Proteomic Evaluation and Validation of Cathepsin D Regulated Proteins in Macrophages Exposed to Streptococcus pneumoniae

Article (PDF Available)inMolecular & Cellular Proteomics 10(6):M111.008193 · June 2011with25 Reads
DOI: 10.1074/mcp.M111.008193 · Source: PubMed
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.
Proteomic Evaluation and Validation of
Cathepsin D Regulated Proteins in
Macrophages Exposed to
Streptococcus pneumoniae*
S
Martin A. Bewley‡, Trong K. Pham§, Helen M. Marriott‡, Josselin Noirel§,
Hseuh-Ping Chu, Saw Y. Ow§, Alexey G. Ryazanov, Robert C. Read‡,‡‡
Moira K. B. Whyte‡‡, Benny Chain¶, Phillip C. Wright§, and David H. Dockrell‡**‡‡
Macrophages are central effectors of innate immune re-
sponses to bacteria. We have investigated how activation
of the abundant macrophage lysosomal protease, cathep-
sin D, regulates the macrophage proteome during killing
of Streptococcus pneumoniae. Using the cathepsin D in-
hibitor pepstatin A, we demonstrate that cathepsin D dif-
ferentially 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 (
0.05). This dataset was enriched for
proteins regulating the mitochondrial pathway of apopto-
sis or inhibiting competing death programs. Five proteins
were selected for further analysis. Western blotting, fol-
lowed by pharmacological inhibition or genetic manipula-
tion of cathepsin D, verified cathepsin D-dependent reg-
ulation 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 mitochon-
dria. Eukaryotic elongation factor (eEF2), a regulator of
protein translation, was also down-regulated by cathep-
sin 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 mu-
rine model that maintaining eEF2 function is associated
with impaired macrophage apoptosis-associated killing
of Streptococcus pneumoniae. These findings demon-
strate that cathepsin D regulates multiple proteins control-
ling the mitochondrial pathway of macrophage apoptosis or
competing death processes, facilitating intracellular bacte-
rial killing. Molecular & Cellular Proteomics 10: 10.1074/
mcp.M111.008193, 1–14, 2011.
Tissue macrophages are central effectors of innate immu-
nity against pathogenic micro-organisms, complementing the
action of other components of the innate immune response
(1). Transcriptomic and proteomic analysis confirms the broad
range of genes that are activated in macrophages by micro-
organisms (2–5). Phagocytosis and intracellular killing in
phagolysosomes activates multiple signal transduction path-
ways whose concerted activity leads to a regulated host
response to pathogens (6).
A central component of the macrophages capacity to han-
dle ingested bacteria is its highly developed phagolysosomal
system, which distinguishes the cell from other phagocytes (7,
8). Phagosomes fuse with lysosomes containing proteases, of
which cathepsin D is one of the most abundant in macro-
phages (9). Cathepsin D expression in macrophages is highly
differentiation dependent (10, 11). Cathepsin D is an aspartic
protease, which possesses activity against a broad range of
substrates (12). It plays a role in neurologic development,
immune homeostasis, apoptosis initiation, and tumor devel-
opment (12). Although cathepsin D was formerly believed to
act principally within the low pH environment of the lysosome,
it has become apparent that it can have residual activity
outside the lysosomal compartment (13). The role of cathep-
sin D in host defense is currently unclear, although it has been
documented to cleave the cholesterol-dependent cytolysin
listerolysin produced by Listeria monocytogenes (14). We
have recently identified a critical role for cathepsin D in the
host response of macrophages against Streptococcus pneu-
moniae (15). Cathepsin D activation was observed in macro-
phages following ingestion of S. pneumoniae into phagolyso-
somes and played a role in the induction of macrophage
apoptosis, which contributed to microbial killing. Cathepsin D
From the ‡Medical School ‡‡Sheffield Teaching Hospitals and
§ChELSI Institute, Department of Chemical and Processing Engineer-
ing, University of Sheffield, Sheffield, UK.; ¶Division of Infection and
Immunity, University College London, London, UK; Department of
Pharmacology University of Medicine and Dentistry of New Jersey-
Robert Wood Johnson Medical School, New Jersey, USA
Received January 26, 2011, and in revised form, April 1, 2011
Author’s Choice—Final version full access.
Published, MCP Papers in Press, April 7, 2011, DOI 10.1074/
mcp.M111.008193
Research
Author’s Choice © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
This paper is available on line at http://www.mcponline.org
Molecular & Cellular Proteomics 10.6 10.1074/mcp.M111.008193–1
is well positioned to transduce signals from the phagolyso-
some during the intracellular killing of bacteria, but it is unclear
how such an effect might be mediated.
In the current study, we applied an isobaric tag for relative
and absolute quantitation (iTRAQ)
1
proteomic approach to
identify proteins that were differentially expressed in macro-
phages during infection with S. pneumoniae (pneumococci) in
the presence of an aspartic protease inhibitor, pepstatin A, as
compared with vehicle control. We identified a number of
differentially expressed proteins and confirmed their differen-
tial expression, after both pharmacological inhibition and ge-
netic manipulation of cathepsin D, relating these to recog-
nized features of pneumococcal infection in macrophages.
We demonstrate that a number of these proteins regulate
aspects of macrophage apoptosis, a process we have previ-
ously described as being critical to the successful control of
pneumococcal infection in macrophages (
16–18). In particu
-
lar, we find that proteins known to regulate oxidant stress,
endoplasmic reticulum (ER) stress, the expression of short-
lived anti-apoptotic proteins and mitochondrial outer mem-
brane permeabilization were differentially regulated in the
presence of cathepsin D inhibition.
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 OD
610 nm
of 0.6 was reached. Prior to
infection, thawed aliquots were opsonized in RPMI (Sigma-Aldrich)
containing 10% v/v antipneumococcal immune serum (
16). For
mouse experiments type 1 S. pneumoniae (WHO reference laboratory
strain SSISP; Statens Serum Institut) were handled under identical
conditions but were not opsonized before instillation. Bacterial num-
bers were assessed by the surface viable count method after inocu-
lation on blood agar (
16).
Cells and Infection—THP-1 cells were cultured in RPMI plus 10%
v/v FCS (complete media). THP-1 cells were differentiated to a mac-
rophage phenotype by treating 0.4 10
6
cell/ml with 200 nM phorbol
12-myristate 13-acetate for 3 days, after which the phorbol 12-my-
ristate 13-acetate was removed, and the cells left to rest for a further
5 days after which cell numbers were determined. These cells have a
phenotype similar to monocyte-derived macrophages (MDM), as ev-
idenced by nuclear to cytoplasmic ratio, concentration of mitochon-
dria and lysosomes, cell surface markers, phagocytic capacity, cyto-
kine generation to Toll-like receptor agonists, and susceptibility to
apoptosis (
19). Human MDMs were isolated from whole blood do
-
nated by healthy volunteers as previously described with informed
consent as approved by the South Sheffield Regional Ethics Com-
mittee of Royal Hallamshire Hospital (Sheffield, United Kingdom) (
16).
After 14 days, representative wells were scraped to determine cell
numbers. Murine bone-marrow derived macrophages (BMDM)s were
isolated by culturing marrow from mice deficient in cathepsin D (
20) or
eukaryotic elongation factor 2 kinase (eEF2k) (
21), or from the corre
-
sponding wild-type littermates. BMDMs were plated at 0.5 10
6
cells/ml for 14 d in Dulbecco’s modified Eagles medium containing
10% FCS and 10% conditioned L929 media (
17). All cell types were
infected with opsonized S. pneumoniae at a multiplicity of infection of
10, or mock-infected (MI) as described elsewhere (
16). Cells were
incubated with 100
M of the aspartic protease inhibitor pepstatin A
or dimethylsulfoxide vehicle control.
SDS-PAGE and Western Immunoblotting—Whole-cell extracts and
cytosolic fractions were isolated as previously described (
18). Blots
were incubated overnight at 4 °C with antibodies against either gelso-
lin (rabbit polyclonal, 1:1000; Abcam, Cambridge, MA), SOD-2 (rabbit
polyclonal 1:1000; Abcam), heat shock protein (Hsp) A5/glucose-
regulated protein (Grp)78/BiP (rabbit polyclonal 1:200; Abcam), S100
calcium binding protein A6/calcyclin (rabbit polyclonal 1:1000; Ab-
cam), murine induced myeloid cell leukemia myeloid cell leukemia
sequence 1 (Mcl-1), (rabbit polyclonal, 1:1000; Rockland, Rockland
ME), cytochrome c (mouse monoclonal, 1:1000; BD Biosciences),
cathepsin D (goat polyclonal, 1:1000; R&D Systems, Minneapolis,
MN), phospho-eukaryotic elongation factor (eEF) 2 (Thr56), eEF2
(both from Cell signaling, Danvers, MA; 1:1000), actin (rabbit poly-
clonal 1:5000; Sigma-Aldrich), or tubulin (mouse polyclonal 1:1000;
Sigma-Aldrich). Protein detection was with horseradish peroxidase
conjugated secondary antibodies (1:2000; Dako) and ECL (Amersham
Biosciences Pharmacia). Bands were quantified using Image J 1.32
software (National Institutes of Health) and fold change from mock-
infected, calculated and normalized to the fold change in tubulin or
actin (
18).
Cathepsin D Activity Assay—Cathepsin D activity was measured in
cell lysates using a fluorometric cathepsin D activity assay kit (Abcam)
in accordance with the manufacturer’s instructions. Fluorescence
was measured on a Packard Bioscience Fusion
TM
microplate ana
-
lyzer. Cathepsin D activity in each lysed sample was expressed as a
percentage of a comparative sample that had been treated with an
excess (500
M) of pepstatin A to act as a negative control.
Sample Preparation and iTRAQ labeling—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 resus
-
pended 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 micro-
grams 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 de-
scribed (
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
m 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 mlmin
1
with an injection volume of 120
l. The mobile
phase comprised buffers A and B. Buffer A contained 10 m
M KH
2
PO
4
,
25% acetonitrile at pH 3, and buffer B consisted of 10 m
M KH
2
PO
4
,
25% acetonitrile, and 500 m
M 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
1
The abbreviations used are: iTRAQ, isobaric tag for relative and
absolute quantitation; BMDM, bone marrow-derived macrophage;
eEF2, eukaryotic elongation factor 2; FLIP, Fas-associated protein
with death domain-like interleukin-1 beta converting enzyme inhibi-
tary protein; Hsp, heat shock protein; MDM, monocyte-derived mac-
rophage; ROS, reactive oxygen species; SOD, superoxide dismutase;
m
, mitochondrial inner transmembrane potential.
Cathepsin D Regulated Proteins
10.1074/mcp.M111.008193–2 Molecular & Cellular Proteomics 10.6
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 C
18
Discovery DSC-18 SPE column (100
g capacity, Supelco,
Sigma) as detailed by Chong and Wright (
24) before submission to the
mass spectrometry instrument.
LC-MS/MS UHR-TOF Analysis—Tandem mass spectrometry of
liquid chromatography (LC)-MS iTRAQ labeled samples was carried
out on a maXis hybrid ultra-high resolution quadrupole time-of-flight
system (Bruker Daltonics, Coventry UK) coupled to an Ultimate 3000
nano-flow HPLC (Dionex, Surrey, UK) (
25). All LC-MS iTRAQ samples
were first desalted online using a 5 mm, 300
m ID LC-Packings C
18
PepMap trap cartridge under 0.1% trifluoroacetic acid and 3% ace-
tonitrile (ACN) for 15 min, and eluted to a 15 cm, 75
mIDLC-
Packings C
18
PepMap analytical column in 0.1% formic acid, with an
ACN gradient extending from 3% to 95%. Elution was performed on
a predefined 70-min gradient program (3–35% ACN) with a 20% wash
step (35–95% ACN) as described (
26). TOF-MS screening measure
-
ments were performed on a predefined 50–2200 m/z acquisition
window at 2500 TOF summations (approx. 2 Hz) at R (resolution)
40000 at 622 m/z. Collision-induced dissociation MS/MS acquisition
were performed over the same 50–2200 m/z window at r 40,000 at
622 m/z with three intensity binned precursors of charge 2to4
with at least 2500 counts between 250–1400 m/z, deriving from the
TOF-MS screening experiment. Accumulation times for tandem MS
(MS/MS) were also intensity binned at a maximum of 5000 summa-
tions (approximately 1 Hz, if precursor 2.5 10
3
ion counts) and a
minimum of 2000 summations (approximately 2.5 Hz, if precursor
2 10
4
ion counts). An optimized set of isolation windows was used
based on the precursor m/z to achieve at least 90% precursor recov-
ery; 1.5 m/z for 300 m/z,3m/z for 500 m/z,4m/z for 1000 m/z
and 5.5 m/z for 1400 m/z. Selected precursors after fragmentation
twice were actively excluded for 90 s from further analysis. To overcome
the increased stability because of the iTRAQ labels, collision energies
were increased by 10% to optimize for peptide fragmentation.
MS/MS Data Analysis—Data processing of LC-MS/MS samples
were first parsed using proprietary vendor analysis software, mi-
croTOF Control v 2.3 Service Pack 1, and processing module, Data
Analysis v 4.0 Service Pack 2 (all from: Bruker Daltonik GmbH, Bre-
men Germany). MS/MS data recovery to MGF was processed via an
embedded daMGF script. Data were then searched against the Inter-
national Protein Index (IPI) human database (downloaded from http://
www.ebi.ac.uk, on September 2009, with 84,032 protein entries) us-
ing a local Phenyx v2.5 (Genebio, Geneva Switzerland) processing
cluster at the ChELSI Institute. The search parameters were set as
follows: MS tolerance was 0.4 Da and MS/MS tolerances were set at:
peptide tolerance 0.2 Da, charge 1, 2, 3, and 4. Minimum
peptide length, z-score, maximum p value, and AC score were 6, 5.5,
10
6
, and 6, respectively. The enzyme mode for searching was tryp
-
sin, permitting up to two missed cleavages. Modifications were per-
formed as follows: eight-plex iTRAQ mass shifts (304 Da, K and
N-term) as fixed modification, cys CAM (57 Da) as fixed modifica-
tion on the C residue, and oxidation of methionine (16 Da) as
variable modification on the M residue. These data were then
searched for within the reversed IPI human database to estimate the
false positive peptide discovery rate using the formula, false positive
peptide discovery rate 2 decoy_hits/(decoy_hits true_hits), as
detailed elsewhere (
27). Processed data were exported to Excel (Mi
-
crosoft 2008, USA) for further analyses via Phenyx local export to
retrieve peptide identification data and reporter intensity values for
each separately identified peptide. Peptide data and intensity values
were parsed locally to Mathematica v7.0 (Wolfram Research, Oxford-
shire UK) with more details found in Pham et al. (
28). From this, only
peptides with 2 peptide identifications were used for both identifi-
cation and quantitation. Functional classification of the identified pro-
teins was generated through use of Ingenuity Pathway Analysis (In-
genuity ® Systems, www.ingenuity.com).
Measurement of Reactive Oxygen Species (ROS)—Production of
intracellular ROS was measured using the cell permeable molecule 2,
7-dichloro-dihydrofluorescein diacetate (DCF; Sigma-Aldrich) (
29).
Macrophages were pre-incubated with 10
M DCF for 30 min before
infection for the indicated time periods. Cells were washed in phos-
phate-buffered saline (PBS) then analyzed by flow cytometry.
Measurement of Mitochondrial Inner Transmembrane Potential
(
m
)—To detect loss of
m
at the required time-points, cells were
incubated with 10
M 5,5, 6,6-tetrachloro-1, 1, 3,3 tetraethylben-
zimidazolocarbocyanine iodide (JC-1; Sigma-Aldrich) for 15 min and
analyzed by flow cytometry. Loss of
m
was demonstrated by a loss
of fluorescence on the FL-2 channel as previously described (
18).
Mouse Infection Model—eEF2 kinase knockout or littermate con-
trol, female C57Bl6 mice at 8–14 weeks of age, received 5 10
5
colony forming units of type 1 S. pneumoniae via intratracheal instil-
lation and 24 h lungs were harvested, homogenized and bacterial
colony counts obtained by the surface viable count method (
17). All
experiments were performed in accordance with the UK Animals Act,
authorized under a UK Home Office License, and approved by the
animal project review committee of the University of Sheffield.
Statistical Analysis—Macrophage data was recorded as means
S.E. of the mean (se) unless otherwise stated. Statistical testing was
performed using Prism
®
5.02 software (GraphPad Software Inc.) with
relevant statistical tests described in the figure legends. Significance
was defined as p 0.05.
Proteomic values were recorded as median values. For the pro-
teomic analysis we used two biological triplicates (for D39 and
D39PepstatinA). To identify differentially expressed proteins we
performed t test comparisons between the iTRAQ reporter ions’ in-
tensities (considering all MS/MS spectrum for a given protein). There-
fore significance increased with the number of MS/MS spectra ob-
tained for a protein. To be significant all t-tests of each pairwise
comparison were less than
0.05, with a false discovery rate of
1% (data not shown).
RESULTS
Macrophage Ingestion of S. pneumoniae is Associated with
Cathepsin D Activation—Following phagocytosis of Strepto-
coccus pneumoniae, the fusion of lysosomes with the phago-
some, generates a phagolysosome where bacteria are killed
(
30). Of the proteolytic enzymes present, cathepsin D is the
FIG.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.
Cathepsin D Regulated Proteins
Molecular & Cellular Proteomics 10.6 10.1074/mcp.M111.008193–3
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 West-
ern 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).
A Quantitative Proteomic Approach Demonstrates a Wide
Range of Proteins are Regulated by Cathepsin D Following
Exposure to S. pneumoniae—Cathepsin D activates multiple
cellular pathways (
33–35). In view of the broad substrate range
of cathepsin D (
36), we opted to perform analysis using iTRAQ
to allow an unbiased quantitative proteomic approach to exam-
ine how cathepsin D activation regulated the macrophage pro-
teome during S. pneumoniae infection. Macrophages were ex-
posed to S. pneumoniae (D39) in the presence or absence of the
cathepsin D inhibitor pepstatin A (PepA). Cells were analyzed
16 h postinfection, in the late stages of the antimicrobial re-
sponse, a time at which a program of apoptosis is initiated and
provides a late increment to bacterial killing in this model (
18). All
MS/MS data were submitted to our Phenyx server and as a
result, 15,734 peptides corresponding to 679 proteins were
identified (supplemental Tables S1 and S2). A false positive
peptide discovery rate of 1.1% was detected. The numbers of
peptides contributing to each detected protein are also pre-
sented (supplemental Table S1). Example spectra for iTRAQ
labeled peptide fragmentation of high and low intensities are
also shown (supplemental Fig. 1).
To better assess cathepsin D regulated proteins involved in
apoptosis, we used a statistical method based on peptide-
level intensities of iTRAQ reporters to determine which pro-
teins show differential regulation to statistically significant
levels during infection in the presence or absence of cathep-
sins D activation. The application of median-corrected inten-
sities at the peptide level, rather than protein level, provides
more accuracy for distinguishing smaller changes in protein
expression (
28). All proteins quantified here are presented
showing the relationship between relative abundance ratios
and p values (from t test) to give an indication of statistical
significance.
We focused our attention on the comparison of biological
triplicates of S. pneumoniae infection in the presence or ab-
sence of pepstatin A (D39pepstatin A versus D39) to identify
cathepsin D regulated proteins. We assumed that most ca-
thepsin D regulated proteins would in fact also be differentially
regulated by infection, because our previous data showed
there was little cathepsin D activation in unstimulated macro-
phages in the absence of exposure to S. pneumoniae (
15).
Although we planned to use subsequent Western blotting to
validate this our experimental protocol using an 8-plex iTRAQ
technique allowed us to include a biological duplicate of
mock-infected cells for comparison with the biological tripli-
cate of S. pneumoniae challenged cells incubated without
pepstatin A (MI versus D39) (see Fig. 1). If we considered
proteins identified by at least two distinct peptides and ratios
with p values 0.05, 18 proteins with differential expression
regulated by cathepsin D were identified (Table I and sup-
plementary Table S3). Using these criteria, we identified 26
proteins that were differentially expressed following exposure
to S. pneumoniae (supplemental Table S4).
Of the 18 proteins (2.7% of the 679) that showed cathepsin
D mediated regulation (Table I), ten proteins were elevated
and eight proteins reduced in D39 pepstatin A samples
versus D39. Functional classification of these 18 proteins
using Ingenuity Pathway Analysis showed that they could be
grouped into a number of different functional categories, in-
cluding cellular assembly and organization, and cell morphol-
ogy (Table II). However, the most significant classification
grouping was that of cell death. Indeed, 12 (66.7%) of the 18
differentially expressed proteins were identified as being di-
rectly or indirectly involved in cell death processes (Table II).
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 influ-
ence pathways involved in the regulation of macrophage apo-
ptosis during S. pneumoniae exposure.
Validation of Proteomic Data—Differentiated THP-1 cells,
under the same conditions as used for the iTRAQ analysis,
were lysed and Western blots performed to verify the pro-
FIG.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.
Cathepsin D Regulated Proteins
10.1074/mcp.M111.008193–4 Molecular & Cellular Proteomics 10.6
teomics findings. In total, five proteins were examined further,
all known to have links to cell death processes. In every case,
Western blot analysis with densitometry performed on four
biological replicates confirmed the findings of the iTRAQ anal-
ysis, providing evidence, not only that each protein was dif-
ferentially expressed in the presence of pepstatin A, but also
that each protein was differentially regulated by infection.
Superoxide dismutase (SOD)-2 (decreased 1.68-fold, p
0.005 in D39pepstatin A versus D39 by iTRAQ analysis) and
glucose sensitive heat shock protein (Hsp) A5, also termed
glucose-regulated protein of 78 kDa (Grp78) or BiP, (de-
creased 1.15-fold, p 0.006 in D39pepstatin A versus D39
by iTRAQ analysis), were both elevated in infection, but up-
regulation was reversed by pepstatin A indicating a role for
cathepsin D in protein up-regulation (Figs. 3A and 3B). In
contrast, the actin regulatory protein gelsolin (up-regulated
1.22-fold, p 0.0005 in D39pepstatin A versus D39 by
iTRAQ analysis) and the translation factor eukaryotic elonga-
tion factor (eEF)2 (up-regulated 2.30-fold, p 0.00005 in
D39pepstatin A versus D39 by iTRAQ analysis) were down-
regulated by Western blot following S. pneumoniae exposure,
a reduction that was blocked by pepstatin, implicating ca-
thepsin D in the down-regulation observed (Figs. 4A and 4B).
A fifth protein, the calcium binding protein, which enhances
the transcriptional activity of the tumor suppressor p53 (
37)
and can increase transcription of caspase 3 (
38), S100A6
TABLE I
Proteins showing significant differential expression in D39 pepstatin A samples versus D39. A list of identifying peptide sequences can be
found in supplemental Table S1
AC Name Gene
# MS/MS
spectra
# distinct
peptides
D39Pepstatin
A vs D39 (log)
D39Pepstatin A vs
D39 (fold change)
p value
IPI00003362 HSPA5 PROTEIN HSPA5 265 27 0.135813434 1.145468168 0.005871795
IPI00010796 PROTEIN DISULFIDE-ISOMERASE P4HB 168 29 0.182633409 1.200374281 9.08145E-05
IPI00027463 PROTEIN S100-A6 S100A6 34 2 0.621278866 1.861306883 3.12998E-07
IPI00032313 PROTEIN S100-A4 S100A4 35 5 0.542251927 1.719875539 1.98609E-06
IPI00186290 EEF2 ELONGATION FACTOR 2 EEF2 5 2 0.83084303 2.295252891 5.45704E-05
IPI00217467 HISTONE H1.4 HIST1H1E 28 10 0.251455004 1.285895038 0.001929287
IPI00414676 HEAT SHOCK PROTEIN HSP 90-BETA HSP90AB1 162 16 0.183289861 1.201162528 0.001015541
IPI00418471 VIMENTIN VIM 786 40 0.194527203 1.214736527 7.26613E-10
IPI00453473 HISTONE H4 HIST1H4L 232 8 0.117458357 1.124634796 0.010240815
IPI00639931 ISOFORM 2 OF ADENYLYL CYCLASE-
ASSOCIATED PROTEIN 1
CAP1 317 5 0.222439155 1.249119814 5.35533E-07
IPI00646773 ISOFORM 2 OF GELSOLIN GSN 130 20 0.202269345 1.22417769 0.000514948
IPI00784295 ISOFORM 1 OF HEAT SHOCK PROTEIN
HSP 90-ALPHA
HSP90AA1 88 8 0.196686965 1.217362904 0.006951707
IPI00894365 CDNA FLJ52842, HIGHLY SIMILAR TO
ACTIN, CYTOPLASMIC 1
ACTB 205 13 0.272662307 1.313456624 5.37642E-07
IPI00896370 SUPEROXIDE DISMUTASE MN,
MITOCHONDRIAL
SOD2 18 4 0.517671504 1.678115611 0.004875778
IPI00908876 CDNA FLJ50830, HIGHLY SIMILAR TO
SERUM ALBUMIN
ALB 8 3 0.610283479 1.840953196 0.001745351
IPI00922693 CDNA FLJ53662, HIGHLY SIMILAR TO
ACTIN, ALPHA SKELETAL MUSCLE
ACTB 139 2 0.229450796 1.257908972 5.01643E-06
IPI00930226 CDNA FLJ57283, HIGHLY SIMILAR TO
ACTIN, CYTOPLASMIC 2
ACTG1 374 24 0.211742014 1.235829017 1.46192E-12
IPI00939159 ADENYLYL CYCLASE-ASSOCIATED
PROTEIN
CAP1 268 4 0.193093886 1.212996672 1.47374E-05
TABLE II
Top five functional classifications (by p value) of the 18 proteins regulated by cathepsin D in infection (D39 pepstatin A vs. D39), as analysed
by Ingenuity Pathway Analysis
Category No. of proteins p value Molecules
Cell Death 12 4.34E-09-3.09E-02 P4HB, SOD2, S100A6, S100A4, HSP90AA1,
ACTB, HSP90AB1, VIM, ALB, GSN,
HSPA5, EEF2
Cell Morphology 8 5.85E-07-4.52E-02 CAP1, SOD2, S100A4, HSP90AA1, ACTB,
VIM, ALB, GSN
Cellular Development 9 4.01E-06-4.43E-02 CAP1, SOD2, S100A4, HSP90AA1, VIM,
ALB, GSN, HSPA5, HIST4H4
Cellular Assembly and Organization 11 1.2E-05-4.52E-02 ACTG1, CAP1, SOD2, S100A4, HSP90AA1,
ACTB, ALB, HIST1H1E, VIM, GSN,
HSPA5
Post-Translational Modification 7 3.4E-05-3.63E-02 P4HB, SOD2, HSP90AA1, HSP90AB1, ALB,
GSN, HSPA5
Cathepsin D Regulated Proteins
Molecular & Cellular Proteomics 10.6 10.1074/mcp.M111.008193–5
or calcyclin (up-regulated 1.86-fold, p 0.0000003 in
D39pepstatin A versus D39 by iTRAQ analysis), was up-
regulated during infection, but in this case pepstatin A treat-
ment resulted in further up-regulation (Fig. 4C). These results
indicate that the iTRAQ analysis appeared to have identified
potential cathepsin D targets with known roles in the regula-
tion of cell survival.
Pharmacological inhibition of cathepsin D could however in
theory have off-target effects. To confirm our findings further
we also repeated Western blots and densitometry on BMDM
derived from cathepsin D deficient infant mice or their wild-
type littermates. This confirmed cathepsin D dependent up-
regulation of SOD-2 and HspA5 (Figs. 5A and 5B) and down-
regulation of gelsolin and eEF2 (Figs. 6A and 5B) following
infection, with an increase in S100A6 following infection that
was further increased in the absence of cathepsin D (Fig. 6C).
Superoxide Dismutase (SOD)-2 is Up-regulated in Macro-
phages During S. pneumoniae Infection in a Cathepsin D
Dependent Manner—The first protein of interest to be inves-
tigated further was SOD-2. SOD-2 is a protein which protects
mitochondria against oxidative stress (
39). The iTRAQ analy
-
sis, verified by Western blots, documented cathepsin D-de-
pendent SOD-2 up-regulation. To validate these results in
primary cells, and to elucidate the potential kinetics of this
up-regulation, a timecourse was performed using MDM.
Western blot analysis demonstrated that S. pneumoniae in-
fection dramatically increased SOD-2 levels from 8 h postin-
fection (Fig. 7A). S. pneumoniae exposed cells treated with
pepstatin A showed reduced SOD-2 expression compared with
nontreated cells. Enhanced expression of SOD-2 is consid-
ered one of the first lines of defense against excess levels of
ROS, which are produced by activated macrophages (
40,
41), and SOD-2 is known to be differentially expressed
following bacterial infection (
42, 43). To confirm enhanced
ROS production in our model and relate its kinetics to
SOD-2 up-regulation, MDM production of ROS was meas-
ured using a fluorescent reporter for ROS, DCF. ROS pro-
duction increased from 8 h after exposure to S. pneu-
moniae. (Fig. 7B). The kinetics of ROS production mirrored
the up-regulation of mitochondrial SOD-2.
Gelsolin is Down-regulated by Cathepsin D in Macrophages
After S. pneumoniae Infection—The next protein of interest to
be studied further was the actin regulatory protein gelsolin
(
44). Gelsolin reduces apoptosis both at the level of mitochon
-
drial outer membrane permeabilization (
45) and caspase ac
-
tivation (
46, 47). A time course of gelsolin expression in MDM
revealed down-regulation in a cathepsin D-dependent fashion
from 12 h post-infection, with maximal down-regulation ob-
served at 16 h (Fig. 8A). The maximal down-regulation coin-
cided with cytochrome c translocation to the cytosol (Fig. 8B),
a hallmark of mitochondrial outer membrane permeabilization,
which is widely recognized as being the point of no return in
an apoptotic program of death (
48). These data suggest a
model in which cathepsin D acts to down-regulate gelsolin,
thus contributing to the destabilization of the mitochondria,
leading to apoptosis.
FIG.3.Validation of iTRAQ analysis for SOD-2 and HspA5 in differentiated THP-1 macrophages. Representative Western blots of total
protein from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn) differentiated THP-1 macrophages, 16 h after infection,
cultured in the presence () or absence (-) of pepstatin A (PepA), probed for (A) superoxide dismutase-2 (SOD-2) or (B) heat shock protein A5
(HspA5). Densitometry was carried out on each Western blot and each protein’s fold change was compared relative to the mock-infected (MI)
level after adjustment for any fold change in tubulin, n 4* p 0.05, 1-way ANOVA with Bonferroni post-test.
Cathepsin D Regulated Proteins
10.1074/mcp.M111.008193–6 Molecular & Cellular Proteomics 10.6
Cathepsin D Induces Eukaryotic Elongation Factor 2 (eEF2)
Down-regulation Enhancing Apoptosis and Bacterial Killing—
eEF2 was also identified as a factor down-regulated by ca-
thepsin 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 phos-
phorylation 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 macro-
phages 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
m
after S. pneumoniae
infection (Figs. 9D and 9E). We conclude from these results
that inactivation of eEF2-dependent protein translation is re-
quired for Mcl-1 down-regulation after S. pneumoniae infec-
tion and that cathepsin D induced down-regulation of eEF2
can drive apoptosis by reducing the translation of Mcl-1.
One of the consequences of macrophage apoptosis during
S. pneumoniae infection is to increase bacterial killing (
18). To
test whether our proteomic screen was identifying targets that
could link macrophage apoptosis with bacterial killing, we
measured bacterial clearance in the eEF2 kinase
/
mice that
had preservation of Mcl-1 and delayed macrophage apopto-
sis. Pneumococcal clearance was reduced by 0.5 log in
FIG.4.Validation of iTRAQ analysis for gelsolin, eukaryotic elongation factor 2 (eEF2) and calcyclin (S100A6) in differentiated THP-1
macrophages. Representative Western blots of total protein from mock-infected (Spn-) or Streptococcus pneumoniae exposed (Spn)
differentiated THP-1 macrophages in the presence () or absence (-) of pepstatin A (PepA) probed for (A) gelsolin, (B) eukaryotic elongation
factor 2 (eEF2) or (C) calcyclin (S100A6) 16 h postinfection. Densitometry was carried out and each protein’s fold change was compared relative
to the mock-infected (MI) level after adjustment for any fold change in tubulin, n 4* p 0.05, 1-way ANOVA with Bonferroni’s post-test.
Cathepsin D Regulated Proteins
Molecular & Cellular Proteomics 10.6 10.1074/mcp.M111.008193–7
eEF2 kinase
/
mouse lungs (Fig. 10
). This difference reflects
the relatively modest effect on apoptosis of maintaining active
eEF2 by inhibition of eEF2 phosphorylation, because cathep-
sin D activation still induced eEF2 down-regulation and also
the fact that cathepsin D can exert its effects on macrophage
survival at several points, as this screen has shown. However
the reduction in bacterial killing did establish that our screen
identified proteins that not only influenced macrophage apo-
ptosis but also bacterial clearance in vivo.
DISCUSSION
To better understand how the co-ordination of antimicrobial
host defense in the phagolysosome regulates macrophage
survival we have examined how activation of one of the most
abundant lysosomal proteases, cathepsin D, influences the
macrophage proteome (
9). 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. Western blotting val-
idated a number of the proteins identified by proteomic anal-
ysis and we found evidence that these changes fit well with
the temporal sequence of molecular events that are associ-
ated with macrophage apoptosis during Streptococcus pneu-
moniae infection, a process we have recently demonstrated
involves cathepsin D activation (
15).
To identify cathepsin D regulated proteins after the inter-
nalization of S. pneumoniae into macrophage phagolyso-
somes we have focused on comparison of biological tripli-
cates of macrophages exposed to S. pneumoniae in the
presence or absence of the cathepsin D inhibitor pepstatin A.
We focused on this comparison because our previous data
showed that there was limited activation of cathepsin D in
unstimulated macrophages in the absence of exposure to S.
pneumoniae (
15). Our approach was not based on a fold
cut-off because our previous results suggest this approach
can have limitations (
28). Instead our statistical approach
involved recently described methodology for quantitative pro-
teomic analysis (
25, 54, 55). The use of median-corrected
intensities and t-tests at the peptide level rather than at the
protein level offer higher significance levels and allow the
detection of smaller fold changes (
28). We believe this was an
appropriate approach in the current study. Small fold changes
in regulators of apoptosis can have significant effects on
phenotype. For example we previously found that a 1.3-fold
change in the anti-apoptotic protein Mcl-1 was associated
with a prosurvival effect for the early stages of microbial killing
by macrophages and helped the cell withstand the proapop-
totic effects of increased cellular stress (
18). We were there
-
fore keen not to exclude proteins with low levels of differential
fold regulation in our model. In addition we have shown iTRAQ
can suppress fold change (
25, 56) so we elected to identify
targets with the iTRAQ approach, without excluding those
with lower fold changes, but to validate these vigorously by
Western blotting.
The validation approach was also critical to prove that the
differentially regulated proteins we identified in cells exposed
to S. pneumoniae, in the presence or absence of pepstatin A,
were also differentially regulated by infection itself. Although
FIG.5.Cathepsin D deficient macrophages validate iTRAQ findings for SOD-2 and HspA5. Representative Western blots for (A) SOD-2
and (B) HspA5 from wild-type (WT) and Cathepsin D knockout (KO) BMDMs 16 h after mock- infection (Spn-) or exposure to Streptococcus
pneumoniae (Spn), in the presence () or absence (-) of pepstatin A (PepA). Blots are representative of three independent experiments.
Densitometry was carried out and each protein’s fold change was calculated relative to the mock-infected (MI) level after adjustment for any
fold change in tubulin, n 3* p 0.05, 2-way ANOVA with Bonferroni post-test.
Cathepsin D Regulated Proteins
10.1074/mcp.M111.008193–8 Molecular & Cellular Proteomics 10.6
we believed there was not significant cathepsin D activity in
unstimulated mock-infected cells our comparison of the
mock-infected biological replicate versus the S. pneumoniae
exposed triplicate only identified two of our five targets (with
a third having a p value that almost reached statistical signif-
icance p 0.058, data not shown) but this appeared to reflect
the stringency of our statistical approach because all five
proteins were differentially regulated by Western blotting.
Thus our approach showed it could identify relevant targets
when biological triplicates were compared but its sensitivity
was reduced when a biological duplicate was used. On the
other hand the variation between the biological duplicates or
triplicates was much less than the variation between experi-
mental conditions suggesting that those differentially regu-
lated proteins represented were unlikely to be false positives
(supplemental Fig. S2).
Studies of the macrophage transcriptome in response to
Streptococcus spp. reveal early up-regulation of multiple
genes involved in immune responses, including those in-
volved in the regulation of apoptosis (
57, 58). At our late time
point after bacterial challenge our analysis did not identify
many proteins with primary host defense functions, but
confirmed that many proteins regulating cell death path-
ways were prominently regulated. Similar findings have
been found following challenge of macrophages with vi-
ruses such as Influenza A virus and epithelial cells with
respiratory viruses, with several regulators of apoptosis,
that we identified, also showing differential regulation in
these studies (
59, 60).
Cathepsin D activity is maximal at low pH with a broad
substrate range but the majority of the proteins we identified
as being regulated by cathepsin D are not localized to the
phagolysosmal compartment (
36, 61). The proteomic ap
-
proach we employed does not identify cleavage events and
therefore does not characterize the cell degradome, unlike
some recently described approaches (
62). The changes to the
cell proteome are therefore likely to reflect a broad range of
indirect effects. Many of the proteins were up-regulated, em-
phasizing that direct enzymatic degradation of targets was
not the sole mechanism. The findings are in line with recent
data which emphasize that key cell proteases active during
apoptosis, such as caspases, can induce extensive changes
FIG.6.Cathepsin D deficient macrophages validate iTRAQ findings for gelsolin, eEF2 and S100A6. Representative Western blots
for (A) gelsolin (B) eukaryotic elongation factor 2 (eEF2), and (C) S100A6 from wild-type (WT) and Cathepsin D knockout (KO) BMDMs 16 h
after mock-infection (Spn-) or Streptococcus pneumoniae exposure (Spn), in the presence () or absence (-) of pepstatin A. Blots are
representative of three independent experiments. Densitometry was carried out and each protein’s fold change was calculated relative to
the mock-infected (MI) levels after adjustment for any fold change in tubulin, n 3* p 0.05, 2-way ANOVA with Bonferroni post-test.
Cathepsin D Regulated Proteins
Molecular & Cellular Proteomics 10.6 10.1074/mcp.M111.008193–9
to the cell proteome not just through the degradation of
protein targets but also indirectly by altering gene transcrip-
tion and protein-protein interactions (
62).
The data set of proteins regulated by cathepsin D is en-
riched for proteins predicted to influence a variety of cell
death pathways. We have previously shown ROS are not
required for effective killing of S. pneumoniae (
63). SOD-2
converts superoxide anions into hydrogen peroxide (
41), but
will not influence bacterial killing. Converting superoxide to
hydrogen peroxide does not prevent apoptosis since both
species can induce apoptosis (
64, 65). However by limiting
the concentration of superoxide SOD-2 may enable preferen-
tial permeabilization of the outer mitochondrial membrane
FIG.7.Streptococcus pneumoniae infection induces cathepsin
D dependent up-regulation of superoxide dismutase-2 (SOD-2).
A, Representative Western blots probed for superoxide dismutase-2
(SOD-2) from mock-infected (Spn-) or Streptococcus pneumoniae
exposed (Spn) monocyte-derived macrophages (MDM) cultured in
the presence () or absence (-) of pepstatin A (PepA) at the desig-
nated time points after challenge. Densitometry was carried out and
fold change was calculated relative to the mock-infected (MI) level
after adjustment for any fold change in tubulin, n 3. B, Intracellular
ROS was measured at the indicated timepoints in MDM, mock-
infected (MI) or Streptococcus pneumoniae exposed (D39), in the
presence of vehicle control or pepstatin A (P). Data are from six
separate donors. * p 0.05, *** p 0.001, 2-way ANOVA with
Bonferroni post-test, comparing MI versus D39.
FIG.8.Streptococcus pneumoniae infection induces cathepsin
D dependent down-regulation of gelsolin. A, Representative West-
ern blots probed for gelsolin from mock-infected (Spn-) or Strepto-
coccus pneumoniae exposed (Spn) monocyte-derived macro-
phages (MDM) cultured in the presence () or absence (-) of pepstatin
A (PepA) for the indicated time periods. Densitometry was carried
out and fold change was calculated using mock-infected (MI) levels
after adjustment for any fold change in tubulin, n 3, ** p 0.01,
2-way ANOVA with Bonferroni post-test. B, Cytosolic fractions were
obtained from mock-infected (Spn-) or Streptococcus pneumoniae
exposed (Spn) monocyte-derived macrophages (MDM) at the des-
ignated time points. Western blots were probed for cytochrome c,
and actin was used as a cytosolic loading control. The blots are
representative of three independent experiments.
Cathepsin D Regulated Proteins
10.1074/mcp.M111.008193–10 Molecular & Cellular Proteomics 10.6
and therefore apoptosis development, rather than permeabi-
lization of the inner mitochondrial membrane (
66), which
would trigger an alternative non-apoptotic caspase-inde-
pendent programmed cell death (
67–69). The induction of
SOD-2 fitted well with the kinetics of ROS generation. SOD-2
generation did not result in a decrease in overall ROS, be-
FIG.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 (
m
), n 3, * p 0.05, 2-way ANOVA with Bonferroni post-test.
Cathepsin D Regulated Proteins
Molecular & Cellular Proteomics 10.6 10.1074/mcp.M111.008193–11
cause it regulates mitochondrial ROS (
41) and because ROS
was measured by DCF, which measures a variety of ROS
species (
70) but in this case it is likely to be the kind of
ROS and its specific location rather than the overall quantity
which influences the death program. Another of the proteins
that was up-regulated in response to cathepsin D activation,
HspA5, is a marker of ER stress (
71). It is induced by oxidant
stress and protects against certain forms of ROS-induced cell
death so potentially could also dampen down the effects of
excessive mitochondrial injury and necroptosis. Although
HspA5 can limit induction of apoptosis in certain circum-
stances (
71), in the context of a model with prominent apo
-
ptotic cell death, these observations suggest that cathepsin D
may regulate the tension between different cell death path-
ways during the cell stress associated with innate immune
responses in macrophages.
Interestingly, our study revealed another protein, S100A6 or
calcyclin, was up-regulated in infection, but in this case pep-
statin A treatment did not reverse the change but further
increased up-regulation. S100A6 enhances the transcriptional
activity of the tumor suppressor p53 (
37) and elevated levels
of S100A6 enhance apoptosis by inducing transcriptional up-
regulation of caspase 3 (
38). It could therefore increase the
susceptibility to apoptosis after infection. It therefore might
seem counter-intuitive that cathepsin D inhibition further in-
creased S100A6 expression but cathepsin D might be acting
to slow down the increase of S100A6, putting a brake on the
onset of apoptosis and allowing the bactericidal function of
the macrophage to continue.
Although cathepsin D inhibited competing death pathways,
we found evidence that cathepsin D contributed to regulation
of the mitochondrial apoptosis pathway at two key points. We
have previously shown that Mcl-1 levels determine the onset
of mitochondrial outer membrane permeabilization in this
model (
18, 72). Gelsolin regulates caspase activation (46, 47)
and prevents mitochondrial outer membrane permeabilization
and translocation of cytochrome c to the cytosol, reducing
activation of caspase 9 (
45). We documented that gelsolin
levels declined immediately before cytochrome c transloca-
tion. Moreover, we also observed that cathepsin D down-
regulated eEF2, which catalyzes a key translocation step dur-
ing protein translation, and is therefore an important regulator
of protein synthesis (
73, 74). Mcl-1, which has a short half-life
of only 20–30 mins is critically dependent on translation to
maintain intracellular levels (
50). We showed using BMDM
lacking eEF2 kinase, a negative regulator of eEF2, that eEF2
activity allows maintenance of Mcl-1 expression, delays the
mitochondrial apoptosis pathway and also delays apoptosis-
associated macrophage killing of S. pneumoniae, in a murine
model in which apoptosis contributes to bacterial clearance
(
17). eEF2 is also down-regulated following human metapneu
-
movirus, respiratory syncytial virus, and HIV-1 infection, with
increased apoptosis susceptibility (
59, 75).
Modulation of protein translation via eEF2 is not a recog-
nized point of regulation for innate host responses so this is a
novel finding of our study. Microbial factors such as diphtheria
toxin and the exotoxin A from Pseudomonas aeruginosa ex-
ploit manipulation of protein translation by targeting eEF2 for
ADP-ribosylation (
74), emphasizing the importance of this cell
function to host responses. Protein translation is highly efficient
in alveolar macrophages and maintenance of factors regulating
protein translation, in their optimal phosphorylation state, is one
mechanism accounting for the longevity of differentiated tissue
macrophages (
76). Modulation of protein translation is a potent
point of regulation for macrophage commitment to apoptosis,
which is manifested by alteration in molecules with very short
half-lives, such as FLIP (
73), or in our model Mcl-1 (18).
In conclusion, our results suggest that cathepsin D acti-
vation during host defense has multiple effects on the mac-
rophage proteome with convergence on those pathways
regulating cell death. These alterations stimulate the mito-
chondrial pathway of apoptosis but inhibit competing cell
death pathways. This emphasizes the complexity of regu-
lation of cell death during innate responses and emphasizes
that regulation of cell death involves appropriate control of
competing pathways to allow the co-ordinated induction of
cell death. These results emphasize the potential of pro-
teomics to identify novel points of regulation of death pro-
cesses, as exemplified by our novel finding that regulation
of protein translation is a key molecular switch controlling
the commitment to apoptosis during the execution of anti-
microbial host defense.
* This work is supported by a Wellcome Trust Senior Clinical Fel-
lowship to DHD, #076945, a British Lung Foundation Fellowship to
HMM, F05/7, a BBSRC grant (BB/D005469/1) to BC and from the
EPSRC (EP/E036252/1) to PCW under the ChELSI initiative.
S This article contains supplemental Figs. S1 and S2 and
Tables S1 to S4.
** To whom correspondence should be addressed: Department of
Infection and Immunity, The University of Sheffield Medical School,
FIG. 10. Eukaryotic elongation factor 2 (eEF2) kinase deficient
mice have reduced bacterial clearance. Bacterial counts were es-
timated in lungs from wild-type (WT) or eukaryotic elongation factor 2
kinase deficient (KO) mice infected with 5 10
5
colony forming units
of type one Streptococcus pneumoniae for 24 h, n 11 per group, *
p 0.05, Mann-Whitney U test. Two WT mice and one eEF2k KO
mouse cleared the bacteria completely.
Cathepsin D Regulated Proteins
10.1074/mcp.M111.008193–12 Molecular & Cellular Proteomics 10.6
Beech Hill Rd, Sheffield, S10 2RX, UK. Tel: 44 (0) 114 226 1427;
Fax: 44 (0) 114 226 8898; E-mail: d.h.dockrell@sheffield.ac.uk.
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Cathepsin D Regulated Proteins
10.1074/mcp.M111.008193–14 Molecular & Cellular Proteomics 10.6
    • "In ochronotic arthropathy, macrophages surround the pigmented areas [13] and HGA-treated chondrocytes and synoviocytes show phagocytic features [27]. Interestingly, both gelsolin and cathepsin D are present in macrophages and gelsolin is down-regulated by cathepsin D [46] , but we did not evaluate the presence of gelsolin in AKU deposits . This may be relevant not only for the initiation of fibril formation in AKU, but also for the dynamic balance proteinaceous amyloid deposits undergo. "
    [Show abstract] [Hide abstract] ABSTRACT: Alkaptonuria (AKU) is an ultra-rare inborn error of metabolism developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces an HGA-melanin ochronotic pigment, of hitherto unknown composition. Besides the accumulation of HGA, the potential role and presence of unidentified proteins has been hypothesized as additional causal factors involved in ochronotic pigment deposition. Evidence has been provided on the presence of serum amyloid A (SAA) in several AKU tissues, which allowed classifying AKU as a novel secondary amyloidosis. In this paper, we will briefly review all direct and indirect lines of evidence related to the presence of amyloidosis in AKU. We also report the first data on abnormal SAA serum levels in a cohort of AKU patients.
    Full-text · Article · Apr 2015
    • "Later, azurophilic granules are mobilized. They contain cathepsins, including cathepsin D as the most common [23]. The number of cells expressing cathepsin D in the liver of mice infected with A/H5N1 virus also reached its maximum value on day 3 of the disease with a gradual decrease to day 10 (Figure 5). "
    [Show abstract] [Hide abstract] ABSTRACT: Highly pathogenic avian influenza H5N1 (HPAI H5N1) viruses can infect mammals, including humans, causing severe systemic disease with the inhibition of the immune system and a high mortality rate. In conditions of lymphoid tissue depletion, the liver plays an important role in host defence against viruses. The changes in mice liver infected with HPAI H5N1 virus A/goose/Krasnoozerskoye/627/05 have been studied. It has been shown that the virus persistence in the liver leads to the expression of proinflammatory cytokines (TNF- α , IL-6) and intracellular proteases (lysozyme, cathepsin D, and myeloperoxidase) by Kupffer cells. Defective antiviral response exacerbates destructive processes in the liver accelerating the development of liver failure.
    Full-text · Article · Dec 2013
    • "In ochronotic arthropathy, macrophages surround the pigmented areas [13] and HGA-treated chondrocytes and synoviocytes show phagocytic features [27]. Interestingly, both gelsolin and cathepsin D are present in macrophages and gelsolin is down-regulated by cathepsin D [46] , but we did not evaluate the presence of gelsolin in AKU deposits . This may be relevant not only for the initiation of fibril formation in AKU, but also for the dynamic balance proteinaceous amyloid deposits undergo. "
    [Show abstract] [Hide abstract] ABSTRACT: Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated human chondrocytes and cartilage. SAA and SAP deposition was examined using immunofluorescence and their levels were evaluated in the patients' plasma by ELISA. 2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular tissues. SAA and SAP composition of the deposits assessed secondary type of amyloidosis. High levels of SAA and SAP were found in AKU patients' plasma. Systemic amyloidosis was assessed by Congo Red staining of patients' abdominal fat and salivary gland. AKU is the second pathology after Parkinson's disease where amyloid is associated with a form of melanin. Aberrant expression of proteins involved in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a novel type II AA amyloidosis open new important perspectives for its therapy, since methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid aggregates.
    Full-text · Article · Jul 2012
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