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Structure of geldanamycin in heat shock protein chaperone complex
cultured keratinocytes that have subsequently been transferred as autologous grafts to wounds on the flanks of large white pigs. Up to three weeks later, the entire wounds were excised and incubated with X-gal to detect the surviving grafted cells. The authors took thin sections through areas that stained blue macroscopically; of 184 sections examined, 15% showed blue cells in the deep layers of the epidermis and 85% showed blue cells in the superficial layers. In one of the specimens that had been grafted for three weeks, a column of cells stretching from the basal layer all the way to the surface was observed and the authors speculated that this represented the porcine equivalent of an epidermal proliferative unit, which had previously been defined in mice. In the second study, Lu and co-workers were interested in comparing the efficiency of gene delivery using topical application of viral vectors with delivery using the ‘gene gun’ approach (whereby cells are transduced by bombardment with gold particles onto which the gene construct of interest is bound). Initial experiments showed that topical application alone did not result in any significant gene transfer to skin keratinocytes. However, after chemical depilation and several rounds of tape-stripping (application and removal of sticky tape to strip off dead cells at the surface), the epidermis became more receptive to gene transfer. Indeed, using an adenovirus vector to deliver the /acZgene, the authors were able to transduce cells throughout the infected area, including some hair follicles. The inflammatory response to the virus was mild, involving mononuclear and polymorphonuclear cell infiltration. Staining could be detected for up to ten days after infection. The authors were less successful when usmg viruses based on a herpes simplex virus type 1 amplicon. The infected area appeared to be transduced but histological examination revealed that the infected cells were necrotic and there was a much greater inflammatory infiltrate. The adenoviral system proved to give a 1Cfold better gene delivery than the gene gun when the same /acZconstruct was used. The authors concluded that topical application of adenoviruses gave the highest efficiency of transducfion. Comparing these two studies, it is clear that for prolonged gene expression (for example, when keratinocytes are used to deliver secreted proteins systemically), it is probably better to infect dividing keratinocytes ex viva using retroviruses and then graft them back to the donor. However, for gene therapy of skin diseases, this option will only work if the elusive keratinocyte stem cell can be identified and infected, and if the gene to be delivered is small enough. Failing this, topical application of adenoviruses might
Copyright
01997
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Ltd. All rights rewved.
prove a viable alternative. particularly for shortterm therapy. Ian A. McKay MA, PhD Senior Lecturer in Molecular Oncology, Depts of Dermatology and Morbid Anatomy, St Bartholomew’s and The Royal London School of Medicine and Dentistry, 2 Newark Street, London, UK El 2AT.
Structure of geldanamycin in heat shock protein chaperone complex Crystal structure of an HspSO-geldanamycincomplex: targeting of a protein chaperone by an anti-tumour agent Stebbins, C.E. ef al. (1997) Cell 89,239-250 Geldanamycin belongs to a group of naturally occurring anti-turnour antibiotics and has potent activity in vitro. not only against a number of tumour cell lines but also in some mouse tumour models in viva For this reason, the US National Cancer Institute has selected it for pre-clinical development as an anti-tumour agent. Geldanamycin depletes cells of two main classes of signalling proteins whose deregulated activity has been implicated in human cancers; these are the proto-oncogenic protein kinases (such as ERBB2 and EGF receptor tyrosine kinases) and the nuclear hormone receptor family. This activity might underlie the anti-tumour effects of geldanamycin. Geldanamycin was thought to be a nonspecific kinase inhibitor but, intriguingly, recent studies have shown that it targets the heat shock protein Hsp90 and its endoplasmic reticulum homologue GP96. Hsp90, in concert with other factors, mediates ATP-dependent refolding of heatdenatured proteins in vitro and in viva. The nuclear hormone receptors are well-studied examples of proteins that require the Hsp90 system to acquire or maintain a conformational state that is capable of binding its ligand. When a hormone is bound to these receptors, the receptor is released as an active transcription factor. Geldanamycin blocks the generation of the competent hormone-binding complex, resulting in failure of activation and proteolytic degradation
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of the immaturely folded hormone receptor. Geldanamycin can also induce rapid proteolysis of protein kinases by interfering with the activity of Hsp90, and can inhibit HspSO-mediated refolding of thermally denatured substrates. The mechanism whereby geldanamycin mediates these effects has until now been unclear. In an elegant study, Stebbins and co-workers have provided new insight into the function of Hsp90 in maturation and refolding of its substrate, and additionally the mechanism of the inhibitory activity of geldanamycin. They have identified the geldanamycin-binding domain (GBD) of Hsp90 and determined the crystal structures of this domain and of its complex with geldanamycin at high resolution (1.65-2.2 A). The GBD was defined by limited proteolysis experiments followed by reverse-phase highperformance liquid chromatography (HPLC) to detect bound drug. Recombinant human HspSO-GBD was then overexpressed in Escherichia co/i and the recombinant domains purified by anion exchange and gel filtration chromatography. Crystals were grown using the method of hanging-drop vapour diffusion. Strikingly, the structure of HspSO-GBD reveals a pronounced pocket (15 A deep) that is highly conserved across species. Mutations that map to this region in the yeast and Drosophila homologues of Hsp90 also support a functional significance for the pocket; the structure suggests that most of these mutations would disrupt the structural integrity of the pocket. Remarkably, geldanamycin binds within this pocket, adopting a compact structure similar to that of a polypeptide chain in a ‘turn conformation’. The authors point out the similarities between the pocket and a typical substrate-binding site. They suggest that the endogenous substrate might be a segment of the protein whose conformational maturation/refolding is being mediated by the Hsp90 chaperone system, and that the pocket might participate directly in this reaction. Geldanamycin, by binding in the pocket, specifically inhibits the HspSO-catalysed conformational reaction. In this way, knowledge of the crystal structure of the geldanamycin-pocket complex has implications for understanding the function of protein chaperones, and also illustrates a novel targeting strategy for cancer therapeutics, providing a rational basis for the development of geldanamycin derivatives with enhanced antitumour activity. Alan J. Warren MRCP, MRCPath, PhD MRC Clinician Scientist, Box 234, Dept of Haematology, Addenbrooke’s NHS Trust, Hills Road, Cambridge, UK CB2 2QQ.
281
Copyright
01997
Elsevm
Science
Ltd. All rights rewved.
prove a viable alternative. particularly for shortterm therapy. Ian A. McKay MA, PhD Senior Lecturer in Molecular Oncology, Depts of Dermatology and Morbid Anatomy, St Bartholomew’s and The Royal London School of Medicine and Dentistry, 2 Newark Street, London, UK El 2AT.
Structure of geldanamycin in heat shock protein chaperone complex Crystal structure of an HspSO-geldanamycincomplex: targeting of a protein chaperone by an anti-tumour agent Stebbins, C.E. ef al. (1997) Cell 89,239-250 Geldanamycin belongs to a group of naturally occurring anti-turnour antibiotics and has potent activity in vitro. not only against a number of tumour cell lines but also in some mouse tumour models in viva For this reason, the US National Cancer Institute has selected it for pre-clinical development as an anti-tumour agent. Geldanamycin depletes cells of two main classes of signalling proteins whose deregulated activity has been implicated in human cancers; these are the proto-oncogenic protein kinases (such as ERBB2 and EGF receptor tyrosine kinases) and the nuclear hormone receptor family. This activity might underlie the anti-tumour effects of geldanamycin. Geldanamycin was thought to be a nonspecific kinase inhibitor but, intriguingly, recent studies have shown that it targets the heat shock protein Hsp90 and its endoplasmic reticulum homologue GP96. Hsp90, in concert with other factors, mediates ATP-dependent refolding of heatdenatured proteins in vitro and in viva. The nuclear hormone receptors are well-studied examples of proteins that require the Hsp90 system to acquire or maintain a conformational state that is capable of binding its ligand. When a hormone is bound to these receptors, the receptor is released as an active transcription factor. Geldanamycin blocks the generation of the competent hormone-binding complex, resulting in failure of activation and proteolytic degradation
13.57
4310/97/$17
00
of the immaturely folded hormone receptor. Geldanamycin can also induce rapid proteolysis of protein kinases by interfering with the activity of Hsp90, and can inhibit HspSO-mediated refolding of thermally denatured substrates. The mechanism whereby geldanamycin mediates these effects has until now been unclear. In an elegant study, Stebbins and co-workers have provided new insight into the function of Hsp90 in maturation and refolding of its substrate, and additionally the mechanism of the inhibitory activity of geldanamycin. They have identified the geldanamycin-binding domain (GBD) of Hsp90 and determined the crystal structures of this domain and of its complex with geldanamycin at high resolution (1.65-2.2 A). The GBD was defined by limited proteolysis experiments followed by reverse-phase highperformance liquid chromatography (HPLC) to detect bound drug. Recombinant human HspSO-GBD was then overexpressed in Escherichia co/i and the recombinant domains purified by anion exchange and gel filtration chromatography. Crystals were grown using the method of hanging-drop vapour diffusion. Strikingly, the structure of HspSO-GBD reveals a pronounced pocket (15 A deep) that is highly conserved across species. Mutations that map to this region in the yeast and Drosophila homologues of Hsp90 also support a functional significance for the pocket; the structure suggests that most of these mutations would disrupt the structural integrity of the pocket. Remarkably, geldanamycin binds within this pocket, adopting a compact structure similar to that of a polypeptide chain in a ‘turn conformation’. The authors point out the similarities between the pocket and a typical substrate-binding site. They suggest that the endogenous substrate might be a segment of the protein whose conformational maturation/refolding is being mediated by the Hsp90 chaperone system, and that the pocket might participate directly in this reaction. Geldanamycin, by binding in the pocket, specifically inhibits the HspSO-catalysed conformational reaction. In this way, knowledge of the crystal structure of the geldanamycin-pocket complex has implications for understanding the function of protein chaperones, and also illustrates a novel targeting strategy for cancer therapeutics, providing a rational basis for the development of geldanamycin derivatives with enhanced antitumour activity. Alan J. Warren MRCP, MRCPath, PhD MRC Clinician Scientist, Box 234, Dept of Haematology, Addenbrooke’s NHS Trust, Hills Road, Cambridge, UK CB2 2QQ.
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