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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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Infection with Streptococcus pneumoniae is associated with activation of cathepsin D in macrophages. A, Cathepsin D activity was measured in whole-cell lysates at 16 h in mock-infected (Spn-), or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages, n = 4, *** = p < 0.001, Student's t test. B, Western blot of differentiated THP-1 cells 16 h after mock-infection (Spn-) or exposure to Streptococcus pneumoniae (Spn+). The blot is representative of three independent infections.
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Figure 2: Infection with Streptococcus pneumoniae is associated with activation of cathepsin D in macrophages. A, Cathepsin D activity was measured in whole-cell lysates at 16 h in mock-infected (Spn-), or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages, n = 4, *** = p < 0.001, Student's t test. B, Western blot of differentiated THP-1 cells 16 h after mock-infection (Spn-) or exposure to Streptococcus pneumoniae (Spn+). The blot is representative of three independent infections.

Mentions: Following phagocytosis of Streptococcus pneumoniae, the fusion of lysosomes with the phagosome, generates a phagolysosome where bacteria are killed (30). Of the proteolytic enzymes present, cathepsin D is the most abundant of the cathepsin proteases (9). To confirm that S. pneumoniae infection activated cathepsin D in MDM, a fluorogenic substrate of cathepsin D was used as a marker of activation. D39 infected cells showed significant activation of cathepsin D at 16 h postinfection, compared with mock-infected cells (Fig. 2A). This result was corroborated by Western blot on differentiated THP-1 cells 16 h postinfection (Fig. 2B). Infection caused an increase in the active 35 kDa form of cathepsin D (31, 32).


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)

Infection with Streptococcus pneumoniae is associated with activation of cathepsin D in macrophages. A, Cathepsin D activity was measured in whole-cell lysates at 16 h in mock-infected (Spn-), or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages, n = 4, *** = p < 0.001, Student's t test. B, Western blot of differentiated THP-1 cells 16 h after mock-infection (Spn-) or exposure to Streptococcus pneumoniae (Spn+). The blot is representative of three independent infections.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Infection with Streptococcus pneumoniae is associated with activation of cathepsin D in macrophages. A, Cathepsin D activity was measured in whole-cell lysates at 16 h in mock-infected (Spn-), or Streptococcus pneumoniae exposed (Spn+) monocyte-derived macrophages, n = 4, *** = p < 0.001, Student's t test. B, Western blot of differentiated THP-1 cells 16 h after mock-infection (Spn-) or exposure to Streptococcus pneumoniae (Spn+). The blot is representative of three independent infections.
Mentions: Following phagocytosis of Streptococcus pneumoniae, the fusion of lysosomes with the phagosome, generates a phagolysosome where bacteria are killed (30). Of the proteolytic enzymes present, cathepsin D is the most abundant of the cathepsin proteases (9). To confirm that S. pneumoniae infection activated cathepsin D in MDM, a fluorogenic substrate of cathepsin D was used as a marker of activation. D39 infected cells showed significant activation of cathepsin D at 16 h postinfection, compared with mock-infected cells (Fig. 2A). This result was corroborated by Western blot on differentiated THP-1 cells 16 h postinfection (Fig. 2B). Infection caused an increase in the active 35 kDa form of cathepsin D (31, 32).

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