Label-Free Quantitative Proteomics Reveals Survival Mechanisms

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Label-free quantitative proteomics reveals survival mechanisms developed by hypertrophic chondrocytes under ER stress Mateusz Kudelko, Cecilia WL Chan, Rakesh Sharma, Qing Yao, Edward Lau, Ivan K Chu, Kathryn S.E. Cheah, Julian Alexander Tanner, and Danny Chan J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00537 • Publication Date (Web): 20 Nov 2015 Downloaded from http://pubs.acs.org on November 24, 2015

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Journal of Proteome Research

Label-free quantitative proteomics reveals survival mechanisms developed by hypertrophic chondrocytes under ER stress

Mateusz Kudelko1, Cecilia W. L. Chan1, Rakesh Sharma1,3, Qing Yao1, Edward Lau2, Ivan K. Chu2, Kathryn S.E. Cheah1, Julian A. Tanner1 and Danny Chan1*

1

Department of Biochemistry, 2Department of Chemistry, The University of Hong Kong,

Pokfulam, Hong Kong, China 3

Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue,

Hong Kong, China

KEYWORDS Glycolysis,

mitochondrial

membrane

polarity,

calcium

chondrodysplasia type Schmid .

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signaling,

metaphyseal


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ABSTRACT Emerging evidence implicates ER stress caused by unfolded mutant proteins in chondrocytes as the underlying pathology of chondrodysplasias. ER stress is triggered in hypertrophic chondrocytes (HCs) in a mouse model (13del) of metaphyseal chondrodysplasia type Schmid (MCDS) caused by misfolded mutant collagen X proteins, but the HCs do not undergo apoptosis, rather chondrocyte differentiation is altered causing skeletal abnormality. How 13del HCs can escape from apoptosis and survive ER stress is not understood. Here, we compared the proteomes of HCs isolated from 13del growth plates with normal HCs, using a label-free quantitative mass spectrometry approach. Pathway enrichment analyses of differentially expressed proteins showed significant changes in glycolysis and ERmitochondria pathways in 13del HCs, as well as in ATDC5 cell lines expressing wt and 13del collagen X. In vivo, we showed expression of mitochondrial calcium channels was reduced while mitochondrial membrane polarity was maintained in 13del chondrocytes, while in vitro, glucose uptake was maintained. We propose 13del HCs survive by a mechanism whereby changes in ER-mitochondria communication reduce import of calcium coupled with maintenance of mitochondrial membrane polarity. These findings provide the initial insights to our understanding of growth plate changes caused by protein misfolding in the pathogenesis of chondrodysplasias


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INTRODUCTION A cell experiences both intrinsic and extrinsic stress conditions during its life. Increased protein folding load in the endoplasmic reticulum (ER stress) is one of the most studied and characterized types of stress that occurs during developmental processes and disease states such as type II diabetes (1, 2), neurodegenerative diseases (3, 4), Parkinson’s disease (5), cardiovascular diseases (6) as well as skeletal dysplasias (7-9). For cell survival, specific or integrated stress pathways, collectively termed the unfolded protein response (UPR), are activated to alleviate the stress and adapt to the new condition; otherwise, the cell will die. Three mechanistically distinct branches of the UPR have been described with IRE1, PERK and ATF6 as sensors which are kept inactive by the binding of BiP, but are activated when BiP is sequestered away in the presence of unfolded proteins (10, 11). Thus the UPR is a cell adaptive mechanism that helps cells to survive by restoring ER homeostasis, but can also lead to cell death if the ER dysfunction cannot be mitigated and homeostasis cannot be reestablished (12, 13). While the general mechanisms that link UPR and apoptosis are well characterized and described today (14), the events that occur during prolonged ER stress remain largely unknown. Skeletal dysplasias are a group of more than 450 clinically distinct pathologies that are caused by abnormal cartilage and/or bone development, and commonly result in dwarfism and bone anomaly (15). Among them metaphyseal chondrodysplasia type Schmid (MCDS) is a disease associated with heterozygous mutations in the COL10A1 gene encoding the 1(X) chain of collagen X, affecting folding and trimeric assembly of the collagen X molecule. MCDS is characterized by a mild short stature and hypertrophic zone elongation of the growth plate (16-18), and evidence indicates activation of ER stress as the pathogenesis for MCDS (8, 9).



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Comprehending how cells respond to ER stress has important implications in developmental processes and disease mechanisms. Previous studies have used mainly in vitro cell systems and chemical agents such as tunicamycin and thapsigargin to activate ER stress (19-21), with the limitation that ER stress in vitro normally leads to apoptosis and changes observed in dying cells may not apply in an in vivo context. Here, we use a mouse model (13del) for MCDS, a skeletal dysplasia characterized by endogenous activation of ER-stress in hypertrophic chondrocytes (HCs) of the growth plate (8). 13del mice express Col10a1 with a 13-base pair deletion within the NC1 encoding domain of collagen X in hypertrophic chondrocytes. This mutation alters collagen X folding, triple helix assembly, and secretion from the ER resulting in the natural activation of ER-stress in HCs (22, 23). Surprisingly, activation of ER stress did not lead to apoptosis of the HCs in 13del mice; instead they survive, re-differentiate and proliferate (8) providing a unique model to address the natural consequences of ER stress in vivo. It has been reported that growth plate chondrocytes survive hypoxic stress during development probably through the cytoprotective actions of HIF-1α, PERK and eIF2α (2426). Furthermore, recent findings showed that some mutations causing ER stress and cell death in vitro, may not necessarily induce cell death in vivo indicating that the in vivo environment provides specific pro-survival signals such as the hypoxic stress pathway (25). Thus, hypertrophic chondrocytes in the growth plate appear to have a distinct response to ER stress that allows survival instead of triggering apoptosis. In the present study, we utilize the 13del mouse model as well as an in vitro cell culture system to investigate the adaptive mechanism(s) developed by HCs to facilitate their survival under an ER stress environment. We undertook an in vivo proteomic approach to unravel the mechanisms of the chondrocytic ER stress response. Cartilage proteomics is challenging due to the limited available tissue and dominance of poorly soluble matrix 4 Environment ACS Paragon Plus

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components (27). Thus, previous studies were based on microarray analysis of microdissected mouse cartilage zones (28, 29) despite the fact that ER stress also affects translation (30) and therefore protein expression. Furthermore, we extend from a study which uses a ‘temporal’ approach based on the analysis of normal mouse femoral head cartilage at different development stages (31) that was unable to address changes in the differentiation programme of chondrocytes within the growth plate. Using a ‘spatial’ strategy through specific labeling of hypertrophic chondrocytes with GFP in mice and dissection of hypertrophic cartilage, we performed comparative proteomics revealing altered glucose utility and mitochondrial function as part of the survival mechanism.



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EXPERIMENTAL DETAILS Hypertrophic chondrocyte dissection and protein extraction 13del transgenic mice were crossed to Col10a1-eGFP mice to generate 13del;Col10a1eGFP. HCs from proximal and distal tibia were obtained from 10-day postnatal (P10) wtCol10a1-eGFP and 13del;Col10a1-eGFP mice in accordance with Institutional Animal Ethics guidelines. The dissected cartilage was rinsed in PBS, frozen immediately into liquid nitrogen and stored at -80oC. The P10 HCs was obtained by dissecting the green fluorescent zone from tibia and removing the bone collar under fluorescent microscope. We used five biological replicates per genotype, where each replicate sample comprised HCs from proximal and distal tibia pooled from three to four P10 mice. Each replicate was then cryosectioned and transferred to Eppendorf tubes containing 100µl of Triethyl ammonium bicarbonate (0.5 M) and 2% (w/v) SDS. Cell disruption was performed by a combination of 10 freeze-thaw cycles followed by sonication (Sonics, Vibra-Cell, USA) on ice for 30 min (10s bursts with intensity ~40% and 10s breaks). The suspension was centrifuged at 1000g for 10 min and supernatants were collected.

Protein reduction, alkylation and in-solution trypsin digestion Protein samples for LC-MS/MS analysis were sequentially reduced, by adding 2 µl of 50 mM tris-(2-carboxyethyl) phosphine (TCEP) to each samples and incubating at 60ºC for 1 h, and then alkylated by addition of 1 μl of 200 mM iodoacetamide (30 min at 25ºC in the dark) following manufacturer’s instruction (AB Sciex, CA, USA). Protein concentrations were estimated using the linear Bradford assay (32) after removal of detergent using Pierce detergent removal resin and columns (Thermo Scientific). 20µg of dried proteins per sample were reconstituted into 25mM ammonium bicarbonate followed by overnight trypsin (Promega) digestion. 6 Environment ACS Paragon Plus

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LC-MS/MS analysis Each sample was analyzed in triplicate by LC-MS/MS using an UltiMate 3000 HPLC system (Dionex) connected online with an LTQ-Orbitrap Velos (Thermo Scientific, Bremen, Germany). Tryptic peptides were loaded onto a self-packed PicoTip® column (360 μm outer diameter, 75 μm inner diameter, 15 μm tip, New Objective) packed with 10 cm length of C18 material (ODS-A C18 5 μm beads, YMC) with a high-pressure injection pump (Next Advance) at 1 μl/min in 98% solvent A (0.1% (v/v) formic acid) and 2% solvent B (0.1% (v/v) formic acid in acetonitrile) for 6 min and subsequently eluted with a linear gradient B from 2% to 40% for 120 min at a flow rate of 300 nL min-1. The LTQ-Orbitrap Velos was controlled using Xcalibur, version 2.0.7 (Thermo Fisher Scientific) and operated in datadependent acquisition mode whereby the survey scan was acquired in the Orbitrap with a resolving power set to 60,000 (at 400 m/z). MS/MS fragment ions were detected in LTQ mass analyzer, for each duty cycle 20 most intense precursor ions from a survey scan were selected for MS/MS. Ion injection times for the MS and MS/MS scans were 350 ms and 150 ms, respectively. The automatic gain control targets for MS (FT) and MS/MS (LTQ) were set to 1 million and 10,000, respectively. The mass range for precursor ion selection was selected between 350 m/z-1700 m/z. Fragmentation was carried out using CID mode with 35% normalized collision energy, with activation of 0.25 and 10ms activation time. Ions selected for fragmentation were dynamically excluded for a period of 30s from MS/MS analysis. For MS/MS analysis, monoisotopic precursor mass was selected and singly charged ions were rejected from analysis. The lock mass was enabled for accurate mass measurements using Polydimethylcyclosiloxane (m/z, 445.12) ions.

Criteria for Protein identification and validation



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The acquired MS/MS data were converted to Mascot Generic format with Proteome Discoverer 1.3 software (Thermo Fisher Scientific). All MS/MS data were analysed using MASCOT version 2.2.06 (Matrix Science) against UniProtKB mouse database of canonical sequences (Oct 2014; 16,480 entries) and sequences for common contaminants appended (downloaded from the Max Planck Institute; http://maxquant.org). By default Mascot decoy database search was performed for all the data set. Enzyme specificity was set to trypsin with maximum two missed cleavages; S-carboxamidomethylation of cysteine residues specified as a fixed modification and oxidation of methionine specified as variable modification. Parent ion and fragment ion mass tolerance were set to 10 ppm and 0.5 Da, respectively. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (33) via the PRIDE partner repository with the dataset identifier PXD002125. The Mascot search results (.dat files) were incorporated in Scaffold 4 (version 4.4.3, Proteome Software) for protein validation and to assign probabilities to peptide and protein matches (34). Peptide-spectrum matches were accepted if the peptide was assigned a probability > 0.95 as specified by the Peptide Prophet algorithm (35). Protein identifications were accepted if the protein contained at least two unique peptide counts and the protein was assigned a probability > 0.99 by the Protein Prophet algorithm and the FDR was < 1%. The minimal list of proteins that satisfy the principal of parsimony is reported (36). Proteins identified as exogenous contaminants, such as keratin or immunoglobulin, were excluded from the analysis.

Statistical and bioinformatics analysis of MS/MS data Spectral count data obtained from biological and technical replicates (wt and 13del) were recombined using the Scaffold Mudpit function. Protein identified across five biological replicate was attributed a total spectral count value which was further normalized to obtain


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quantitation value. Label-free quantitation was done based on spectral counting wherein the numbers of MS/MS scans are attributed to the same peptide ion (31). The frequency of these MS/MS scans correlates with the abundance of a given peptide in each sample (wt and 13del). Proteins differentially expressed in 13del HCs were chosen based on a two-step selection. First level of selection was based on fold change (FC). FC was directly determined by calculating the ratio between experimental spectral count values obtained for each protein in 13del and wt HCs. A two-fold change in abundance was used as threshold to select potential differentially expressed proteins. A second level of selection based on a p-value was determined by Fisher's Exact test. Proteins with absolute log2 FC >1 and p value < 0.05 were finally selected as differentially expressed. Proteins were annotated with GO terms from NCBI (downloaded Apr 24, 2015) (37) in concordance with gene ontology (GO) standards. Functional annotation and enrichment analysis on differentially expressed proteins was performed using Qiagen’s Ingenuity pathway analysis software (IPA, Ingenuity Systems, Inc., 2014, Redwood City, CA, USA; http://www.ingenuity.com). IPA determines biologically enriched pathways and functions based on direct and indirect relationships in published literature. The p-value is calculated using a right-sided Fisher’s exact test and measures the statistical significance (p-value 0.99 (0.1% FDR) (Supplementary Tables 1A, 2A and 2B). Out of 1078 proteins identified, 969 were quantified in both wt and 13del HCs (Figure 2A), with 109 proteins with quantitation values only in wt or/13del (Supplementary Table 1C). We selected differentially expressed proteins based on fold change (FC13del/wt ≥2) and p-value

Label-Free Quantitative Proteomics Reveals Survival Mechanisms

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