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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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Streptococcus pneumoniae infection induces cathepsin D dependent up-regulation of superoxide dismutase-2 (SOD-2). A, Representative Western blots probed for superoxide dismutase-2 (SOD-2) from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages (MDM) cultured in the presence (+) or absence (-) of pepstatin A (PepA) at the designated time points after challenge. Densitometry was carried out and fold change was calculated relative to the mock-infected (MI) level after adjustment for any fold change in tubulin, n = 3. B, Intracellular ROS was measured at the indicated timepoints in MDM, mock-infected (MI) or Streptococcus pneumoniae exposed (D39), in the presence of vehicle control or pepstatin A (+P). Data are from six separate donors. * p < 0.05, *** p < 0.001, 2-way ANOVA with Bonferroni post-test, comparing MI versus D39.
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Figure 7: Streptococcus pneumoniae infection induces cathepsin D dependent up-regulation of superoxide dismutase-2 (SOD-2). A, Representative Western blots probed for superoxide dismutase-2 (SOD-2) from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages (MDM) cultured in the presence (+) or absence (-) of pepstatin A (PepA) at the designated time points after challenge. Densitometry was carried out and fold change was calculated relative to the mock-infected (MI) level after adjustment for any fold change in tubulin, n = 3. B, Intracellular ROS was measured at the indicated timepoints in MDM, mock-infected (MI) or Streptococcus pneumoniae exposed (D39), in the presence of vehicle control or pepstatin A (+P). Data are from six separate donors. * p < 0.05, *** p < 0.001, 2-way ANOVA with Bonferroni post-test, comparing MI versus D39.

Mentions: The first protein of interest to be investigated further was SOD-2. SOD-2 is a protein which protects mitochondria against oxidative stress (39). The iTRAQ analysis, verified by Western blots, documented cathepsin D-dependent SOD-2 up-regulation. To validate these results in primary cells, and to elucidate the potential kinetics of this up-regulation, a timecourse was performed using MDM. Western blot analysis demonstrated that S. pneumoniae infection dramatically increased SOD-2 levels from 8 h postinfection (Fig. 7A). S. pneumoniae exposed cells treated with pepstatin A showed reduced SOD-2 expression compared with nontreated cells. Enhanced expression of SOD-2 is considered one of the first lines of defense against excess levels of ROS, which are produced by activated macrophages (40, 41), and SOD-2 is known to be differentially expressed following bacterial infection (42, 43). To confirm enhanced ROS production in our model and relate its kinetics to SOD-2 up-regulation, MDM production of ROS was measured using a fluorescent reporter for ROS, DCF. ROS production increased from 8 h after exposure to S. pneumoniae. (Fig. 7B). The kinetics of ROS production mirrored the up-regulation of mitochondrial SOD-2.


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)

Streptococcus pneumoniae infection induces cathepsin D dependent up-regulation of superoxide dismutase-2 (SOD-2). A, Representative Western blots probed for superoxide dismutase-2 (SOD-2) from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages (MDM) cultured in the presence (+) or absence (-) of pepstatin A (PepA) at the designated time points after challenge. Densitometry was carried out and fold change was calculated relative to the mock-infected (MI) level after adjustment for any fold change in tubulin, n = 3. B, Intracellular ROS was measured at the indicated timepoints in MDM, mock-infected (MI) or Streptococcus pneumoniae exposed (D39), in the presence of vehicle control or pepstatin A (+P). Data are from six separate donors. * p < 0.05, *** p < 0.001, 2-way ANOVA with Bonferroni post-test, comparing MI versus D39.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Streptococcus pneumoniae infection induces cathepsin D dependent up-regulation of superoxide dismutase-2 (SOD-2). A, Representative Western blots probed for superoxide dismutase-2 (SOD-2) from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages (MDM) cultured in the presence (+) or absence (-) of pepstatin A (PepA) at the designated time points after challenge. Densitometry was carried out and fold change was calculated relative to the mock-infected (MI) level after adjustment for any fold change in tubulin, n = 3. B, Intracellular ROS was measured at the indicated timepoints in MDM, mock-infected (MI) or Streptococcus pneumoniae exposed (D39), in the presence of vehicle control or pepstatin A (+P). Data are from six separate donors. * p < 0.05, *** p < 0.001, 2-way ANOVA with Bonferroni post-test, comparing MI versus D39.
Mentions: The first protein of interest to be investigated further was SOD-2. SOD-2 is a protein which protects mitochondria against oxidative stress (39). The iTRAQ analysis, verified by Western blots, documented cathepsin D-dependent SOD-2 up-regulation. To validate these results in primary cells, and to elucidate the potential kinetics of this up-regulation, a timecourse was performed using MDM. Western blot analysis demonstrated that S. pneumoniae infection dramatically increased SOD-2 levels from 8 h postinfection (Fig. 7A). S. pneumoniae exposed cells treated with pepstatin A showed reduced SOD-2 expression compared with nontreated cells. Enhanced expression of SOD-2 is considered one of the first lines of defense against excess levels of ROS, which are produced by activated macrophages (40, 41), and SOD-2 is known to be differentially expressed following bacterial infection (42, 43). To confirm enhanced ROS production in our model and relate its kinetics to SOD-2 up-regulation, MDM production of ROS was measured using a fluorescent reporter for ROS, DCF. ROS production increased from 8 h after exposure to S. pneumoniae. (Fig. 7B). The kinetics of ROS production mirrored the up-regulation of mitochondrial SOD-2.

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