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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 406-418 |
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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,
[email protected]
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抯 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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