Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rb protein is a critical regulator of entry into the cell cycle, and loss of Rb function by deletions, mutations, or interaction with DNA viral oncoproteins leads to oncogenic transformation. We have shown that the human papilloma virus (HPV)-16 E7 gene is sufficient to induce the immortalization of mammary epithelial cells (MECs). Surprisingly, the steady-state level of Rb protein in these immortal cells was drastically decreased. Here, we used pulse-chase analysis to show that the in vivo loss of Rb protein in E7-immortalized MECs is a consequence of enhanced degradation. Expression of HPV16 E7 in a cell line with a temperature-sensitive mutation in the E1 enzyme of the ubiquitin pathway demonstrated that degradation of Rb was ubiquitin dependent. Treatment of E7-immortalized MECs with aldehyde inhibitors of proteasome-associated proteases led to a marked stabilization of Rb protein, particularly the hypophosphorylated form. Taken together, our results provide evidence for HPV-16 E7-induced enhanced degradation of Rb protein via a ubiquitin-proteasome pathway and suggest a second mechanism of oncogenic transformation by E7, in addition to its previously identified ability to sequester Rb from E2F. Our analyses also show that normal Rb levels are regulated by the ubiquitin-proteasome degradation pathway.
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PMID:E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway. 884 Sep 74

Ubiquitin-mediated proteolysis is involved in the turnover of many short-lived regulatory proteins. This pathway leads to the covalent attachment of one or more multiubiquitin chains to target substrates which are then degraded by the 26S multicatalytic proteasome complex. Multiple classes of regulatory enzymes have been identified that mediate either ubiquitin conjugation or ubiquitin deconjugation from target substrates. Timed destruction of cellular regulators by the ubiquitin-proteasome pathway plays a critical role in ensuring normal cellular processes. This review provides multiple examples of key growth regulatory proteins whose levels are regulated by ubiquitin-mediated proteolysis. Pharmacological intervention which alters the half-lives of these cellular proteins may have wide therapeutic potential. Specifically, prevention of p53 ubiquitination (and subsequent degradation) in human papilloma virus positive tumors, and perhaps all tumors retaining wild-type p53 but lacking the retinoblastoma gene function, should lead to programmed cell death. Specific inhibitors of p27 and cyclin B ubiquitination are predicted to be potent antiproliferative agents. Inhibitors of IkappaB ubiquitination should prevent NFkappaB activation and may have utility in a variety of autoimmune and inflammatory conditions. Finally, we present a case for deubiquitination enzymes as novel, potential drug targets.
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PMID:The ubiquitin-mediated proteolytic pathway as a therapeutic area. 902 Mar 79

Rapid degradation of wild-type p53 in the human uterine cervix is induced by the infection of high-risk human papilloma virus (HPV) types 16 and 18. HPV-E6 protein plays a critical role in the poly-ubiquitination of wild-type p53 by mediating the association of p53 with E6-associated protein (E6AP). As a result, the poly-ubiquitinated p53 is rapidly and selectively degraded by the 26S proteasome. We have established a high throughput assay system to monitor poly-ubiquitination of wild-type p53 using a new fluorescence homogeneous technology known as Homogeneous Time-Resolved Fluorescence (HTRFTM). The Europium Cryptate [Eu(K)]-labeled ubiquitins are incorporated into poly-ubiquitin chains conjugated with the biotinylated p53. In the HTRF assay, Europium cryptate-labeled ubiquitin and streptavidin-labeled allophycocyanin (XL665) are used as the fluorescence donor and acceptor, respectively. The biotinylated p53 is ubiquitinated by ubiquitination enzymes, then by the addition of streptavidin-labeled XL665, the donor and acceptor molecules are brought in close proximity, thereby generating fluorescent signals. This time-resolved fluorescence assay system shows a sufficient signal for its application in synthetic compound screening and having almost the same level of sensitivity as that monitored by the scintillation proximity assay (SPA) using 125I-labeled ubiquitin. The detection of poly-ubiquitination of wild-type p53 by using the HTRFTM or SPA systems described here is much easier and quicker than by using conventional methods. Therefore, these new systems would be appropriate for high throughput screening of compounds for the discovery of new inhibitors of poly-ubiquitination of wild-type p53.
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PMID:Application of homogeneous time-resolved fluorescence (HTRFTM) to monitor poly-ubiquitination of wild-type p53. 1053 89

In higher eukaryotic cells, the p53 protein is degraded by the ubiquitin-26S proteasome system mediated by Mdm2 or the human papilloma virus E6 protein. Here we show that COP9 signalosome (CSN)-specific phosphorylation targets human p53 to ubiquitin-26S proteasome-dependent degradation. As visualized by electron microscopy, p53 binds with high affinity to the native CSN complex. p53 interacts via its N-terminus with CSN subunit 5/Jab1 as shown by far-western and pull-down assays. The CSN-specific phosphorylation sites were mapped to the core domain of p53 including Thr155. A phosphorylated peptide, Deltap53(145-164), specifically inhibits CSN-mediated phosphorylation and p53 degradation. Curcumin, a CSN kinase inhibitor, blocks E6-dependent p53 degradation in reticulocyte lysates. Mutation of Thr155 to valine is sufficient to stabilize p53 against E6-dependent degradation in reticulocyte lysates and to reduce binding to Mdm2. The p53T155V mutant accumulates in both HeLa and HL 60 cells and exhibits a mutant (PAb 240+) conformation. It induces the cyclin-dependent inhibitor p21. In HeLa and MCF-7 cells, inhibition of CSN kinase by curcumin or Deltap53(145-164) results in accumulation of endogenous p53.
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PMID:COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system. 1128 27

In cervical carcinomas abnormalities in the MHC class I surface expression are a frequent event, which are often associated with the deficient expression of the peptide transporter subunit TAP1 thereby resulting in impaired T cell response. In order to understand the role of other components of the MHC class I antigen processing machinery (APM) in the immune escape, 16 surgically removed primary cervical carcinoma lesions were analyzed for their mRNA expression of the heterodimeric peptide transporter TAP, the constitutive and interferon (IFN)-gamma inducible proteasome subunits and their activators PA28alpha/beta, various chaperones as well as MHC class I antigens. High expression levels of all APM components were detected in normal cervical tissue, whereas 15/16 of cervical carcinoma lesions exhibited an impaired expression of at least one APM component, including the proteasome subunits, their activators PA28alpha/beta, the peptide transporter subunits TAP1 and TAP2, different chaperones, HLA class I heavy chains and beta2-microglobulin (beta2-m). In particular, calnexin expression was strongly downregulated in 69% of cervical cancer lesions analyzed. Such abnormalities were neither associated with a specific human papilloma virus (HPV) or HLA class I phenotype nor with tumor grading and staging. Analysis of five cervical carcinoma cell lines demonstrated a reduced MHC class I surface expression due to deficient expression and function of TAP, LMP subunits or specific HLA-alleles which could be mostly corrected by IFN-gamma treatment. The high frequency of abnormalities of APM component expression together with their potential negative influence on T cell-mediated immune recognition emphasize the need to evaluate the antigen processing pathway in cervical carcinoma patients, particularly in those selected for T-cell-based immunotherapies.
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PMID:Deficient expression of components of the MHC class I antigen processing machinery in human cervical carcinoma. 1171 91

In cervical carcinogenesis, the p53 tumor suppressor pathway is disrupted by HPV (human papilloma virus) E6 oncogene expression. E6 targets p53 for rapid proteasome-mediated degradation. We therefore investigated whether proteasome inhibition by MG132 could restore wild-type p53 levels and sensitize HPV-positive cervical cancer cell lines to apoptotic stimuli such as rhTRAIL (recombinant human TNF-related apoptosis inducing ligand). In a panel of cervical cancer cell lines, CaSki was highly, HeLa intermediate and SiHa not sensitive to rhTRAIL-induced apoptosis. MG132 strongly sensitized HeLa and SiHa to rhTRAIL-induced apoptosis in a caspase-dependent and time-dependent manner. MG132 massively induced TRAIL receptor DR4 and DR5 membrane expression in HeLa, whereas in SiHa only DR5 membrane expression was upregulated from almost undetectable to high levels. Antagonistic DR4 antibody partially inhibited apoptosis induction by rhTRAIL and MG132 in HeLa but had no effect on apoptosis in SiHa. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in elevated levels of active p53 as demonstrated by p53 small interfering RNA (siRNA) sensitive p21 upregulation. Although p53 siRNA partially inhibited MG132-induced DR5 upregulation in HeLa and SiHa, no effect on rhTRAIL-induced apoptosis was observed. MG132 plus rhTRAIL enhanced caspase 8 and caspase 3 activation and concomitant cleavage of X-linked inhibitor of apoptosis (XIAP), particularly in HeLa. In addition, caspase 9 activation was only observed in HeLa. Downregulation of XIAP using siRNA in combination with rhTRAIL induced high levels of apoptosis in HeLa, whereas MG132 had to be added to the combination of XIAP siRNA plus rhTRAIL to induce apoptosis in SiHa. In conclusion, proteasome inhibition sensitized HPV-positive cervical cancer cell lines to rhTRAIL independent of p53. Our results indicate that not only DR4 and DR5 upregulation but also XIAP inactivation contribute to rhTRAIL sensitization by MG132 in cervical cancer cell lines. Combining proteasome inhibitors with rhTRAIL may be therapeutically useful in cervical cancer treatment.
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PMID:Proteasome inhibitor MG132 sensitizes HPV-positive human cervical cancer cells to rhTRAIL-induced apoptosis. 1628 99

Although HIV protease inhibitor (PI) drugs predominantly target HIV proteases 1 and 2, it is also known that part of their efficacy is due to selective inhibition of the proteasome. The pathogenicity of high-risk human papilloma virus (HPV) is dependent on expression of viral E6 proteins which inappropriately activate the 26S proteasome to degrade p53 and other cellular proteins that are detrimental to viral replication. Comparison of the ability of the PIs indinavir, ritonavir, amprenavir, lopinavir, atazanavir, nelfinavir and saquinavir to inhibit E6-mediated proteasomal degradation of mutant p53 in E6-transfected C33A cells showed that 15 microM lopinavir, 1 mM indinavir or 125 microM ritonavir treatment for 24 h produced a stable increase in the level of nuclear p53 in these cells with minimal cell death. After 4 h exposure of HPV16+ve SiHa cells to 15 microM lopinavir, a transient increase in wild-type p53 expression was observed associated with a 7% reduction in the chymotryptic activity of the 205 proteasome and apoptosis after 24h. Comparison of growth rates of PI treated SiHa, CaSki, C33A, C33A-E6 and non-transformed NIH/3T3 cells showed that SiHa were the most sensitive, whereas NIH/3T3 were least affected. In conclusion, these data show that specific HIV PIs such as lopinavir and possibly indinavir, can induce selective toxicity of HPV-transformed cervical carcinoma cells expressing wild-type p53 and may form the basis of a topically applied alternative to surgery for the treatment of HPV-related premalignant lesions of the cervix.
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PMID:Specific HIV protease inhibitors inhibit the ability of HPV16 E6 to degrade p53 and selectively kill E6-dependent cervical carcinoma cells in vitro. 1731 Aug 26

Inactivation of tumor suppressor p53 accompanies the majority of malignant diseases in humans. Restoration of p53 functions in tumor results in death of cancer cells, which can be used in cancer therapy. In cervical cancer a product of E6 gene of the human papilloma virus promotes accelerated degradation of p53 in proteasome system. Therefore, one of the approaches to reactivation of p53 in cervical carcinoma cells could be the use of small molecules that inhibit functions of viral proteins. By using as a test system human cervical carcinoma cells (HeLa cell line bearing human papilloma virus type 18, HPV-18) with introduced reporter construct that expresses beta-galactosidase under control of a p53-dependent promoter we carried out screening of a library of small molecules to select small molecules capable of reactivating transcriptional activity of p53. We then characterized the effects of two most active compounds in cell lines that differ in the status of p53-dependent signaling pathway. Both of the compounds caused specific activation of p53 in the cell lines expressing HPV-18, to a lesser extent--HPV-16, and do not cause any effect in control p53 negative cells, or in the cells with undisrupted p53 pathway. Activation of p53 in cervical carcinoma cells was accompanied by the induction of the p53-dependent gene CDKN1 (p21), by inhibition of proliferation, and by the induction of apoptosis. Both of the compounds were capable of deep inhibition of transcription from the HPV genome, which apparently was the cause for p53 reactivation in response to decreased expression of the E6 protein. The observed low toxicity for normal cells allows considering these chemical compounds as prototypes for future anticancer drugs.
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PMID:[Transcriptional inhibition of human papilloma virus in cervical carcinoma cells reactivates functions of the tumor suppressor p53]. 1768 29

DNA vaccination represents an attractive strategy for cancer immunotherapy combining vaccine stability, cost-effectiveness, and safety. However, a major problem of genetic vaccination is the limited potency, due to intrinsic lack of amplifying and spreading abilities in vivo and to the suboptimal intracellular processing/presentation of tumor antigens. We explored the therapeutic antitumor potency of DNA vaccines based on a mutated, nontransforming form of the E7 gene (E7GGG gene) of human papilloma virus 16 (HPV-16) fused, with or without a linker, to the potato virus X (PVX) coat protein sequence (PVX-CP). Transfection of mammalian cells demonstrated expression of the E7GGG protein, while the fusion proteins were detected only in the presence of proteasome inhibitors, suggesting increased instability and faster degradation via the proteasome. The DNA fusion vaccines, administered intramuscularly to C57BL/6 mice after challenging with a tumorigenic dose of E7-expressing TC-1 cells, inhibited the growth of tumors in vivo better than the E7GGG gene alone and induced both humoral and cell-mediated immune responses. Therefore, fusion of the HPV-16 E7GGG gene with a plant virus coat protein gene might be a valid strategy to induce antitumor immunity in a safe setting by a novel genetic vaccine targeting cervical carcinoma.
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PMID:Antitumor activity of DNA vaccines based on the human papillomavirus-16 E7 protein genetically fused to a plant virus coat protein. 1843 24

Proof of principle that molecularly targeted therapy is a valid therapeutic approach for squamous cell carcinoma of the head and neck (SCCHN) has emerged with epidermal growth factor receptor targeting agents. Other interesting targets, such as Src, insulin-like growth factor 1 receptor, and the proteasome, have been shown in vitro to play key roles in SCCHN, and their inhibition is currently being studied in phase II trials. Identification of predictive biomarkers of resistance or sensitivity to these therapies remains one of the main challenges in the optimal selection of patients most likely to benefit from them. However, clinical trials with these novel agents need to be designed rationally to improve the overall outcome of patients. Given the emerging evidence that human papilloma virus-related SCCHN is a distinct disease, it should be studied in specific trials.
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PMID:Molecularly targeted agents in the treatment of recurrent or metastatic squamous cell carcinomas of the head and neck. 1901 Feb 69


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