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)

Long-term culture of phorbol ester (TPA)-differentiated and growth-arrested human U937 leukemia cells was associated with expression of c-jun transcription factors and vimentin intermediate filaments until the cells entered a retrodifferentiation program. This retrodifferentiation process revealed a reversion of the senecent differentiated cells back to undifferentiated and proliferative active young cells. A significant protein ubiquitination was detectable before retrodifferentiation and rejuvenation indicating a proteolytic down-modulation of differentiation markers. Thus, proteolytic activity significantly increased during retrodifferentiation, however, proteasomal protein expression remained unaltered. In order to investigate proteasomal associates, (ADP-ribose)polymerase-1 (PARP-1) expression progressively increased to maximal levels at the time of retrodifferentiation suggesting a possible regulatory association. Indeed, PARP-1 immunoprecipitations demonstrated a co-immunoprecipitation of proteolytically active 20S proteasome with maximal levels during retrodifferentiation. Inhibition of PARP and the proteasome by 3-aminobenzamide and MG-132, respectively, revealed about 90% of apoptotic cells by cell cycle analysis at the time of retrodifferentiation whereas control cells doubled. In contrast, a similar PARP and proteasome inhibition within 5d after TPA-induced differentiation demonstrated little if any apoptotic effects. More specifically, down-modulation of PARP-1 by an antisense PARP-1 vector construct underwent a rapid differentiation and aging and revealed no detectable retrodifferentiation in contrast to control vector-transfected U937 cells. In conclusion, retrodifferentiation of growth-arrested U937 monocytic cells requires proteasomal protein degradation and activity of PARP-1.
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PMID:Retrodifferentiation and rejuvenation of senescent monocytic cells requires PARP-1. 1731 23

Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.
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PMID:Dynamics of mutated GFAP aggregates revealed by real-time imaging of an astrocyte model of Alexander disease. 1760 20

GT-1 murine neuronal cells exposed to an experimental proteasome inhibitor (EPI) for 24h showed increased cell death via a non-apoptotic mechanism, as assessed by TUNEL and DNA fragmentation assays. Immunofluorescence staining demonstrated that EPI induced reorganization and relocation of non-ubiquinated actin microfilaments and microtubules to the perinuclear region in EPI treated cells. Immunohistochemistry analysis also demonstrated that other non-cytoskeletal proteins became ubiquitinated and/or upregulated including ubiquitin and other stress proteins. Perinuclear-centrosomal accumulation of gamma-tubulin and vimentin, key components of aggresomes, was observed in the EPI treated cells. Biochemical analysis indicated that EPI-induced accumulation of ubiquitinated protein aggregates in GT-1 cells was detergent - and mechanical - disruption resistant, a feature of aggresomes. Similar results were observed in GT-1 cells treated with lactacystin, a prototypical proteasome inhibitor, which is structurally dissimilar to EPI indicating a pharmacologic effect. In conclusion, EPI causes cytoskeletal reorganization and accumulation of diverse ubiquitinated and non-ubiquitinated proteins in the perinuclear region and potentially overloads the endoplasmic reticulum-dependent quality control mechanism. These processes acting alone, or in combination, are hypothesized to affect axonal transport or other aspects of cellular homeostasis and thus, represent events potentially relevant to the development of peripheral neuropathy associated with administration of proteasome inhibitors in nonclinical studies.
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PMID:Effect of an experimental proteasome inhibitor on the cytoskeleton, cytosolic protein turnover, and induction in the neuronal cells in vitro. 1815 69

Vimentin is the main intermediate filament (IF) protein of mesenchymal cells and tissues. Unlike other IF-/- mice, vimentin-/- mice provided no evidence of an involvement of vimentin in the development of a specific disease. Therefore, we generated two transgenic mouse lines, one with a (R113C) point mutation in the IF-consensus motif in coil1A and one with the complete deletion of coil 2B of the rod domain. In epidermal keratins and desmin, point mutations in these parts of the alpha-helical rod domain cause keratinopathies and desminopathies, respectively. Here, we demonstrate that substoichiometric amounts of vimentin carrying the R113C point mutation disrupted the endogenous vimentin network in all tissues examined but caused a disease phenotype only in the eye lens, leading to a posterior cataract that was paralleled by the formation of extensive protein aggregates in lens fibre cells. Unexpectedly, central, postmitotic fibres became depleted of aggregates, indicating that they were actively removed. In line with an increase in misfolded proteins, the amounts of Hsp70 and ubiquitylated vimentin were increased, and proteasome activity was raised. We demonstrate here for the first time that the expression of mutated vimentin induces a protein-stress response that contributes to disease pathology in mice, and hypothesise that vimentin mutations cause cataracts in humans.
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PMID:A dominant vimentin mutant upregulates Hsp70 and the activity of the ubiquitin-proteasome system, and causes posterior cataracts in transgenic mice. 1894 Sep 12

Hepatitis C virus (HCV) core protein is essential for virus particle formation. Using HCV core-expressing and non-expressing Huh7 cell lines, Uc39-6 and Uc321, respectively, we performed comparative proteomic studies of proteins in the 0.5% Triton X-100-insoluble fractions of cells, and found that core-expressing Uc39-6 cells had much lower vimentin content than Uc321 cells. In experiments using vimentin-overexpressing and vimentin-knocked-down cells, we demonstrated that core protein levels were affected by cellular vimentin content. When vimentin expression was knocked-down, there was no difference in mRNA level of core protein; but proteasome-dependent degradation of the core protein was strongly reduced. These findings suggest that the turnover rate of core protein is regulated by cellular vimentin content. HCV production was also affected by cellular vimentin content. Our findings together suggest that modulation of hepatic vimentin expression might enable the control of HCV production.
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PMID:Cellular vimentin content regulates the protein level of hepatitis C virus core protein and the hepatitis C virus production in cultured cells. 1901 28

Cataracts are characterized by an opacification of the eye lens, often caused by protein misfolding and aggregation. The intermediate filament protein vimentin, which is highly expressed in lens fiber cells and in mesenchymal tissues, is a main structural determinant in these cells forming a membrane-connected cytoskeleton. Additional functions of vimentin remain to be identified. Here, we demonstrate that a mutation in VIM causes a dominant, pulverulent cataract. We sequenced the complete human VIM gene in 90 individuals suffering from congenital cataract and found a G596A change in exon 1 in a single individual, causing the missense mutation E151K in coil 1B of vimentin. The mutant vimentin formed an aberrant vimentin cytoskeleton and increased the proteasome activity in transfected cells. Furthermore, this mutation causes a severe kinetic defect in vimentin assembly both in vitro and in vivo. Hence, in conjunction with available mouse and cell culture models, our results reveal for the first time an important functional role for vimentin in the maintenance of lens integrity. Finally, this invites novel therapy approaches for cataracts.
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PMID:Dominant cataract formation in association with a vimentin assembly disrupting mutation. 1912 78

Gigaxonin mutations cause the fatal human neurodegenerative disorder giant axonal neuropathy (GAN). Broad deterioration of the nervous system in GAN patients is accompanied by massive disorganization of intermediate filaments (IFs) both in neurons and many non-neuronal cells. With newly developed antibodies, gigaxonin is now shown to be expressed at extremely low levels throughout the nervous system. In lymphoblast cell lines derived from severe and mild forms of GAN, mutations in gigaxonin are shown to yield highly unstable proteins, thereby permitting a rapid diagnostic test for the spectrum of GAN mutations as an alternative to invasive nerve biopsy or systematic sequencing of the GAN gene. Gigaxonin has been proposed as a substrate adaptor for an E3 ubiquitin ligase, which affects proteasome-dependent degradation of microtubule-related proteins including MAP1B, MAP8 and the tubulin folding chaperone TBCB. We demonstrate that, unlike its counterpart TBCE, TBCB only moderately destabilizes microtubules. Neither TBCB abundance nor microtubule organization or densities are altered in GAN mutant fibroblasts, thus demonstrating that altered TBCB levels are not primary determinants of IF disorganization in GAN. Characteristic GAN mutant-induced ovoid aggregates of vimentin are not produced in normal fibroblasts after disrupting microtubule assembly, either by TBCE overexpression or depolymerizing drugs. Thus, IF disorganization in GAN fibroblasts is independent of TBCB and microtubule loss and must be regulated by a yet unidentified mechanism.
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PMID:Gigaxonin controls vimentin organization through a tubulin chaperone-independent pathway. 1916 53

Advanced gallbladder cancer has an extremely poor prognosis because of metastasis. Identification of metastasis-related biomarkers is essential to improve patient survival. In the present study, metastasis-associated proteins were identified by comparative proteomic analysis and the metastasis-related function of the candidate protein, chloride intracellular channel 1 (CLIC1), was further elucidated. Two cell lines with high or low metastatic potential (termed GBC-SD18H and GBC-SD18L, respectively), originating from the same parental gallbladder carcinoma GBC-SD cell line, were identified by spontaneous metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach comprised of two-dimensional gel electrophoresis analysis and mass spectroscopy was used to identify and compare the protein expression patterns between GBC-SD18L and GBC-SD18H. Twenty-six proteins were identified and further verified by one-dimensional Western blotting and semiquantitative reverse transcriptase polymerase chain reaction analysis. It was determined that CLIC1, ezrin, vimentin, annexin A3, WD repeat domain 1, triosephosphate isomerase, C1-tetrahydrofolate synthase, Rho GDP-dissociation inhibitor 1, T-complex protein 1, heterogeneous nuclear ribonucleoprotein K, glutamate dehydrogenase 1, proteasome activator complex subunit 3 and Rab GDP-dissociation inhibitor beta were significantly up-regulated in the highly metastatic GBC-SD18H cell line compared to the poorly metastatic GBC-SD18L cell line. However, phosphoglycerate kinase 1 and programmed cell death protein 8 were significantly down-regulated in the highly metastatic GBC-SD18H cell line compared to GBC-SD18L. Considering that CLIC1 was profuse in highly metastatic GBC-SD18H but scarce in poorly metastatic GBC-SD18L, the association of CLIC1 with metastasis was further elucidated by the overexpression and RNA interference of CLIC1 in GBC-SD18L cells and GBC-SD18H cells, respectively. The results demonstrated that the overexpression of CLIC1 promoted cell motility and invasion of GBC-SD18L in vitro, while RNA interference of CLIC1 remarkably decreased cell motility and invasive potency of GBC-SD18H in vitro, indicating that CLIC1 might play an important role in metastasis of gallbladder carcinoma.
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PMID:Identification of metastasis-associated proteins involved in gallbladder carcinoma metastasis by proteomic analysis and functional exploration of chloride intracellular channel 1. 1929 76

Increased expression of the astrocytic intermediate filament protein glial fibrillary acidic protein (GFAP) is a characteristic of astrogliosis. This process occurs in the brain during aging and neurodegeneration and coincides with impairment of the ubiquitin proteasome system. Inhibition of the proteasome impairs protein degradation; therefore, we hypothesized that the increase in GFAP may be the result of impaired proteasomal activity in astrocytes. We investigated the effect of proteasome inhibitors on GFAP expression and other intermediate filament proteins in human astrocytoma cells and in a rat brain model for astrogliosis. Extensive quantitative RT-PCR, immunocytochemistry, and Western blot analysis resulted unexpectedly in a strong decrease of GFAP mRNA to <4% of control levels [Control (DMSO) 100+/-19.2%; proteasome inhibitor (epoxomicin) 3.5+/-1.3%, n=8; P < or = 0.001] and a loss of GFAP protein in astrocytes in vitro. We show that the proteasome alters GFAP promoter activity, possibly mediated by transcription factors as demonstrated by a GFAP promoter-luciferase assay and RT(2) Profiler PCR array for human transcription factors. Most important, we demonstrate that proteasome inhibitors also reduce GFAP and vimentin expression in a rat model for induced astrogliosis in vivo. Therefore, proteasome inhibitors could serve as a potential therapy to modulate astrogliosis associated with CNS injuries and disease.
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PMID:Intermediate filament transcription in astrocytes is repressed by proteasome inhibition. 1933 45

Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common known cause of Parkinson disease, but how this protein results in the pathobiology of Parkinson disease is unknown. Moreover, there is variability in pathology among cases, and alpha-synuclein (alpha-syn) neuronal inclusions are often present, but whether LRRK2 is present in these pathological inclusions is controversial. This study characterizes novel LRRK2 antibodies, some of which preferentially recognize an aggregated form of LRRK2, as observed in cell culture models. Large perinuclear aggregates containing LRRK2 were promoted by proteasome inhibition and prevented by microtubule polymerization inhibition. Furthermore, they were vimentin- and gamma-tubulin- but not lamp1-immunoreactive, suggesting that these structures fit the definition of aggresomes. Inhibition of heat shock protein 90 led to the degradation of only the soluble/cytosolic pool of LRRK2, suggesting that the aggresomes formed independent of the stability provided by the heat shock protein 90. Although these novel anti-LRRK2 antibodies identified aggregates in model cell systems, they did not immunostain pathological inclusions in human brains. Furthermore, coexpression of LRRK2 and alpha-syn did not recruit alpha-syn into aggresomes in cultured cells, even in the presence of proteasome inhibition. Thus, although LRRK2 is a model system for aggresome formation, LRRK2 is not present in alpha-syn pathological inclusions.
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PMID:Leucine-rich repeat kinase 2 expression leads to aggresome formation that is not associated with alpha-synuclein inclusions. 1953 93


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