Serpins 2005 – fun between the b-sheetsMeeting report based upon presentations made at the 4th Interna-tional Symposium on Serpin Structure, Function and Biology Cairns, Australia James
Trang 1Serpins 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 Whisstock1,2,3, Stephen P Bottomley1, Phillip I Bird1, Robert N Pike1and Paul Coughlin4
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.
Trang 2in 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.
Trang 3Meeting 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 nastruc-tural 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
Trang 4in 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 concon-trol 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
Trang 5support of the meeting The authors thank the
NHMRC, the ARC and the Victorian State
Govern-ment for research support
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