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Identification of Conus peptidylprolyl cis-trans isomerases (PPIases) and assessment of their role in the oxidative folding of conotoxins.

ABSTRACT: Peptidylprolyl cis-trans isomerases (PPIases) are ubiquitous proteins that catalyze the cis-trans isomerization of prolines. A number of proteins, such as Drosophila rhodopsin and the human immunodeficiency viral protein HIV-1 Gag, have been identified as endogenous substrates for PPIases. However, very little is known about the interaction of PPIases with small, disulfide-rich peptides. Marine cone snails synthesize a wide array of cysteine-rich peptides, called conotoxins, many of which contain one or more prolines or hydroxyprolines. To identify whether PPIase-associated cis-trans isomerization of these residues affects the oxidative folding of conotoxins, we identified, sequenced, and expressed three functionally active isoforms of PPIase from the venom gland of Conus novaehollandiae, and we characterized their ability to facilitate oxidative folding of conotoxins in vitro. Three conotoxins, namely mu-GIIIA, mu-SIIIA, and omega-MVIIC, derived from two distinct toxin gene families were assayed. Conus PPIase significantly increased the rate of appearance of the native form of mu-GIIIA, a peptide containing three hydroxyprolines. In contrast, the presence of PPIase had no effect on the folding of mu-SIIIA and omega-MVIIC, peptides containing no or one proline residue, respectively. We further showed that an endoplasmic reticulum-resident PPIase isoform facilitated folding of mu-GIIIA more efficiently than two cytosolic isoforms. This is the first study to demonstrate PPIase-assisted folding of conotoxins, small disulfide-rich peptides with unique structural properties.

SUBMITTER: Safavi-Hemami H

PROVIDER: S-EPMC2857115 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Identification of Conus peptidylprolyl cis-trans isomerases (PPIases) and assessment of their role in the oxidative folding of conotoxins.

Safavi-Hemami Helena H Bulaj Grzegorz G Olivera Baldomero M BM Williamson Nicholas A NA Purcell Anthony W AW

The Journal of biological chemistry 20100210 17

Peptidylprolyl cis-trans isomerases (PPIases) are ubiquitous proteins that catalyze the cis-trans isomerization of prolines. A number of proteins, such as Drosophila rhodopsin and the human immunodeficiency viral protein HIV-1 Gag, have been identified as endogenous substrates for PPIases. However, very little is known about the interaction of PPIases with small, disulfide-rich peptides. Marine cone snails synthesize a wide array of cysteine-rich peptides, called conotoxins, many of which contai ...[more]

PMID: 20147296

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Project description:Free in solution, the immunosuppressive compounds cyclosporin A (CsA), FK506, ascomycin and rapamycin are present in many solvents in various slowly interconverting conformations. Together with their cellular receptor proteins, cyclophilin (CyP) and FK506-binding protein (FKBP), however, these inhibitors have been shown to have a homogeneous conformation. The existence of a slow cis-trans interconversion of an imidic bond in the inhibitor molecule during the course of the formation of the CsA-CyP18cy complex (where CyP18cy is human 18 kDa cytosolic CyP) prompted us to investigate the reaction of the peptidomacrolides FK506, ascomycin and rapamycin with two specific binding-proteins in more detail. Since formation of the FK506-FKBP complex results in the inhibition of the peptidylprolyl cis-trans-isomerase activity of the binding protein, we used the enzyme's decrease in enzymic activity to monitor binding of the inhibitors to their enzyme targets. For FK506, the kinetics of inhibition of human 12 kDa cytosolic FKBP (FKBP12cy) were clearly dependent on time. Subsequent to a rapid inactivation reaction, not resolved in its kinetics due to manual mixing, a slow dominant first-order inactivation process with a relaxation time of 1163 s at 10 degrees C was observed. Concomitantly the Ki value of the slow phase dropped 2.6-fold within the first 60 min of incubation. Using the FKBP12cy homologue 25 kDa membrane FKBP (FKBP25mem), a bacterial peptidylprolyl cis-trans-isomerase, the rate and amplitudes of the inhibition reactions were very similar to FKBP12cy. On the other hand, the kinetics and amplitudes of the inhibition of FKBP12cy varied significantly if rapamycin was used as an inhibitor instead of FK 506. Owing to reduced conformation transition in rapamycin upon binding to FKBP12cy, the slow phase during inhibition was significantly decreased in amplitude. A likely reason for this became apparent when the activation-enthalpy and the pH-dependence of the rate constants of the slow phase were determined. We conclude that the cis to trans interconversion of the pipecolinyl bond of the three peptidomacrolides may be responsible for the slow process. There was no indication of a suicide catalysis of this process by FKBPs.

Dataset Information

Identification of Conus peptidylprolyl cis-trans isomerases (PPIases) and assessment of their role in the oxidative folding of conotoxins.

Publications

Identification of Conus peptidylprolyl cis-trans isomerases (PPIases) and assessment of their role in the oxidative folding of conotoxins.

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