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Pathogen-mediated proteolysis of the cell death regulator RIPK1 and the host defense modulator RIPK2 in human aortic endothelial cells.

Madrigal AG, Barth K, Papadopoulos G, Genco CA - PLoS Pathog. (2012)

Bottom Line: RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2.Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed.We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Porphyromonas gingivalis is the primary etiologic agent of periodontal disease that is associated with other human chronic inflammatory diseases, including atherosclerosis. The ability of P. gingivalis to invade and persist within human aortic endothelial cells (HAEC) has been postulated to contribute to a low to moderate chronic state of inflammation, although how this is specifically achieved has not been well defined. In this study, we demonstrate that P. gingivalis infection of HAEC resulted in the rapid cleavage of receptor interacting protein 1 (RIPK1), a mediator of tumor necrosis factor (TNF) receptor-1 (TNF-R1)-induced cell activation or death, and RIPK2, a key mediator of both innate immune signaling and adaptive immunity. The cleavage of RIPK1 or RIPK2 was not observed in cells treated with apoptotic stimuli, or cells stimulated with agonists to TNF-R1, nucleotide oligomerization domain receptor 1(NOD1), NOD2, Toll-like receptor 2 (TLR2) or TLR4. P. gingivalis-induced cleavage of RIPK1 and RIPK2 was inhibited in the presence of a lysine-specific gingipain (Kgp) inhibitor. RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2. Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed. We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor. Our studies thus reveal an important role for pathogen-mediated modification of cellular kinases as a potential strategy for bacterial persistence within target host cells, which is associated with low-grade chronic inflammation, a hallmark of pathogen-mediated chronic inflammatory disorders.

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Cleavage of recombinant RIPK2 kinase by P. gingivalis in the absence of host cell proteins.P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES (none), DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase for 1 h at 37°C. Reactions were stopped by the addition of SDS-PAGE loading dye and analyzed by Western blot analysis with an antibody to the N′-terminal kinase domain of RIPK2. Top panel: reaction with recombinant protein and P. gingivalis; bottom panel: 10% of reaction prior to incubation with P. gingivalis (untreated recombinant protein, i.e., gel loading control).
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ppat-1002723-g010: Cleavage of recombinant RIPK2 kinase by P. gingivalis in the absence of host cell proteins.P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES (none), DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase for 1 h at 37°C. Reactions were stopped by the addition of SDS-PAGE loading dye and analyzed by Western blot analysis with an antibody to the N′-terminal kinase domain of RIPK2. Top panel: reaction with recombinant protein and P. gingivalis; bottom panel: 10% of reaction prior to incubation with P. gingivalis (untreated recombinant protein, i.e., gel loading control).

Mentions: To determine if P. gingivalis was capable of cleaving RIPK2 in the absence of host cell proteins, recombinant RIPK2 kinase domain (rRIPK2 kd) was incubated with P. gingivalis and analyzed by Western blot for the detection of cleavage products with an antibody targeting the N′-terminal kinase domain. As shown in Figure 10, rRIPK2 kd migrates at ∼40-kDa and was cleaved in the presence of P. gingivalis 381. Furthermore, KYT-1 partially inhibited P. gingivalis-induced cleavage of RIPK2 kd. Surprisingly, the lowest molecular weight band was of a similar sized fragment ∼20-kDa as observed in endothelial cells treated with P. gingivalis (Figure 1B-left panel). Proteolysis of rRIPK2 kd was further inhibited with KYT-36, and completely inhibited in the presence of both KYT-1 and KYT-36, or with TLCK. We also observed that pretreatment of P. gingivalis with caspase inhibitors prevented proteolysis, with zVAD-fmk activity having a greater effect than BocD-fmk activity. The ability of these peptide inhibitors to prevent P. gingivalis-induced proteolysis of RIPK2 in a cell-free system is similar to our findings observed in endothelial cell culture. These findings support the notion that P. gingivalis may bypass cellular activation mechanisms and directly induce the proteolysis of RIPK2 and other intracellular proteins via Kgp-specific cleavage.


Pathogen-mediated proteolysis of the cell death regulator RIPK1 and the host defense modulator RIPK2 in human aortic endothelial cells.

Madrigal AG, Barth K, Papadopoulos G, Genco CA - PLoS Pathog. (2012)

Cleavage of recombinant RIPK2 kinase by P. gingivalis in the absence of host cell proteins.P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES (none), DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase for 1 h at 37°C. Reactions were stopped by the addition of SDS-PAGE loading dye and analyzed by Western blot analysis with an antibody to the N′-terminal kinase domain of RIPK2. Top panel: reaction with recombinant protein and P. gingivalis; bottom panel: 10% of reaction prior to incubation with P. gingivalis (untreated recombinant protein, i.e., gel loading control).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369954&req=5

ppat-1002723-g010: Cleavage of recombinant RIPK2 kinase by P. gingivalis in the absence of host cell proteins.P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES (none), DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase for 1 h at 37°C. Reactions were stopped by the addition of SDS-PAGE loading dye and analyzed by Western blot analysis with an antibody to the N′-terminal kinase domain of RIPK2. Top panel: reaction with recombinant protein and P. gingivalis; bottom panel: 10% of reaction prior to incubation with P. gingivalis (untreated recombinant protein, i.e., gel loading control).
Mentions: To determine if P. gingivalis was capable of cleaving RIPK2 in the absence of host cell proteins, recombinant RIPK2 kinase domain (rRIPK2 kd) was incubated with P. gingivalis and analyzed by Western blot for the detection of cleavage products with an antibody targeting the N′-terminal kinase domain. As shown in Figure 10, rRIPK2 kd migrates at ∼40-kDa and was cleaved in the presence of P. gingivalis 381. Furthermore, KYT-1 partially inhibited P. gingivalis-induced cleavage of RIPK2 kd. Surprisingly, the lowest molecular weight band was of a similar sized fragment ∼20-kDa as observed in endothelial cells treated with P. gingivalis (Figure 1B-left panel). Proteolysis of rRIPK2 kd was further inhibited with KYT-36, and completely inhibited in the presence of both KYT-1 and KYT-36, or with TLCK. We also observed that pretreatment of P. gingivalis with caspase inhibitors prevented proteolysis, with zVAD-fmk activity having a greater effect than BocD-fmk activity. The ability of these peptide inhibitors to prevent P. gingivalis-induced proteolysis of RIPK2 in a cell-free system is similar to our findings observed in endothelial cell culture. These findings support the notion that P. gingivalis may bypass cellular activation mechanisms and directly induce the proteolysis of RIPK2 and other intracellular proteins via Kgp-specific cleavage.

Bottom Line: RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2.Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed.We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Porphyromonas gingivalis is the primary etiologic agent of periodontal disease that is associated with other human chronic inflammatory diseases, including atherosclerosis. The ability of P. gingivalis to invade and persist within human aortic endothelial cells (HAEC) has been postulated to contribute to a low to moderate chronic state of inflammation, although how this is specifically achieved has not been well defined. In this study, we demonstrate that P. gingivalis infection of HAEC resulted in the rapid cleavage of receptor interacting protein 1 (RIPK1), a mediator of tumor necrosis factor (TNF) receptor-1 (TNF-R1)-induced cell activation or death, and RIPK2, a key mediator of both innate immune signaling and adaptive immunity. The cleavage of RIPK1 or RIPK2 was not observed in cells treated with apoptotic stimuli, or cells stimulated with agonists to TNF-R1, nucleotide oligomerization domain receptor 1(NOD1), NOD2, Toll-like receptor 2 (TLR2) or TLR4. P. gingivalis-induced cleavage of RIPK1 and RIPK2 was inhibited in the presence of a lysine-specific gingipain (Kgp) inhibitor. RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2. Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed. We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor. Our studies thus reveal an important role for pathogen-mediated modification of cellular kinases as a potential strategy for bacterial persistence within target host cells, which is associated with low-grade chronic inflammation, a hallmark of pathogen-mediated chronic inflammatory disorders.

Show MeSH
Related in: MedlinePlus