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

Bewley, M. A., Pham, T. K., Marriott, H. M., Noirel, J., Chu, H.P., Ow, S. Y., Ryazanov, A. G., Read, R. C., Whyte, M. K. B., Chain, B., Wright, P. C. and Dockrell, D. H. (2011) Proteomic evaluation and validation of cathepsin D regulated proteins in macrophages exposed to Streptococcus pneumoniae. Molecular & cellular proteomics : MCP, 10 (6). M111.008193. ISSN 1535-9484

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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.

Item Type: Article
Institution: The University of Sheffield
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > School of Medicine (Sheffield) > Department of Infection & Immunity
Depositing User: Miss Anthea Tucker
Date Deposited: 29 Mar 2012 09:41
Last Modified: 29 Mar 2012 09:41
Published Version: http://dx.doi.org/10.1074/mcp.M111.008193
Status: Published
Publisher: American Society for Biochemistry and Molecular Biology
Identification Number: 10.1074/mcp.M111.008193
Related URLs:
URI: http://eprints.whiterose.ac.uk/id/eprint/43791

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