Limits...
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.

Show MeSH

Related in: MedlinePlus

General caspase inhibitors z-VAD-FMK and Boc-D-FMK alter P. gingivalis-induced modification of RIPK1 and RIPK2 in HUVEC.HUVEC were pretreated (Pre-Tx) with medium (M), 0.25% DMSO vehicle control (C), 25 µM z-VAD-FMK (VAD), or 100 µM Boc-D-FMK (Boc) with for 1.5 h. HUVEC were then treated with medium (M) or P. gingivalis strain 381 (MOI 100, 381) for 2 h. Whole cell lysates were analyzed for (A) RIPK1 or (B) RIPK2 and GAPDH. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3369954&req=5

ppat-1002723-g005: General caspase inhibitors z-VAD-FMK and Boc-D-FMK alter P. gingivalis-induced modification of RIPK1 and RIPK2 in HUVEC.HUVEC were pretreated (Pre-Tx) with medium (M), 0.25% DMSO vehicle control (C), 25 µM z-VAD-FMK (VAD), or 100 µM Boc-D-FMK (Boc) with for 1.5 h. HUVEC were then treated with medium (M) or P. gingivalis strain 381 (MOI 100, 381) for 2 h. Whole cell lysates were analyzed for (A) RIPK1 or (B) RIPK2 and GAPDH. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa.

Mentions: Previous reports have identified a caspase-mediated cleavage of RIPK1, RIPK3 and RIPK4 during death receptor-mediated apoptosis [22], [23], [24], [25]. Despite these reports, prior to our study there have been no reports demonstrating the cleavage of RIPK2 under any condition. To determine if caspase inhibitors prevented the proteolysis of RIPK1 and RIPK2 in cells treated with P. gingivalis, HUVEC were pretreated with the general caspase inhibitors zVAD-FMK, Boc-D-FMK or vehicle control prior to addition of live organism to the cell culture. We confirmed that the general caspase inhibitors abrogated caspase activity as monitored by inhibition of staurosporine-induced PARP cleavage (Figure S6-lower panel). Treatment of cells with general caspase inhibitors interfered with P. gingivalis-mediated proteolysis of RIPK1 (Figure 5A, Figure S6-top panel) and RIPK2 (Figure 5B and Figure S6-mid panel). While these findings suggest that caspases are involved in the proteolysis of RIPK2, FMK-linked general and enzyme-specific caspase inhibitors have been demonstrated to inhibit the proteolytic activity of the P. gingivalis cysteine protease Kgp, but not the arginine-specific gingipains [50]. Importantly, both caspases and gingipains are classified under the same clan of cysteine proteases (C25), as they share similar active sites, structural motifs and are subject to similar modes of chemical inhibition [51]. Thus, the ability of caspase inhibitors to interfere with P. gingivalis-induced RIPK2 proteolysis by caspases inhibitors may reflect an inhibition of host caspase activity, P. gingivalis Kgp activity, or both.


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)

General caspase inhibitors z-VAD-FMK and Boc-D-FMK alter P. gingivalis-induced modification of RIPK1 and RIPK2 in HUVEC.HUVEC were pretreated (Pre-Tx) with medium (M), 0.25% DMSO vehicle control (C), 25 µM z-VAD-FMK (VAD), or 100 µM Boc-D-FMK (Boc) with for 1.5 h. HUVEC were then treated with medium (M) or P. gingivalis strain 381 (MOI 100, 381) for 2 h. Whole cell lysates were analyzed for (A) RIPK1 or (B) RIPK2 and GAPDH. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369954&req=5

ppat-1002723-g005: General caspase inhibitors z-VAD-FMK and Boc-D-FMK alter P. gingivalis-induced modification of RIPK1 and RIPK2 in HUVEC.HUVEC were pretreated (Pre-Tx) with medium (M), 0.25% DMSO vehicle control (C), 25 µM z-VAD-FMK (VAD), or 100 µM Boc-D-FMK (Boc) with for 1.5 h. HUVEC were then treated with medium (M) or P. gingivalis strain 381 (MOI 100, 381) for 2 h. Whole cell lysates were analyzed for (A) RIPK1 or (B) RIPK2 and GAPDH. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa.
Mentions: Previous reports have identified a caspase-mediated cleavage of RIPK1, RIPK3 and RIPK4 during death receptor-mediated apoptosis [22], [23], [24], [25]. Despite these reports, prior to our study there have been no reports demonstrating the cleavage of RIPK2 under any condition. To determine if caspase inhibitors prevented the proteolysis of RIPK1 and RIPK2 in cells treated with P. gingivalis, HUVEC were pretreated with the general caspase inhibitors zVAD-FMK, Boc-D-FMK or vehicle control prior to addition of live organism to the cell culture. We confirmed that the general caspase inhibitors abrogated caspase activity as monitored by inhibition of staurosporine-induced PARP cleavage (Figure S6-lower panel). Treatment of cells with general caspase inhibitors interfered with P. gingivalis-mediated proteolysis of RIPK1 (Figure 5A, Figure S6-top panel) and RIPK2 (Figure 5B and Figure S6-mid panel). While these findings suggest that caspases are involved in the proteolysis of RIPK2, FMK-linked general and enzyme-specific caspase inhibitors have been demonstrated to inhibit the proteolytic activity of the P. gingivalis cysteine protease Kgp, but not the arginine-specific gingipains [50]. Importantly, both caspases and gingipains are classified under the same clan of cysteine proteases (C25), as they share similar active sites, structural motifs and are subject to similar modes of chemical inhibition [51]. Thus, the ability of caspase inhibitors to interfere with P. gingivalis-induced RIPK2 proteolysis by caspases inhibitors may reflect an inhibition of host caspase activity, P. gingivalis Kgp activity, or both.

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