Time-dependent inhibition of peptidylprolyl <i>cis-trans</i>-isomerases by FK506 is probably due to <i>cis-trans</i> isomerization of the inhibitor's imide bond

Zarnt, Toralf; Lang, Kurt; Burtscher, Helmut; Fischer, Gunter

doi:10.1042/bj3050159

Cited by 14 publications

(15 citation statements)

References 32 publications

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“…This variant (EK-FKBP12) possesses an amino-terminal extension of 16 amino acids, composed of the first 6 residues of ␤-galactosidase, a tetrahistidine tag and the recognition site for enterokinase. It was used in the recombinant production of FKBP12, and the mature protein was obtained from this fusion protein (EK-FKBP12) by cleaving it with the protease enterokinase (25). EK-FKBP12 is a stably folded protein.…”

Section: Resultsmentioning

confidence: 99%

“…EK-FKBP12 contained an amino-terminal extension of the following 16 residues: TMITNSMHHHHDDDDK. Residues 1-6 represent a fragment from the NH 2 terminus of ␤-galactosidase, they are followed by a tetrahistidine tag and by the recognition site for cleavage by enterokinase (25). FK506 was a gift of Fujisawa Corporation.…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant human FKBP12 and the fusion protein EK-FKBP12 were expressed and purified as described by Zarnt et al (25). The concentrations of the two proteins were determined spectrophotometrically by using an absorption coefficient of 9530 M Ϫ1 cm Ϫ1 at 280 nm (25). EK-FKBP12 contained an amino-terminal extension of the following 16 residues: TMITNSMHHHHDDDDK.…”

Section: Methodsmentioning

confidence: 99%

“…Prolyl Isomerase Assays-The procedure described by Zarnt et al (25) was used to assay the prolyl isomerase activities of FKBP12 and EK-FKBP12. Suc-Ala-Phe-Pro-Phe-4-nitroanilide served as the assay peptide.…”

Section: Methodsmentioning

confidence: 99%

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Autocatalytic Folding of the Folding Catalyst FKBP12

Schölz

Zarnt²,

Kern³

et al. 1996

Journal of Biological Chemistry

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Prolyl isomerases are folding enzymes and thus have the potential to catalyze their own folding. We show here that the folding of cytosolic FKBP12 (FK 506 binding protein) is an autocatalytic process both for the mature protein and for a fusion protein with an aminoterminal extension of 16 residues. Native FKBP contains seven trans-prolyl peptide bonds, and the cis-to-trans isomerizations of some or all of them constitute the slow, rate-limiting events in folding. The rate of an autocatalytic reaction increases with reactant concentration, because the product catalyzes its own formation. Accordingly, the folding of the fusion protein was more than 10-fold accelerated when the protein concentration was increased from 0.05 M to 10 M. At high concentrations of both forms of FKBP12 autocatalysis was very efficient, and the observed folding rate seemed to approach the rate of the fast direct folding reaction of the protein molecules with the correct (all trans) peptidyl-prolyl bond conformation.Protein folding in vitro and in vivo is often decelerated because it is coupled with prolyl isomerization (1-5). Cis/trans equilibria are usually established at the prolyl peptide bonds in an unfolded polypeptide chain, and, as a consequence, denatured proteins are heterogeneous mixtures of species which refold with different rates. Only the molecules with the nativelike prolines 1 can refold directly in a rapid reaction. The molecules with incorrect prolines refold in slow reactions that are limited in rate by the cis/trans isomerizations of these prolines. Proline-limited folding reactions are catalyzed in vitro and in vivo by peptidyl-prolyl cis/trans isomerases (3, 4, 6 -12). Four families of these ubiquitous proteins are now known (13-16), and their functions are not confined to catalyzing slow steps in protein folding (13,17 (24) and refolding can be followed by a change in protein fluorescence and by the regain of the prolyl isomerase activity.In an autocatalytic reaction the product accelerates its own formation and thus its rate is expected to increase with reactant concentration. For a proline-limited folding reaction, autocatalysis should thus lead to an increase in rate with protein concentration until the rate of direct fast refolding is approached. Therefore, in our refolding experiments, we varied the concentration of FKBP12 in a wide range, and in some folding experiments FK 506 was added to inhibit the prolyl isomerase activity of the refolded molecules. We found that autocatalysis contributes indeed to the refolding of most unfolded FKBP12 molecules. A strong autocatalytic rate enhancement was found in the folding of a variant of FKBP12 for which the direct and the proline-limited folding reactions differ vastly in rate. EXPERIMENTAL PROCEDURESMaterials-Urea (ultrapure) and guanidinium chloride (ultrapure) were from ICN Biomedicals, ␣-chymotrypsin and recombinant human cytoplasmic Cyp18 were from Boehringer-Mannheim, LiCl and CF 3 CH 2 OH were from Fluka, the assay peptide Suc-Ala-Phe-Pro-Phe-4-nitroanilide w...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Autocatalytic Folding of the Folding Catalyst FKBP12

Schölz

Zarnt²,

Kern³

et al. 1996

Journal of Biological Chemistry

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“…Other drug effects may result from the facilitated dissociation of receptor⅐PPIase complexes (8,9) and the slow rate of formation of the PPIase⅐drug complexes (10,11). In addition, the PPIase-mediated intracellular accumulation of the drugs prevents the reliable analysis of dose-response curves.…”

mentioning

confidence: 99%

Reversible Inhibition of Calcineurin by the Polyphenolic Aldehyde Gossypol

Baumgrass

Weiwad

Erdmann

et al. 2001

Journal of Biological Chemistry

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The reversible inhibition of calcineurin (CaN), which is the only Ca 2؉ /calmodulin-dependent protein Ser/Thr phosphatase, is thought to be a key functional event for most cyclosporin A (CsA)-and tacrolimus (FK506)-mediated biological effects. In addition to CaN inhibition, however, CsA and FK506 have multiple biochemical effects because of their action in a gain-of-function model that requires prior binding to immunophilic proteins. We screened a small molecule library for direct inhibitors of CaN using CaN-mediated dephosphorylation of 33 P-labeled 19-residue phosphopeptide substrate (RII phosphopeptide) as an assay and found the polyphenolic aldehyde gossypol to be a novel CaN inhibitor. Unlike CsA and FK506, gossypol does not require a matchmaker protein for reversible CaN inhibition with an IC 50 value of 15 M. Gossypolone, a gossypol analog, showed improved inhibition of both RII phosphopeptide and p-nitrophenyl phosphate dephosphorylation with an IC 50 of 9 and 6 M, respectively. In contrast, apogossypol hexaacetate was inactive. Gossypol acts noncompetitively, interfering with the binding site for the cyclophilin 18⅐CsA complex in CaN. In contrast to CsA and FK506, gossypol does not inactivate the peptidyl-prolyl-cis/ trans-isomerase activity of immunophilins. Similar to CsA and FK506, T cell receptor signaling induced by phorbol 12-myristate 13-acetate/ionomycin is inhibited by gossypol in a dose-dependent manner, demonstrated by the inhibition of nuclear factor of activated T cell (NFAT) c1 translocation from the cytosol into the nucleus and suppression of NFAT-luciferase reporter gene activity.In vivo inhibition by membrane-penetrable low molecular mass compounds plays an important role in evaluating the biological function of enzymes involved in protein phosphorylation/dephosphorylation. The functional discrimination with specific inhibitors of the four major protein Ser/Thr phosphatases, protein phosphatase 1 (PP1), 1 protein phosphatase 2A (PP2A), protein phosphatase 2B (PP2B, calcineurin, CaN), and protein phosphatase 2C (PP2C), has proven to be successful (1). Okadaic acid and microcystin are strong inhibitors of PP1 and PP2A, but they exhibit poor inhibition of the Ca 2ϩ /calmodulinregulated CaN and PP2C. It was difficult to identify CaN function in cell signaling until the membrane-penetrable cyclopeptide cyclosporin A (CsA) and the peptidomacrolide FK506 were found to inhibit CaN specifically under certain conditions (2). Currently, most reports about CaN involvement in cellular processes are based on CsA and FK506 susceptibility of the appropriate bioassays. CaN inhibition by these drugs is characterized by their prior binding to the 18-kDa cyclophilin (Cyp18) and 12-kDa FK506-binding protein (FKBP12), respectively, indicating a gain-of-function mechanism (3). These mammalian prototypes of two different families of the enzyme class of peptidyl-prolyl-cis/trans-isomerases (EC 5.2.1.8; PPIases) function as molecular matchmakers because the drugs cannot bind to CaN on their own. The prototypic P...

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FK506‐Binding Proteins

Fischer

2002

Wiley Encyclopedia of Molecular Medicine

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Time-dependent inhibition of peptidylprolyl cis-trans-isomerases by FK506 is probably due to cis-trans isomerization of the inhibitor's imide bond

Cited by 14 publications

References 32 publications

Autocatalytic Folding of the Folding Catalyst FKBP12

Autocatalytic Folding of the Folding Catalyst FKBP12

Reversible Inhibition of Calcineurin by the Polyphenolic Aldehyde Gossypol

FK506‐Binding Proteins

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