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α1-Antitrypsin deficiency, liver disease and emphysema
The International Journal of Biochemistry & Cell Biology 35 (2003) 1009–1014
Medicine in focus
␣1-Antitrypsin deficiency, liver disease and emphysema Helen Parfrey∗ , Ravi Mahadeva, David A. Lomas Respiratory Medicine Unit, Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, UK Received 13 March 2002; received in revised form 13 July 2002; accepted 13 August 2002
Abstract
␣1 -Antitrypsin is a member of the serine proteinase inhibitor (serpin) superfamily and a potent inhibitor of neutrophil elastase. The most important deficiency variant of ␣1 -antitrypsin arises from the Z mutation (Glu342Lys). This mutation perturbs the protein’s tertiary structure to promote a precise, sequential intermolecular linkage that results in polymer formation. These polymers accumulate within the endoplasmic reticulum of the hepatocyte forming inclusion bodies that are associated with neonatal hepatitis, juvenile cirrhosis and adult hepatocellular carcinoma. The resultant secretory defect leads to plasma deficiency of ␣1 -antitrypsin. This exposes lung tissue to uncontrolled proteolytic attack from neutrophil elastase, culminating in alveolar destruction. Thus, the Z ␣1 -antitrypsin homozygote is predisposed to developing early onset basal, panacinar emphysema. In this review, we summarise the current understanding of the pathobiology of ␣1 -antitrypsin deficiency and the associated liver cirrhosis and emphysema. We show how this knowledge has led to the development of novel therapeutic approaches to treat this condition. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: ␣1 -Antitrypsin; Loop–sheet polymerisation; Emphysema; Liver cirrhosis
1. Introduction ␣1 -Antitrypsin is the most abundant circulating proteinase inhibitor and the archetypal member of the serine proteinase inhibitor (serpin) superfamily [1]. It is a highly polymorphic 52 kDa acute phase glycoprotein encoded by a single gene on the long arm of chromosome 14. Synthesis of protein occurs principally by hepatocytes and monocytes, reaching the lungs by passive diffusion. In addition, there is local production of ␣1 -antitrypsin within the lung by alveolar macrophages and epithelial cells. Here, its role is to protect the alveolar matrix from proteolytic attack ∗ Corresponding author. Tel.: +44-1223-762818; fax: +44-1223-336827. E-mail address: [email protected] (H. Parfrey).
by neutrophil elastase that has been liberated from activated neutrophils. Biochemical and crystallographic studies have revealed the mechanism by which ␣1 -antitrypsin functions as a proteinase inhibitor. The tertiary structure of ␣1 -antitrypsin comprises a dominant five stranded -sheet A, which supports a mobile reactive centre loop [1] (Fig. 1). The loop presents the key P1 –P1 methionine-serine residues as bait for the target proteinase, neutrophil elastase. Upon docking the proteinase cleaves at the P1 –P1 bond and is swung to the lower pole of the molecule as the reactive loop inserts between strands 3 and 5 of -sheet A [2]. This manoeuvre distorts and inactivates the proteinase making it more susceptible to degradation. The ␣1 -antitrypsin–proteinase complex is then cleared from the circulation by hepatocytes. The
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Fig. 1. The crystal structure of native ␣1 -antitrypsin (M) displays its key features of the mobile reactive loop (red) and -sheet A (green). Mutations that destabilise -sheet A lie in the shutter region (blue circle) and at the junction between the head of strand 5A and the base of the reactive loop (Z mutation, black arrow). These mutations open -sheet A and allow partial reactive-loop insertion giving rise to the polymerogenic intermediate M∗ . Polymers (P) form when the loop inserts into -sheet A of another ␣1 -antitrypsin molecule producing a dimer. This process repeats producing long chain polymers. The individual antitrypsin molecules in the polymer are shown in red, yellow and blue.
reactive loop–-sheet A interaction is essential for serpin inhibitory behaviour. Paradoxically, this is also its Achilles heel making the molecule vulnerable to aberrant loop–sheet linkage, whereby intermolecular insertion can occur to form polymers [2] (Fig. 1). Over 75 allelic variants of ␣1 -antitrypsin have been reported and classified according to their migratory distance on isoelectric focusing analysis [3]. Normal ␣1 -antitrypsin migrates in the middle (M) of the gel whilst the clinically important S and Z deficiency variants have a more anodal migration owing to changes in their overall charge. The S allele (Glu264Val) occurs in 28% of southern Europeans and although it reduces plasma levels of ␣1 -antitrypsin to 60% of normal, there are no associated pulmonary or hepatic sequelae [3]. The Z mutation arises from a single point mutation (Glu342Lys) that is found in 4% of the North European Caucasian population with 1:1700 being homozygotes. This results in a profound plasma deficiency of ␣1 -antitrypsin as the protein is retained as periodic acid schiff (PAS) positive, diastase resistant inclusions within the rough endoplasmic reticulum of the hepatocyte [4]. It is these insoluble inclusion bodies (Fig. 2a) that are associated with juvenile hepatitis, cirrhosis and hepatocellular carcinoma. As over 85%
of ␣1 -antitrypsin is retained within the liver, the diminished plasma levels are inadequate to protect the lung from indiscriminate proteolytic attack. Thus the Z homozygote has a propensity for early onset panacinar, basal emphysema [5]. This observation by Laurell and Eriksson, together with the almost simultaneous finding that intratracheal instillation of the plant proteinase papain produced emphysema in rats [6], gave rise to the proteinase: antiproteinase hypothesis of lung injury. This continues to dominate current thinking.
2. Pathogenesis 2.1. Liver disease The mutation in Z ␣1 -antitrypsin is located at the head of strand 5A and the base of the mobile reactive loop (Fig. 1). We hypothesised this would open -sheet A between strands 3 and 5 to favour the incorporation of the reactive loop from a second ␣1 -antitrypsin molecule to produce a dimer. This can then extend by the same process to form chains of loop–sheet polymers. The demonstration of spontaneous polymerisation of Z ␣1 -antitrypsin at 37 ◦ C,
H. Parfrey et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 1009–1014
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Fig. 2. Electron micrograph of a hepatic inclusion body (black arrow) obtained from the liver of a child with Z ␣1 -antitrypsin deficiency (reproduced from [7] with permission). The rough endoplasmic reticulum is distended and filled with an amorphous material that is PAS positive and diastase resistant (a). These hepatic inclusions are composed of long chains of ␣1 -antitrypsin polymers as displayed in this electron micrograph (b). Each “bead on the string” represents one ␣1 -antitrypsin molecule. (Reproduced with permission from Lomas et al. [16].)
whilst the normal M variant remained in its native conformation, supported this concept. Polymerisation of Z ␣1 -antitrypsin was accelerated by elevating both temperature and protein concentration and could be
competitively blocked by a 13 mer exogenous reactive loop peptide that competed with the intact reactive loop for annealing to -sheet A [7]. In vitro polymers of Z ␣1 -antitrypsin form “beads on a string” when
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examined by electron microscopy. The identical appearance of polymers from a cirrhotic liver of a Z ␣1 -antitrypsin homozygote established polymerisation as the mechanism responsible for ␣1 -antitrypsin deficiency [2,7] (Fig. 2b). Within the hepatocyte, the Z ␣1 -antitrypsin is degraded by both proteosome dependent and independent pathways, and there is evidence of inefficient clearance in those susceptible to liver disease [8,9]. Nevertheless, the process whereby the accumulation of the Z ␣1 -antitrypsin polymers within the hepatocyte leads to cell death and cirrhosis remains to be elucidated. Biophysical studies have demonstrated that the initial step in polymerisation is the conversion of native ␣1 -antitrypsin into a polymerogenic intermediate (M∗ ), predicted to involve protein unfolding and the partial insertion of the reactive loop into -sheet A (Fig. 1). The second, slower phase is due to loop–sheet interaction that results in polymer formation [3]. Our recent data for Z ␣1 -antitrypsin suggests that this protein is present in the M∗ (partial loop insertion) conformation [10], which facilitates polymer formation (Fig. 1). Loop–sheet linkage has been shown to be responsible for the severe plasma deficiency and hepatic inclusions associated with other ␣1 -antitrypsin mutants— Siiyama (Ser53Phe) and Mmalton (Phe52). These mutations all lie within the shutter domain of the molecule that controls the conformation and movement of -sheet A [2] (Fig. 1). The extent to which mutations destabilise this region regulates the rate of polymerisation. This is best exemplified by other shutter domain mutations, the S (Glu264Val) and I (Arg39Cys) variants of ␣1 -antitrypsin, which polymerise slowly resulting in a milder plasma deficiency and no clinical phenotype. Furthermore, if either of these mutants is inherited with the rapidly polymerising Z variant then the two interact to produce heteropolymers that are associated with more severe hepatic disease [11]. This intermolecular polymerisation is not unique to ␣1 -antitrypsin as it can occur in variants of other members of the serpin family, such as antithrombin, C1 inhibitor and ␣1 -antichymotrypsin to cause plasma deficiency and thrombosis, angio-oedema and emphysema, respectively [3]. It also accounts for a novel inclusion body dementia, familial encephalopathy with neuroserpin inclusion bodies or FENIB [2]. These conditions have
been termed conformational diseases as they arise from a structural rearrangement within a protein that transforms it into a pathological species [2].
3. Lung disease Smoking is the most important risk factor for the development of emphysema in Z ␣1 -antitrypsin homozygotes [3]. The premature lung disease in these individuals is primarily attributed to the lack of circulating Z ␣1 -antitrypsin resulting in uncontrolled proteolytic attack from host proteinases. To confound this problem, the Z ␣1 -antitrypsin that reaches the lungs is approximately five-fold less effective at inhibiting neutrophil elastase compared to the normal M variant. Recently, we have identified polymers in bronchial lavage fluid from Z ␣1 -antitrypsin homozygotes [3]. Polymer formation inactivates ␣1 -antitrypsin and thereby further decreases the already limited antiproteinase defence within the lung, increasing the risk of tissue destruction. Moreover, the conformational transition from monomer to polymer converts ␣1 -antitrypsin into a chemoattractant for neutrophils [12]. This novel function may partly explain the excess of neutrophils in the lungs of Z homozygotes [13], which will undoubtedly increase the proteolytic burden on the already strained defence mechanisms. Studies are ongoing to determine the effect of smoking and infection on Z ␣1 -antitrypsin polymer formation and the role of these polymers in disease progression.
4. Treatment Understanding the structural basis of ␣1 -antitrypsin deficiency opens the way for new strategies to block polymerisation that may potentially attenuate the associated liver and lung disease. Exogenous reactive loop peptides can anneal as strand 4 of -sheet A to inhibit polymerisation [7]. These peptides were 11-13 amino acids in length, could bind to more than one serpin and thus were unsuitable as therapeutic agents. We have recently developed a 6-mer peptide, comprising residues P7 –P2 of the reactive loop, which specifically binds to Z ␣1 -antitrypsin and in doing so inhibits polymerisation [10] (Fig. 3). The current challenge is to deliver this small peptide or peptide mimetic to
H. Parfrey et al. / The International Journal of Biochemistry & Cell Biology 35 (2003) 1009–1014
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Fig. 3. In Z ␣1 -antitrypsin the reactive loop (red) is partially inserted into -sheet A (green), which opens the sheet between strands 3 and 5. The 6-mer peptide (yellow) comprises P7 –P2 of the reactive loop and inserts into the open -sheet A as shown. This stabilises -sheet A and blocks polymer formation. In the absence of this peptide, the reactive loop of one Z ␣1 -antitrypsin molecule inserts into the open -sheet A of another molecule forming a dimer.
the endoplasmic reticulum of the hepatocyte where it can exert its inhibitory effect on polymer formation and alleviate the associated liver disease. This novel peptide offers a potential therapy for Z ␣1 -antitrypsin hepatic disease for which liver transplantation is the only current curative procedure. However, it converts ␣1 -antitrypsin into an inactive conformation and so additional measures will be required to treat the associated lung disease. Other techniques to prevent polymer formation target protein folding. The naturally occurring osmolyte, trimethylamine-N-oxide, reduces polymerisation of Z ␣1 -antitrypsin in vitro and the chemical chaperone, 4-phenylbutyric acid may have a similar effect in vivo [14,15]. The ability of such chemical chaperones to enhance the secretion of Z ␣1 -antitrypsin is being assessed in clinical trials.
An alternative strategy is to correct the plasma deficiency of ␣1 -antitrypsin with intravenous replacement therapy. Whilst this will have no effect on the liver disease, it may slow the progression of the emphysema. Although this is attractive in theory, it remains to be proven in large clinical trials. At present, the best option remains avoidance of deleterious factors, such as cigarette smoking, along with conventional medical treatments for emphysema including lung transplantation for those individuals with end-stage disease.
5. Conclusion Z ␣1 -antitrypsin deficiency results from a unique, sequential interaction between the reactive loop of
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one molecule and -sheet A of another, culminating in polymer formation. These polymers accumulate within the hepatocyte and are associated with liver disease. Polymers also form spontaneously in lung tissue, thereby inactivating the ␣1 -antitrypsin. This not only diminishes the already compromised antiproteinase shield but may also lead to recruitment of neutrophils to propagate tissue inflammation and injury, the end result being emphysema. Biochemical and crystallographic structures have played a significant role in understanding the biological function of ␣1 -antitrypsin and determining the mechanism whereby pathological variants cause disease. This knowledge has been central to the development of potential therapies to impede polymerisation and thus prevent the liver and lung disease. Acknowledgements This work is supported by the Medical Research Council (UK) and the Wellcome Trust (UK). HP is an MRC Clinical Training Fellow and RM is a Wellcome Trust Advanced Clinical Fellow. References [1] G.A. Silverman, P.I. Bird, R.W. Carrell, P.B. Coughlin, P.G. Gettins, J.I. Irving, D.A. Lomas, C.J. Luke, R.W. Moyer, P.A. Pemberton, E. Remold-O’Donnell, G.S. Salvesen, J. Travis, J.C. Whisstock, The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature, J. Biol. Chem. 276 (2001) 33293– 33296. [2] R.W. Carrell, D.A. Lomas, Alpha-1 antitrypsin deficiency— a model for conformational diseases, N. Engl. J. Med. 346 (2002) 45–53. [3] D.A. Lomas, R. Mahadeva, Alpha-1 antitrypsin polymerisation and the serpinopathies: pathobiology and prospects for therapy, J. Clin. Invest., in press. [4] H.L. Sharp, Alpha1 -antitrypsin deficiency, Hosp. Pract. 6 (1971) 83–96. [5] C.B. Laurell, S. Eriksson, The electrophoretic ␣1 -globulin pattern of serum in ␣1 -antitrypsin deficiency, Scand. J. Clin. Lab. Invest. 15 (1963) 132–140.
[6] P. Gross, E.A. Pfitzer, E. Tolker, M.A. Babyak, M. Kaschak, Experimental emphysema. Its production with papain in normal and silicotic rats, Arch. Environ. Health 11 (1965) 50–58. [7] D.A. Lomas, D.L. Evans, J.T. Finch, R.W. Carrell, The mechanism of Z alpha-1 antitrypsin accumulation in the liver, Nature 357 (1992) 605–607. [8] J.H. Teckman, J. Burrows, T. Hidvegi, B. Schmidt, P.D. Hale, D.H. Perlmutter, The proteasome participates in degradation of mutant ␣1 -antitrypsin Z in the endoplasmic reticulum of hepatoma-derived hepatocytes, J. Biol. Chem. 276 (48) (2001) 44865–44872. [9] C.M. Cabral, P. Choudhury, Y. Liu, R.N. Sifers, Processing by endoplasmic reticulum mannosidases partitions a secretion-impaired glycoprotein into distinct disposal pathways, J. Biol. Chem. 275 (2000) 25015–25022. [10] R. Mahadeva, T.R. Dafforn, R.W. Carrell, D.A. Lomas, Six-mer peptide selectively anneals to a pathogenic serpin conformation and blocks polymerisation: implications for the prevention of Z ␣1 -antitrypsin related cirrhosis, J. Biol. Chem. 277 (9) (2002) 6771–6774. [11] R. Mahadeva, W.S.W. Chang, T.R. Dafforn, D.J. Oakley, R.C. Foreman, J. Calvin, D. Wight, D.A. Lomas, Heteropolymerisation of S, I and Z ␣1 -antitrypsin and liver cirrhosis, J. Clin. Invest. 103 (1999) 999–1006. [12] J.S. Parmar, R. Mahadeva, B.J. Reed, N. Farahi, K.A. Cadwallader, M.T. Keogan, E.R. Chilvers, D.A. Lomas, Polymers of ␣1 -antitrypsin are chemotactic for human neutrophils. A new paradigm for the pathogenesis of emphysema, Am. J. Respir. Cell. Mol. Biol. 26 (2002) 723– 730. [13] H.M. Morrison, J.A. Kramps, D. Burnett, R.A. Stockley, Lung lavage fluid from patients with ␣1 -proteinase inhibitor deficiency or chronic obstructive bronchitis: anti-elastase function and cell profile, Clin. Sci. 72 (1987) 373–381. [14] J.A.J. Burrows, L.K. Willis, D.H. Perlmutter, Chemical chaperones mediate increased secretion of mutant ␣1 -antitrypsin (␣1 -AT) Z: a potential pharmacological strategy for prevention of liver injury and emphysema in ␣1 -AT deficiency, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 1796– 1801. [15] G.L. Devlin, H. Parfrey, D.J. Tew, D.A. Lomas, S.P. Bottomley, Prevention of polymerisation of M and Z ␣1 -antitrypsin (␣1 -AT) with trimethylamine-N-oxide. Implications for the treatment of ␣1 -AT deficiency, Am. J. Respir. Cell. Mol. Biol. 24 (2001) 727–732. [16] D.A. Lomas, J.T. Firich, K. Seyama, T. Nukiwa, R.W. Carrell, Alpha-1 antitrypsin Siiyama (Ser53 Phe); further evidence for intracellular loop–sheet polymerisation, J. Biol. Chem. 268 (1993) 15333–15335.
Medicine in focus
␣1-Antitrypsin deficiency, liver disease and emphysema Helen Parfrey∗ , Ravi Mahadeva, David A. Lomas Respiratory Medicine Unit, Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, UK Received 13 March 2002; received in revised form 13 July 2002; accepted 13 August 2002
Abstract
␣1 -Antitrypsin is a member of the serine proteinase inhibitor (serpin) superfamily and a potent inhibitor of neutrophil elastase. The most important deficiency variant of ␣1 -antitrypsin arises from the Z mutation (Glu342Lys). This mutation perturbs the protein’s tertiary structure to promote a precise, sequential intermolecular linkage that results in polymer formation. These polymers accumulate within the endoplasmic reticulum of the hepatocyte forming inclusion bodies that are associated with neonatal hepatitis, juvenile cirrhosis and adult hepatocellular carcinoma. The resultant secretory defect leads to plasma deficiency of ␣1 -antitrypsin. This exposes lung tissue to uncontrolled proteolytic attack from neutrophil elastase, culminating in alveolar destruction. Thus, the Z ␣1 -antitrypsin homozygote is predisposed to developing early onset basal, panacinar emphysema. In this review, we summarise the current understanding of the pathobiology of ␣1 -antitrypsin deficiency and the associated liver cirrhosis and emphysema. We show how this knowledge has led to the development of novel therapeutic approaches to treat this condition. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: ␣1 -Antitrypsin; Loop–sheet polymerisation; Emphysema; Liver cirrhosis
1. Introduction ␣1 -Antitrypsin is the most abundant circulating proteinase inhibitor and the archetypal member of the serine proteinase inhibitor (serpin) superfamily [1]. It is a highly polymorphic 52 kDa acute phase glycoprotein encoded by a single gene on the long arm of chromosome 14. Synthesis of protein occurs principally by hepatocytes and monocytes, reaching the lungs by passive diffusion. In addition, there is local production of ␣1 -antitrypsin within the lung by alveolar macrophages and epithelial cells. Here, its role is to protect the alveolar matrix from proteolytic attack ∗ Corresponding author. Tel.: +44-1223-762818; fax: +44-1223-336827. E-mail address: [email protected] (H. Parfrey).
by neutrophil elastase that has been liberated from activated neutrophils. Biochemical and crystallographic studies have revealed the mechanism by which ␣1 -antitrypsin functions as a proteinase inhibitor. The tertiary structure of ␣1 -antitrypsin comprises a dominant five stranded -sheet A, which supports a mobile reactive centre loop [1] (Fig. 1). The loop presents the key P1 –P1 methionine-serine residues as bait for the target proteinase, neutrophil elastase. Upon docking the proteinase cleaves at the P1 –P1 bond and is swung to the lower pole of the molecule as the reactive loop inserts between strands 3 and 5 of -sheet A [2]. This manoeuvre distorts and inactivates the proteinase making it more susceptible to degradation. The ␣1 -antitrypsin–proteinase complex is then cleared from the circulation by hepatocytes. The
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Fig. 1. The crystal structure of native ␣1 -antitrypsin (M) displays its key features of the mobile reactive loop (red) and -sheet A (green). Mutations that destabilise -sheet A lie in the shutter region (blue circle) and at the junction between the head of strand 5A and the base of the reactive loop (Z mutation, black arrow). These mutations open -sheet A and allow partial reactive-loop insertion giving rise to the polymerogenic intermediate M∗ . Polymers (P) form when the loop inserts into -sheet A of another ␣1 -antitrypsin molecule producing a dimer. This process repeats producing long chain polymers. The individual antitrypsin molecules in the polymer are shown in red, yellow and blue.
reactive loop–-sheet A interaction is essential for serpin inhibitory behaviour. Paradoxically, this is also its Achilles heel making the molecule vulnerable to aberrant loop–sheet linkage, whereby intermolecular insertion can occur to form polymers [2] (Fig. 1). Over 75 allelic variants of ␣1 -antitrypsin have been reported and classified according to their migratory distance on isoelectric focusing analysis [3]. Normal ␣1 -antitrypsin migrates in the middle (M) of the gel whilst the clinically important S and Z deficiency variants have a more anodal migration owing to changes in their overall charge. The S allele (Glu264Val) occurs in 28% of southern Europeans and although it reduces plasma levels of ␣1 -antitrypsin to 60% of normal, there are no associated pulmonary or hepatic sequelae [3]. The Z mutation arises from a single point mutation (Glu342Lys) that is found in 4% of the North European Caucasian population with 1:1700 being homozygotes. This results in a profound plasma deficiency of ␣1 -antitrypsin as the protein is retained as periodic acid schiff (PAS) positive, diastase resistant inclusions within the rough endoplasmic reticulum of the hepatocyte [4]. It is these insoluble inclusion bodies (Fig. 2a) that are associated with juvenile hepatitis, cirrhosis and hepatocellular carcinoma. As over 85%
of ␣1 -antitrypsin is retained within the liver, the diminished plasma levels are inadequate to protect the lung from indiscriminate proteolytic attack. Thus the Z homozygote has a propensity for early onset panacinar, basal emphysema [5]. This observation by Laurell and Eriksson, together with the almost simultaneous finding that intratracheal instillation of the plant proteinase papain produced emphysema in rats [6], gave rise to the proteinase: antiproteinase hypothesis of lung injury. This continues to dominate current thinking.
2. Pathogenesis 2.1. Liver disease The mutation in Z ␣1 -antitrypsin is located at the head of strand 5A and the base of the mobile reactive loop (Fig. 1). We hypothesised this would open -sheet A between strands 3 and 5 to favour the incorporation of the reactive loop from a second ␣1 -antitrypsin molecule to produce a dimer. This can then extend by the same process to form chains of loop–sheet polymers. The demonstration of spontaneous polymerisation of Z ␣1 -antitrypsin at 37 ◦ C,
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Fig. 2. Electron micrograph of a hepatic inclusion body (black arrow) obtained from the liver of a child with Z ␣1 -antitrypsin deficiency (reproduced from [7] with permission). The rough endoplasmic reticulum is distended and filled with an amorphous material that is PAS positive and diastase resistant (a). These hepatic inclusions are composed of long chains of ␣1 -antitrypsin polymers as displayed in this electron micrograph (b). Each “bead on the string” represents one ␣1 -antitrypsin molecule. (Reproduced with permission from Lomas et al. [16].)
whilst the normal M variant remained in its native conformation, supported this concept. Polymerisation of Z ␣1 -antitrypsin was accelerated by elevating both temperature and protein concentration and could be
competitively blocked by a 13 mer exogenous reactive loop peptide that competed with the intact reactive loop for annealing to -sheet A [7]. In vitro polymers of Z ␣1 -antitrypsin form “beads on a string” when
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examined by electron microscopy. The identical appearance of polymers from a cirrhotic liver of a Z ␣1 -antitrypsin homozygote established polymerisation as the mechanism responsible for ␣1 -antitrypsin deficiency [2,7] (Fig. 2b). Within the hepatocyte, the Z ␣1 -antitrypsin is degraded by both proteosome dependent and independent pathways, and there is evidence of inefficient clearance in those susceptible to liver disease [8,9]. Nevertheless, the process whereby the accumulation of the Z ␣1 -antitrypsin polymers within the hepatocyte leads to cell death and cirrhosis remains to be elucidated. Biophysical studies have demonstrated that the initial step in polymerisation is the conversion of native ␣1 -antitrypsin into a polymerogenic intermediate (M∗ ), predicted to involve protein unfolding and the partial insertion of the reactive loop into -sheet A (Fig. 1). The second, slower phase is due to loop–sheet interaction that results in polymer formation [3]. Our recent data for Z ␣1 -antitrypsin suggests that this protein is present in the M∗ (partial loop insertion) conformation [10], which facilitates polymer formation (Fig. 1). Loop–sheet linkage has been shown to be responsible for the severe plasma deficiency and hepatic inclusions associated with other ␣1 -antitrypsin mutants— Siiyama (Ser53Phe) and Mmalton (Phe52). These mutations all lie within the shutter domain of the molecule that controls the conformation and movement of -sheet A [2] (Fig. 1). The extent to which mutations destabilise this region regulates the rate of polymerisation. This is best exemplified by other shutter domain mutations, the S (Glu264Val) and I (Arg39Cys) variants of ␣1 -antitrypsin, which polymerise slowly resulting in a milder plasma deficiency and no clinical phenotype. Furthermore, if either of these mutants is inherited with the rapidly polymerising Z variant then the two interact to produce heteropolymers that are associated with more severe hepatic disease [11]. This intermolecular polymerisation is not unique to ␣1 -antitrypsin as it can occur in variants of other members of the serpin family, such as antithrombin, C1 inhibitor and ␣1 -antichymotrypsin to cause plasma deficiency and thrombosis, angio-oedema and emphysema, respectively [3]. It also accounts for a novel inclusion body dementia, familial encephalopathy with neuroserpin inclusion bodies or FENIB [2]. These conditions have
been termed conformational diseases as they arise from a structural rearrangement within a protein that transforms it into a pathological species [2].
3. Lung disease Smoking is the most important risk factor for the development of emphysema in Z ␣1 -antitrypsin homozygotes [3]. The premature lung disease in these individuals is primarily attributed to the lack of circulating Z ␣1 -antitrypsin resulting in uncontrolled proteolytic attack from host proteinases. To confound this problem, the Z ␣1 -antitrypsin that reaches the lungs is approximately five-fold less effective at inhibiting neutrophil elastase compared to the normal M variant. Recently, we have identified polymers in bronchial lavage fluid from Z ␣1 -antitrypsin homozygotes [3]. Polymer formation inactivates ␣1 -antitrypsin and thereby further decreases the already limited antiproteinase defence within the lung, increasing the risk of tissue destruction. Moreover, the conformational transition from monomer to polymer converts ␣1 -antitrypsin into a chemoattractant for neutrophils [12]. This novel function may partly explain the excess of neutrophils in the lungs of Z homozygotes [13], which will undoubtedly increase the proteolytic burden on the already strained defence mechanisms. Studies are ongoing to determine the effect of smoking and infection on Z ␣1 -antitrypsin polymer formation and the role of these polymers in disease progression.
4. Treatment Understanding the structural basis of ␣1 -antitrypsin deficiency opens the way for new strategies to block polymerisation that may potentially attenuate the associated liver and lung disease. Exogenous reactive loop peptides can anneal as strand 4 of -sheet A to inhibit polymerisation [7]. These peptides were 11-13 amino acids in length, could bind to more than one serpin and thus were unsuitable as therapeutic agents. We have recently developed a 6-mer peptide, comprising residues P7 –P2 of the reactive loop, which specifically binds to Z ␣1 -antitrypsin and in doing so inhibits polymerisation [10] (Fig. 3). The current challenge is to deliver this small peptide or peptide mimetic to
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Fig. 3. In Z ␣1 -antitrypsin the reactive loop (red) is partially inserted into -sheet A (green), which opens the sheet between strands 3 and 5. The 6-mer peptide (yellow) comprises P7 –P2 of the reactive loop and inserts into the open -sheet A as shown. This stabilises -sheet A and blocks polymer formation. In the absence of this peptide, the reactive loop of one Z ␣1 -antitrypsin molecule inserts into the open -sheet A of another molecule forming a dimer.
the endoplasmic reticulum of the hepatocyte where it can exert its inhibitory effect on polymer formation and alleviate the associated liver disease. This novel peptide offers a potential therapy for Z ␣1 -antitrypsin hepatic disease for which liver transplantation is the only current curative procedure. However, it converts ␣1 -antitrypsin into an inactive conformation and so additional measures will be required to treat the associated lung disease. Other techniques to prevent polymer formation target protein folding. The naturally occurring osmolyte, trimethylamine-N-oxide, reduces polymerisation of Z ␣1 -antitrypsin in vitro and the chemical chaperone, 4-phenylbutyric acid may have a similar effect in vivo [14,15]. The ability of such chemical chaperones to enhance the secretion of Z ␣1 -antitrypsin is being assessed in clinical trials.
An alternative strategy is to correct the plasma deficiency of ␣1 -antitrypsin with intravenous replacement therapy. Whilst this will have no effect on the liver disease, it may slow the progression of the emphysema. Although this is attractive in theory, it remains to be proven in large clinical trials. At present, the best option remains avoidance of deleterious factors, such as cigarette smoking, along with conventional medical treatments for emphysema including lung transplantation for those individuals with end-stage disease.
5. Conclusion Z ␣1 -antitrypsin deficiency results from a unique, sequential interaction between the reactive loop of
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one molecule and -sheet A of another, culminating in polymer formation. These polymers accumulate within the hepatocyte and are associated with liver disease. Polymers also form spontaneously in lung tissue, thereby inactivating the ␣1 -antitrypsin. This not only diminishes the already compromised antiproteinase shield but may also lead to recruitment of neutrophils to propagate tissue inflammation and injury, the end result being emphysema. Biochemical and crystallographic structures have played a significant role in understanding the biological function of ␣1 -antitrypsin and determining the mechanism whereby pathological variants cause disease. This knowledge has been central to the development of potential therapies to impede polymerisation and thus prevent the liver and lung disease. Acknowledgements This work is supported by the Medical Research Council (UK) and the Wellcome Trust (UK). HP is an MRC Clinical Training Fellow and RM is a Wellcome Trust Advanced Clinical Fellow. References [1] G.A. Silverman, P.I. Bird, R.W. Carrell, P.B. Coughlin, P.G. Gettins, J.I. Irving, D.A. Lomas, C.J. Luke, R.W. Moyer, P.A. Pemberton, E. Remold-O’Donnell, G.S. Salvesen, J. Travis, J.C. Whisstock, The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature, J. Biol. Chem. 276 (2001) 33293– 33296. [2] R.W. Carrell, D.A. Lomas, Alpha-1 antitrypsin deficiency— a model for conformational diseases, N. Engl. J. Med. 346 (2002) 45–53. [3] D.A. Lomas, R. Mahadeva, Alpha-1 antitrypsin polymerisation and the serpinopathies: pathobiology and prospects for therapy, J. Clin. Invest., in press. [4] H.L. Sharp, Alpha1 -antitrypsin deficiency, Hosp. Pract. 6 (1971) 83–96. [5] C.B. Laurell, S. Eriksson, The electrophoretic ␣1 -globulin pattern of serum in ␣1 -antitrypsin deficiency, Scand. J. Clin. Lab. Invest. 15 (1963) 132–140.
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