Substrate recognition by the zinc metalloprotease effector NleC from enteropathogenic Escherichia coli

Cristina Giogha, Tania Wong Fok Lung, Sabrina Mühlen, Jaclyn S. Pearson, Elizabeth L. Hartland

Research output: Contribution to journalArticleResearchpeer-review

10 Citations (Scopus)

Abstract

Upon infection of epithelial cells, enteropathogenic Escherichiacoli suppresses host cell inflammatory signalling in a type III secretion system (T3SS) dependent manner. Two key T3SS effector proteins involved in this response are NleE and NleC. NleC is a zinc metalloprotease effector that degrades the p65 subunit of NF-κB. Although the site of p65 cleavage by NleC is now well described, other areas of interaction have not been precisely defined. Here we constructed overlapping truncations of p65 to identify regions required for NleC cleavage. We determined that NleC cleaved both p65 and p50 within the Rel homology domain (RHD) and that two motifs, E22IIE25 and P177VLS180, within the RHD of p65 were important for recognition and binding by NleC. Alanine substitution of one or both of these motifs protected p65 from binding and degradation by NleC. The E22IIE25 and P177VLS180 motifs were located within the structurally distinct N-terminal subdomain of the RHD involved in DNA binding by p65 on adjacent, parallel strands. Although these motifs have not been recognized previously, both were needed for the correct localization and function of p65. In summary, this work has identified two regions of p65 within the RHD needed for binding and cleavage by NleC and provides further insight into the molecular basis of substrate recognition by a T3SS effector.

Original languageEnglish
Pages (from-to)1766-1778
Number of pages13
JournalCellular Microbiology
Volume17
Issue number12
DOIs
Publication statusPublished - Dec 2015
Externally publishedYes

Cite this

@article{c6ea14d34e8349f2b0cee07cff2a7279,
title = "Substrate recognition by the zinc metalloprotease effector NleC from enteropathogenic Escherichia coli",
abstract = "Upon infection of epithelial cells, enteropathogenic Escherichiacoli suppresses host cell inflammatory signalling in a type III secretion system (T3SS) dependent manner. Two key T3SS effector proteins involved in this response are NleE and NleC. NleC is a zinc metalloprotease effector that degrades the p65 subunit of NF-κB. Although the site of p65 cleavage by NleC is now well described, other areas of interaction have not been precisely defined. Here we constructed overlapping truncations of p65 to identify regions required for NleC cleavage. We determined that NleC cleaved both p65 and p50 within the Rel homology domain (RHD) and that two motifs, E22IIE25 and P177VLS180, within the RHD of p65 were important for recognition and binding by NleC. Alanine substitution of one or both of these motifs protected p65 from binding and degradation by NleC. The E22IIE25 and P177VLS180 motifs were located within the structurally distinct N-terminal subdomain of the RHD involved in DNA binding by p65 on adjacent, parallel strands. Although these motifs have not been recognized previously, both were needed for the correct localization and function of p65. In summary, this work has identified two regions of p65 within the RHD needed for binding and cleavage by NleC and provides further insight into the molecular basis of substrate recognition by a T3SS effector.",
author = "Cristina Giogha and {Wong Fok Lung}, Tania and Sabrina M{\"u}hlen and Pearson, {Jaclyn S.} and Hartland, {Elizabeth L.}",
year = "2015",
month = "12",
doi = "10.1111/cmi.12469",
language = "English",
volume = "17",
pages = "1766--1778",
journal = "Cellular Microbiology",
issn = "1462-5814",
publisher = "Wiley-Blackwell",
number = "12",

}

Substrate recognition by the zinc metalloprotease effector NleC from enteropathogenic Escherichia coli. / Giogha, Cristina; Wong Fok Lung, Tania; Mühlen, Sabrina; Pearson, Jaclyn S.; Hartland, Elizabeth L.

In: Cellular Microbiology, Vol. 17, No. 12, 12.2015, p. 1766-1778.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Substrate recognition by the zinc metalloprotease effector NleC from enteropathogenic Escherichia coli

AU - Giogha, Cristina

AU - Wong Fok Lung, Tania

AU - Mühlen, Sabrina

AU - Pearson, Jaclyn S.

AU - Hartland, Elizabeth L.

PY - 2015/12

Y1 - 2015/12

N2 - Upon infection of epithelial cells, enteropathogenic Escherichiacoli suppresses host cell inflammatory signalling in a type III secretion system (T3SS) dependent manner. Two key T3SS effector proteins involved in this response are NleE and NleC. NleC is a zinc metalloprotease effector that degrades the p65 subunit of NF-κB. Although the site of p65 cleavage by NleC is now well described, other areas of interaction have not been precisely defined. Here we constructed overlapping truncations of p65 to identify regions required for NleC cleavage. We determined that NleC cleaved both p65 and p50 within the Rel homology domain (RHD) and that two motifs, E22IIE25 and P177VLS180, within the RHD of p65 were important for recognition and binding by NleC. Alanine substitution of one or both of these motifs protected p65 from binding and degradation by NleC. The E22IIE25 and P177VLS180 motifs were located within the structurally distinct N-terminal subdomain of the RHD involved in DNA binding by p65 on adjacent, parallel strands. Although these motifs have not been recognized previously, both were needed for the correct localization and function of p65. In summary, this work has identified two regions of p65 within the RHD needed for binding and cleavage by NleC and provides further insight into the molecular basis of substrate recognition by a T3SS effector.

AB - Upon infection of epithelial cells, enteropathogenic Escherichiacoli suppresses host cell inflammatory signalling in a type III secretion system (T3SS) dependent manner. Two key T3SS effector proteins involved in this response are NleE and NleC. NleC is a zinc metalloprotease effector that degrades the p65 subunit of NF-κB. Although the site of p65 cleavage by NleC is now well described, other areas of interaction have not been precisely defined. Here we constructed overlapping truncations of p65 to identify regions required for NleC cleavage. We determined that NleC cleaved both p65 and p50 within the Rel homology domain (RHD) and that two motifs, E22IIE25 and P177VLS180, within the RHD of p65 were important for recognition and binding by NleC. Alanine substitution of one or both of these motifs protected p65 from binding and degradation by NleC. The E22IIE25 and P177VLS180 motifs were located within the structurally distinct N-terminal subdomain of the RHD involved in DNA binding by p65 on adjacent, parallel strands. Although these motifs have not been recognized previously, both were needed for the correct localization and function of p65. In summary, this work has identified two regions of p65 within the RHD needed for binding and cleavage by NleC and provides further insight into the molecular basis of substrate recognition by a T3SS effector.

UR - http://www.scopus.com/inward/record.url?scp=84947934413&partnerID=8YFLogxK

U2 - 10.1111/cmi.12469

DO - 10.1111/cmi.12469

M3 - Article

VL - 17

SP - 1766

EP - 1778

JO - Cellular Microbiology

JF - Cellular Microbiology

SN - 1462-5814

IS - 12

ER -