Watching proteases in action.

encoding proteases within any genome are easily identified, and three-dimensional structures of many proteases have been solved. Most proteolytic cleavage mechanisms and pro-tease substrate specificities have been established (the latter in some detail) 1. We are also able to block proteolytic activities with potent inhibi-tors. Transgenic approaches have been used for targeted deletions of protease-or protease inhibitor-encoding genes, providing important insights into the biological significance of proteases 2. Given that we know a fair amount about these enzymes, why do we still not know when and where proteases cleave their natural substrates? The answer is simply that watching proteases in action is not a trivial task. In this issue of Nature Chemical Biology, Matthew Bogyo and colleagues describe elegantly how to use chemistry for the design of quenched activity based probes (qABPs) that can be applied to visualize the dynamics of active proteases 3. The new probes enable imaging proteases in real time and at the point of their action. The next generation of these probes may prove suitable for whole-body imaging of proteases for biomedical diagnostics. Why should we be interested in exploring the labyrinth of proteolysis in vivo and in such detail? Cells use proteases for many critical cellular functions, including protein degradation, prohormone processing 4 , antigen presentation and induction of programmed cell death. Proteolysis is rapid and with increased expression of the cytoskeletal protein ezrin, for which expression levels are correlated with those of hPR 9. The second interesting effect was that inhibition of hPR-B transcription also led to reduced expression of hPR-A, even though the transcription start site for the latter is 760 bases downstream and is not complementary to the anti-hPR-B PNA. This implies the existence of a feedback mechanism in which downregulation of hPR-B leads to downregulation of hPR-A. The second paper from Corey's laboratory describes the use of duplex RNA to interfere with transcription. The RNA duplexes, reminiscent of siRNA, targeted regions consisting of 19 nucleotides in the vicinity of transcription start sites and inhibited transcription at low-nanomolar concentrations. In addition to hPR, specific antigene effects were observed for three other genes when targeted by RNA duplexes having the appropriate sequence. Sequence selectivity was very good, with two mismatches being sufficient to preclude knockdown. In contrast to the antigene PNAs, which most likely exert their effects through formation of PNA-DNA duplexes with the open complex, the inhibition mechanism for the antigene RNA …