or to outfit them with an increasingly sophis-ticated set of accessories.

1. Lin, Y.A., Chalker, J.M., Floyd, N., Bernardes, G.J.L. & Davis, B.G. J. Am. Chem. Soc. 130, 9642–9643 (2008).

2. Bernardes, G.J.L., Chalker, J.M., Errey, J.C. & Davis, B.G. J. Am. Chem. Soc. 130, 5052–5053 (2008).

3. Carrico, I.S. Chem. Soc. Rev. 37, 1423–1431 (2008).4. Laughlin, S.T., Baskin, J.M., Amacher, S.L. & Bertozzi,

C.R. Science 320, 664–667 (2008).5. Binder, J.B., Blank, J.J. & Raines, R.T. Org. Lett. 9,

4885–4888 (2007).6. Jordan, J.P. & Grubbs, R.H. Angew. Chem. Int. Ed. 46,

5152–5155 (2007).7. Grubbs, R.H. Angew. Chem. Int. Ed. 45, 3760–3765

(2006).8. Hoveyda, A.H. et al. Org. Biomol. Chem. 2, 8–23

(2004).9. Saxon, E. & Bertozzi, C.R. Science 287, 2007–2010

(2000).10. Dieterich, D.C., Link, A.J., Graumann, J., Tirrell, D.A.

& Schuman, E.M. Proc. Natl. Acad. Sci. USA 103, 9482–9487 (2006).

Escherichia coli, evidently as an analog of methi-onine. Presumably, the suitable positioning of S-allylcysteine with this approach would permit direct modification of such proteins through cross-metathesis.

Until now, the goal of chemists has been to identify any rudimentary bioorthogonal meth-ods suitable for labeling proteins. With the increased ability to use different conjugating strategies, we can now be more discriminating in the choice of linkers available for protein conjugation. The protein chemist now has a choice, for example, between using a polar tri-azole ring obtained by azide-alkyne cycload-dition or a nonpolar alkene group available from cross-metathesis. These tools for protein tailoring permit chemical biologists to accept biopolymers as they are provided ‘off the rack’

previously, and use of these catalysts may allow metathesis on proteins under com-pletely aqueous conditions6.

If the metathesis catalyst can be fully opera-tional under physiological conditions, the cross-metathesis reaction may be extended to in vivo applications. One strategy for biopoly-mer modification in vivo uses the biosynthetic machinery to introduce new amino acids or sac-charides incorporating non-natural functional-ity, permitting their conjugations through the Staudinger ligation9 or azide-alkyne cycload-dition reactions4,10. The direct incorporation of allylsulfides into protein structures may facilitate the use of the metathesis reaction for in vivo applications. In this regard, it is notable that the authors report some incorporation of S-allylcysteine into proteins expressed in

20S ways to apoptosisDavid J McConkey

Chemical inhibitors of the proteasome have received substantial attention owing to the success of bortezomib in the treatment of multiple myeloma. A recent whole-cell screen identified the proteasome inhibitor argyrin A and suggests a new role for p27Kip-1 in regulating apoptosis.

The proteasome is a large protease that functions as one of the two major routes of bulk protein degradation in cells1. Although the concept was initially received with strong scientific skepti-cism, proteasome inhibition is now a validated therapeutic approach in cancer2. The peptide boronate proteasome inhibitor bortezomib (Velcade, formerly PS-341) has unmatched antitumor activity in people with multiple myeloma (MM)3. Interestingly, for reasons that are not entirely clear, preclinical studies have concluded that bortezomib and other protea-some inhibitors kill MM cells via distinct mech-anisms, and so combining the two drugs leads to synergistic cell killing, even in MM cells that are bortezomib-resistant4. A recent natural-product screen for compounds that promote p27 accu-mulation identified argyrin A as a new protea-some inhibitor that blocks tumor cell growth via a mechanism distinct from that of bortezomib5. The implication of p27 accumulation will cause many in the field to reconsider their ideas about how proteasome inhibition causes cell killing.

Two general hypotheses have been advanced to explain the cytotoxic effects of proteasome inhibitors. One posits that proteasome inhi-bition causes the accumulation of particular proteins that induce cell death, while the other suggests that the general accumulation of junk protein that occurs when the proteasome is blocked triggers apoptosis. For example, some previous studies have implicated stabilization of IκBα, the physiological inhibitor of the inflammation-associated transcription factor NFκB6, expression of Myc7, or accumulation of proapoptotic members of the BCL-2 family (Bim, Bik and Noxa)7,8 in bortezomib-induced apoptosis, whereas others concluded that the drug kills cells by stimulating the unfolded pro-tein response (UPR) and endoplasmic reticular (ER) stress.

The p27 protein blocks cell cycle progres-sion by inhibiting cyclin-dependent kinases9. Although p27 is rarely inactivated by mutation, its expression level is often reduced in cancers, and Nickeleit et al.5 have predicted that small molecules that restore p27 expression in can-cers would have antitumor activity. To test this hypothesis, they developed a high-throughput whole-cell assay to screen a focused natural-product library of myxobacterial metabo-lites at a relatively high level of stringency (70 nM). Their screen identified argyrin A, and

subsequent functional studies demonstrated that it promotes p27 accumulation by inhibiting the proteasome. Intraperitoneal administration of the compound produced strong proteasome inhibition in vivo and decreased tumor growth in preclinical models. Importantly, it was effi-cacious not only in tumors derived from cells that were killed by the drug in vitro but also in tumors derived from bortezomib-resistant cells. Mechanistic studies demonstrated that all of the effects of argyrin A are dependent on accumula-tion of p27 (Fig. 1). However, experiments with a mutant form of p27 revealed that its familiar function as a cyclin-dependent kinase inhibi-tor was not involved, which indicates that some new, unidentified mechanism is at play.

Several independent lines of evidence sup-port their conclusion that argyrin A is a more specific inhibitor of the proteasome than bortezomib. Like argyrin A, proteasome sub-unit knockdown induced apoptosis via a p27-dependent mechanism, whereas bortezomib did not. Furthermore, gene expression pro-filing studies demonstrated that the effects of argyrin A are more similar to proteasome subunit knockdown than bortezomib’s effects are. Finally, the authors ruled out stabilization of IkBα and Myc accumulation in argyrin A’s effects, and their gene expression profiling results did not support a role for ER stress in

David J. McConkey is in the Departments of Urology and Cancer Biology, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. e-mail: dmcconke@mdanderson.org

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that proteasome subunit knockdown in a Drosophila melanogaster eye model of neuro-degeneration results in a cytotoxic response that is mediated by protein aggregate accu-mulation10.

Argyrin A, bortezomib and other protea-some inhibitors will be particularly valuable as chemical probes to study the potential role of bulk protein buildup and its cellular conse-quences in more detail. For instance, because the proteasome is one of two major protein degradation systems in the cell (in conjunction with autophagy and lysosomal degradation), it is possible that proteasome inhibition might cause excessive protein buildup and/or rerout-ing of protein aggregates to lysosomes, and these effects could induce cellular stress responses. Delineating these mechanisms will be invaluable for our efforts to ‘tailor’ proteasome inhibitor-based therapies to particular tumors and will simultaneously augment our knowledge of an important biological process.

1. Goldberg, A.L. Biochem. Soc. Trans. 35, 12–17 (2007).

2. Adams, J. Cancer Cell 5, 417–421 (2004).3. Richardson, P.G. et al. N. Engl. J. Med. 352, 2487–

2498 (2005).4. Chauhan, D. et al. Cancer Cell 8, 407–419 (2005).5. Nickeleit, I. et al. Cancer Cell 14, 23–35 (2008).6. Keats, J.J. et al. Cancer Cell 12, 131–144 (2007).7. Nikiforov, M.A. et al. Proc. Natl. Acad. Sci. USA 104,

19488–19493 (2007).8. Tan, T.T. et al. Cancer Cell 7, 227–238 (2005).9. Sherr, C.J. & Roberts, J.M. Genes Dev. 9, 1149–1163

(1995).10. Pandey, U.B. et al. Nature 447, 859–863 (2007).

will have to be revisited as they may have been caused by off-target effects of the drug. It will be especially important to explore the roles of bulk protein accumulation, the UPR and ER stress, as elegant recent work has shown

the effects of either argyrin A or proteasome subunit knockdown. Therefore, previous con-clusions about the molecular mechanisms underlying the effects of proteasome inhibi-tion derived from studies with bortezomib

Figure 1 Mechanisms of tumor growth inhibition by argyrin A. The 26S proteasome is a barrel-like structure consisting of two 19S cap complexes and a 20S catalytic core. Substrates, including p27 and misfolded or damaged proteins, are ‘tagged’ with polymers of ubiquitin (Ub), which allows them to be recognized by the 19S cap complexes and degraded by the proteasome. Proteasome inhibition with argyrin A (but not bortezomib) results in stabilization and accumulation of p27 and other proteins, leading to angiogenesis inhibition and cell death.

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Chloroplast SRP takes another roadLaurent Nussaume

The signal recognition particle (SRP), a ribonucleoprotein complex that is conserved across all organisms, is essential for cotranslational insertion of proteins into membranes. A three-dimensional structure of cpSRP43 provides insights into how plants have adapted the SRP for post-translational targeting of membrane proteins.

Protein targeting mechanisms deliver pro-teins to specific locations inside or outside the cell. This process is essential for evo-lution as it creates a multitude of internal

compartments contributing to the acquisi-tion of new functions by cells. The labora-tory of Günther Blobel, who won the 1999 Nobel prize for his pioneering work on pro-tein sorting1, showed that the SRP complex is required for the recognition and transport of specific proteins to the endoplasmic reticu-lum in eukaryotes and to the plasma mem-brane in prokaryotes. Cytosolic SRPs bind to hydrophobic signal sequences located at the N terminus of newly synthesized polypep-tides as they emerge from the ribosome. In bacteria, they are minimally composed of a 54-kDa GTPase bound to an RNA moiety2. The SRP RNA, with its eight helical elements,

is one of the most conserved RNA structures known. As in prokaryotes, the chloroplast SRP (cpSRP) is involved in the targeting of integral membrane proteins. One would expect that chloroplasts, owing to their prokaryotic origin, would also contain an SRP RNA. Surprisingly, they do not. Instead of having a conserved RNA (ref. 3), the cpSRP harbors a plant-specific protein called cpSRP43 (refs. 4,5). A new crystal structure of cpSRP43 (ref. 6) reveals that this protein serves as an RNA mimic in SRP complexes and re-routes the SRP for post-translational targeting of membrane proteins involved in light-harvesting complexes.

Laurent Nussaume is in the Plant Biology Development Laboratory, Department of Plant Biology and Environmental Microbiology, Institute of Environmental Biology and Biotechnology, Direction des Sciences du Vivant, Commissariat à l’Energie Atomique, Centre National de la Recherche Scientifique, Université Aix-Marseille, Saint Paul lez Durance F-13108, France. e-mail: lnussaume@cea.fr

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