Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.31 (beta-glucuronidase)
 7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The tobacco etch potyvirus (TEV) genome encodes a polyprotein that is processed by three virus-encoded proteinases. Although replication of TEV likely occurs in the cytoplasm, two replication-associated proteins, VPg-proteinase (nuclear inclusion protein a) (NIa) and RNA-dependent RNA polymerase (nuclear inclusion protein b) (NIb), accumulate in the nucleus of infected cells. The 6-kDa protein is located adjacent to the N terminus of NIa in the TEV polyprotein, and, in the context of a 6-kDa protein/NIa (6/NIa) polyprotein, impedes nuclear translocation of NIa (M. A. Restrepo-Hartwig and J. C. Carrington, J. Virol. 66:5662-5666, 1992). The 6-kDa protein and three polyproteins containing the 6-kDa protein were identified by affinity chromatography of extracts from infected plants. Two of the polyproteins contained NIa or the N-terminal VPg domain of NIa linked to the 6-kDa protein. To investigate the role of the 6-kDa protein in vivo, insertion and substitution mutagenesis was targeted to sequences coding for the 6-kDa protein and its N- and C-terminal cleavage sites. These mutations were introduced into a TEV genome engineered to express the reporter protein beta-glucuronidase (GUS), allowing quantitation of virus amplification by a fluorometric assay. Three-amino-acid insertions at each of three positions in the 6-kDa protein resulted in viruses that were nonviable in tobacco protoplasts. Disruption of the N-terminal cleavage site resulted in a virus that was approximately 10% as active as the parent, while disruption of the C-terminal processing site eliminated virus viability. The subcellular localization properties of the 6-kDa protein were investigated by fractionation and immunolocalization of 6-kDa protein/GUS (6/GUS) fusion proteins in transgenic plants. Nonfused GUS was associated with the cytosolic fraction (30,000 x g centrifugation supernatant), while 6/GUS and GUS/6 fusion proteins sedimented with the crude membrane fraction (30,000 x g centrifugation pellet). The GUS/6 fusion protein was localized to apparent membranous proliferations associated with the periphery of the nucleus. These data suggest that the 6-kDa protein is membrane associated and is necessary for virus replication.
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PMID:The tobacco etch potyvirus 6-kilodalton protein is membrane associated and involved in viral replication. 813 25

A two component system, consisting of a fusion protein and an appropriate prodrug, suited to perform selective tumor therapy in vivo is presented. The fusion protein, due to its humanized carcinoembryonic antigen-specific variable region, specifically binds to carcinoembryonic antigen-expressing tumors and has an enzymatic activity comparable to that of human beta-glucuronidase. The prodrug is a nontoxic glucuronide-spacer derivative of doxorubicin decomposing to doxorubicin by enzymatic deglucuronidation. In vivo studies in nude mice bearing human carcinoembryonic antigen-expressing tumor xenografts revealed that 7 days after injection of 20 mg/kg fusion protein a high specificity ratio (> 100:1) was obtained between tumor and plasma or tumor and normal tissues. Injection of 250 mg/kg of prodrug at day 7 resulted in tumor therapeutic effects superior to those of conventional chemotherapy without any detectable toxicity. These superior therapeutic effects which were observed using established human tumor xenografts can be explained by the approximately 4-12-fold higher doxorubicin concentrations found in tumors of mice treated with fusion protein and prodrug than in those treated with the maximal tolerable dose of drug alone. The nondetectable toxicity in the animals treated with fusion protein and prodrug is probably caused by up to 5-fold lower drug concentrations in normal tissues compared to the animals treated with doxorubicin. Thus, a more tumor-selective therapy, resulting in stronger therapeutic effects and reduced toxicity seems to be possible by the appropriate use of the humanized nontoxic fusion protein and the nontoxic prodrug.
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PMID:Tumor-selective prodrug activation by fusion protein-mediated catalysis. 817 22