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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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Cathepsin D-mediated down-regulation of eukaryotic elongation factor 2 (eEF2) has functional consequences for the regulation of the mitochondrial pathway of apoptosis. A, Western blots of total protein from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) differentiated THP-1 cells at the designated time points in the presence (+) or absence (-) of pepstatin A (PepA) were probed for eukaryotic elongation factor 2 (eEF2). The blots depicted are representative of three independent experiments. Densitometry was carried out and fold change was calculated using the mock-infected (MI) level after adjustment for any fold change in actin, n = 3, * p < 0.05, 2-way ANOVA with Bonferroni post-test. B, Western blots probed for phospho-eEF2 (peEF2) from Spn- or Spn+ differentiated THP-1 cells at the indicated time points after bacterial challenge. The blots are representative of three independent experiments. C, Western blot of protein probed for myeloid cell leukemia sequence (Mcl)-1 from wild-type (WT) or eEF2 kinase knock-out (KO) bone marrow-derived macrophages (BMDM), mock-infected (Spn-) or challenged with Streptococcus pneumoniae (Spn+), in the presence (+) or absence (-) of pepstatin A (PepA) and cultured for 16 h. The blots are representative of three independent experiments. Densitometry was carried out and fold change was calculated using mock-infected (MI) levels after adjustment for any fold change in actin, n = 3, * p < 0.05, 1-way ANOVA with Dunnett's post-test versus MI. D, Representative histograms and (E) pooled data from JC-1 staining of BMDMs expressing (WT) or deficient (KO) in eEF2 kinase. In the histograms dark gray fill represents Spn-, light gray fills Spn+. The pooled data shows the percentage of cells showing loss of inner mitochondrial transmembrane potential (Δψm), n = 3, * = p < 0.05, 2-way ANOVA with Bonferroni post-test.
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Figure 9: Cathepsin D-mediated down-regulation of eukaryotic elongation factor 2 (eEF2) has functional consequences for the regulation of the mitochondrial pathway of apoptosis. A, Western blots of total protein from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) differentiated THP-1 cells at the designated time points in the presence (+) or absence (-) of pepstatin A (PepA) were probed for eukaryotic elongation factor 2 (eEF2). The blots depicted are representative of three independent experiments. Densitometry was carried out and fold change was calculated using the mock-infected (MI) level after adjustment for any fold change in actin, n = 3, * p < 0.05, 2-way ANOVA with Bonferroni post-test. B, Western blots probed for phospho-eEF2 (peEF2) from Spn- or Spn+ differentiated THP-1 cells at the indicated time points after bacterial challenge. The blots are representative of three independent experiments. C, Western blot of protein probed for myeloid cell leukemia sequence (Mcl)-1 from wild-type (WT) or eEF2 kinase knock-out (KO) bone marrow-derived macrophages (BMDM), mock-infected (Spn-) or challenged with Streptococcus pneumoniae (Spn+), in the presence (+) or absence (-) of pepstatin A (PepA) and cultured for 16 h. The blots are representative of three independent experiments. Densitometry was carried out and fold change was calculated using mock-infected (MI) levels after adjustment for any fold change in actin, n = 3, * p < 0.05, 1-way ANOVA with Dunnett's post-test versus MI. D, Representative histograms and (E) pooled data from JC-1 staining of BMDMs expressing (WT) or deficient (KO) in eEF2 kinase. In the histograms dark gray fill represents Spn-, light gray fills Spn+. The pooled data shows the percentage of cells showing loss of inner mitochondrial transmembrane potential (Δψm), n = 3, * = p < 0.05, 2-way ANOVA with Bonferroni post-test.

Mentions: eEF2 was also identified as a factor down-regulated by cathepsin D. eEF2 was reduced 16–20 h post-infection in a cathepsin D-dependent fashion (Fig. 9A). Proteins with short half-lives, such as Mcl-1, a key regulator of macrophage susceptibility to apoptosis, including during S. pneumoniae infection, are exquisitely sensitive to alterations in protein translation (18, 49, 50). eEF2 activity is negatively regulated by eEF2 kinase, which prevents protein translation via the phosphorylation of eEF2 (51). During cellular stress, eEF2 kinase is activated with the purpose of reducing protein synthesis and conserving cellular energy sources (21, 52). We found an increase in eEF2 kinase activity 12–16 h post-infection (Fig. 9B), but there was no evidence that this was regulated by cathepsin D activation (data not shown). By studying macrophages deficient in eEF2 kinase we could examine the role of eEF2 using a system where eEF2 activity is inappropriately prolonged in the absence of the normal inhibitory effect of phosphorylation. As compared with wild-type BMDM, BMDM from eEF2 kinase−/− mice (53), maintained Mcl-1 levels (Fig. 9C) and had delayed dissipation of Δψm after S. pneumoniae infection (Figs. 9D and 9E). We conclude from these results that inactivation of eEF2-dependent protein translation is required for Mcl-1 down-regulation after S. pneumoniae infection and that cathepsin D induced down-regulation of eEF2 can drive apoptosis by reducing the translation of Mcl-1.


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-mediated down-regulation of eukaryotic elongation factor 2 (eEF2) has functional consequences for the regulation of the mitochondrial pathway of apoptosis. A, Western blots of total protein from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) differentiated THP-1 cells at the designated time points in the presence (+) or absence (-) of pepstatin A (PepA) were probed for eukaryotic elongation factor 2 (eEF2). The blots depicted are representative of three independent experiments. Densitometry was carried out and fold change was calculated using the mock-infected (MI) level after adjustment for any fold change in actin, n = 3, * p < 0.05, 2-way ANOVA with Bonferroni post-test. B, Western blots probed for phospho-eEF2 (peEF2) from Spn- or Spn+ differentiated THP-1 cells at the indicated time points after bacterial challenge. The blots are representative of three independent experiments. C, Western blot of protein probed for myeloid cell leukemia sequence (Mcl)-1 from wild-type (WT) or eEF2 kinase knock-out (KO) bone marrow-derived macrophages (BMDM), mock-infected (Spn-) or challenged with Streptococcus pneumoniae (Spn+), in the presence (+) or absence (-) of pepstatin A (PepA) and cultured for 16 h. The blots are representative of three independent experiments. Densitometry was carried out and fold change was calculated using mock-infected (MI) levels after adjustment for any fold change in actin, n = 3, * p < 0.05, 1-way ANOVA with Dunnett's post-test versus MI. D, Representative histograms and (E) pooled data from JC-1 staining of BMDMs expressing (WT) or deficient (KO) in eEF2 kinase. In the histograms dark gray fill represents Spn-, light gray fills Spn+. The pooled data shows the percentage of cells showing loss of inner mitochondrial transmembrane potential (Δψm), n = 3, * = p < 0.05, 2-way ANOVA with Bonferroni post-test.
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Figure 9: Cathepsin D-mediated down-regulation of eukaryotic elongation factor 2 (eEF2) has functional consequences for the regulation of the mitochondrial pathway of apoptosis. A, Western blots of total protein from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn+) differentiated THP-1 cells at the designated time points in the presence (+) or absence (-) of pepstatin A (PepA) were probed for eukaryotic elongation factor 2 (eEF2). The blots depicted are representative of three independent experiments. Densitometry was carried out and fold change was calculated using the mock-infected (MI) level after adjustment for any fold change in actin, n = 3, * p < 0.05, 2-way ANOVA with Bonferroni post-test. B, Western blots probed for phospho-eEF2 (peEF2) from Spn- or Spn+ differentiated THP-1 cells at the indicated time points after bacterial challenge. The blots are representative of three independent experiments. C, Western blot of protein probed for myeloid cell leukemia sequence (Mcl)-1 from wild-type (WT) or eEF2 kinase knock-out (KO) bone marrow-derived macrophages (BMDM), mock-infected (Spn-) or challenged with Streptococcus pneumoniae (Spn+), in the presence (+) or absence (-) of pepstatin A (PepA) and cultured for 16 h. The blots are representative of three independent experiments. Densitometry was carried out and fold change was calculated using mock-infected (MI) levels after adjustment for any fold change in actin, n = 3, * p < 0.05, 1-way ANOVA with Dunnett's post-test versus MI. D, Representative histograms and (E) pooled data from JC-1 staining of BMDMs expressing (WT) or deficient (KO) in eEF2 kinase. In the histograms dark gray fill represents Spn-, light gray fills Spn+. The pooled data shows the percentage of cells showing loss of inner mitochondrial transmembrane potential (Δψm), n = 3, * = p < 0.05, 2-way ANOVA with Bonferroni post-test.
Mentions: eEF2 was also identified as a factor down-regulated by cathepsin D. eEF2 was reduced 16–20 h post-infection in a cathepsin D-dependent fashion (Fig. 9A). Proteins with short half-lives, such as Mcl-1, a key regulator of macrophage susceptibility to apoptosis, including during S. pneumoniae infection, are exquisitely sensitive to alterations in protein translation (18, 49, 50). eEF2 activity is negatively regulated by eEF2 kinase, which prevents protein translation via the phosphorylation of eEF2 (51). During cellular stress, eEF2 kinase is activated with the purpose of reducing protein synthesis and conserving cellular energy sources (21, 52). We found an increase in eEF2 kinase activity 12–16 h post-infection (Fig. 9B), but there was no evidence that this was regulated by cathepsin D activation (data not shown). By studying macrophages deficient in eEF2 kinase we could examine the role of eEF2 using a system where eEF2 activity is inappropriately prolonged in the absence of the normal inhibitory effect of phosphorylation. As compared with wild-type BMDM, BMDM from eEF2 kinase−/− mice (53), maintained Mcl-1 levels (Fig. 9C) and had delayed dissipation of Δψm after S. pneumoniae infection (Figs. 9D and 9E). We conclude from these results that inactivation of eEF2-dependent protein translation is required for Mcl-1 down-regulation after S. pneumoniae infection and that cathepsin D induced down-regulation of eEF2 can drive apoptosis by reducing the translation of Mcl-1.

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