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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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Related in: MedlinePlus

Cathepsin D deficient macrophages validate iTRAQ findings for SOD-2 and HspA5. Representative Western blots for (A) SOD-2 and (B) HspA5 from wild-type (WT) and Cathepsin D knockout (KO) BMDMs 16 h after mock- infection (Spn-) or exposure to Streptococcus pneumoniae (Spn+), in the presence (+) or absence (-) of pepstatin A (PepA). Blots are representative of three independent experiments. Densitometry was carried out and each protein's fold change was calculated relative to the mock-infected (MI) level after adjustment for any fold change in tubulin, n = 3 * = p < 0.05, 2-way ANOVA with Bonferroni post-test.
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Figure 5: Cathepsin D deficient macrophages validate iTRAQ findings for SOD-2 and HspA5. Representative Western blots for (A) SOD-2 and (B) HspA5 from wild-type (WT) and Cathepsin D knockout (KO) BMDMs 16 h after mock- infection (Spn-) or exposure to Streptococcus pneumoniae (Spn+), in the presence (+) or absence (-) of pepstatin A (PepA). Blots are representative of three independent experiments. Densitometry was carried out and each protein's fold change was calculated relative to the mock-infected (MI) level after adjustment for any fold change in tubulin, n = 3 * = p < 0.05, 2-way ANOVA with Bonferroni post-test.

Mentions: Pharmacological inhibition of cathepsin D could however in theory have off-target effects. To confirm our findings further we also repeated Western blots and densitometry on BMDM derived from cathepsin D deficient infant mice or their wild-type littermates. This confirmed cathepsin D dependent up-regulation of SOD-2 and HspA5 (Figs. 5A and 5B) and down-regulation of gelsolin and eEF2 (Figs. 6A and 5B) following infection, with an increase in S100A6 following infection that was further increased in the absence of cathepsin D (Fig. 6C).


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)

Cathepsin D deficient macrophages validate iTRAQ findings for SOD-2 and HspA5. Representative Western blots for (A) SOD-2 and (B) HspA5 from wild-type (WT) and Cathepsin D knockout (KO) BMDMs 16 h after mock- infection (Spn-) or exposure to Streptococcus pneumoniae (Spn+), in the presence (+) or absence (-) of pepstatin A (PepA). Blots are representative of three independent experiments. Densitometry was carried out and each protein's fold change was calculated relative to the mock-infected (MI) level after adjustment for any fold change in tubulin, n = 3 * = p < 0.05, 2-way ANOVA with Bonferroni post-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Cathepsin D deficient macrophages validate iTRAQ findings for SOD-2 and HspA5. Representative Western blots for (A) SOD-2 and (B) HspA5 from wild-type (WT) and Cathepsin D knockout (KO) BMDMs 16 h after mock- infection (Spn-) or exposure to Streptococcus pneumoniae (Spn+), in the presence (+) or absence (-) of pepstatin A (PepA). Blots are representative of three independent experiments. Densitometry was carried out and each protein's fold change was calculated relative to the mock-infected (MI) level after adjustment for any fold change in tubulin, n = 3 * = p < 0.05, 2-way ANOVA with Bonferroni post-test.
Mentions: Pharmacological inhibition of cathepsin D could however in theory have off-target effects. To confirm our findings further we also repeated Western blots and densitometry on BMDM derived from cathepsin D deficient infant mice or their wild-type littermates. This confirmed cathepsin D dependent up-regulation of SOD-2 and HspA5 (Figs. 5A and 5B) and down-regulation of gelsolin and eEF2 (Figs. 6A and 5B) following infection, with an increase in S100A6 following infection that was further increased in the absence of cathepsin D (Fig. 6C).

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