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NEWS AND VIEWS et al. show3, amputation of the fin in adult zebrafish provides a suitable model for studying angiogenesis during tissue regeneration. Thus, the combination of (chemo-) genetics and rapid phenotyping assays promises to provide a powerful new armamentarium for future angiogenic gene and drug discovery (Fig. 1). However, critics point out that studying vascular development in a piscine or amphibian embryo is irrelevant for gaining insight in angiogenesis in human disease. In addition, for a new small-animal angiogenesis model to provide an attractive alternative to the assays available in mice (the preferred model of choice for preclinical cancer studies), vessel growth in this model should resemble angiogenesis in mice and humans. Thus, how reliable and predictive is the adult zebrafish model of regenerative angiogenesis developed by Bayliss and colleagues3? They observed that, in adult zebrafish, blood vessels in the regener-ating fin start to grow rapidly after fin amputation and initially form a vascular plexus of fragile channels lined with naked endothelial cells; these nascent vessels subsequently matured into a more stable vasculature, in part through coverage of endothelial cells by mural smooth muscle cells3. This sequence of events is markedly similar to what occurs in mice and humans1 and thus suggests a strong resemblance to angiogenesis in mammals. The findings of Bayliss et al. also raise a number of questions. For instance, tissue regeneration in zebrafish relies partly on an initial dedifferentiation step whereby the injured tissue dedifferentiates to a blastema, which subsequently proliferates and gives rise to the different cell lineages in the regenerating tissue10. Do endothelial cells in this adult zebrafish model of fin regeneration also differentiate from such a primitive blastema, or do they divide locally from neighboring vessels, as occurs
in mammals? Some previous work suggests that the latter mechanism may be operating. Myeloid cells are known to accumulate in the regenerating fin11—do these hematopoietic cells also contribute to vessel regeneration? Indeed, recent studies in mice show that angiocompetent hematopoietic (stem) cells are mobilized from the bone marrow, home to sites of active vessel growth, extravasate and stimulate angiogenesis by releasing angiogenic factors2. Do endothelial precursors exist in adult zebrafish and are hematopoietic progenitors mobilized from their kidney niche (the equivalent of the bone marrow niche in mammals)12 to stimulate vessel regeneration as well? The present regenerating fin angiogenesis model, in combination with available protocols for hematopoietic progenitor cell transplantation and the powerful genetic toolbox available in zebrafish, promises to be useful for addressing many of these questions. Bayliss et al. also found that VEGF inhibitors induce very similar effects in adult zebrafish and in mice and humans. Indeed, administration of a VEGF receptor inhibitor inhibited growth of vessels, especially of the fragile new vessels with naked endothelial cells, and allowed regeneration of only a small avascular region of the fin3—all reminiscent of what is seen in vertebrates. Moreover, much as has been observed in cancer patients treated with VEGF (receptor) inhibitors2, VEGF levels were compensatorily upregulated in adult zebrafish treated with a VEGF receptor inhibitor3. Recent studies in mice documented that VEGF (receptor) antagonists prune pre-existing fenestrated microvessels by as much as 70%; it will be interesting to see whether this is also the case in zebrafish. If so, the zebrafish model will also offer an opportunity to study the safety and toxicity profile of new anti-angiogenic compounds. When gene targeting tools in mice became available some 17 years ago, phenotyping assays
and models had to be developed and miniaturized for use in this small rodent. Skeptics argued that the mouse would never become a suitable model for studying certain human disorders, such as atherosclerosis—a premature critique that was rapidly silenced when the apoE-deficient mouse was generated. As of 2006, powerful genetic tools have become available for studying zebrafish and tadpoles; unfortunately, (adult) disease models are still largely lacking, permitting the most critical of us to question whether these primitive animal models will be ever useful for studying angiogenic human disorders. However, as Bayliss’ pioneering initiative3 shows, the future of zebrafish and tadpoles for accelerated anti-angiogenic drug discovery may soon be brighter than was ever expected. Tadpoles also offer exciting opportunities, as their tail regenerates after clipping, similar to the fin in the adult zebrafish, and they have been previously used for screens of chemical compounds for cardiovascular development. The prospect of combining disease models with chemogenetic approaches in zebrafish and tadpoles should only further raise our appetite for future fishing and frogging expeditions aimed at locating anti-angiogenic drugs. 1. Carmeliet, P. Nat. Med. 9, 653–660 (2003). 2. Carmeliet, P. Nature 438, 932–936 (2005). 3. Bayliss, P.E. et al. Nat. Chem. Biol 2, 265–273 (2006). 4. Ny, A. et al. Nat. Med. 11, 998–1004 (2005). 5. Ny, A., Autiero, M. & Carmeliet, P. Exp. Cell Res. 312, 684–693 (2006). 6. Chan, J., Bayliss, P.E., Wood, J.M. & Roberts, T.M. Cancer Cell 1, 257–267 (2002). 7. Thummel, R. et al. Dev. Dyn. 235, 336–346 (2006). 8. Berghmans, S. et al. Biotechniques 39, 227–237 (2005). 9. Lawson, N.D. & Weinstein, B.M. Dev. Biol. 248, 307– 318 (2002). 10. Poss, K.D. et al. Dev. Biol. 222, 347–358 (2000). 11. Lieschke, G.J., Oates, A.C., Crowhurst, M.O., Ward, A.C. & Layton, J.E. Blood 98, 3087–3096 (2001). 12. de Jong, J.L. & Zon, L.I. Annu. Rev. Genet. 39, 481–501 (2005).
Metalloproteases see the light Matthew Bogyo Small-molecule probes that chemically tag targets by virtue of their enzymatic activities offer a means to focus system-wide experiments and provide functional information for entire families of proteins. Recent advances in the design and application of light-activated probes that target metalloproteases have created the opportunity to study this medically important family of enzymes in unprecedented detail. Matthew Bogyo is in the Departments of Pathology and Microbiology and Immunology, Stanford University, 300 Pasteur Drive, Stanford, California 94305, USA. e-mail:
[email protected]
“In the middle of every difficulty lies opportunity.” This statement, made by Albert Einstein decades ago, seems to perfectly address the issues at the heart of the field of proteomics. Researchers in this area continue to face great
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challenges and difficulties in finding ways to monitor potentially hundreds of thousands of proteins that are present at levels that can span as many as six orders of magnitude in any given proteome1. Yet a system-wide understanding
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NEWS AND VIEWS of protein regulation and function provides vast opportunities, ranging from identification of new therapeutic targets and biomarkers for human disease to mapping of the fundamental pathways of cellular survival. New tools and methodologies that enable meaningful proteomic studies are the keys to helping this field realize these opportunities. Motivated by this, Cravatt and co-workers describe their global look at a key enzyme superfamily in this issue of Nature Chemical Biology2. Arguably one of the greatest advances in our ability to globally monitor protein regulation and function has been the development of methods to enrich samples for subsets of proteins or protein families, thereby reducing problems associated with sample complexity. One widely used method adopts smallmolecule tags that chemically modify protein targets, allowing them to be rapidly isolated for proteomic analysis. The ability to selectively enrich samples for proteins of interest is particularly relevant because important regulatory proteins such as transcription factors and enzymes are often expressed at exceedingly low levels1. Thus studies of families of enzymatic proteins such as proteases require the development of probes that can selectively fish out these targets from the sea of other proteins. A second important issue facing proteomic research is the need to devise ways to monitor not just the expression levels but also the functional regulation of proteins. Virtually all global proteomic methods only provide information about overall abundance, yet most proteins involved in critical biological processes are regulated through tightly controlled post-translational mechanisms. For example, proteases are synthesized as inactive zymogens that must be activated in a spatially and temporally controlled manner. Therefore, the ability to monitor the dynamics of the regulation of their activity is necessary for understanding their function in both normal and disease processes. Cravatt and co-workers describe the synthesis and application of a small library of lightactivated probes that specifically target the active forms of metalloproteases2. These reagents represent an important addition to an evergrowing toolbox of reagents known as activitybased probes (ABPs) that both enable enrichment of a specific target class of proteins and provide dynamic information on the regulation of their enzymatic activity. Although a number of ABPs that target proteases have found widespread applications in studies of protease function (for reviews see refs. 3–6), this technology has remained difficult to apply to metalloproteases owing to the fact that these enzymes do not form stable covalent bonds with a substrate
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Figure 1 Mechanisms of protease active site labeling by activity-based probes. (a) Probes that target cysteine proteases make use of the catalytic thiol residue to form a permanent covalent bond (dashed box) between the probe and target enzyme. (b) Metalloproteases do not use a catalytic amino acid side chain as the primary nucleophile. Therefore, probes bind the catalytic zinc ion but require a light-activated crosslinker to chemically modify (dashed box) the target enzyme.
(that is, acyl enzyme intermediates) during catalysis. Most ABPs make use of chemically reactive functional groups that are capable of specific modification of reactive nucleophiles within the enzyme active site (Fig. 1). In the case of serine and cysteine proteases, this nucleophile is a catalytic hydroxyl or thiol and the reaction results in stable chemical linkage to the enzyme. For metalloproteases the primary catalytic nucleophile is a bound water molecule. Therefore ABPs that target this family of enzymes must form stable linkages with potentially unreactive amino acid residues in or around the active site of the enzyme. Cravatt and co-workers have solved this problem by developing metalloprotease ABPs carrying a light-activated cross-linker that facilitate permanent covalent modification of target proteases upon exposure to UV light (Fig. 1). Because the probes bind only when a protease active site is properly formed and free of inhibitors, the resulting labeling provides an indirect indication of the levels of active proteases within a sample7. By making small libraries of primary peptide sequences, the authors show that it is possible to identify probe sets that effectively target a wide range of metalloprotease targets. So why target metalloproteases? Proteases make up nearly 2% of the human genome8 and represent 5–10% of all known potential drug targets9. In particular, there are nearly 200
distinct metalloproteases whose functions are linked to a number of clinically relevant conditions, most notably cancer. For this reason many large pharmaceutical companies initiated clinical trials with matrix metalloprotease inhibitors for treatment of late-stage cancer patients. Unfortunately, none of these trials produced promising results and most have now been abandoned9. It is becoming clear that the trials may have failed in part because of a lack of understanding of the complex functional roles of metalloproteases in disease progression. Thus ABPs such as the ones described in this issue are likely to provide valuable new information that could aid future attempts to target this family of proteases for therapeutic gain. 1. Ghaemmaghami, S. et al. Nature 425, 737–741 (2003). 2. Sieber, S. A., Niessen, S., Hoover, H. S. & Cravatt, B. F. Nat. Chem. Biol. 2, 274–281 (2006). 3. Berger, A.B., Vitorino, P.M. & Bogyo, M. Am. J. Pharmacogenomics 4, 71–81 (2004). 4. Jessani, N. & Cravatt, B.F. Curr. Opin. Chem. Biol. 8, 54–59 (2004). 5. Speers, A.E. & Cravatt, B.F. ChemBioChem 5, 41–47 (2004). 6. Jeffery, D.A. & Bogyo, M. Curr. Opin. Biotechnol. 14, 87–95 (2003). 7. Saghatelian, A., Jessani, N., Joseph, A., Humphrey, M. & Cravatt, B.F. Proc. Natl. Acad. Sci. USA 101, 10000– 10005 (2004). 8. López-Otín, C. & Overall, C.M. Nat. Rev. Mol. Cell Biol. 3, 509–519 (2002). 9. Overall, C.M. & Kleifeld, O. Nat. Rev. Cancer 6, 227– 239 (2006).
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