Phage display of functional human TNF-alpha converting enzyme catalytic domain: a rapid method for the production of stabilized proteolytic proteins for assay development and high-throughput screening.

The catalytic domain of human tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) was expressed in a phage display system to determine whether stable and active enzyme could be made for high-throughput screening (HTS). This would address many issues around screening of proteases in this class. The phage-displayed TACE catalytic domain (PDT) properly cleaved the fusion protein of glutathione S-transferase (GST)-pro-TNF-alpha to generate the mature TNF-alpha in vitro. To determine the utility of the PDT in HTS, the authors further demonstrated that PDT was able to generate a strong reproducible fluorescence signal by cleaving a fluorogenic TNF-alpha-specific peptide in vitro. More important, the catalytic activity of the PDT was inhibited by a broad-spectrum matrix metalloprotease (MMP) inhibitor but not by an MMP-I specific inhibitor, illustrating the potential utility of PDT for HTS. The PDT was also compared with baculovirus-expressed TACE (BET) in these assays to establish the relative efficacy of PDT. Both PDT and BET showed a similar specific cleavage profile against the defined substrates. Activity of the BET, however, was stable at 4 degrees C for less than 24 h. In contrast, the PDT exhibited remarkable stability, losing very little activity even after 2 years at 4 degrees C. On the basis of these results, the authors concluded that the phage display system might be a useful tool for expressing proteins that have stability issues related to auto-proteolytic activity. Furthermore, the ease and low cost of large-scale production of phage should make it suitable for assay development and HTS.

The central cannabinoid CB1 receptor is required for diet-induced obesity and rimonabant's antiobesity effects in mice.

Cannabinoid receptor CB1 is expressed abundantly in the brain and presumably in the peripheral tissues responsible for energy metabolism. It is unclear if the antiobesity effects of rimonabant, a CB1 antagonist, are mediated through the central or the peripheral CB1 receptors. To address this question, we generated transgenic mice with central nervous system (CNS)-specific knockdown (KD) of CB1, by expressing an artificial microRNA (AMIR) under the control of the neuronal Thy1.2 promoter. In the mutant mice, CB1 expression was reduced in the brain and spinal cord, whereas no change was observed in the superior cervical ganglia (SCG), sympathetic trunk, enteric nervous system, and pancreatic ganglia. In contrast to the neuronal tissues, CB1 was undetectable in the brown adipose tissue (BAT) or the liver. Consistent with the selective loss of central CB1, agonist-induced hypothermia was attenuated in the mutant mice, but the agonist-induced delay of gastrointestinal transit (GIT), a primarily peripheral nervous system-mediated effect, was not. Compared to wild-type (WT) littermates, the mutant mice displayed reduced body weight (BW), adiposity, and feeding efficiency, and when fed a high-fat diet (HFD), showed decreased plasma insulin, leptin, cholesterol, and triglyceride levels, and elevated adiponectin levels. Furthermore, the therapeutic effects of rimonabant on food intake (FI), BW, and serum parameters were markedly reduced and correlated with the degree of CB1 KD. Thus, KD of CB1 in the CNS recapitulates the metabolic phenotype of CB1 knockout (KO) mice and diminishes rimonabant's efficacy, indicating that blockade of central CB1 is required for rimonabant's antiobesity actions.