RACK1 Protein Interacts with Helicobacter pylori VacA Cytotoxin: The Yeast Two-Hybrid Approach

doi:10.1006/bbrc.2001.5950

Biochemical and Biophysical Research Communications

Volume 289, Issue 1, 23 November 2001, Pages 103-110

https://doi.org/10.1006/bbrc.2001.5950 Get rights and content

Abstract

The VacA toxin is the major virulence factor of Helicobacter pylori. The studies on VacA intracellular expression suggest that it interacts with cytosolic proteins and that this interaction contributes significantly to vacuolization. The aim of this study was to identify the host protein(s) that interacts with the VacA protein. We used the fragments of VacA protein fused with GAL4-BD as the baits in the yeast two-hybrid approach. The yeast transformed with plasmids encoding bait proteins were screened with human gastric mucosa cDNA library, encoded C-terminal fusion proteins with GAL4-AD. Three independent His-β-Gal-positive clones were identified in VacA-b1 screen; they matched two different lengths of cDNA encoding RACK1 protein. The specific activity of β-galactosidase found in the yeast expressing both VacA-b1 and RACK1 fusion proteins was 12–19 times higher compared to all negative controls used. VacA is capable of binding the RACK1 in vitro as was confirmed by the pull-down assay with GST fusion VacA protein and [³⁵S]Met-labeled RACK1 protein fragments.

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In ribosomes, RACK1 is required for efficient global translation via the recruitment of eIF4E to 40S, for the release of eIF6 from 60S to promote binding between 40S and 60S [73] and for the phosphorylation of eIF4G mediated by PKCβII [76,77]. In addition, numerous proteins, including other kinases, such as SRC and JNK, have been reported to bind RACK1, allowing participation in different cellular processes, from autophagy to cell adhesion [78–98]. RACK1 has been linked to the activity of P53/P73, proteins involved in the control of the cell cycle and carcinogenesis as transcription factors for key cell cycle genes, including some ribonuclear proteins that are related to RACK1 binding [90].
Tight control of mRNA expression is required for cell differentiation; imbalanced regulation may lead to developmental disorders and cancer. The activity of the translational machinery (including ribosomes and translation factors) regulates the rate (slow or fast) of translation of encoded proteins, and the quality of these proteins highly depends on which mRNAs are available for translation. Specific RNA-binding and ribosomal proteins seem to play a key role in controlling gene expression to determine the differentiation fate of the cell. This demonstrates the important role of RNA-binding proteins, specific ribosome-binding proteins and microRNAs as key molecules in controlling the specific proteins required for the differentiation or dedifferentiation of cells. This delicate balance between specific proteins (in terms of quality and availability) and post-translational modifications occurring in the cytoplasm is crucial for cell differentiation, dedifferentiation and oncogenic potential. In this review, we report how defects in the regulation of mRNA translation can be dependent on specific proteins and can induce an imbalance between differentiation and dedifferentiation in cell fate determination.
Downregulation of tumor suppressor RACK1 by Helicobacter pylori infection promotes gastric carcinogenesis through the integrin β-1/NF-κB signaling pathway
2019, Cancer Letters
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H. pylori is a major risk factor for the development of GC, and it has been estimated that at least 90% of GC is attributable to H. pylori infection [6]. CagA and VacA are thought to be the major virulence factors of H. pylori [30], and a previous study confirmed that the RACK1 protein interacts with H. pylori VacA [31]. Our in vitro results showed that H. pylori infection led to decreased RACK1 expression in a time- and MOI-dependent manner, however, this effect was independent of the virulence factors CagA and VacA.
Receptor of activated protein kinase C 1 (RACK1) is downregulated in gastric cancer and is involved in modulating NF-κB signaling pathway activity. However, the underlying molecular mechanisms regulating RACK1 expression are unclear. In this study, we demonstrated that downregulated expression of RACK1 was observed in gastric cancer tissue compared to adjacent normal tissue and was correlated with poor prognosis in patients. Helicobacter pylori (H. pylori) infection downregulated RACK1 expression in concert with canonical NF-κB signaling pathway activation in vivo and in vitro. RACK1 overexpression suppressed NF-κB signaling pathway activation as well as the release of downstream proinflammatory cytokines. In addition, RACK1 downregulation increased integrin β-1 expression, while integrin β-1 silencing decreased NF-κB signaling activation. Moreover, H. pylori infection downregulated RACK1 but upregulated integrin β-1 expression at the precancerous lesion stages in human subjects. Our data indicate that H. pylori infection promotes the upregulation of integrin β-1 expression via downregulation of RACK1 expression, which subsequently leads to the elevated activation of the NF-κB signaling pathway, an essential step in H. pylori-induced carcinogenesis.
Helicobacter pylori vacuolating toxin
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The vacuolating toxin VacA is a key virulence factor of the Gram-negative bacterium Helicobacter pylori, which colonizes the stomachs of approximately 50% of the world’s populations. H. pylori infection causes chronic gastric inflammation that can evolve into more severe diseases, such as atrophic gastritis, peptic ulcer, gastric lymphoma, or adenocarcinoma. VacA is currently classified as a nonconventional pore-forming toxin. Although not all its biological effects seem to be dependent on its channel activity, VacA could be envisaged as a cell-invasive chloride channel or even a prototype of a new class of intracellular-acting A–B toxins in which the A subunit exhibits pore-forming instead of enzymatic activity. This chapter discusses the current knowledge about VacA genetic and production, clinical relevance, structure, mechanisms of interaction with host cells that lead to its different biological activities, and functional relationship with another key virulence factor of H. pylori, CagA.
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VacA toxin from the cancer-inducing bacterium Helicobacter pylori is currently classified as a pore-forming toxin but is also considered a multifunctional toxin, apparently causing many pleiotropic cell effects. However, an increasing body of evidence suggests that VacA could be the prototype of a new class of monofunctional A–B toxins in which the A subunit exhibits pore-forming instead of enzymatic activity. Thus, VacA may use a peculiar mechanism of action, allowing it to intoxicate the human stomach. By combining the action of a cell-binding domain, a specific intracellular trafficking pathway and a novel mitochondrion-targeting sequence, the VacA pore-forming domain is selectively delivered to the inner mitochondrial membrane to efficiently kill target epithelial cells by apoptosis.
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The receptor for activated protein kinase C 1 (RACK1) is an intracellular adaptor protein. Accumulating evidence attributes to this member of the tryptophan-aspartate (WD) repeat family the role of regulating several major nervous system pathways. Structurally, RACK1 is a seven-bladed-β-propeller, interacting with diverse proteins having distinct structural folds. When bound to the IP₃ receptor, RACK1 regulates intracellular Ca²⁺ levels, potentially contributing to processes such as learning, memory and synaptic plasticity. By binding to the NMDA receptor, it dictates neuronal excitation and sensitivity to ethanol. When bound to the stress-induced acetylcholinesterase variant AChE-R, RACK1 is implicated in stress responses and behavior, compatible with reports of RACK1 modulations in brain ageing and in various neurodegenerative diseases. This review sheds new light on both the virtues and the variety of neuronal RACK1 interactions and their physiological consequences.
Helicobacter pylori vacuolating toxin
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¹: To whom the correspondence and reprint requests should be addressed at Department of Gastroenterology, Medical Center for Postgraduate Education in Cancer Center, Roentgena 5 str, 02-781 Warsaw, Poland. Fax: (48-22) 644-7601. E-mail: [email protected].

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Regular ArticleRACK1 Protein Interacts with Helicobacter pylori VacA Cytotoxin: The Yeast Two-Hybrid Approach

Abstract

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

FEBS Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biophys. J.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Helicobacter pylori and the pathogenesis of gastroduodenal inflammation

J. Infect. Dis.

Helicobacter pylori

Am. J. Gastroenterol.

Helicobacter pylori: A bacterial cause of gastritis, peptic ulcer disease, and gastric cancer

Am. Soc. Microbiol. News

Gastric carcinoma and gastric lymphoma

Curr. Opinion Gastroenterol.

Gene structure of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease

J. Exp. Med.

Genetic analysis of the Helicobacter pylori vacuolating cytotoxin: Structural similarities with the IgA protease type of exporter protein

Mol. Microbiol.

Oligomeric and subunit structure of the Helicobacter pylori vacuolating cytotoxin

J. Cell Biol.

Carboxy-terminal proteolytic processing of Helicobacter pylori vacuolating toxin

Infect. Immun.

Regular Article
RACK1 Protein Interacts with Helicobacter pylori VacA Cytotoxin: The Yeast Two-Hybrid Approach