Activation of the membrane-bound Nrf1 transcription factor by USP19, a ubiquitin-specific protease C-terminally anchored in the endoplasmic reticulum.

The membrane-bound transcription factor Nrf1 (encoded by Nfe2l1) is activated by sensing glucose deprivation, cholesterol abundance, proteasomal inhibition and oxidative stress and then mediates distinct signaling responses to maintain cellular homeostasis. Herein, we found that Nrf1 stability and transactivity are both enhanced by USP19, a ubiquitin-specific protease tail-anchored in the endoplasmic reticulum (ER) through its C-terminal transmembrane domain. Further experiments revealed that USP19 directly interacts with Nrf1 in proximity to the ER and topologically acts as a deubiquitinating enzyme to remove ubiquitin moieties from this protein, which allow it to circumvent potential proteasomal degradation. This USP19-mediated effect takes place only after Nrf1 is retro-translocated by p97 out of the ER membrane to dislocate the cytoplasmic side. Conversely, knockout of USP19 causes significant decreases in the abundance of Nrf1 and the entrance of its active isoform into the nucleus, which result in the downregulation of its target proteasomal subunits and a modest reduction in USP19-/--derived tumor growth in xenograft mice when compared with wild-type controls. Altogether, these results demonstrate that USP19 serves as a novel mechanistic modulator of Nrf1, but not Nrf2, thereby enabling Nrf1 to be rescued from the putative ubiquitin-directed ER-associated degradation pathway. In turn, our additional experimental evidence has revealed that transcriptional expression of endogenous USP19 and its promoter-driven reporter genes is differentially regulated by Nrf2, as well by Nrf1, at distinct layers within a complex hierarchical regulatory network.

Generation of the tumor-suppressive secretome from tumor cells.

Rationale: The progression of cancer cells depends on the soil and building an inhibitory soil might be a therapeutic option. We previously created tumor-suppressive secretomes by activating Wnt signaling in MSCs. Here, we examined whether the anti-tumor secretomes can be produced from tumor cells. Methods: Wnt signaling was activated in tumor cells by overexpressing ß-catenin or administering BML284, a Wnt activator. Their conditioned medium (CM) was applied to cancer cells or tissues, and the effects of CM were evaluated. Tumor growth in the mammary fat pad and tibia in C57BL/6 female mice was also evaluated through µCT imaging and histology. Whole-genome proteomics analysis was conducted to determine and characterize novel tumor-suppressing proteins, which were enriched in CM. Results: The overexpression of ß-catenin or the administration of BML284 generated tumor-suppressive secretomes from breast, prostate and pancreatic cancer cells. In the mouse model, ß-catenin-overexpressing CM reduced tumor growth and tumor-driven bone destruction. This inhibition was also observed with BML284-treated CM. Besides p53 and Trail, proteomics analysis revealed that CM was enriched with enolase 1 (Eno1) and ubiquitin C (Ubc) that presented notable tumor-suppressing actions. Importantly, Eno1 immunoprecipitated CD44, a cell-surface adhesion receptor, and its silencing suppressed Eno1-driven tumor inhibition. A pan-cancer survival analysis revealed that the downregulation of MMP9, Runx2 and Snail by CM had a significant impact on survival outcomes (p < 0.00001). CM presented a selective inhibition of tumor cells compared to non-tumor cells, and it downregulated PD-L1, an immune escape modulator. Conclusions: The tumor-suppressive secretome can be generated from tumor cells, in which ß-catenin presented two opposing roles, as an intracellular tumor promoter in tumor cells and a generator of extracellular tumor suppressor in CM. Eno1 was enriched in CM and its interaction with CD44 was involved in Eno1's anti-tumor action. Besides presenting a potential option for treating primary cancers and metastases, the result indicates that aggressive tumors may inhibit the growth of less aggressive tumors via tumor-suppressive secretomes.

Ubiquitin C decrement plays a pivotal role in replicative senescence of bone marrow mesenchymal stromal cells.

Human bone marrow-mesenchymal stromal cells (hBM-MSCs) undergo cellular senescence during in vitro culture. In this study, we defined this replicative senescence as impaired proliferation, deterioration in representative cell characteristics, accumulated DNA damage, and decreased telomere length and telomerase activity with or without genomic abnormalities. The UBC gene expression gradually decreased during passaging along with the reduction in series of molecules including hub genes; CDK1, CCNA2, MCM10, E2F1, BRCA1, HIST1H1A and HIST1H3B. UBC knockdown in hBM-MSCs induced impaired proliferation in dose-dependent manner and showed replicative senescence-like phenomenon. Gene expression changes after UBC knockdown were similar to late passage hBM-MSCs. Additionally, UBC overexpession improved the proliferation activity of hBM-MSCs accompanied by increased expression of the hub genes. Consequently, UBC worked in higher-order through regulation of the hub genes controlling cell cycle and proliferation. These results indicate that the decrement of UBC expression plays a pivotal role in replicative senescence of hBM-MSCs.

Effect of gestational cadmium exposure on fetal growth, polyubiquitinated protein and monoubiqutin levels in the fetal liver of mice.

Cadmium (Cd) is an environmental pollutant present in contaminated water, food and soil. Cd adversely affects fetal development. We exposed pregnant mice to daily oral doses of 5 and 10 mg/kg Cd and examined fetal growth. It was demonstrated that the exposure to Cd (10 mg/kg) during gestation caused fetal growth retardation (FGR). Investigation of the ubiquitin-proteasome system in fetal livers of mice exposed to gestational Cd revealed increased polyubiquitinated protein accumulation, contrasting with decreased levels of monoubiquitin protein. Moreover, the expression level of Ubc (encoding polyubiquitin C protein) was significantly decreased in 5 and 10 mg/kg Cd-treated groups in comparison with the control group. Therefore, we propose that decrease of monoubiquitin level and accumulation of polyubiquitinated protein in the fetal liver may be important factors in Cd-induced FGR.

Role of RPL39 in Metaplastic Breast Cancer.

Background: Metaplastic breast cancer is one of the most therapeutically challenging forms of breast cancer because of its highly heterogeneous and chemoresistant nature. We have previously demonstrated that ribosomal protein L39 (RPL39) and its gain-of-function mutation A14V have oncogenic activity in triple-negative breast cancer and this activity may be mediated through inducible nitric oxide synthase (iNOS). The function of RPL39 and A14V in other breast cancer subtypes is currently unknown. The objective of this study was to determine the role and mechanism of action of RPL39 in metaplastic breast cancer. Methods: Both competitive allele-specific and droplet digital polymerase chain reaction were used to determine the RPL39 A14V mutation rate in metaplastic breast cancer patient samples. The impact of RPL39 and iNOS expression on patient overall survival was estimated using the Kaplan-Meier method. Co-immunoprecipitation and immunoblot analyses were used for mechanistic evaluation of RPL39. Results: The RPL39 A14V mutation rate was 97.5% (39/40 tumor samples). High RPL39 (hazard ratio = 0.71, 95% confidence interval = 0.55 to 0.91, P = 006) and iNOS expression (P = 003) were associated with reduced patient overall survival. iNOS inhibition with the pan-NOS inhibitor NG-methyl-L-arginine acetate decreased in vitro proliferation and migration, in vivo tumor growth in both BCM-4664 and BCM-3807 patient-derived xenograft models (P = 04 and P = 02, respectively), and in vitro and in vivo chemoresistance. Mechanistically, RPL39 mediated its cancer-promoting actions through iNOS signaling, which was driven by the RNA editing enzyme adenosine deaminase acting on RNA 1. Conclusion: NOS inhibitors and RNA editing modulators may offer novel treatment options for metaplastic breast cancer.

Endogenous Reference Genes for Gene Expression Studies on Bicuspid Aortic Valve Associated Aortopathy in Humans.

Bicuspid aortic valve (BAV) disease is the most common congenital cardiac abnormality and predisposes patients to life-threatening aortic complications including aortic aneurysm. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most commonly used methods to investigate underlying molecular mechanisms involved in aortopathy. The accuracy of the gene expression data is dependent on normalization by appropriate housekeeping (HK) genes, whose expression should remain constant regardless of aortic valve morphology, aortic diameter and other factors associated with aortopathy. Here, we identified an appropriate set of HK genes to be used as endogenous reference for quantifying gene expression in ascending aortic tissue using a spin column-based RNA extraction method. Ascending aortic biopsies were collected intra-operatively from patients undergoing aortic valve and/or ascending aortic surgery. These patients had BAV or tricuspid aortic valve (TAV), and the aortas were either dilated (≥4.5cm) or undilated. The cohort had an even distribution of gender, valve disease and hypertension. The expression stability of 12 reference genes were investigated (ATP5B, ACTB, B2M, CYC1, EIF4A2, GAPDH, SDHA, RPL13A, TOP1, UBC, YWHAZ, and 18S) using geNorm software. The most stable HK genes were found to be GAPDH, UBC and ACTB. Both GAPDH and UBC demonstrated relative stability regardless of valve morphology, aortic diameter, gender and age. The expression of B2M and SDHA were found to be the least stable HK genes. We propose the use of GAPDH, UBC and ACTB as reference genes for gene expression studies of BAV aortopathy using ascending aortic tissue.

Alpha-enolase is upregulated on the cell surface and responds to plasminogen activation in mice expressing a ∆133p53α mimic.

The p53 protein is a master regulator of the stress response. It acts as a tumor suppressor by inducing transcriptional activation of p53 target genes, with roles in apoptosis, cell cycle arrest and metabolism. The discovery of at least 12 isoforms of p53, some of which have tumor-promoting properties, has opened new avenues of research. Our previous work studied tumor phenotypes in four mouse models with different p53 backgrounds: wild-type p53, p53 null, mutant p53 lacking the proline domain (mΔpro), and a mimic for the human Δ133p53α p53 isoform (Δ122p53). To identify the major proteins affected by p53 function early in the response to DNA damage, the current study investigated the entire proteome of bone marrow, thymus, and lung in the four p53 models. Protein extracts from untreated controls and those treated with amsacrine were analyzed using two-dimensional fluorescence difference gel electrophoresis. In the bone marrow, reactive proteins were universally decreased by wild-type p53, including α-enolase. Further analysis of α-enolase in the p53 models revealed that it was instead increased in Δ122p53 hematopoietic and tumor cell cytosol and on the cell surface. Alpha-enolase on the surface of Δ122p53 cells acted as a plasminogen receptor, with tumor necrosis factor alpha induced upon plasminogen stimulation. Taken together, these data identified new proteins associated with p53 function. One of these proteins, α-enolase, is regulated differently by wild-type p53 and Δ122p53 cells, with reduced abundance as part of a wild-type p53 response and increased abundance with Δ122p53 function. Increased cell surface α-enolase on Δ122p53 cells provides a possible explanation for the model's pro-inflammatory features and suggests that p53 isoforms may direct an inflammatory response by increasing the amount of α-enolase on the cell surface.

Activation of Cre recombinase alone can induce complete tumor regression.

The Cre/loxP system is a powerful tool for generating conditional genomic recombination and is often used to examine the mechanistic role of specific genes in tumorigenesis. However, Cre toxicity due to its non-specific endonuclease activity has been a concern. Here, we report that tamoxifen-mediated Cre activation in vivo induced the regression of primary lymphomas in p53-/- mice. Our findings illustrate that Cre activation alone can induce the regression of established tumors.

Brain site-specific proteome changes in aging-related dementia.

This study is aimed at gaining insights into the brain site-specific proteomic senescence signature while comparing physiologically aged brains with aging-related dementia brains (for example, Alzheimer's disease (AD)). Our study of proteomic differences within the hippocampus (Hp), parietal cortex (pCx) and cerebellum (Cb) could provide conceptual insights into the molecular mechanisms involved in aging-related neurodegeneration. Using an isobaric tag for relative and absolute quantitation (iTRAQ)-based two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-LC-MS/MS) brain site-specific proteomic strategy, we identified 950 proteins in the Hp, pCx and Cb of AD brains. Of these proteins, 31 were significantly altered. Most of the differentially regulated proteins are involved in molecular transport, nervous system development, synaptic plasticity and apoptosis. Particularly, proteins such as Gelsolin (GSN), Tenascin-R (TNR) and AHNAK could potentially act as novel biomarkers of aging-related neurodegeneration. Importantly, our Ingenuity Pathway Analysis (IPA)-based network analysis further revealed ubiquitin C (UBC) as a pivotal protein to interact with diverse AD-associated pathophysiological molecular factors and suggests the reduced ubiquitin proteasome degradation system (UPS) as one of the causative factors of AD.

Identification of suitable reference genes for gene expression measurement in uterine sarcoma and carcinosarcoma tumors.

OBJECTIVE: To date no suitable reference genes have been identified in carcinosarcomas and non-epithelial malignant tumors of corpus uteri for normalizing real-time RT-PCR (qRT-PCR) assays. The purpose of this study was to select appropriate references for gene expression studies in these tumors. MATERIALS AND METHODS: We used RNA extracts from 75 tissue samples, representing 50 tumors and 25 fragments of normal uterine tissues obtained from 50 patients treated for mixed tumors, smooth muscle sarcoma and stromal sarcoma of the uterus. qRT-PCR for five potential reference (housekeeping) genes, namely B2M, HMBS, HPRT1, TBP and UBC, was performed. The expression stability of these genes was assayed using geNorm software application. RESULTS: The analysis of gene expression data with geNorm identified HPRT1 as the most stable reference gene, followed by UBC and HMBS, for all the investigated tissues. When stratified by disease, the results still pointed at HPRT1 as the gene that retained the greatest robustness in mixed tumors as well as in smooth muscle and stromal sarcomas. CONCLUSIONS: Our work is the first report on reference gene selection for qRT-PCR applications in mixed tumors, smooth muscle sarcoma and stromal sarcoma of the uterus. A ranking of candidate genes' stability values for the three types of tumors is provided and might serve as a valuable guide for future gene expression studies of these rare entities.

Selection of housekeeping genes for gene expression studies in a rat model of irinotecan-induced mucositis.

BACKGROUND/AIMS: Mucositis is the term used to describe damage caused by chemotherapy to mucous membranes of the alimentary tract. RT-PCR has recently been utilised to determine the molecular events that occur in mucositis. As this method relies on the use of a validated endogenous control, this study aims to validate commonly used housekeeping genes in an irinotecan-induced mucositis model. METHODS: Rats were administered irinotecan and sacrificed at different time points, in particular 1, 24, 72 and 144 h following treatment. Histopathological damage was assessed by haematoxylin and eosin staining. RT-PCR was used to evaluate the expression of 11 housekeeping genes. Expression stability was determined by the Normfinder program. Matrix metalloproteinase 2 was used as a target gene to validate the appropriateness of the top-ranking housekeeping gene. RESULTS: For normalisation to multiple housekeeping genes, the most stable combination across all time points in the jejunum was Ywhaz/UBC and in the colon UBC/ß-actin. SDHA and GAPDH were the most variable genes in the jejunum and colon where they were 4.4 and 3.2 fold upregulated following irinotecan, respectively. CONCLUSIONS: For normalisation of irinotecan-induced mucositis gene expression studies, a combination of Ywhaz/UBC and UBC/ß-actin should be used in the jejunum and colon, respectively. UBC is the most favourable if restricted to a single housekeeping gene across all time points.

Proteasome inhibitor lactacystin induces cholinergic degeneration.

OBJECTIVE: Ubiquitin proteasome system dysfunction is believed to play an important role in the development of Parkinson's disease (PD), and almost all studies till now have mainly focused on the susceptibility of dopaminergic neurons to proteasome inhibition. However, in fact, there are many other types of neurons such as cholinergic ones involved in PD. In our present study, we attempt to figure out what effect the failure of ubiquitin proteasome function would execute on cholinergic cells in culture. METHODS: We treated cholinergic cells in culture with various doses of lactacystin. Then MTT assay was used to evaluate the cellular viability and the AnnexinV-PI method was used to detect apoptosis. Both cellular soluble and insoluble polyubiquitinated proteins were detected by western blot. Furthermore, the mitochondrial membrane potential was analyzed using JC-1 and the intracellular production of reactive oxygen species (ROS) was determined using the fluorescent probe CM-H2DCFDA. RESULTS: We found that low doses of lactacystin were enough to induce significant apoptotic cell death, disturb the mitochondrial membrane potential, and cause oxidative stress. We also found that the amounts of polyubiquitinated proteins dramatically increased with high doses, although the loss of cells did not increase accordingly. CONCLUSIONS: Our results suggest that cholinergic cells are sensitive to ubiquitin proteasome system dysfunction, which exerts its toxic effect by causing mitochondrial dysfunction and subsequent oxidative stress, not through polyubiquitinated proteins accumulation.

The structure of the UbcH8-ubiquitin complex shows a unique ubiquitin interaction site.

Ubiquitin-mediated proteolysis utilizes a series of three key enzymes (E1, E2, and E3) to transfer and then covalently modify a substrate with ubiquitin. E2 conjugating enzymes are central proteins in this pathway responsible for the acceptance of a ubiquitin from the E1 enzyme and association with an E3 protein. All E2 enzymes covalently bind ubiquitin through a thiolester linkage between a conserved active-site cysteine on E2 and the C-terminal glycine on ubiquitin. It is not known whether E2 enzymes utilize similar surfaces and residues to coordinate a ubiquitin molecule and how this might contribute to any substrate specificity. In this work, we determined the structure of the human E2 enzyme UbcH8 (UBE2L6) covalently bound to ubiquitin by NMR spectroscopy. A disulfide bond mimicking the short-lived thiolester was formed between the two proteins providing a stable complex. Overall, the structure of UbcH8 does not undergo a significant conformational change upon forming a complex with ubiquitin. Chemical shift perturbation and cross-saturation experiments were used to identify contacts between UbcH8 and ubiquitin and those contacts used as inputs for HADDOCK molecular docking to produce the structure of the UbcH8-ubiquitin complex. An ensemble of 16 structures (root-mean-square deviation of 0.83 A) showed that ubiquitin interacts with the linker region prior to the alpha5 helix as well as residues near the catalytic site. This region corresponds to an area of negative potential on the UbcH8 surface and is considerably different from other E2-ubiquitin interaction sites. Our findings indicate the positioning of ubiquitin on UbcH8 would still allow interaction with E1 and E3 enzymes. Together, the results suggest the UbcH8-ubiquitin complex may provide an additional level of specificity in the ubiquitination pathway.

Control of cell growth by the SCF and APC/C ubiquitin ligases.

The ubiquitin-proteasome system plays key roles in the control of cell growth. The cell cycle, in particular, is highly regulated by the functions of the SCF and APC/C ubiquitin ligases, and perturbation of their function can result in tumorigenesis. Although the SCF and APC/C complexes are well established in growth control pathways, many aspects of their function remain unknown. Recent studies have shed light on the mechanism of SCF-mediated ubiquitination and new functions for the SCF complex and APC/C. Our expanding understanding of the roles of the SCF and APC/C complexes highlight the potential for targeted molecular therapies.

Time course of housekeeping gene expression changes in diffuse alveolar damage induced by hyperoxia exposure in mice.

We have found diffuse alveolar damage (DAD) has taken place in some patients under mechanical ventilation with high-inspired oxygen concentrations. To clarify the molecular pathophysiology of this, the time course of gene expression changes induced by hyperoxia exposure in mouse lungs was examined using real-time quantitative polymerase chain reaction (real-time qPCR). Our raw data and those normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) showed that: (1) there is a decrease in levels of mRNAs for surfactant-associated protein C (SFTPC), cytochrome P450, 2F2 (CYP2F2), Claudin 1 (CLDN1), membrane-associated zonula occludens protein-1 (ZO-1), lysozyme (LYZS), and this suggests alveolar dysfunction and a disruption of the immune system, (2) we confirmed apoptotic conditions, such as significant up-regulations of mRNA levels in Myc and Galectin-3, and (3) hyperoxic conditions probably yielded reactive oxygen species (ROS), which resulted in a malignant cycle of ROS production by Myc overexpression [Shimada I, Matsui K, Brinkmann B, Hohoff C, Hiraga K, Tabuchi Y, et al. Novel transcript profiling of diffuse alveolar damage induced by hyperoxia exposure in mice: normalization by glyceraldehyde 3-phosphate dehydrogenase. Int J Legal Med 2008;122:373-83]. In this experiment, GAPDH was up-regulated when hyperoxia exposure was continued. Therefore, we reexamined our data and found that: (1) mRNA levels of other housekeeping genes, including beta(2)-microglobulin (beta2M), ribosomal protein: large P2 (RPLP2), and importin 8 (IPO8) altered to a lesser extent, (2) mRNA levels of beta2M and IPO8 were down-regulated when hyperoxia exposure was continued, and (3) our previous work was validated by normalization with these three housekeeping genes.

Mechanisms of Cdc48/VCP-mediated cell death: from yeast apoptosis to human disease.

The evolutionary conserved protein Cdc48/VCP is involved in various cellular processes, such as protein degradation, membrane fusion and chaperone activity. Increased levels of Cdc48/VCP correlate with cancer, whereas Cdc48/VCP at endogenous levels has been proposed to be a pathological effector in protein deposition diseases. Upon mutation Cdc48/VCP triggers the multisystem disorder 'inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia' (IBMPFD). The roles of Cdc48/VCP under these diverse pathological conditions, especially its function in decreased and increased incidences of cell death underlying these diseases, are poorly understood. Mutation of yeast CDC48 (cdc48(S565G)) results in yeast cells demonstrating morphological markers of apoptotic cell death. In other species it has been confirmed that mutations and depletion of Cdc48/VCP cause apoptosis, whereas increased levels of this protein provide an anti-apoptotic effect. This review critically compares mechanisms of Cdc48/VCP-mediated apoptosis observed in yeast and other species. Cdc48/VCP plays a triple role in cell death. At first, loss-of-function of Cdc48/VCP due to mutation or depletion causes ER stress and oxidative stress, triggering apoptosis. Secondly, upon exogenously applied ER stress functional Cdc48/VCP is important in the processing of caspases and plays therewith a pro-apoptotic role. Finally, Cdc48/VCP protects cells from apoptosis through mediating and activating pro-survival signaling pathways, namely Akt and NFkappaB signaling. This complex role in cell death pathways could correspond with the various pathophysiological conditions Cdc48/VCP is involved in.

Enhanced upregulation of smooth muscle related transcripts by TGF beta2 in asthmatic (myo) fibroblasts.

BACKGROUND: Transforming growth factor beta (TGF beta) upregulates a number of smooth muscle specific genes in (myo)fibroblasts. As asthma is characterised by an increase in airway smooth muscle, we postulated that TGFbeta(2) favours differentiation of asthmatic (myo)fibroblasts towards a smooth muscle phenotype. METHODS: Primary fibroblasts were grown from bronchial biopsy specimens from normal (n = 6) and asthmatic (n = 7) donors and treated with TGF beta2 to induce myofibroblast differentiation. The most stable genes for normalisation were identified using RT-qPCR and the geNorm software applied to a panel of 12 "housekeeping" genes. Expression of alpha-smooth muscle actin (alpha SMA), heavy chain myosin (HCM), calponin 1 (CPN 1), desmin, and gamma-actin were measured by RT-qPCR. Protein expression was assessed by immunocytochemistry and western blotting. RESULTS: Phospholipase A2 and ubiquitin C were identified as the most stably expressed and practically useful genes for normalisation of gene expression during myofibroblast differentiation. TGF beta2 induced mRNA expression for all five smooth muscle related transcripts; alpha SMA, HCM and CPN 1 protein were also increased but desmin protein was not detectable. Although there was no difference in basal expression, HCM, CPN 1 and desmin were induced to a significantly greater extent in asthmatic fibroblasts than in those from normal controls (p = 0.041 and 0.011, respectively). CONCLUSIONS: Although TGF beta2 induced the transcription of several smooth muscle related genes, not all were translated into protein. Thus, while TGF beta2 is unable to induce a bona fide smooth muscle cell phenotype, it may "prime" (myo)fibroblasts for further differentiation, especially if the cells are derived from asthmatic airways.

Regulation of proteolysis by cytokines in the human intestinal epithelial cell line HCT-8: role of IFNgamma.

Protein metabolism contributes in the regulation of gut barrier function, which may be altered during inflammatory states. There are three major proteolytic pathways in mammalian cells: lysosomal, Ca(2+)-activated and ubiquitin-proteasome. The regulation of proteolytic activities during inflammation remains unknown in intestine. Intestinal epithelial cells, HCT-8, were stimulated by IL-1beta, IFNgamma and TNFalpha each alone or in combination (Cytomix). Proteolytic activities were assessed using fluorogenic substrates and specific inhibitors, protein expressions by Western blot. Lysosomal and Ca(2+)-activated pathways were not significantly altered by any treatment. In contrast, the activity of ubiquitin-proteasome system was stimulated by IFNgamma and Cytomix (155, 160 versus 100, P<0.05, respectively) but remained unaffected by IL-1beta and TNFalpha. Free ubiquitin expression, but not ubiquitinated proteins, was enhanced by IFNgamma and Cytomix. The expression of proteasome 20S alpha1 subunit, a constitutive proteasome 20S subunit, was not altered, beta5 subunit expression was weakly decreased by Cytomix and inducible beta5i subunit expression was markedly increased in response to IFNgamma and to Cytomix (202, 206 versus 100, P<0.05, respectively). In conclusion, lysosomal, Ca(2+)-activated and constitutive proteasome activities were not affected by IL-1beta, IFNgamma and TNFalpha alone or in combination, in HCT-8 cells. These results suggest that IFNgamma, but not IL-1beta and TNFalpha, increases immunoproteasome, which might contribute to enhanced antigen presentation during inflammatory bowel diseases.

Role of leptin and melanocortin signaling in uremia-associated cachexia.

The pathogenesis of cachexia in patients with uremia is unknown. We tested the hypothesis that uremia-associated cachexia is caused by leptin signaling through the hypothalamic melanocortin receptor 4 (MC4-R). We performed either subtotal nephrectomy (N) or sham operations in WT, leptin receptor-deficient (db/db), and MC4-R knockout (MC4-RKO) mice. The animals were on 17% protein diets, and none of the uremic animals were acidotic. WT-N mice produced a classic syndrome of cachexia characterized by decreased food intake, increased metabolic rate, and loss of lean body mass. Corrected leptin levels were elevated. db/db mice and MC4-RKO mice resisted the cachexic effects of uremia on weight gain, body composition, and metabolic rate. Likewise, treatment of WT mice with intracranial agouti-related peptide reversed the cachexic effects of uremia on appetite, weight gain, body composition, and metabolic rate. Gene expression of ubiquitin C and proteasome subunits C2, C3, and C9 was not changed in the uremic animals, suggesting that other pathways are involved in this model of nonacidotic uremic cachexia. The results of this study suggest that elevated circulating levels of cytokines such as leptin may be an important cause of uremia-associated cachexia via signaling through the central melanocortin system.

Why do cells require heat shock proteins to survive heat stress?

The cellular response to heat stress includes the induction of a group of proteins called the Heat Shock Proteins, whose functions include the synthesis of the thermoprotectant trehalose, refolding of denatured proteins, and ubiquitin- and proteasome-dependent degradation. Recent studies show that simply increasing the activity of ubiquitin- and proteasome-dependent degradation can replace the essential functions played by the induction of heat shock proteins during a heat stress. These results suggest that accumulation of denatured or aggregated proteins is the reason for the loss of cell viability due to heat stress.