Protease Inhibitor MG132 Research Articles

Liver X receptor β increases aquaporin 2 protein level via a posttranscriptional mechanism in renal collecting ducts.

Liver X receptors (LXRs) including LXRα and LXRβ are nuclear receptor transcription factors and play an important role in lipid and glucose metabolism. It has been previously reported that mice lacking LXRβ but not LXRα develop a severe urine concentrating defect, likely via a central mechanism. Here we provide evidence that LXRβ regulates water homeostasis through increasing aquaporin 2 (AQP2) protein levels in renal collecting ducts. LXRβ-/- mice exhibited a reduced response to desmopressin (dDAVP) stimulation, suggesting that the diabetes insipidus phenotype is of both central and nephrogenic origin. AQP2 protein abundance in the renal inner medulla was significantly reduced in LXRβ-/- mice but with little change in AQP2 mRNA levels. In vitro studies showed that AQP2 protein levels were elevated upon LXR agonist treatment in both primary cultured mouse inner medullary duct cells (mIMCD) and the mIMCD3 cell line with stably expressed AQP2. In addition, LXR agonists including TO901317 and GW3965 failed to induce AQP2 gene transcription but diminished its protein ubiquitination in primary cultured mIMCD cells, thereby inhibiting its degradation. Moreover, LXR activation-induced AQP2 protein expression was abolished by the protease inhibitor MG132 and the ubiquitination-deficient AQP2 (K270R). Taken together, the present study demonstrates that activation of LXRβ increases AQP2 protein levels in the renal collecting ducts via a posttranscriptional mechanism. As such, LXRβ represents a key regulator of body water homeostasis.

A regulatory subunit of protein phosphatase 2A, PPP2R5E, regulates the abundance of microtubule crosslinking factor 1.

Dynamic changes in microtubule organization are regulated by numerous microtubule-associating proteins and their post-translational modification. Microtubule crosslinking factor 1 (MTCL1) is a recently identified protein that regulates microtubule organization. To obtain further insight into its functions, we searched for proteins that associate with it using mass spectrometry analysis. We found that PPP2R5E, a regulatory subunit of protein phosphatase 2A, interacted with MTCL1. Depletion of PPP2R5E reduced the abundance of MTCL1 abundance, whereas exogenous expression of PPP2R5E increased endogenous MTCL1. Furthermore, inhibition of phosphatase activity by okadaic acid reduced MTCL1, which was restored by the addition of the protease inhibitor MG132. Finally, we show that cells depleted of PPP2R5E and MTCL1 exhibited defects in microtubule organization. Our results suggest that the PPP2R5E phosphatase may contribute to microtubule organization by stabilizing MTCL1.

Human sperm degradation of zona pellucida proteins contributes to fertilization

BackgroundThe mammalian oocyte extracellular matrix known as the zona pellucida (ZP) acts as a barrier to accomplish sperm fusion with the female gamete. Although penetration of the ZP is a limiting event to achieve fertilization, this is one of the least comprehended stages of gamete interaction. Even though previous studies suggest that proteases of sperm origin contribute to facilitate the passage of sperm through the ZP, in human this process is not yet fully understood. The aim of this study was to determine the ability of human sperm to degrade recombinant human ZP (rhZPs) proteins and to characterize the proteases involved in this process.MethodsPurified rhZP2, rhZP3 and rhZP4 proteins were incubated with capacitated sperm and the proteolytic activity was determined by Western blot analysis. To further characterize the proteases involved, parallel incubations were performed in the presence of the protease inhibitors o-phenanthroline, benzamidine and MG-132 meant to block the activity of metalloproteases, serine proteases and the proteasome, respectively. Additionally, protease inhibitors effect on sperm-ZP binding was evaluated by hemizona assay.ResultsThe results showed that rhZPs were hydrolyzed in the presence of capacitated sperm. O-phenanthroline inhibited the degradation of rhZP3, MG-132 inhibited the degradation of rhZP4 and benzamidine inhibited the degradation of the three proteins under investigation. Moreover, hemizona assays demonstrated that sperm proteasome inhibition impairs sperm interaction with human native ZP.ConclusionsThis study suggests that sperm proteasomes could participate in the degradation of ZP, particularly of the ZP4 protein. Besides, metalloproteases may be involved in specific degradation of ZP3 while serine proteases may contribute to unspecific degradation of the ZP. These findings suggest that localized degradation of ZP proteins by sperm is probably involved in ZP penetration and may be of help in understanding the mechanisms of fertilization in humans.

Platelet derived growth factor receptor α-phospholipase Cγ1-protein kinase Cα signal transductionin in human lung cancer cell GLC-82 for combination of the drugs tumor necrosis factor-related apoptosis-inducing ligand and proteasome inhibitor MG132

Objective To tumor necrosis factor- related apoptosis- inducing ligand(TRAIL)combined with a protease inhibitor MG132 observed in human lung adenocarcinoma cell line GLC- 82 inhibition of proliferation and platelet derived growth factor receptor α(PDGFRα)- phospholipase Cγ1(PLCγ1)-protein kinase Cα(PKCα)signal transduction pathway. Methods Methyl thiazol tetrazolium(MTT)assay inhibition of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell growth detection; using flow cytometry to detect the effects of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell apoptosis, using SP immunohistochemical detection of protease inhibitors MG132 joint expression of TRAIL- associated protein PDGFRα, PLCγ1,PKCα's, Western blotting to determine the effect of protease inhibitors MG132 TRAIL combined for GLC- 82 cells PDGFRα, PLCγ1, PKCαprotein expression. Results MTT assay inhibition of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell growth detection. The inhibitory rates were(34.92±3.95)%,(33.17±1.78)% and(76.96±1.28)%, respectively; using flow cytometry to detect the effects of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell apoptosis. Apoptosis rates of the four group were 6.78%, 38.72%, 45.60% and 91.74%, respectively. Using SPimmunohistochemical detection of protease inhibitors MG132 joint expression of TRAIL- associated protein PDGFRα, PLCγ1, PKCα's, Western blotting to determine the effect of protease inhibitors MG132 TRAIL combined for GLC- 82 cells PDGFRα, PLCγ1, PKCα protein expression. Conclusion TRAIL combined protease inhibitor MG132 on human lung adenocarcinoma GLC- 82 cells significantly inhibited the growth, can reduce the level of GLC- 82 cells PDGFRα- PLCγ1- PKCα each protein signaling pathway, the incidence of lung cancer and PDGFRα- PLCγ1- PKCα enhanced signal transduction pathways are closely related. Key words: Tumor necrosis factor-related apoptosis-inducing ligand; MG132; Lung adenocarcinoma; Signal transduction

Altered Nrf2 signaling mediates hypoglycemia-induced blood-brain barrier endothelial dysfunction in vitro.

Hypoglycemia impairs blood-brain barrier (BBB) endothelial function; a major hallmark in the pathogenesis of various CNS disorders. Previously, we have demonstrated that prolonged hypoglycemic exposure down-regulated BBB endothelial NF-E2 related factor-2 (Nrf2) expression; a redox-sensitive transcriptional factor that regulates endothelial function. Here, we sought to determine the functional role of Nrf2 in preserving BBB integrity and molecular mechanisms underlying hypoglycemia-induced Nrf2 down-regulation in vitro using human cerebral microvascular endothelial cell line (hCMEC/D3). Cell monolayers were exposed to normal or hypoglycemic (5.5 or 2.2mM D-glucose) media for 3-24h. Pharmacological or gene manipulation (by silencing RNA) approaches were used to investigate specific molecular pathways implicated in hypoglycemia-induced Nrf2 degradation. BBB integrity was assessed by paracellular permeability to labeled dextrans of increasing molecular sizes (4-70kDa). Silencing Nrf2 expression in hCMEC/D3 cells abrogated the expression of claudin-5 and VE-cadherin, while ZO-1 was up-regulated. These effects were paralleled by a decrease in electrical resistance of hCMEC/D3 monolayers and potential increase in permeability to all labeled dextrans. Hypoglycemic exposure (3-24h) led to progressive and sustained down-regulation of Nrf2 (without affecting mRNA) and its target, NQO-1, with a concomitant increase in the cytosolic pool of E3 ubiquitin ligase, Siah2 (but not Keap1). Pretreatment with protease inhibitor MG132, or selective knock-down of Siah2 (but not Keap1) significantly attenuated hypoglycemia-induced Nrf2 destabilization. While hypoglycemic exposure triggered a significant increase in BBB permeability to dextrans, silencing Siah2 gene abrogated the effects of hypoglycemia and restored BBB integrity. In summary, our data indicate a potential role for Nrf2 signaling in regulating tight junction integrity and maintaining BBB function. Nrf2 suppression by increased Siah2-driven proteasomal degradation mediates hypoglycemia-evoked endothelial dysfunction and loss of BBB integrity. Overall, this study suggests that sustained activation of endothelial Nrf2 signaling could have therapeutic potential to prevent hypoglycemia-induced cerebrovascular dysfunction.

Effect of the protease inhibitor MG132 on the transforming growth factor-β/Smad signaling pathway in HSC-T6 cells

The activation of hepatic stellate cells (HSCs) and their transformation to myofibroblasts are the key steps in the pathological progress of liver fibrosis. The transforming growth factor-β (TGFβ)/Smad pathway is involved in the proliferation and collagen synthesis of HSCs. This study aimed to examine the effect of the protease inhibitor MG132 on the signaling pathway of TGFβ/Smad in HSC-T6 cells and seek a novel therapeutic approach for liver fibrosis. The HSC-T6 cells were treated with MG132 at different concentrations (0-10 μmol/L). Cell proliferation was detected by MTT method. The mRNA and protein expression levels of TGFβ1, Smad3 and Smad7 were determined in HSC-T6 cells by real-time PCR and Western blotting, respectively, after treatment with MG132 at different concentrations (1, 2, 3 μmol/L) or RPMI1640 alone (serving as control). The results showed that MG132 could inhibit the proliferation of HSC-T6 cells in a dose-dependent manner, and the IC(50) of MG132 was 6.84 μmol/L. After treatment with MG132 at 1, 2 or 3 μmol/L for 24 h, the mRNA expression levels of TGF-β1 and Smad3 were significantly decreased (P<0.05), but the Smad7 mRNA expression had no significant change (P>0.05). There was also a significant decrease in the protein expression level of TGF-β1 and Smad3 (P<0.05). However, the expression of Smad7 protein was substantially increased when compared with the control group (P<0.05). It was concluded that the inhibition of TGFβ/Smad pathway in HSC-T6 cells by MG132 can reduce the production of profibrosis factors (TGFβ1, Smad3) and promote the expression of anti-fibrosis factor (Smad7), suggesting that MG132 may become a potential therapeutic alternative for liver fibrosis.

The F‐box protein CPR1/CPR30 negatively regulates R protein SNC1 accumulation

Disease resistance (R) proteins, as central regulators of plant immunity, are tightly regulated for effective defense responses and to prevent constitutive defense activation under non-pathogenic conditions. Here we report the identification of an F-box protein CPR1/CPR30 as a negative regulator of an R protein SNC1 likely through SCF (Skp1-cullin-F-box) mediated protein degradation. The cpr1-2 (cpr30-1) loss-of-function mutant has constitutive defense responses, and it interacts synergistically with a gain-of function mutant snc1-1 and a bon1-1 mutant where SNC1 is upregulated. The loss of SNC1 function suppresses the mutant phenotypes of cpr1-2 and cpr1-2 bon1-1, while overexpression of CPR1 rescues mutant phenotypes of both bon1-1 and snc1-1. Furthermore, the amount of SNC1 protein is upregulated in the cpr1-2 mutant and down-regulated when CPR1 is overexpressed. The regulation of SNC1 by CPR1 is dependent on the 26S proteosome as a protease inhibitor MG132 stabilizes SNC1 and reverses the effect of CPR1 on SNC1. Interestingly, CPR1 is induced after infection of both virulent and avirulent pathogens similarly to the other negative defense regulator BON1. Thus, this study reveals a new mechanism in R protein regulation likely through protein degradation and suggests negative regulation as a critical component in fine control of plant immunity.

USP4 inhibits p53 through deubiquitinating and stabilizing ARF-BP1

Tumour suppressor p53 levels in the cell are tightly regulated by controlled degradation through ubiquitin ligases including Mdm2, COP1, Pirh2, and ARF-BP1. The ubiquitination process is reversible via deubiquitinating enzymes, such as ubiquitin-specific peptidases (USPs). In this study, we identified ubiquitin-specific peptidase 4 (USP4) as an important regulator of p53. USP4 interacts directly with and deubiquitinates ARF-BP1, leading to the stabilization of ARF-BP1 and subsequent reduction of p53 levels. Usp4 knockout mice are viable and developmentally normal, but showed enhanced apoptosis in thymus and spleen in response to ionizing radiation. Compared with wild-type mouse embryonic fibroblasts (MEFs), Usp4-/- MEFs exhibited retarded growth, premature cellular senescence, resistance to oncogenic transformation, and hyperactive DNA damage checkpoints, consistent with upregulated levels and activity of p53 in the absence of USP4. Finally, we showed that USP4 is overexpressed in several types of human cancer, suggesting that USP4 is a potential oncogene.

Statins reduce the androgen sensitivity and cell proliferation by decreasing the androgen receptor protein in prostate cancer cells

Statins (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors) are cholesterol-lowering drugs that are widely used to prevent and treat atherosclerotic cardiovascular disease. Recent epidemiological studies suggest that statins reduce serum prostate-specific antigen (PSA) levels and decrease the risk of prostate cancer. In the present study, we determined the molecular mechanisms related to the regulation of PSA, androgen receptor (AR) and cell proliferation in prostate cancer cell lines by statins. Western blotting, quantitative real-time polymerase chain reaction, cytotoxicity analysis and a cell proliferation assay were used to resolve the regulatory role of statins (mevastatin and simvastatin) in three prostate cancer cell lines, RWPE-1, 22Rv1, and LNCaP. Western blotting revealed that both mevastatin and simvastatin downregulated AR and PSA protein. However, these statins did not downregulate AR mRNA expression, while they decreased PSA mRNA. The protease inhibitor MG132 inhibited the downregulation of AR protein which suggested that statins decreased AR protein levels by increasing AR proteolysis. Furthermore, statins reduced cell proliferation in AR positive cells but not in AR negative cells, suggesting that statins regulate cell proliferation via AR expression. In addition, cell proliferation assay at various concentrations of dihydrotestosterone (DHT) showed that statins decreased androgen sensitivity in LNCaP cells. Statins decreased AR protein by proteolysis but not mRNA transcription. The drop in AR levels resulted in a reduction in androgen sensitivity and a decrease in cell proliferation in AR positive prostate cancer cells.

Antioxidant-induced Phosphorylation of Tyrosine 486 Leads to Rapid Nuclear Export of Bach1 That Allows Nrf2 to Bind to the Antioxidant Response Element and Activate Defensive Gene Expression

Antioxidants cause stabilization and nuclear translocation of NF-E2-related factor 2 (Nrf2), where it binds to the antioxidant response element (ARE) and induces up-regulation of defensive genes that protect cells against oxidative and electrophilic stress. Bach1, the negative regulator of Nrf2, competes with Nrf2 for binding to the ARE in the human NQO1 promoter. In this study, we demonstrate that Bach1 exits the nucleus within 1-2 h upon antioxidant treatment. Genistein, an inhibitor of tyrosine kinases, blocked nuclear export of Bach1. Site-directed mutagenesis and immunoprecipitation assays identified tyrosine 486 that was phosphorylated in response to the antioxidant and was essential for nuclear export of Bach1. Chromatin immunoprecipitation assays revealed a competitive interplay between Bach1 and Nrf2 at 1-2 and 4 h for binding to the human NQO1 ARE. Luciferase and real time PCR assays showed a significant decrease in antioxidant induction of reporter activity and mRNA levels in cells transfected with mutant Bach1 compared with wild type. This decrease was due to the absence of nuclear export of the mutant protein. Bach1 levels inside the nucleus returned to normal at 4 h after antioxidant treatment in the absence but not in the presence of protein synthesis inhibitor cycloheximide. In addition, antioxidant treatment increased the transcription of Bach1 as shown by pulse chase and real time PCR experiments. Taken together, these results indicate that increased synthesis of Bach1 restored its nuclear levels to normal at 4 h. In conclusion, antioxidant-induced tyrosine 486 phosphorylation leads to nuclear exit of Bach1, thus allowing Nrf2 access to the ARE.

Ongoing DNA synthesis in the rat cerebral cortex is regulated by a proteolytic pathway independent of the proteasome and calpains

By using mini-units of tissue and protease inhibitors in short term incubation (0-180 min), we studied the role of proteolysis for ongoing DNA replication in the developing rat cerebral cortex. The protease inhibitors TLCK, TPCK, PMSF, MG-132 and PSI markedly inhibited DNA synthesis. The inhibitory effects were concentration-dependent and of early onset (within 60 min). The most selective proteasome inhibitors lactacystin and clasto-lactacystin-beta-lactone as well as the calpain inhibitor I and II had no or minimal effects on DNA synthesis. Only high concentrations of calpain inhibitor I (>or= 250 microM) and calpain inhibitor II (>or= 500 microM) gave a DNA synthesis inhibition. These results suggest that (1) ongoing DNA replication is regulated by proteolysis and (2) the proteolytic pathways involved are neither the proteasome nor the calpains.

Expressional Control of a Cadmium-transporting P1B-type ATPase by a Metal Sensing Degradation Signal

Cadmium is a highly toxic environmental contaminant implicated in various diseases. Our previous data demonstrated that Pca1, a P1B-type ATPase, plays a critical role in cadmium resistance in yeast S. cerevisiae by extruding intracellular cadmium. This illustrates the first cadmium-specific efflux pump in eukaryotes. In response to cadmium, yeast cells rapidly enhance expression of Pca1 by a post-transcriptional mechanism. To gain mechanistic insights into the cadmium-dependent control of Pca1 expression, we have characterized the pathway for Pca1 turnover and the mechanism of cadmium sensing that leads to up-regulation of Pca1. Pca1 is a short-lived protein (t1/2 < 5 min) and is subject to ubiquitination when cells are growing in media lacking cadmium. Distinct from many plasma membrane transporters targeted to the vacuole for degradation via endocytosis, cells defective in this pathway did not stabilize Pca1. Rather, Pca1 turnover was dependent on the proteasome. These data suggest that, in the absence of cadmium, Pca1 is targeted for degradation before reaching the plasma membrane. Mapping of the N terminus of Pca1 identified a metal-responding degradation signal encompassing amino acids 250-350. Fusion of this domain to a stable protein demonstrated that it functions autonomously in a metal-responsive manner. Cadmium sensing by cysteine residues within this domain circumvents ubiquitination and degradation of Pca1. These data reveal a new mechanism for substrate-mediated control of P1B-type ATPase expression. Cells have likely evolved this mode of regulation for a rapid and specific cellular response to cadmium.

Nox1‐mediated EGFR transactivation increases N‐cadherin shedding and smooth muscle migration

The transmembrane glycoprotein N‐cadherin (N‐cad) has a critical role in the dynamic rearrangement of cell to cell adhesion. Reduction in the level of cell surface N‐cad by proteolytic shedding increases transcriptional activity of genes involved in cell migration. Although NADPH oxidases participate in cell growth, their role in shedding of N‐cad is unknown. We hypothesized that the NADPH oxidase subunit Nox1 contributes to shedding of N‐cad and migration of smooth muscle cells (SMC). Studies were performed in SMC cultured from wildtype (WT) and Nox1 null mice. Treatment of WT SMC with thrombin reduced membrane levels of N‐cad, which was prevented by the protease inhibitor MG132, suggesting N‐cad shedding. Pretreatment with antisense to Nox1 abolished the thrombin‐mediated shedding of N‐cad. Similarly, thrombin failed to reduce N‐cad levels in Nox1 null SMC. In WT SMC, cleavage of N‐cad and activation of matrix‐metalloproteinase (MMP)‐9 by thrombin was dependent on transactivation of EGFR. Activation of EGFR and MMP‐9 were significantly reduced in Nox1 null SMC. Finally, WT SMC migration was attenuated by the EGFR inhibitor AG1478 or by Nox1 antisense. These data suggest that Nox1‐mediated EGFR transactivation promotes proteolysis of N‐cad, potentially by MMP‐9, disrupting cell‐cell adhesions important in SMC migration.

Prickle-1 Negatively Regulates Wnt/β-Catenin Pathway by Promoting Dishevelled Ubiquitination/Degradation in Liver Cancer

Aberrant activation of Wnt signaling due to accumulation of beta-catenin has been linked to tumorigenesis. Mutations of beta-catenin, APC, and axins are important but not frequent enough to be accountable for the accumulation of beta-catenin in human hepatocellular carcinoma (HCC). In this study, we characterized the roles of Prickle-1, a Dishevelled (Dvl)-associated protein, in regulation of Wnt/beta-catenin activity in HCC. The expression levels of human Prickle-1 and Dvl3 were examined in HCC cell lines and human HCC samples. The interaction and effects of Prickle-1 on Dvl3, the Wnt/beta-catenin pathway, and cell growth were assessed in HCC cell lines. We showed that Prickle-1 bound with Dvl3 and facilitated Dvl3 ubiquitination/degradation, and this was through its destruction box (D-box) motifs. Enforced expression of Prickle-1 significantly reduced the Wnt/beta-catenin activity and tumorigenic properties of HCC cells. Clinicopathologic analysis showed that underexpression of Prickle-1 was significantly associated with overexpression of Dvl3, beta-catenin accumulation (P = .023), and larger tumor size (P = .030). Our results have elucidated a novel mechanistic relationship between Prickle-1 and Dvl3 in the Wnt/beta-catenin pathway. The facilitation of Prickle-1 on Dvl3 degradation and the suppression of beta-catenin activity and cell growth suggest that Prickle-1 is a negative regulator of the Wnt/beta-catenin signaling pathway and is a putative tumor suppressor in human HCCs.

Contribution of GADD45 Family Members to Cell Death Suppression by Cellular Bcl-x L and Cytomegalovirus vMIA

Mammalian cells and viruses encode inhibitors of programmed cell death that localize to mitochondria and suppress apoptosis initiated by a wide variety of inducers. Mutagenesis was used to probe the role of a predicted alpha-helical region within the hydrophobic antiapoptotic domain (AAD) of cytomegalovirus vMIA, the UL37x1 gene product. This region was found to be essential for cell death suppression activity. A screen for proteins that interacted with the AAD of functional vMIA but that failed to interact with mutants identified growth arrest and DNA damage 45 (GADD45alpha), a cell cycle regulatory protein activated by genotoxic stress, as a candidate cellular binding partner. GADD45alpha interaction required the AAD alpha-helical character that also dictated GADD45alpha-mediated enhancement of death suppression. vMIA mutants that failed to interact with GADD45alpha were completely nonfunctional in cell death suppression, and any of the three GADD45 family members (GADD45alpha, GADD45beta/MyD118, or GADD45gamma/OIG37/CR6/GRP17) was able to cooperate with vMIA; however, none influenced cell death when introduced into cells alone. GADD45alpha was found to increase vMIA protein levels comparably to treatment with protease inhibitors MG132 and ALLN. Targeted short interfering RNA knockdown of all three GADD45 family members maximally reduced vMIA activity, and this reduction was abrogated by additional GADD45alpha. Interestingly, GADD45 family members were also able to bind and enhance cell death suppression by Bcl-xL, a member of the Bcl-2 family of cell death suppressors, suggesting a direct cooperative link between apoptosis and the proteins that regulate the DNA damage response.

The kinase p38 Regulates Peroxisome Proliferator Activated Receptor-γ in Human Trophoblasts

Deficiency of either the mitogen-activated protein kinase p38 or the nuclear receptor PPARgamma results in disrupted vasculogenesis and abnormal development of the murine placenta. In addition, PPARgamma regulates differentiation of human trophoblasts. Here we tested the hypothesis that p38 plays an important role in the regulation of PPARgamma in primary human trophoblasts. We initially confirmed that cultured trophoblasts derived from normal term human placentas express p38 as well as its functional phosphorylated form. Whereas PPARgamma did not alter p38 expression, p38 inhibitors diminished the transcriptional activity of PPARgamma in primary trophoblasts. In addition, inhibition of p38 resulted in marked attenuation of PPARgamma-stimulated hCG production by cultured trophoblast. Our data support an effect of p38 on PPARgamma protein stability because p38 inhibition led to reduced expression of PPARgamma protein without a significant effect on PPARgamma mRNA, and this reduction was blocked by the protease inhibitor MG-132. Together, these data indicate that p38 regulates PPARgamma expression and activity in term human trophoblasts. Cross talk between p38 and PPARgamma signaling may play a role in modulating differentiation and function of the human placenta.

E1AF degradation by a ubiquitin–proteasome pathway

E1AF is a member of the ETS family of transcription factors. In mammary tumors, overexpression of E1AF is associated with tumorigenesis, but E1AF protein has hardly been detected and its degradation mechanism is not yet clear. Here we show that E1AF protein is stabilized by treatment with the 26S protease inhibitor MG132. We found that E1AF was modified by ubiquitin through the C-terminal region and ubiquitinated E1AF aggregated in nuclear dots, and that the inhibition of proteasome-activated transcription from E1AF target promoters. These results suggest that E1AF is degraded via the ubiquitin–proteasome pathway, which has some effect on E1AF function.

Over-expression of human UREB1 in colorectal cancer: HECT domain of human UREB1 inhibits the activity of tumor suppressor p53 protein

Many fundamental processes, including oncogenesis, have implicated HECT domain proteins with ubiquitin ligase activity. The protein human upstream regulatory element binding protein 1 (hUREB1) is a HECT domain protein whose function is not defined yet. Here, we investigate the function of hUREB1 as a ubiquitin–protein ligase in human colorectal cells. Ectopic expression of the HECT domain of hUREB1 reduces the protein level and transcriptional activity of the p53 tumor suppressor, which is abrogated by the deletion in the HECT domain or point mutations in the essential residues of the HECT domain. The ubiquitination and destabilization of p53 is observed in cells treated with the protease inhibitor MG132, implying that the HECT domain of hUREB1 suppresses the transcriptional activity of p53 through a ubiquitin-dependent degradation pathway. Based on the results of Northern blot analysis, RT-PCR, and immunohistochemical analyses, the over-expression of hUREB1 is associated with colorectal carcinoma. Moreover, protein levels of hUREB1 and p53 were inversely correlated. These findings suggest that hUREB1 can function, at least in part, as a negative regulator of p53 during the colorectal carcinoma progression through the ubiquitination pathway mediated by the HECT domain.

Cell-cycle-related variation in proteins in suspension-cultured rice cells.

To understand the cell cycle process in plants, we searched for proteins that quantitatively change during the cell cycle in suspension-cultured rice ( Oryza sativa L.) cells. The proteins were analyzed by a two-dimensional polyacrylamide gel electrophoresis image-analysis system. We detected 11 proteins that quantitatively changed during the cell cycle, among which beta-tubulins and a calreticulin-like protein were identified. The amounts of beta-tubulin proteins were low in the M phase and high in the G1 phase. In contrast, mRNAs for two of the three types of beta-tubulin were high in the M phase of the cell cycle. The addition of protease inhibitors MG132 or E64d to the cells decreased the beta-tubulin proteins during 24 h, suggesting that beta-tubulin proteins are degraded in vivo by proteases other than those whose activities are inhibited by MG132 or E64d.

Development of an in vitro assay for the proteolytic processing of the CDP/Cux transcription factor.

The CDP/Cux transcription factor was previously shown to be proteolytically processed at the G1/S transition. In view of characterizing and eventually identifying the protease responsible for CDP/Cux processing, we have established an in vitro proteolytic processing assay. CDP/Cux recombinant proteins expressed in mammalian or bacterial cells were efficiently processed in vitro using as a source of protease either whole cell extracts, the nuclear or the cytoplasmic fraction. Processing was found to take place optimally at a lower pH, to be insensitive to variations in salt concentration, and to be inhibited by the protease inhibitors MG132 and E64D. Interestingly, the bacterially-produced substrate was more efficiently processed than the substrate purified from mammalian cells. Moreover, processing in vitro was more efficient when CDP/Cux substrates were purified from populations of cells enriched in the S phase than in the G1 phase of the cell cycle. Altogether, these results suggest that posttranslational modifications of CDP/Cux in mammalian cells inhibits processing and contributes to the cell cycle-dependent regulation of processing. The in vitro processing assay described in this study will provide a useful tool for the purification and identification of the protease responsible for the processing of CDP/Cux.