Different ratios in 20 S proteasomes and regulatory subunit complexes in two isoforms of the 26 S proteasome purified from rabbit skeletal muscle

Sawada, Hitoshi; Muto, Kazuko; Fujimuro, Masahiro; Akaishi, Takahiro; Sawada, Minoru; Yokosawa, Hideyoshi; Goldberg, Alfred L.

doi:10.1016/0014-5793(93)80731-9

Cited by 30 publications

(17 citation statements)

References 18 publications

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“…We have previously reported that the 26s proteasome can be collected in a pellet fraction by centrifugation at I00000 g for 5 h, while the ubiquitinating enzyme system is recovered in a supernatant fraction [38,45]. Crude extracts obtained from HeLa cells, which had been previously cultured at 43°C or 45°C for 30min and subsequently at 37°C for various periods, were centrifuged at 105 000 g for 5 h. The resultant supernatant and precipitate fractions were used as the ubiquitinating enzyme system fraction and the 26s proteasome fraction, respectively, and their activities toward heat-denatured '2sI-lysozyme and multiubiquitinated, heat-denatured 'Z51-lysozyme, respectively, were assayed (Fig.…”

Section: Subcellular Distribution Of Multi-ubiquitinated Proteins In mentioning

confidence: 99%

Dynamics of Ubiquitin Conjugation during Heat‐Shock Response Revealed by using a Monoclonal Antibody Specific to Multi‐Ubiquitin Chains

Fujimuro

Sawada

Yokosawa

1997

European Journal of Biochemistry

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Levels of intracellular multi-ubiquitinated proteins in heat-shocked HeLa cells were investigated using a monoclonal antibody specific to multi-ubiquitin chains. After heat-shock treatment at 42-44 "C for 30 min, the level of multi-ubiquitinated proteins increased within the first 2 h at 37°C and returned to the initial level within the following 2 h. The accumulation of multi-ubiquitin conjugates was elevated by increasing the temperature, while the opposite was the case for the level of ubiquitinated histone H2A. Immunocytochemical analysis revealed that the amount of ubiquitin conjugates rapidly increased in the cytosol and concomitantly decreased in the nucleus under heat-shock conditions. The heat-shock treatment elicited little apparent change in the activity of the 26s proteasome, but it did induce a gradual increase in activity of the ubiquitinating enzyme system. These results strongly suggest that the level of cytoplasmic multi-ubiquitinated proteins and that of nuclear ubiquitinated histone H2A increases and decreases, respectively, in response to heat shock and that the heat-shock-induced accumulation of multiubiquitinated proteins is caused by activation of the ubiquitinating enzyme system rather than inactivation of the 26s proteasome.Keywords: heat shock; proteasome; multi-ubiquitin chain; histone 2A; ubiquitin.A small set of universally conserved genes is activated when an organism is exposed to stress such as heat shock, heavy metals, ischemia, and irradiation [l-31. The proteins encoded by these genes are called stress proteins or heat-shock proteins. These proteins play key roles not only in the stress response, but also in the normal physiological response without stress [4].Ubiquitin, a heat-shock protein comprising 76 amino acid residues, is highly conserved and widely distributed in eukaryotes [5, 61. The ubiquitin gene contains a heat-shock promoter and is expressed by heat shock in eukaryotic cells such as chicken fibroblasts [7-91 and yeasts [lo, 111. ligase). Multi-ubiquitinated proteins produced in this way are degraded in an ATP-dependent manner by the 26s proteasome, a ubiquitin/ATP-dependent protease complex [see reviews 6, 27 -291.Although ubiquitin acts as a signal for protein degradation, several intracellular proteins that are not destined to be degraded are also ubiquitinated. The most abundant proteins are ubiquitinated histone H2A and H2B [30]. Approximately 10% of H2A is ubiquitinated at a ratio of one histone molecule to less than five ubiquitin molecules, and 2% of H2B is ubiquitinated at a ratio of one to less than four [31, 321. However, it is unclear whether these ubiquitin molecules are attached to histones via the multi-ubiquitin chain or not. Moreover, it is still uncertain why and how ubiquitinated histones are deubiquitinated in response to heat shock [33, 341 and in chromosome condensation during the mitotic cell cycle [35].When cells are exposed to heat shock, many aberrant proteins are produced from heat-sensitive proteins, nascent polypeptides, or partially ...

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Section: Subcellular Distribution Of Multi-ubiquitinated Proteins In mentioning

confidence: 99%

Dynamics of Ubiquitin Conjugation during Heat‐Shock Response Revealed by using a Monoclonal Antibody Specific to Multi‐Ubiquitin Chains

Fujimuro

Sawada

Yokosawa

1997

European Journal of Biochemistry

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“…Furthermore, proteasomes are activated during in vivo Xenopus egg activation induced by treatment with the calcium ionophore A23187 (22). The 26 S proteasome consists of at least two subunits: one is a 700-kDa proteolytic core complex called the 20 S proteasome with 28 subunits, and the other is a 700 -1000-kDa regulatory subunit complex made up of ϳ20 subunits (31)(32)(33)(34)(35)(36)(37)(38). Although there are many reports that several proteasome subunits can be phosphorylated, there is little or no direct effect on proteasome activity from these modifications (39 -42).…”

Section: Fig 4 Effects Of Protease Inhibitors and Herbimycin A On Imentioning

confidence: 99%

Transient Nuclear Factor κB (NF-κB) Activation Stimulated by Interleukin-1β May Be Partly Dependent on Proteasome Activity, but Not Phosphorylation and Ubiquitination of the IκBα Molecule, in C6 Glioma Cells

Uehara

Matsuno

Kaneko

et al. 1999

Journal of Biological Chemistry

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We previously reported that several stresses can induce cytokine-induced neutrophil chemoattractant expression in a nuclear factor B (NF-B)-dependent manner. In this study, we focused further on the regulation of NF-B. The activation of NF-B and the subsequent cytokine-induced neutrophil chemoattractant induction in response to interleukin-1␤ (IL-1␤) were inhibited by proteasome inhibitors, MG132 and proteasome inhibitor I. Translocation of NF-B into nuclei occurs by the phosphorylation, multi-ubiquitination, and degradation of IB␣, a regulatory protein of NF-B. Nascent IB␣ began to degrade 5 min after treatment with IL-1␤ and disappeared completely after 15 min. However, IB␣ returned to basal levels after 45-60 min. Interestingly, resynthesized IB␣ was already phosphorylated at Ser-32. These results suggest that 1) the upstream signals are still activated, although the translocation of NF-B peaks at 15 min; and 2) the regulated protein(s) acts downstream of IB␣ phosphorylation. Western blotting showed that the resynthesized and phosphorylated IB molecules were also upward-shifted by multi-ubiquitination in response to IL-1␤ treatment. On the other hand, ATP-dependent Leu-Leu-Val-Tyr cleaving activity transiently increased, peaked at 15 min, and then decreased to basal levels at 60 min. Furthermore, the cytosolic fraction that was stimulated by IL-1␤ for 15 min, but not for 0 and 60 min, could degrade phosphorylated and multi-ubiquitinated IB␣. These results indicate that the transient translocation of NF-B in response to IL-1␤ may be partly dependent on transient proteasome activation. Nuclear factor B (NF-B)1 participates in the regulation of the expression of multiple immediate-early genes involved in immune, acute-phase, and inflammatory responses (1). NF-B is a heterodimer protein of the Rel family of transcription factors. In mammalian cells, the factors include p65 (RelA), RelB, c-Rel, p50/p105 (NF-B1), and p52/p100 (NF-B2). NF-B proteins are constitutively present in cells and are retained in the cytoplasm associated with the inhibitory protein IB (2, 3). Activated NF-B complexes, typically composed of p50 and p65, are translocated to the nucleus in response to several cytokines (TNF-␣, IL-1␤, and IL-2), bacterial endotoxin, and stresses (UV, H 2 O 2 ).(1, 4 -6). The activation of NF-B appears to require the phosphorylation and degradation of the IB proteins, thereby allowing the rapid translocation of NF-B from the cytoplasm to the nucleus (4, 7-9). In particular, it has been shown that the phosphorylation of Ser-32 and Ser-36 and the ubiquitination at Lys-21 and Lys-22 are essential for targeting IB for signal-induced degradation by the ubiquitin/proteasome system (10). The ubiquitin-dependent degradation of regulatory shortlived proteins plays an important role in cellular processes, including the cell cycle, immune system functions, inflammatory responses, and tissue differentiation. A key element in the regulation process is E3, a member of the ubiquitin-substrate ligase family of enzymes. After bin...

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“…ATP-dependent degradation of poly-Ubprotein conjugates by the 26 S proteasome was measured as follows; a reaction mixture (200 PI), which contained 50 mM Tris-HCI (pH 7.8), 10 mM MgCI,, 2 mM ATP, 1 mM DTT, 0.2 pg of ['2SI]proteins (18,000 cpm) tagged with multi-Ub chains and 3 lug of the 26 S proteasome purified from rabbit skeletal muscle as described previously [35], was incubated at 37°C for 2 h, and the reaction was stopped by the addition of 25 ~1 of cold 10% bovine serum albumin and 600 ~1 of cold 10% trichloroacetic acid. After standing for more than 10 min on ice, the acid-soluble radioactivity was measured by a y-counter.…”

Section: Degradation Of Poly-uandprotein Conjugatesmentioning

confidence: 99%

Production and characterization of monoclonal antibodies specific to multi‐ubiquitin chains of polyubiquitinated proteins

1994

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Polyubiquitinated proteins tagged with multi-ubiquitin chains are substrates preferred by the 26 S proteasome (a ubiquitin/ATP-dependent proteolytic complex). Here, we developed a simple method for the efficient preparation of polyubiquitinated proteins which are degraded by the 26 S proteasome in an ATP-dependent manner. Our efficient method enabled us to produce ten monoclonal antibodies that recognized the multi-ubiquitin chains of the polyubiquitinated proteins, but not free ubiquitin or the protein moieties. Eight of the antibodies recognized only the multi-ubiquitin chains of the polyubiquitinated proteins, while the other two antibodies cross-reacted with mono-ubiquitin and methyl-ubiquitin, both of which are linked to proteins via an isopeptide bond, as well as with the multi-ubiquitin chains. Thus these antibodies are novel and useful tools for the identification and quantification of polyubiquitinated proteins in various cells and tissues under physiological and pathological conditions.

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Different ratios in 20 S proteasomes and regulatory subunit complexes in two isoforms of the 26 S proteasome purified from rabbit skeletal muscle

Cited by 30 publications

References 18 publications

Dynamics of Ubiquitin Conjugation during Heat‐Shock Response Revealed by using a Monoclonal Antibody Specific to Multi‐Ubiquitin Chains

Dynamics of Ubiquitin Conjugation during Heat‐Shock Response Revealed by using a Monoclonal Antibody Specific to Multi‐Ubiquitin Chains

Transient Nuclear Factor κB (NF-κB) Activation Stimulated by Interleukin-1β May Be Partly Dependent on Proteasome Activity, but Not Phosphorylation and Ubiquitination of the IκBα Molecule, in C6 Glioma Cells

Production and characterization of monoclonal antibodies specific to multi‐ubiquitin chains of polyubiquitinated proteins

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