Chemical and biological consequences of sugar incorporation into a potential N-glycosylation site in the tubulin-binding repeat of tau protein

Ötvös, László; Craik, David J.; Bokonyi, Krisztina; Varga, I.; Pease, Anne Marie; Wade, John D.; Hoffmann, Ralf

doi:10.1007/0-306-46881-6_306

Cited by 2 publications

(2 citation statements)

References 6 publications

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“…We also know that fluorescein-K-pyrrhocoricin kills E. coli strains (with an IC 50 of approximately 10 µM against E. coli D22) (14). As a negative control fluorescein-labeled peptide, we used the same β-tubulin fragment as during the Western blotting, just without the addition of the spacer unit (27). The same heat shock proteins, controls, and LPS preparations were used as in the Western blotting, except DnaJ was not studied.…”

Section: Resultsmentioning

confidence: 99%

Interaction between Heat Shock Proteins and Antimicrobial Peptides

Ötvös

Me²,

Pj³

et al. 2000

Biochemistry

328

283

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Drosocin, pyrrhocoricin, and apidaecin, representing the short (18−20 amino acid residues) proline-rich antibacterial peptide family, originally isolated from insects, were shown to act on a target bacterial protein in a stereospecific manner. Native pyrrhocoricin and one of its analogues designed for this purpose protect mice from bacterial challenge and, therefore, may represent alternatives to existing antimicrobial drugs. Furthermore, this mode of action can be a basis for the design of a completely novel set of antibacterial compounds, peptidic or peptidomimetic, if the interacting bacterial biopolymers are known. Recently, apidaecin was shown to enter Escherichia coli and subsequently kill bacteria through sequential interactions with diverse target macromolecules. In this paper report, we used biotin- and fluorescein-labeled pyrrhocoricin, drosocin, and apidaecin analogues to identify biopolymers that bind to these peptides and are potentially involved in the above-mentioned multistep killing process. Through use of a biotin-labeled pyrrhocoricin analogue, we isolated two interacting proteins from E. coli. According to mass spectrometry, Western blot, and fluorescence polarization, the short, proline-rich peptides bound to DnaK, the 70-kDa bacterial heat shock protein, both in solution and on the solid-phase. GroEL, the 60-kDa chaperonin, also bound in solution. Control experiments with an unrelated labeled peptide showed that while binding to DnaK was specific for the antibacterial peptides, binding to GroEL was not specific for these insect sequences. The killing of bacteria and DnaK binding are related events, as an inactive pyrrhocoricin analogue made of all-d-amino acids failed to bind. The pharmaceutical potential of the insect antibacterial peptides is underscored by the fact that pyrrhocoricin did not bind to Hsp70, the human equivalent of DnaK. Competition assay with unlabeled pyrrhocoricin indicated differences in GroEL and DnaK binding and a probable two-site interaction with DnaK. In addition, all three antibacterial peptides strongly interacted with two bacterial lipopolysaccharide (LPS) preparations in solution, indicating that the initial step of the bacterial killing cascade proceeds through LPS-mediated cell entry.

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Section: Resultsmentioning

confidence: 99%

Interaction between Heat Shock Proteins and Antimicrobial Peptides

Ötvös

Me²,

Pj³

et al. 2000

Biochemistry

328

283

View full text Add to dashboard Cite

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“…Other peptides included the negative control conantokin G, an N-methyl-D-aspartate (NMDA) receptor antagonist (13); pyrrhocoricin made of all D-amino acids; magainin 2, an antibacterial peptide that kills bacteria by disintegrating the membrane (14); cecropin A, another membrane-active antimicrobial peptide (15); buforin II, an antibacterial peptide that binds to bacterial DNA (5); the N-terminal 1-9 and C-terminal 10-20 halves of pyrrhocoricin; biotin-labeled Land D-pyrrhocoricin (12); fluorescein-labeled pyrrhocoricin, drosocin, and apidaecin (11); the pyrrhocoricin two halves also labeled with fluorescein; fluorescein-and biotin-labeled β-tubulin fragment 434-445 serving as negative controls (16,17); and another negative control fluorescein-labeled peptide with the sequence NTDGSTDYGILQINSR.…”

Section: Syntheticmentioning

confidence: 99%

The Antibacterial Peptide Pyrrhocoricin Inhibits the ATPase Actions of DnaK and Prevents Chaperone-Assisted Protein Folding

et al. 2001

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Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat shock protein DnaK, and peptide binding to DnaK can be correlated with antimicrobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to Escherichia coli DnaK. Biologically active pyrrhocoricin made of L-amino acids diminished the ATPase activity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit the DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP). The effect of pyrrhocoricin on DnaK's other significant biological function, the refolding of misfolded proteins, was studied by assaying the alkaline phosphatase and beta-galactosidase activity of live bacteria. Remarkably, both enzyme activities were reduced upon incubation with L-pyrrhocoricin or drosocin. D-Pyrrhocoricin, magainin 2, or buforin II, an antimicrobial peptide involved in binding to bacterial nucleic acids, had only negligible effect. According to fluorescence polarization and dot blot analysis of synthetic DnaK fragments and labeled pyrrhocoricin analogues, pyrrhocoricin bound with a K(d) of 50.8 microM to the hinge region around the C-terminal helices D and E, at the vicinity of amino acids 583 and 615. Pyrrhocoricin binding was not observed to the homologous DnaK fragment of Staphylococcus aureus, a pyrrhocoricin nonresponsive strain. In line with the lack of ATPase inhibition, drosocin binding appears to be slightly shifted toward the D helix. Our data suggest that drosocin and pyrrhocoricin binding prevents the frequent opening and closing of the multihelical lid over the peptide-binding pocket of DnaK, permanently closes the cavity, and inhibits chaperone-assisted protein folding. The biochemical results were strongly supported by molecular modeling of DnaK-pyrrhocoricin interactions. Due to the prominent sequence variations of procaryotic and eucaryotic DnaK molecules in the multihelical lid region, our findings pave the road for the design of strain-specific antibacterial peptides and peptidomimetics. Far-fetched applications of the species-specific inhibition of chaperone-assisted protein folding include the control of not only bacteria but also fungi, parasites, insects, and perhaps rodents.

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Chemical and biological consequences of sugar incorporation into a potential N-glycosylation site in the tubulin-binding repeat of tau protein

Cited by 2 publications

References 6 publications

Interaction between Heat Shock Proteins and Antimicrobial Peptides

Interaction between Heat Shock Proteins and Antimicrobial Peptides

The Antibacterial Peptide Pyrrhocoricin Inhibits the ATPase Actions of DnaK and Prevents Chaperone-Assisted Protein Folding

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