α<sub>1</sub>-Antitrypsin Polymerization:  A Fluorescence Correlation Spectroscopic Study

Purkayastha, Pradipta; Klemke, Jason W.; Lavender, Stacey; Oyola, Rolando; Cooperman, Barry S.; Gai, Feng

doi:10.1021/bi048662e

Cited by 33 publications

(44 citation statements)

References 51 publications

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“…␣ 1 -AT polymers were labeled with deuterium for different time periods to investigate the stability and solvent accessibility of monomers within the polymers in a region-specific manner. Because dissociation of the polymer occurs much slower than our deuterium labeling time (as a previous study demonstrated (19)), dilution of the polymeric sample into D 2 O does not affect the structural integrity of the polymer within our experimental time scale. Fig.…”

Section: Structure and Dynamics Of The Monomeric Intermediate Pre-mentioning

confidence: 76%

The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

Tsutsui

Kuri

Sengupta

et al. 2008

Journal of Biological Chemistry

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The serpinopathies are a group of inherited disorders that share as their molecular basis the misfolding and polymerization of serpins, an important class of protease inhibitors. Depending on the identity of the serpin, conditions arising from polymerization include emphysema, thrombosis, and dementia. The structure of serpin polymers is thus of considerable medical interest. Wild-type ␣ 1 -antitrypsin will form polymers upon incubation at moderate temperatures and has been widely used as a model system for studying serpin polymerization. Using hydrogen/deuterium exchange and mass spectrometry, we have obtained molecular level structural information on the ␣ 1 -antitrypsin polymer. We found that the flexible reactive center loop becomes strongly protected upon polymerization. We also found significant increases in protection in the center of ␤-sheet A and in helix F. These results support a model in which linkage between serpins is achieved through insertion of the reactive center loop of one serpin into ␤-sheet A of another. We have also examined the heat-induced conformational changes preceding polymerization. We found that polymerization is preceded by significant destabilization of ␤-sheet C. On the basis of our results, we propose a mechanism for polymerization in which ␤-strand 1C is displaced from the rest of ␤-sheet C through a binary serpin/serpin interaction. Displacement of strand 1C triggers further conformational changes, including the opening of ␤-sheet A, and allows for subsequent polymerization.2 is a member of the serpin class of protease inhibitors. Serpins inhibit their target proteases via a unique mechanism ( Fig. 1) (1). Cleavage of a serpin's reactive center loop (RCL) by a protease triggers a massive conformational change in which the RCL inserts into the central ␤-sheet A, becoming a sixth strand. This process disrupts the protease active site and traps the acyl-enzyme intermediate, rendering the protease inactive. The active, loop-expelled form of ␣ 1 -AT is significantly less stable than the loop-inserted form, and as a result of this metastability, the serpin structure is readily disrupted by mutations that result in inappropriate conformational changes. It has been established that a number of serious diseases associated with serpin mutations are caused by the formation and accumulation of serpin polymers (2). The details of the polymerization mechanism are therefore of considerable interest.Serpin polymerization has been studied extensively by a variety of methods, including circular dichroism, fluorescence spectroscopy, electron microscopy, and x-ray crystallography (3-5). The most generally accepted model of pathological serpin polymerization is one in which the RCL of one serpin anneals between ␤-strands 3 and 5A of another (1). Two major lines of evidence support this model. The first is that RCLmimicking peptides have been shown to anneal between strands 3 and 5A and have also been shown to block polymerization in vitro (6). The second comes from fluorescence spectroscopy. Dist...

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Section: Structure and Dynamics Of The Monomeric Intermediate Pre-mentioning

confidence: 76%

The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

Tsutsui

Kuri

Sengupta

et al. 2008

Journal of Biological Chemistry

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“…6,7 The patent b-sheet A then accepts the reactive loop of a second a 1 -antitrypsin molecule to form a dimer, which can extend into chains of reactive center loop-b-sheet A polymers. 2,6,[8][9][10][11][12] The recent crystal structure of a dimer of another serpin, antithrombin, demonstrated a linkage between a b-hairpin of the reactive loop and strand 5A of one molecule and b-sheet A of another. This dimer was used as the basis of a novel model for the polymer in which helix I is unravelled and the proteins are linked by a b-hairpin containing the reactive center loop and strand 5A.…”

mentioning

confidence: 99%

A Novel Monoclonal Antibody to Characterize Pathogenic Polymers in Liver Disease Associated with α1-Antitrypsin Deficiency†

et al. 2010

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Alpha 1 -antitrypsin is the most abundant circulating protease inhibitor. The severe Z deficiency allele (Glu342Lys) causes the protein to undergo a conformational transition and form ordered polymers that are retained within hepatocytes. This causes neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. We have developed a conformation-specific monoclonal antibody (2C1) that recognizes the pathological polymers formed by a 1 -antitrypsin. This antibody was used to characterize the Z variant and a novel shutter domain mutant (His334Asp; a 1 -antitrypsin King's) identified in a 6-week-old boy who presented with prolonged jaundice. His334Asp a 1 -antitrypsin rapidly forms polymers that accumulate within the endoplasmic reticulum and show delayed secretion when compared to the wild-type M a 1 -antitrypsin. The 2C1 antibody recognizes polymers formed by Z and His334Asp a 1 -antitrypsin despite the mutations directing their effects on different parts of the protein. This antibody also recognized polymers formed by the Siiyama (Ser53Phe) and Brescia (Gly225Arg) mutants, which also mediate their effects on the shutter region of a 1 -antitrypsin. Conclusion: Z and shutter domain mutants of a 1 -antitrypsin form polymers with a shared epitope and so are likely to have a similar structure. (HEPATOLOGY 2010;52:1078-1088 T he serpinopathies are conformational diseases characterized by the polymerization and intracellular retention of members of the serine protease inhibitor or serpin superfamily of proteins.1 The best known is a 1 -antitrypsin deficiency, with the most common severe deficiency allele being the Z mutation (Glu342Lys). This mutation results in the retention of ordered polymers of a 1 -antitrypsin as periodic acid Schiff positive inclusion bodies within the endoplasmic reticulum (ER) of hepatocytes.2 These inclusions predispose the individual homozygous for the Z variant of the a 1 -antitrypsin protease inhibitor (PI*Z) to neonatal hepatitis, cirrhosis, and rarely, hepatocellular carcinoma.3 Deficiency of circulating a 1 -antitrypsin results in early onset panlobular emphysema.

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“…This perturbation in structure allows the formation of an unstable intermediate (M Ã ) and a sequential β-strand linkage between the reactive center loop of one molecule and β-sheet A of another (Fig. 1A) (3)(4)(5)(6)(7)(8). However this model has recently been challenged by the crystal structure of a self-terminating dimer of another serpin, antithrombin (9).…”

mentioning

confidence: 99%

Defining the mechanism of polymerization in the serpinopathies

Ekeowa

Freeke

Miranda

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

126

144

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The serpinopathies result from the ordered polymerization of mutants of members of the serine proteinase inhibitor (serpin) superfamily. These polymers are retained within the cell of synthesis where they cause a toxic gain of function. The serpinopathies are exemplified by inclusions that form with the common severe Z mutant of α 1 -antitrypsin that are associated with liver cirrhosis. There is considerable controversy as to the pathway of serpin polymerization and the structure of pathogenic polymers that cause disease. We have used synthetic peptides, limited proteolysis, monoclonal antibodies, and ion mobility-mass spectrometry to characterize the polymerogenic intermediate and pathological polymers formed by Z α 1 -antitrypsin. Our data are best explained by a model in which polymers form through a single intermediate and with a reactive center loop-β-sheet A linkage. Our data are not compatible with the recent model in which polymers are linked by a β-hairpin of the reactive center loop and strand 5A. Understanding the structure of the serpin polymer is essential for rational drug design strategies that aim to block polymerization and so treat α 1 -antitrypsin deficiency and the serpinopathies.alpha-1-antitrypsin deficiency | protein folding | serpins | polymers | cirrhosis

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α₁-Antitrypsin Polymerization: A Fluorescence Correlation Spectroscopic Study

Cited by 33 publications

References 51 publications

The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

A Novel Monoclonal Antibody to Characterize Pathogenic Polymers in Liver Disease Associated with α1-Antitrypsin Deficiency†

Defining the mechanism of polymerization in the serpinopathies

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α1-Antitrypsin Polymerization: A Fluorescence Correlation Spectroscopic Study

Cited by 33 publications

References 51 publications

The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

A Novel Monoclonal Antibody to Characterize Pathogenic Polymers in Liver Disease Associated with α1-Antitrypsin Deficiency†

Defining the mechanism of polymerization in the serpinopathies

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α₁-Antitrypsin Polymerization: A Fluorescence Correlation Spectroscopic Study