The Biochemistry of Hemolysin Toxin Activation:  Characterization of HlyC, an Internal Protein Acyltransferase

Trent, M. Stephen; Worsham, Lesa M.S.; Ernst-Fonberg, Mary Lou

doi:10.1021/bi971588y

Cited by 37 publications

(132 citation statements)

References 40 publications

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“…In general, HlyC has been estimated to be a single protein that participates in the acylation of HlyA hemolysin secreted by E. coli strains (23). Electrophoretic separation in combination with immunochemical detection, MALDI-mass spectrometry, and peptide mass mapping demonstrated that the four protein spots correspond to HlyC isoforms.…”

Section: Discussionmentioning

confidence: 99%

“…Based on these results and our data, we postulate that a conformational change of HlyC occurs upon interaction with pro-HlyA, generating specific sites for different protease systems to process the active HlyC proteins. Considering that activation of one molecule of pro-HlyA seems to require one molecule of HlyC (16,22,23), it is likely that degradation of HlyC is necessary to prevent its intracellular accumulation as HlyA is continuously secreted throughout growth.…”

Section: Fig 7 Model For Hlyc Degradationmentioning

confidence: 99%

“…At present, only HlyC from E. coli and CyaC from B. pertussis have been shown to be involved in the fatty acylation of their corresponding RTX toxin (13,21). Recently, a novel type of fatty acyltransferase activity, that catalyzes the formation of an internal protein amide bond has been described for HlyC (22,23), despite the fact that HlyC does not show any significant similarity to known acyl transferases (13,19), the protein seems to acylate HlyA in an equimolar fashion, and it appears to be functionally consumed during the reaction (16,22). In this respect, it is also notable that HlyC and HlyA are encoded in the same mRNA and therefore synthesized at nearly equimolar amounts (24,25).…”

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confidence: 99%

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In Vivo Proteolytic Degradation of the Escherichia coli Acyltransferase HlyC

Guzmán-Verri

Chaves-Olarte

Garcı́a

et al. 2001

Journal of Biological Chemistry

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Escherichia coli hemolysin (HlyA) is the prototype toxin of a major family of exoproteins produced by Gram-negative bacteria known as "repeats in toxins." Only fatty acid-acylated HlyA molecules at residues Lys 564 and Lys 690 are able to damage the target cell membrane. Fatty acylation of pro-HlyA is dependent on the co-synthesized acyltransferase HlyC and the acylated form of acyl-carrier protein. By using a collection of hlyA and hlyC mutant strains, the processing of HlyC was investigated. HlyC was not detected by Western blot in an E. coli strain encoding hlyC and hlyA, but it was present in a strain encoding only hlyC. The hlyC mRNA pattern, however, was similar in both strains indicating that the turnover of HlyC does not occur at the transcriptional level. HlyC was detected in Western blots of cell lysates from an E. coli strain encoding HlyC and a HlyA derivative where both acylation sites were substituted. Similar results were obtained when HlyC was expressed in a hlyA mutant strain lacking part of a putative HlyC binding domain, indicating that this particular HlyA region affects HlyC stability. We did not detect HlyC in cell lysates from hlyC mutants with different abilities to acylate pro-HlyA, suggesting that the degradation of HlyC is not related to the HlyA acylation process. The protease systems ClpAP, ClpXP, and FtsH were found to be responsible for the HlyA-dependent processing of HlyC.

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Section: Discussionmentioning

confidence: 99%

Section: Fig 7 Model For Hlyc Degradationmentioning

confidence: 99%

mentioning

confidence: 99%

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In Vivo Proteolytic Degradation of the Escherichia coli Acyltransferase HlyC

Guzmán-Verri

Chaves-Olarte

Garcı́a

et al. 2001

Journal of Biological Chemistry

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“…HlyC uses the fatty-acyl residues carried by acylcarrier protein (ACP) to form a covalent acyl-HlyC intermediate, which species then transfers the fatty-acyl residues to the ε-amino groups of the Lys 564 and Lys 690 residues of proHlyA (Worsham et al, 2001(Worsham et al, , 2005. ACPs carrying various fatty-acyl residuesincluding palmitate (16:0) and palmitoleate (16:1), the most common in E. coli-could be efficiently used in vitro as acyl donors for the modification of HlyA ( (Issartel et al, 1991), (Trent et al, 1998)). In vivo, however, HlyC exhibits a high selectivity for myristic acid (14:0), which species was found to constitute about 68% of the acyl chains covalently linked to Lys 564 and Lys 690 of the native HlyA (Lim et al, 2000).…”

Section: The Posttranslational Activation Of Hlyamentioning

confidence: 99%

E. coli Alpha Hemolysin and Properties

Bakás¹,

Sabina²,

Romina³

et al. 2012

Biochemistry

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“…A model of the reaction mechanism was proposed, and formation of an intermediary acyl-ACP⅐HlyC complex was demonstrated (22-25). Various acyl-ACP-carrying fatty acids, including the most common in E. coli, the palmitoyl (C16:0) and pamitoleil (C16:1) residues, were found to be efficiently used in vitro as acyl donors for modification of HlyA (18,26). In vivo, however, HlyC exhibits a high selectivity for C14:0 myristic acid, which represents only about 2% of total E. coli fatty acids but was found to constitute about 68% of the acyl chains covalently linked to lysines 564 and 690 of native HlyA (27).…”

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confidence: 99%

Acylation of Lysine 983 Is Sufficient for Toxin Activity of Bordetella pertussis Adenylate Cyclase

Basar¹,

Havlı́ček²,

Bezoušková³

et al. 2001

Journal of Biological Chemistry

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The capacity of adenylate cyclase toxin (ACT) to penetrate into target cells depends on post-translational fatty-acylation by the acyltransferase CyaC, which can palmitoylate the conserved lysines 983 and 860 of ACT. Here, the in vivo acylating capacity of a set of mutated CyaC acyltransferases was characterized by two-dimensional gel electrophoresis and mass spectrometric analyses of the ACT product. Substitutions of the potentially catalytic serine 20 and histidine 33 residues ablated acylating activity of CyaC. Conservative replacements of alanine 140 by glycine (A140G) and valine (A140V) residues, however, affected selectivity of CyaC for the two acylation sites on ACT. Activation by the A140G variant of CyaC generated a mixture of bi-and monoacylated ACT molecules, modified either at both Lys-860 and Lys-983, or only at Lys-860, respectively. In contrast, the A140V CyaC produced a nearly 1:1 mixture of nonacylated pro-ACT with ACT monoacylated almost exclusively at Lys-983. The respective proportion of toxin molecules acylated at Lys-983 correlated well with the cell-invasive activity of both ACT mixtures, which was about half of that of ACT fully acylated on Lys-983 by intact CyaC. These results show that acylation of Lys-860 alone does not confer cell-invasive activity on ACT, whereas acylation of Lys-983 is necessary and sufficient.The whooping cough agent, Bordetella pertussis, secretes a 1706-residue-long RTX 1 adenylate cyclase toxin-hemolysin (ACT, AC-Hly, or CyaA), which can invade a variety of eukaryotic cells (1, 2). ACT delivers into cells a catalytic adenylate cyclase domain (AC) that is activated by intracellular calmodulin and catalyzes unregulated conversion of ATP to cAMP (3-6). This impairs microbicidal functions of immune effector cells and induces apoptosis of lung macrophages (7,8). In addition, ACT has the capacity to form small cation-selective membrane channels that account for its weak hemolytic activity (7-13).The capacity of ACT to form hemolytic channels and to penetrate target cell membranes and deliver the AC domain (cell-invasive activity) depends on a covalent post-translational fatty-acyl modification (14 -16). This is catalyzed by a dedicated protein acyltransferase, CyaC, which can acylate the ⑀-amino groups of two internal lysine residues of ACT, Lys-983 and Lys-860, located within conserved RTX acylation sites (14 -17). The mechanism of this novel type of protein acylation was recently analyzed in substantial detail for the prototype RTX toxin-activating and acyl-ACP-dependent protein acyltransferase HlyC, which acylates the homologous lysines 564 and 690 of the Escherichia coli ␣-hemolysin HlyA (18 -21). Several residues, including Ser-20 and His-23, were identified as being potentially involved in acyl transfer catalysis by . A model of the reaction mechanism was proposed, and formation of an intermediary acyl-ACP⅐HlyC complex was demonstrated (22-25). Various acyl-ACP-carrying fatty acids, including the most common in E. coli, the palmitoyl (C16:0) and pamitoleil (C16:...

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The Biochemistry of Hemolysin Toxin Activation: Characterization of HlyC, an Internal Protein Acyltransferase

Cited by 37 publications

References 40 publications

In Vivo Proteolytic Degradation of the Escherichia coli Acyltransferase HlyC

In Vivo Proteolytic Degradation of the Escherichia coli Acyltransferase HlyC

E. coli Alpha Hemolysin and Properties

Acylation of Lysine 983 Is Sufficient for Toxin Activity of Bordetella pertussis Adenylate Cyclase

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