Internal Lysine Palmitoylation in Adenylate Cyclase Toxin from
            <i>Bordetella pertussis</i>

Hackett, Murray; Guo, Lin; Shabanowitz, Jeffrey; Hunt, Donald F.; Hewlett, Erik L.

doi:10.1126/science.7939682

Cited by 224 publications

(207 citation statements)

References 34 publications

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“…There have been few reports describing MS/MS characterization of acylated proteins or peptides. In identifying the novel palmitoylations on internal lysine residues in adenylate cyclase toxins (ACT) [34,35], Hackett and coworkers showed that the amide-linked palmitoyl groups are retained on the backbone fragments (b, y) during CAD in a triplequadrupole mass spectrometer.…”

Section: Ecd Versus Cadmentioning

confidence: 99%

Identification and localization of the fatty acid modification in ghrelin by electron capture dissociation

Guan

2002

J. Am. Soc. Mass Spectrom.

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Electron capture dissociation (ECD) has been demonstrated to be an effective fragmentation technique for characterizing the site and structure of the fatty acid modification in ghrelin, a 28-residue growth-hormone-releasing peptide that has an unusual ester-linked n-octanoyl (C8:0) modification at Ser-3. ECD cleaves 21 of 23 possible backbone amine bonds, with the product ions (c and z⅐ ions) covering a greater amino acid sequence than those obtained by collisionally activated dissociation (CAD). Consistent with the ECD nonergodic mechanism, the ester-linked octanoyl group is retained on all backbone cleavage product ions, allowing for direct localization of this labile modification. In addition, ECD also induces the ester bond cleavage to cause the loss of octanoic acid from the ghrelin molecular ion; the elimination process is initiated by the capture of an electron at the protonated ester group, which is followed by the radical-site-initiated reaction known as ␣-cleavage. emerged as a promising new tool for tandem mass spectrometry (MS/MS) of multiply charged (protonated) proteins and peptides generated by electrospray ionization (ESI) [6] using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry [7,8]. ECD typically causes extensive fragmentation of backbone amine bonds to produce c and z⅐ fragment ions, in contrast to the b and y fragment ions produced from the amide bond cleavage by conventional energetic fragmentation methods, such as collisionally activated dissociation (CAD) [9 -14], surface-induced dissociation (SID) [15], infrared multiphoton dissociation (IRMPD) [16], blackbody infrared dissociation (BIRD) [17,18]. Although c and z⅐ ions were initially observed in the 193 nm ultraviolet photodissociation (UVPD) mass spectra of multiply charged protein ions [19], they were the products of unexpected ECD processes: The low-energy electrons were ejected from metal surfaces by 193 nm photons (6.4 eV), and were captured by the protein ions trapped in a FTICR cell [1]. In addition, ECD is a nonergodic process (i.e., cleavage happens prior to energy randomization); the ECD backbone cleavages impart very little internal energy into the fragments, as a result, the labile side-chain modifications can be retained on the backbone fragment ions. This is in sharp contrast to the conventional ergodic fragmentation methods that typically eject the labile modifications before cleaving the backbone bonds. These unique attributes of ECD in providing extensive, nonergodic peptide backbone fragmentation have been demonstrated to be advantageous for localizing labile posttranslational modifications, such as ␥-carboxylation [20], sulfonation [20], glycosylation [21][22][23], and phosphorylation [24,25].In this study we examine ECD of ghrelin, a posttranslationally acylated peptide hormone that has an unusual C8:0 acylation (octanoic acid modification) at Ser-3 residue [26,27]. The ester-linked C8:0 fatty acyl moiety is essential for the activities of ghrelin, which include growth hormone secretion, feeding ...

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Section: Ecd Versus Cadmentioning

confidence: 99%

Identification and localization of the fatty acid modification in ghrelin by electron capture dissociation

Guan

2002

J. Am. Soc. Mass Spectrom.

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“…In support of this decision, studies of other proteins have shown that sites of palmitoylation in vivo and in vitro are equivalent (Bizzozero et al, 1987;Gutierrez and Magee, 1991;Stanley et al, 1994). Different methodologies for protein structure analysis have been used to identify palmitoylated amino acids (Papac et al, 1992;Weimbs and Stoffel, 1992;Bizzozero et al, 1994a;Hackett et al, 1994 (Ozols, 1984), necessitating the use of gel filtration on Sephadex LH60. Similar problems with recovery of palmitoylated peptides from HPLC columns have been described in other studies (Ovchinnikov et al, 1988 Palmitoylated Cysteine Residues of ea-Tubulin Luduefia and Roach (1981) determined that nondenatured a-and ,B-tubulin from brain, in the form of heterodimers, each contain three or four cysteine residues that are accessible for alkylation, implying that these cysteine residues are at the surface of the molecules.…”

Section: Proteinsmentioning

confidence: 99%

“…Cysteine residues are the primary sites of palmitoylation. However, serine, threonine, and lysine residues may also be palmitoylated (for review, see Bizzozero et al, 1994b;Hackett et al, 1994;Stanley et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation.

Ozols

Caron

1997

MBoC

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As shown in the companion article, tubulin is posttranslationally modified in vivo by palmitoylation. Our goal in this study was to identify the palmitoylation sites by protein structure analysis. To obtain quantities of palmitoylated tubulin required for this analysis, a cell-free system for enzymatic [3Hlpalmitoylation was developed and characterized in our companion article. We then developed a methodology to examine directly the palmitoylation of all 451 amino acids of a-tubulin. 3H-labeled palmitoylated a-tubulin was cleaved with cyanogen bromide (CNBr). The CNBr digest was resolved according to Veptide size by gel filtration on Sephadex LH60 in formic acid:ethanol. The position of H-labeled palmitoylated amino acids in peptides could not be identified by analysis of the Edman degradation sequencer product because the palmitoylated sequencer products were lost during the final derivatization step to phenylthiohydantoin derivatives. Modification of the gas/liquid-phase sequencer to deliver the intermediate anilinothiozolinone derivative, rather than the phenylthiohydantoin derivative, identified the cycle containing the 3H-labeled palmitoylated residue. Therefore, structure analysis of peptides obtained from gel filtration necessitated dual sequencer runs of radioactive peptides, one for sequence analysis and one to identify H-labeled palmitoylated amino acids. Further cleavage of the CNBr peptides by trypsin and Lys-C protease, followed by gel filtration on Sephadex LH60 and dual sequencer runs, positioned the 3H-labeled palmitoylated amino acid residues in peptides. Integration of all the available structural information led to the assignment of the palmitoyl moiety to specific residues in a-tubulin. The palmitoylated residues in a-tubulin were confined to cysteine residues only. The major site for palmitoylation was cysteine residue 376.

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“…The CyaA toxin is synthesized as an inactive precursor, proCyaA, which is converted into the active toxin form (CyaA) on specific acylation of two lysine residues (26,27). Then CyaA is secreted across the bacterial envelope by a dedicated type I secretion machinery and binds to the CD11b/CD18 integrin expressed by a subset of leukocytes including neutrophils, macrophages, and dendritic cells (22,(28)(29)(30).…”

mentioning

confidence: 99%

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

Veneziano

Rossi

Chenal

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Significance Many bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, although how they pass through a lipid bilayer remains largely unknown. Bordetella pertussis adenylate cyclase (CyaA) toxin delivers its catalytic domain directly across the cell membrane. To characterize this unique translocation process, we designed an in vitro assay based on a tethered lipid bilayer assembled over a biosensor surface derivatized with calmodulin, a natural activator of the toxin. CyaA activation by calmodulin provided a highly sensitive readout for toxin translocation across the bilayer. CyaA translocation was calcium- and membrane potential-dependent but independent of any additional eukaryotic protein. This biomimetic membrane will permit in vitro studies of protein translocation in precisely defined conditions.

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Internal Lysine Palmitoylation in Adenylate Cyclase Toxin from Bordetella pertussis

Cited by 224 publications

References 34 publications

Identification and localization of the fatty acid modification in ghrelin by electron capture dissociation

Identification and localization of the fatty acid modification in ghrelin by electron capture dissociation

Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation.

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

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