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)

The identification and characterization of the CFTR gene and protein have provided not only a major impetus to the dissection of the molecular pathophysiology of cystic fibrosis (CF) but also a new perspective on the structure and function of the large superfamily of membrane transport proteins to which it belongs. While the mechanism of the active vectorial translocation of many hydrophobic substrates by several of these transporters remains nearly as perplexing as it has for several decades, considerable insight has been gained into the control of the bidirectional permeation of chloride ions through a single CFTR channel by the phosphorylation of the R-domain and ATP interactions at the two nucleotide binding domains. However, details of these catalytic and allosteric mechanisms remain to be elucidated and await the replacement of two-dimensional conceptualizations with three dimensional structure information. Secondary and tertiary structure determination is required both for the understanding of the mechanism of action of the molecule and to enable a more complete appreciation of the misfolding and misprocessing of mutant CFTR molecules. This is the primary cause of the disease in the majority of the patients and hence understanding the details of the cotranslational interactions with multiple molecular chaperones, the ubiquitin-proteasome pathway and other components of the quality control machinery at the endoplasmic reticulum could provide a basis for the development of new therapeutic interventions.
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PMID:Cystic fibrosis: channel, catalytic, and folding properties of the CFTR protein. 951 28

Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.
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PMID:Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome. 984 94

Although the number of pathologies known to arise from the inappropriate folding of proteins continues to grow, mechanisms underlying the recognition and ultimate disposition of misfolded polypeptides remain obscure. For example, how and where such substrates are identified and processed is unknown. We report here the identification of a specific subcellular structure in which, under basal conditions, the 20S proteasome, the PA700 and PA28 (700- and 180-kD proteasome activator complexes, respectively), ubiquitin, Hsp70 and Hsp90 (70- and 90-kD heat shock protein, respectively) concentrate in HEK 293 and HeLa cells. The structure is perinuclear, surrounded by endoplasmic reticulum, adjacent to the Golgi, and colocalizes with gamma-tubulin, an established centrosomal marker. Density gradient fractions containing purified centrosomes are enriched in proteasomal components and cell stress chaperones. The centrosome-associated structure enlarges in response to inhibition of proteasome activity and the level of misfolded proteins. For example, folding mutants of CFTR form large inclusions which arise from the centrosome upon inhibition of proteasome activity. At high levels of misfolded protein, the structure not only expands but also extensively recruits the cytosolic pools of ubiquitin, Hsp70, PA700, PA28, and the 20S proteasome. Thus, the centrosome may act as a scaffold, which concentrates and recruits the systems which act as censors and modulators of the balance between folding, aggregation, and degradation.
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PMID:Dynamic association of proteasomal machinery with the centrosome. 1022 50

Impaired biosynthetic processing of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse-chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well.
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PMID:COOH-terminal truncations promote proteasome-dependent degradation of mature cystic fibrosis transmembrane conductance regulator from post-Golgi compartments. 1138 Oct 82

Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examination of P23H and K296E mutant opsin processing in COS-7 cells, we have shown that the mutant protein does not accumulate in the Golgi, as previously thought, instead it forms aggregates that have many of the characteristic features of an aggresome. The aggregates form close to the centrosome and lead to the dispersal of the Golgi apparatus. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. The degradation of mutant opsin is dependent on the proteasome machinery. Unlike the situation with DeltaF508-CFTR, proteasome inhibition does not lead to a marked increase in aggresome formation but increases the retention of the protein within the ER, suggesting that the proteasome is required for the efficient retrotranslocation of the mutant protein. Inhibition of N-linked glycosylation with tunicamycin leads to the selective retention of the mutant protein within the ER and increases the steady state level of mutant opsin. Glycosylation, however, has no influence on the biogenesis and targeting of wild-type opsin in cultured cells. This demonstrates that N-linked glycosylation is required for ER-associated degradation of the mutant protein but is not essential for the quality control of opsin folding. The addition of 9-cis-retinal to the media increased the amount of P23H, but not K296E, that was soluble and reached the plasma membrane. These data show that rhodopsin autosomal dominant retinitis pigmentosa is similar to many other neurodegenerative diseases in which the formation of intracellular protein aggregates is central to disease pathogenesis, and they suggest a mechanism for disease dominance.
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PMID:The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation. 1208 51

Aggresomes are pericentrosomal cytoplasmic structures into which aggregated, ubiquitinated, misfolded proteins are sequestered. Misfolded proteins accumulate in aggresomes when the capacity of the intracellular protein degradation machinery is exceeded. Previously, we demonstrated that an intact microtubule cytoskeleton is required for the aggresome formation [Johnston et al., 1998: J. Cell Biol. 143:1883-1898]. In this study, we have investigated the involvement of microtubules (MT) and MT motors in this process. Induction of aggresomes containing misfolded DeltaF508 CFTR is accompanied by a redistribution of the retrograde motor cytoplasmic dynein that colocalizes with aggresomal markers. Coexpression of the p50 (dynamitin) subunit of the dynein/dynactin complex prevents the formation of aggresomes, even in the presence of proteasome inhibitors. Using in vitro microtubule binding assays in conjunction with immunogold electron microscopy, our data demonstrate that misfolded DeltaF508 CFTR associate with microtubules. We conclude that cytoplasmic dynein/dynactin is responsible for the directed transport of misfolded protein into aggresomes. The implications of these findings with respect to the pathogenesis of neurodegenerative disease are discussed.
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PMID:Cytoplasmic dynein/dynactin mediates the assembly of aggresomes. 1221 Nov 13

Pharmacologic- and gene-based therapies have historically been developed as two independent therapeutic platforms for cystic fibrosis (CF) lung disease. Inhibition of the dysregulated epithelial Na channel (ENaC) is one pharmacologic approach to enhance airway clearance in CF. We investigated pharmacologic approaches to enhance CFTR gene delivery with recombinant adeno-associated virus (rAAV) and identified compounds that significantly improved viral transduction while simultaneously inhibiting ENaC activity through an unrelated mechanism. Treatment of human CF airway epithelia with proteasome modulating agents (LLnL and doxorubicin) at the time of rAAV2 or rAAV2/5 infection dramatically enhanced CFTR gene delivery and correction of CFTR-mediated short-circuit currents. Surprisingly, these agents also facilitated long-term (15-day) functional inhibition of ENaC currents independent of CFTR vector administration. Inhibition of ENaC activity was predominantly attributed to a doxorubicin-dependent decrease in gamma-ENaC subunit mRNA expression and an increase in gamma-ENaC promoter methylation. This is the first report to describe the identification of compounds with dual therapeutic action that are able to enhance the efficacy of CFTR gene therapy to the airway while simultaneously ameliorating primary aspects of CF disease pathophysiology. The identification of such compounds mark a new area for drug development, not only for CF, but also for other gene therapy disease targets.
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PMID:Dual therapeutic utility of proteasome modulating agents for pharmaco-gene therapy of the cystic fibrosis airway. 1556 31

The limited packaging capacity of adeno-associated virus (AAV) precludes the design of vectors for the treatment of diseases associated with larger genes. Autonomous parvoviruses, such as minute virus of mice and B19, while identical in size (25 nm), are known to package larger genomes of 5.1 and 5.6 kb, respectively, compared to AAV genomes of 4.7 kb. One primary difference is the fact that wild-type (wt) AAV utilizes three capsid subunits instead of two to form the virion shell. In this study, we have characterized the packaging capacity of AAV serotypes 1 through 5 with and without the Vp2 subunit. Using reporter transgene cassettes that range in size from 4.4 to 6.0 kb, we determined that serotypes 1 through 5 with and without Vp2 could successfully package, replicate in, and transduce cells. Dot blot analysis established that packaging efficiency was similar for all vector cassettes and that the integrity of encapsidated genomes was intact regardless of size. Although physical characterization determined that virion structures were indistinguishable from wt, transduction experiments determined that all serotype vectors carrying larger genomes (5.3 kb and higher) transduced cells less efficiently (within a log) than AAV encapsidating wt size genomes. This result was not unique to reporter genes and was observed for CFTR vector cassettes ranging in size from 5.1 to 5.9 kb. No apparent advantage in packaging efficiency was observed when Vp2 was present or absent from the virion. Further analysis determined that a postentry step was responsible for the block in infection and specific treatment of cells upon infection with proteasome inhibitors increased transduction of AAV encapsidating larger DNA templates to wt levels, suggesting a preferential degradation of virions encapsidating larger-than-wt genomes. This study illustrates that AAV is capable of packaging and protecting recombinant genomes as large as 6.0 kb but the larger genome-containing virions are preferentially degraded by the proteasome and that this block can be overcome by the addition of proteasome inhibitors.
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PMID:Packaging capacity of adeno-associated virus serotypes: impact of larger genomes on infectivity and postentry steps. 1601 54

Mutations in the AAA+ protein (ATPase associated with a variety of cellular activities) p97/VCP (valosin-containing protein) cause a dominantly inherited syndrome of inclusion body myopathy with Paget's disease of the bone and fronto-temporal dementia (IBMPFD). p97/VCP is a ubiquitously expressed protein that participates in a number of cellular processes including endoplasmic reticulum-associated degradation (ERAD). p97/VCP aids in the extraction of ubiquitinated proteins from the endoplasmic reticulum (ER) and facilitates their delivery to the proteasome. This study focuses on the effects of disease-associated p97/VCP mutations on this pathway. We show that p97/VCP containing the most prevalent IBMPFD-associated mutation, R155H, has normal ATPase activity and hexameric structure. However, when expressed in cultured cells, both this and a second IBMPFD-associated p97/VCP mutant increase the overall level of ubiquitin-conjugated proteins and specifically impair degradation of mutant DeltaF508-CFTR handled by the ERAD pathway. These effects are similar to those previously described for an ATPase deficient p97/VCP mutant and suggest that IBMPFD mutations impair p97/VCP cellular function. In a subset of cells, IBMPFD mutations also promote formation of aggregates that contain p97/VCP, ubiquitin conjugates and ER-resident proteins. Undegraded mutant DeltaF508-CFTR also accumulates in these aggregates. We conclude that IBMPFD mutations in p97/VCP disrupt ERAD and that this may contribute to the pathogenesis of IBMPFD.
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PMID:Inclusion body myopathy-associated mutations in p97/VCP impair endoplasmic reticulum-associated degradation. 1632 91

The 26S proteasome is the primary protease responsible for degrading misfolded membrane proteins in the endoplasmic reticulum. Here we examine the specific role of beta subunit function on polypeptide cleavage and membrane release of CFTR, a prototypical ER-associated degradation substrate with 12 transmembrane segments. In the presence of ATP, cytosol and fully active proteasomes, CFTR was rapidly degraded and released into the cytosol solely in the form of trichloroacetic acid (TCA)-soluble peptide fragments. Inhibition of proteasome beta subunits markedly decreased CFTR degradation but surprisingly, had relatively minor effects on membrane extraction and release. As a result, large TCA-insoluble degradation intermediates derived from multiple CFTR domains accumulated in the cytosol where they remained stably bound to inhibited proteasomes. Production of TCA-insoluble fragments varied for different proteasome inhibitors and correlated inversely with the cumulative proteolytic activities of beta1, beta2 and beta5 subunits. By contrast, ATPase inhibition decreased CFTR release but had no effect on the TCA solubility of the released fragments. Our results indicate that the physiologic balance between membrane extraction and peptide cleavage is maintained by excess proteolytic capacity of the 20S subunit. Active site inhibitors reduce this capacity, uncouple ATPase and peptidase activities, and generate cytosolic degradation intermediates by allowing the rate of unfolding to exceed the rate of polypeptide cleavage.
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PMID:Uncoupling proteasome peptidase and ATPase activities results in cytosolic release of an ER polytopic protein. 1639 Aug 70


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