Hsp90 chaperones protein folding in vitro

Wiech, Hans; Büchner, Johannes; Zimmermann, Richard; Jakob, Ursula

doi:10.1038/358169a0

Cited by 496 publications

(308 citation statements)

References 13 publications

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“…Previously we have demonstrated that the GroE chaperone system of Escherichia coli consisting of GroEL, GroES, and ATP (Schmidt & Buchner, 1992) and eukaryotic Hsp90 (Wiech et al, 1992) influence the refolding of MAK 33 Fab. Both chaperones do not accelerate the folding process.…”

Section: Discussionmentioning

confidence: 99%

Prolyl isomerases catalyze antibody folding in vitro

et al. 1993

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Some slow-folding phases in the in vitro refolding of proteins originate from the isomerization of prolyl-peptide bonds, which can be accelerated by a class of enzymes called prolyl isomerases (PPIs). We used the in vitro folding of an antibody Fab fragment as a model system to study the effect of PPI on a folding reaction that is only partially reversible. We show here that members of both subclasses of PPIs, cyclophilin and FK 506 binding protein (FKBP), accelerate the refolding process and increase the yield of correctly folded molecules. An acceleration of folding was not observed in the presence of the specific inhibitor cyclosporin A, but still the yield of correctly folded molecules was increased. Bovine serum albumin (BSA) increased the yield comparable to cyclophilin but, in contrast, did not influence the rate of reactivation. These effects were observed only when cyclophilin or BSA were present during the first few seconds of refolding. However, the rate-limiting reactivation reaction is still accelerated when PPI is added several minutes after starting refolding. In contrast, the prokaryotic chaperone GroEL influences the refolding yield when added several minutes after initiating refolding. The results show that PPIs influence the folding of Fab in two different ways. (1) They act as true catalysts of protein folding by accelerating the rate-limiting isomerization of Xaa-Pro peptide bonds. Proline isomerization is obviously a late folding step and has no influence on the formation of aggregates within the first seconds of the refolding reaction. (2) PPI and BSA are able to increase the yield of refolding of Fab by reducing the formation of aggregates or the adsorption to the surface of the reaction vessel in an unspecific manner. This behavior is clearly distinct from the mechanism of action observed with the chaperone GroEL.Keywords: antibodies; BSA; chaperone; cyclophilin; Fab fragment; folding catalyst; GroE; protein folding; peptidyl-prolyl isomerase Peptidyl-prolyl-cis-trans-isomerases are conserved and abundant proteins located in the cytosol and organelles of eukaryotic cells and in the cytosol and periplasm of bacteria. They were first described as PPIs by Fischer et al. (1984) and Handschumacher et al. (1984). There are two unrelated subclasses of PPIs: cyclophilins, which are specifically inhibited by the immunosuppressive cyclic peptide cyclosporin A, and FK 506 binding proteins, which are inhibited by the immunosuppressive drug FK 506 (cf.

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Section: Discussionmentioning

confidence: 99%

Prolyl isomerases catalyze antibody folding in vitro

et al. 1993

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“…Early evidence for the role of Hsp90 in protein folding came from in vitro studies where it was demonstrated that chemically denatured substrates like citrate synthase (mainly α-helical structure) and Fab fragment of a monoclonal antibody (only β-sheets present) can be efficiently refolded with a high yield in the presence of purified Hsp90 (Wiech et al, 1992). It was also shown in vitro that Hsp90 primarily functions in preventing aggregation of stress-denatured proteins and maintains them in a folding competent state (Freeman and Morimoto, 1996;Yonehara et al, 1996).…”

Section: Ii41122 the Hsp90 Chaperone Systemmentioning

confidence: 99%

Firefly luciferase mutants as sensors of proteome stress

et al. 2011

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“…In eukaryotes, the cytoplasmic HSP90s act as specific chaperones for a wide range of client proteins such as the cytoplasmic receptor steroid hormones [5]. HSP90 alone can act to prevent protein aggregation and promote refolding in itro [6], but in i o it is functionally associated in multiprotein complexes with a range of associated proteins such as p23 [7], HSP70\HSP40 [8], FKBP52 [9], Hop (p60\Sti1) [10], Cdc37\p50 [11] and Cyp40 (cyclophilin 40) [12].…”

Section: Introductionmentioning

confidence: 99%

A novel chaperone-activity-reducing mechanism of the 90-kDa molecular chaperone HSP90

Itoh

Ogura

Komatsuda

et al. 1999

Biochemical Journal

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The 90-kDa heat shock protein (HSP90) acts as a specific molecular chaperone in the folding and regulates a wide range of associated proteins such as steroid hormone receptors. It is known that HSP90 possesses two different chaperone sites, both in the N-and C-domains, and that the chaperone activity of HSP90 is blocked by binding of geldanamycin (GA) to the Ndomain, the same as the ATP-binding site. Here we show that Cisplatin [cis-diamminedichloroplatinum (II), CDDP], an antineoplastic agent, associates with HSP90 and reduces its chaperone activity. In order to analyse the binding proteins, bovine brain cytosols were applied to a CDDP-affinity column and binding proteins were eluted by CDDP. In the elutants, only 90-kDa protein bands were detected on SDS\PAGE, and the protein was cross-reacted with the anti-HSP90 antibody on immunoblotting. No protein bands were detected in the elutants

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Hsp90 chaperones protein folding in vitro

Cited by 496 publications

References 13 publications

Prolyl isomerases catalyze antibody folding in vitro

Prolyl isomerases catalyze antibody folding in vitro

Firefly luciferase mutants as sensors of proteome stress

A novel chaperone-activity-reducing mechanism of the 90-kDa molecular chaperone HSP90

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