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Inhibition of anthrax lethal toxin-induced cytolysis of RAW264.7 cells by celastrol.

Chapelsky S, Batty S, Frost M, Mogridge J - PLoS ONE (2008)

Bottom Line: Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin.We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro.This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT

Background: Bacillus anthracis is the bacterium responsible for causing anthrax. The ability of B. anthracis to cause disease is dependent on a secreted virulence factor, lethal toxin, that promotes survival of the bacteria in the host by impairing the immune response. A well-studied effect of lethal toxin is the killing of macrophages, although the molecular mechanisms involved have not been fully characterized.

Methodology/principal findings: Here, we demonstrate that celastrol, a quinone methide triterpene derived from a plant extract used in herbal medicine, inhibits lethal toxin-induced death of RAW264.7 murine macrophages. Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin. Surprisingly, celastrol conferred almost complete protection when it was added up to 1.5 h after intoxication, indicating that it could rescue cells in the late stages of intoxication. Since the activity of the proteasome has been implicated in intoxication using other pharmacological agents, we tested whether celastrol blocked proteasome activity. We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro. Furthermore, celastrol blocked stimulation of IL-18 processing, indicating that celastrol acted upstream of inflammasome activation.

Conclusions/significance: This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway.

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Celastrol is not a potent inhibitor of the proteasome in vitro.(A) The chymotrypsin-like (black bars), caspase-like (white bars), and trypsin-like (grey bars) activities of purified 26S proteasome were assessed in the presence or absence of celastrol or MG132. (B) The chymotrypsin-like activity of purified 20S proteasome was assessed in the presence or absence of celastrol or MG132. (C) The chymotrypsin-like activity of the proteasome in RAW264.7 cell lysate was assessed in the presence or absence of celastrol or MG132. Values represent the means of three experiments±SEM.
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pone-0001421-g004: Celastrol is not a potent inhibitor of the proteasome in vitro.(A) The chymotrypsin-like (black bars), caspase-like (white bars), and trypsin-like (grey bars) activities of purified 26S proteasome were assessed in the presence or absence of celastrol or MG132. (B) The chymotrypsin-like activity of purified 20S proteasome was assessed in the presence or absence of celastrol or MG132. (C) The chymotrypsin-like activity of the proteasome in RAW264.7 cell lysate was assessed in the presence or absence of celastrol or MG132. Values represent the means of three experiments±SEM.

Mentions: We next assessed whether celastrol inhibited the proteolytic activity of the proteasome. The 26S proteasome is a large multi-subunit complex consisting of a 20S barrel-shaped core complex and two 19S regulatory complexes [36]. The regulatory complexes ensure that only ubiquitylated proteins access the inside of the barrel where the proteolytic sites reside. The proteasome exhibits three distinct proteolytic activities, chymotrypsin-like, trypsin-like, and caspase-like, which can be assayed individually using fluorogenic substrates [37]. The chymotrypsin-like site is thought to be the most important of the three, although the trypsin-like and caspase-like activities must also be inhibited to reduce the degradation of most proteins by 50% [37]. We mixed purified 26S proteosome with the fluorogenic substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC) to measure chymotrypsin-like activity; t-butoxycarbonyl-Leu-Arg-Arg-7-amido-4-methylcoumarin (Boc-LRR-AMC) to measure trypsin-like activity; or acetyl-L-norleucyl-L-prolyl-L-norleucyl-L-aspartyl-methylcoumarylamide (Ac-nLPnLD-AMC) to measure caspase-like activity. Whereas MG132 inhibited cleavage of the three substrates, neither 3 µM nor 10 µM celastrol was able to inhibit cleavage of the substrates by the 26S proteasome (Fig. 4A). Celastrol showed some inhibitory activity against the chymotryptic acivity of the 20S proteasome at 10 µM, but not at 3 µM (Fig. 4B), a concentration that was sufficient to cause the accumulation of ubiquitylated proteins in vivo (Fig. 2B). To test further whether celastrol inhibited proteasome activity, we incubated RAW264.7 cell lysates with Suc-LLVY-AMC. MG132 reduced the rate of cleavage of this substrate, but celastrol exhibited little inhibitory activity (Fig. 4C).


Inhibition of anthrax lethal toxin-induced cytolysis of RAW264.7 cells by celastrol.

Chapelsky S, Batty S, Frost M, Mogridge J - PLoS ONE (2008)

Celastrol is not a potent inhibitor of the proteasome in vitro.(A) The chymotrypsin-like (black bars), caspase-like (white bars), and trypsin-like (grey bars) activities of purified 26S proteasome were assessed in the presence or absence of celastrol or MG132. (B) The chymotrypsin-like activity of purified 20S proteasome was assessed in the presence or absence of celastrol or MG132. (C) The chymotrypsin-like activity of the proteasome in RAW264.7 cell lysate was assessed in the presence or absence of celastrol or MG132. Values represent the means of three experiments±SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2170518&req=5

pone-0001421-g004: Celastrol is not a potent inhibitor of the proteasome in vitro.(A) The chymotrypsin-like (black bars), caspase-like (white bars), and trypsin-like (grey bars) activities of purified 26S proteasome were assessed in the presence or absence of celastrol or MG132. (B) The chymotrypsin-like activity of purified 20S proteasome was assessed in the presence or absence of celastrol or MG132. (C) The chymotrypsin-like activity of the proteasome in RAW264.7 cell lysate was assessed in the presence or absence of celastrol or MG132. Values represent the means of three experiments±SEM.
Mentions: We next assessed whether celastrol inhibited the proteolytic activity of the proteasome. The 26S proteasome is a large multi-subunit complex consisting of a 20S barrel-shaped core complex and two 19S regulatory complexes [36]. The regulatory complexes ensure that only ubiquitylated proteins access the inside of the barrel where the proteolytic sites reside. The proteasome exhibits three distinct proteolytic activities, chymotrypsin-like, trypsin-like, and caspase-like, which can be assayed individually using fluorogenic substrates [37]. The chymotrypsin-like site is thought to be the most important of the three, although the trypsin-like and caspase-like activities must also be inhibited to reduce the degradation of most proteins by 50% [37]. We mixed purified 26S proteosome with the fluorogenic substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC) to measure chymotrypsin-like activity; t-butoxycarbonyl-Leu-Arg-Arg-7-amido-4-methylcoumarin (Boc-LRR-AMC) to measure trypsin-like activity; or acetyl-L-norleucyl-L-prolyl-L-norleucyl-L-aspartyl-methylcoumarylamide (Ac-nLPnLD-AMC) to measure caspase-like activity. Whereas MG132 inhibited cleavage of the three substrates, neither 3 µM nor 10 µM celastrol was able to inhibit cleavage of the substrates by the 26S proteasome (Fig. 4A). Celastrol showed some inhibitory activity against the chymotryptic acivity of the 20S proteasome at 10 µM, but not at 3 µM (Fig. 4B), a concentration that was sufficient to cause the accumulation of ubiquitylated proteins in vivo (Fig. 2B). To test further whether celastrol inhibited proteasome activity, we incubated RAW264.7 cell lysates with Suc-LLVY-AMC. MG132 reduced the rate of cleavage of this substrate, but celastrol exhibited little inhibitory activity (Fig. 4C).

Bottom Line: Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin.We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro.This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT

Background: Bacillus anthracis is the bacterium responsible for causing anthrax. The ability of B. anthracis to cause disease is dependent on a secreted virulence factor, lethal toxin, that promotes survival of the bacteria in the host by impairing the immune response. A well-studied effect of lethal toxin is the killing of macrophages, although the molecular mechanisms involved have not been fully characterized.

Methodology/principal findings: Here, we demonstrate that celastrol, a quinone methide triterpene derived from a plant extract used in herbal medicine, inhibits lethal toxin-induced death of RAW264.7 murine macrophages. Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin. Surprisingly, celastrol conferred almost complete protection when it was added up to 1.5 h after intoxication, indicating that it could rescue cells in the late stages of intoxication. Since the activity of the proteasome has been implicated in intoxication using other pharmacological agents, we tested whether celastrol blocked proteasome activity. We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro. Furthermore, celastrol blocked stimulation of IL-18 processing, indicating that celastrol acted upstream of inflammasome activation.

Conclusions/significance: This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway.

Show MeSH
Related in: MedlinePlus