Abstract:Helicobacter pylori contributes to the development of peptic ulcers and atrophic gastritis. Furthermore, H. pylori strains carrying the cagA gene are more virulent than cagA-negative strains and are associated with the development of gastric adenocarcinoma. The cagA gene product, CagA, is translocated into gastric epithelial cells and localizes to the inner surface of the plasma membrane, in which it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif. Tyrosine-phosphorylated CagA speci… Show more
“…The structure of CagA presented here provides a clear definition of the domain organization of the large, stable N-terminal region of CagA, covering about two-thirds of the whole CagA protein. Some of these domains share different degrees of similarities with prokaryotic or eukaryotic proteins, the aim of which might be protein mimicry, which could explain how different domains of CagA can affect location of the protein in H. pylori as well as the eukaryotic target cell (26)(27)(28)(29). Another remarkable characteristic of CagA N-terminal region is its flexibility, in particular the domain D1 (residues 1-300).…”
Infection with the gastric pathogen
Helicobacter pylori
is a risk factor for the development of gastric cancer. Pathogenic strains of
H. pylori
carry a type IV secretion system (T4SS) responsible for the injection of the oncoprotein CagA into host cells.
H. pylori
and its
cag
-T4SS exploit α5β1 integrin as a receptor for CagA translocation. Injected CagA localizes to the inner leaflet of the host cell membrane, where it hijacks host cell signaling and induces cytoskeleton reorganization. Here we describe the crystal structure of the N-terminal ∼100-kDa subdomain of CagA at 3.6 Å that unveils a unique combination of folds. The core domain of the protein consists of an extended single-layer β-sheet stabilized by two independent helical subdomains. The core is followed by a long helix that forms a four-helix helical bundle with the C-terminal domain. Mapping of conserved regions in a set of CagA sequences identified four conserved surface-exposed patches (CSP1–4), which represent putative hot-spots for protein–protein interactions. The proximal part of the single-layer β-sheet, covering CSP4, is involved in specific binding of CagA to the β1 integrin, as determined by yeast two-hybrid and in vivo competition assays in
H. pylori
cell-culture infection studies. These data provide a structural basis for the first step of CagA internalization into host cells and suggest that CagA uses a previously undescribed mechanism to bind β1 integrin to mediate its own translocation.
“…The structure of CagA presented here provides a clear definition of the domain organization of the large, stable N-terminal region of CagA, covering about two-thirds of the whole CagA protein. Some of these domains share different degrees of similarities with prokaryotic or eukaryotic proteins, the aim of which might be protein mimicry, which could explain how different domains of CagA can affect location of the protein in H. pylori as well as the eukaryotic target cell (26)(27)(28)(29). Another remarkable characteristic of CagA N-terminal region is its flexibility, in particular the domain D1 (residues 1-300).…”
Infection with the gastric pathogen
Helicobacter pylori
is a risk factor for the development of gastric cancer. Pathogenic strains of
H. pylori
carry a type IV secretion system (T4SS) responsible for the injection of the oncoprotein CagA into host cells.
H. pylori
and its
cag
-T4SS exploit α5β1 integrin as a receptor for CagA translocation. Injected CagA localizes to the inner leaflet of the host cell membrane, where it hijacks host cell signaling and induces cytoskeleton reorganization. Here we describe the crystal structure of the N-terminal ∼100-kDa subdomain of CagA at 3.6 Å that unveils a unique combination of folds. The core domain of the protein consists of an extended single-layer β-sheet stabilized by two independent helical subdomains. The core is followed by a long helix that forms a four-helix helical bundle with the C-terminal domain. Mapping of conserved regions in a set of CagA sequences identified four conserved surface-exposed patches (CSP1–4), which represent putative hot-spots for protein–protein interactions. The proximal part of the single-layer β-sheet, covering CSP4, is involved in specific binding of CagA to the β1 integrin, as determined by yeast two-hybrid and in vivo competition assays in
H. pylori
cell-culture infection studies. These data provide a structural basis for the first step of CagA internalization into host cells and suggest that CagA uses a previously undescribed mechanism to bind β1 integrin to mediate its own translocation.
“…The tyrosine phosphorylation site of CagA is characterized by the Glu-Pro-IleTyr-Ala (EPIYA) motif, which is present in multiple numbers in the carboxy-terminal polymorphic region (EPIYA-repeat region) of the protein (Higashi et al, 2002a, b). On the basis of sequences flanking the EPIYA motifs, four distinct EPIYA segments, EPIYA-A, -B, -C and -D, each of which contains a single EPIYA motif, have been identified in the EPIYA-repeat region (Higashi et al, 2002b(Higashi et al, , 2005Naito et al, 2006) ( Figure 1). The representative CagA proteins of Western H. pylori isolates (Western CagA) possess the EPIYA-A and EPIYA-B segments followed by the EPIYA-C segment.…”
Section: Translocation Of H Pylori Caga Into Gastric Epithelial Cellsmentioning
“…More specifically, tyrosine-phosphorylated CagA seems to bind and deregulate the activity of Src homology 2-containing protein tyrosine phosphatase 2 via the Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D site and of carboxyl-terminal Src kinase via the EPIYA-A or EPIYA-B site (22). In parallel, CagA EPIYA motifs have been suggested to play an essential role for the tethering of CagA to the membrane in a phosphorylation-independent manner (18). Consequently, CagA variability with reference to EPIYA motifs may play an important role in H. pylori pathogenesis.…”
Cytotoxin-associated gene A (CagA) diversity with regard to EPIYA-A, -B, -C, or -D phosphorylation motifs may play an important role in Helicobacter pylori pathogenesis, and therefore determination of these motifs in H. pylori clinical isolates can become a useful prognostic tool. We propose a strategy for the accurate determination of CagA EPIYA motifs in clinical strains, based upon one-step PCR amplification using primers that flank the EPIYA coding region. We thus analyzed 135 H. pylori isolates derived from 75 adults and 60 children Greek patients. A total of 34 cases were found to be EPIYA PCR negative and were consequently verified as cagA negative by cagA-specific PCR, empty-site cagA PCR, and Western blotting. Sequencing of the remaining 101 PCR-positive amplicons confirmed that an accurate prediction of the number of EPIYA motifs on the basis of size distribution of the PCR products was feasible in all cases. Furthermore, our assay could identify closely related H. pylori subclones within the same patient, harboring different numbers of EPIYA repeats. The prevalence of CagA proteins with three EPIYA motifs (ABC) or four EPIYA motifs (ABCC) was the same within the adult and children groups. However, CagA species with more than four EPIYA motifs were observed exclusively within adults (8.6%), suggesting that CagA-positive strains may acquire additional EPIYA-C motifs throughout adulthood. Our strategy requires no initial cagA screening of the clinical isolates and can accurately predict the number of EPIYA repeats in single or multiple closely related subclones bearing different numbers of EPIYA motifs in their CagA, which may coexist within the same patient.
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