Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human papillomavirus type 18 (HPV-18) has recently been closely linked with human malignant cervical lesions. The early region of the genome of the related bovine papillomavirus (BPV) has been shown to be important for the production of the transformed phenotype. BPV E6 has been shown to be a transforming protein. We report the primary structure of the HPV-18 E6 open reading frame and its predicted amino acid sequence. Both E6 protein and E6-beta-galactosidase fusion protein were synthesized in bacteria. Antisera were raised against the E6-beta-galactosidase fusion protein and against an E6 N-terminal peptide which was 14 amino acids long. We show that these antisera reacted on Western blots against E6 synthesized in bacteria. The HPV E6 antigen and antibodies described here will be useful in understanding HPV expression and its association with human malignancies and may also be diagnostically useful.
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PMID:The expression of human papillomavirus type 18 E6 protein in bacteria and the production of anti-E6 antibodies. 301 29

Previous work has shown that a fusion protein bearing a "nonremovable" N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last approximately 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues). We also show that a Lys --> Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to beta-galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the UbR29 but not by the UbR48 moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys29, whereas ufd2 mutants are impaired in the ubiquitination at Lys48. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys48 but also Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.
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PMID:A proteolytic pathway that recognizes ubiquitin as a degradation signal. 761 50

Immortal human fibroblasts, SVts8 cells, which express a heat-labile SV40 large T antigen, induces a senescence-like phenomenon in response to upward shift in temperature. Cells with arrested division show strong induction of senescence-associated beta-galactosidase. We examined how p53 and pRB are involved in this phenomenon since they are major targets of the T antigen. Transfection of cells with plasmids encoding the wild-type T antigen or human papilloma virus type 16 E6/E7 proteins completely abolished the arrest in cell division, a plasmid encoding the E6 protein suppressed it markedly, while a plasmid encoding E7 had no effect. Plasmids encoding dominant-negative p53 mutants also suppressed the arrest in cell division to various degrees. Upon temperature shift, p21 mRNA was upregulated 10-fold in SVts8 cells, but only slightly in clones expressing the wild-type T antigen or dominant-negative p53 mutants. These data demonstrate that p53 plays a major role in this senescence-like phenomenon.
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PMID:The introduction of dominant-negative p53 mutants suppresses temperature shift-induced senescence in immortal human fibroblasts expressing a thermolabile SV40 large T antigen. 1005 41

We describe a novel vector-host system suitable for the efficient preparation of fluorescent single-chain antibody Fv fragments (scFv) in Escherichia coli. The previously described pscFv1F4 vector used for the bacterial expression of functional scFv to the E6 protein of human papillomavirus type 16 was modified by appending to its C-terminus the green fluorescent protein (GFP). The expression of the scFv1F4-GFP fusion proteins was monitored by analyzing of the typical GFP fluorescence of the transformed cells under UV illumination. The brightest signal was obtained when scFv1F4 was linked to the cycle 3 GFP variant (GFPuv) and expressed in the cytoplasm of AD494(DE3) bacteria under control of the arabinose promoter. Although the scFv1F4 expressed under these conditions did not contain disulfide bridges, about 1% of the molecules were able to bind antigen. Fluorescence analysis of antigen-coated agarose beads incubated with the cytoplasmic scFv-GFP complexes showed that a similar proportion of fusions retained both E6-binding and green-light-emitting activities. The scFv1F4-GFPuv molecules were purified by affinity chromatography and successfully used to detect viral E6 protein in transfected COS cells by fluorescence microscopy. When an anti-beta-galactosidase scFv, which had previously been adapted to cytoplasmic expression at high levels, was used in this system, it was possible to produce large amounts of functional fluorescent antibody fragments. This indicates that these labeled scFvs may have many applications in fluorescence-based single-step immunoassays.
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PMID:Production of fluorescent single-chain antibody fragments in Escherichia coli. 1068 42

Angelman syndrome (AS), characterized by motor dysfunction, mental retardation, and seizures, is caused by several genetic etiologies involving chromosome 15q11-q13, including mutations of the UBE3A gene. UBE3A encodes UBE3A/E6-AP, a ubiquitin-protein ligase, and shows brain-specific imprinting, with brain expression predominantly from the maternal allele. Lack of a functional maternal allele of UBE3A causes AS. In order to understand the causal relationship between maternal UBE3A mutations and AS, we have constructed a mouse model with targeted inactivation of Ube3a. The inactive allele contains a lacZ reporter gene for analysis of brain-specific imprinting. Maternal, but not paternal, transmission of the targeted allele leads to beta-galactosidase activity in hippocampal and cerebellar neurons. Maternal inheritance of the Ube3a mutant allele also causes impaired performance in tests of motor function and spatial learning, as well as abnormal hippocampal EEG recordings. As predicted from the dependence of UBE3A-mediated ubiquitination of p53 on HPV E6 protein, our maternal-deficient mice show normal brain p53 levels.
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PMID:Neurobehavioral and electroencephalographic abnormalities in Ube3a maternal-deficient mice. 1189 68

The E6 protein of the high-risk human papillomavirus type 16 (HPV-16) is involved in the tumorigenesis of human cervical cells by targeting numerous cellular proteins. We characterized new anti-E6 monoclonal antibodies and used them for precise localization of the E6 oncoprotein within carcinoma cells. Overexpressed E6 protein was predominantly detected in the nucleus of transiently transfected HaCaT cells. While mostly localized at the periphery of condensed chromatin, E6 was also associated with nuclear ribonucleoproteic ultrastructures and with some ribosomal areas in the cytoplasm of SiHa and CaSki cells. The chimeric beta-galactosidase-E6 protein expressed in transfected HeLa cells was essentially localized in the nuclear compartment. Together, these data indicate that the E6 sequence of HPV-16 may encode a nuclear localization signal. The preferential nuclear distribution of this viral oncoprotein in HPV-transformed cells correlates with its activities at the transcriptional level.
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PMID:Preferential nuclear localization of the human papillomavirus type 16 E6 oncoprotein in cervical carcinoma cells. 1286 40

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