Proteomic Characterization of Aggregating Proteins after the

ARTICLE pubs.acs.org/jpr

Proteomic Characterization of Aggregating Proteins after the Inhibition of the Ubiquitin Proteasome System Inga B. Wilde, Maria Brack, Jason M. Winget, and Thibault Mayor* Department of Biochemistry and Molecular Biology, Centre for High-Throughput Biology, University of British Columbia, 2125 East Mall, Vancouver, British Columbia, V6T 1Z4, Canada

bS Supporting Information ABSTRACT: Protein aggregation, which is associated with the impairment of the ubiquitin proteasome system, is a hallmark of many neurodegenerative diseases. To better understand the contribution of proteasome inhibition in aggregation, we analyzed which proteins may potentially localize in chemically induced aggregates in human neuroblastoma tissue culture cells. We enriched for proteins in high-density structures by using a sucrose gradient in combination with stable isotope labeling with amino acids in cell culture (SILAC). The quantitative analysis allowed us to distinguish which proteins were specifically affected by the proteasome inhibition. We identified 642 potentially aggregating proteins, including the p62/sequestosome 1 and NBR1 ubiquitin-binding proteins involved in aggregation. We also identified the ubiquitin-associated protein 2 like (UBAP2L). We verified that it cofractionated with ubiquitin in the highdensity fraction and that it was colocalized in the ubiquitin-containing aggregates after proteasome inhibition. In addition, we identified several chaperone proteins and used data from protein interaction networks to show that they potentially interact with distinct subgroups of proteins within the aggregating structures. Several other proteins associated with neurodegenerative diseases, like UCHL1, were identified, further underlining the potential of our analysis to better understand the aggregation process and proteotoxic stress caused by proteasome inhibition. KEYWORDS: aggregation, proteasome, ubiquitin, neurodegenerative disease, proteomics

’ INTRODUCTION Protein aggregation is a feature of the most common age-related neuro-degenerative diseases like Huntington’s and Parkinson’s diseases.1,2 In addition to containing symptomatic proteins (e.g., alphasynuclein in Parkinson’s), these aggregates also contain various other proteins.2 One protein that most distinct aggregates have in common is ubiquitin.3,4 Ubiquitin can be covalently attached to other proteins and a major function of this post-translation modification is to target substrates for proteolysis via the proteasome.5 Impairment of the ubiquitin proteasome system, the machinery responsible for degrading most short-lived proteins in the cell, has been linked to many aggregation-related diseases.6 Indeed, inhibition of the proteasome causes the formation of a large array of aggregate structures containing disease-specific proteins.7-10 It is unclear which other proteins aggregate during the proteotoxic stress caused by proteasome inhibition. The 26S proteasome is a large multimeric protease complex composed of at least 32 different proteins and has three catalytic sites for proteolysis.11 Proteins to be degraded by the proteasome are targeted by a covalent post-translational modification with ubiquitin, a conserved 76 amino acids protein.12 The covalent attachment of ubiquitin (called ubiquitination) is mediated by an ATP-dependent three-step enzymatic cascade involving the ubiquitin-activating enzyme (E1), a ubiquitin conjugating enzyme (E2) that accepts ubiquitin from the E1, and a ubiquitin-ligase (E3) that r 2011 American Chemical Society

catalyzes the transfer of ubiquitin onto a lysine-residue of the substrate. The level of complexity in the ubiquitin system is underlined by the number of verified or putative E2 and E3 enzymes that are encoded by the human genome (40 and over 600, respectively).13,14 As well, there are over 90 deubiquitinating enzymes (DUBs) that can cleave off ubiquitin from the substrates. There are 7 conserved lysine residues on ubiquitin that can be conjugated to subsequent ubiquitin moieties to form polyubiquitinchains. The targeting signal for proteasomal degradation is mainly comprised of polyubiquitin-chains of at least 4 ubiquitin molecules conjugated via specific lysine residues (e.g., K48 and K29).5 A large fraction of proteasome substrates is composed of shortlived misfolded proteins. For instance, up to 30% of newly translated proteins are targeted for degradation by the ubiquitin proteasome system shortly after their synthesis.15 Errors during translation and misfolded trapped intermediates may account for a large fraction of the aberrant proteins to be degraded. In addition, mutations or specific insults like oxidative damage can favor misfolding that is followed by proteolysis. Interestingly, the activity of the proteasome is known to decrease in aging organisms.16,17 One possibility is that the efficacy of the degradation machinery diminishes in aging cells, leading to a build up of aberrant proteins.18 Similarly, amyloid Received: August 22, 2010 Published: January 05, 2011 1062

dx.doi.org/10.1021/pr1008543 | J. Proteome Res. 2011, 10, 1062–1072

Journal of Proteome Research

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Figure 1. Enrichment of high-density fractions containing ubiquitin after proteasome inhibition. (A) Schematic representation of the enrichment procedure. After SILAC labeling and MG132-treatment of heavy-labeled cells, both cell populations were mixed together. The total cell lysate1 was centrifuged and low solubility proteins were collected on a sucrose cushion,2 and then transferred on top of a sucrose gradient. After ultracentrifugation, proteins in high-density fractions containing poly ubiquitinated proteins3 were analyzed by mass spectrometry. (B) Cell lysate from MG132-treated cells were centrifuged on a sucrose-cushion. Proteins from the total cell lysate (T), supernatant (S; a and b denote top and bottom portions) or cushion interface (C) were analyzed by antiubiquitin immunoblot (left) or silver-staining (right). (C) Western blot of proteins in fractions of the sucrosegradient (top to bottom) with antiubiquitin antibody. (D) Percentage of proteins above the log2 values a given SILAC ratio (heavy: MG132/light: DMSO) from the indicated samples. Ratios corresponding to ubiquitin are indicated by arrows.

aggregates formed by prion, huntingtin or CFTR proteins and alpha-synuclein reduce proteasome activity,19-23 which then may lead to the accumulation of other proteasome substrates. Extended solvent exposure of misfolded and/or hydrophobic domains of these aberrant proteins would then result in their aggregation. Proteasome inhibition has been shown to induce the formation of various aggregate structures in different cell types, even in the absence of any apparent ectopic expression of disease-specific proteins.24-28 This suggests that there is a vast reservoir of aggregation-prone proteins in the cell. We showed that chemical inhibition of the proteasome causes the accumulation of ubiquitinated proteins that are poorly soluble, concurrently leading to the formation of ubiquitin-containing aggregates in neuroblastoma tissue culture cells.29 To gain further insight into the aggregation mechanism and composition of these structures, we devised an approach to enrich for proteins displaying low solubility after proteasome inhibition, in combination with stable isotope labeling with amino acids in cell culture (SILAC) for mass spectrometry analysis.

’ MATERIAL AND METHODS Cell Culture

SH-SY5Y cells (ATCC number CRL-2266) were cultured in Dulbecco’s modified Eagle Medium Nutrient Mixture F12 (1:1)

supplemented with 10% FBS and 1% Pen/Strep (Invitrogen) and maintained at 37 °C and 5% CO2. Cells were seeded either in culture dishes or on HCl pretreated coverslips at least 24 h prior to treatment with 10 μM (Figure 1A-C, 3A, B) or 20 μM (Figure 1D, 2, 3C) MG132. Experiments were typically performed at 70-90% cell confluence. SH-SY5Y cells stably expressing GFP-ubiquitin were described elsewhere.29 For SILAC, cells were grown for 7 cell divisions in arginine- and lysine-free DMEM/F12 (ThermoFisher) supplemented with 10% dialyzed FBS (Invitrogen), 0.1 units/L penicillin and streptomycin, and 73 mg/L L-lysine and 122 mg/L L-arginine as previously described.30 We used normal isotopic Arg and Lys (SigmaAldrich, Oakville, ON) and 13C6-Arg and D4-Lys (Cambridge Isotope Laboratories, Andover, MA). Equal amounts of light and heavy labeled cells were mixed together prior to lysis. Enrichment of Ubiquitin-Containing Fractions on a Sucrose-Gradient

Cells were either treated with MG132 or mock-treated with DMSO for 8 h (Figure 1) and 12 h (Figure 2) and subsequently trypsinized, washed with ice-cold TBS and frozen at -80 °C. Cells were lysed in RIPA buffer (50 mM TrisHCl pH 7.2, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% DOC, 1 mM PMSF, 1 protease inhibitor cocktail, Roche, 25 mM Mg2Cl). Nuclei and 1063

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Figure 2. Identification of proteins specifically enriched in high-density fractions after proteasome inhibition. (A) Percentage of proteins above a given SILAC ratio (Log2 of MG132/DMSO) from the indicated samples from two independent experiments (I and II). (B) Full m/z scans of representative peptides of p62/sequestosome 1, UBAP2L, HSP70 and HSPH1 (peptide sequences and calculated ratio are indicated). Arrows point to the monoisotopic masses of each labeled peptide. (C) Average percentage of proteins in the two experiments above a given SILAC ratio for the indicated samples.

cell debris were spun down at 800 g for 10 min at 4 °C, and the lysate was filtered through medical gauze and incubated with 10 μg/mL DNaseI. The lysate was afterward loaded on a 50% (w/v) sucrose cushion and centrifuged at 42 000 g for 20 min at 4 °C. After discarding most of the supernatant, about 1 mL of the remaining top interface and 1 mL of the cushion were mixed and loaded on a 5%-step sucrose-gradient ranging from 30 to 70% sucrose in gradient-buffer (0.1% Triton X-100, 0.1% β-Mercaptoethanol, 10 mM Pipes pH 7.2) and topped up with 25% sucrose. The gradient was centrifuged at 110 000 g for 105 min at 4 °C. Subsequently the different 1 mL fractions from the gradient were collected at ambient temperature, snap-frozen and stored at -80 °C. To remove the sucrose, the fractions were washed in 6 mL 10 mM Pipes pH 7.2 and proteins centrifuged at 42 000 g for 2 h at 4 °C. Western Blot Analysis

Proteins were diluted in 3x Laemmli sample buffer, heated for 10 min at 70 °C and separated on a continuous 4-20% polyacrylamide gel (Nusep Inc.), prior to wet transfer onto 0.45 μM nitrocellulose membranes (Biorad). Blotting was performed with

P4G7 antiubiquitin (Santa Cruz), mouse anti-Hsp70 antibody (gift from Barak Rotblat, BCCRC), mouse anti-p62 antibody (Santa-Cruz), or polyclonal rabbit anti-UBAP2L (Sigma), followed by incubation with a secondary HRP-conjugated antibody (Biorad) and ECL (Perkin-Elmer). Immunofluorescence Microscopy

For fluorescence microscopy GFP-ubiquitin expressing cells were fixed with cold methanol and subsequently incubated with the indicated antibodies followed by AlexaFluor568 antirabbit/ mouse and Hoechst 33342 (Molecular Probes). The coverslips were mounted with Prolonggold (Invitrogen). Images were taken with the AxioObserverZ1 in combination with the ColibriLED-system (365 nm, 470 nm and 590 nm) and an AxioCam HRm and analyzed with the AxioVision rel 4.7 software (Zeiss).

Mass Spectrometry and Data Analyses

Unless otherwise stated, all chemicals were of ACS grade and solvents of HPLC grade (ThermoFisher and Sigma). Soluble proteins from total cell lysates were chloroform-methanol precipitated.31 Protein pellets resolved in 8 M urea, 50 mM Hepes, pH 8, reduced with 3 μM TCEP for 20 min and alkylated 1064



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Figure 3. Validation of selected aggregating proteins. (A) Western-blot analysis of samples from total cell-lysates and sucrose-gradient fractions from MG132- and DMSO-treated cells with antiubiquitin, anti-p62, anti-UBAP2L and anti-HSP70 antibodies. (B) SH-SY5Y cells stably expressing GFPubiquitin treated with MG132 were labeled with antibodies against p62, UBAP2L and HSP70 and followed by Alexa568 secondary antibodies and Hoechst staining.

with 55 mM chloroacetamide for 30 min at room temperature, predigested with endolysine C (Roche) for 3 h at 37 °C, before dilution with 100 mM Tris pH 8.5. One mM CaCl2 was added before Trypsin (Roche) for 16 h at 37 °C prior to the addition of 10% formic acid. Peptides were fractionated by STAGE tips32 on C18-SCX (strong cationic exchange) using a 4-step ammonium acetate or sodium-chloride elution (100 mM, 350 mM, 500 mM and 1 M) followed by a separate C18 elution step in 80% acetonitrile, 0.5% acetic acid, dried in a vacuum concentrator, and resuspended in 3.2 μL sample buffer (1%TFA, 0.5% acetic acid, 3% acetonitrile). For liquid chromatography-tandem mass spectrometry, samples were analyzed on a LTQ-FT (Figure 1D) or a LTQ-Orbitrap (Figure 2; Thermo Fisher Scientific, Bremen, Germany) coupled online to an Agilent 1100 Series nanoflow HPLC instrument using a nanospray ionization source (Proxeon Biosystems) as previously described.33 Raw MS spectra-files were processed to MASCOT generic format using DTA Supercharge (v.1.37) and peak lists were searched against a database containing 72,082 human protein sequences in the International Proteome Index 3.47 using MASCOT (Matrix-Science). Search parameters (as indicated in Supplementary Table 2, Supporting Information) included two missed cleavage sites, fixed carbamidomethyl modification on cysteine, variable methionine oxidation, peptide mass tolerance was of 20

ppm, MS/MS tolerance of 0.6 Da and decoy analysis. Individual MASCOT files were combined using a custom script on Proteus (GenoLogics Life Sciences Software, Victoria, BC, Canada). Only proteins identified by two of more unique peptides of at least 5 amino acids, ranked first and with a MASCOT ion score equal or greater than 25 were considered and analyzed with MSQuant (v1.4.3a59) for iterative mass recalibration and determining relative peptide SILAC ratio. p-like values were estimated by dividing the portion of proteins in the total cell lysate (corresponding to the maximum false positive rate determined empirically) with the portion of protein in the ubiquitin-containing fraction that were above a given SILAC ratio. On the basis of this, we assigned p-like values of 0.05 to proteins with log2 ratio above 0.5 and 0.03 with ratios above 1, while all other proteins were assigned 1. We next multiplied the p-like values determined in the two experiments. We assigned a score of 1 for proteins with combined p-like values of 9 10-4, a score of 2 for 1.5 10-3 < p-like < 3 10-2 and 3 for p-like of 5 10-2, while proteins with combined p-like value of 1 (i.e., not enriched) were discarded. Number of peptides when the protein was enriched were summed up. We assigned a score of 1 for proteins with 15 or more peptides, a score of 2 for 5 to 14 peptides, and a score of 3 for less than 5 peptides. Both scores were added and proteins with scores of (A) 2 or 3 were 1065



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considered enriched with very strong confidence, (B) 4 or 5 with strong confidence, and (C) 6 with low confidence. Proteininteraction networks were generated in cytoscape34 using protein interaction data retrieved using MIMI.35

’ RESULTS Ubiquitin-Containing Aggregates Induced upon Proteasome Inhibition Can Be Enriched for Quantitative Mass Spectrometry analysis

We sought to specifically identify aggregating proteins after proteasome inhibition in SH-SY5Y neuroblastoma cells using SILAC and density-based fractionations (Figure 1A). We first determined that poorly soluble ubiquitin-conjugated proteins present after 12 h of MG132 treatment were specifically enriched on a sucrose cushion after a high-speed centrifugation, while the majority of the proteins remained in the supernatant (Figure 1B). We further separated the proteins with a discontinuous sucrose gradient, and found an enrichment of poly ubiquitinated proteins in the high-density fractions typically corresponding to 55% to 60% sucrose (Figure 1C). We applied SILAC to compare mock-treated and MG132treated cells to specifically distinguish by mass spectrometry between proteins in the induced aggregates and in other unrelated high-density complexes. We reasoned that aggregating proteins should only be enriched in proteasome-inhibited cells (heavy SILAC-labeled), while unspecific proteins should display no particular enrichment. We performed a first experiment in which we identified, by using liquid chromatography tandem mass spectrometry (LC-MS), proteins present (1) in total cell lysate, (2) in the sucrose cushion, and (3) in the two combined high-density ubiquitin-containing fractions, prior to determining their SILAC ratio. Proteins identified with at least 2 peptides were quantified with MSQuant (Supplementary Table 1, Supporting Information). All ratios were normalized according to the median ratio value obtained in the total cell lysate sample, to account for possible error in the sample mixing before lysis. Over 98% of the proteins quantified in the total cell lysate displayed Log2 ratio values smaller than one (Figure 1D), indicating that the MG132 treatment did not cause a major change in the composition of the identified portion of the proteome within the time frame of the experiment. The ratio distribution of proteins identified in the cushion sample displayed similar features (Figure 1D). Notably, ubiquitin was found increased in MG132-treated cells (log2(ratio): 1.9), indicating that there was a specific enrichment of ubiquitinated proteins, although their abundance was likely too low to be detected among other nonspecific proteins. In contrast to the two previous samples, distribution of the ratio of proteins in the ubiquitin-containing fractions displayed a higher enrichment in the MG132-treated cells and the scattering of the ratio produced a steeper slope (Figure 1D); hence the ubiquitin-containing fractions mainly consisted of proteins enriched after the inhibition of the proteasome. These results indicate that the combination of SILAC and density gradient can be employed to specifically identify which proteins accumulate with ubiquitinated conjugates in high-density structures during proteasome inhibition. High-Density Ubiquitin-Containing Fractions Proteins Found in Aggregate Structures

Include

We repeated the analysis using a larger number of cells to identify proteins coenriched with ubiquitin in high-density structures after proteasome inhibition. We collected and processed about 4 107

cells (four 15 cm dishes) from each label in each of two independent experiments. About half of the peptides derived from proteins coenriched with ubiquitin in the high-density fractions were fractionated by strong cationic exchange and analyzed in four LC-MS runs. In each experiment, we identified 3041 and 4145 peptides with MASCOT with a false discovery rate below 0.5% (Supplementary Table 2, Supporting Information). Seven-hundred fifty proteins identified with at least two unique peptides in one of the two experiments were quantified with MSQuant (510 and 603, respectively). In parallel, we performed the same analysis with an aliquot of the total cell lysate using a single LC-MS run, and normalized all ratios according to the respective median ratio values in these samples (Figure 2A, Suppplementary Table 3, Supporting Information). We found that a large number of proteins were enriched in low solubility structures induced by MG132 and show representative full scans of m/z spectra of selected enriched peptides (Figure 2B). To assemble a comprehensive list of potential proteins aggregating during proteasome inhibition, we merged the data from the two experiments. We compared the SILAC ratio obtained in the high-density fractions and in the control total cell lysates to determine the optimum enrichment threshold. We then scored the aggregating candidate proteins according to their enrichment values and peptide numbers. In the two total cell lysates, we found a small number of proteins enriched in the MG132-treated cells (Figure 2A). These proteins could be either truly enriched in the cell due to the MG132 treatment, or they could be false positives due to the labeling or the analysis itself. We therefore used the ratio values in total cell lysates to estimate the maximum possible false positive rates determined empirically (mFPRe, Figure 2C). We then reported the percentage of proteins enriched in MG132-treated cells for a given threshold and used the mFPRe to estimate the portion of possible false positives in our analysis (p-like, Figure 2C). Based on these estimates, we had good confidence that proteins with log2 ratio values above 0.5 are most likely truly enriched (p-like 0.05). We also previously determined that there was a higher confidence in ratios calculated with a higher number of peptides.36 Based on a novel scoring scheme (see Material and Methods), we identified 644 potential proteins enriched in low solubility structures upon proteasome inhibition, including 542 proteins with strong confidence (Supplementary Table 4, Supporting Information). In this scheme, a protein detected in two experiments with a high SILAC enrichment received a better score then a protein identified in one experiment with a lower enrichment. In addition to ubiquitin, we identified among these 542 proteins several proteins involved in the ubiquitin system like the HERC2 and UBR4 E3-ligases and the UCHL1, UCHL5 and USP5 deubiquitinating enzymes (Table 1). UCHL1 is an abundant protein expressed in neurons and its down-regulation as been associated with Parkinson’s and Alzheimer diseases.37 It was also identified in previous mass spectrometry analyses of Lewy Bodies.38,39 When overexpressed, UCHL1 aggregates in the absence of proteasome activity.40 Our results confirmed that endogenous UCHL1 is enriched in low solubility structures induced by proteasome inhibition. We detected 55 proteins that were previously identified in proteomic analyses of aggregates in tissues derived from patients with Parkinson’s or Alzheimer diseases (Supplementary Table 5, Supporting Information).38,39,41 More specifically, we identified 43 of the 156 proteins (28%) identified with at least two peptides after performing laser capture microdissection of Lewy Bodies in the temporal cortex of dementia 1066



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Table 1. Ubiquitin-Related Proteins Identified in High-Density Fractions Containing Ubiquitinated Proteins identifier

name

weighted ratioa

total peptidesb

1.3

14

E1 IPI00645078

UBA1 E3

IPI00005826 IPI00640981

HERC2 UBR4

2.2 1.5

6 2

IPI00179298

HUWE1

2.0

3

IPI00219513

UCHL5

1.4

2

IPI00024664

USP5

1.1

4

IPI00018352

UCHL1

1.5

15

DUB

ubiquitin-binding (verified or putative) IPI00783855

NBR1

3.3

3

IPI00179473 IPI00022694

SQSTM1 (p62) PSMD4 (S5a)

2.9 1.8

10 2

IPI00029019

UBAP2L

1.4

5

a

SILAC ratio in log2 were weighted based on the number of identified peptides if a protein was identified in both SILAC-experiments. b Sum of peptides identified in one or both SILAC-experiments.

patients38 and 21 of the 40 proteins (53%) that were found coenriched with alpha-synuclein in purified Lewy Bodies from brain tissues of patients diagnosed with Lewy body variant of Alzheimer’s disease.39 These proteins include the amyloid beta A4 precursor protein (APP) and several other cytoskeletal proteins (e.g., dynein, clathrin and spectrin). We also identified proteins involved in spinocerebellar ataxia (Ataxin-10),42 and amyotrophic lateral sclerosis (TDP43, FUS).43 An important fraction of the identified proteins (39%; 216/542) were also found ubiquitinated in previous studies44-46 or annoted in the Ubiprot database47 (Supplementary Table 6, Supporting Information), suggesting that there may be a strong prevalence of ubiquitinated proteins in the induced aggregates. The protein half-lives of several polypeptides identified in the induced aggregates were assessed in a previous study.48 Among these proteins, less than 20% (47/270) have short half-lives ( 0.05) processes are shown, graphed as the -log(P-value), black bars. In cases where all proteins in our data set mapping to one biological process were also entirely a subset of another process, we have removed the smaller category for clarity. Gray bars represent the percentage of coverage of each process (the number of process genes in our data set divided by the total number of genes annotated to that process). (B) Percentage of chaperone proteins above a given SILAC ratio (log2 of heavy/light).

2.0

20

IPI00414676 HSP90AB1 (Hsp90) IPI00027230 HSP90B1

2.1 1.4

50 18

IPI00030275 TRAP1

0.7

10

IPI00784154 HSPD1

0.6

27

IPI00290566 TCP1

1.4

22

IPI00297779 CCT2

1.3

12

IPI00552715 CCT3

2.0

23

IPI00302927 CCT4

1.1

4

IPI00010720 CCT5 IPI00027626 CCT6A

1.5 1.4

4 14

IPI00018465 CCT7

1.3

9

IPI00302925 CCT8

1.4

9

Chaperonins

Chaperone Proteins Enriched in the High-Density Fractions

We identified 41 chaperones and related proteins enriched in the MG132-treated cells compared to mock-treated cells including the Hsp70-superfamily, the Hsp40-family, the small heat shock-proteins, the Hsp90-family and the chaperonins (Table 2). Additionally we found calnexin and calreticulin, prefoldin 2 and TBCB. Many of these abundant proteins were also identified and quantified in the total cell lysate. While most chaperone proteins were only enriched in the high-density fractions after MG132 treatment, a few also displayed a total cellular higher abundance after proteasome inhibition, like Hsp70 (Figure 4B). Transcription of Hsp70 is induced after proteasome inhibition61 and induction of Hsp70 can reduce toxicity linked to aggregation.62 We confirmed that Hsp70 cofractionated with ubiquitin in the high-density fractions after MG132 treatment (Figure 3A) and that it colocalized with ubiquitin in the induced aggregates (Figure 3B). As well, we also observed that Hsp70 levels were higher in MG132-treated cells compared to mock-treated cells (Figure 3A; compare TCL lanes). Similarly, Hsp105 (HSPH1) was found enriched in the cell (Figure 4B) whereas other heat shock proteins were not affected. This indicates that a specific

IPI00382470 HSP90AA1 (Hsp90)

Other (putative) chaperones/cochaperones IPI00303207 ABCE1

1.1

7

IPI00020984 CANX

3.1

7

IPI00020599 CALR

2.2

7

IPI00549248 NPM1 IPI00000877 HYOU1

0.6 0.8

6 6

IPI00010796 P4HB

2.5

3

IPI00025252 PDIA3

1.6

18

IPI00006052 PFDN2

2.4

2

IPI00419585 PPIA

1.4

16

IPI00646304 PPIB

1.2

11

IPI00007019 PPIL1

1.1

2

IPI00789698 PTGES3 IPI00032140 SERPINH1

1.7 2.6

7 2

IPI00013949 SGTA

1.6

5

IPI00293126 TBCB

1.8

3

a

SILAC ratio in log2 were weighted based on the number of identified peptides if a protein was identified in both SILAC-experiments. b Sum of peptides identified in one or both SILAC-experiments. 1068


Journal of Proteome Research subset of chaperone proteins is up-regulated during the formation of ubiquitin-containing aggregates caused by proteasome inhibition.

’ DISCUSSION Intracellular ubiquitin-containing aggregates are characteristics of many age-related neurodegenerative diseases. The herein described enrichment-procedure combined with SILAC allowed the identification of proteins that were present in high-density structures that cofractionated with ubiquitin conjugates after proteasome inhibition. We reasoned that these fractions were enriched with ubiquitinated misfolded proteins and their associated proteins (e.g., chaperone proteins) that accumulate and aggregate during proteasome inhibition. However, we cannot exclude that we identified other proteins in large complexes depleted of ubiquitin, which may also form during MG132 treatment. A large fraction of the proteins identified in the high-density fractions displayed a higher SILAC ratio for MG132-treated cells compared to proteins identified in total cell lysates (74% and 4% for log2(ratio) > 0.5). This suggests that most proteins present in these fractions displayed lower solubility after treatment with MG132. Proteins not displaying any SILAC enrichment could be part of other unrelated large protein complexes. Alternatively, they could represent highly dynamic aggregating proteins that rapidly cycled with the cytosol (that contains both light and heavy labeled polypeptides after cell lysis). Several proteins identified were also found in aggregates related to Parkinson’s and Alzheimer’s diseases: UCHL1, UBA1, Hsp70, Hsc70, Hsp90, proteasomal subunits and 14-3-3 proteins.38,39,41,63,64 This indicates that protein aggregates induced upon proteasome inhibition are in part similar to other aggregates isolated from deceased patients with neurodegenerative diseases. Many of these proteins were previously found to be important in the aggregation process. Therefore, in addition to misfolded proteins, the induced aggregates likely contain a large portion of cofactors involved in aggregation. We detected several proteins associated with the ubiquitin system that, to our best knowledge, have not yet been identified in other aggregates, like HERC2, UBAP2L and UBR4 (a component of the N-end-rule pathway). We verified that the endogenous UBAP2L protein colocalized with the ubiquitin-containing aggregates while it resides both in the cytoplasm and the nucleus in unstressed cells. Further analysis will be required to determine whether UBAP2L is involved in the aggregation process. Protein folding is the most significantly enriched biological process among aggregating proteins and we identified several chaperone proteins in the high-density fraction following proteasome inhibition. This was not surprising given that many misfolded proteins should localize in the induced aggregates. We also found that levels of both Hsp70 and Hsp105 in the cell were higher in MG132-treated cells. After proteasome inhibition, there is an increase of both hsp70 mRNA and protein levels.24,61 More recently, Hsp105 was also found up-regulated after proteasome inhibition,65 and was also shown to reduce toxicity associated with aggregation.66 In contrast, SILAC analysis did not reveal any increase of Hsp60 protein levels in MG132-treated cells, while it was enriched in the induced aggregates. As for Hsp70 and Hsp105, mRNA levels of Hsp60 are up-regulated after heat stress by the HSF1 transcription factor.67 This suggests that proteasome inhibition may cause a stress distinct from heat

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shock, and that, besides HSF1, other regulation factors may be involved in this proteotoxic stress response. The chaperone proteins directly interacted with a subset of MG132-enriched proteins identified in the high-density fractions, and formed a highly interconnected network with these proteins (Figure S2A, Supporting Information). Interestingly, each chaperone directly interacts with distinct subgroups of proteins with few overlaps (Figure S2B-E, Supporting Information). In this particular case, the related Hsp70 (HSPA1A, B; Figure S2C, Supporting Information) and Hsc70 (HSP1A8; Figure S2D, Supporting Information) chaperone proteins clustered in the center of the network but with separate partners. Also, a few chaperone cofactors identified in the total cell lysates (e.g., St13 hsc70-interacting protein) were not detected in the induced aggregates. Both proteins can have discrete cellular functions68,69 and our analysis suggests that they may interact with different subsets of proteins within the ubiquitin-containing aggregates. It will be important to delineate this network in the future, to better understand the respective role of these proteins in aggregation and disease progression. We found that proteins involved in pre-mRNA processing (e.g., splicing, end-processing) were significantly enriched among aggregating proteins (Figure 4A). Many of these proteins also belong to a large protein complex (see Figure S1, Supporting Information). It is unclear whether the whole assembled complex is exported from the nuclei or whether several partially assembled or unassembled cytosolic components aggregate after proteasome inhibition. Interestingly, several mRNA processing factors have been shown to be involved in neurodegeneration.70 We also found several cytoskeletal proteins enriched in the aggregates. The prevalence of this class of proteins was not surprising as the cytoskeleton plays a central role in the aggregation process.71 Importantly, mutations or disruption of numerous cytoskeleton proteins identified in our analysis were found associated with proteopathies. For instance, mutations in the actin-binding protein emerin, as well as in lamin, can cause Emery-Dreifuss muscular dystrophy in which nuclear inclusions can be found.72,73 Myopathy myofibrillar filamin C-related are caused by defects in filamin C.74 The loss of synaptic proteins, like synaptopodin has an impact on the synaptic dysfunctions detected in Alzheimer’s disease.75 Profilin has been suggested to regulate polyglutamine aggregation.76 We also find motorproteins, like dynein1, that are likely to have an impact on aggregate-formation; loss of dynein-function for instance promotes aggregate-formation of aggregate-prone proteins.77,78 Further analysis of cytoskeletal proteins that localize in the induced aggregates may reveal additional factors associated with aggregation diseases. Overall, we identified a large array of proteins in the aggregates induced during proteasome inhibition. Not surprising, an important fraction of the proteins in the aggregates were found ubiquitinated in previous studies. As well, a large portion of these aggregating proteins have long half-lives. One possibility is that a significant portion of proteins identified in the aggregates are misfolded polypeptides from stable and abundant proteins that are targeted for degradation. These abundant proteins may also be further enriched because they are more prone to misfolding and aggregation. Future analysis will be required to determine which aggregating proteins correspond to misfolded and ubiquitinated polypeptides vs those involved in the process of aggregate formation. 1069



’ ASSOCIATED CONTENT

bS

Supporting Information Supplementary tables. Figure S1. Interaction networks of MG132-enriched proteins in high-density fractions. The 542 enriched proteins were input into MIMI to identify those with interactions in the data set. The resulting 354 proteins were shown. Proteins that were highly interconnected were separated into subgroups using MCODE. Names of several genes are indicated. Ubiquitin-related proteins are colored red, chaperones in blue and 14-3-3 proteins in green. Note that UBB and UBC are two different genes coding for the same ubiquitin protein. Figure S2. Chaperone networks among aggregating proteins. (A) Chaperones and their direct interactors extracted from Figure 4. Chaperones shown as filled circles and their partners as empty circles. (B-E) Selected chaperones from (B) shown with their direct interactors. The chaperones are depicted in dark gray and their direct interacting partners in black. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone: 1 604 822 5144.

’ ACKNOWLEDGMENT We thank B. Rotblat (BCCRC) and J. Gsponer (UBC) for discussions, L. Foster and N. Stoynov (UBC) for help with mass spectrometry, and the members of the Mayor lab for their support. The work was supported by a Small Projects Health Research Grant from the British Columbia Protein Network and a Pilot Project Grant of the Canadian Institute of Health Research. ’ REFERENCES (1) Chiti, F.; Dobson, C. M. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 2006, 75, 333–66. (2) Ross, C. A.; Poirier, M. A. Protein aggregation and neurodegenerative disease. Nat. Med. 2004, No. 10 Suppl, S10–7. (3) Lowe, J.; Blanchard, A.; Morrell, K.; Lennox, G.; Reynolds, L.; Billett, M.; Landon, M.; Mayer, R. J. Ubiquitin is a common factor in intermediate filament inclusion bodies of diverse type in man, including those of Parkinson’s disease, Pick’s disease, and Alzheimer’s disease, as well as Rosenthal fibres in cerebellar astrocytomas, cytoplasmic bodies in muscle, and mallory bodies in alcoholic liver disease. J. Pathol. 1988, 155 (1), 9–15. (4) Alves-Rodrigues, A.; Gregori, L.; Figueiredo-Pereira, M. E. Ubiquitin, cellular inclusions and their role in neurodegeneration. Trends Neurosci. 1998, 21 (12), 516–20. (5) Weissman, A. M. Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell. Biol. 2001, 2 (3), 169–78. (6) Lecker, S. H.; Goldberg, A. L.; Mitch, W. E. Protein degradation by the ubiquitin-proteasome pathway in normal and disease states. J. Am. Soc. Nephrol. 2006, 17 (7), 1807–19. (7) Hyun, D. H.; Lee, M.; Halliwell, B.; Jenner, P. Proteasomal inhibition causes the formation of protein aggregates containing a wide range of proteins, including nitrated proteins. J. Neurochem. 2003, 86 (2), 363–73. (8) Bardag-Gorce, F.; Riley, N. E.; Nan, L.; Montgomery, R. O.; Li, J.; French, B. A.; Lue, Y. H.; French, S. W. The proteasome inhibitor, PS341, causes cytokeratin aggresome formation. Exp. Mol. Pathol. 2004, 76 (1), 9–16. (9) Yoshimoto, Y.; Nakaso, K.; Nakashima, K. L-dopa and dopamine enhance the formation of aggregates under proteasome inhibition in PC12 cells. FEBS Lett. 2005, 579 (5), 1197–202.

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