Proteomic evaluation and validation of cathepsin D regulated proteins in macrophages exposed to Streptococcus pneumoniae.

Martin A Bewley,Alexey G Ryazanov,Josselin Noirel,Hseuh-Ping Chu,Moira K.B Whyte,Robert C Read,Phillip C Wright,Helen M Marriott,David H Dockrell,Trong K Pham,Benny Chain,Saw Y Ow

doi:10.1074/mcp.m111.008193

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.

Highlights

From the ‡Medical School ‡‡Sheffield Teaching Hospitals and §ChELSI Institute, Department of Chemical and Processing Engineering, University of Sheffield, Sheffield, UK.; ¶Division of Infection and Immunity, University College London, London, UK; ʈDepartment of Pharmacology University of Medicine and Dentistry of New JerseyRobert Wood Johnson Medical School, New Jersey, USA
Given that cathepsin D has previously been implicated in the regulation of apoptosis pathways in myeloid cells [13], and we have shown macrophage apoptosis in response to S. pneumoniae is necessary for bactericidal clearance in this model [17, 18], several of these proteins were investigated further to determine how cathepsin D activation might influence pathways involved in the regulation of macrophage apoptosis during S. pneumoniae exposure
We show that a range of proteins are differentially expressed in the presence of cathepsin D, with a high proportion predicted to impact the regulation of the mitochondrial pathway of apoptosis

Summary

EXPERIMENTAL PROCEDURES

Bacteria—Type 2 S. pneumoniae (D39 strain, NCTC 7466) were grown in Brain Heart Infusion (BHI) media supplemented with 20% v/v fetal calf serum (FCS) until an OD610 nm of 0.6 was reached. Modifications were performed as follows: eight-plex iTRAQ mass shifts (ϩ304 Da, K and N-term) as fixed modification, cys CAM (ϩ57 Da) as fixed modification on the C residue, and oxidation of methionine (ϩ16 Da) as variable modification on the M residue These data were searched for within the reversed IPI human database to estimate the false positive peptide discovery rate using the formula, false positive peptide discovery rate ϭ 2 decoy_hits/(decoy_hits ϩ true_hits), as detailed elsewhere [27]. Measurement of Mitochondrial Inner Transmembrane Potential (⌬␺m)—To detect loss of ⌬␺m at the required time-points, cells were incubated with 10 ␮M 5,5Ј, 6,6Ј-tetrachloro-1, 1Ј, 3,3Ј tetraethylbenzimidazolocarbocyanine iodide (JC-1; Sigma-Aldrich) for 15 min and analyzed by flow cytometry. To be significant all t-tests of each pairwise comparison were less than ␣ ϭ 0.05, with a false discovery rate of Յ1% (data not shown)

RESULTS

Cellular Assembly and Organization

DISCUSSION