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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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Experimental schematic. A biological duplicate of mock-infected (MI) cells and two biological triplicates of Streptococcus pneumoniae exposed cells incubated with vehicle control (D39) or with pepstatin A (D39 + Pepstatin A) were used for analysis.
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Figure 1: Experimental schematic. A biological duplicate of mock-infected (MI) cells and two biological triplicates of Streptococcus pneumoniae exposed cells incubated with vehicle control (D39) or with pepstatin A (D39 + Pepstatin A) were used for analysis.

Mentions: Protein samples were precipitated using ice-cold acetone at −20 °C overnight, harvested by centrifugation at 21,000 × g at 4 °C for 20 min (22) and resuspended in 1 m Triethyloammoniumbicarbonate at pH 8.5. Total protein quantification involved the Rc-Dc Quantification Assay (Bio-Rad; UK) according to the manufacturer's instructions. One hundred micrograms of each sample was used for the eight-plex iTRAQ technique (Applied Biosystems, Foster City, CA). These samples were reduced, alkylated, digested, and labeled with iTRAQ reagents according to the manufacturer's protocol (Applied Biosystems), as previously described (22). The labeling of samples was carried out with 15 sets for data analysis. Two independent biological triplicates (D39 labeled with reagents 115, 116, 117, and D39 with pepstatin A labeled with reagents 118, 119, 121) and one biological duplicate (MI, labeled with reagents 113 and 114) were applied (Fig. 1). After incubation at room temperature, labeled samples were combined before being dried in a vacuum concentrator. Fractionation of samples using strong cation exchange on a BioLC HPLC system (Dionex, UK) was used to clean the samples, as well as to reduce their complexity (23). The strong cation exchange fractionation was carried out using a PolySulfoethyl A Column (PolyLC, USA) with a 5 μm particle size, 20 cm length × 2.1 mm diameter, and 200 Å pore size. The system was operated at a flow rate of 0.2 ml·min−1 with an injection volume of 120 μl. The mobile phase comprised buffers A and B. Buffer A contained 10 mm KH2PO4, 25% acetonitrile at pH 3, and buffer B consisted of 10 mm KH2PO4, 25% acetonitrile, and 500 mm KCl, at pH 3. A 60-min gradient was used, which was 5 min at 100% buffer A, followed by ramping from 5% to 30% buffer B over 40 min, then 30% to 100% buffer B over 5 min, and finally holding at 100% buffer A for 5 min. A UV detector UVD170U and Chromeleon Software (Dionex, The Netherlands) were used to record the chromatogram. Labeled peptide fractions were collected every minute, and then each fraction was dried in a vacuum concentrator. These dried labeled-peptides were then cleaned up using C18 Discovery DSC-18 SPE column (100 μg capacity, Supelco, Sigma) as detailed by Chong and Wright (24) before submission to the mass spectrometry instrument.


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)

Experimental schematic. A biological duplicate of mock-infected (MI) cells and two biological triplicates of Streptococcus pneumoniae exposed cells incubated with vehicle control (D39) or with pepstatin A (D39 + Pepstatin A) were used for analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Experimental schematic. A biological duplicate of mock-infected (MI) cells and two biological triplicates of Streptococcus pneumoniae exposed cells incubated with vehicle control (D39) or with pepstatin A (D39 + Pepstatin A) were used for analysis.
Mentions: Protein samples were precipitated using ice-cold acetone at −20 °C overnight, harvested by centrifugation at 21,000 × g at 4 °C for 20 min (22) and resuspended in 1 m Triethyloammoniumbicarbonate at pH 8.5. Total protein quantification involved the Rc-Dc Quantification Assay (Bio-Rad; UK) according to the manufacturer's instructions. One hundred micrograms of each sample was used for the eight-plex iTRAQ technique (Applied Biosystems, Foster City, CA). These samples were reduced, alkylated, digested, and labeled with iTRAQ reagents according to the manufacturer's protocol (Applied Biosystems), as previously described (22). The labeling of samples was carried out with 15 sets for data analysis. Two independent biological triplicates (D39 labeled with reagents 115, 116, 117, and D39 with pepstatin A labeled with reagents 118, 119, 121) and one biological duplicate (MI, labeled with reagents 113 and 114) were applied (Fig. 1). After incubation at room temperature, labeled samples were combined before being dried in a vacuum concentrator. Fractionation of samples using strong cation exchange on a BioLC HPLC system (Dionex, UK) was used to clean the samples, as well as to reduce their complexity (23). The strong cation exchange fractionation was carried out using a PolySulfoethyl A Column (PolyLC, USA) with a 5 μm particle size, 20 cm length × 2.1 mm diameter, and 200 Å pore size. The system was operated at a flow rate of 0.2 ml·min−1 with an injection volume of 120 μl. The mobile phase comprised buffers A and B. Buffer A contained 10 mm KH2PO4, 25% acetonitrile at pH 3, and buffer B consisted of 10 mm KH2PO4, 25% acetonitrile, and 500 mm KCl, at pH 3. A 60-min gradient was used, which was 5 min at 100% buffer A, followed by ramping from 5% to 30% buffer B over 40 min, then 30% to 100% buffer B over 5 min, and finally holding at 100% buffer A for 5 min. A UV detector UVD170U and Chromeleon Software (Dionex, The Netherlands) were used to record the chromatogram. Labeled peptide fractions were collected every minute, and then each fraction was dried in a vacuum concentrator. These dried labeled-peptides were then cleaned up using C18 Discovery DSC-18 SPE column (100 μg capacity, Supelco, Sigma) as detailed by Chong and Wright (24) before submission to the mass spectrometry instrument.

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