Proteasome inhibition induces differential heat shock protein response but not unfolded protein response in HepG2 cells

Liver, a central organ responsible for the metabolism of carbohydrates, proteins, and lipoproteins, is exposed to various kinds of physiological, pathological, and environmental stresses. We hypothesized that blockage of proteasome degradation pathway induces heat shock protein (HSP) response and unfolded protein response in the liver cells. In this study, we have characterized cellular responses to proteasome inhibition in HepG2 cells, a well-differentiated human hepatoma cells. We found that proteasome inhibition induced differential response among cytosolic HSPs, that is, increased expression of HSP70, but no change in HSP40, HSC70, and HSP90. However, proteasome inhibition did not induce typical unfolded protein response as indicated by absence of stimulation of GRP78 and GRP94 proteins. Upon proteasome inhibition, inclusion bodies were accumulated, and ubiquitin-conjugated proteins appeared in insoluble fraction, together with HSP40, HSP70, HSC70, and HSP90. After proteasome inhibition, misfolded proteins were increased in the cytosol and in the ER compartment as evaluated by examining ubiquitin-conjugated proteins. However, essentially all ER-associated ubiquitin-conjugated proteins were located on the surface of the ER, which explains why proteasome inhibition does not induce unfolded protein response. In conclusion, proteasome inhibition induces differential HSP response, but not unfolded protein response in HepG2 cells. Our study also suggests that HSPs play important roles in directing proteasomal degradation and protein aggregate formation.     

The human ubiquitin conjugating enzyme UBE2J2 (Ubc6) is a substrate for proteasomal degradation

The human Ube2J2 enzyme functions in the ubiquitination of proteins at the ER. Here we demonstrate that it, and a second ubiquitin conjugating (Ubc) enzyme Ube2G2, are unstable, and incubation of transfected cells with proteasome inhibitors increased steady-state protein levels. For Ube2J2, pharmacological induction of the unfolded protein response (UPR) did not significantly alter ectopic protein levels, however the effect of proteasomal inhibition was abolished if the enzyme was inactivated or truncated to disrupt its ER-localization. These results suggest for the first time that the steady state expression of Ubcs’ may be important in regulating the degradation of ER proteins in mammalian cells.     

The inhibition of microsomal triglyceride transfer protein activity in rat hepatoma cells promotes proteasomal and nonproteasomal degradation of apoprotein b100

In the human hepatic cell line, HepG2, apolipoprotein B100 (apoB100) degradation is increased by inhibiting lipid transfer mediated by the microsomal triglyceride transfer protein (MTP) and is predominantly accomplished by the ubiquitin-proteasome pathway. In the current study, we determined whether this degradative pathway was restricted to HepG2 cells or was of more general importance in hepatic apoB100 metabolism. Rat hepatoma McArdle RH7777 cells (McA), compared to HepG2 cells, secrete a large fraction of apoB100 associated with VLDL particles, as does the normal mammalian liver. In McA cells studied under basal conditions, the proteasome inhibitor lactacystin (LAC) increased apoB100 recovery, indicating that the role of the proteasome in apoB100 metabolism is not restricted to HepG2 cells. When apoB100 lipidation was blocked by an inhibitor of MTP (MTPI), recovery of cellular apoB100 was markedly reduced, but LAC was only partially ( approximately 50%) effective in reversing the induced degradation. This partial effectiveness of LAC may have represented either (1) incomplete inhibition by LAC of its preferred target, the chymotrypsin-like activity of the proteasome, (2) the presence of an apoB100 proteolytic activity of the proteasome resistant to LAC, or (3) a nonproteasomal proteolytic pathway of apoB100 degradation. By studying immunoisolated proteasomes and McA cells treated with LAC and/or MTPI and a variety of protease inhibitors, we determined that the proteasomal component of apoB100 degradation was entirely attributable to the chymotrypsin-like catalytic activity, but only accounted for part of apoB100 degradation induced by MTPI. The nonproteasomal apoB100 degradative pathway was nonlysosomal and resistant to E64d, DTT, and peptide aldehydes such as MG132 or ALLN but was partially sensitive to the serine protease inhibitor APMSF. Furthermore, when the protein trafficking inhibitor, brefeldin A, was used to block endoplasmic reticulum (ER) to Golgi transport in MTPI-treated McA cells, degradative activity resistant to LAC was increased, suggesting that the nonproteasomal pathway is associated with the ER.     

Cellular tolerance of prion protein PrP in yeast involves proteolysis and the unfolded protein response

Secretory proteins undergo a stringent quality control process in the endoplasmic reticulum (ER). Misfolded ER proteins are returned to the cytosol and destroyed by the proteasome. Prion protein PrP is degraded by the proteasome in mammalian cells. However, the significance of proteolysis on PrP-induced cell death is controversial. Moreover, the specific pathway involved in PrP degradation remains unknown. Here, we demonstrate that the unglycosylated form of human PrP is subjected to the ER-associated protein degradation (ERAD) process in the yeast Saccharomyces cerevisiae. We also show that unglycosylated PrP is degraded by the Hrd1-Hrd3 pathway. Accumulation of misfolded proteins triggers the unfolded protein response (UPR), which promotes substrate refolding. Interestingly, we find that the expression of PrP leads to growth impairment in cells deficient in UPR and ERAD. These findings raise the possibility that decreased UPR activity and proteolysis may contribute to the pathogenesis of some prion-related diseases.     

Degradation of trafficking-defective long QT syndrome type II mutant channels by the ubiquitin-proteasome pathway

Mutations in the human ether-a-go-go-related gene (hERG) cause chromosome 7-linked long QT syndrome type II (LQT2). We have shown previously that LQT2 mutations lead to endoplasmic reticulum (ER) retention and rapid degradation of mutant hERG proteins. In this study we examined the role of the ubiquitin-proteasome pathway in the degradation of the LQT2 mutation Y611H. We showed that proteasome inhibitors N-acetyl-L-leucyl-L-leucyl-L-norleucinal and lactacystin but not lysosome inhibitor leupeptin inhibited the degradation of Y611H mutant channels. In addition, ER mannosidase I inhibitor kifunensine and down-regulation of EDEM (ER degradation-enhancing alpha-mannosidase-like protein) also suppressed the degradation of Y611H mutant channels. Proteasome inhibition but not mannosidase inhibition led to the accumulation of full-length hERG protein in the cytosol. The hERG protein accumulated in the cytosol was deglycosylated. Proteasome inhibition also resulted in the accumulation of polyubiquitinated hERG channels. These results suggest that the degradation of LQT2 mutant channels is mediated by the cytosolic proteasome in a process that involves mannose trimming, polyubiquitination, and deglycosylation of mutant channels.     

ER stress induces alternative nonproteasomal degradation of ER proteins but not of cytosolic ones

Inhibition of protein folding in the endoplasmic reticulum (ER) causes ER stress, which triggers the unfolded protein response (UPR). To decrease the biosynthetic burden on the ER, the UPR inhibits in its initial stages protein synthesis. At later stages it upregulates components of ER-associated degradation (ERAD) and of the ubiquitin/proteasome system, which targets ER as well as cytosolic proteins for disposal. Here we report that, at later stages, the UPR also activates an alternative nonproteasomal pathway of degradation, which is resistant to proteasome inhibitors and is specific for ER substrates (assessed with uncleaved precursor of asialoglycoprotein receptor H2a and unassembled CD3delta) and not for cytosolic ones (p53). To mimic the initial inhibition of translation during UPR, we incubated cells with cycloheximide. After this treatment, degradation of ERAD substrates was no longer effected by proteasomal inhibition, similarly to the observed outcome of UPR. The degradation also became insensitive to abrogation of ubiquitination in a cell line carrying a thermosensitive E1 ubiquitin activating enzyme mutant. Of all protease inhibitors tested, only the metal chelator o-phenanthroline could block this nonproteasomal degradation. Preincubation of o-phenanthroline with Mn2+ or Co2+, but not with other cations, reversed the inhibition. Our results suggest that, upon inhibition of translation, an alternative nonproteasomal pathway is activated for degradation of proteins from the ER. This involves a Mn2+/Co2+-dependent metalloprotease or other metalloprotein. The alternative pathway selectively targets ERAD substrates to reduce the ER burden, but does not affect p53, the levels of which remain dependent on proteasomal control.     

Increased expression and altered subunit composition of proteasomes induced by continuous proteasome inhibition establish apoptosis resistance and hyperproliferation of Burkitt lymphoma cells

The proteasome is the main protease for extralysosomal protein degradation in eukaryotic cells, and constitutes a sophisticated high molecular mass proteinase complex underlying a tightly coordinated expression and assembly of multiple subunits and subcomplexes. Here we show that continuous inhibition of proteasomal chymotrypsin-like peptidase activity by the proteasome inhibitor bortezomib induces in human Namalwa Burkitt lymphoma cells increased de novo biogenesis of proteasomes accompanied by increased expression of the proteasome maturation protein POMP, increased expression of 19S-20S-19S proteasomes, and abrogation of expression of beta 1i, beta 2i and beta 5i immunosubunits and PA28 in favor of increased expression of constitutive proteolytic beta1, beta2 and beta 5 subunits and 19S regulatory complexes. These alterations of proteasome expression and subunit composition are accompanied by an increase in proteasomal caspase-like, trypsin-like and chymotrypsin-like peptidase activities, not inhibitable by high doses of bortezomib. Cells harboring these proteasomal alterations display rapid proliferation and cell cycle progression, and acquire resistance to apoptosis induced by proteasome inhibitors, gamma-irradiation and staurosporine. This acquired apoptosis resistance is accompanied by de novo expression of anti-apoptotic Hsp27 protein and the loss of ability to accumulate and stabilize pro-apoptotic p53 protein. Thus, increased expression, altered subunit composition and increased activity of proteasomes constitute a hitherto unknown adaptive and autoregulatory feedback mechanism to allow cells to survive the lethal challenge of proteasome inhibition and to establish a hyperproliferative and apoptosis-resistant phenotype.     

Enhanced ubiquitin-proteasome activity in calreticulin deficient cells: a compensatory mechanism for cell survival

Calreticulin is a lectin chaperone essential for intracellular calcium homeostasis. Deletion of calreticulin gene compromises the overall quality control within the endoplasmic reticulum (ER) leading to activation of the unfolded protein response. However, the ER structure of calreticulin deficient cells (crt-/-) is not altered due to accumulation of misfolded proteins. Therefore, the aim of this study was to determine whether the ubiquitin-proteasome pathway is activated in crt-/- cells as a compensatory mechanism for cell survival. Here we show a significant increase in the expression of genes involved in ER associated degradation and activation of the ubiquitin-proteasome system in crt-/- cells. We also demonstrated that the ubiquitination of two proteins processed in ER, connexin 43 and A1AT NHK (alpha1-antitrypsin mutant) are increased in crt-/- cells. Furthermore, we showed that the increased proteasome activity in the crt-/- cells could be rescued upon re-introduction of calreticulin or calsequestrin (a muscle calcium binding protein). We also illustrated that increased cytosolic Ca2+ enhances the proteasome activity. Interestingly, suppression of calnexin function using siRNA further elevated the proteasome activity in crt-/- cells. This is the first report to show that loss of calreticulin function enhances the ubiquitin-proteasome activity which could function as a compensatory mechanism for cell survival.     

Simultaneous inhibition of the ubiquitin-proteasome system and autophagy enhances apoptosis induced by ER stress aggravators in human pancreatic cancer cells

In contrast to normal tissue, cancer cells display profound alterations in protein synthesis and degradation. Therefore, proteins that regulate endoplasmic reticulum (ER) homeostasis are being increasingly recognized as potential therapeutic targets. The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. However, interactions between autophagy, the proteasome, and ER stress pathways in cancer remain largely undefined. This study demonstrated that withaferin-A (WA), the biologically active withanolide extracted from Withania somnifera, significantly increased autophagosomes, but blocked the degradation of autophagic cargo by inhibiting SNARE-mediated fusion of autophagosomes and lysosomes in human pancreatic cancer (PC) cells. WA specifically induced proteasome inhibition and promoted the accumulation of ubiquitinated proteins, which resulted in ER stress-mediated apoptosis. Meanwhile, the impaired autophagy at early stage induced by WA was likely activated in response to ER stress. Importantly, combining WA with a series of ER stress aggravators enhanced apoptosis synergistically. WA was well tolerated in mice, and displayed synergism with ER stress aggravators to inhibit tumor growth in PC xenografts. Taken together, these findings indicate that simultaneous suppression of 2 key intracellular protein degradation systems rendered PC cells vulnerable to ER stress, which may represent an avenue for new therapeutic combinations for this disease.     

Anti-atherogenic antioxidants regulate the expression and function of proteasome alpha-type subunits in human endothelial cells

It has been proposed that phenolic antioxidants such as probucol exert their anti-atherogenic effects through scavenging lipid-derived radicals. In this study the potential for genomics to reveal unanticipated pharmacological properties of phenolic antioxidants is explored. It was found that two anti-atherogenic compounds, BO-653 and probucol, inhibited the expression of three alpha-type proteasome subunits, PMSA2, PMSA3, and PMSA4 in human umbilical vein endothelial cells. Here we report that both BO-653 and probucol caused not only inhibition of the mRNA levels of these three subunits but also inhibition of both the gene expression and protein synthesis of the alpha-type subunit, PMSA1. Other subunit components of the proteasome such as the beta-type subunits (PMSB1, PMSB7), the ATPase subunit of 19 S (PMSC6), the non-ATPase subunit of 19 S (PMSD1), and PA28 (PMSE2) were not significantly affected by treatment with these compounds. The specific inhibition of alpha-type subunit expression in response to these antioxidants resulted in functional alterations of the proteasome with suppression of degradation of multiubiquitinated proteins and IkappaBalpha. These results suggest that certain compounds previously classified solely as antioxidants are able to exert potentially important modulatory effects on proteasome function.