination early in protein biosynthesis.
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PMID:Cotranslational ubiquitination of cystic fibrosis transmembrane conductance regulator in vitro. 951 8

The DeltaF508 mutation leads to retention of cystic fibrosis transmembrane conductance regulator (CFTR) in the endoplasmic reticulum and rapid degradation by the proteasome and other proteolytic systems. In stably transfected LLC-PK1 (porcine kidney) epithelial cells, DeltaF508 CFTR conforms to this paradigm and is not present at the plasma membrane. When LLC-PK1 cells or human nasal polyp cells derived from a DeltaF508 homozygous patient are grown on plastic dishes and treated with an epithelial differentiating agent (DMSO, 2% for 4 days) or when LLC-PK1 cells are grown as polarized monolayers on permeable supports, plasma membrane DeltaF508 CFTR is significantly increased. Moreover, when confluent LLC-PK1 cells expressing DeltaF508 CFTR were treated with DMSO and mounted in an Ussing chamber, a further increase in cAMP-activated short-circuit current (i.e., approximately 7 microA/cm2; P < 0.00025 compared with untreated controls) was observed. No plasma membrane CFTR was detected after DMSO treatment in nonepithelial cells (mouse L cells) expressing DeltaF508 CFTR. The experiments describe a way to augment DeltaF508 CFTR maturation in epithelial cells that appears to act through a novel mechanism and allows insertion of functional DeltaF508 CFTR in the plasma membranes of transporting cell monolayers. The results raise the possibility that increased epithelial differentiation might increase the delivery of DeltaF508 CFTR from the endoplasmic reticulum to the Golgi, where the DeltaF508 protein is shielded from degradative pathways such as the proteasome and allowed to mature.
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PMID:Activation of DeltaF508 CFTR in an epithelial monolayer. 968 15

Endoplasmic reticulum (ER) degradation pathways can selectively route proteins away from folding and maturation. Both soluble and integral membrane proteins can be targeted from the ER to proteasomal degradation in this fashion. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral, multidomain membrane protein localized to the apical surface of epithelial cells that functions to facilitate Cl- transport. CFTR was among the first membrane proteins for which a role of the proteasome in ER-related degradation was described. However, the signals that route CFTR to ubiquitination and subsequent degradation are not known. Moreover, limited information is available concerning the subcellular localization of polyubiquitinated CFTR or mechanisms underlying retrograde dislocation of CFTR from the ER membrane to the proteasome either before or after ubiquitination. In the present study, we show that proteasome inhibition with clasto-lactacystin beta-lactone (4 microM, 1 h) stabilizes the presence of a deglycosylated CFTR intermediate for up to 5 h without increasing the core glycosylated (band B) form of CFTR. Deglycosylated CFTR is present under the same conditions that result in accumulation of polyubiquitinated CFTR. Moreover, the deglycosylated form of both wild type and DeltaF508 CFTR can be found in the cytosolic fraction. Both the level and stability of cytosolic, deglycosylated CFTR are increased by proteasome blockade. During retrograde translocation from the ER to the cytosol, CFTR associates with the Sec61 trimeric complex. Sec61 is the key component of the mammalian co-translational protein translocation system and has been proposed to function as a two way channel that transports proteins both into the ER and back to the cytosol for degradation. We show that the level of the Sec61.CFTR complexes are highest when CFTR degradation proceeds at the greatest rate (approximately 90 min after pulse labeling). Quantities of Sec61.CFTR complexes are also increased by inhibition of the proteasome. Based on these results, we propose a model in which complex membrane proteins such as CFTR are transported through the Sec61 trimeric complex back to the cytosol, escorted by the beta subunit of Sec61, and degraded by the proteasome or by other proteolytic systems.
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PMID:The mechanism underlying cystic fibrosis transmembrane conductance regulator transport from the endoplasmic reticulum to the proteasome includes Sec61beta and a cytosolic, deglycosylated intermediary. 979 4

Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.
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PMID:Aggresomes: a cellular response to misfolded proteins. 986 62

The ubiquitin-proteasome pathway has been implicated in the degradation of newly synthesized, misfolded and unassembled proteins in the endoplasmic reticulum (ER). Using a cell-free reticulocyte lysate system we have examined the relationship between biosynthesis and ER-associated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR), a polytopic protein with 12 predicted transmembrane segments. Our results provide direct evidence that full-length, glycosylated and membrane-integrated CFTR is a substrate for degradation and that degradation involves polyubiquitination and cytosolic proteolytic activity. CFTR ubiquitination was both temperature- and ATP-dependent. Degradation was significantly inhibited by EDTA, apyrase, and the proteasome inhibitors hemin and MG132. Degradation was inhibited to a lesser extent by clasto-lactacystin beta-lactone, ALLN, and Nalpha-tosyl-L-phenylalanine chloromethyl ketone and was relatively unaffected by lactacystin and N-tosyl lysyl chloromethyl ketone. In the presence of hemin, polyubiquitinated CFTR remained tightly associated with ER microsomes. However, membrane-bound ubiquitinated CFTR could be subsequently degraded into trichloroacetic acid-soluble fragments upon incubation in hemin-free, ATP-containing lysate. Thus ER-associated degradation of CFTR occurs via a membrane-bound, rather than cytosolic, intermediate and likely involves recruitment of degradation machinery to the ER membrane. Our data suggest a model in which the degradation of polytopic proteins such as CFTR is coupled to retrograde translocation and removal of the polypeptide from the lipid bilayer.
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PMID:Evidence that endoplasmic reticulum (ER)-associated degradation of cystic fibrosis transmembrane conductance regulator is linked to retrograde translocation from the ER membrane. 991 89

The most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), DeltaF508, is a trafficking mutant that has prolonged associations with molecular chaperones and is rapidly degraded, at least in part by the ubiquitin-proteasome system. Sodium 4-phenylbutyrate (4PBA) improves DeltaF508-CFTR trafficking and function in vitro in cystic fibrosis epithelial cells and in vivo. To further understand the mechanism of action of 4PBA, we tested the hypothesis that 4PBA modulates the targeting of DeltaF508-CFTR for ubiquitination and degradation by reducing the expression of Hsc70 in cystic fibrosis epithelial cells. IB3-1 cells (genotype DeltaF508/W1282X) that were treated with 0.05-5 mM 4PBA for 2 days in culture demonstrated a dose-dependent reduction in Hsc70 protein immunoreactivity and mRNA levels. Immunoprecipitation with Hsc70-specific antiserum demonstrated that Hsc70 and CFTR associated under control conditions and that treatment with 4PBA reduced these complexes. Levels of immunoreactive Hsp40, Hdj2, Hsp70, Hsp90, and calnexin were unaffected by 4PBA treatment. These data suggest that 4PBA may improve DeltaF508-CFTR trafficking by allowing a greater proportion of mutant CFTR to escape association with Hsc70.
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PMID:Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of DeltaF508-CFTR. 1066 19

Inefficient delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) to the surface of cells contributes to disease in the majority of cystic fibrosis patients. Analysis of cystic fibrosis-associated missense mutations in the first nucleotide binding domain (NBD1), including A455E, S549R, Y563N, and P574H, revealed reduced levels of mature CFTR with elevated levels of carboxyl-terminal polypeptide fragments of 105 and 90 kDa. These fragments appear early in biogenesis and degrade rapidly in four distinct cell types tested including the bronchial epithelial IB3-1 cell line. They were detected at highest levels with CFTRA455E where the 105-kDa fragment accounted for 40% of newly synthesized polypeptide but for only 20 and 7% of nascent wild type and mutant DeltaF508 proteins, respectively. The bands represent core- and unglycosylated forms of the same CFTR fragment supporting that precursor forms are correctly inserted into the membrane of the endoplasmic reticulum. Proteolytic cleavage would be predicted to occur on the cytosolic face of the endoplasmic reticulum within the NBD1-R domain segment, but pharmacological testing did not support involvement of the 26 S proteasome. The examined missense mutations in NBD1 manifest differently than the major mutant, DeltaF508, and highlight a critical conformational aspect of biogenesis of CFTR.
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PMID:Cystic fibrosis mutations lead to carboxyl-terminal fragments that highlight an early biogenesis step of the cystic fibrosis transmembrane conductance regulator. 1076 88

The DeltaF508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR) is a trafficking mutant, which is retained and degraded in the endoplasmic reticulum by the ubiquitin-proteasome pathway. The mutant protein fails to reach a completely folded conformation that is no longer a substrate for ubiquitination ("stable B"). Wild type protein reaches this state with 25% efficiency. In this study the rabbit reticulocyte lysate with added microsomal membranes has been used to reproduce the post-translational events in the folding of wild type and DeltaF508 CFTR. In this system wild type CFTR does not reach the stable B form if the post-translational temperature is 37 degrees C, whereas at 30 degrees C the behavior of both wild type and mutant proteins mimics that observed in the cell. Geldanamycin stabilizes DeltaF508 CFTR with respect to ubiquitination only when added post-translationally. The interaction of wild type and mutant CFTR with the molecular chaperones heat shock cognate 70 (hsc70) and heat shock protein 90 (hsp90) has been assessed. Release of wild type protein from hsc70 coincides with the cessation of ubiquitination and formation of stable B. Geldanamycin immediately prevents the binding of hsp90 to DeltaF508 CFTR, and after a delay releases it from hsc70. Release of mutant protein from hsc70 also coincides with the formation of stable B DeltaF508 CFTR.
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PMID:Post-translational disruption of the delta F508 cystic fibrosis transmembrane conductance regulator (CFTR)-molecular chaperone complex with geldanamycin stabilizes delta F508 CFTR in the rabbit reticulocyte lysate. 1098 7


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