A novel entamoeba histolytica cysteine proteinase, EhCP4, is key for invasive amebiasis and a therapeutic target

Chen He, George Nora, Eric Schneider, Iain Kerr, Elizabeth Hansell, Ken Hirata, David Gonzalez, Mohameed Sajid, Sarah Elizabeth Boyd, Petr Hurz, Eduardo Cobo, Christine Le, Wei-ting Liu, Lars Eckmann, Pieter Dorrestein, Eric Houpt, Linda Brinen, Charles Craik, William Roush, James McKerrow & 1 others Sharon Reed

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Abstract

Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design
Original languageEnglish
Pages (from-to)18516 - 18527
Number of pages12
JournalJournal of Biological Chemistry
Volume285
Issue number24
DOIs
Publication statusPublished - 2010
Externally publishedYes

Cite this

He, Chen ; Nora, George ; Schneider, Eric ; Kerr, Iain ; Hansell, Elizabeth ; Hirata, Ken ; Gonzalez, David ; Sajid, Mohameed ; Boyd, Sarah Elizabeth ; Hurz, Petr ; Cobo, Eduardo ; Le, Christine ; Liu, Wei-ting ; Eckmann, Lars ; Dorrestein, Pieter ; Houpt, Eric ; Brinen, Linda ; Craik, Charles ; Roush, William ; McKerrow, James ; Reed, Sharon. / A novel entamoeba histolytica cysteine proteinase, EhCP4, is key for invasive amebiasis and a therapeutic target. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 24. pp. 18516 - 18527.
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title = "A novel entamoeba histolytica cysteine proteinase, EhCP4, is key for invasive amebiasis and a therapeutic target",
abstract = "Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design",
author = "Chen He and George Nora and Eric Schneider and Iain Kerr and Elizabeth Hansell and Ken Hirata and David Gonzalez and Mohameed Sajid and Boyd, {Sarah Elizabeth} and Petr Hurz and Eduardo Cobo and Christine Le and Wei-ting Liu and Lars Eckmann and Pieter Dorrestein and Eric Houpt and Linda Brinen and Charles Craik and William Roush and James McKerrow and Sharon Reed",
year = "2010",
doi = "10.1074/jbc.M109.086181",
language = "English",
volume = "285",
pages = "18516 -- 18527",
journal = "Journal of Biological Chemistry",
issn = "1083-351X",
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He, C, Nora, G, Schneider, E, Kerr, I, Hansell, E, Hirata, K, Gonzalez, D, Sajid, M, Boyd, SE, Hurz, P, Cobo, E, Le, C, Liu, W, Eckmann, L, Dorrestein, P, Houpt, E, Brinen, L, Craik, C, Roush, W, McKerrow, J & Reed, S 2010, 'A novel entamoeba histolytica cysteine proteinase, EhCP4, is key for invasive amebiasis and a therapeutic target', Journal of Biological Chemistry, vol. 285, no. 24, pp. 18516 - 18527. https://doi.org/10.1074/jbc.M109.086181

A novel entamoeba histolytica cysteine proteinase, EhCP4, is key for invasive amebiasis and a therapeutic target. / He, Chen; Nora, George; Schneider, Eric; Kerr, Iain; Hansell, Elizabeth; Hirata, Ken; Gonzalez, David; Sajid, Mohameed; Boyd, Sarah Elizabeth; Hurz, Petr; Cobo, Eduardo; Le, Christine; Liu, Wei-ting; Eckmann, Lars; Dorrestein, Pieter; Houpt, Eric; Brinen, Linda; Craik, Charles; Roush, William; McKerrow, James; Reed, Sharon.

In: Journal of Biological Chemistry, Vol. 285, No. 24, 2010, p. 18516 - 18527.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - A novel entamoeba histolytica cysteine proteinase, EhCP4, is key for invasive amebiasis and a therapeutic target

AU - He, Chen

AU - Nora, George

AU - Schneider, Eric

AU - Kerr, Iain

AU - Hansell, Elizabeth

AU - Hirata, Ken

AU - Gonzalez, David

AU - Sajid, Mohameed

AU - Boyd, Sarah Elizabeth

AU - Hurz, Petr

AU - Cobo, Eduardo

AU - Le, Christine

AU - Liu, Wei-ting

AU - Eckmann, Lars

AU - Dorrestein, Pieter

AU - Houpt, Eric

AU - Brinen, Linda

AU - Craik, Charles

AU - Roush, William

AU - McKerrow, James

AU - Reed, Sharon

PY - 2010

Y1 - 2010

N2 - Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design

AB - Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design

UR - http://www.jbc.org/content/285/24/18516.short

U2 - 10.1074/jbc.M109.086181

DO - 10.1074/jbc.M109.086181

M3 - Article

VL - 285

SP - 18516

EP - 18527

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 1083-351X

IS - 24

ER -