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Titolo:
Different structural requirements for plasminogen activator inhibitor 1 (PAI-1) during latency transition and proteinase inhibition as evidenced by phage-displayed hypermutated PAI-1 libraries
Autore:
Stoop, AA; Eldering, E; Dafforn, TR; Read, RJ; Pannekoek, H;
Indirizzi:
Univ Amsterdam, Acad Med Ctr, Dept Biochem, NL-1105 AZ Amsterdam, Netherlands Univ Amsterdam Amsterdam Netherlands NL-1105 AZ Z Amsterdam, Netherlands Univ Cambridge, Dept Haematol, Wellcome Trust Ctr Mol Mechanisms Dis, Cambridge Inst Med Res, Cambridge CB2 2XY, England Univ Cambridge Cambridge England CB2 2XY Res, Cambridge CB2 2XY, England
Titolo Testata:
JOURNAL OF MOLECULAR BIOLOGY
fascicolo: 4, volume: 305, anno: 2001,
pagine: 773 - 783
SICI:
0022-2836(20010126)305:4<773:DSRFPA>2.0.ZU;2-L
Fonte:
ISI
Lingua:
ENG
Soggetto:
MOLECULAR-WEIGHT INHIBITOR; CELL-ADHESION; IN-VITRO; FUNCTIONAL STABILITY; UROKINASE RECEPTOR; DIRECTED EVOLUTION; VARIABLE REGION-1; SUICIDE SUBSTRATE; BINDING-SITE; VITRONECTIN;
Keywords:
PAI-1; protein stability; DNA shuffling; phage-display; RCL insertion;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
52
Recensione:
Indirizzi per estratti:
Indirizzo: Pannekoek, H Univ Amsterdam, Acad Med Ctr, Dept Biochem, Meibergdreef 9, NL-1105 AZ Amsterdam, Netherlands Univ Amsterdam Meibergdreef 9 Amsterdam Netherlands NL-1105 AZ
Citazione:
A.A. Stoop et al., "Different structural requirements for plasminogen activator inhibitor 1 (PAI-1) during latency transition and proteinase inhibition as evidenced by phage-displayed hypermutated PAI-1 libraries", J MOL BIOL, 305(4), 2001, pp. 773-783

Abstract

Plasminogen activator inhibitor type 1 (PAI-1) is a member of the serine protease inhibitor (serpin) superfamily. Its highly mobile reactive-center loop (RCL) is thought to account for both the rapid inhibition of tissue-type plasminogen activator (t-PA), and the rapid and spontaneous transition ofthe unstable, active form of PAI-1 into a stable, inactive (latent) conformation (t(1/2) at 37 degreesC, 2.2 hours). We determined the amino acid residues responsible for the inherent instability of PAI-1, to assess whether these properties are independent and, consequently, whether the structural basis for inhibition and latency transition is different. For that purpose,a hypermutated PAI-1 library that is displayed on phage was pre-incubated for increasing periods (20 to 72 hours) at 37 degreesC, prior to a stringent selection for rapid t-PA binding. Accordingly, four rounds of phage-display selection resulted in the isolation of a stable PAI-1 variant (st-44: t(1/2) 450 hours) with 11 amino acid mutations. Backcrossing by DNA shufflingof this stable mutant with wt PAI-1 was performed to eliminate non-contributing mutations. It was shown that the combination of mutations at positions 50, 56, 61, 70, 94, 150, 222, 223, 264 and 331 increases the half-life ofPAI-1 245-fold. Furthermore, within the Limits of detection the stable mutants isolated are functionally indistinguishable from wild-type PAI-1 with respect to the rate of inhibition of t-PA, cleavage by t-PA, and binding tovitronectin. These stabilizing mutations constitute largely reversions to the stable "serpin consensus sequence" and are located in areas implicated in PAI-1 stability (e.g, the vitronectin-binding domain and the proximal hinge). Collectively, our data provide evidence that the structural requirements for PAI-1 loop insertion during latency transition and target proteinase inhibition can be separated. (C) 2001 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 23/09/20 alle ore 12:33:18