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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 down-regulation of gelsolin. A, Representative Western blots probed for gelsolin from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages (MDM) cultured in the presence (+) or absence (-) of pepstatin A (PepA) for the indicated time periods. Densitometry was carried out and fold change was calculated using mock-infected (MI) levels after adjustment for any fold change in tubulin, n = 3, ** p < 0.01, 2-way ANOVA with Bonferroni post-test. B, Cytosolic fractions were obtained from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages (MDM) at the designated time points. Western blots were probed for cytochrome c, and actin was used as a cytosolic loading control. The blots are representative of three independent experiments.
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Figure 8: Streptococcus pneumoniae infection induces cathepsin D dependent down-regulation of gelsolin. A, Representative Western blots probed for gelsolin from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages (MDM) cultured in the presence (+) or absence (-) of pepstatin A (PepA) for the indicated time periods. Densitometry was carried out and fold change was calculated using mock-infected (MI) levels after adjustment for any fold change in tubulin, n = 3, ** p < 0.01, 2-way ANOVA with Bonferroni post-test. B, Cytosolic fractions were obtained from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages (MDM) at the designated time points. Western blots were probed for cytochrome c, and actin was used as a cytosolic loading control. The blots are representative of three independent experiments.

Mentions: The next protein of interest to be studied further was the actin regulatory protein gelsolin (44). Gelsolin reduces apoptosis both at the level of mitochondrial outer membrane permeabilization (45) and caspase activation (46, 47). A time course of gelsolin expression in MDM revealed down-regulation in a cathepsin D-dependent fashion from 12 h post-infection, with maximal down-regulation observed at 16 h (Fig. 8A). The maximal down-regulation coincided with cytochrome c translocation to the cytosol (Fig. 8B), a hallmark of mitochondrial outer membrane permeabilization, which is widely recognized as being the point of no return in an apoptotic program of death (48). These data suggest a model in which cathepsin D acts to down-regulate gelsolin, thus contributing to the destabilization of the mitochondria, leading to apoptosis.


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 down-regulation of gelsolin. A, Representative Western blots probed for gelsolin from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages (MDM) cultured in the presence (+) or absence (-) of pepstatin A (PepA) for the indicated time periods. Densitometry was carried out and fold change was calculated using mock-infected (MI) levels after adjustment for any fold change in tubulin, n = 3, ** p < 0.01, 2-way ANOVA with Bonferroni post-test. B, Cytosolic fractions were obtained from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages (MDM) at the designated time points. Western blots were probed for cytochrome c, and actin was used as a cytosolic loading control. The blots are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Streptococcus pneumoniae infection induces cathepsin D dependent down-regulation of gelsolin. A, Representative Western blots probed for gelsolin from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages (MDM) cultured in the presence (+) or absence (-) of pepstatin A (PepA) for the indicated time periods. Densitometry was carried out and fold change was calculated using mock-infected (MI) levels after adjustment for any fold change in tubulin, n = 3, ** p < 0.01, 2-way ANOVA with Bonferroni post-test. B, Cytosolic fractions were obtained from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages (MDM) at the designated time points. Western blots were probed for cytochrome c, and actin was used as a cytosolic loading control. The blots are representative of three independent experiments.
Mentions: The next protein of interest to be studied further was the actin regulatory protein gelsolin (44). Gelsolin reduces apoptosis both at the level of mitochondrial outer membrane permeabilization (45) and caspase activation (46, 47). A time course of gelsolin expression in MDM revealed down-regulation in a cathepsin D-dependent fashion from 12 h post-infection, with maximal down-regulation observed at 16 h (Fig. 8A). The maximal down-regulation coincided with cytochrome c translocation to the cytosol (Fig. 8B), a hallmark of mitochondrial outer membrane permeabilization, which is widely recognized as being the point of no return in an apoptotic program of death (48). These data suggest a model in which cathepsin D acts to down-regulate gelsolin, thus contributing to the destabilization of the mitochondria, leading to apoptosis.

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