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Jul 19, 2016 - Spemann Graduate School of Biology and Medicine (SGBM), and. #. Faculty of. Biology, University of Freiburg, 79104 Freiburg, Germany...
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Proteomic Profiling of Cardiomyocyte-Specific Cathepsin A Overexpression Links Cathepsin A to the Oxidative Stress Response Agnese Petrera,† Ursula Kern,† Dominik Linz,§ Alejandro Gomez-Auli,†,⊥,# Mathias Hohl,§ Johann Gassenhuber,‡ Thorsten Sadowski,‡ and Oliver Schilling*,†,∥ †

Institute of Molecular Medicine and Cell Research, University of Freiburg, Stefan Meier Strasse 17, 79104 Freiburg, Germany Diabetes Division, Sanofi-Aventis Deutschland GmbH, 65926 Frankfurt, Germany § Klinik für Innere Medizin III, Universitätsklinikum des Saarlandes, 66424 Homburg/Saar, Germany ∥ BIOSS Centre for Biological Signaling Studies, ⊥Spemann Graduate School of Biology and Medicine (SGBM), and #Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany ‡

S Supporting Information *

ABSTRACT: Cathepsin A (CTSA) is a lysosomal carboxypeptidase present at the cell surface and secreted outside the cell. Additionally, CTSA binds to β-galactosidase and neuraminidase 1 to protect them from degradation. CTSA has gained attention as a drug target for the treatment of cardiac hypertrophy and heart failure. Here, we investigated the impact of CTSA on the murine cardiac proteome in a mouse model of cardiomyocyte-specific human CTSA overexpression using liquid chromatography−tandem mass spectrometry in conjunction with an isotopic dimethyl labeling strategy. We identified up to 2000 proteins in each of three biological replicates. Statistical analysis by linear models for microarray data (limma) found >300 significantly affected proteins (moderated p-value ≤0.01), thus establishing CTSA as a key modulator of the cardiac proteome. CTSA strongly impaired the balance of the proteolytic system by upregulating several proteases such as cathepsin B, cathepsin D, and cathepsin Z while down-regulating numerous protease inhibitors. Moreover, cardiomyocyte-specific human CTSA overexpression strongly reduced the levels of numerous antioxidative stress proteins, i.e., peroxiredoxins and protein deglycase DJ-1. In vitro, using cultured rat cardiomyocytes, ectopic overexpression of CTSA resulted in accumulation of reactive oxygen species. Collectively, our proteomic and functional data strengthen an association of CTSA with the cellular oxidative stress response. KEYWORDS: proteolysis, oxidative stress, transgenic mouse, mass spectrometry

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INTRODUCTION Cathepsin A (CTSA) is a serine carboxypeptidase widely distributed in mammalian tissues with high expression in kidney, endothelium, liver, lung, and placenta and is also abundantly present in human heart tissue.1,2 Mainly localized in the lysosomes, CTSA is also present at the cell surface and found secreted outside the cell.3 As a lysosomal protease, CTSA exerts carboxypeptidase activity in the acidic range at pH 5.5. At a neutral pH, however, CTSA functions as deamidase, converting amide-blocked peptides into the acidic form as well as an esterase.4 Distinct from its catalytic functions, CTSA has an important protective function in the lysosome by stabilizing the high molecular weight enzyme complex consisting of β-galactosidase (GLB1) and neuraminidase 1 (NEU1) to protect its binding partners from proteolytic degradation.5 CTSA deficiency or mutations affecting the structural integrity of CTSA lead to the severe genetic lysosomal storage disease galactosialidosis (OMIM # 256540).6 Several peptide hormones have been described as © XXXX American Chemical Society

in vitro substrates for CTSA such as bradykinin, substance P, oxytocin, and angiotensin I.7 However, the physiological role of CTSA has not yet been entirely understood. In vivo, CTSA is involved in the physiological inactivation of the potent vasoconstrictor peptide endothelin-1, as demonstrated in genetically modified mice expressing enzymatically inactive CTSA.8 In this mouse model, the reduced degradation rate of endothelin-1 leads to significantly increased arterial blood pressure.8 A relevant body of work reported a cardiovascular role of CTSA, suggesting a new avenue for therapeutic strategies against cardiomyopathies. Studies in mice models of heart failure with a novel inhibitor of CTSA revealed a remarkable beneficial effect against cardiac hypertrophy and atrial fibrillation likely attributed to a local increase of bradykinin in the cardiac tissue where CTSA is highly expressed.2,9 Very recently, pharmacological inhibition of Received: May 6, 2016

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Journal of Proteome Research

[d(2)13 C] in the presence of 20 mM sodium cyanoborohydride. After the reaction was quenched with glycine, samples were combined in pairs in a 1:1 ratio. Following desalting by C18 solid phase extraction (Sep-Pak C18 Plus Light Cartridge, Waters, Frankfurt, Germany), samples (∼300 μg) were fractionated by strong cation exchange chromatography and analyzed by LC−MS/MS.

CTSA has also been proven to be cardioprotective in a myocardial infarction mouse model.10 Increased CTSA expression has been reported in different cardiovascular disorders in both human and animal models.11,12 CTSA gene expression and activity are significantly increased in an animal model for type 2 diabetes, i.e., Zucker Diabetic Fatty rats.13 In this model, CTSA inhibition suppressed the development of atrial structural changes and atrial fibrillation promotion.13 In the same study, Linz et al. characterized the phenotype of a transgenic mouse model for CTSA cardio-specific overexpression, showing that an overexpression of CTSA is associated with increased atrial fibrillation susceptibility. In light of these results and to support further studies on CTSA as a putative drug target for cardiovascular diseases, we strove to study the impact of CTSA on the cardiac proteome. In the current work, we investigated the whole cardiac proteome of transgenic mice carrying a cardiomyocyte-specific overexpression of CTSA. We applied liquid chromatography−tandem mass spectrometry (LC−MS/MS) in conjunction with an isotopic dimethyl labeling strategy to profile proteome changes in CTSA overexpressing hearts compared to wild-type (WT) controls. We identified a large number of significantly affected proteins (limma, moderated p-value ≤0.01) with functional clusters, including cellular proteolysis and oxidative stress response.

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LC−MS/MS Analysis

For mass spectrometry analysis, a Q-Exactive plus system (Thermo Scientific, Bremen, Germany) was used coupled to an Easy nanoLC 1000 (Thermo Scientific) with a flow rate of 300 nL/min. Buffer A was 0.5% formic acid, and buffer B was 0.5% formic acid in 100% acetonitrile (water and acetonitrile were at least HPLC gradient grade quality). A gradient of increasing organic proportion was used for peptide separation (5−40% acetonitrile in 80 min). The analytical column was an Acclaim PepMap column (Thermo Scientific, 2 μm particle sizes, 100 Å pore sizes, length 150 mm, i.d. 50 μM). The mass spectrometer was operated in data dependent mode with a top 10 method at a mass range of 300−2000. Data Analysis

MS files were analyzed by MaxQuant version 1.3.0.5 with the Uniprot mouse database downloaded in October 2014, counting 43393 entries. The sequence of human cathepsin A was included in the search database. MaxQuant analysis included an initial search with a precursor mass tolerance of 20 ppm for mass recalibration. In the main Andromeda search precursor, mass and fragment mass were searched with an initial mass tolerance of 6 and 20 ppm, respectively. The search included variable modifications of methionine oxidation and Nterminal acetylation and a fixed modification of carbamidomethyl cysteine. Tryptic cleavage specificity with up to two missed cleavages was allowed, and minimal peptide length was set to seven amino acids. A minimum of one peptide was required for protein identification. The false discovery rate (FDR) was set to 0.05 for peptide and protein identifications in individual analyses; however, we considered only proteins that were identified in all three replicates. For comparison between samples, we used a labeling scheme based on a multiplicity of two: dimethLys0/dimethNter0 (light label) and dimethLys6/ dimethNter6 (heavy label). A minimum of two ratio counts was used to determine the normalized protein intensity. Files obtained by MaxQuant were further processed using RStudio v.0.99.446 as an IDE for R (R Foundation for Statistical Computing, Vienna, Austria) as previously described.15 Briefly, reverse and potential contaminant entries were removed, and only proteins identified in all three replicates were considered. Overlap of identified proteins was determined using R and BioVenn 2007. Ratios were log2 transformed and normalized by centering, and a linear model was fitted using the limma package16 followed by a moderated t-test. Proteins with an altered abundance were considered by having a log2 fold change of more or less than 50% and a pvalue ≤0.01. Average fold change (fc) values are expressed ± SEM (n = 3). Protein entries were batch queried with the UniProt web interface, and their gene ontology (GO) molecular function and biological process terms were retrieved. Proteins were then manually classified into relevant functional categories. Ingenuity Pathway Analysis (QIAGEN) was used for enrichment analysis; STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) was used to annotate functional interaction of proteins.17

EXPERIMENTAL SECTION

Animals

The generation of transgenic mice overexpressing human CTSA specifically in cardiomyocytes (hCTSA-tg) has been described by Linz et al.13 Age-matched wild-type littermates were used as a control. Animals were housed in an airconditioned room with a 12 h dark/light cycle and received a standard mouse diet with free access to tap water. They were allowed seven days to adjust to the new environment before starting the experiments. All animal studies conformed to the German law for the protection of animal guidelines and the guide for the care and use of laboratory animals published by the United States National Institutes of Health (NIH Publication No. 85-23, revised 1996) as well as to SanofiAventis Ethical Committee guidelines. Preparation of Tissue Samples for Proteomic Analysis

In hCTSA-tg and wild-type mice, hearts were rapidly removed during general anesthesia by intraperitoneal injection of pentobarbital (100 mg/kg ip), trimmed free from noncardiac tissues, and weighed. Snap frozen fresh whole mouse hearts (150−200 mg) were lysed in 500 μL of homogenization buffer (100 mM Na-acetate, 5 mM EDTA, 1 mM dithiothreitol, 0.01 mM trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64), 1 mM phenyl-methanesulfonyl fluoride, 0.05% Brij, pH 5.5) using an Ultra-Turrax and centrifuged at 1000 g for 15 min at 4 °C. Protein concentrations were determined via the bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific). Quantitative Proteome Comparison

For proteome comparison, the whole hearts from hCTSA-tg and wild-type mice were prepared. Sample preparation was performed as described previously.14 Briefly, proteins were precipitated with trichloroacetic acid (TCA), solubilized, trypsinized, reduced, and alkylated. Samples were then labeled with either 20 mM formaldehyde light [d(0)12 C] or heavy B

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Figure 1. Cardiac proteome profiling of hCTSA-tg mice. (A) Venn diagram representing the number of identified proteins in each replicate and their overlap; 1317 proteins were commonly found within the three replicates. (B) Distribution and geometric mean (horizontal bar) of fold change values (log2 CTSA/WT ratio) of proteins from each replicate. (C) Volcano plot visualizing the proteome alteration in the hCTSA-tg mice. Moderated t-test p-values are plotted against mean protein fold changes. Data points in the red and green quadrants indicate significant negative and positive changes in protein abundance, respectively. CTSA and GLB1 are highlighted in the green quadrant.

Western Blot Analysis

Cell Culture and Overexpression of hCTSA

Thirty micrograms of heart lysate was loaded on to 12% SDSpolyacrylamide gels. GAPDH served as an internal loading control. After electrophoretic separation, proteins were transferred on polyvinylidene fluoride membranes using a semidry blot system (Bio-Rad, Munich, Germany). After being blocked, the membranes were exposed to the primary antibodies (GAPDH, 1:1000; human cathepsin A, 1:1000; mouse cathepsin B, 1:500; mouse cathepsin Z, 1:500; human cathepsin D, 1:250; mouse LAMP-1, 1:500) overnight at 4 °C. After being washed, the membranes were incubated for 2 h with the secondary antibody. The membranes were washed and developed with the West Pico Chemiluminescent substrate (Pierce). Peroxidase activity was detected with a LumiImager device (Roche Applied Science, Mannheim, Germany). The primary antibodies were purchased from Abcam (Cambridge, MA) (GAPDH: Cat. No. ab9484; mouse LAMP-1: Cat. No. ab25245), R&D Systems (Minneapolis, MN) (human cathepsin A: Cat. No. AF1049; mouse cathepsin B: Cat. No. AF965; mouse cathepsin Z: Cat. No. AF1033; human cathepsin D: Cat. No. AF1014). Western blots were quantified using the Fusioncapt advance software (Vilber Lourmat, Eberhardzell, Germany).

H9c2 rat cardiac myoblasts were purchased from ATCC (Cat. No. CRL-1446) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; PAN) supplemented with 10% fetal calf serum (FCS; PAN), 1% nonessential amino acids, 1% MEM vitamins, and 1% penicillin/streptomycin at 37 °C in humidified air containing 5% CO2. To prevent the loss of differentiation potential, cells were not allowed to become confluent. H9c2 cells were transfected with hCTSA cloned into pCMV6-Entry expression vector purchased from OriGene, Rockville, MD (Cat. No.RC213409) or pCMV6-Entry empty vector as a control using SuperFect (Qiagen). For positive selection after transfection, 800 μL/mL Geneticin (Thermo Fischer Scientific) was added to the culture medium for 3 weeks. Intracellular Reactive Oxygen Species (ROS) Measurement

Intracellular production of ROS was measured by monitoring the oxidation of the cell-permeable fluorogenic reagent CellROX Deep Red Reagent (Molecular Probes, Carlsbad, CA). Cells (2 ×105) were treated with 1 mM or 100 uM hydrogen peroxide in serum-free medium for 2 or 24 h, respectively. Cells were incubated for 30 min at 37 °C with 5 uM CellROX Deep Red, and fluorescence intensity was measured by flow cytometry (FACScalibur, Fortessa) and evaluated with FlowJo (Tree Star, Inc.). At least 50000 events were analyzed. Dead cells were excluded from the analysis by size.

LAMP-1 Immunostaining and Quantification in Tissue

For pretreatment, slices with 4 μm-thick deparaffinised sections were placed in Coplin jars with 0.05% citraconic anhydride solution (pH 7.4) for 1 h at 98 °C and then incubated overnight at 4 °C with the first antibody followed by the appropriate secondary antibody at 37 °C for 40 min. Immunofluorescence studies were performed by applying polyclonal antibody against LAMP-1 (Abcam, Cat. No. ab24170), diluted to 1:300. FITC-conjugated secondary antibodies (1:30) were added and incubated for 2 h at 37 °C (Dianova, Germany). To perform wash steps, 1× PBS-Tween buffer was used. Sections were counterstained with DAPI (Calbiochem, Germany) and mounted with fluorescent mounting medium (Vectashield, Vector Laboratories, Burlingame, CA, United States) for fluorescence microscopic analysis. All sections were evaluated using a Nikon E600 epifluorescence microscope (Nikon, Germany) with appropriate filters. Relative fluorescence intensity was determined using ImageJ2x (Rawak Software, Inc.).

Statistical Analysis

Statistical analysis using linear models for microarray data (limma)16 allows for the use of linear models to assess differential expression in the context of multifactor designed experiments. In addition, limma has the ability to analyze complex experiments involving comparisons between many peptides simultaneously in a small sample size. Following a moderated-t test, the p-value threshold was set to ≤0.01. Average fc values are expressed ± SEM. For cell culture experiments, data are expressed as mean ± SEM. Statistical significance was calculated for at least three independent experiments employing Student’s t-test using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA). Values of p < 0.05 were considered significant. C

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The mass spectrometry data have been deposited to the ProteomeXchange Consortium18 PRIDE partner repository with data set identifier PXD003860. Annotated spectra can be accessed using the following URLs: replicate 1 (http:// prospector2.ucsf.edu/prospector/cgi-bin/mssearch.cgi?report_ title=MS-Viewer&search_key=tmarisg7rw&search_name= msviewer; search key is tmarisg7rw); replicate 2 (http:// prospector2.ucsf.edu/prospector/cgi-bin/mssearch.cgi?report_ title=MS-Viewer&search_key=m56gfdau4u&search_name= msviewer; search key is m56gfdau4u); replicate 3 (http:// prospector2.ucsf.edu/prospector/cgi-bin/mssearch.cgi?report_ title=MS-Viewer&search_key=owfrn6rqb7&search_name= msviewer; search key is owfrn6rqb7).

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RESULTS AND DISCUSSION

Global Impact of CTSA on the Cardiac Proteome

CTSA is emerging as a promising drug target for the treatment of heart failure. Recently, a mouse model was engineered to specifically overexpress human CTSA in the murine heart.13 We used this mouse model to study the role of CTSA in cardiac biology and its impact on the cardiac proteome. Hence, we quantitatively compared the cardiac proteome of mice with cardiomyocyte-specific overexpression of human CTSA (hCTSA-tg, abbreviated CTSA) to WT mice. Three biological replicates were performed, resulting in a total of six mice. We applied a labeling strategy with stable formaldehyde isotopes light [d(0)12 C] and heavy [d(2)13 C] in combination with LC−MS/MS. We identified between 1821 and 2027 proteins in the different replicates (Tables S1−S3), of which 1317 were consistently identified in all three replicates (Figure 1A). Protein ratios were normalized and expressed as fc values (fc: log2 of CTSA/WT ratio). All replicates showed a near-normal distribution and an average fc close to zero, indicating that the majority of the proteins were not altered (Figure 1B). Overexpression of human cathepsin A was also observed in all three replicates, confirming the validity of the method (average fc CTSA/WT= 2.8 ± 0.4, ∼7-fold increase) (Figure 1B). To identify proteins with an altered abundance, we applied a linear model fitting strategy, i.e., limma16 (Table S4). Proteins were considered to be significantly changed upon the following strict criteria: (a) average increase or decrease of protein abundance by more than 50% and (b) limma-moderated t-test p-value of ≤0.01. Of the proteins, 307 met these criteria and were therefore considered as being significantly affected by CTSA overexpression in the cardiac tissue. Among the significantly regulated proteins, 148 increased (positive log2 fc value of CTSA/WT ratio), while 159 decreased (negative log2 fc value of CTSA/WT ratio) in hCTSA-tg mice (Figure 1C). Along with the overexpression of CTSA, we observed an upregulation of the interaction partner GLB1 (average fc CTSA/WT= 2.4 ± 0.2, ∼5-fold increase), further supporting the biological validity of our method (Figure 1C). The complete list of the significantly altered proteins can be found in Table S5. This large number of significantly affected proteins highlights CTSA as a major modulator of the cardiac proteome.

Figure 2. Biological processes and pathways affected in hCTSA-tg mice. (A) Pie chart indicating functional categories represented by the significantly affected proteins (moderated t-test p-value ≤0.01; at least 50% increased or decreased abundance) and percentages of assigned proteins. For annotations of proteins, see the Results and Discussion. (B) Number of proteins with increased or decreased abundance within each functional category. (C) Top five most significantly dysregulated biological functions in hCTSA-tg mice according to the Ingenuity Knowledge Database. Z-scores are calculated as a prediction of up- or downregulation of biological functions. An absolute z-score of ≥2 is considered significant. A biological function is increased if the z-score is ≥2 (green) and decreased if the z-score is ≤ −2 (red).

on their biological functions (Figure 2A). The proteins were manually examined for their known gene ontology biological process and molecular function (GO terms) and grouped into their respective functional category. The majority of proteins were related to protein biosynthesis pathways (27%). Energy metabolism, including oxidative phosphorylation, glucose, and lipid metabolisms, included 24% of the affected proteins. A substantial proportion of the differentially regulated proteins fell into the category of proteolysis (12%). To a lower extent, cytoskeleton/ECM and signaling proteins were represented (10 and 8%, respectively). STRING analysis of the proteins belonging to these two groups highlighted functional connectivity for >50% of them, pointing toward regulation of actin cytoskeleton organization (GO Biological Processes, pvalue 4.1 × 10−5) (Figure S1). Smaller classes of proteins included metal ion binding (5%), heart contraction (4%), stress response (4%), vesicle trafficking, and protein transport (3% each). As shown in Figure 2B, some functional categories, such as signaling, metal ion binding, and more pronouncedly, stress response proteins, comprised predominantly downregulated

Biological Processes and Pathways Affected by Cardiac CTSA Overexpression

To explore the biological processes and functions that are represented by the affected proteins, we classified them based D

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Figure 3. Impact of CTSA on intracellular proteolytic systems. A. STRING protein functional association network of proteins that were found to be significantly affected in hCTSA-tg mice and belong to the proteolysis group. STRING was employed using “high confidence”. Disconnected nodes are not shown. Connections are shown using the standard STRING coloring scheme as highlighted in the legend. (B) The balance between proteases and protease inhibitors appears shifted toward proteolysis in hCTSA-tg mice. (C) Western blot analysis further corroborated the increased abundance of cathepsin B (here detected the mature form; ∼31 kDa) and cathepsin Z (∼34 kDa) upon overexpression of CTSA in the cardiac tissue.

proteins in the hCTSA-tg mice. The opposite trend, i.e., a predominant upregulation, was shown for proteins involved in vesicle trafficking. Enrichment analysis with Ingenuity Pathway Analysis (IPA) allowed the linking of alterations observed within the single functional category in a global picture of the significantly affected pathways in the hCTSA-tg mice hearts. The IPA Downstream Effects Analysis enabled visualizing the top five most significantly dysregulated biological functions in our data set (z-score >2 or < −2) (Figure 2C). Catabolism of proteins, proteolysis, cell death, and quantity of reactive oxygen species were predicted to increase in the hCTSA-tg mice hearts (positive z-score). On the contrary, proliferation of muscle cells and microtubule dynamics were predicted to decrease in the transgenic mice (negative z-score). For subsequent experiments, we focused on proteolysis and stress response proteins. CTSA Expression Strongly Influences the “Protease Web”

Figure 4. Lysosomal phenotype in hCTSA-tg mice. (A) Immunofluorescent staining of cardiac tissue from hCTSA-tg and WT mice with the lysosomal marker LAMP-1 (green). Nuclei are stained with DAPI (blue). Relative fluorescence was quantified. Results are expressed as mean ± SEM (n = 3, Student’s t-test, *p ≤ 0.05). (B) Accumulation of LAMP-1 positive vesicles was confirmed by immunoblotting for LAMP-1. Western blot quantification by integrated optical density (IOD) was normalized to GAPDH as a loading control. Results are expressed as mean ± SEM (n = 4, Student’s t-test, *p < 0.05, **p ≤ 0.01).

We aimed to understand how CTSA shapes the biological network of proteases and protease inhibitors, the so-called “protease web”.19 STRING analysis revealed the interconnection between the identified proteins in the functional category proteolysis, defining four enriched biological clusters (according to KEGG pathways nomenclature): complement and coagulation cascades, lysosomes, proteasomes, and the calpain/calpastatin system (Figure 3A). The latter is of utmost importance in the pathophysiology of cardiac remodeling and E

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Figure 5. Cathepsin A overexpression is associated with intracellular ROS accumulation. (A) IPA Downstream Effects Analysis-based network associated with the high-ranking biological function of quantity of reactive oxygen species. Nodes and edges are color-coded based on the predicted relationship as represented in the prediction legend. Numbers below protein symbols represent the fold changes (fc: log2 of CTSA/WT ratio) and pvalues. (B) CTSA overexpression in H9c2 cells. Generation of stably expressing CTSA-H9c2 cells (abbreviated as CTSA) was confirmed by Western blot. Here, we detected the single chain (55 kDa) and heavy chain (32 kDa) forms of human cathepsin A. (C) Flow cytometry analysis of CellROX Deep Red stained CTSA-H9c2 and wt cells upon short-term oxidative stress (1.0 mM H202 for 2 h) or long-term oxidative stress (0.1 mM H202 for 24 h). Geometric mean deep red fluorescence values are calculated ± SEM (n = 3, Student’s t-test, *p < 0.05). Representative histogram for FL-4 height (deep red) is shown from the long-term oxidative stress experiment (0.1 mM H202 for 24 h).

heart failure.20 Calpains are calcium-activated cysteine proteases, and their activity is tightly controlled by the endogenous inhibitor calpastatin.21 It has been previously shown that cardiac overexpression of calpain-1 in transgenic mice leads to significant proteolytic activity in unstressed myocardium, increasing ubiquitination of cardiac proteins and proteasomal activity.22 We observed increased levels of calpain1 (fc CTSA/WT = 0.9 ± 0.03; ∼2-fold increase) along with decreased levels of calpastatin (fc CTSA/WT= −0.9 ± 0.1; ∼2fold decrease) in hCTSA-tg mice. In line with an increased potential for calpain proteolytic activity, we also observed increased abundance of proteasomal subunits (Pmsd6, Pmsc5, Pmsb4, and Pmsb5) and lysosomal enzymes such as cathepsin B, cathepsin D, and especially cathepsin Z (cathepsin Z fc CTSA/WT = 1.9 ± 0.5 ; ∼4-fold increase) in hCTSA-tg mice. Previous reports highlighted the strong influence of cysteine cathepsin expression on the abundance of further proteases and protease inhibitors; altered expression of individual cathepsins has been often related to dysregulation of the proteolysis balance, e.g., as shown for cathepsin B and cathepsin L.14,15,23 Our proteomic data suggest that overexpression of CTSA strongly affects the levels of several proteases and protease inhibitors (Figure 3B). Specifically, upregulation of CTSA leads to increased levels of other cathepsins and catabolic enzymes, while decreasing the abundance of a multitude of protease inhibitors, among these are some broad-spectrum inhibitors (α2-macroglobulin and murinoglobulin-1) and numerous serine endopeptidase inhibitors involved in the blood

coagulation cascade (i.e., several members of the serpins family). The observed alteration of protein levels within the cathepsins family is especially interesting in view of their lysosomal localization. We further corroborated increased levels of cathepsin B and cathepsin Z in the cardiac tissue of hCTSAtg mice by Western blotting (Figure 3C). We also assessed the protein expression levels of LAMP-1 to determine if the increased abundance of lysosomal cathepsins is associated with an increase of lysosome-like organelles. Immunostaining for the lysosomal marker LAMP-1 in cardiac samples of wild-type and hCTSA-tg mice showed an increase of LAMP-1 positive vesicles in hCTSA-tg mice (Figure 4A). This result was further corroborated by immunoblotting for LAMP-1 (Figure 4B). These findings suggest that cardiac-specific overexpression of CTSA leads to an increased proteolytic activity likely attributed to higher levels of lysosomal and proteosomal activities along with a partial repression of the protease inhibitor pool. CTSA Expression Impairs the Cellular Oxidative Stress Defense in Vivo and in Vitro

As outlined above, we identified a group of oxidative stress response proteins within the array of significantly affected proteins. This includes several proteins involved in the cellular protection against oxidative stress and cell death, such as peroxiredoxin-1, peroxiredoxin-2, thioredoxin-like protein 1, protein deglycase DJ-1, and glutathione S-transferase A4. As shown in Figure 5A, these cytoprotective proteins displayed decreased abundance in hCTSA-tg mice. We hypothesized that the CTSA-induced proteome alteration might equally affect the F

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Journal of Proteome Research quantity of ROS on the cell-autonomous level. To further explore the link of CTSA with the cellular oxidative stress response and cellular ROS levels, we used the cell-autonomous system of cultured rat cardiomyoblast H9c2 cells. We stably transfected rat cardiomyoblast H9c2 cells to achieve an overexpression of human CTSA, as shown in Figure 5B. We then investigated ROS production in the H9c2 cells by fluorescence analysis of the oxidative dye CellROX Deep Red Reagent. Short-term (1.0 mM H202 for 2 h) exposure to H2O2 resulted in significantly increased ROS levels in comparison to untreated cells (Figure 5C) while lower H2O2 concentrations (0.1 mM H202 for 24 h) over an extended period of time did not significantly increase the already elevated cellular ROS levels (likely due to the long incubation in serum-free medium, which increases intracellular stress in H9c2 cells24). CTSA overexpression significantly yielded increased ROS levels in comparison to the control cells, both in untreated (short-term) and H2O2-treated cells (long-term). Ambient oxygen alone leads to mildly increased ROS levels upon CTSA overexpression when cells are incubated in serum-free medium (Figure S2). This effect was rescued by adding as little as 1% fetal calf serum to the cell culture medium. Collectively, this data substantiates the functional link of CTSA to the cellular oxidative stress response, as highlighted by the cardiac proteome investigation. Interestingly, in a cell-autonomous system, CTSA overexpression is not associated with a lysosomal phenotype, as shown by equal LAMP-1 and cathepsin D expression in CTSA-H9c2 compared to control cells (Figure S3). Thus, the higher sensitivity to oxidative stress upon CTSA overexpression might be functionally uncoupled from the effect of CTSA overexpression on lysosome abundance.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], Tel.: +49 761 203 9615. Notes

The authors declare the following competing financial interest(s): T.S. and J.G. are employees of Sanofi. O.S. has received funding from Sanofi.

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ACKNOWLEDGMENTS We thank Franz Jehle for mass spectrometry technical support. O.S. acknowledges support from the Deutsche Forschungsgemeinschaft (Grants SCHI 871/5, SCHI 871/6, GR 1748/6, INST 39/900-1, and SFB850-Project B8), the European Research Council (Grant ERC-2011-StG 282111-ProteaSys), and the Excellence Initiative of the German Federal and State Governments (Grant EXC 294, BIOSS). This study was supported in part by the Excellence Initiative of the German Research Foundation (Grant GSC-4, Spemann Graduate School).

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CONCLUSIONS Our quantitative shotgun proteomic approach highlights the strong impact of CTSA on the cardiac proteome with an influence on lysosomal and protease biology as well as on the cellular oxidative stress response. Protein catabolism and oxidative stress responses are two tightly coupled processes. Increased lysosomal proteolysis often promotes intracellular ROS production;25,26 likewise, free radicals and ROS activate intracellular proteolytic systems such as the ubiquitin/ proteasome and lysosomal/autophagy systems.27,28 We identified CTSA as a player in the proteolysis-oxidative stress response axis in murine cardiac tissue. It is noteworthy that, in a cell-autonomous system, CTSA overexpression is also linked to the oxidative stress response but does not affect lysosomal abundance. Pharmaceutical CTSA inhibition is presently being considered for the attenuation of myocardial infarction-induced heart failure, and small molecule CTSA inhibitors were found to have a favorable safety profile in healthy young and elderly human subjects.29 Our results may assist in further understanding the role of CTSA in cardiovascular biology and pathology.

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of CTSA overexpressing cells in serum-free medium; Figure S3: lysosomal markers in CTSA-H9c2 cells (PDF) Table S1: proteins identified via MaxQuant in replicate 1; Table S2: proteins identified via MaxQuant in replicate 2; Table S3: proteins identified via MaxQuant in replicate 3; Table S4: proteins identified in all three replicates statistically analyzed by limma; Table S5: list of significantly affected proteins in hCTSA-tg mice (PDF)

REFERENCES

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.6b00413. Figure S1: STRING interaction analysis of cytoskeleton/ ECM and signaling proteins; Figure S2: ROS production G

DOI: 10.1021/acs.jproteome.6b00413 J. Proteome Res. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.jproteome.6b00413 J. Proteome Res. XXXX, XXX, XXX−XXX