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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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Validation of iTRAQ analysis for gelsolin, eukaryotic elongation factor 2 (eEF2) and calcyclin (S100A6) in differentiated THP-1 macrophages. Representative Western blots of total protein from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) differentiated THP-1 macrophages in the presence (+) or absence (-) of pepstatin A (PepA) probed for (A) gelsolin, (B) eukaryotic elongation factor 2 (eEF2) or (C) calcyclin (S100A6) 16 h postinfection. Densitometry was carried out and each protein's fold change was compared relative to the mock-infected (MI) level after adjustment for any fold change in tubulin, n = 4 * = p < 0.05, 1-way ANOVA with Bonferroni's post-test.
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Figure 4: Validation of iTRAQ analysis for gelsolin, eukaryotic elongation factor 2 (eEF2) and calcyclin (S100A6) in differentiated THP-1 macrophages. Representative Western blots of total protein from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) differentiated THP-1 macrophages in the presence (+) or absence (-) of pepstatin A (PepA) probed for (A) gelsolin, (B) eukaryotic elongation factor 2 (eEF2) or (C) calcyclin (S100A6) 16 h postinfection. Densitometry was carried out and each protein's fold change was compared relative to the mock-infected (MI) level after adjustment for any fold change in tubulin, n = 4 * = p < 0.05, 1-way ANOVA with Bonferroni's post-test.

Mentions: Differentiated THP-1 cells, under the same conditions as used for the iTRAQ analysis, were lysed and Western blots performed to verify the proteomics findings. In total, five proteins were examined further, all known to have links to cell death processes. In every case, Western blot analysis with densitometry performed on four biological replicates confirmed the findings of the iTRAQ analysis, providing evidence, not only that each protein was differentially expressed in the presence of pepstatin A, but also that each protein was differentially regulated by infection. Superoxide dismutase (SOD)-2 (decreased 1.68-fold, p = 0.005 in D39+pepstatin A versus D39 by iTRAQ analysis) and glucose sensitive heat shock protein (Hsp) A5, also termed glucose-regulated protein of 78 kDa (Grp78) or BiP, (decreased 1.15-fold, p = 0.006 in D39+pepstatin A versus D39 by iTRAQ analysis), were both elevated in infection, but up-regulation was reversed by pepstatin A indicating a role for cathepsin D in protein up-regulation (Figs. 3A and 3B). In contrast, the actin regulatory protein gelsolin (up-regulated 1.22-fold, p = 0.0005 in D39+pepstatin A versus D39 by iTRAQ analysis) and the translation factor eukaryotic elongation factor (eEF)2 (up-regulated 2.30-fold, p = 0.00005 in D39+pepstatin A versus D39 by iTRAQ analysis) were down-regulated by Western blot following S. pneumoniae exposure, a reduction that was blocked by pepstatin, implicating cathepsin D in the down-regulation observed (Figs. 4A and 4B). A fifth protein, the calcium binding protein, which enhances the transcriptional activity of the tumor suppressor p53 (37) and can increase transcription of caspase 3 (38), S100A6 or calcyclin (up-regulated 1.86-fold, p = 0.0000003 in D39+pepstatin A versus D39 by iTRAQ analysis), was up-regulated during infection, but in this case pepstatin A treatment resulted in further up-regulation (Fig. 4C). These results indicate that the iTRAQ analysis appeared to have identified potential cathepsin D targets with known roles in the regulation of cell survival.


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)

Validation of iTRAQ analysis for gelsolin, eukaryotic elongation factor 2 (eEF2) and calcyclin (S100A6) in differentiated THP-1 macrophages. Representative Western blots of total protein from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) differentiated THP-1 macrophages in the presence (+) or absence (-) of pepstatin A (PepA) probed for (A) gelsolin, (B) eukaryotic elongation factor 2 (eEF2) or (C) calcyclin (S100A6) 16 h postinfection. Densitometry was carried out and each protein's fold change was compared relative to the mock-infected (MI) level after adjustment for any fold change in tubulin, n = 4 * = p < 0.05, 1-way ANOVA with Bonferroni's post-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Validation of iTRAQ analysis for gelsolin, eukaryotic elongation factor 2 (eEF2) and calcyclin (S100A6) in differentiated THP-1 macrophages. Representative Western blots of total protein from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) differentiated THP-1 macrophages in the presence (+) or absence (-) of pepstatin A (PepA) probed for (A) gelsolin, (B) eukaryotic elongation factor 2 (eEF2) or (C) calcyclin (S100A6) 16 h postinfection. Densitometry was carried out and each protein's fold change was compared relative to the mock-infected (MI) level after adjustment for any fold change in tubulin, n = 4 * = p < 0.05, 1-way ANOVA with Bonferroni's post-test.
Mentions: Differentiated THP-1 cells, under the same conditions as used for the iTRAQ analysis, were lysed and Western blots performed to verify the proteomics findings. In total, five proteins were examined further, all known to have links to cell death processes. In every case, Western blot analysis with densitometry performed on four biological replicates confirmed the findings of the iTRAQ analysis, providing evidence, not only that each protein was differentially expressed in the presence of pepstatin A, but also that each protein was differentially regulated by infection. Superoxide dismutase (SOD)-2 (decreased 1.68-fold, p = 0.005 in D39+pepstatin A versus D39 by iTRAQ analysis) and glucose sensitive heat shock protein (Hsp) A5, also termed glucose-regulated protein of 78 kDa (Grp78) or BiP, (decreased 1.15-fold, p = 0.006 in D39+pepstatin A versus D39 by iTRAQ analysis), were both elevated in infection, but up-regulation was reversed by pepstatin A indicating a role for cathepsin D in protein up-regulation (Figs. 3A and 3B). In contrast, the actin regulatory protein gelsolin (up-regulated 1.22-fold, p = 0.0005 in D39+pepstatin A versus D39 by iTRAQ analysis) and the translation factor eukaryotic elongation factor (eEF)2 (up-regulated 2.30-fold, p = 0.00005 in D39+pepstatin A versus D39 by iTRAQ analysis) were down-regulated by Western blot following S. pneumoniae exposure, a reduction that was blocked by pepstatin, implicating cathepsin D in the down-regulation observed (Figs. 4A and 4B). A fifth protein, the calcium binding protein, which enhances the transcriptional activity of the tumor suppressor p53 (37) and can increase transcription of caspase 3 (38), S100A6 or calcyclin (up-regulated 1.86-fold, p = 0.0000003 in D39+pepstatin A versus D39 by iTRAQ analysis), was up-regulated during infection, but in this case pepstatin A treatment resulted in further up-regulation (Fig. 4C). These results indicate that the iTRAQ analysis appeared to have identified potential cathepsin D targets with known roles in the regulation of cell survival.

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