Preface

Coligan, John E.; Dunn, Ben M.; Ploegh, Hidde L.; Speicher, David W.; Wingfield, Paul T.

doi:10.1002/0471140864.psprefs24

Cited by 94 publications

(117 citation statements)

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“…Standard SDS͞PAGE was performed according to Laemmli (15). Proteins were detected by the Coomassie G or silver staining methods (16). Total cell lysates for two-dimensional gel electrophoresis (2DGE) were obtained from overnight cultures.…”

Section: Methodsmentioning

confidence: 99%

“…First dimensions were performed on Immobiline DryStrip gels (Pharmacia Biotech) according to the manufacturer's instructions. After second-dimension separation by SDS͞PAGE and silver staining (16), gels were dried, scanned, and compared using Z3 software (Compugen, Jamesburg, NJ). For comparative purposes, at least five independent 2DGE analyses were performed with each material.…”

Section: Methodsmentioning

confidence: 99%

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The two-component system BvrR/BvrS essential for Brucella abortus virulence regulates the expression of outer membrane proteins with counterparts in members of the Rhizobiaceae

Guzmán-Verri

Manterola

Sola‐Landa

et al. 2002

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

157

167

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The Brucella BvrR͞BvrS two-component regulatory system is homologous to the ChvI͞ChvG systems of Sinorhizobium meliloti and Agrobacterium tumefaciens necessary for endosymbiosis and pathogenicity in plants. BvrR͞BvrS controls cell invasion and intracellular survival. Probing the surface of bvrR and bvrS transposon mutants with monoclonal antibodies showed all described major outer membrane proteins (Omps) but Omp25, a protein known to be involved in Brucella virulence. Absence of Omp25 expression was confirmed by two-dimensional electrophoresis of envelope fractions and by gene reporter studies. The electrophoretic analysis also revealed reduction or absence in the mutants of a second set of protein spots that by matrix-assisted laser desorption ionization MS and peptide mass mapping were identified as a non-previously described Omp (Omp3b). Because bvrR and bvrS mutants are also altered in cell-surface hydrophobicity, permeability, and sensitivity to surface-targeted bactericidal peptides, it is proposed that BvrR͞BvrS controls cell envelope changes necessary to transit between extracellular and intracellular environments. A genomic search revealed that Omp25 (Omp3a) and Omp3b belong to a family of Omps of plant and animal cellassociated ␣-Proteobacteria, which includes Rhizobium leguminosarum RopB and A. tumefaciens AopB. Previous work has shown that RopB is not expressed in bacteroids, that AopB is involved in tumorigenesis, and that dysfunction of A. tumefaciens ChvI͞ChvG alters surface properties. It is thus proposed that the BvrR͞BvrS and Omp3 homologues of the cell-associated ␣-Proteobacteria play a role in bacterial surface control and host cell interactions.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The two-component system BvrR/BvrS essential for Brucella abortus virulence regulates the expression of outer membrane proteins with counterparts in members of the Rhizobiaceae

Guzmán-Verri

Manterola

Sola‐Landa

et al. 2002

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

157

167

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“…The mutant protein would be expected to undergo a mobility shift in the opposite direction, because abnormal retardation of migration on SDS-PAGE is usually considered to be associated with a high proportion of basic amino acids (49). Although the reason for this discrepancy is presently unknown, it may be due to intramolecular hydrophobic interactions, which can sometimes affect the mobility of proteins in SDS-PAGE (49 (38,39). Both results strongly support the notion that the OPCA motif in PKC PB1 interacts with the conserved lysine residue in ZIP/ p62 PB1 as a typical PB1-PB1 interaction.…”

Section: Identification Of the Apkc Pb1 Domain Boundaries Required Fomentioning

confidence: 99%

Solution Structure of Atypical Protein Kinase C PB1 Domain and Its Mode of Interaction with ZIP/p62 and MEK5

Hirano

Yoshinaga

Ogura

et al. 2004

Journal of Biological Chemistry

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Atypical protein kinase C (aPKC) has been implicated in several signaling pathways such as cell polarity, cell survival, and cell differentiation. In contrast to other PKCs, aPKC is unique in having the PB1 (Phox and Bem 1) domain in the N terminus. The aPKC PB1 domain binds with ZIP/p62, Par6, or MEK5 through a PB1-PB1 domain interaction that controls the localization of aPKC. Here, we determined the three-dimensional structure of the PB1 domain of PKC by NMR and found that the PB1 domain adopts a ubiquitin fold. The OPCA (OPR, PC, and AID) motif inserted into the ubiquitin fold was presented as a ␤␤␣ fold in which the side chains of conserved Asp residues were oriented to the same direction to form an acidic surface. This structural feature suggested that the acidic surface of the PKC PB1 domain interacted with the basic surface of the target PB1 domains, and this was confirmed in the case of the PKC -ZIP/p62 complex by mutational analysis. Interestingly, in the PKC PB1 domain a conserved lysine residue was located on the side opposite to the OPCA motifpresenting surface, suggesting dual roles for the PKC PB1 domain in that it could interact with either the conserved lysine residue or the acidic residues on the OPCA motif of the target PB1 domains.As part of the ongoing study of atypical protein kinase C (aPKC), 1 isoform PKC was first cloned in 1988 (1), followed by the cloning of isoform PKC / (2, 3). Now, aPKC has been suggested as playing a crucial role in signal transduction pathways such as cell polarity, cell differentiation, and cell survival (4, 5). In contrast to conventional PKC, aPKC contains only a single C1 domain but is devoid of a C2 domain in the Nterminal regulatory region. A tandem repeat of the C1 domain is known to be a target of diacylglycerol, and the C2 domain is a binding site for Ca 2ϩ . However, because of the lack of a C2 domain and the incomplete C1 domain in aPKC, neither Ca 2ϩ nor diacylglycerol activates aPKC. Instead, aPKC contains a PB1 domain at the N terminus.In PKC signaling, the regulation of both the catalytic activity and the spatial localization of PKC is essential. PKC is known to translocate to a specific region in response to activation signals and then phosphorylate specific target proteins. Such spatial localization of PKC in cells is partially regulated by either PKC-binding scaffold proteins or anchoring proteins (6). In particular, aPKC binds with PB1 domain-containing proteins such as ZIP/p62 (7-9), Par6 (10 -13), or MEK5 (14) through the PB1-PB1 domain interaction. Thus, the PB1-mediated interaction between aPKC and the scaffolding or anchoring proteins plays a crucial role in exerting the biological functions of aPKC (4).ZIP/p62 homologues were isolated in different biological contexts as ZIP, p62, and EBIAP (7,15,16). Recently, it has been demonstrated that ZIP/p62 is involved in the NF B activation pathway in association with aPKC. ZIP/p62 is a scaffold protein of aPKC for the activation of NF B downstream of extracellular signals such as tumor necrosis fac...

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“…The samples were centrifuged at 1600 ϫ g for 5 min, and the pellet was resuspended in 1 ml of immunoprecipitation buffer. After three washing steps, samples were resuspended in 50 l of SDS-PAGE sample buffer for further analysis on a 12.5% SDS-PAGE and silver staining as described (37).…”

Section: Construction Of Recombinant Dna Expression and Purification Ofmentioning

confidence: 99%

“…The lower electrode solution was 13.6% H 3 PO 4 and the upper electrode solution, 20 mM NaOH. After isoelectric focusing separation in a miniature two-dimensional electrophoresis cell, the proteins were separated in the second dimension by SDS-PAGE and stained with Coomassie Brilliant Blue as described (33,37).…”

Section: Construction Of Recombinant Dna Expression and Purification Ofmentioning

confidence: 99%

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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Preface

Cited by 94 publications

References 0 publications

The two-component system BvrR/BvrS essential for Brucella abortus virulence regulates the expression of outer membrane proteins with counterparts in members of the Rhizobiaceae

The two-component system BvrR/BvrS essential for Brucella abortus virulence regulates the expression of outer membrane proteins with counterparts in members of the Rhizobiaceae

Solution Structure of Atypical Protein Kinase C PB1 Domain and Its Mode of Interaction with ZIP/p62 and MEK5

In Vivo Proteolytic Degradation of the Escherichia coli Acyltransferase HlyC

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