Do Hee Lee

Proteasome inhibitors: valuable new tools for cell biologists.

Proteasomes are major sites for protein degradation in eukaryotic cells. The recent identification of selective proteasome inhibitors has allowed a definition of the roles of the ubiquitin-proteasome pathway in various cellular processes, such as antigen presentation and the degradation of regulatory or membrane proteins. This review describes the actions of these inhibitors, how they can be used to investigate cellular responses, the functions of the proteasome demonstrated by such studies and their potential applications in the future.

Trehalose Accumulation during Cellular Stress Protects Cells and Cellular Proteins from Damage by Oxygen Radicals

The disaccharide trehalose, which accumulates dramatically during heat shock and stationary phase in many organisms, enhances thermotolerance and reduces aggregation of denatured proteins. Here we report a new role for trehalose in protecting cells against oxygen radicals. Exposure of Saccharomyces cerevisiae to a mild heat shock (38 °C) or to a proteasome inhibitor (MG132) induced trehalose accumulation and markedly increased the viability of the cells upon exposure to a free radical-generating system (H2O2/iron). When cells were returned to normal growth temperature (28 °C) or MG132 was removed from the medium, the trehalose content and resistance to oxygen radicals decreased rapidly. Furthermore, a mutant unable to synthesize trehalose was much more sensitive to killing by oxygen radicals than wild-type cells. Providing trehalose exogenously enhanced the resistance of mutant cells to H2O2. Exposure of cells to H2O2 caused oxidative damage to amino acids in cellular proteins, and trehalose accumulation was found to reduce such damage. After even brief exposure to H2O2, the trehalose-deficient mutant exhibited a much higher content of oxidatively damaged proteins than wild-type cells. Trehalose accumulation decreased the initial appearance of damaged proteins, presumably by acting as a free radical scavenger. Therefore, trehalose accumulation in stressed cells plays a major role in protecting cellular constituents from oxidative damage.

Selective Inhibitors of the Proteasome-dependent and Vacuolar Pathways of Protein Degradation in Saccharomyces cerevisiae

We have studied whether various agents that inhibit purified yeast and mammalian 26 S proteasome can suppress the breakdown of different classes of proteins in Saccharomyces cerevisiae. The degradation of short-lived proteins was inhibited reversibly by peptide aldehyde inhibitors of proteasomes, carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132) and carbobenzoxyl-leucinyl-leucinyl-norvalinal (MG115), in a yeast mutant with enhanced permeability, but not in wild-type strains. Lactacystin, an irreversible proteasome inhibitor, had no effect, but the β-lactone derivative of lactacystin, which directly reacts with proteasomes, inhibited the degradation of short-lived proteins. These inhibitors also blocked the rapid ubiquitin-dependent breakdown of a β-galactosidase fusion protein and caused accumulation of enzymatically active molecules in cells. The degradation of the bulk of cell proteins, which are long-lived molecules, was not blocked by proteasome inhibitors, but could be blocked by phenylmethylsulfonyl fluoride. This agent, which inhibits multiple vacuolar proteases, did not affect the proteasome or breakdown of short-lived proteins. These two classes of inhibitors can thus be used to distinguish the cytosolic and vacuolar proteolytic pathways and to increase the cellular content of short-lived proteins.

Proteasome Inhibitors Cause Induction of Heat Shock Proteins and Trehalose, Which Together Confer Thermotolerance in Saccharomyces cerevisiae

ABSTRACT An accumulation in cells of unfolded proteins is believed to be the common signal triggering the induction of heat shock proteins (hsps). Accordingly, in Saccharomyces cerevisiae, inhibition of protein breakdown at 30°C with the proteasome inhibitor MG132 caused a coordinate induction of many heat shock proteins within 1 to 2 h. Concomitantly, MG132, at concentrations that had little or no effect on growth rate, caused a dramatic increase in the cells’ resistance to very high temperature. The magnitude of this effect depended on the extent and duration of the inhibition of proteolysis. A similar induction of hsps and thermotolerance was seen with another proteasome inhibitor, clasto-lactacystin β-lactone, but not with an inhibitor of vacuolar proteases. Surprisingly, when the reversible inhibitor MG132 was removed, thermotolerance decreased rapidly, while synthesis of hsps continued to increase. In addition, exposure to MG132 and 37°C together had synergistic effects in promoting thermotolerance but did not increase hsp expression beyond that seen with either stimulus alone. Although thermotolerance did not correlate with hsp content, another thermoprotectant trehalose accumulated upon exposure of cells to MG132, and the cellular content of this disaccharide, unlike that of hsps, quickly decreased upon removal of MG132. Also, MG132 and 37°C had additive effects in causing trehalose accumulation. Thus, the resistance to heat induced by proteasome inhibitors is not just due to induction of hsps but also requires a short-lived metabolite, probably trehalose, which accumulates when proteolysis is reduced.

Involvement of the molecular chaperone Ydj1 in the ubiquitin-dependent degradation of short-lived and abnormal proteins in Saccharomyces cerevisiae.

In Escherichia coli and mitochondria, the molecular chaperone DnaJ is required not only for protein folding but also for selective degradation of certain abnormal polypeptides. Here we demonstrate that in the yeast cytosol, the homologous chaperone Ydj1 is also required for ubiquitin-dependent degradation of certain abnormal proteins. The temperature-sensitive ydj1-151 mutant showed a large defect in the overall breakdown of short-lived cell proteins and abnormal polypeptides containing amino acid analogs, especially at 38 degrees C. By contrast, the degradation of long-lived cell proteins, which is independent of ubiquitin, was not altered nor was cell growth affected. The inactivation of Ydj1 markedly reduced the rapid, ubiquitin-dependent breakdown of certain beta-galactosidase (beta-gal) fusion polypeptides. Although degradation of N-end rule substrates (arginine-beta-gal and leucine-beta-gal) and the B-type cyclin Clb5-beta-gal occurred normally, degradation of the abnormal polypeptide ubiquitin-proline-beta-gal (Ub-P-beta-gal) and that of the short-lived normal protein Gcn4 were inhibited. As a consequence of reduced degradation of Ub-P-beta-gal, the beta-gal activity was four to five times higher in temperature-sensitive ydj1-151 mutant cells than in wild-type cells; thus, the folding and assembly of this enzyme do not require Ydj1 function. In wild-type cells, but not in ydj1-151 mutant cells, this chaperone is associated with the short-lived substrate Ub-P-beta-gal and not with stable beta-gal constructs. Furthermore, in the ydj1-151 mutant, the ubiquitination of Ub-P-beta-gal was blocked and the total level of ubiquitinated protein in the cell was reduced. Thus, Ydj1 is essential for the ubiquitin-dependent degradation of certain proteins. This chaperone may facilitate the recognition of unfolded proteins or serve as a cofactor for certain ubiquitin-ligating enzymes.

Oxidative stress-enhanced SUMOylation and aggregation of ataxin-1: Implication of JNK pathway.

Although the polyglutamine protein ataxin-1 is modified by SUMO at multiple sites, the functions of such modification or how it is regulated are still unknown. Here we report that SUMO-1 or Ubc9 over-expression stimulated the aggregation of ataxin-1 and that oxidative stress, such as hydrogen peroxide treatment, further enhanced SUMO conjugation and aggregation of ataxin-1. Accordingly, co-treatment with antioxidant N-acetyl-cysteine attenuated the effect of oxidative stress. Ataxin-1, which can activate c-Jun N-terminal kinase (JNK) pathway by itself, strongly associated with apoptosis signal-regulating kinase 1 (ASK1) while not interacting with JNK. Finally, treatment of JNK-specific inhibitor caused a reduction in the oxidant-enhanced SUMOylation and aggregation of ataxin-1. Together these results indicate that SUMO modification of ataxin-1 promotes the aggregation of ataxin-1 and that oxidative stress and JNK pathway play roles in this process.

NSC-87877, inhibitor of SHP-1/2 PTPs, inhibits dual-specificity phosphatase 26 (DUSP26)

Protein phosphorylation plays critical roles in many regulatory mechanisms controlling cell activities and thus involved in various diseases. The cellular equilibrium of phosphorylation is regulated through the actions of protein kinases and phosphatases. Therefore, these regulatory proteins have emerged as promising targets for drug development. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by 8-hydroxy-7-(6-sulfonaphthalen-2-yl)diazenyl-quinoline-5-sulfonic acid (NSC-87877), a potent inhibitor of SHP-1 and SHP-2 PTPs. Phosphatase activity of dual-specificity protein phosphatase 26 (DUSP26) was decreased by the inhibitor in a dose-dependent manner. Kinetic studies with NSC-87877 and DUSP26 revealed a competitive inhibition. NSC-87877 effectively inhibited DUSP26-mediated dephosphorylation of p38, a member of mitogen-activated protein kinase (MAPK) family. Since DUSP26 is involved in survival of anaplastic thyroid cancer (ATC) cells, NSC-87877 could be a therapeutic reagent for treating ATC.

PROTEIN DEGRADATION BY THE PROTEASOME AND DISSECTION OF ITS IN VIVO IMPORTANCE WITH SYNTHETIC INHIBITORS

Despite extensive progress in recent years in knowledge about the structure, catalytic mechanism, and peptidase activities of the 20S proteasome [1], many fundamental questions about its function remain unclear. For example, the mechanisms by which protein substrates are hydrolyzed by the 20S and 26S proteasomes and the nature of the peptides generated have not been systematically investigated, although these questions have important biological implications. Our recent studies have established that these particles degrade each protein in a highly processive manner to generate small peptides. This behavior raises many mechanistic questions and clearly distinguishes it from conventional proteases. In addition, much remains to be learned concerning the physiological importance of this particle in intracellular proteolysis both in mammalian cells and lower eukaryotes. Until recently, the physiological importance of these particles has been quite unclear, in large part due to the lack of selective inhibitors of its activity. The present article summarizes recent progress made in our knowledge about this processive mechanism and also about the proteasome’s function in vivo obtained through the use of inhibitors that enter cells and selectively block its activity. Many of the present studies have utilized the 20S proteasome from Thermoplasma acidophilum, which has proven a very valuable model for understanding proteasome structure. However, its enzymatic properties have not been explored in depth. The Thermoplasma particle offers many advantages for studies of the mechanism of the 20S proteasome in digesting proteins. Unlike the eukaryotic proteasome, it contains only one type of -subunit and one type of -subunit, and its structure has been resolved by X-ray diffraction [2]. The important X-ray diffraction and mutagenesis studies by Baumeister, Huber and coworkers have suggested a novel proteolytic mechanism, in which the nucleophilic attack on the peptide bond is initiated by the hydroxyl group on the N-terminal threonine of the -subunit [2, 3]. Our initial studies were undertaken to define its catalytic properties more precisely than previously. The recombinant archael proteasome, which contains only one type of active site, was found to exhibit not only strong ‘chymotrypsin-like’ activity (as had been found previously), but also some ‘post-glutamyl’ and some ‘trypsin-like’ activities. Thus, its one active site has rather broad specificity. In contrast to prior reports, we found that the Thermoplasma particle is relatively inactive when isolated, but can be activated 2to 4-fold by ionic detergents (SDS) and also by guanidine HCl. In this feature, the archael particle resembles the 20S particles from eukaryotes. Its activity was inhibited reversibly by peptide aldehydes and especially well by several novel peptide aldehydes with large hydrophobic residues in the P4 position, which seems to be an important residue in determining activity. Like the mammalian particle, this proteasome was inactivated irreversibly by 3,4 dichloroisocoumarin (DCI), but quite resistant to lactacystin or its -lactone derivative, which inhibits eukaryotic proteasomes. Sequence analysis indicated that DCI covalently modified the hydroxyl group of the N-terminal threonine of the subunit, the presumed active site nucleophile.

Activation of autophagy during glutamate-induced HT22 cell death

Recent evidence suggests that autophagy plays a role in oxidative injury-induced cell death. Here we examined whether glutamate-mediated oxidative toxicity induces autophagy in murine hippocampal HT22 cells and if autophagy induction affects the molecular events associated with cell death. Markers for autophagy induction including LC3 conversion, suppression of mTOR pathway, and GFP-LC3 dot formation were enhanced by glutamate treatment. By contrast, autophagy inhibition blocked glutamate-induced LC3 conversion and consequently reduced cell death. Activation of ERK1/2, a hallmark of glutamate-induced cytotoxicity, was also decreased by autophagy inhibition. Interestingly, autophagy inhibition also affected the expression of chaperones including Hsp60 and Hsp70, which are differentially regulated during HT22 cell death. Conversely, knock-down of Hsp60 greatly decreased LC3 conversion. Together these results suggest that glutamate-induced cytotoxicity involves autophagic cell death and chaperones may play a role in this process.

Positive regulation of apoptosis signal-regulating kinase 1 by dual-specificity phosphatase 13A

Apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP kinase kinase kinase, is activated by several death stimuli and is tightly regulated by several mechanisms such as interactions with regulatory proteins and post-translational modifications. Here, we report that dual-specificity phosphatase 13A (DUSP13A) functions as a novel regulator of ASK1. DUSP13A interacts with the N-terminal domain of ASK1 and induces ASK1-mediated apoptosis through the activation of caspase-3. DUSP13A enhances ASK1 kinase activity and thus its downstream factors. Small interfering RNA (siRNA) analyses show that knock-down of DUSP13A in human neuroblastoma SK-N-SH cells reduces ASK1 kinase activity. The phosphatase activity of DUSP13A is not required for the regulation of ASK1. This regulatory action of DSUP13 on ASK1 activity involves competition with Akt1, a negative regulator of ASK1, for binding to ASK1. Taken together, this study provides novel insights into the role of DUSP13A in the precise regulation of ASK1.