Effect of heat shock on protein degradation in mammalian cells: involvement of the ubiquitin system.

Parag, H A; Raboy, Bilha; Kulka, Richard G.

doi:10.1002/j.1460-2075.1987.tb04718.x

Cited by 231 publications

(121 citation statements)

References 36 publications

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“…Heat-shock treatment stimulates ubiyuitin conjugation to denatured proteins with the consumption of free (mono)ubiquitin (Carlson et al, 1987;Parag et al, 1987). In the present study, heat-shock treatment of HeLa cells increased the urea-soluble multi-ubiyuitin chains (Table 2).…”

Section: Discussionsupporting

confidence: 53%

Immunoassay for the Quantification of Intracellular Multi‐Ubiquitin Chains

Takada

Nasu

Hibi

et al. 1995

European Journal of Biochemistry

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A sandwich ELISA has been developed to measure intracellular levels of multi-ubiquitin chains. The mixture of multi-ubiquitin chains, prepared in vitro by incubation of ubiquitin (plus "'I-ubiquitin) and lysozyme with ubiquitin-ligating enzymes and ATP, was partially purified and established as a standard named the multi-ubiquitin-chain reference preparation 1 (MUCRPI). The concentration of MUCRPI was calculated from the recovered radioactivity of "'I-ubiquitin. All measurements by the ELISA were expressed in terms of MUCRPI. The ELISA showed good sensitivity (98 pg/ml), precision (intra-assays < 6%) and reproducibility (interassay < 9%). In addition, there was no substantial cross-reaction with mono-, di-and tri-ubiquitin, or mono-ubiquitinated and di-ubiquitinated lysozyme in the ELISA, and large multi-ubiquitin chains ( n > approximately 6 ) may be fully reactive. These results combined with excellent results in the recovery and dilution tests guarantee accurate measurement of multi-ubiquitin chains in cell extracts prepared with a lysis buffer (water soluble) or the buffer supplemented 8 M urea (urea soluble). The level of the water-soluble multi-ubiquitin chains in reticulocytes was lower than that of erythrocytes, but the urea-soluble chain level was higher in the reticulocytes. Heat-shock treatment of HeLa cells increased the urea-soluble multi-ubiquitin chains. These data indicate that this ELISA provides a useful and reliable approach to the study of intracellular multi-ubiquitin-conjugate turnover.Keywords: ELISA ; heat shock ; multi-ubiquitin chain; reticulocyte; ubiquitin.Ubiquitin is found in either a free form or a form bound covalently to intracellular proteins in vivo (Hershko, 1988;Finley and Chau, 1991). In the latter form, the ubiquitin C-terminus is ligated to &-amino groups of appropriate Lys residues in target proteins by a family of ubiquitin-conjugating enzymes: a ubiquitin-activating enzyme (El), a ubiquitin-carrier protein (E2), and a ubiquitin-protein ligase (E3) (Hershko et al., 1983;Hershko and Ciechanover, 1992). In this process, several ubiquitin moieties are usually ligated sequentially to the initial acceptor protein, forming multi-ubiquitin chains in which the ubiquitin Cterminus is conjugated to the &-amino group of Lys48 of another ubiquitin moiety (Chau et al., 1989). The multi-ubiquitin chain acts as a signal to induce degradation of target proteins by 2 6 s proteasome (Chau et al., 1989;Hershko and Ciechanover, 1992). Therefore, the chain structure is essential for ubiquitin-dependent proteolysis.The ubiquitin-dependent proteolysis has been implicated in a variety of cellular processes, including stress response (Finley et al., 1987;Carlson et a]., 1987), cell-cycle control (Glotzer et al., 1991;Nishizawa et al., 1993) and apoptosis (Delic et al., 1993). Intracellular levels of ubiquitin and ubiquitin-protein conjugates are changed in response to heat shock (Parag et al., Abbreviations. MUCRPI , multi-ubiquitin chain reference preparation 1 ; E l , ubiquitin-activating enzy...

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Section: Discussionsupporting

confidence: 53%

Immunoassay for the Quantification of Intracellular Multi‐Ubiquitin Chains

Takada

Nasu

Hibi

et al. 1995

European Journal of Biochemistry

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“…It has been reported that the change in the ubiquitin-H2A level in rat hepatoma cells in response to heat shock was not inhibited with cycloheximide [37]. In our experiments, a similar result was obtained using cycloheximide at a final concentration of 0.1 mM (data not shown), which suggests that the behavior of ubiquitin and the formation of ubiquitin conjugates, shown in this report, are not due to the post-heat-shock synthesis of heat-shock proteins including ubiquitin and other proteins.…”

Section: Four Fragments Gave Sequences Of T I T L E V E P S D T I E Nmentioning

confidence: 98%

“…A temperature-sensitive El-mutant, the ts85 cell, failed to degrade aberrant proteins or truncated nascent polypeptides accumulated at a nonpermissive temperature [36]. In addition, it has been reported that ubiquitinated proteins are rapidly produced by heat-shock treatment in rat hepatoma cells and decrease during the subsequent cultivation at normal temperature [37]. However, it is still not known whether these ubiquitinated proteins have multi-ubiquitinated or mono-ubiquitinated structures and whether the accumulation of multi-ubiquitinated proteins is caused by activation of the ubiquitinating enzyme system or inactivation of the 26s proteasome.…”

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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“…Proteasome may play a generalized, constitutive role in 'mopping up' target proteins made redundant in the normal course of metabolism. A significant body of direct and indirect evidence has accumulated suggesting that environmental stress and resulting damage and metabolic shifts result in increased protein ubiquitination and ultimate removal of such conjugates by the proteasome and related proteolytic particles (7,8,19,21,23,25). Seeds represent some of the most environmental stress-tolerant structures in nature and provide an ideal system for the study of the function of proteasome and related reactions and proteins.…”

Section: Resultsmentioning

confidence: 99%

“…The precise regulation of the targeting of cytoplasmic proteins for the ubiquitination pathway is not understood. Ubiquitin was shown to be one of the heat-shock proteins (2,7,17,19) and ubiquitination tends to increase following environmental stress (7,23,25). Damage to, or redundancy of, target proteins are generally considered to be signals for ubiquitin conjugation (18).…”

mentioning

confidence: 99%

Dry Pea Seed Proteasome

Skoda¹,

Malek²

1992

Plant Physiol.

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Proteasomes were isolated from mature, dry pea seeds (Pisum sativum L.). They appear to be similar to proteasomes from other sources in that they are cylindrical (shown by negative staining), have a molecular mass greater than 600 kilodaltons (by gel permeation chromatography), and consist of several subunits between 25 and 31 kilodaltons. The seed proteasomes possess three characteristic partial activities (trypsin-like, chymotrypsin-like, and peptidyl glutamyl peptidase) as determined with fluorogenic peptide substrates. Activation and inhibition by various effectors, and particularly sensitivity to porphyrins, also match characteristics of proteasomes described for other organisms. The potential role of the proteasome in seed biology is discussed.

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Effect of heat shock on protein degradation in mammalian cells: involvement of the ubiquitin system.

Cited by 231 publications

References 36 publications

Immunoassay for the Quantification of Intracellular Multi‐Ubiquitin Chains

Immunoassay for the Quantification of Intracellular Multi‐Ubiquitin Chains

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

Dry Pea Seed Proteasome

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