The University of Southampton
University of Southampton Institutional Repository

Proteomic evaluation and validation of cathepsin D regulated proteins in macrophages exposed to Streptococcus pneumoniae

Proteomic evaluation and validation of cathepsin D regulated proteins in macrophages exposed to Streptococcus pneumoniae
Proteomic evaluation and validation of cathepsin D regulated proteins in macrophages exposed to Streptococcus pneumoniae
Macrophages are central effectors of innate immune responses to bacteria. We have investigated how activation of the abundant macrophage lysosomal protease, cathepsin D, regulates the macrophage proteome during killing of Streptococcus pneumoniae. Using the cathepsin D inhibitor pepstatin A, we demonstrate that cathepsin D differentially regulates multiple targets out of 679 proteins identified and quantified by eight-plex isobaric tag for relative and absolute quantitation. Our statistical analysis identified 18 differentially expressed proteins that passed all paired t-tests (α = 0.05). This dataset was enriched for proteins regulating the mitochondrial pathway of apoptosis or inhibiting competing death programs. Five proteins were selected for further analysis. Western blotting, followed by pharmacological inhibition or genetic manipulation of cathepsin D, verified cathepsin D-dependent regulation of these proteins, after exposure to S. pneumoniae. Superoxide dismutase-2 up-regulation was temporally related to increased reactive oxygen species generation. Gelsolin, a known regulator of mitochondrial outer membrane permeabilization, was down-regulated in association with cytochrome c release from mitochondria. Eukaryotic elongation factor (eEF2), a regulator of protein translation, was also down-regulated by cathepsin D. Using absence of the negative regulator of eEF2, eEF2 kinase, we confirm that eEF2 function is required to maintain expression of the anti-apoptotic protein Mcl-1, delaying macrophage apoptosis and confirm using a murine model that maintaining eEF2 function is associated with impaired macrophage apoptosis-associated killing of Streptococcus pneumoniae. These findings demonstrate that cathepsin D regulates multiple proteins controlling the mitochondrial pathway of macrophage apoptosis or competing death processes, facilitating intracellular bacterial killing.
1535-9476
M111.008193
Bewley, Martin A.
1412f184-67e0-4fe7-8692-db253d411471
Pham, Trong K.
a46afc99-84e3-4aca-ac5a-3552bcf3bbe4
Marriott, Helen M.
34ca8904-d637-4081-b34c-12be45ecef9f
Noirel, Josselin
1bec6448-6e2f-4bf2-a7f8-2f740b10ab59
Chu, Hseuh-Ping
efd7e903-8ab6-4ae7-b93e-b3032d05047a
Ow, Saw Y.
970802ba-0620-40d0-b591-697e4d1e633b
Ryazanov, Alexey G.
cd5922b4-e04c-47c2-b9bc-b02693f7be0c
Read, Robert C.
b5caca7b-0063-438a-b703-7ecbb6fc2b51
Whyte, Moira K.B.
7c8b9870-17db-4359-ac5c-57ba9197ed82
Chain, Benny
c83e3462-a8af-446b-9533-3bf440d0a6a3
Wright, Phillip C.
80e9e2f7-13d4-43ee-a5d9-633305d25a90
Dockrell, David H.
a068c9bf-35b8-4c10-8f91-58639cfeca0b
Bewley, Martin A.
1412f184-67e0-4fe7-8692-db253d411471
Pham, Trong K.
a46afc99-84e3-4aca-ac5a-3552bcf3bbe4
Marriott, Helen M.
34ca8904-d637-4081-b34c-12be45ecef9f
Noirel, Josselin
1bec6448-6e2f-4bf2-a7f8-2f740b10ab59
Chu, Hseuh-Ping
efd7e903-8ab6-4ae7-b93e-b3032d05047a
Ow, Saw Y.
970802ba-0620-40d0-b591-697e4d1e633b
Ryazanov, Alexey G.
cd5922b4-e04c-47c2-b9bc-b02693f7be0c
Read, Robert C.
b5caca7b-0063-438a-b703-7ecbb6fc2b51
Whyte, Moira K.B.
7c8b9870-17db-4359-ac5c-57ba9197ed82
Chain, Benny
c83e3462-a8af-446b-9533-3bf440d0a6a3
Wright, Phillip C.
80e9e2f7-13d4-43ee-a5d9-633305d25a90
Dockrell, David H.
a068c9bf-35b8-4c10-8f91-58639cfeca0b

Bewley, Martin A., Pham, Trong K., Marriott, Helen M., Noirel, Josselin, Chu, Hseuh-Ping, Ow, Saw Y., Ryazanov, Alexey G., Read, Robert C., Whyte, Moira K.B., Chain, Benny, Wright, Phillip C. and Dockrell, David H. (2011) Proteomic evaluation and validation of cathepsin D regulated proteins in macrophages exposed to Streptococcus pneumoniae. Molecular & Cellular Proteomics, 10 (6), M111.008193. (doi:10.1074/mcp.M111.008193). (PMID:21474794)

Record type: Article

Abstract

Macrophages are central effectors of innate immune responses to bacteria. We have investigated how activation of the abundant macrophage lysosomal protease, cathepsin D, regulates the macrophage proteome during killing of Streptococcus pneumoniae. Using the cathepsin D inhibitor pepstatin A, we demonstrate that cathepsin D differentially regulates multiple targets out of 679 proteins identified and quantified by eight-plex isobaric tag for relative and absolute quantitation. Our statistical analysis identified 18 differentially expressed proteins that passed all paired t-tests (α = 0.05). This dataset was enriched for proteins regulating the mitochondrial pathway of apoptosis or inhibiting competing death programs. Five proteins were selected for further analysis. Western blotting, followed by pharmacological inhibition or genetic manipulation of cathepsin D, verified cathepsin D-dependent regulation of these proteins, after exposure to S. pneumoniae. Superoxide dismutase-2 up-regulation was temporally related to increased reactive oxygen species generation. Gelsolin, a known regulator of mitochondrial outer membrane permeabilization, was down-regulated in association with cytochrome c release from mitochondria. Eukaryotic elongation factor (eEF2), a regulator of protein translation, was also down-regulated by cathepsin D. Using absence of the negative regulator of eEF2, eEF2 kinase, we confirm that eEF2 function is required to maintain expression of the anti-apoptotic protein Mcl-1, delaying macrophage apoptosis and confirm using a murine model that maintaining eEF2 function is associated with impaired macrophage apoptosis-associated killing of Streptococcus pneumoniae. These findings demonstrate that cathepsin D regulates multiple proteins controlling the mitochondrial pathway of macrophage apoptosis or competing death processes, facilitating intracellular bacterial killing.

Full text not available from this repository.

More information

Published date: June 2011
Organisations: Clinical & Experimental Sciences

Identifiers

Local EPrints ID: 346755
URI: https://eprints.soton.ac.uk/id/eprint/346755
ISSN: 1535-9476
PURE UUID: 56e99e40-529e-4e53-a386-a63f0116417b
ORCID for Robert C. Read: ORCID iD orcid.org/0000-0002-4297-6728

Catalogue record

Date deposited: 09 Jan 2013 11:51
Last modified: 27 Jun 2018 00:30

Export record

Altmetrics

Contributors

Author: Martin A. Bewley
Author: Trong K. Pham
Author: Helen M. Marriott
Author: Josselin Noirel
Author: Hseuh-Ping Chu
Author: Saw Y. Ow
Author: Alexey G. Ryazanov
Author: Robert C. Read ORCID iD
Author: Moira K.B. Whyte
Author: Benny Chain
Author: Phillip C. Wright
Author: David H. Dockrell

University divisions

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of https://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×