Ubiquitin-proteasome Complex Research Articles

Cachexia in heart disease: highlights from the ESC 2010

Cardiac cachexia is a co-morbidity that may develop in terminal stages of chronic heart failure (CHF). Up to 15% of ambulatory patients with heart failure are affected. Over the last decades, cardiac cachexia and alterations in muscle metabolism in heart disease have received increasing research interest. This article highlights some recent studies of cardiac cachexia that were presented at the annual meeting of the European Society of Cardiology in September 2010 in Stockholm, Sweden. Studies presented here were focused on effects of exercise training and protein degradation, particularly into the role of the ubiquitin–proteasome complex and its ubiquitin ligases MuRF-1 and MAFbx. Exercise training in patients with CHF was found to increase maximal oxygen consumption and to reduce MuRF-1 expression. Lysosomal muscle degradation does not seem to play a major role in patients with CHF, however, inflammatory cytokines such as tumor necrosis factor-a trigger muscle protein degradation. Other studies found that the serum levels of the adipokine adiponectin are elevated in patients with CHF and that these levels may be correlated with muscle mass, muscle strength in the arms, or with trunk fat mass. Another study showed that the expression of myostatin in skeletal muscle, a negative regulator of muscle growth that is essential for normal regulation of muscle mass, is decreased in spontaneously hypertensive rats with heart failure compared with control animals. This is also true for follistatin, a powerful antagonist, and its potential as a biomarker of muscle wasting. These findings may pave the way for effective treatment approaches to cardiac cachexia.

Attenuation of proteolysis and muscle wasting by curcumin c3 complex in MAC16 colon tumour-bearing mice

Muscle wasting or cachexia is caused by accelerated muscle protein breakdown via the ubiquitin-proteasome complex. We investigated the effect of curcumin c3 complex (curcumin c3) on attenuation of muscle proteolysis using in vitro and in vivo models. Our in vitro data indicate that curcumin c3 as low as 0.50 microg/ml was very effective in significantly inhibiting (30 %; P < 0.05) tyrosine release from human skeletal muscle cells, which reached a maximum level of inhibition of 60 % (P < 0.05) at 2.5 microg/ml. Curcumin c3 at 2.5 microg/ml also inhibited chymotrypsin-like 20S proteasome activity in these cells by 25 % (P < 0.05). For in vivo studies, we induced progressive muscle wasting in mice by implanting the MAC16 colon tumour. The in vivo data indicate that low doses of curcumin c3 (100 mg/kg body weight) was able to prevent weight loss in mice bearing MAC16 tumours whereas higher doses of curcumin c3 (250 mg/kg body weight) resulted in approximately 25 % (P < 0.05) weight gain as compared with the placebo-treated animals. Additionally, the effect of curcumin c3 on preventing and/or reversing cachexia was also evident by gains in the weight of the gastrocnemius muscle (30-58 %; P < 0.05) and with the increased size of the muscle fibres (30-65 %; P < 0.05). Furthermore, curcumin inhibited proteasome complex activity and variably reduced expression of muscle-specific ubiquitin ligases: atrogin-1/muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MURF-1). In conclusion, oral curcumin c3 results in the prevention and reversal of weight loss. The data imply that curcumin c3 may be an effective adjuvant therapy against cachexia.

Cigarette smoke-induced emphysema in A/J mice is associated with pulmonary oxidative stress, apoptosis of lung cells, and global alterations in gene expression

Cigarette smoking is the major risk factor for developing chronic obstructive pulmonary disease, the fourth leading cause of deaths in the United States. Despite recent advances, the molecular mechanisms involved in the initiation and progression of this disease remain elusive. We used Affymetrix Gene Chip arrays to determine the temporal alterations in global gene expression during the progression of pulmonary emphysema in A/J mice. Chronic cigarette smoke (CS) exposure caused pulmonary emphysema in A/J mice, which was associated with pronounced bronchoalveolar inflammation, enhanced oxidative stress, and increased apoptosis of alveolar septal cells. Microarray analysis revealed the upregulation of 1,190, 715, 260, and 246 genes and the downregulation of 1,840, 730, 442, and 236 genes in the lungs of mice exposed to CS for 5 h, 8 days, and 1.5 and 6 mo, respectively. Most of the genes belong to the functional categories of phase I genes, Nrf2-regulated antioxidant and phase II genes, phase III detoxification genes, and others including immune/inflammatory response genes. Induction of the genes encoding multiple phase I enzymes was markedly higher in the emphysematous lungs, whereas reduced expression of various cytoprotective genes constituting ubiquitin-proteasome complex, cell survival pathways, solute carriers and transporters, transcription factors, and Nrf2-regulated antioxidant and phase II-responsive genes was noted. Our data indicate that the progression of CS-induced emphysema is associated with a steady decline in the expression of various genes involved in multiple pathways in the lungs of A/J mice. Many of the genes discovered in this study could rationally play an important role in the susceptibility to CS-induced emphysema.

The Two Isoforms of the Na+/Ca2+Exchanger, NCX1 and NCX3, Constitute Novel Additional Targets for the Prosurvival Action of Akt/Protein Kinase B Pathway

The proteins NCX1, NCX2, and NCX3 expressed on the plasma membrane of neurons play a crucial role in ionic regulation because they are the major bidirectional system promoting the efflux and influx of Na(+) and Ca(2+) ions. Here, we demonstrate that NCX1 and NCX3 proteins are novel additional targets for the survival action of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. Indeed, the doxycycline-dependent overexpression of constitutively active Akt1 in tetracycline (Tet)-Off PC-12 positive mutants and the exposure of Tet-Off PC-12 wild type to nerve growth factor induced an up-regulation of NCX1 and NCX3 proteins. NCX1 up-regulation induced by Akt1 activation occurred at the transcriptional level because NCX1 mRNA increased, and it was counteracted by cAMP response element-binding protein 1 inhibition through small interfering RNA strategy. In contrast, Akt1-induced NCX3 up-regulation recognized a post-transcriptional mechanism occurring at the proteasome level because 1) NCX3 transcript did not increase and 2) the proteasome inhibitor N-benzyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) did not further enhance NCX3 protein levels in Akt1 active mutants as it would be expected if the ubiquitin-proteasome complex was not already blocked by Akt1 pathway. As expected, in PC-12 Tet-Off wild-type cells MG-132 enhanced NCX3 protein levels. This up-regulation produced an increased activity of NCX function. Furthermore, NCX1 and NCX3 up-regulation contributed to the survival action of Akt1 during chemical hypoxia because both the silencing of NCX1 or NCX3 and the pharmacological paninhibition of NCX isoforms reduced the prosurvival property of Akt1. Together, these results indicated that NCX1 and NCX3 represent novel additional molecular targets for the prosurvival action of PI3-K/Akt pathway.

The Proteasome Inhibitor BSc2118 Causes Growth Inhibition, Cell Cycle Arrest and Cyclin D1 Degradation in Mantle Cell Lymphoma Cell Lines.

The ubiquitin-proteasome complex has recently been identified as a novel therapeutic target, particularly in hematological malignancies. An increased proteasome activity has been described in certain types of malignant cells including mantle cell lymphoma (MCL). The proteasome inhibitor bortezomib has shown in vitro activity in MCL cell lines and also clinical efficacy in patients with refractory or relapsed MCL. We previously described a novel tripeptide compound, BSc2118 which inhibits all three proteolytic activities of the 20S proteasome and has anti-tumor activity in melanoma and multiple myeloma (Cancer Res 2006; 66: 7598–7605). We investigated the anti-tumor effects of BSc2118 in the MCL cell lines HBL-2, JeKo-1 and Granta-519 and studied its effects on cell cycle progression and the expression of the cell cycle regulatory proteins p21, p27 and cyclin D1. Furthermore the inhibition of intracellular proteasome and NF-kappa B activity was analyzed in MCL cells. In MCL cell lines HBL-2, JeKo-1 and Granta-519, BSc2118 caused a time- and dose-dependent growth inhibition, an induction of apoptosis and a dose-dependent inhibition of intracellular proteasome activity. After 24 and 48 hours of incubation with 40 – 260 nM BSc2118 we found a time- and dose-dependent cell cycle arrest in G2/M phase in all three MCL cell lines. Furthermore we could demonstrate a dose-dependent stabilization of p21 and a degradation of cyclin D1 in western blot analysis. No significant changes could be seen in p27 expression under proteasome inhibition with BSc2118. After TNF-alpha stimulation we found a low NF-kappa B activity also in untreated MCL cells, which could be partially inhibited by preincubation with BSc2118. In this study we show the effects of the novel proteasome inhibitor BSc2118 on cell cycle and growth inhibition in MCL cells lines and furthermore demonstrate that inhibition of proteasome activity, cyclin D1 degradation and p21 stabilization are crucial mechanisms of action for this compound in MCL. Since recent trials have shown a clinical efficacy of proteasome inhibition in MCL, our preclinical data suggest to consider BSc2118 as a novel agent in drug development against MCL.

Protein gene product 9.5 and ubiquitin are expressed in metabolically active epithelial cells of normal and pathologic human kidney

In a study initially designed to evaluate the specific protein gene product 9.5 expression in parietal epithelial cells of Bowman's capsule, a marked positivity was also observed in the tubular and collecting duct epithelial cells. Since protein gene product 9.5 is an important enzyme in the ubiquitin system of proteolysis, and plays a regulatory role in cell cycle and proliferation, its presence in specific segments of the nephron was of considerable interest. We investigated protein gene product 9.5 and ubiquitin expression in both normal and pathologic renal samples (more than 100 cases) using an immunohistochemical technique. We found that protein gene product 9.5 and ubiquitin were constantly present in Bowman's capsule parietal cells and tubular/collecting duct epithelial cells, with the strongest positivity in metabolically active and proliferative conditions, such as tubular hypertrophy, cellular regeneration and crescent formation. Conversely, the expression of these molecules was attenuated in atrophic tubules. Podocytes were negative. The diffuse presence of the protein gene product 9.5 and ubiquitin in normal and pathologic metabolically active epithelial cells of the nephron suggests that these proteins (and likely the whole ubiquitin-proteasome complex) play a fundamental role in the mechanism upregulating protein metabolism of the kidney and that its expression is correlated with activated cellular functions, like proliferation.

Three Emerging New Drugs for NSCLC: Pemetrexed, Bortezomib, and Cetuximab

Despite advances made in cytotoxic chemotherapy, the prognosis for patients with non-small cell lung cancer (NSCLC) continues to be poor. New, more effective drugs must be identified and developed to improve the outcome of these patients. Three drugs with promising activity in NSCLC are pemetrexed (Alimta; Eli Lilly and Company, Indianapolis, IN, http://www.lilly.com), bortezomib (Velcade; Millennium Pharmaceuticals, Inc., Cambridge, MA, http://www.mlnm.com), and cetuximab (Erbitux; ImClone Systems, Inc., New York, NY, http://www.imclone.com). Pemetrexed inhibits thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyl transferase, enzymes necessary for purine and pyrimidine synthesis, thus causing cell-cycle arrest in the S phase. Bortezomib, a proteasome inhibitor, interferes with the cytosolic protein degradation machinery, namely the ubiquitin-proteasome complex, causing breakdown of cell-cycle regulators and cell-cycle arrest. Cetuximab is a chimeric mouse-human antibody that inhibits ligand-dependent activation of the epidermal growth factor receptor, resulting in receptor internalization and inhibition of downstream pathways that, in turn, causes cell growth and progression. All three drugs are approved for different tumor types, and studies defining their role in NSCLC are under way.

Protein Misfolding in Disease: Cause or Response?

Misfolding of newly formed proteins not only results in a loss of physiological function of the protein but also may lead to the intra- or extra- cellular accumulation of that protein. A number of diseases have been shown to be characterised by the accumulation of misfolded proteins, notable examples being Alzheimers disease and the tauopathies. The obvious inference is that these proteinaceous deposits are pathogenic features of the disease. However, systems such as the unfolded protein response and ubiquitin-proteasome complex are in place in the cell to target misfolded proteins for degradation and clearance. Evidence suggests that in disease states, these protein-handling systems may be overwhelmed and the misfolded proteins accumulate as either extracellular deposits (eg. senile plaques in Alzheimers disease) or intracellular inclusions (as in Lewy bodies in Parkinsons disease). These accumulations may be the direct cause of the particular pathology associated with the diseases or they may be inert “packages” designed to protect the cell from toxic insult. Keywords: Protein, proteinaceous, toxic

Proteasome Inhibitors Induce Cerebellar Granule Cell Death

Many activities of neuronal cells, such as synaptic transmission, inflammation, neuroprotection, and neurotoxicity, are regulated by the activity of the transcription factor nuclear factor-kappaB (NF-kappaB). In resting cells, NF-kappaB activity is present both in the cytoplasm, as an inducible-inactive complex, and in the nucleus as a constitutive form. The activation of its inducible form is related to processing of IkappaB(s), which occurs through the proteasome. To understand whether NF-kappaB is involved in the survival of cerebellar granule cells (CGCs) maintained under conditions of mild depolarization (25 mM KCl), these cells were treated with different proteasome inhibitors. The results presented show that these pharmacological tools reduce CGC survival with changes in nuclear morphology and induction of apoptosis. Furthermore, we demonstrate that PSI-induced apoptosis is reverted by inhibitors of transcription and translation, as well as by specific caspase inhibitors. These issues are also associated with a redistribution of NF-kappaB, in that a reduced amount of nuclear NF-kappaB and an increased p65 cytoplasmic level have been observed. Finally, we propose that, at least in part, p65 metabolism could also be regulated by the ubiquitin-proteasome complex. Altogether, the results presented define an important role for NF-kappaB in maintainig CGC survival.

The Role of the Ubiquitin–Proteasome Pathway in the Formation of Mallory Bodies

The dynamics of Mallory body (MB) formation are difficult to follow in vivo. Because of the lack of an in vitro mouse hepatocyte culture model, a cellular extract approach was developed. In this model an immunoprecipitate was obtained using an antibody to cytokeratin-8 (CK-8). The isolate contained a large number of compounds: CK-8, ubiquitin, a frameshift mutation of ubiquitin (UBB +1), proteasomal subunits β5 (a catalytic subunit of the 20S proteasome) and Tbp7 (an ATPase subunit of the 26S proteasome), transglutaminase, tubulin, heat shock proteins 90 and 70, and MBs. In Western blots, CK-8 immunoprecipitates showed colocalization of these components in a complex of proteins colocalized in a high-molecular-weight smear. When the CK-8 immunoprecipitate was incubated with the isolate of proteasomes and an energy generating source (ATP), the components of the ubiquitinated protein smear increased. These observations taken together with the in vivo observation that these proteins colocalized at the edge of the MB shown in the present study suggest that these proteins form aggregates through covalent binding of CK-8, ubiquitin, and the proteasomes. Covalent aggregation is suggested by the fact that the protein complex found in the high-molecular-weight smear that forms in vitro fails to dissociate in SDS. This protein complex is present in the CK-8 immunoprecipitates of livers forming MBs but not in control livers. In conclusion, the results support the concept that Mallory bodies are aggresomes which form as the result of the failure of the ubiquitin–proteasome complex to adequately eliminate cytokeratins destined for proteolysis.

Inhibition of IκB-α and IκB-β Proteolysis by Calpain Inhibitor I Blocks Nitric Oxide Synthesis

Lipopolysaccharide (LPS) stimulates the induction of the inducible isoform of nitric oxide synthase (iNOS) in part by inducing the nuclear translocation of the transcription factor nuclear factor-κ B (NF-κB). LPS induces ubiquination and phosphorylation of the IκB inhibitory subunit of NF-κB. Subsequently, the ubiquitin–proteasome multicatalytic enzyme complex catalyzes the proteolytic degradation of IκB with resultant nuclear translocation of NF-κB. Our results demonstrate that the proteasome inhibitor calpain inhibitor I dose-dependently inhibited LPS-induced nitric oxide synthesis in RAW macrophages. The inhibitor was found to block iNOS transcription and protein translation as noted by Northern analysis and Western blotting, respectively. LPS stimulated rapid proteolytic degradation of IκB-α which was inhibited by approximately 50% in the presence of calpain inhibitor I. In contrast, LPS induced the delayed proteolytic degradation of IκB-β which was almost totally inhibited by calpain inhibitor I. Calpain inhibitor I also decreased the LPS-induced nuclear translocation of NF-κB. These results demonstrate that the ubiquitin–proteasome complex has an important role in induction of iNOS in response to stimuli which act via the NF-κB/IκB signal transduction pathway. Furthermore, the results suggest that the ubiquitin–proteasome complex is important in the degradation of IκB-β as well as IκB-α. Finally, we have demonstrated that there is a marked difference in the extent of proteolysis of IκB-α and IκB-β when the ubiquitin–proteasome complex is inhibited with calpain inhibitor I.