[24] Multicatalytic endopeptidase complex: Proteasome

Rivett, A. Jennifer; Savory, Peter; Djaballah, Hakim

doi:10.1016/0076-6879(94)44026-3

Cited by 76 publications

(66 citation statements)

References 52 publications

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“…This activity could be devoted to cleave a particular substrate that promotes cell death, for example a pro-caspase family member into an active form, or could degrade regulatory proteins that normally control the apoptotic pathway. This hypothesis is strengthened by the observation that proteasomes can cleave after Asp in synthetic peptide substrates (Rivett et al, 1994;Kisselev et al, 1999) and that proteasomes are the major ICE-like proteinase in P19 cells treated with retinoic acid (Kobayashi et al, 1996). On the basis of such considerations and of previous findings, it is tempting to place the proteasome activity in CGCs undergoing apoptosis upstream of the caspase activation, as already shown in sympathetic neurons deprived of NGF (Sadoul et al, 1996) and in thymocytes treated with dexamethasone, ␥-irradiation, or etoposide (Grimm et al, 1996;Hirsch et al, 1998;Stefanelli et al, 1998).…”

Section: Discussionmentioning

confidence: 91%

Proteasome Involvement and Accumulation of Ubiquitinated Proteins in Cerebellar Granule Neurons Undergoing Apoptosis

et al. 2000

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We investigated the potential role of the ubiquitin proteolytic system in the death of cerebellar granule neurons induced by reduction of extracellular potassium. Inhibitors of proteasomal function block apoptosis if administered at onset of this process, but they do not exert such effect when added 2-3 hr later. The same inhibitors also prevent caspase-3 activity and calpain-caspase-3-mediated processing of tau protein, suggesting that proteasomes are involved upstream of the caspase activation. Although the proteasomes seem to play an early primary role in programmed cell death, we found that with progression of apoptosis, during the execution phase, a perturbation in normal ubiquitin-proteasome function occurs, and high levels of ubiquitinated proteins accumulate in the cytoplasm of dying cells. Such accumulation correlates with a progressive decline of proteasome chymotrypsin and trypsinlike activities and, to a lower extent, of postacidic-like activity. Both intracytoplasmic accumulation of ubiquitinated proteins and decline of proteasome function are reversed by the pancaspase inhibitor Z-VAD-fmk. The decline in proteasome function is accompanied by, and likely attributable to, a marked and progressive decline of deubiquitinating activities. The finding that the proteasomes are early involved in apoptosis and that ubiquitinated proteins accumulate during this process prospect granule neurons as a model system aimed at correlating these events with neurodegenerative diseases.

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Section: Discussionmentioning

confidence: 91%

Proteasome Involvement and Accumulation of Ubiquitinated Proteins in Cerebellar Granule Neurons Undergoing Apoptosis

et al. 2000

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“…Proteasome activity was determined as the difference between the total activity of crude extracts or fractions and the remaining activity in the presence of 20 M MG132. Assays of 26 S proteasomes were carried out in 25 mM Tris/HCl buffer, pH 7.5, containing 5 mM ATP and assays of 20 S proteasomes were performed in 50 mM Hepes buffer, pH 7.5, containing 0.02% SDS as described previously (32). Fluorescence was measured using a PerkinElmer Life Sciences 650-40 fluorescence spectrophotometer.…”

Section: Methodsmentioning

confidence: 99%

Central Role of the Proteasome in Senescence and Survival of Human Fibroblasts

Chondrogianni

Stratford

Trougakos

et al. 2003

Journal of Biological Chemistry

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302

111

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Normal human fibroblasts undergo a limited number of divisions in culture and progressively they reach a state of irreversible growth arrest, a process termed as replicative senescence. The proteasome is the major cellular proteolytic machinery, the function of which is impaired during replicative senescence. However, the exact causes of its malfunction in these conditions are unknown. Using WI38 fibroblasts as a model for cellular senescence we have observed reduced levels of proteasomal peptidase activities coupled with increased levels of both oxidized and ubiquitinated proteins in senescent cells. We have found the catalytic subunits of the 20 S complex and subunits of the 19 S regulatory complex to be down-regulated in senescent cells. This is accompanied by a decrease in the level of both 20 S and 26 S complexes. Partial inhibition of proteasomes in young cells caused by treatment with specific inhibitors induced a senescence-like phenotype, thus demonstrating the fundamental importance of the proteasome for retaining cellular maintenance and homeostasis. Stable overexpression of ␤ 1 and ␤ 5 subunits in WI38 established cell lines was shown to induce elevated expression levels of ␤ 1 subunit in ␤ 5 transfectants and vice versa.

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“…Proteasomes were purified from rat liver [39,40]. Polyclonal anti-(rat liver proteasome) antibodies (pAb544) were raised in rabbits as described previously using dinitrophenol-modified proteasomes [39] and purified IgC preparations (pre-immune and anti-proteasome) were obtained using protein-A-agarose.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of Proteasomes in Mammalian Cells

Mason

Hendil²,

Rivett

1996

European Journal of Biochemistry

117

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The proteasome, a multimeric protease, plays an important role in nonlysosomal pathways of intracel-M a r protein degradation. This study was undertaken to determine which subunits of mammalian proteasomes are phosphorylated and to investigate the possible role of phosphorylation in regulating proteasome activity and the association with regulatory components. Rat-I fibroblasts were grown in the presence of ['*P]phosphate and proteasomes were immunoprecipitated from cell lysates with proteasome-specific polyclonal antibodies. Subsequent analysis by two-dimensional polyacrylamide gel electrophoresis showed two radiolabeled proteasome subunits which were identified using monoclonal antibodies as C8 and C9. Treatment of human embryonic lung cells (L-132), under identical conditions, also showed the same two phosphorylated subunits. Phosphoamino acid analysis revealed phosphoserine to be present in both C8 and C9. Examination of the sequence of C9 showed a potential cCMP-dependent phosphorylation site (-Arg3-Arg-Tyr-Asp-&-Arg8-), whilst C8 contains several potential casein kinase I1 phosphorylation sites. Following immunoprecipitation by a monoclonal antibody and dephosphorylation by acid phosphatase, proteasomes were observed to have significantly lower activities when compared to phosphorylated proteasomes, implying that phosphorylation may be an important mechanism of regulating proteasome function. Free proteasomes were separated by gel-filtration from those complexed with regulatory complexes to form the 26s proteinase. The ratio of phosphorylation of C8 and C9 was found to be very similar in the two complexes but the level of phosphorylation was higher in the 26s proteinase than in free proteasomes.Keywords: proteasome; 26s protease; phosphorylation ; pcptidase activity ; regulation.Proteasomes (multicatalytic proteinase complexes) are highmolecular-mass (700 kDa) multi-subunit cylindrical particles which are found in the nucleus and cytoplasm of eukaryotic cells (for reviews see [I -41). Eukaryotic proteasomes are composed of at least 14 non-identical subunits which are encoded by members of the same gene family. They can be divided into two groups a and p, based on their relationship to the a and /I subunits, respectively, of the simpler proteasome isolated from Abbrevintiorzs.AAF-NH-Mec, Ala-Ala-Phe-7-amino-4-1nethyl-coumarin; Boc-LSTR-NH-Mec, terf-butoxycarbonyl-Leu-Ser-Thr-Arg-7-amino-4-methylcoumarin; DMEM, Dulbecco's modified Eagle's medium; FCS, foetal calf serum; M-DMEM, methionine-free DMEM ; MHC, major histocornpatability complex: NaCIP,, 137 mM NaCI, 1.5 mM KH,PO,, 20 mM Na,HPO,, 2.7 mM KCI, pH 7.2; P-DMEM, phosphate-free DMEM: RIPA, 50 mM Tris pH 8.0, 150 mM NaCI, 1 % (masslvol.) Nonidet P-40, 0.1 % SDS, 0.5% sodium deoxycholate; Suc-LLVY-NH-Mec, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin: TrislNaCUEDTA. 50 mM Tris/HCl pH 7.0,150 mM NaCl, 5 mM EDTA, 0.1 % BSA, 0.1 % SDS, 0.1 % sodium deoxycholate, 0.5% (masslvol.) Nonidet-P40.Enzynrs. Proteasome (EC 3.4.99.46) ; potato acid phosphatase (EC ...

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[24] Multicatalytic endopeptidase complex: Proteasome

Cited by 76 publications

References 52 publications

Proteasome Involvement and Accumulation of Ubiquitinated Proteins in Cerebellar Granule Neurons Undergoing Apoptosis

Proteasome Involvement and Accumulation of Ubiquitinated Proteins in Cerebellar Granule Neurons Undergoing Apoptosis

Central Role of the Proteasome in Senescence and Survival of Human Fibroblasts

Phosphorylation of Proteasomes in Mammalian Cells

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