The present study demonstrates that even brief inhibition of degradation by the 26S proteasome inhibits global protein synthesis, mediated through increased phosphorylation of eIF2alpha (eukaryotic translational initiation factor 2alpha) by the HRI (haem-regulated inhibitor) kinase. Exposure of COS-7 cells to the proteasome inhibitor MG-132 (the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-leucinal) for 4 h resulted in a 55-60% decrease in protein synthesis rate compared with control cells. This repression of protein synthesis after treatment with MG-132 is not due to induction of apoptosis, which is known to occur after longer periods of 26S inhibition. Instead, we observed a significantly increased phosphorylation of eIF2alpha, which is known to repress global protein synthesis. In three MEF (mouse embryonic fibroblast) knockout cell lines lacking one of the four kinases known to phosphorylate eIF2alpha, increased phosphorylation of eIF2alpha still occurred after inhibition of the 26S proteasome. These three cell lines included a deletion of the PKR (double-stranded-RNA-dependent protein kinase); a deletion of the PERK (PKR-like endoplasmic reticulum resident kinase); or a deletion of the GCN2 (positive general control of transcription-2) kinase, indicating that none of these kinases was primarily responsible for the observed phosphorylation of eIF2alpha. In contrast, in a fourth MEF knockout cell line, HRI(-/-) cells lacking the HRI kinase failed to increase eIF2alpha phosphorylation upon proteasome inhibitor treatment (MG-132 or various doses of Bortezomib), indicating that the HRI kinase is the primary kinase activated by brief treatment of MEFs with 26S proteasome inhibitors.

Abstract. Velcade (also known as PS-341 or Bortezomib) is a highly selective and reversible inhibitor of the 26S proteasome and is approved for the treatment of patients with advanced multiple myeloma. Here we investigated the antiproliferative effect of Velcade on 4T1 breast cancer and B16F10 melanoma cells and evaluated the mechanism of action. It was found that two cell lines are differentially sensitive to proteasome inhibitor Velcade. The IC 50 concentrations for B16F10 and 4T1 were 2.5 nM and 71 nM, respectively, indicating that B16F10 cells are more sensitive to proteasomal inhibition. Velcade was equally potent in inhibiting the chymotrypsin-like activity of the proteasome in both cell lines. It was determined that B16F10 cells proliferate more rapidly than 4T1 cells; doubling time (T d ) = 14.2 h versus T d =22.9 h, suggesting that a rapid proliferation rate may be an important factor in cellular resistance towards proteasomal inhibition. We observed for the first time that p53 and p21 proteins were increased in B16F10 cells but not in 4T1 following Velcade-treatment, demonstrating that p53 and p21 may enhance Velcade sensitivity. Furthermore, it was observed that caspase-3 proenzyme was reduced bỹ 20% in B16F10 melanoma cells, but not in 4T1 cells in response to 26S proteasomal inhibition by Velcade. Altogether, we concluded that p53 protein plays a central role in higher sensitivity of B16F10 cells to Velcade by inducing the accumulation of p21, a cell cycle inhibitor, as well as by stimulating the mitochondrial pathway of apoptosis through caspase-3 activation. IntroductionThe 26S proteasome, is an intracellular ATP-dependent multicatalytic protease involved in the degradation of shortlived proteins under normal metabolic conditions. It is also involved in the degradation of long-lived proteins, the processing of certain proteins (e.g., transcription factor NF-κB), and antigen presentation (1,2). The 26S proteasome has a molecular mass of ~2.5 MDa and is formed by the assembly of one 20S proteasome complex (the core particle) with 2 molecules of the 19S complex, the regulatory complex. The 19S regulatory complexes are attached to both ends of the 20S proteasome. The eukaryotic 20S proteasome is made up of 2 copies of 7 distinct α and 7 distinct ß-type subunits. The α subunits make up the 2 outer layers, and the ß subunits the 2 inner rings of the structure [i.e. (α 1 -α 7 )(ß 1 -ß 7 )(ß 1 -ß 7 )(α 1 -α 7 )] (3,4).Studies of the proteasome inhibitors MG132 and lactacystin indicated that the proteasome is responsible for the degradation of >80% of intracellular proteins. Among the key regulatory proteins degraded by the 26S proteasome are the transcription factor c-Fos, M-, S-, and G1-phase specific cyclins, cyclin-dependent kinase inhibitors, p53, polyamine biosynthetic enzymes ornithine decarboxylase, S-adenosylmethionine decarboxylase, and a variety of oncoproteins (1,(5)(6)(7)(8). For targeting the 26S proteasome, substrate proteins are usually conjugated to a 76 amino acid protein ubiquitin, a mul...

An important hallmark of cancer cells is acquired resistance toward apoptosis. The apoptotic pathway is the most well-defined cell death program and is characterized by several morphological and biochemical features. The tumor suppressor protein p53 is a critical regulator of apoptosis in many cell types. p53 stimulates a wide network of signals that act through either extrinsic or intrinsic pathways of apoptosis. However, a number of studies have shown that apoptosis can be induced in a p53-independent manner as well. In this study, we examined the mechanism of apoptosis in p53-null breast cancer cells in response to the proteasome inhibitor bortezomib. Initially, we determined the p53 status of 4T1 breast carcinoma and 4THMpc (a highly mestatic derivative of 4T1) cells and verified that both cells are p53 deficient. It was subsequently shown that apoptosis can be induced in both cells in a dose-dependent manner in response to bortezomib treatment, based on DNA fragmentation evidence. Western blot analyses of ubiquitin-protein conjugates additionally showed that the proteasome is potently inhibited by bortezomib in p53-null 4T1 and 4THMpc cells. The results presented in the current study also show that caspase-3 is significantly activated in response to the treatment with bortezomib, implying that induction of apoptosis in these p53-deficient cells is occurring via caspase-3. The additional results presented here suggest that the pro-apoptotic proteins Bad, Noxa, and Puma are not critical regulators of apoptosis induction in p53-null 4T1 and 4THMpc cells. Similarly, there was no difference in the expression level of Mcl-1 in treated cells, suggesting that this anti-apoptotic protein is also uninvolved in the apoptotic response resulting from bortezomib treatment. In contrast, a very significant upregulation of the anti-apoptotic protein Hsp25/27 was detected in these p53-deficient cells after treatment with bortezomib. If the increased expression of Hsp25/27 protein levels are muting the apoptotic effects of the bortezomib treatment, then the apoptosis-inducing effects of such proteasome inhibitors might be increased with approaches simultaneously inhibiting Hsp25/27 protein in p53-deficient cells.

–  We examined seasonal variations in liver condition factor, total lipid and fatty acid composition of maturing pikeperch (Sander lucioperca) from Lake Eğirdir, Turkey. The spawning of pikeperch in Eğirdir Lake started in March, continued very fast during May and gradually ended in July as determined by measuring the gonadosomatic index. The total lipid content of male and female liver reached its highest level in September and November (the end of dense feeding period during which temperature decreased sharply), but declined to the lowest level in May (just after spawning). The highest level of liver condition factor was observed in January and March during which gonads mature rapidly. Palmitic acid was the predominant saturated fatty acid (SFA) in female and male liver. The ratio of monounsaturated fatty acids (MUFA) was higher than that of polyunsaturated fatty acids (PUFA) in both sexes. Docosahexaenoic acid (22:6n‐3), eicosopentaenoic acid (20:5n‐3) and arachidonic acid (20:4n‐6) were the predominant PUFA. Seasonal variations in PUFA were more evident than that in other fatty acids. PUFA, especially the n‐3 fatty acids ratio, increased to the maximum level during the sharp decreases in temperature (in November). However, the amount of PUFA decreased to the lowest level in reproductive period. The results suggest that the ratio of pikeperch liver fatty acids is influenced by gonad maturation and temperature variations and that pikeperch requires a large amount of PUFA and the n‐3 fatty acids for the development of gonads.

The short-lived enzyme S-adenosylmethionine decarboxylase uses a covalently bound pyruvoyl cofactor to catalyze the formation of decarboxylated S-adenosylmethionine, which then donates an aminopropyl group for polyamine biosynthesis. Here we demonstrate that S-adenosylmethionine decarboxylase is ubiquitinated and degraded by the 26 S proteasome in vivo, a process that is accelerated by inactivation of S-adenosylmethionine decarboxylase by substrate-mediated transamination of its pyruvoyl cofactor. Proteasome inhibition in COS-7 cells prevents the degradation of S-adenosylmethionine decarboxylase antigen; however, even brief inhibition of the 26 S proteasome caused substantial losses of S-adenosylmethionine decarboxylase activity despite accumulation of S-adenosylmethionine decarboxylase antigen. Levels of the enzyme's substrate (S-adenosylmethionine) increased rapidly after 26 S proteasome inhibition, and this increase in substrate level is consistent with the observed loss of activity arising from an increased rate of inactivation by substrate-mediated transamination. Evidence is also presented that this substrate-mediated transamination accelerates normal degradation of S-adenosylmethionine decarboxylase, as the rate of degradation of the enzyme was increased in the presence of AbeAdo (5 -([(Z)-4-amino-2-butenyl]methylamino]-5 -deoxyadenosine) (a substrate analogue that transaminates the enzyme); conversely, when the intracellular substrate level was reduced by methionine deprivation, the rate of degradation of the enzyme was decreased. Ubiquitination of S-adenosylmethionine decarboxylase is demonstrated by isolation of His-tagged AdoMetDC (S-adenosylmethionine decarboxylase) from COS-7 cells co-transfected with hemagglutinin-tagged ubiquitin and showing bands that were immunoreactive to both anti-AdoMetDC antibody and anti-hemagglutinin antibody. This is the first study to demonstrate that AdoMetDC is ubiquitinated and degraded by the 26 S proteasome, and substrate-mediated acceleration of degradation is a unique finding.