Dataset Information

Modification of heterotrimeric G-proteins in Swiss 3T3 cells stimulated with Pasteurella multocida toxin.

ABSTRACT: Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G(q), G(12) and G(i). PMT has been shown by others to lead to the deamidation of recombinant G?(i) at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native G? subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the G? subunit of the G(q) and G(i) families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these G? subunits. Surprisingly, PMT also induced a similar modification of G?(11), a member of the G(q) family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of G?(13) was observed following PMT treatment of cells, suggesting differential modification of this G? subunit by PMT. G(s) was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated G?(i), but not G?(q). We also show that PMT inhibits the GTPase activity of G(q).

SUBMITTER: Babb RC

PROVIDER: S-EPMC3489841 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Modification of heterotrimeric G-proteins in Swiss 3T3 cells stimulated with Pasteurella multocida toxin.

Babb Rebecca C RC Homer Karen A KA Robbins Jon J Lax Alistair J AJ

PloS one 20121105 11

Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G(q), G(12) and G(i). PMT has been shown by others to lead to the deamidation of recombinant Gα(i) at Gln-205 to inhibit its intrinsic GTPa ...[more]

PMID: 23144805

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Project description:Bone remodeling is a continuous process to retain the structural integrity and function of the skeleton. A tight coupling is maintained between osteoclast-mediated resorption of old or damaged bones and osteoblast-mediated formation of new bones for bone homeostasis. While osteoblasts differentiate from mesenchymal stem cells, osteoclasts are hematopoietic in origin and derived from myeloid precursor cells. Osteoclast differentiation is driven by two cytokines, cytokine receptor activator of NF-κB ligand (RANKL), and macrophage colony-stimulating factor. Imbalances in the activity of osteoblasts and osteoclasts result in the development of bone disorders. Bacterially caused porcine atrophic rhinitis is characterized by a loss of nasal ventral conche bones and a distortion of the snout. While Bordetella bronchiseptica strains cause mild and reversible symptoms, infection of pigs with toxigenic Pasteurella multocida strains causes a severe and irreversible decay. The responsible virulence factor Pasteurella multocida toxin (PMT) contains a deamidase activity in its catalytical domain that constitutively activates specific heterotrimeric G proteins to induce downstream signaling cascades. While osteoblasts are inhibited by the toxin, osteoclasts are activated, thus skewing bone remodeling toward excessive bone degradation. Still, the mechanism by which PMT interferes with bone homeostasis, and the reason for this unusual target tissue is not yet well understood. Here, we show that PMT has the potential to differentiate bone marrow-derived macrophages into functional osteoclasts. This toxin-mediated differentiation process is independent of RANKL, a cytokine believed to be indispensable for triggering osteoclastogenesis, as addition of osteoprotegerin to PMT-treated macrophages does not show any effect on PMT-induced osteoclast formation. Although RANKL is not a prerequisite, toxin-primed macrophages show enhanced responsiveness to low concentrations of RANKL, suggesting that the PMT-generated microenvironment offers conditions where low concentrations of RANKL lead to an increase in the number of osteoclasts resulting in increased resorption. PMT-mediated release of the osteoclastogenic cytokines such as IL-6 and TNF-α, but not IL-1, supports the differentiation process. Although the production of cytokines and the subsequent activation of signaling cascades are necessary for PMT-mediated differentiation into osteoclasts, they are not sufficient and PMT-induced activation of G protein signaling is essential for efficient osteoclastogenesis.

Dataset Information

Modification of heterotrimeric G-proteins in Swiss 3T3 cells stimulated with Pasteurella multocida toxin.

Publications

Modification of heterotrimeric G-proteins in Swiss 3T3 cells stimulated with Pasteurella multocida toxin.

Similar Datasets

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