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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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KYT inhibitors specifically inhibit P. gingivalis gingipain activity and do not alter host 3 caspase activity.Effect of gingipain inhibitors on P. gingivalisA) Rgp or B) Kgp protease activity. P. gingivalis was untreated (none) or pretreated with 10 µM Rgp-specific inhibitor KYT-1, 10 µM Kgp-specific inhibitor KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 10 min and monitored for arginine-X-specific or lysine-X-specific protease activity. Effect of inhibitors on P. gingivalis is presented as percent Rgp-X activity or Kgp-X activity relative to untreated P. gingivalis. C) HUVEC were untreated or treated with 2 µM staurosporine (STS) for 5 h. Whole cell lysates were analyzed for caspase-3 activity in the presence of KYT-36 gingipain inhibitor (3 µM). Activity is represented as fold change relative to untreated. A reversible caspase inhibitor was included to demonstrate observed fluorescence is specific to caspase-3 like proteases. Statistical analysis was performed using unpaired T-test (α = 0.05), **p<0.001, NS = no significance.
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ppat-1002723-g007: KYT inhibitors specifically inhibit P. gingivalis gingipain activity and do not alter host 3 caspase activity.Effect of gingipain inhibitors on P. gingivalisA) Rgp or B) Kgp protease activity. P. gingivalis was untreated (none) or pretreated with 10 µM Rgp-specific inhibitor KYT-1, 10 µM Kgp-specific inhibitor KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 10 min and monitored for arginine-X-specific or lysine-X-specific protease activity. Effect of inhibitors on P. gingivalis is presented as percent Rgp-X activity or Kgp-X activity relative to untreated P. gingivalis. C) HUVEC were untreated or treated with 2 µM staurosporine (STS) for 5 h. Whole cell lysates were analyzed for caspase-3 activity in the presence of KYT-36 gingipain inhibitor (3 µM). Activity is represented as fold change relative to untreated. A reversible caspase inhibitor was included to demonstrate observed fluorescence is specific to caspase-3 like proteases. Statistical analysis was performed using unpaired T-test (α = 0.05), **p<0.001, NS = no significance.

Mentions: A number of host cell surface proteins and proteins in the extracellular milieu are cleaved by the cysteine proteases of P. gingivalis[44], [52], [53]. To evaluate if P. gingivalis gingipains played a role in the proteolysis of RIPK1 and RIPK2, we first evaluated previously established gingipain-specific inhibitors KYT-1 (arginine-specific gingipain inhibitor) and KYT-36 (lysine-specific gingipain inhibitor) [54], [55] as well as TLCK, which inhibits both arginine-specific and lysine-specific protease activity [56] in our studies. We confirmed the ability of the protease inhibitors to reduce the arginine-specific activity by KYT-1, the lysine-specific activity by KYT-36 and both arginine- and lysine-specific protease activity by TLCK in a substrate-based assay (Figure 7A and B). Treatment of P. gingivalis 381 with KYT-1 or vehicle control (DMSO) did not alter the ability of P. gingivalis 381 to induce proteolysis of RIPK1 (Figure 8A) or RIPK2 (Figure 8B) in HAEC. However, treatment of P. gingivalis with KYT-36 alone or in combination with KYT-1 blocked P. gingivalis-induced proteolysis of RIPK1 and RIPK2. TLCK also inhibited the P. gingivalis-induced proteolysis of RIPK1 and RIPK2. Pretreatment of P. gingivalis with KYT-1, KYT-36, TLCK and vehicle controls did not alter bacterial viability (data not shown), nor did KYT-36 alter host caspase 3 activity (Figure 7C). These findings indicate that Kgp activity was responsible for P. gingivalis-mediated proteolysis of RIPK1 and RIPK2.


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)

KYT inhibitors specifically inhibit P. gingivalis gingipain activity and do not alter host 3 caspase activity.Effect of gingipain inhibitors on P. gingivalisA) Rgp or B) Kgp protease activity. P. gingivalis was untreated (none) or pretreated with 10 µM Rgp-specific inhibitor KYT-1, 10 µM Kgp-specific inhibitor KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 10 min and monitored for arginine-X-specific or lysine-X-specific protease activity. Effect of inhibitors on P. gingivalis is presented as percent Rgp-X activity or Kgp-X activity relative to untreated P. gingivalis. C) HUVEC were untreated or treated with 2 µM staurosporine (STS) for 5 h. Whole cell lysates were analyzed for caspase-3 activity in the presence of KYT-36 gingipain inhibitor (3 µM). Activity is represented as fold change relative to untreated. A reversible caspase inhibitor was included to demonstrate observed fluorescence is specific to caspase-3 like proteases. Statistical analysis was performed using unpaired T-test (α = 0.05), **p<0.001, NS = no significance.
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Related In: Results  -  Collection

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ppat-1002723-g007: KYT inhibitors specifically inhibit P. gingivalis gingipain activity and do not alter host 3 caspase activity.Effect of gingipain inhibitors on P. gingivalisA) Rgp or B) Kgp protease activity. P. gingivalis was untreated (none) or pretreated with 10 µM Rgp-specific inhibitor KYT-1, 10 µM Kgp-specific inhibitor KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 10 min and monitored for arginine-X-specific or lysine-X-specific protease activity. Effect of inhibitors on P. gingivalis is presented as percent Rgp-X activity or Kgp-X activity relative to untreated P. gingivalis. C) HUVEC were untreated or treated with 2 µM staurosporine (STS) for 5 h. Whole cell lysates were analyzed for caspase-3 activity in the presence of KYT-36 gingipain inhibitor (3 µM). Activity is represented as fold change relative to untreated. A reversible caspase inhibitor was included to demonstrate observed fluorescence is specific to caspase-3 like proteases. Statistical analysis was performed using unpaired T-test (α = 0.05), **p<0.001, NS = no significance.
Mentions: A number of host cell surface proteins and proteins in the extracellular milieu are cleaved by the cysteine proteases of P. gingivalis[44], [52], [53]. To evaluate if P. gingivalis gingipains played a role in the proteolysis of RIPK1 and RIPK2, we first evaluated previously established gingipain-specific inhibitors KYT-1 (arginine-specific gingipain inhibitor) and KYT-36 (lysine-specific gingipain inhibitor) [54], [55] as well as TLCK, which inhibits both arginine-specific and lysine-specific protease activity [56] in our studies. We confirmed the ability of the protease inhibitors to reduce the arginine-specific activity by KYT-1, the lysine-specific activity by KYT-36 and both arginine- and lysine-specific protease activity by TLCK in a substrate-based assay (Figure 7A and B). Treatment of P. gingivalis 381 with KYT-1 or vehicle control (DMSO) did not alter the ability of P. gingivalis 381 to induce proteolysis of RIPK1 (Figure 8A) or RIPK2 (Figure 8B) in HAEC. However, treatment of P. gingivalis with KYT-36 alone or in combination with KYT-1 blocked P. gingivalis-induced proteolysis of RIPK1 and RIPK2. TLCK also inhibited the P. gingivalis-induced proteolysis of RIPK1 and RIPK2. Pretreatment of P. gingivalis with KYT-1, KYT-36, TLCK and vehicle controls did not alter bacterial viability (data not shown), nor did KYT-36 alter host caspase 3 activity (Figure 7C). These findings indicate that Kgp activity was responsible for P. gingivalis-mediated proteolysis of RIPK1 and RIPK2.

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