DUB-3, a Cytokine inducible Deubiquitinating Enzyme That Blocks Profileration

Previous studies have identified the DUB family of cytokine-regulated murine deubiquitinating enzymes, which play a role in the control of cell proliferation and survival. Through data base analyses and cloning, we have identified a human cDNA (DUB-3) that shows significant homology to the known murine DUB family members. Northern blotting has shown expression of this gene in a number of tissues including brain, liver, and muscle, with two transcripts being apparent (1.6 and 1.7 kb). In addition, expression was observed in cell lines including those derived from a number of hematopoietic tumors such as the Burkitt's lymphoma cell line RAJI. We have also demonstrated that DUB-3, which was shown to be an active deubiquitinating enzyme, is induced in response to interleukin-4 and interleukin-6 stimulation. Finally, we have demonstrated that constitutive expression of DUB-3 blocks proliferation and can initiate apoptosis in both IL-3-dependent Ba/F3 cells and NIH3T3 fibroblasts. These findings suggest that human DUB-3, like the murine DUB family members, is transiently induced in response to cytokines and can, when constitutively expressed, block growth factor-dependent proliferation.

ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture

Replication of the ~30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

Cytochrome P450 1B1 expression in rat esophageal tumorigenesis promoted by gastric and duodenal reflux.

Cytochrome P450 1B1 (CYP1B1) mRNA is constitutively expressed in most normal extra-hepatic tissues; however the protein is not detectable in these tissues but is expressed in a wide variety of tumors. CYP1B1 is responsible for the activation of a number of carcinogens present in tobacco smoke and food. A surgical model of rat esophageal tumorigenesis, promoted by gastric or duodenal reflux was used to determine CYP1B1 expression in premalignant esophageal tissue. Immunohistochemistry was performed using a modified amplified fluorescein tyramide protocol. CYP1B1 was not observed in normal esophageal mucosa, submucosa, or muscularis mucosa. Animals exposed to gastric reflux developed mild hyperplasia. Varying degrees of hyperplasia were observed in the duodenal reflux group. All regions of hyperplasia showed moderate or strong CYP1B1 immunoreactivity. Duodenal reflux induced a small number of premalignant changes: immunoreactivity was absent from the epithelium of squamous dysplasia (0/10), Barrett's esophagus (0/7), and majority of dysplastic Barrett's esophagus (1/4). Moderate or strong immunoreactivity was observed in the majority (7/8) of squamous cell carcinomas (SCCs) in situ. Immunoreactivity was also observed in the lamina propria and submucosa in association with inflammation, regardless of the severity of inflammation. The expression of CYP1B1 in hyperplasia, SCCs in situ, or in association with inflammation may increase the production of carcinogenic metabolites, which may promote esophageal tumorigenesis.