Báo cáo khoa học: Serpins 2005 – fun between theb-sheets Meeting report based upon presentations made at the 4th Interna-tional Symposium on Serpin Structure, Function and Biology (Cairns, Australia) doc

REVIEW ARTICLE Serpins 2005 – fun between the b-sheets Meeting report based upon presentations made at the 4th Interna- tional Symposium on Serpin Structure, Function and Biology (Cairns, Australia) James C. Whisstock 1,2,3 , Stephen P. Bottomley 1 , Phillip I. Bird 1 , Robert N. Pike 1 and Paul Coughlin 4 1 The Department of Biochemistry and Molecular Biology, 2 ARC Centre for Structural and Functional Microbial Genomics, and 3 Victorian Bioinformatics Consortium, Monash University, Clayton Campus, Melbourne, Victoria, Australia 4 Australian Centre for Blood Diseases, Monash University, Prahran, Victoria, Australia Introduction Serpins are the largest family of protease inhibitors identified to date and the only protease inhibitor fam- ily that can be found in all superkingdoms (Eukarya, Bacteria and Archaea) as well as certain viruses [1,2]. Most serpins function as inhibitors of chymotrypsin- like serine proteases, although several cross-class serpin inhibitors of papain-like cysteine proteases and cas- pases have been identified [3–5]. Inhibitory serpins function both extracellularly and intracellularly. Extracellular serpins play important roles in control- ling proteolytic cascades in plasma (for example the coagulation and the inflammatory response pathways) and intracellular serpins generally perform cytoprotec- tive roles and guard against inappropriate release of cytotoxic proteases (e.g., protease inhibitor-9 inhibits the pro-apoptotic protease granyzme B [6]). Numerous serpins have evolved functions distinct from protease inhibition; noninhibitory serpins include the human hormone delivery serpins cortisol binding globulin and thyroxine binding globulin, the tumour suppressor maspin, and the 47 kDa molecular chaperone heat shock protein (HSP) 47 [7]. One of the central tenets of inhibitory serpin function is the ability of the molecule to undergo a dramatic con- formational change, termed the ‘stressed’ to ‘relaxed’ (S to R) transition, that is also accompanied by a change Keywords conformational disease; protease; serpin; serpinopathies Correspondence J. Whisstock, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia E-mail: james.whisstock@med.monash.edu.au (Received 26 July 2005, revised 16 August 2005, accepted 18 August 2005) doi:10.1111/j.1742-4658.2005.04927.x Serpins are the largest family of protease inhibitors and are fundamental for the control of proteolysis in multicellular eukaryotes. Most eukaryote serpins inhibit serine or cysteine proteases, however, noninhibitory mem- bers have been identified that perform diverse functions in processes such as hormone delivery and tumour metastasis. More recently inhibitory ser- pins have been identified in prokaryotes and unicellular eukaryotes, never- theless, the precise molecular targets of these molecules remains to be identified. The serpin mechanism of protease inhibition is unusual and involves a major conformational rearrangement of the molecule concomit- ant with a distortion of the target protease. As a result of this requirement, serpins are susceptible to mutations that result in polymerization and con- formational diseases such as the human serpinopathies. This review reports on recent major discoveries in the serpin field, based upon presentations made at the 4th International Symposium on Serpin Structure, Function and Biology (Cairns, Australia). Abbreviations HCII, heparin cofactor II; HSP, heat shock protein; MENT, myeloid and erythroid nuclear termination stage specific protein; PAI-1, plasminogen activator inhibitor-1; PEDF, pigment epithelium-derived factor; R, relaxed; RCL, reactive centre loop; S, stressed. 4868 FEBS Journal 272 (2005) 4868–4873 ª 2005 FEBS in topology. During this rearrangement, the region responsible for interaction with the target protease, the reactive centre loop (RCL), moves from an exposed position to one in which it forms an extra strand in the centre of the A b-sheet (Fig. 1). The S to R transition is required for protease inhibition; the structure of the final serpin enzyme complex revealed that the serpin adopts the relaxed conformation and that the protease is distorted into a partially unfolded state which is cova- lently attached to the serpin via an acyl bond [8]. Any complex machine is vulnerable to breakdown and serpins are no exception. Serpins are particularly susceptible to destabilizing mutations that result in misfolding and the formation of pathogenic conform- ers. In particular, serpins are able to polymerize; dur- ing this process the RCL of one molecule ‘domain swaps’ and inserts into the A b-sheet of another to form a loop-sheet linkage [9–11]. Serpin polymeriza- tion can result in human disease (or serpinopathies) via two mechanisms. First, serpin polymers can no longer function as protease inhibitors and serpin defi- ciency results in a failure to properly control proteoly- sis. Secondly, the retention of the long chain polymers in the endoplasmic reticulum of cells that synthesize serpins can result in cell death and tissue destruction. The molecular processes underlying the serpinopathies share striking similarities with those of other conform- ational diseases, including prion, Huntington’s and Alzheimer’s diseases. Serpinopathies identified to date include cirrhosis and emphysema (antitrypsin defici- ency ⁄ polymerization), dementia (neuroserpin polymer- ization) and thromboembolic disease (antithrombin polymerization ⁄ deficiency) [12]. Thus, serpins repre- sent important targets for therapeutics and in addition represent an excellent model system for the broader study of conformational disease processes. Fig. 1. (A) Cartoon of the X-ray crystal structure of native Manduca sexta serpin-1K in complex with inactive rat trypsin (PDB identifier 1K90 [35]). The RCL is highlighted in magenta at the top of the molecule, the body of the serpin is in green and the protease is in cyan. (B) Car- toon of the final human a 1 –antitrypsin–enzyme complex (1EZX [8]) [colouring as for (A)]; the serpin has undergone the S to R transition, the RCL is buried in the central A b-sheet and the distorted protease (bovine trypsin) remains attached to the serpin RCL via an acyl bond. J. C. Whisstock et al. Serpins 2005. Fun between the b-sheets FEBS Journal 272 (2005) 4868–4873 ª 2005 FEBS 4869 Meeting Report The 4th International Symposium on Serpin Structure, Function and Biology was held in Cairns, Australia from 4–9 June 2005. Over 110 delegates from 13 coun- tries attended the conference, which comprised 40 oral presentations and 70 posters exploring a wide range of serpin biology. Here we summarize some of the high- lights of the meeting. On the face of it, serpins appear to represent an extraordinarily complex method of inhibiting proteas- es. Serine and cysteine protease inhibition can be achieved by relatively simple molecules that bind tightly to and block the protease active site (e.g., basic pancreatic trypsin inhibitor). The opening plenary presentation by Dan Lawrence (University of Michi- gan Medical School, USA) provided insight into the ‘‘why so complex?’’ question. Dan highlighted that ser- pins not only function as protease inhibitors, but also provide cells with molecular sensors of proteolysis as a result of the conformational rearrangement that the serpin undergoes upon complex formation with a tar- get protease. Furthermore, the ability of serpins to adopt a relatively inactive ‘partially inserted’ confor- mation (e.g., antithrombin) provides a mechanism for serpin activation in the presence of specific cofactors (e.g., heparin). Supporting this theme, Steven Olson (University of Illinois at Chicago, USA) presented work demonstrating the crucial role of the heparin binding site of cleaved antithrombin in antiangiogenic activity [13] and Peter Andreasen (Aarhus University, Denmark) explored the relationship between conform- ational change in plasminogen activator inhibtor-1 (PAI-1) and cancer. The serpin field has long been supported strongly by protein crystallography and this meeting proved no exception; in addition to published work, 10 unpub- lished serpin structures were presented affording major new insights into serpin function and providing a strong structural theme throughout the meeting. James Huntington (Cambridge Institute for Medical Research, UK) presented the structure of the antithrombin– thrombin–heparin ternary complex [14]. Together with the structures of antithrombin and heparin cofactor II (HCII), as well as other serpin complexes, these data start to reveal a complete molecular picture of serpin function and dysfunction in the coagulation cascade. In a related talk, Daniel Johnson (Cambridge Institute for Medical Research, UK) provided an elegant struc- tural explanation for dysfunction of a natural human mutation of antithrombin, the Truro variant [15,16]. Alexey Dementiev (University of Illinois at Chicago, USA) together with Peter Gettins (University of Illinois at Chicago, USA) presented the structure of a final serpin–enzyme complex, only the second such structure determined to date; their data revealed exquisite variation in the way serpins inhibit target proteases. Several new X-ray structures and biophysi- cal studies of thermophilic prokaryote serpins were presented by Ashley Buckle (Monash University, Australia) and Lisa Cabrita (Monash University, Aus- tralia) [17–19]. In addition to revealing a novel serpin conformation, these data provide detailed molecular insight into how serpins can survive in an extreme environment. There were many new insights into the structure and biochemistry of serpins with extra-inhibitory and cross-class inhibitory functions. Guy Salvesen (The Burnham Institute, USA) presented a study of cross- class inhibition of caspases by viral serpins and the control of cell death [20]. Continuing on the theme of cross-class inhibition, Sheena McGowan (Monash University, Australia) presented several X-ray crystal structures of the myeloid and erythroid nuclear ter- mination stage specific protein (MENT), with these data revealing a possible mechanism by which this unusual nuclear cysteine protease inhibitor can interact with DNA and chromatin [3,21]. Sergei Grigoryev (Pennsylvania State University College of Medicine, USA) presented a cellular view of MENT function, in particular exploring the link between cathepsin inhibitory activity and MENT positioning on chromatin. One of the major questions in the field of serpin bio- logy is the precise role and mechanism of function of three noninhibitory human serpins – maspin, pigment epithelium-derived factor (PEDF) and HSP47. James Irving (Monash University, Australia) and Peter Get- tins (University of Illinois at Chicago, USA) presented the X-ray crystal structure of the noninhibitory human tumor suppressor maspin [22,23]. Talks from Ming Zhang (Baylor College of Medicine, USA) and Sally Twining (Medical College of Wisconsin, USA) explored the role of maspin in development and in pre- venting tumour invasion using a battery of site-direc- ted mutants [24,25]. It is hoped that the use of model organisms together with structural insight will serve to drive our understanding of this important human tumour suppressor. Patricia Becerra (NIH-NEI, USA) presented an extensive study on PEDF, highlighting novel intracellular binding partners and relating these data back to the strong antiangiogenic function of this unusual molecule. Finally, Kaz Nagata (Kyoto Univer- sity Institute for Frontier Medical Sciences, Japan) and Tim Dafforn (Birmingham University, UK) both pre- sented talks on the essential serpin HSP47 and the way Serpins 2005. Fun between the b-sheets J. C. Whisstock et al. 4870 FEBS Journal 272 (2005) 4868–4873 ª 2005 FEBS in which this serpin promotes the folding of collagen and other molecules. Prior to the meeting only two partially inserted native serpins had been structurally characterized. Analysis of unpublished data reveal that rather than being a rare exception, numerous serpins are able to adopt the partially inserted serpin conformation and that serpin activation by cofactors may be far more common than previously thought. In particular, Anita Horvath (Monash University, Australia) presented the structure of murine antichymotrypsin, these data sug- gesting that the antichymotrypsin-like serpins are under conformational control. Another theme of the meeting was the use of model organisms to understand serpin function. Using HCII knockout mice, Doug Tollefsen (Washington Univer- sity Medical School, USA) presented data that sugges- ted that dermatan sulfate present in the blood vessel wall activates HCII and helps prevent neointimal hyperplasia after endothelial injury [26]. Using an array of thrombin variants, Frank Church (The Uni- versity of North Carolina at Chapel Hill, USA) provi- ded insight into sites on thrombin that were crucial for glycosaminoglycan binding and HCII inhibition. The role of serpins in complement and inflammation was highlighted by Al Davis (Centre for Blood Research Institute, Harvard University, USA). It was demon- strated that the highly glycosylated N-terminal domain of C1-inhibitor, whose function was previously enig- matic, provided a distinct anti-inflammatory function to this serpin via its ability to both bind bacterial lipo- polysaccharide and prevent neutrophil rolling prior to extravasation [27]. Gary Silverman (Magee-Womens Hospital, USA) presented knockout data for all Caenorhabditis elegans serpins and provided seminal insight into the role of serpins in worm development and homeostasis [28]. Mike Kanost (Kansas State University, USA) and Jean-Marc Reichhart (University Louis Pasteur, France) explored the role of insect serpins in the con- trol of immune protease cascades and in the control of Toll signalling, respectively. Jean-Marc demonstrated that the fly serpin-27A is absolutely required for dor- sal-ventral polarity, providing an interesting counter- part to the role of maspin in embryogenesis [29]. A large proportion of the meeting was devoted to understanding and controlling inappropriate conform- ational change in serpins. Stephen Bottomley (Monash University, Australia) presented a global overview of his groups’ work on serpin folding, unfolding and mis- folding [30]. Patrick Wintrode (Case Western Reserve University, USA) presented a hydrogen exchange mass spectrometry-based approach for understanding and monitoring serpin conformational change. The work presented at the conference revealed that much pro- gress is being made in combating serpin aggregation. David Lomas (Cambridge Institute for Medical Research, UK) and his colleagues presented their recent work on neuroserpin and the use of the Droso- phila to understand conformational disease processes [31]. Robin Carrell (Cambridge Institute for Medical Research, UK), Aiwu Zhou (Cambridge Institute for Medical Research, UK) and Mary Pearce (Monash University, Australia) focused on antitrypsin and the development of therapeutics that specifically prevent conformational change [32]. So where is the serpin field headed and what are the major questions we hope to see answered by Serpins 2008? One clear gap in our knowledge is the molecular mechanism of serpin–protease complex interaction with cell surface receptors – how do serpins alert cells to the presence of proteolytic activity? The structure of PAI-1 and vitronectin has been determined [33], and it is hoped that further advances in this field will lead to a detailed structural understanding of how serpins interact with receptors such as the low-density lipopro- tein related receptor. Much valuable information has already been gleaned from the study of serpins in model organisms such as the mouse, fly and worm, and more exciting discoveries are no doubt on the way. On the other hand, the function of plant serpins represents an obvious deficiency in our global under- standing of the serpin superfamily. Serpins from higher plants have been shown to be capable of inhibiting proteases, however, plants do not contain close puta- tive homologs of chymotrypsin-like serine proteases and their role remains relatively obscure. It has been suggested that plant serpins perform a role in defence against insect and pathogen attack [34]. Specific knock- outs in model organisms such as Arabidopsis thaliana may prove invaluable for understanding the role of this branch of the family. Indeed the study of plant serpins as well as serpins from prokaryotes may pro- vide insight into new functions in multicellular eukary- otes. Finally we hope that advances will be made in the development of small molecule therapeutics, which result in molecules that are effective in preventing serpin polymerization in vivo. We look forward to the next meeting in Europe in three years with the expectation that the field will continue to expand exponentially. Acknowledgements We thank Mike Pickford from ASN Events Pty Ltd (Melbourne, Australia) for conference organization and Jim Balmer from BMG Labtech for generous J. C. Whisstock et al. Serpins 2005. Fun between the b-sheets FEBS Journal 272 (2005) 4868–4873 ª 2005 FEBS 4871 support of the meeting. The authors thank the NHMRC, the ARC and the Victorian State Govern- ment for research support. References 1 Irving JA, Pike RN, Lesk AM & Whisstock JC (2000) Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. 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