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Plasminogen activator inhibitor type 2: a unique serpin with two mobile loops
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The superfamily of serine protease inhibitors (serpins) is a large group of proteins with diverse functions but a common tertiary structure. Active serpins are highly metastable molecules. Metastability is the property underlying the success and ubiquitousness of serpins. However, serpin metastability also accounts for improper conformational changes in serpin mutants which may result in pathological serpin polymerization. Plasminogen activator inhibitor type 2 (PAI-2) is a member of the subfamily of ov-serpins. It is the only wild-type (wt) serpin that spontaneously polymerizes under physiological conditions. Another unique feature of PAI-2 is the loop connecting helices C and D (the CD-loop), which is the longest among known serpins and is involved in interactions with the environment.

Two partially overlapping goals were achieved during this thesis. The first was to study the molecular determinants involved in PAI-2 polymerization. By using in vitro mutagenesis in combination with biochemical and fluorescence methods, we have shown that wt PAI-2 exists both in stable monomeric and in polymerogenic conformations. The polymerogenic conformation can spontaneously form loop-sheet polymers and does not require conformational rearrangements prior to polymerization. The polymerogenic conformation of PAI-2 has an open β-sheet A, and it is stabilized by a disulfide bond formed between the unique CD-loop of PAI-2 and the bottom of the molecule. Under reducing conditions, the polymerogenic conformation of PAI-2 converts to the stable monomeric form. The polymerogenic and the stable monomeric forms are fully interconvertible, depending on the redox status of the environment. The stable monomeric conformation can be stabilized by vitronectin disulfide-bound to the CD-loop of PAI-2. The most populated conformation of the stable monomeric form of PAI-2 is that with the CD-loop folded on the side of the molecule. However, a mall fraction of stable monomeric PAI-2 can also exist with the CD-loop oriented toward the bottom of the molecule. Thus, the CD-loop of PAI-2 is a mobile molecular switch that regulates PAI-2 conformation.

The second goal of the thesis was to use PAI-2 as a model protein to develop methods for intramolecular distance measurements. An improved purification procedure, the stability of the protein and our understanding of its structure make PAI-2 an attractive candidate for use as a model protein. In this context, we have used PAI-2 successfully to measure distances by the previously used DDEM and newly developed PDDEM methods.

Place, publisher, year, edition, pages
Umeå: Medicinsk biokemi och biofysik , 2004. , 61 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 870
Identifiers
URN: urn:nbn:se:umu:diva-223ISBN: 91-7305-572-7 (print)OAI: oai:DiVA.org:umu-223DiVA: diva2:142721
Public defence
2004-01-30, 00:00
Available from: 2004-03-30 Created: 2004-03-30 Last updated: 2010-08-05Bibliographically approved
List of papers
1. The spontaneous polymerization of plasminogen activator inhibitor type-2 and Z-antitrypsin are due to different molecular aberrations
Open this publication in new window or tab >>The spontaneous polymerization of plasminogen activator inhibitor type-2 and Z-antitrypsin are due to different molecular aberrations
2003 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 537, no 1-3, 11-16 p.Article in journal (Refereed) Published
Abstract [en]

The wild-type form of plasminogen activator inhibitor type-2 (PAI-2) and the pathogenic Z-mutant of alpha(1)-antitrypsin (alpha(1)AT) are serpins that spontaneously polymerize by the loop-sheet mechanism. Compared to the consensus serpin sequence, both PAI-2 and Z-alpha(1)AT have deviations in the so-called breach region located at the top of the A beta-sheet. In the case of Z-alpha(1)AT, conformational perturbations caused by a single amino acid substitution result in polymerization in vivo and predisposes to disease. To test whether the polymerization of PAI-2 is due to aberrations in the breach region, we constructed substitution mutants of PAI-2 with conserved residues in this region. Analysis of the mutants revealed that deviations in the breach region modulate but are not the major cause of PAI-2 polymerization. Rather, PAI-2 exists in a highly polymerogenic conformation and does not require conformational rearrangements before polymerization can take place.

Keyword
Serpin, Loop–sheet polymerization, Plasminogen activator inhibitor type-2
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-3836 (URN)10.1016/S0014-5793(03)00057-7 (DOI)12606023 (PubMedID)
Available from: 2004-03-30 Created: 2004-03-30 Last updated: 2017-12-14Bibliographically approved
2. A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization.
Open this publication in new window or tab >>A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization.
2003 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 22, no 8, 1753-1761 p.Article in journal (Refereed) Published
Abstract [en]

Plasminogen activator inhibitor type 2 (PAI-2) is the only wild-type serpin that polymerizes spontaneously under physiological conditions. We show that PAI-2 loses its ability to polymerize following reduction of thiol groups, suggesting that an intramolecular disulfide bond is essential for the polymerization. A novel disulfide bond was identified between C79 (in the CD-loop) and C161 (at the bottom of helix F). Substitution mutants in which this disulfide bond was broken did not polymerize. Reactive center loop peptide insertion experiments and binding of bis-ANS to hydrophobic cavities indicate that the C79-C161 disulfide bond stabilizes PAI-2 in a polymerogenic conformation with an open A-beta-sheet. Elimination of this disulfide bond causes A-beta-sheet closure and abrogates the polymerization. The finding that cytosolic PAI-2 is mostly monomeric, whereas PAI-2 in the secretory pathway is prone to polymerize, suggests that the redox status of the cell could regulate PAI-2 polymerization. Taken together, our data suggest that the CD-loop functions as a redox-sensitive switch that converts PAI-2 between an active stable monomeric and a polymerogenic conformation, which is prone to form inactive polymers.

Keyword
PAI-2, polymerization, redox, serpin
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-3837 (URN)10.1093/emboj/cdg178 (DOI)12682008 (PubMedID)
Available from: 2004-03-30 Created: 2004-03-30 Last updated: 2017-12-14Bibliographically approved
3. Plasminogen activator inhibitor type 2 is a unique serpin with two mobile functional loops.
Open this publication in new window or tab >>Plasminogen activator inhibitor type 2 is a unique serpin with two mobile functional loops.
Show others...
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-3838 (URN)
Available from: 2004-03-30 Created: 2004-03-30 Last updated: 2010-01-13Bibliographically approved
4. Partial donor-donor energy migration (PDDEM): a novel fluorescence method for internal protein distance measurements
Open this publication in new window or tab >>Partial donor-donor energy migration (PDDEM): a novel fluorescence method for internal protein distance measurements
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2004 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 6, no 11, 3001-3008 p.Article in journal (Refereed) Published
Abstract [en]

We show that the photophysics of chemically identical but photophysically non-identical fluorescent pairs can be used for measuring distances within proteins. For this purpose, the theory of partial donor-donor energy migration (PDDEM, S. Kalinin, J. G. Molotkovsky and L. B.-Angstrom. Johansson, Spectrochim. Acta, Part A, 2002, 58, 1057-1097) was applied for distance measurements between BODIPY groups covalently linked to cystein residues in plasminogen activator inhibitor of type 2 (PAI-2). Two sulfhydryl specific derivatives of BODIPY were used namely: N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl) iodoacetamide and N-(4.4-difluoro-5.7-ditriethyl-4-bona-3a,4a-diaza-s-indacene-3-yl) methyl iodoacetamide. To determine distances, the time-resolved fluorescence relaxation for two singly labelled forms of PAI-2, as well as the corresponding doubly labelled protein were combined and analysed in a global manner. Fluorescence depolarisation experiments on the labelled mutants were also analysed. The distances determined by PDDEM were in good agreement to those obtained from donor-donor energy migration (DDEM) experiments and structural data on PAI-2. The PDDEM approach allows for the use of very different fluorescent probes, which enables wide range of distances to be measured. The PDDEM model also provides a rational explanation to why previous observations of polyfluorophore-labelled proteins exhibit a shorter average fluorescence lifetime compared to the arithmetic average of lifetimes obtained for the corresponding single labelled proteins.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-14351 (URN)10.1039/b403264k (DOI)
Available from: 2007-06-26 Created: 2007-06-26 Last updated: 2017-12-14Bibliographically approved

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