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

Bewley MA, Pham TK, Marriott HM, Noirel J, Chu HP, Ow SY, Ryazanov AG, Read RC, Whyte MK, Chain B, Wright PC, Dockrell DH - Mol. Cell Proteomics (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Medical School, University of Sheffield, Sheffield, UK.

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.

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Eukaryotic elongation factor 2 (eEF2) kinase deficient mice have reduced bacterial clearance. Bacterial counts were estimated in lungs from wild-type (WT) or eukaryotic elongation factor 2 kinase deficient (KO) mice infected with 5 × 105 colony forming units of type one Streptococcus pneumoniae for 24 h, n = 11 per group, * p < 0.05, Mann-Whitney U test. Two WT mice and one eEF2k KO mouse cleared the bacteria completely.
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Figure 10: Eukaryotic elongation factor 2 (eEF2) kinase deficient mice have reduced bacterial clearance. Bacterial counts were estimated in lungs from wild-type (WT) or eukaryotic elongation factor 2 kinase deficient (KO) mice infected with 5 × 105 colony forming units of type one Streptococcus pneumoniae for 24 h, n = 11 per group, * p < 0.05, Mann-Whitney U test. Two WT mice and one eEF2k KO mouse cleared the bacteria completely.

Mentions: One of the consequences of macrophage apoptosis during S. pneumoniae infection is to increase bacterial killing (18). To test whether our proteomic screen was identifying targets that could link macrophage apoptosis with bacterial killing, we measured bacterial clearance in the eEF2 kinase−/− mice that had preservation of Mcl-1 and delayed macrophage apoptosis. Pneumococcal clearance was reduced by ∼0.5 log in eEF2 kinase−/− mouse lungs (Fig. 10). This difference reflects the relatively modest effect on apoptosis of maintaining active eEF2 by inhibition of eEF2 phosphorylation, because cathepsin D activation still induced eEF2 down-regulation and also the fact that cathepsin D can exert its effects on macrophage survival at several points, as this screen has shown. However the reduction in bacterial killing did establish that our screen identified proteins that not only influenced macrophage apoptosis but also bacterial clearance in vivo.


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

Bewley MA, Pham TK, Marriott HM, Noirel J, Chu HP, Ow SY, Ryazanov AG, Read RC, Whyte MK, Chain B, Wright PC, Dockrell DH - Mol. Cell Proteomics (2011)

Eukaryotic elongation factor 2 (eEF2) kinase deficient mice have reduced bacterial clearance. Bacterial counts were estimated in lungs from wild-type (WT) or eukaryotic elongation factor 2 kinase deficient (KO) mice infected with 5 × 105 colony forming units of type one Streptococcus pneumoniae for 24 h, n = 11 per group, * p < 0.05, Mann-Whitney U test. Two WT mice and one eEF2k KO mouse cleared the bacteria completely.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108842&req=5

Figure 10: Eukaryotic elongation factor 2 (eEF2) kinase deficient mice have reduced bacterial clearance. Bacterial counts were estimated in lungs from wild-type (WT) or eukaryotic elongation factor 2 kinase deficient (KO) mice infected with 5 × 105 colony forming units of type one Streptococcus pneumoniae for 24 h, n = 11 per group, * p < 0.05, Mann-Whitney U test. Two WT mice and one eEF2k KO mouse cleared the bacteria completely.
Mentions: One of the consequences of macrophage apoptosis during S. pneumoniae infection is to increase bacterial killing (18). To test whether our proteomic screen was identifying targets that could link macrophage apoptosis with bacterial killing, we measured bacterial clearance in the eEF2 kinase−/− mice that had preservation of Mcl-1 and delayed macrophage apoptosis. Pneumococcal clearance was reduced by ∼0.5 log in eEF2 kinase−/− mouse lungs (Fig. 10). This difference reflects the relatively modest effect on apoptosis of maintaining active eEF2 by inhibition of eEF2 phosphorylation, because cathepsin D activation still induced eEF2 down-regulation and also the fact that cathepsin D can exert its effects on macrophage survival at several points, as this screen has shown. However the reduction in bacterial killing did establish that our screen identified proteins that not only influenced macrophage apoptosis but also bacterial clearance in vivo.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Medical School, University of Sheffield, Sheffield, UK.

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.

Show MeSH
Related in: MedlinePlus