Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 406-418
doi:10.1111/j.1745-7270.2008.00416.x
Chaperone proteins identified
from synthetic proteasome inhibitor-induced inclusions in PC12 cells by proteomic
analysis
Xing’an Li1,2,
Yingjiu Zhang2, Yihong Hu1,
Ming Chang1, Tao Liu3,
Danping Wang1, Yu Zhang1,
Lei Zhang1, and Linsen Hu1*
1 Laboratory for Proteomics, Department of
Neurology, The First Affiliated Hospital of Jilin University, Changchun 130021,
China
2 Key Laboratory for Molecular Enzymology and
Engineering, Ministry of Education (Jilin University), Changchun 130021,
China
3 College of Life Science, Jilin University,
Changchun 130021, China
Received: February
20, 2008
Accepted: March 18,
2008
This work was
supported by a grant from the Natural Science Foundation of Jilin Province (No.
200505200)
*Corresponding
author: Tel, 86-431-85612419; Fax, 86-431-85637090; E-mail,
Chaperone
proteins are significant in Lewy bodies, but the profile of chaperone proteins
is incompletely unraveled. Proteomic analysis is used to determine protein
candidates for further study. Here, to identify potential chaperone proteins
from agent-induced inclusions, we carried out proteomic analysis of
artificially synthetic proteasome inhibitor (PSI)-induced inclusions formed in
PC12 cells exposed to 10 mM PSI for 48 h. Using biochemical fractionation,
2-D electrophoresis, and identification through peptide mass fingerprints
searched against multiple protein databases, we repeatedly identified eight
reproducible chaperone proteins from the PSI-induced inclusions. Of these, 58
kDa glucose regulated protein, 75 kDa glucose regulated protein, and
calcium-binding protein 1 were newly identified. The other five had been
reported to be consistent components of Lewy bodies. These findings suggested
that the three potential chaperone proteins might be recruited to PSI-induced
inclusions in PC12 cells under proteasome inhibition.
Keywords chaperone proteins;
proteomic analysis; PSI-induced inclusions
Lewy body (LB) diseases are neurodegenerative and include at least
three clinical syndromes, idiopathic Parkinson?
disease (PD), PD dementia, and dementia with LBs [1]. Ninety
percent of PD cases occur sporadically and are characterized pathologically by
cytoplasmic inclusions, LBs stained with eosin or anti-a-synuclein (a-SYN) antibody
[2,3], in substantia nigra pars compacta [4]. Although the direct role of LBs
in the disease is still a subject of debate, the development of LBs is
substantially a process of protein aggregation related to the pathogenesis of
PD [5,6]. Having similar molecular components as LBs [4], LB-like inclusions
(LIs) have been described in some rare cases of neurodegenerative disease
[7,8], and also created in some animal models of PD by both inhibition of
mitochondria or proteasomes [9–11] and excessive transgenic expression of human wild-type a-SYN [3,12].
Based on attractive progress in the knowledge about the biochemical mechanisms
of LBs and LIs, other investigators have attempted to replicate LBs in a
variety of cellular models of PD using proteasome inhibitors [13–15]. For
example, one report showed that artificially synthetic proteasome inhibitor
(PSI) can induce a progressive cell death coupled with appearance of
cytoplasmic inclusions in remaining cells [15]. These cell culture-based works
do not only offer evidence to support the concept that LBs could represent
aggresome-like structures, just like aggresomes forming at the centrosome in
response to proteolytic stress [16,17], but also extensively provide
alternative protein candidates associated with protein components of LBs for
further investigation [18]. As a constituent of the endoplasmic reticulum (ER)-associated
degradation (ERAD) machinery in cytoplasm, proteasome is essential to prevent
proteolytic stress, and proteasome inhibition can cause loss of ERAD leading to
ER stress [19–21]. Under the loss of ERAD, up-regulation of ER chaperone proteins
in cells increases as a compensatory mechanism to prevent protein aggregation
[16,22]. For example, proteins aggregated peripherally in cytoplasm are
typically subjected to chaperone proteins such as heat shock proteins (HSPs).
If the compensatory mechanism is not effective, however, the aggregated
proteins assisted with HSPs are recruited in aggresomes where protein
degradation is enhanced [16]. HSPs are therefore termed “aggresome-related
chaperone proteins” [18]. In the protein composition of LBs, a
considerable number of components have been identified as chaperone proteins
[23], such as a B-crystallin, 70 kDa heat shock protein 1A/1B (HSP70), 71 kDa heat
shock cognate protein (HSC70), and 14-3-3 z [5,24,25]. Despite their common pathways of controlling protein aggregation
[16,17], chaperone proteins recruited to aggresomes are variable and the
recruitments vary depending on the type of aggregated proteins, the state of
“the host cell”, and the localization of chaperone proteins in
cytoplasm. Recently, two proteomic analyses have indicated that a better way to
understand potential chaperone proteins in LBs and LIs is to examine the
respective contents of their intermediate organelles [5,18]. In the present work, using 2-D electrophoresis followed by
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS), we attempted to characterize the proteomic features of
PSI-induced inclusions purified from PC12 cells under proteasome inhibition by
biochemical fractionation. Then in the proteomic context we mainly focused on a
portion of chaperone proteins.
Materials and Methods
Chemicals
All reagents of analytically pure and cell culture grade were
purchased from Amersham Biosciences (Uppsala, Sweden) unless specified
otherwise. Artificially synthesized PSI, Z-lle-Glu (OtBu)-Ala-Leu-al or N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-Leu-al,
was from EMD Biosciences (an affiliate of Merck Chemicals, Darmstadt, Germany).
Cell culture plastics, media, and related chemicals were from Gibco (Grand
Island, USA). DNase I and RNase A were from TaKaRa Biotechnology (Dalian,
China). Percoll (a density of 1.131 g/ml) and proteinase inhibitors
[4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin A, E-64, bestatin,
leupeptin, and aprotinin] were from Sigma (St. Louis, USA).
Cell culture and PSI induction
PC12 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai,
China) were maintained in Dulbecco’s modified Eagle’s medium supplemented with
10% fetal bovine serum (V/V), 20 g/L glutamine, 60 U/ml
penicillin, and 100 mg/ml streptomycin. To produce agent-induced inclusions in cells
under proteasome inhibition, PSI, which blocks proteolytic activity of 26S
proteasome without influencing its ATPase or isopeptidase activities and has
several features advantageous for cell biology [26], was particularly
considered. Ten micromoles per liter of PSI was selected by reference to a
previous report [15]. Cells in log phase were split to a density of 1–2?105 viable cells per
milliliter/ml and further cultured for 24 h.
After 48 h following exposure to PSI in dimethylsulfoxide, cells were collected
by centrifugation at 836 g for 5 min. The eosinophilic feature of
PSI-induced inclusions in the cells was assessed by the hematoxylin––eosin (HE) method as described below.
Purification of PSI-induced
inclusions
PSI-induced inclusions were purified as described previously
[6,18,27–29] with some modifications, and all subsequent steps of
purification were carried out at 4 ?C unless specified otherwise. Briefly, the
cells were collected at the indicated time and washed in cold Tris-buffered
saline (pH 7.4), then in cold 0.1?Tris-buffered saline containing 80 g/L sucrose. Cell cultures were
treated repeatedly with liquid nitrogen two or three times then homogenized
with lysing buffer [1 mM HEPES (pH 7.2), 0.5 mM MgCl2, 0.5% NP-40 (V/V), 0.1% b-mercaptoethanol (V/V),
and 1% proteinase inhibitors (V/V)]. After suspended repeatedly
by pipetting and shaken vigorously by hand, the lysate was incubated for 30 min
at 37 ?C until cells were thoroughly homogenized. Initial pellets were
collected by low centrifugation at 80 g for 15 min, washed with buffer L
(1 mM HEPES, 0.5 mM MgCl2, and proteinase inhibitors) on
ice for 5 min, and recollected by centrifugation at 836 g for 10 min.
The initial pellets were incubated in 10?DNase I solution (200 U/ml DNase I, 250 mg/mL RNase A, and
proteinase inhibitors) for 24 h, during which the initial pellets were
suspended repeatedly by pipetting and shaken vigorously by hand several times.
The resulting pellets were collected by centrifugation at 836 g for 10
min, washed with 50 mM Tris-HCl buffer (STB; pH 7.4) containing 0.32 M sucrose
supplemented with protease inhibitors, and recollected by centrifugation at
4000 g for 10 min. The eosinophilic feature of PSI-induced inclusions in
the resulting pellets was assessed by the HE method as described below. For further
purification, the resulting pellets were diluted to 600 ml using 12%
Percoll in STB (V/V) and overlaid on 600 ml of 35% Percoll in STB.
The material band just below the interface of the sample and 35% Percoll was
collected by centrifugation at 35,000 g for 30 min, washed in 10 mM Tris
(pH 8.0) containing 250 mM sucrose supplemented with proteinase inhibitors, and
recollected by centrifugation at 4000 g for 30 min. After purification,
the fraction of PSI-induced inclusions were used for 2-D electrophoresis.
Assessment of PSI-induced
inclusions
The fraction of PSI-induced inclusions was stained with the HE
method and examined by an experienced observer not familiar with the sample
identity. At the same time, the PSI-induced inclusions in the sections on
slides were quantified. The number of PSI-induced inclusions was counted in
nine random fields (three fields per section, three different sections on
slides) and expressed as a percentage of nucleus-free to total PSI-induced
inclusions. The 2-test was used to compare the
percentages before and after the procedure of purification. P<0.05 was
accepted as significant. Measurements were repeated at least three times
[30,31].
Protein extraction
The fraction of PSI-induced inclusions was frozen and thawed two or
three times with liquid nitrogen. Lysis buffer [30 mM Tris, 7 M urea, 2 M
thiourea, 40 g/L 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate
(CHAPS), 60 mM dithiothreitol (DTT), 2% pharmalyte (pH 3.0–10.0; V/V),
and proteinase inhibitors] was added to a total volume of 250 ml, and then the
mixture was incubated at room temperature for 1 h followed by sonication at 35%
of amplitude in an ice-cold water bath. Protein extracts were collected by
centrifugation at 25,000 g for 30 min, subjected to a 2-D cClean-uUp
kKit
following the manufacturer’s instructions (Amersham Biosciences), and dissolved
with rehydration solution [8 M urea, 20 g/L CHAPS, 6 g/L DTT, and 0.5%
immobilized pH-gradient buffer (V/V)] at room temperature for 1
h.As controls, the homogenates of whole cells without and with
exposure to PSI before purification were also prepared using liquid nitrogen
and lysis buffer, respectively, and two samples of proteins were extracted from
their respective homogenates. Steps of homogenization and subsequent steps of
protein extraction were carried out as described above.
2-D electrophoresis and image
analysis
2-D electrophoresis was carried out as described previously [32–34] with some
modifications. Briefly, approximately 600 mg protein extracts of the
purified fraction of PSI-induced inclusions, quantified by Bradford assay, were
applied to Immobiline DryStrip gel strips (24 cm, pH 3.0–10.0,
non-linear; Amersham Biosciences) then reswollen at room temperature for 4 h.
The first dimension electrophoresis (or isoelectric focusing) was run on an
Ettan IPGphor II isoelectric focusing unit (50 mA/strip; Amersham
Biosciences) at 20 ?C for 17 h. The strips were then equilibrated for 15 min in
50 mM Tris-HCl (pH 8.8) buffer [6 M urea, 20 g/L SDS, 30% glycerol (V/V),
20 g/L DTT, and a trace of bromophenol blue], re-equilibrated for another 15
min in the same buffer with 40 g/L iodoacetamide but without DTT, and
transferred onto the top of 1 mm-thick separating SDS-polyacrylamide gels [1
g/L SDS, 125 g/L total gel concentration (T, acrylamide plus cross-linking
agent), 2.6% cross-linking agent (C; W/W), 24 cm?20 cm]. Protein markers (14.40–97.00 kDa) were used to
mark the molecular mass. The second dimension electrophoresis was run on an
Ettan DALT six electrophoresis unit (2 W/gel, 600 V, 400 mA; Amersham
Biosciences) at 18 ?C overnight. The gels were incubated in 200 g/L
trichloroacetic acid (TCA) fixing solution for 1 h, stained in 2.5 g/L
Coomassie brilliant blue R 250 for 4 h, and destained in 10% (V/V)
acetic acid until the gel background was clear.
Gels were scanned using a Umax CE scanner (Amersham Biosciences)
with Image Master Labscan version 3.01b (Amersham Biosciences). The images were
analyzed with Image Master 2-D Evolution version 2003.02 (Amersham
Biosciences). Spots in images were densitometrically measured and statistically
evaluated by computer-assisted pattern analysis to detect whether spots
appeared as reproducibly significant in at least three of the four gels. A spot
was considered to be negligible in the present experimental condition if it was
not detectable in three of the four gels. These significant spots were selected
for MS analysis. As controls, two samples of proteins extracted from the
homogenates of whole cells without and with exposure to PSI before purification
were also separated by 2-D electrophoresis as described above.
Protein identification
Proteins were identified by MALDI-TOF MS peptide mass fingerprints
(PMF) as described [33–35], with some modifications. With an Ettan Spot Picker robotic
workstation (Amersham Biosciences), spots (1.0 mm diameter) were excised from
gels. With an Ettan TA Digester robotic workstation (Amersham Biosciences),
spots were in turn destained with Wash I [50% methanol (V/V)
containing 50 mM ammonium acid carbonate], dehydrated with Wash II
(acetonitrile; ACN), desiccated for at least 1 h, and digested at room
temperature with 1 mg/ml modified porcine trypsin (dissolved in 20 mM ammonium acid
carbonate) overnight. Digestion was ended with 50% ACN (V/V)
containing 0.1% trifluoroacetic acid (TFA; V/V). After
desiccation at room temperature for at least 24 h, 0.3 ml digested peptide mixture
[dissolved in a solution of 1:100:100 TFA:ACN:deionized water (V/V/V)]
was spotted on the surface of a specific steel slide (Amersham Biosciences) and
mixed with 0.3 ml of 4 mg/ml a-cyano-4-hydroxy-transcinnamic acid (dissolved in the same solvent)
with an Ettan Spotter robotic workstation. After desiccation at room temperature,
the mixture was subjected to MS analysis. PMF were produced with an Ettan
MALDI-TOF Pro workstation (Amersham Biosciences).To acquire spectra of protein digests in positive reflection ion
mode equipped with a 337 nm nitrogen laser, we set the instrument parameters as
follows: acceleration potential at 20 kV; pulsed extraction at 2000 V; low mass
ion rejection at m/z 500; laser mode of 8 shots per second; and 200 shots for
each spectrum. To process the acquired spectra, we set the instrument parameters
as follows: algorithm mode at centroid; smooth spectra filter for noise
removal; external calibration of the angiotensin III peak at m/z 897.5 and
human adrenal cortex hormone fragment 18–39 peak at m/z 2465.2;
internal calibration of the trypsin autolysis peaks at m/z 842.50 and m/z
2211.10; mass range of peak detectable at m/z 800–2500; mass tolerance at 0.2
Da; monoisotopic cut-off at m/z 3000; and the baseline adjusted automatically.
To identify the spectra processed, we set instrument parameters as follows: one
missed cleavage site per peptide allowed at most; complete amino acid
modification of iodoacetamide; partial amino acid modification of oxidation;
search type of PMF; ProFound search engine; and a maximum expectation of 0.05
and a minimum of 20% coverage of matched peptides. Submission of PMF to the
ProFound search engine (fully integrated in the Ettan MALDI-TOF Pro workstation
or available at http://prowl.rockefeller.edu/prowl-cgi/profound.exe)
against the database of NCBInr (http://www.ncbi.nlm.nih.gov)
led to initial identification. Submission of PMF to the Mascot search engine
against the NCBInr, SwissProt, and MSDB (http://www.matrixscience.com/search_form_select.html)
databases enhanced the accuracy of the initial identification.
Results
Eosinophilic feature of the PSI-induced
inclusions and evaluation of the processes of purification
To examine whether cytoplasmic PSI-induced inclusions are formed in
PC12 cells under proteasome inhibition, we detected the eosinophilic feature of
PSI-induced inclusions using the HE method. Compared to normal cells (data not
shown), as expected, the PC12 cells under proteasome inhibition were
characterized by cytoplasmic PSI-induced inclusions stained with eosin [Fig.
1(A)]. Furthermore, similar to ubiquitin/a-SYN-positive inclusions formed
in PC12 cells exposed to PSI for 24 h [15], the PSI-induced inclusions
displayed a focal, homogeneous morphology, and appeared as two styles
indicative of nucleus-binding PSI-induced inclusions and nucleus-free
PSI-induced inclusions. In addition, some of the PSI-induced inclusions were
observed in the cytoplasm of remaining cells, whereas the others were extruded
into the extracellular space following destruction of the host cells [36]. To examine whether the pure intact PSI-induced inclusions were successfully
isolated from the PC12 cells by the procedure of purification, we also detected
the eosinophilic feature with the HE method after each fraction of PSI-induced
inclusions was prepared from each subsequent process of purification. First, an
abundance of intact PSI-induced inclusions were enriched in initial pellets by
a process of purification, that is, incubation of the cells with lysing buffer
containing 0.5% NP40 and centrifugation at 80 g (data not shown),
applied to produce a centrosome-enriched fraction or an a-SYN
aggregate-enriched fraction [6,18,28]. Besides a majority of the two styles of
PSI-induced inclusions, some particles, such as larger subcellular components,
heavier cellular debris, and cytoplasm membrane fragments, also resided in the
initial pellets. Second, nucleus-free PSI-induced inclusions were enriched in
the resulting pellets by the next process of purification, incubation of the
initial pellets with 10?DNase I
solution containing DNase I mixed with RNase A [Fig. 1(B)], applied to
degrade nuclei in cells [29,37]. Compared to 51% of the nucleus-free
PSI-induced inclusions to total PSI-induced inclusions in normal cells, the
percentage in the resulting pellets collected following the two processes of
purification was significantly increased to 97% (P<0.05) [Fig.
1(C)]. Finally, the articles described above were eliminated by a third
process of purification, separation of the resulting pellets with
centrifugation at 35,000 g in discontinuous Percoll-mediated density
gradients (data not shown), applied to purify organelles from EpH4 cells and
LBs from brain tissue [29,38]. After the three processes of purification, as
expected, an abundance of pure intact PSI-induced inclusions were isolated.
2-D electrophoresis gel map of
PSI-induced inclusions and selection of specific protein spots
To examine whether proteins extracted from the pure intact
PSI-induced inclusions were usable for cell-free assay [38], we carried out 2-D
gel-based analysis of the PSI-induced inclusions. As with the two samples of
proteins extracted from homogenates of whole cells without and with exposure to
PSI, which was easily carried out by 2-D electrophoresis (data not shown), the
sample of proteins extracted from the purified fraction of PSI-induced
inclusions was also achieved without difficulty [Fig. 2(A)]. The
purified fraction of PSI-induced inclusions was dissolved with lysing solution,
rehydrated with rehydration solution, and resolved on 2-D gel. Further,
compared to the two protein spot patterns on the 2-D gel, indicative of their
respective total proteins of whole cells without and with exposure to PSI, the
protein spot pattern on the 2-D gel indicative of the purified fraction of
PSI-induced inclusions was significantly different [38,39]. That is, the 2-D
gel-based performance of the purified fraction of PSI-induced inclusions led to
protein map establishment of the PSI-induced inclusions. Thus, following the
processes of purification, the 2-D electrophoresis system allowed us to build
up a desirable map of the PSI-induced inclusion proteins within a molecular
mass range of 14.40–97.00 kDa and an isoelectric point (pI) range of 3–10. Of all
protein spots focalized with increasing clarity in four gels, 114 spots were
observed reproducibly, and each spot in at least three of the four gels was not
found to be significantly different in terms of appearance, disappearance, and
shift. After the 114 spots were evaluated by proteomic analysis (as
indicated below), eight specific spots were focused on, because of the specific
properties of their chaperone proteins [17,23]. Coincidently, the eight
specific spots were observed to focalize more obviously and distinctly than
most other spots [Fig. 2(B)], except for two that were close to the left
and right sides of one of the eight spots and also observed to focalize clearly
and distinctly on the 2-D gel [Fig. 2(A), No. 6].
MALDI-TOF MS of eight specific
proteins and identification using PMF
To examine whether the 114 selected spots were able to be assigned
as proteins by MS analysis-based identification, we detected production of PMF
and subsequent identification through PMF. For unequivocal identification, we
considered sequence coverage of at least 20%, the expectation of 0.05 at
maximum, at least five matching peptides, and a gap of at least three peptides
between the accepted protein candidate and the first excluded one in the list
of protein candidates provided by the NCBInr database [40]. After the selected
114 spots were excised from the 2-D electrophoresis gel and digested with
trypsin, and MALDI-TOF MS was carried out and the spots were identified using
PMF, we attempted to characterize the proteomic features of the PSI-induced
inclusions. Based on the proteomic context, eight specific proteins were
assigned as chaperone proteins and were expected to be stressed because of
their aberrant expression in cells faced with an environment of proteolytic
stress [4,16,17,23]. For example, the PMFs of eight specific proteins were
produced, and characterized by their specific MS patterns, their highly
reproducible m/z of ion signals, and their relative intensities of ion
signals [Fig. 3(A)]. Following submission of the PMFs to the ProFound
search engine against the NCBInr database, the eight specific proteins were
initially identified as chaperone proteins and characterized by their
respective groups of identification data (Table 1). Each of the eight
chaperone proteins was identified four times and each identification was shown
as a comparable result (Table 1). The eight identified chaperone
proteins were: 58 kDa glucose-regulated protein (GRP58) [Fig. 2(B), No.
1; Fig. 3(A), No. 1]; 75 kDa GRP (GRP75) [Fig. 2(B), No. 2; Fig.
3(A), No. 2]; 27 kDa HSP 1 (HSP27) [Fig. 2(B), No. 3; Fig. 3(A),
No. 3]; valosin-containing protein (VCP) [Fig. 2(B), No. 4; Fig. 3(A),
No. 4]; HSP70 [Fig. 2(B), No. 5; Fig. 3(A), No. 5]; protein
kinase C inhibitor protein 1 (KCIP-1, or 14-3-3 z) [Fig. 2(B), No. 6;
Fig. 3(A), No. 6]; calcium-binding protein 1 (CaBP1) [Fig. 2(B),
No. 7; Fig. 3(A), No. 7]; and HSC70 [Fig. 2(B), No. 8; Fig.
3(A), No. 8].To further enhance the accuracy of the initial identification
through PFM, we applied the probability-based Mowse score to evaluate the
consistency of identification in the multiple protein databases NCBInr,
SwissProt, and MSDB. In this analysis, if the value of the top score for a
protein candidate is greater than the level of significance threshold (61 in
NCBInr, 51 in SwissProt, and 56 in MSDB) and at the same time the value of the
runner-up for another candidate protein is less than the level (P<0.05), the
protein candidate with the top score is identified as the accepted protein
candidate (or termed the “the protein of interest”). For each of the
eight chaperone proteins, the PMF coupled with the most desirable group of
identification data in the four comparable results (indicated in bold text in Table
1) was selected to be a hit in the multiple protein databases by the
probability-based Mowse score. For example, following submission of the PMFs to
the Mascot search engine against one of the multiple protein databases,
SwissProt (significance threshold of 51), the eight chaperone proteins were
identified in the following way: GRP58 was assigned as protein
disulfide-isomerase A3 precursor (PDIA3_RAT) based on a top score of 186
(>51) compared with 33 (<51) of the runner-up [Fig. 3(B), No. 1];
GRP75 was assigned as stress-70 protein, mitochondrial precursor (GRP75_RAT)
based on a top score of 154 (>51) compared with 34 (<51) of the runner-up [Fig. 3(B), No. 2]; HSP27 was assigned as HSP b-1 (HSPB1_RAT) based on a
top score of 79 (>51) compared with 30 (<51) of the runner-up [Fig. 3(B),
No. 3]; VCP was assigned as transitional endoplasmic reticulum ATPase
(TERA_RAT) based on a top score of 101 (>51) compared with 36 (<51) of the runner-up [Fig. 3(B), No. 4]; HSP70 was assigned as 70 kDa HSP
(HSP71_RAT) based on a top score of 170 (>51) compared with 27 (<51) of the runner-up [Fig. 3(B), No. 5]; 14-3-3 z was assigned as 14-3-3
protein z (1433Z_RAT) based on a top score of 69 (>51) compared with 33
(<51) of the runner-up [Fig. 3(B), No. 6]; CaBP1 was assigned as
protein disulfide-isomerase A6 precursor (PDIA6_RAT) based on a top score of 84
(>51) compared with 33 (<51) of the runner-up [Fig. 3(B), No. 7];
and HSC70 was assigned as 71 kDa HSC protein (HSP7C_RAT) based on a top score
of 249 (>51) compared with 47 (<51) of the runner-up [Fig. 3(B),
No. 8]. Similarly, following submission of the PMFs to the Mascot search engine
against the other two multiple protein databases, NCBInr (significance
threshold of 61) and MSDB (significance threshold of 56), the eight chaperone
proteins were shown to have comparable results (data not shown). So, after
identification through PMF was enhanced by analysis using the probability-based
Mowse score, the eight chaperone proteins were ultimately determined. Of the
eight chaperone proteins, HSP27, VCP, HSP70, 14-3-3z, and HSC70 had been
reported to be consistent components of classical LBs in brainstem and cortex
by immunostaining [5,12,18,25,41], but GRP58, GRP75, and CaBP1 had not been
reported as associated components of LBs.In addition, remarkably, based on the proteomic context of
PSI-induced inclusions, another 15 proteins that had been reported to be
consistent protein components of LBs were identified. These include Cu, Zn
superoxide dismutase, tubulin a1C, tubulin b5, heme oxygenase-1 (HO-1), creatine kinase-B, ubiquinol-cytochrome
c reductase core protein I, ATP synthase b subunit,
tyrosine3-monoxygenase activation protein epsilon (or 14-3-3 e), tyrosine
hydroxylase, proteasome subunit b type 5, proteasome 26S subunit ATPase2
(PSMC2), proteasome 26S subunit ATPase5 (PSMC5), proteasome 26S subunit ATPase6
(PSMC6), proteasome 26S subunit non-ATPase11 (PSMD11), and proteasome 26S
subunit non-ATPase13 (PSMD13) [9,12,18,29,42–46]. Of the 15 consistent
components of LBs, both 14-3-3 e [close to the left side of 14-3-3 z on the 2-D gel indicated
in Fig. 2(A), No. 6] and HO-1 (close to the right side of 14-3-3 z on the same 2-D
gel), were shown with their respective identification data from the NCBInr
database [identification data of 14-3-3 e: gi|13928824|ref|NP_113791.1
(accession No.), 0.014 (expectation), 25.5 (coverage), 4.6 (pI), 29.27 (mass),
11 (measured peptides), and 6 (matched peptides); identification data of HO-1:
gi|7767105|pdb|1DVGIB (accession No.), 0.003 (expectation), 28.1 (coverage),
6.0 (pI), 29.89 (mass), 11 (measured peptides), and 5 (matched peptides)].The 20 consistent components of LBs identified from the PSI-induced
inclusions are characterized and categorized as the functional classes of
antioxidant defense, cytoskeleton system, metabolism and mitochondrial
function, neurotransmission, ubiquitin proteasome system, and protein folding
and transport (Table 2) [4,5,12,18].
Discussion
Subcellular proteomic analysis is an efficient approach to reveal potential
components of organelles, and proteomic analysis of pure intact organelles is
able to provide a context in which interesting proteins associated with
intracellular environments are able to be focused on. In the present work, we
recapitulate cytoplasmic PSI-induced inclusions in PC12 cells under proteasome
inhibition. Using biochemical fractionation, 2-D electrophoresis, and protein
identification through PMF, we attempted to characterize the proteomic features
of the PSI-induced inclusions. In the proteomic context we mainly focused on
the profile of potential chaperone proteins. We developed a novel procedure of purification to isolate the
PSI-induced inclusions from PC12 cells. Although the processes of purification
are subjected to contamination of other cytoplasmic proteins, many laboratories
began to combine traditional purification procedures with alternative methods
because of the impossibility of complete purification [38]. Incubation of the
cells with lysing buffer solution followed by low centrifugation contributed to
setting free and enriching an abundance of intact PSI-induced inclusions in the
initial pellets. Although some entities such as heavy mitochondria and
cytoskeleton networks coprecipitated with the PSI-induced inclusions into the
initial pellets [38], it is not coincidence that mitochondria recruited earlier
in aggresomes while cytoskeleton networks such as intermediate filaments and
microtubules also participated in formation of aggresome-related inclusions
[4,16,29]. After incubation of the initial pellets with DNase I mixed with
RNase A contributed to degrading the remaining nuclei, centrifugation of the
initial pellets in discontinuous gradient contributed to eliminating some other
subcellular particles remaining in the resulting pellets [38]. In the proteomic context of
PSI-induced inclusions, we focused on the profile of eight chaperone proteins,
of which three were newly identified. The other five had been reported to be
consistent components of LBs. In addition, we also identified 15 proteins
reported to be consistent components of LBs. The main component of LBs, a-SYN, interestingly, was not identified
in the present work. There are several explanations for this. The total level
of synuclein-1, the rat homolog of human a-SYN,
was at the same level of low expression in normal cells and was not altered by
proteasomal inhibition [15]; rather, excess levels of a-SYN
play the dominant role in the development and formation of a-SYN-positive inclusions, such as LBs
[4,17,23]. Just as in sporadic PD, a-SYN
species (of high and various molecular weights) might have various
post-translation modifications in PC12 cells under proteasome inhibition [4];
low-abundance proteins are either not visible on gel owing to limitations in
sample loading or marked by high-abundance proteins [39]; and not all proteins
can be identified with the current state of MS technology [18]. Thus, as was
indicated in the results, supported by identification through PMF-based
proteomic analysis of substantia nigra of PD patients [40], a-SYN was not identified from the
PSI-induced inclusions in PC12 cells under proteasome inhibition. However, 20
consistent components of LBs were identified from the PSI-induced inclusions.
For example, HO-1 catalyzing rapid degradation of heme to biliverdin in brain
[47], a putative marker of oxidative stress response [48], is an important
cytoplasmic constituent of LBs [49]. HO-1 was intensely shown by immunostaining
in peripheries of LBs [47], and further shown by immunoelectron microscopy to
be in intimate association with filaments of LBs [48]. In brief, to some
extent, the PSI-induced inclusions characterized by the potential 20 consistent
components of LBs could be with constituent protein features of LBs [12,18].
Based on the identification of the potential 20 consistent components of LBs,
the three newly identified chaperone proteins could provide clues of
alternatives for further study.
As supported by growing evidence, the three newly identified
chaperone proteins are expected to be noted as significant. In cells faced with
proteolytic stresses, aggresomes are equipped with a variety of chaperone
proteins recruited from cytoplasm, ER, and nucleus [17,50,51]. GRP58, a Ca2+-binding chaperone protein, GRP75, a member of the HSP70 family, and
CaBP1, a probable resident protein of the ER, are induced in response to
proteolytic stress when homeostasis of cells is disrupted [52–62]. The other
five chaperone proteins reported to be consistent components of LBs were
observed to aberrantly express in response to environments of proteolytic
stress in neurodegenerative disease [5,25,45,46,63–65]. We did not carry out experiments of validation such as
immunostaining or immunoblotting in the present work [5,10]. Next, a major task
for us is to validate the true association of the three chaperone proteins with
their subcellular localization. In conclusion, based on attempts to characterize the proteomic
features of PSI-induced inclusions formed in PC12 cells, we identified eight
chaperone proteins, of which CaBP1, GRP58, and GRP75 were newly identified.
These findings suggest that the three potential chaperone proteins might be
recruited in the PSI-induced inclusions in PC12 cells under proteasome
inhibition. In addition, we presented at the level of proteomics an approach to
understanding the relevance of the aberrant expression of chaperone proteins to
the PSI-induced inclusions in PC12 cells under proteasome inhibition.
Acknowledgements
We thank both Prof. Fengchen Ge and Prof. Yunbo
Xue (Apiculture Science Institute of Jilin Province, Jilin, China) for their
financial help.
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