title: Proteomic evaluation and validation of cathepsin D regulated proteins in macrophages exposed to Streptococcus pneumoniae.
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.
authors.affiliation: Medical School, University of Sheffield, Sheffield, UK.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Bewley
authors.forename: Trong K
authors.initials: TK
authors.lastname: Pham
authors.forename: Helen M
authors.initials: HM
authors.lastname: Marriott
authors.forename: Josselin
authors.initials: J
authors.lastname: Noirel
authors.forename: Hseuh-Ping
authors.initials: HP
authors.lastname: Chu
authors.forename: Saw Y
authors.initials: SY
authors.lastname: Ow
authors.forename: Alexey G
authors.initials: AG
authors.lastname: Ryazanov
authors.forename: Robert C
authors.initials: RC
authors.lastname: Read
authors.forename: Moira K B
authors.initials: MK
authors.lastname: Whyte
authors.forename: Benny
authors.initials: B
authors.lastname: Chain
authors.forename: Phillip C
authors.initials: PC
authors.lastname: Wright
authors.forename: David H
authors.initials: DH
authors.lastname: Dockrell
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D002402:Cathepsin D / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D015169:Colony Count, Microbial; D054736:Elongation Factor 2 Kinase / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000502:physiology; D000091342:Endoplasmic Reticulum Chaperone BiP; D057075:Enzyme Assays; D005260:Female; D018260:Gelsolin / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000382:microbiology; D008264:Macrophages / Q000276:immunology / Q000382:microbiology / Q000502:physiology*; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D018384:Oxidative Stress; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D020543:Proteome / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000276:immunology / Q000502:physiology*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D000099156:Superoxide Dismutase 2
chemical_list: D051017:Apoptosis Regulatory Proteins; D002402:Cathepsin D; D018797:Cell Cycle Proteins; D054736:Elongation Factor 2 Kinase; D000091342:Endoplasmic Reticulum Chaperone BiP; D018260:Gelsolin; D006360:Heat-Shock Proteins; D033681:Mitochondrial Membrane Transport Proteins; D010436:Pepstatins; D011480:Protease Inhibitors; D020543:Proteome; D017382:Reactive Oxygen Species; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; D013482:Superoxide Dismutase; D000099156:Superoxide Dismutase 2; C055351:S100A6 protein, human; C517690:Eef2k protein, mouse; C031375:pepstatin