Skin Barrier Defects Caused by Keratinocyte-Specific Deletion of

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The Skin Barrier Defects Caused by KeratinocyteSpecific Deletion of ADAM17 or EGFR are Based on Highly Similar Proteome and Degradome Alterations Stefan Tholen, Cristina Wolf, Bettina Mayer, Julia Daniela Knopf, Stefanie Löffek, Yawen Qian, Jayachandran N Kizhakkedathu, Martin L. Biniossek, Claus-Werner Franzke, and Oliver Schilling J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00691 • Publication Date (Web): 11 Apr 2016 Downloaded from http://pubs.acs.org on April 19, 2016

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Journal of Proteome Research

The Skin Barrier Defects Caused by Keratinocyte-Specific Deletion of ADAM17 or EGFR are Based on Highly Similar Proteome and Degradome Alterations Stefan Tholen1, Cristina Wolf2,3, Bettina Mayer1, Julia D. Knopf1,4, Stefanie Löffek2,5, Yawen Qian2, Jayachandran N. Kizhakkedathu6, Martin L. Biniossek1, Claus-Werner Franzke2,9,*, Oliver Schilling1,7,8,9,* 1. Institute of Molecular Medicine and Cell Research, University of Freiburg, Germany 2. University Medical Center Freiburg, Department of Dermatology, Freiburg, Germany 3. present address: Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Luxembourg 4. present address: Centre for Molecular Biology, University of Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Heidelberg, Germany 5. present address:, Skin Cancer Unit of the Dermatology Department, Medical Faculty, University Duisburg-Essen, Germany 6. Department of Pathology and Laboratory Medicine and Department of Chemistry, Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada 7. BIOSS Centre for Biological Signaling Studies, University of Freiburg, D-79104 Freiburg, Germany 8. German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany 9. to whom correspondence should be addressed: PD Dr Oliver Schilling

PD Dr Claus-Werner Franzke

Institute of Molecular Medicine and Cell Research

University Freiburg Medical Center

University of Freiburg

Department of Dermatology

Stefan Meier Strasse 17

Hauptstr. 7,

79104 Freiburg, Germany

79104 Freiburg, Germany

Tel: +49 761 203 9615

Tel: +49 761 270 67850

[email protected]

[email protected]

*

equal contribution

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Abstract

Keratinocyte-specific deletion of ADAM17 in mice impairs terminal differentiation of keratinocytes leading to severe epidermal barrier defects. Mice deficient for ADAM17 in keratinocytes phenocopy mice with a keratinocyte-specific deletion of EGFR, highlighting the role of ADAM17 as a “ligand sheddase” of EGFR ligands. In this study we aim for the first proteomic / degradomic approach to characterize the disruption of the ADAM17-EGFR signaling axis and its consequences for epidermal barrier formation. Proteomic profiling of the epidermal proteome of mice deficient for either ADAM17 or EGFR in keratinocytes at postnatal days 3 and 10 revealed highly similar protein alterations for ADAM17 and EGFR deficiency. These include massive proteome alterations of structural and regulatory components important for barrier formation, like transglutaminases, involucrin, filaggrin, and filaggrin-2. Cleavage site analysis using TAILS revealed increased proteolytic processing of S100 fused-type proteins, including filaggrin-2. Alterations in proteolytic processing are supported by altered abundance of numerous proteases upon keratinocyte-specific Adam17 or Egfr deletion, among them kallikreins, cathepsins and their inhibitors. This study highlights the essential role of proteolytic processing for maintenance of a functional epidermal barrier. Furthermore it suggests that most defects in formation of the postnatal epidermal barrier upon keratinocyte-specific ADAM17 deletion are mediated via EGFR.

Keywords Epidermal barrier formation, epidermis, proteomics, proteases, ADAM17, EGFR, filaggrin-2, ectodomain shedding, TAILS, degradomics


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1 Introduction

A disintegrin and metalloproteinase (ADAM)17, also known as tumor necrosis factor α-converting enzyme (TACE), belongs to the transmembrane zinc metalloproteinases

1

that cleave their

membrane-anchored precursor substrates in a process defined as ectodomain shedding

2, 3

. Next

to tumor necrosis factor (TNF)-α, a number of additional shedding substrates have been described for ADAM17. These include cell adhesion proteins like L-selectin

2

, cytokine receptors like

interleukin-6 receptor (IL-6R) 4, and most of the ligands for the epidermal growth factor receptor (EGFR) family like, transforming growth factor-α (TGF-α), epiregulin, amphiregulin and heparinbinding epidermal growth factor (HB-EGF) 5-8. Mice deficient for ADAM17 die at birth due to defects in heart development and show abnormalities in other organs, such as eyes, lung and skin

6-8

. Thereby Adam17-/- mice phenocopy Egfr-/- mice.

Moreover, mice deficient for the EGFR ligands TGF-α, amphiregulin and HB-EGF display similar phenotypes

6-8

, further suggesting the relevance of ADAM17 as an essential modulator of the

EGFR signaling pathway in vivo 9. Mice deficient for EGFR display defects in hair follicle development and epidermal differentiation comprising inflammatory skin reactions EGFR inhibitors in cancer therapy

15

10-14

, which resemble dermatological side effects using

. To investigate the role of ADAM17 and EGFR in skin

homeostasis, mice with a conditional keratinocyte-specific deletion were generated

16

. Adam17ΔKC

mice phenocopy EgfrΔKC mice showing curly whiskers, 10% penetrance of open eyelids, altered hair growth and a dry scaly skin. Getting born with an intact epidermal barrier, most of the mice in both knockout mouse systems die after the third week of life due to severe epidermal barrier defects. Surviving animals develop chronic dermatitis and systemic myeloproliferative disease as adults 16. The epidermal barrier protects the organism from water loss, mechanical stress, invading microorganisms, and foreign substances. To maintain its function the multilayered epidermis, consisting of the basal, the spinous, the granular, and the cornified cell layer, is continuously 3 ACS Paragon Plus Environment


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regenerated by terminally differentiating keratinocytes. During this tightly regulated process the differentiation-committed basal keratinocytes were translocated within the suprabasal cell layers and eventually form the outermost cornified cell layer (stratum corneum) in a process called cornification

17

. The cornification process consists of three key steps, (a) the replacement of

intracellular organelles and content by polymerized keratin intermediate filaments and a water retaining matrix, (b) the crosslinking of proteins by transglutamination at the cell periphery to form the cornified envelope (CE) and (c) the linkage of dead corneocytes into a multicellular barrier by intercellular lipid lamellae and corneodesmosomes

18

. Most structural and regulatory components

important for epidermal differentiation are expressed in a gene cluster called the ‘epidermal differentiation complex’ located on the human chromosome 1q21. Among these are structural components like involucrin, loricrin, profilaggrin and the small proline-rich proteins, which are cross-linked by transglutaminases (TGMs) to maintain the mechanical resistance and insolubility of the CE 17, 19. We have previously shown that the ADAM17–EGFR signaling axis in keratinocytes is critical for the tightly regulated expression of epidermal differentiation proteins, regulation of TGM activity and finally for the maintenance of the postnatal epidermal barrier

16

. To define the systemic

consequences of the disruption of the ADAM17–EGFR signaling axis during skin differentiation, we performed a proteomic analysis of epidermal lysates of Adam17ΔKC and EgfrΔKC mice. This analysis consists first of a gel-free quantitative proteomic approach to investigate alterations in protein abundance

20, 21

. Here, to track defects in epidermal barrier formation over time, epidermis lysates

of three and ten days old mice were analyzed. Second ‘terminal amine isotopic labeling of substrates’ (TAILS), an N-terminomic technique for the cell-contextual identification and quantification of native and proteolytically generated protein N-termini, was applied to determine ADAM17 dependent cleavage sites in the epidermis

22

. In this study we show that a keratinocyte-

specific deficiency for ADAM17 as well as EGFR results in numerous protein alterations, especially for structural and regulatory proteins important for CE formation. Furthermore massive alterations can be observed in proteolytic processing. Especially cathepsin proteases are strongly decreased upon disruption of the ADAM17–EGFR signaling axis. TAILS revealed, among other ADAM17 dependent cleavage events, new processing sites for filaggrin and filaggrin-2. Overall, this study 4 ACS Paragon Plus Environment

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underlines the essential role of proteolytic processing in skin and shows that most alterations observed in disturbed formation of the postnatal epidermal barrier upon keratinocyte-specific ADAM17 deletion are mediated via EGFR.

2 Methods 2.1 Animals The generation of Adam17flox/floxKrt14-Cre, Egfrflox/floxKrt14-Cre mice has been described previously

16,

23

. All mice were of mixed genetic background (129Sv, C57BL/6), and all

comparisons were between same-sexed littermates. For the experiments, skin biopsies from the middle back of euthanized male or female animals were taken. The animals were kept according to the

guidelines

of

the

German

Animal

Welfare

association

and

approved

by

the

Regierungspräsidium Freiburg.

2.2 Preparation of Epidermal Samples For the preparation of the protein lysates the back skin epidermis was detached from the dermis by heat separation as described previously

24

. The epidermis was cut into small pieces and

homogenized in 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 % Nonidet P-40, 0.5 % sodium deoxycholate supplemented with 2 mM EDTA, 5 mM 1,10-orthophenanthroline (Sigma-Aldrich) and protease inhibitor cocktail set III (Calbiochem) on ice with a T18 basic Ultra Turrax (Ika, Germany).



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2.3 Histology Skin tissues were fixed in 3.7% buffered formaldehyde or 4% PFA and embedded in paraffin. 5-µm sections were stained with hematoxylin and eosin (H&E).

2.4 Quantitative Proteome Comparison Protein content was measured using the Bradford assay (BIO-RAD, Munich, Germany). 300 µg of protein were used per condition for the quantitative secretome comparison. In each comparison samples of littermates were compared at P3 or P10. Preparation of mass spectrometry samples was performed as described previously, including stable isotope labeling with either d213C-formaldehyde (“heavy”) or d012C formaldehyde (“light”) for quantitative comparison and pre-fractionation by strong cation exchange (SCX) chromatography 25. Briefly, samples were prefractionated by high performance liquid chromatography (HPLC) using a strong cation exchange column (SCX; PolyLC; Colombia, USA). Buffer A was 5 mM KH2PO4, 25% acetonitrile, pH 2.7 and buffer B was 5 mM KH2PO, 1 M KCl, 25 % acetonitrile, pH 2.7. Peptides were eluted in a linear gradient with increasing concentration of buffer B. 10-14 fractions were collected, desalted using self-packed C18 STAGE tips (Empore, 3M, USA), and analyzed by LCMS/MS as described in the corresponding section. Formaldehyde labeling was performed as a label-switch experiment, meaning the wild type sample was labeled light in the first biological replicate and heavy in the second biological replicate. Labeling was performed after tryptic digestion. LC-MS/MS analysis is described in the corresponding section. Data were converted to mzXML format

26

using Proteowizard

27

with

centroiding of MS1 and MS2 data. Peptide sequences were identified by X! Tandem (version 2013.09.01.1)

28

, including cyclic permutation, in conjunction with PeptideProphet (part of version

4.7 of the Trans Proteomic Pipeline)

29

and a decoy search strategy: the complete mouse

proteome database was downloaded from UniProt

30

on Nov 26th 2013. It was appended with an

equal number of randomized sequences, derived from the original mouse proteome entries. The decoy database was generated with DBToolkit

31

. Tryptic cleavage specificity with no missed 6

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cleavage sites was applied. Mass tolerance was 10 ppm for parent ions and 0.3 Da for fragment ions. Static modifications are cysteine carboxyamidomethylation (+57.02 Da), lysine and Nterminal dimethylation (light formaldehyde 28.03 Da; heavy formaldehyde 34.06 Da). X!Tandem results were further validated by PeptideProphet at a confidence level of > 95 %. Corresponding protein identifications are based on the ProteinProphet algorithm with a protein false discovery rate of < 1 %. The relative quantitation for each protein was calculated from the relative areas of the extracted ion chromatograms of the precursor ions and their isotopically distinct equivalents using the XPRESS

32

algorithm. Reported fold change values (Fc; log2 of heavy/light (H/L)) ratio are

based on normalized XPRESS ratios. Proteins were considered to be altered in their abundance if they were (A) identified in both biological replicates of each experimental setup, (B) showed an alteration in abundance of more than 50 % (Fc < -0.58; Fc > 0.58) with consistently increased or decreased abundance, and (C) if their H/L ratio calculated by XPRESS was confirmed by manual inspection of the extracted ions chromatograms.

2.5 Western Blot Analysis For western blot analysis epidermal tissues and keratinocytes were lysed in 50 mM Tris-HCl, 1 % Nonidet P-40, 0.1 % sodium dodecyl sulfate (SDS) and 0.15 M NaCl, pH 8, supplemented with protease inhibitors (5 mM EDTA, 10 µM E64, 1 mM PMSF). For epidermal lysates an Ultra-Turrax was used and cell debris removed by centrifugation at 3000 g for 10 min at 4 °C. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, USA). 45 µg of proteins of epidermal lysates were separated by electrophoresis on 12 % SDS-polyacrylamide gels. α-Tubulin served as internal loading control. After electrophoretic separation proteins were transferred on polyvinylidene fluoride (PVDF) membranes using a semidry blot system (BioRad, Munich, Germany). After blocking, membranes were exposed to the primary antibodies (Ctsb 1:500; Ctsd 1:1000; Ctse 1:500; Ctsl 1:500; involucrin 1:1000; protein S100-A9 1:1000; α-tubulin 1:1000) overnight at 4°C. After washing, the membranes were incubated for 2 h with the secondary antibody. The membranes were washed 7 ACS Paragon Plus Environment


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and developed with the West Pico Chemiluminescent substrate (Pierce, Rockford, USA). Peroxidase activity was detected with a LumiImager device (Roche, Mannheim, Germany). The primary antibodies were purchased from R&D Systems, Minneapolis, USA (Ctsb: Catalog No. AF965; Ctsd: Catalog No. AF1014; Ctse: Catalog No. AF1130; Ctsl: Catalog No. AF1515; protein S100-A9: Catalog No. AF2065), Sigma-Aldrich (α-tubulin: catalog No. T 6199), and Abcam (involucrin: Catalog No. ab28057).

2.6 Cleavage Site Analysis with TAILS TAILS was performed using formaldehyde labeling according to the original publication

22

. 2 mg of

protein were used per condition. Formaldehyde labeling was performed as a label-switch experiment, meaning the wild type sample was labeled light in the first biological replicate and heavy in the second biological replicate. After tryptic digest samples were desalted using a reversed phase C18 column, prefractionated by SCX as described, and desalted using self-packed C18 STAGE tips (Empore, USA)

33

. Briefly, samples were prefractionated by high performance

liquid chromatography (HPLC) using a strong cation exchange column (SCX; PolyLC; Colombia, USA). Buffer A was 5 mM KH2PO4, 25% acetonitrile, pH 2.7 and buffer B was 5 mM KH2PO, 1 M KCl, 25 % acetonitrile, pH 2.7. Peptides were eluted in a linear gradient with increasing concentration of buffer B. 10-14 fractions were collected, desalted using self-packed C18 STAGE tips (Empore, USA), and analyzed by LC-MS/MS as described in the corresponding section. As for the quantitative proteome comparison, data were converted to mzXML format Proteowizard

27

26

using

with centroiding of MS1 and MS2 data. Peptide sequences were identified by X!

Tandem (version 2013.09.01.1)

28

, including cyclic permutation, in conjunction with PeptideProphet

(part of version 4.7 of the Trans Proteomic Pipeline)

29

and a decoy search strategy: the complete

mouse proteome database was downloaded from UniProt

30

on Nov 26th 2013. Semi Arg-C

specificity with up to two missed cleavage sites was applied. Static modifications are (+ 57.02 Da), lysine and N-terminal dimethylation (light formaldehyde + 28.03 Da; heavy formaldehyde + 34.06 Da). For acetylated N-termini (+ 42.01 Da), modifications are cysteine carboxyamidomethylation, N-terminal acetylation, and lysine dimethylation. Mass tolerance was 10 ppm for parent ions and 8 ACS Paragon Plus Environment

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0.02 Da for fragment ions. X!Tandem results were further validated by PeptideProphet at a confidence level of > 95 %. The relative quantification for each peptide was calculated using the XPRESS

32

algorithm as described for “Quantitative Proteome Comparison”. Two biological

replicates comparing wild type and ADAM17ΔKC epidermal lysates of littermates at P3 with independent sample preparation (not the same samples as for quantitative proteome comparison) and mass spectrometric measurement were analyzed. N-termini were considered to be altered in their abundance if they were (A) identified in both biological replicates, (B) showed an alteration in abundance of more than 50 % (Fc < -0.58; Fc > 0.58) in the same direction and (C) if their H/L ratio calculated by XPRESS was confirmed by manual inspection of the extracted ions chromatograms (XIC).

2.7 LC-MS/MS Analysis For nanoflow-LC-MS/MS, MS samples were analyzed on an Orbitrap XL (Thermo Scientific GmbH, Bremen, Germany) mass spectrometer. The instrument was coupled to an Ultimate3000 micro pump (Thermo Scientific) with a flow rate or 300 nl / min. 0.5 % acetic acid and 0.5 % acetic acid in 80 % acetonitrile (water and acetonitrile were at least HPLC gradient grade quality) with a gradient of increasing organic proportion were used for peptide separation (main separation ramp: 3 - 30% B within 70 min, flow rate 300 nL / min). Column-tips with 75 µm inner diameter and a length of 11 cm were self-packed

34

with Reprosil-Pur 120 ODS-3 (Dr. Maisch, Ammerbuch, Germany). The

mass spectrometer was operated in data dependent mode and switched automatically between MS and MS/MS. MS scans were acquired in the mass range of 370 to 1,700 m/z. Each MS scan was followed by a maximum of 5 MS/MS scans. The resolution for the FTMS mode was set to 60.000. The dynamic exclusion time was 30 s. MS/MS parameters were: minimum signal intensity: 1000; isolation width: 2.0 Da; normalized collision energy of 35 %. Lock mass (445.120025) was used for data acquired in MS mode. Automatic gain control target values were 10000.00 for the linear ion trap and 500000.00 for the Orbitrap.



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2.8 Generation and Cultivation of Keratinocytes for Immunofluorecent Staining Keratinocytes were isolated from the skin of Adam17ΔKC or EgfrΔKC mice and their wild type littermates essentially as described

16

. In brief, the epidermis and dermis were separated by

overnight digestion with dispase (STEMCELL Technologies) at 4°C. The epidermis was digested with 0.25 % trypsin (wt/vol) and 2 mM EDTA for 30 min at 37°C and the reaction was stopped with PBS containing 10 % FCS. 105 cells/cm2 were plated in defined serum-free keratinocyte medium (CellnTec) supplemented with 20 U/ml penicillin and 20 µg/ml streptomycin (Invitrogen) and maintained at 37°C in 5 % CO2. Subconfluent cells derived from passages 1–3 were used for the immunofluorescent staining. Keratinocytes used for western blot analysis were derived from primary keratinocytes in passages one or two and immortalized using the SV40 large T antigen. Keratinocytes were grown in suspension culture on polyhydroxyethyl-methacrylate (poly-HEMA)–coated plates as previously described

35

. 6-well plates were coated with poly-HEMA (Sigma-Aldrich), followed by extensive

PBS washes. Suspensions of 1-1.5x106 keratinocytes in 2 ml keratinocyte medium (CnT-07, CELLnTEC, Switzerland) was added in each coated well and incubated in a humidified incubator with 5% CO2 at 37°C for 24 h.

2.9 Immunofluorescent Staining Lamp-1 staining was performed by seeding 45000 cells per well on cover slips in 24- well plates. Cells were treated with the EGFR inhibitor AG1478 (200 nM), with EGF (100 ng/ml) or TGF-α (40 ng/ml) for 7 h, 24 h and 45 h and compared to untreated controls. After incubation cells were fixed with 4 % PFA in PBS for 15 min at room temperature, washed with PBS and permeabilized with 0.2 % Triton X-100 in PBS for 7 min at room temperature. Afterwards cells were treated for 4 min with -20 °C cold acetone, washed with PBS and blocked with 5 % BSA for 30 min. Lamp-1 antibody (1:700; Catalog No. ab25245; Abcam, Cambridge, UK) was applied in 5 % BSA over night at 4 °C. After washing with PBS secondary antibody (Alexa 488 goat anti-rat, 1:1000: Invitrogen, A11006) was applied in 5 % BSA for 1 h at room temperature. Cells were washed with 10 ACS Paragon Plus Environment

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PBS and nuclei stained for 5 min with 2 µg / ml Hoechst (Fluka / Sigma, Munich, Germany). After a final wash with PBS, cover slips were mounted with Permafluor (Labvision, Fremont, CA, USA) and analyzed using the Axio fluorescence microcope (Zeiss).

3 Results and Discussion 3.1 Impact of Keratinocyte-specific Disruption of the ADAM17-EGFR Signaling Axis on the Epidermal Proteome Profile The ADAM17–EGFR signaling axis is essential for maintenance of a healthy postnatal epidermal barrier. To elucidate the consequences of a loss of ADAM17 or EGFR, we compared the epidermal proteome composition of wild type mice and mice either lacking ADAM17 or EGFR in keratinocytes. For this purpose the epidermis was separated from the dermis by heat separation and subsequently homogenized for proteomic analysis. Each quantitative proteome comparison was performed with two biological replicates, each comprising different wild type and different Adam17ΔKC mice or EgfrΔKC mice, respectively. A schematic overview of the proteome comparisons is provided by Fig. 1a. To extent the number of biological replicates, we successfully corroborated protein alterations by immunoblotting using further biological replicates that are distinct from the mice, which were originally investigated by the proteomic experiments. In each comparison littermates were analyzed and stable isotope labeling with either d213C-formaldehyde (“heavy”) or d012C formaldehyde (“light”) in combination with liquid chromatography - tandem mass spectrometry (LC-MS/MS) was applied. To document alterations in protein abundance over time, quantitative proteome comparisons were analyzed at postnatal day 3 (P3) (for ADAM17: Tables S1/2; for EGFR: Tables S3/4), when the epidermal barrier is still intact, and at P10 (for ADAM17: Tables S5/6; for EGFR: Tables S7/8; overview about experimental design in Figure S1), when epidermal barrier defects can be detected for the first time, as evidenced by a reduced granular layer (Figure 1b) as well as elevated transepidermal water loss at the skin surface 16. 11 ACS Paragon Plus Environment


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Quantitative proteome comparison of wild type and Adam17ΔKC epidermis identified 716 proteins in both replicates at P3 and P10, respectively. For the comparison of wild type and EgfrΔKC epidermis 613 proteins were identified in all four experiments at P3 and P10. Alterations of protein abundance in all quantitative proteome comparisons are stated as fold change values (Fc; log2 of heavy/light (H/L) ratio).. All datasets displayed an average Fc close to zero, showing that the majority of proteins are unaltered (box plots in Figure 2A/B, histograms in Figures S1 and S2). We used the APEX method to calculate protein abundances

36, 37

. The resulting APEX scores

displayed good correlation between the different replicates and genotypes at time-points P3 and P10 (Figure 2C/D). To assess the extent of potential proteome contaminants, we further analyzed the p10 datasets for the Adam17ΔKC mice using the mouse sequence database as outlined in the Methods section appended with the contaminant database of MaxQuant and randomized sequences thereof. For replicate 1, we identified only eight proteins stemming from peptides that could not equally be mapped to mouse proteins due to sequence identity. This is less than 0.7 % of the proteome coverage in replicate 1 and falls within the FDR. For replicate 2, we identified only 10 proteins stemming from peptides that could not equally be mapped to mouse proteins due to sequence identity. This is less than 0.5 % of the proteome coverage in replicate 2 and also falls within the FDR. We conclude that our proteome analysis of murine skin is not confounded by putative proteome contaminants. We employed hierarchical clustering (HC) to obtain a global overview of the eight proteome comparisons (Figure 3). HC highlights that replicate samples of the same genotype (Adam17ΔKC or EgfrΔKC) and time point (P3 or P10) bear closest resemblance. At the next level, proteomes of the different time points are grouped together; indicating that proteome alterations between P3 and P10 supersede proteome alterations induced by disruption of either ADAM17 or EGFR. We refrained from using databases of putative contaminants. Precautions were used to prevent cross-contamination. The usage of a chemical labeling strategy yields equal “light” and “heavy” tagging of putative contaminants; unlike metabolic labeling strategies, in which contaminants are always “light”. Moreover, the sequence similarity between human and mouse proteins would likely


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result in an over-estimation of seemingly human proteins if based solely on peptide-to-sequence matches.

3.2 Keratinocyte-specific Deletion of Either Adam17 or Egfr Yields Similar, yet Distinct Proteome Alterations Proteins were considered to be altered in their abundance if they were quantified in both biological replicates of an experimental setup with an alteration of more than 50 % (Fc < -0.58; Fc > 0.58, with both replicates consistently showing either increase or decrease), and if their H/L ratio calculated by XPRESS was confirmed by manual inspection of the extracted ion chromatograms (XIC). These strict cutoff criteria have been successfully used before

25

. To balance the limited

number of replicates, immunoblotting corroboration (see below) was performed on biological replicates that were not part of the initial proteome study. For further analysis we focus on those proteins that are (a) consistently affected for at least one genotype and time-point and (b) are identified in at least six of the eight proteomic experiments. Table S9 summarizes these proteins. A selection is presented in Figure 4. It becomes evident that most alterations in protein abundance observed in Adam17ΔKC epidermis are also observed in EgfrΔKC epidermis; highlighting the importance of the ADAM17-EGFR signaling axis. There are very few proteins that are specifically affected in either Adam17ΔKC or EgfrΔKC epidermis (see also below). Furthermore only few proteins were altered at P3 in at least one genotype but displayed rather normalized ratios at P10. These rare cases include catenin alpha-1, decorin, and hemopexin. We noticed that a higher number of proteins shows different abundance comparing the time points P3 and P10. To test this we calculated the differences of Fc (ΔFc) between P3 and P10. Thereby three cases were distinguished: (a) ΔFc < 0.58 (no substantial alteration), (b) increase in protein abundance by ΔFc ≥ 0.58, and (c) decrease in protein abundance by ΔFc ≥ 0.58. According to this classification

55%

of

all

proteins

fulfilling

the

above

mentioned

cutoff

criteria

were

increasing/decreasing comparing P3 and P10 for Adam17ΔKC epidermis (Figure 5A). For EgfrΔKC epidermis this accounts for 63% (Figure 5B). This finding possibly reflects progression of the 13 ACS Paragon Plus Environment


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epidermal barrier defect. Proteins reflecting these cases include hornerin, kallikrein (KLK) 10, keratin (KRT) 1 and KRT10. However, most changes in protein abundance at P10 are already manifested at P3, prior to the occurrence of the actual epidermal barrier defect. To identify altered protein clusters and interactions upon disruption of the ADAM17-EGFR signaling axis, the online Search Tool for the Retrieval of Interacting Genes (STRING) was used. STRING unravels links between proteins based on published literature and large-scale databases. STRING found connectivity between altered proteins in EgfrΔKC epidermis at P10 and pointed to functional clusters (Figure 5C), among them “keratins and CE modifying enzymes” and “cysteine proteases and their inhibitors”. In the next section we will discuss protein alterations observed for transglutaminases and their substrates, especially CE components such as S100 fused-type proteins and keratins, in either Adam17ΔKC or EgfrΔKC epidermis. The cluster “cysteine proteases and their inhibitors” mainly consist of cathepsins, calpains, and their inhibitors. Their protein alterations upon disruption of the ADAM17-EGFR signaling axis and their role in epidermal barrier formation will be discussed below as well.

3.3 Protein Alterations of Transglutaminases and Their Substrates Observed in Epidermis of Adam17ΔKC and EgfrΔKC Mice Transglutaminases (TGMs) 1 and 3 are essential for proper formation of the CE, since they covalently crosslink its structural components including involucrin, loricrin, filaggrin, and small proline-rich proteins (SPRRs) by catalyzing the formation of Nε-(γ-glutamyl) lysine isopeptide bonds. The physiological relevance of TGMs is documented by mutations in the TGM1 gene that have been identified in patients suffering from lamellar ichthyosis, an autosomal recessive skin disorder with severe keratinization defects skin barrier defects and perinatal lethality epidermal barrier defects

41

38, 39

40

. Accordingly, Tgm1 deletion in mice causes severe

. Tgm3 deficiency in mice did nor result in no gross

, but leads to higher susceptibility to cutaneous inflammation, indicating

that TGM3 contributes to skin barrier stability 42. In EgfrΔKC epidermis, we noticed reduced levels of TGM3 at P10 (Fc = -0.7 and -0.9, respectively). In Adam17ΔKC epidermis, the reduction of TGM3 at P10 was less pronounced (Fc = -0.5 and -0.3, 14 ACS Paragon Plus Environment

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respectively). At P3, for both genotypes, TGM3 did not meet our criteria for quantitative alteration at P3 (Figure 4). TGM1 was only identified in three of the eight experiments and is therefore not displayed in Figure 4 or Table S9. Nevertheless, due to the alterations observed for TGM3, it is worth reporting the observed effects on TGM1, which correspond to the effects observed for TGM3. At P10 TGM1 was found reduced in both EgfrΔKC epidermis replicates (not identified in Adam17ΔKC epidermis). At P3 it was quantified as mildly reduced in one Adam17ΔKC epidermis replicate (not quantified in the second Adam17ΔKC replicate or in the two EgfrΔKC epidermis replicates, Tables S1, 7, 8). In line with our proteomic findings, TGM activity is reduced in EgfrΔKC mice as well as Adam17ΔKC mice at P10 16. A strong decrease of numerous TGM substrates was revealed by our proteomic analysis of Adam17ΔKC epidermis and EgfrΔKC epidermis. Most of them are encoded by the ‘epidermal differentiation complex’ that contains three different protein families. The first protein family consists of a number of precursor proteins important for the formation of the CE. lnvolucrin, which provides a scaffold in early CE formation to which other proteins are subsequently crosslinked 44

43,

, and the SPRR cornifin-A, which functions as a cross-bridging component during all stages of

CE formation

45

, are decreased in both experimental setups, upon Adam17 deletion and Egfr

deletion in keratinocytes, at P3 and P10 (Figure 4). The decrease of involucrin protein levels was corroborated by western blot analysis in epidermal lysates of Adam17ΔKC epidermis (Figure 6A) and EgfrΔKC epidermis, representing biological replicates that are distinct from the specimens of the proteomic study. (Figure 6B). Additionally, involucrin was decreased in immortalized keratinocytes deficient for Adam17 (Figure 6C) highlighting involucrin protein alterations as a cell-autonomous effect. The second protein family consists of EF-hand containing Ca2+-binding proteins (S100), which exhibit various functionalities. Amongst others, members of the S100 protein family regulate cell growth and differentiation processes as well as regulating Ca2+ homeostasis. Ca2+ levels are important cell-regulatory signals during CE formation and control the activity of numerous enzymes including TGMs

46-50

. EGFR deletion has opposing effects on proteins S100-A3 and –A9: whereas

protein S100-A3 is strongly reduced at both P3 and P10, protein S100-A9 displays increased levels. In Adam17ΔKC epidermis, reduced levels of protein S100-A3 at P10 and increased levels of 15 ACS Paragon Plus Environment


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protein S100-A9 at P3 are corroborated by the proteomic analysis; western-blot analysis confirms generally increased S100-A9 levels for both genotypes at both time points (Figure 6A/B). Protein S100-A9 builds up a heterodimeric complex with protein S100-A8, called calprotectin. Calprotectin is strongly expressed in abnormally differentiating keratinocytes, as in psoriasis

51, 52

, and plays a

key role in the induction and propagation of inflammation, as it is secreted by and acts as a chemoattractant for neutrophils, activated monocytes and macrophages

53-55

. In agreement

Adam17ΔKC mice develop a thickened epidermis, dry skin and chronic skin inflammation 16. The third protein family consists of S100 fused-type proteins, which have evolved by fusion of ancestors of the two previously described families, which all possess N-terminal Ca2+-binding EFhand motifs, a unique central repeat domain, and a C-terminal part specific for each protein

19

. In

the quantitative proteome comparisons four members of this family were found to be decreased in Adam17ΔKC and EgfrΔKC epidermis at P3 and P10, namely filaggrin, filaggrin-2, trichohyalin-like protein 1, and cornulin. A fifth member of this family, hornerin, was only affected at P10 in EgfrΔKC epidermis, with incomplete data for Adam17ΔKC (Figure 4). Filaggrin is the best characterized S100 fused-type protein and is an essential component of the stratum corneum as it contributes to the aggregation of keratin filaments into higher molecular structures in the initial phase of CE formation. The aggregation into macrofibrils protects the keratin proteins against proteolytic degradation during keratinocyte terminal differentiation

17, 56, 57

.

In this process the type II keratin KRT1 and its heterodimer type I partner KRT10 are of special interest since they form the major cytoskeleton in suprabasal keratinocytes, which serves as a scaffold for CE components. Hence, KRT1 and KRT10 are the first proteins to be expressed during cornification even preceding the expression of filaggrin and other CE components

17

. Interestingly

several keratin members are decreased at P3 and P10 in Adam17ΔKC and EgfrΔKC epidermis. Among them are KRT1 and KRT10, which are decreased at P10 in the onset of the epidermal barrier defect, but unaltered at P3, when the epidermis appears healthy (Figure 4). Furthermore, it has been reported that absence of KRT1 results in an increase of protein S100-A9

58

, which is

supported by our data (Figure 6A/B). The relevance of KRT1 and KRT10 in epidermal barrier formation is highlighted by mutations in one of these genes resulting in a congenital epidermolytic ichthyosis characterized by skin 16 ACS Paragon Plus Environment

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erosions, hyperkeratosis and barrier defects lethality and a barrier defect higher molecular structures

62

58

59-61

. Hence, loss of KRT1 in mice causes perinatal

. Mutations in filaggrin, which aggregates keratin filaments into

, increase the risk of atopic dermatitis

63-66

and lead to ichthyosis

vulgaris, which is characterized by a hyperkeratosis and a dry scaly skin 67. Altogether, decrease of TGM activity, the decrease of a subset of keratins, as well as the decrease of TGM substrates (including filaggrin) already at P3, could contribute to a large extent to epidermal barrier defects upon disruption of the ADAM17-EGFR signaling axis.

3.4 Alterations of Proteases and Protease Inhibitors Observed in Epidermis of Adam17ΔKC and EgfrΔKC Mice Proteolysis is tightly controlled to maintain skin homeostasis. Generally, cornification requires a massive activation of epidermal proteases, since these are involved in at least three steps of skin differentiation. Firstly, numerous precursors of the CE undergo proteolytic processing before CE formation. Secondly, loss of organelles requires proteolytic clearance. Thirdly, the controlled shedding of the corneocytes at the skin surface, a process called desquamation, depends on proteolysis of corneodesmosomes

17

. In Adam17ΔKC epidermis and EgfrΔKC epidermis, proteases

and protease inhibitors contributing to all of the above mentioned processes are altered in their abundance. Similar to Adam17ΔKC epidermis, filaggrin is decreased in EgfrΔKC epidermis. As mentioned before, the CE precursor filaggrin aggregates keratin filaments into higher molecular parallel structures

62

.

During this process filaggrin is dephosphorylated and cleaved into 37 kDa monomers. Numerous proteases have been postulated as being involved in filaggrin processing

18

, including the non-

lysosomal cysteine protease calpain 63. M-Calpain protein levels are increased in EgfrΔKC epidermis at P10 (Fc = 0.7 and 0.7, respectively), but not altered in Adam17ΔKC epidermis at P10 (Fc = 0.5 and -0.3, respectively (Table S7/8); M-Calpain was not identified in any dataset at P3 and is therefore not displayed in Figure 4 and Table S9). Moreover, protein levels of the calpain inhibitor calpastatin decreased in Adam17ΔKC epidermis and EgfrΔKC epidermis at P10 (Figure 4), thus



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Page 18 of 44

enabling increased calpain activity and elevated filaggrin cleavage. This finding substantiates the role for calpains in filaggrin processing. Furthermore, several members of the serpin family of serine protease inhibitors showed alterations in protein abundance. Serpin a1d presented increased levels in Adam17ΔKC epidermis with inconsistent data for EgfrΔKC epidermis while serpin b3a was consistently increased in EgfrΔKC epidermis at P10 (Figure 4). In line, gene expression data from P10 skin of Adam17ΔKC mice revealed an up-regulation of serpin a3a as well as serpin a3n and a reduction of serine protease activity in the stratum corneum

16

.

Kallikrein serine proteases (KLKs) play important roles in desquamation by cleaving structural components, such as corneodesmosin, desmocollin and desmoglein

68, 69

. We find elevated levels

of KLKs 10 and 6 at P10 for both Adam17ΔKC epidermis and EgfrΔKC epidermis (Figure 4). Possibly they are secreted and have a function in the desquamation process. To our knowledge, a link between ADAM17/EGFR and kallikrein proteases has not been reported before. Next to serine proteases, lysosomal cysteine and aspartic proteases are involved in cornification. Cathepsin D (Ctsd) deficient mice harbor defects in TGM1 activation, less pronounced CEs, and a thickened epidermis

70

. Mice deficient for cathepsin L (Ctsl) show a periodic hair loss together with

epidermal hyperplasia, acanthosis, and hyperkeratosis

71

. We observe a pronounced decrease of

cathepsins B, D, and L at P10 for both genotypes (Figure 4; 6A/B). A link between the ADAM17EGFR signaling axis and cathepsin protein abundance has not been reported before and was also corroborated for Ctsd and Ctsl in cell lysates of immortalized keratinocytes deficient for Adam17 (Figure 6C), thus characterizing this effect as a cell-autonomous feature. Cathepsin E (Ctse) was decreased in both replicates comparing wild type and EgfrΔKC epidermis at P10 (Tables S7/8). Since it was not identified in any other dataset at P3 or P10, Ctse is not included in Figure 4 or Table S9. At P3, only cathepsin B in EgfrΔKC epidermis meets our cutoff criteria for decreased abundance (Figure 4). Cystatins are the main family of cathepsin inhibitors

72

. In the present study, two cystatins display

mixed behavior. There are consistently increased levels of cystatin M/E in Adam17ΔKC epidermis at P10 and consistently decreased levels of cystatin M/E in EgfrΔKC epidermis at P3. For cystatin B, we find consistently decreased levels for both genotypes at P10 (Figure 4). In Ctsl deficient skin, 18 ACS Paragon Plus Environment

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both cystatins were found to accumulate

73

. Generally, deletion of either ADAM17 or EGFR results

in substantial perturbations of lysosomal protease levels as well as affecting their endogenous inhibitors. We further investigated this observation in vitro using cultured murine keratinocytes. Neither application of the EGFR inhibitor AG1478 for up to 45 h nor addition of the EGFR ligands EGF or TGF-α had an effect on lysosomal morphology as evidenced by immunohistochemical analysis for the lysosomal marker Lamp-1 (Figure S3). This finding underlines the complexity of proteolytic networks in vivo. The majority of protein alterations observed in Adam17ΔKC epidermis are observed as well in EgfrΔKC epidermis. Therefore, most proteome alterations observed in Adam17ΔKC epidermis are likely mediated via EGFR. In general, alterations observed in the EgfrΔKC epidermis displayed more extreme Fc values as compared to the Adam17ΔKC epidermis (Table S9), likely reflecting the contribution of further EGFR activators next to ADAM17.

3.5 Proteins Uniquely Affected by Lack of Either ADAM17 or EGFR Keratinocyte-specific Adam17 and Egfr deletion yields highly similar proteome profiles with only few uniquely affected proteins at either P3 or P10. Proteins that are selectively affected in Adam17ΔKC epidermis include death-associated protein-like 1 (increased at P10), eukaryotic translation initiation factor 3 subunit B (decreased at P3 and P10), and platelet-activating factor acetylhydrolase IB subunit alpha (decreased at P10) (Figure 4). In contrast, the present study highlights caspase-14 and dihydropyrimidinase-related protein 2 as proteins that are selectively affected in EgfrΔKC epidermis (decreased at P10). For lactoylglutathione lyase we observed opposing behavior with an increase in Adam17ΔKC at P3 and a decrease in EgfrΔKC epidermis at P10. Although a specific motif of uniquely affected proteins does not emerge, this data shows that Adam17ΔKC epidermis does not entirely phenocopy EgfrΔKC epidermis on the proteome level.



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3.6 ADAM17 Dependent Proteolytic Processing Cornification requires the activity of proteases for degradation of organelles, proteolysis of corneodesmosomes and proteolytic processing of CE precursors, such as filaggrin. To identify stable cleavage products during CE formation dependent on ADAM17, we applied the quantitative N-terminomic technique Terminal Amine Isotopic Labeling of Substrates (TAILS)

74

. TAILS has

already been employed to investigate the impact of Ctsb and/or Ctsl depletion to extracellular proteolysis of mouse embryonic fibroblasts

25, 75

and, more important in this context, to the skin

proteome 73. The TAILS approach uses a negative selection strategy for quantitative comparison of protein N-termini from different biological samples. It allows for the identification of naturally modified (e.g. acetylated) and unmodified N-termini. The latter contain primary amines (-NH2) upon sample harvest and consist of native as well as proteolytically generated N-termini. In the TAILS protocol these naturally unmodified N-termini are chemically dimethylated using isotopically labeled formaldehyde and enriched for MS analysis. In the majority of cases the identified natively unmodified N-termini correspond to cleavage sites of proteolytic processing. TAILS was performed in two biological replicates comparing epidermal lysates of three days old Adam17ΔKC mice. Both replicates showed an average fold change close to zero (box plots in Figure 7A, histograms in Figure S4). Events of proteolytic processing are represented by dimethylated N-termini. Of these, 2475 dimethylated were identified in the first (Table S10) and 2358 in the second TAILS experiment (Table S11). 1432 dimethylated N-termini were identified and quantified in both biological replicates. The majority of these represent internal cleavage sites pointing to events of proteolytic processing (Figure 7B/C). A mild increase can be observed for Ntermini derived from the first 10 % of the full length protein chain, a result in compliance with other studies 76. Like for the quantitative proteome comparisons, N-termini were considered to be altered in their abundance if they were quantified with an alteration in abundance of more than 50 % (Fc < -0.58; Fc > 0.58) in both biological replicates and if their H/L ratio calculated by XPRESS was confirmed by manual inspection of the extracted ion chromatograms. According to these criteria 73 N-termini increased (Table S12) and 21 N-termini decreased (Table S13) in ADAM17ΔKC epidermis at P3. In 20 ACS Paragon Plus Environment

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some cases altered abundance of a proteolytically processed N-terminus rather reflects altered abundance of the corresponding protein than impaired proteolytic processing. For example, several cleavage sites in keratins decrease in the Adam17ΔKC epidermis (Table S13). This finding is best explained by a reduced overall abundance of these keratins, since the abundance of most keratins decreased in the proteome comparisons of Adam17ΔKC epidermis (Figure 4, Table S9).

3.7 Analysis of N-terminal Acetylation Identifies Alternative Translation Start Sites N-terminal acetylation is a co-translational process, which occurs after removal of the initiator methionine

77

. Thus, N-terminal acetylation likely reflects the natively translated N-terminus of a

protein. Independently of our interest in the ADAM17 / EGFR biology, we investigated N-terminal acetylation, which is an integral part of an N-terminomic TAILS experiment. 499 acetylated Ntermini were identified in both replicates (replicate 1: Table S14; replicate 2: Table S15). Sequence specificity of N-terminal acetylation in the present study represents the prototypical profile of small and acidic residues (Figure 7D)

73, 78

. Since N-terminal acetylation is a co-

translational process, it has been used to analyze start sites of protein translation

78

. Here we

focused on the usage of alternative translation start site that differ from the annotated protein Nterminus in Uniprot

79

. We found five acetylated protein N-termini that were identified in both

replicates, display removal of a putative initiator methionine, and map to positions that differ from the canonical, Uniprot-annotated protein N-terminus (Figure 7E). In all cases, the newly found alternative translation start site is close to the annotated protein N-terminus.

3.8 Proteolytic Processing of KRT1, KRT10, Filaggrin and Filaggrin-2 TAILS identified 22 N-termini of KRT1 and 16 N-termini of KRT10 as increased in the Adam17ΔKC epidermis (Table S12). A higher occurrence of these cleavage sites likely reflects increased degradation of KRT1/KRT10 intermediate filaments, which provide stability to the tissue

80

. In line,

KRT1 and KRT10 protein levels decrease in the quantitative proteome comparison of Adam17ΔKC epidermis at P10 (Figure 4), when transepidermal water loss at the skin surface is significantly 21 ACS Paragon Plus Environment


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Page 22 of 44

elevated for the first time. In combination, these findings suggest that increased proteolysis of KRTs 1 and 10 strongly contributes to their decreased protein levels. Interestingly, KRT1 and KRT10 protein levels are not affected at P3, when the skin of Adam17ΔKC mice appears healthy, but TAILS already detects an increased degradation of these proteins at this early state. Besides providing epidermal stability, KRT1/KRT10 filaments are assumed to play an essential role for epidermal barrier formation, because they are highly crosslinked with the CE and 17

serve as a scaffold for further CE components

. Consequently, increased degradation of

KRT1/KRT10 filaments may contribute to the increase in transepidermal water loss observed in Adam17ΔKC mice. We also noticed elevated proteolytic processing of filaggrin together with decreased overall filaggrin levels. TAILS quantified six filaggrin cleavage sites as being more abundant in the Adam17ΔKC epidermis (Table S12). Profilaggrin is expressed as a polyprotein containing 10 to 12 near-identical filaggrin repeats, each of them 324 amino acids long

63

. All N-termini quantified more

abundant in the Adam17ΔKC epidermis are located in filaggrin repeats, indicating increased and faulty proteolytic processing of filaggrin in epidermis depleted for ADAM17 in keratinocytes. Similar results were obtained for filaggrin-2. Six N-termini of filaggrin-2 were quantified more abundant in Adam17ΔKC epidermis (Figure 8A, Table S12). Of these, three N-termini represent cleavage sites in filaggrin repeat domains, indicating increased and faulty proteolytic processing of filaggrin-2 (Figure 8B). Accordingly, filaggrin-2 protein levels are reduced in Adam17ΔKC epidermis (Figure 4). Filaggrin and filaggrin-2 are important structural components in maintaining skin barrier function 82

81,

. Increased proteolytic processing of both S100 fused-type protein members likely contributes to

the defects in epidermal barrier formation observed in Adam17ΔKC mice

4 Conclusion The present study investigated the defects in epidermal barrier formation by a global proteomic and degradomic approach and highlights the essential role of ADAM17-EGFR signaling for the postnatal maintenance of epidermal barrier function. 22 ACS Paragon Plus Environment

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Independent profiling of the epidermal proteome deficient for either ADAM17 or EGFR in keratinocytes of mice revealed highly similar protein alterations with only a few uniquely affected proteins by ADAM17 or EGFR at both time points analyzed (P3, P10). Hence, most alterations observed in Adam17ΔKC epidermis are likely mediated via the ADAM17-EGFR signaling axis. The majority of proteins altered at P10 in both experimental setups, ADAM17 and EGFR, are already altered at P3, prior to the occurrence of severe epidermal barrier defects. Disruption of the ADAM17-EGFR signaling axis results in massive alterations in the proteolytic network and consequently in proteolytic processing. Numerous proteases, among them kallikreins, calpains, cathepsins and their inhibitors, display strong alterations, resulting in (A) decrease of TGM activity and (B) alterations in protein abundance of several regulatory and structural components important for CE formation. Here, proteolytic processing of KRT1/KRT10 filaments, filaggrin and filaggrin-2 are highlighted. Proteolytic processing is essential to maintain skin homeostasis. Disruption of ADAM17 dependent EGFR signaling using EGFR inhibitors leads to dermatological side effects in cancer therapy

15, 83

.

Hence, unveiling the proteolytic network and its effect on signaling pathways in skin should be the future challenge and could considerably contribute to develop novel therapeutic strategies for the treatment of dermatological side effects.

List of abbreviations

ACE: angiotensin-converting enzyme; ADAM17: a disintegrin and metalloproteinase domaincontaining protein; BCA: bicinchoninic acid; BSA: bovine serum albumin; CE: cornified envelope; Ctsb: cathepsin B; Ctsd: cathepsin D; Ctse: cathepsin E; Ctsl: cathepsin L; DPPIV: dipeptidylpeptidase IV; E64d: (2S, 3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester; ECM: extracellular matrix; EDTA: ethylene diamine tetraacetic acid; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; Fc: fold change; GO: gene ontology; H&E: hematoxylin and eosin; HB-EGF: heparin-binding epidermal growth factor; HPLC: highperformance liquid chromatography; IL-6R: interleukin-6 receptor; KLK: kallikrein; KRT: keratin; 23 ACS Paragon Plus Environment


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Lamp: lysosome-associated membrane glycoprotein; LC-MS/MS: liquid chromatography - tandem mass spectrometry; LEKTI: lympho-epithelial kazal-type related inhibitor I; MHC: major histocompatibility complex; MS, mass spectrometry; PBS: phosphate buffered saline; PMSF: phenylmethanesulfonyl fluoride; PVDF: polyvinylidene fluoride; SCX: strong cation exchange; SDS: sodium dodecyl sulfate; STRING: search tool for the retrievel of interacting genes; TACE: tumor necrosis factor α-converting enzyme; TAILS: terminal amine isotopic labeling of substrates; TNF-α: tumor necrosis factor–α; TGF-α: transforming growth factor-α; TGM: transglutaminase; TSLP: thymic stromal lymphopoietin; XIC: extracted ions chromatograms

Data access The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium 84 via the PRIDE partner repository with the dataset identifier PXD002597.

Competing Interests The authors declare no conflict of interest.

Acknowledgements This work was supported by the German Research Foundation (SFB 850/B6) and the FritzThyssen

foundation

(Az.10.14.2.150)

to

C.-W.F.

and

by

grants

of

the

Deutsche

Forschungsgemeinschaft (DFG) (SCHI 871/2 and SCHI 871/5, SCHI 871/6, GR 1748/6, INST 39/900-1, and SFB850 (Project B8), a starting grant of the European Research Council (Programme “Ideas” - Call identifier: ERC-2011- StG 282111-ProteaSys), and the Excellence Initiative of the German Federal and State Governments (EXC 294, BIOSS) to O.S. J.N.K. is 24 ACS Paragon Plus Environment

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recipient of a Career Investigator Scholar award from Michael Smith Foundation for Health Research. The authors thank Franz Jehle for excellent technical assistance with mass spectrometry analysis and Alejandro Gomez-Auli for help with graphical software.

Supporting Information Available

Figure S1: Distribution of fold change values (log2) of proteins identified in wild type and Adam17ΔKC epidermis displayed as histograms at P3 (A) and P10 (B).

Figure S2: Distribution of fold change values (log2) of proteins identified in wild type and Egfr ΔKC epidermis displayed as histograms at P3 (A) and P10 (B).

Figure S3: Immunofluorescent staining of keratinocytes with the lysosomal marker Lamp-1. Immunofluorescent staining of keratinocytes (ctrl), keratinocytes treated with the EGFR inhibitor AG1478 (200 nM), with EGF (100 ng/ml) or TGF-α (40 ng/ml). Staining was performed for Lamp-1 (green) and Nuclei are stained with Hoechst (blue).

Figure S4: Distribution of fold change values (log2) for identified cleavage sites in both TAILS replicates comparing wild type and Adam17ΔKC epidermis at P3 displayed as histograms.

Table S1: List of all identified and quantified proteins in the quantitative proteome comparison of wild type (WT) and Adam17ΔKC epidermal lysates at P3 (replicate 1).


Table S3: List of all identified and quantified proteins in the quantitative proteome comparison of wild type (WT) and EgfrΔKC epidermal lysates at P3 (replicate 1). 25 ACS Paragon Plus Environment


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Page 26 of 44

Table S4: List of all identified and quantified proteins in the quantitative proteome comparison of wild type (WT) and EgfrΔKC epidermal lysates at P3 (replicate 2).





Table S9: List of all proteins (a) altered (FC ≤ -0.58 / FC ≥ 0.58) at least in one experimental setup in both replicates (e.g. both replicates of the proteome comparison WT/ Adam17ΔKC at P3) and (b) identified at least in six quantitative proteome comparisons (WT/ Adam17ΔKC and WT/EGFRΔKC).

Table S10: Dimethylated (naturally unmodified) N-termini identified in the TAILS experiment comparing wild type (WT) and Adam17ΔKC epidermal lysates at P3 (replicate 1). Up to two potential protein IDs per peptide are stated. Prime-site sequences of proteineous cleavage sites were determined experimentally. The corresponding non-prime sequences are derived bioinformatically by database searches. Modifications are lysine and N-terminal dimethylation (light formaldehyde + 28.03 Da; heavy formaldehyde + 34.06 Da) and cysteine carboxyamidomethylation(+ 57.02 Da).

Table S11: Dimethylated (naturally unmodified) N-termini identified in the TAILS experiment comparing wild type (WT) and Adam17ΔKC epidermal lysates at P3 (replicate 2). Up to two potential 26 ACS Paragon Plus Environment

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protein IDs per peptide are stated. Prime-site sequences of proteineous cleavage sites were determined experimentally. The corresponding non-prime sequences are derived bioinformatically by database searches. Modifications are lysine and N-terminal dimethylation (light formaldehyde + 28.03 Da; heavy formaldehyde + 34.06 Da) and cysteine carboxyamidomethylation(+ 57.02 Da).

Table S12: Dimethylated (naturally unmodified) N-termini increased by more than 50 % in abundance (FC ≥ 0.58) in both biological replicates of the TAILS experiments comparing wild type (WT) and Adam17ΔKC epidermis at P3.

Table S13: Dimethylated (naturally unmodified) N-termini decreased by more than 50 % in abundance (FC ≤ -0.58) in both biological replicates of the TAILS experiments comparing wild type (WT) and Adam17ΔKC epidermis at P3.

Table S14: Acetylated (naturally modified) N-termini identified in the TAILS experiment comparing wild type (WT) and Adam17ΔKC epidermal lysates at P3 (replicate 1). Up to two potential protein IDs per peptide are stated. Prime-site sequences of proteineous cleavage sites were determined experimentally. The corresponding non-prime sequences are derived bioinformatically by database searches. Modifications are N-terminal acetylation 42.01 Da, lysine dimethylation (light formaldehyde

+

28.03

Da;

heavy

formaldehyde

+

34.06

Da)

and

cysteine

carboxyamidomethylation (+ 57.02 Da).

Table S15: Acetylated (naturally modified) N-termini identified in the TAILS experiment comparing wild type (WT) and Adam17ΔKC epidermal lysates at P3 (replicate 2). Up to two potential protein IDs per peptide are stated. Prime-site sequences of proteineous cleavage sites were determined experimentally. The corresponding non-prime sequences are derived bioinformatically by database searches. Modifications are N-terminal acetylation 42.01 Da, lysine dimethylation (light formaldehyde

+

28.03

Da;

heavy

formaldehyde

+

carboxyamidomethylation (+ 57.02 Da).


34.06

Da)

and

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Figure Legends

Figure 1: (A) Overview of experimental setup. Each quantitative proteome comparison and TAILS experiment was performed in two biological replicates (2x), each comprising different wild type and different Adam17ΔKC mice or EgfrΔKC mice. Quantitative proteome comparisons were performed at P3, when the epidermal barrier is still intact, and at P10, when epidermal barrier defects can be detected for the first time. TAILS was performed in two biological replicates comparing wild-type and Adam17ΔKC epidermis at P3. (B) Adam17 KC show first evidences of Δ

defects in epidermal barrier integrity at P10. H&E staining of paraffin-embedded back skin sections of Adam17 KC (A17ΔKC) mice show no histologic differences in epidermal architecture Δ

compared to their littermate controls (WT) at postnatal day 3 (P3), while at P10 reduced stratum granulosum layers were detected.


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Figure 2: Quantitative proteome comparisons. (A) Distribution of fold change values (log2) of proteins identified in wild type and Adam17 KC epidermis displayed as Box-Whisker plot. Dashed Δ

lines indicate alteration in abundance of more than 50 % (Fc < -0.58; Fc > 0.58). IQR = Interquartile Range (B) Distribution of fold change values (log2) of proteins identified in wild type and Egfr KC epidermis displayed as Box-Whisker plot. Dashed lines indicate alteration in Δ

abundance of more than 50 % (Fc < -0.58; Fc > 0.58). IQR = Interquartile Range (C) Correlation analysis of APEX protein abundance scores for all datasets comparing wild type and Adam17 KC Δ

epidermis as well as wild type and Egfr17 KC epidermis at P3. r = pearson coefficient (D) Δ

Correlation analysis of APEX protein abundance scores for all datasets comparing wild type and Adam17 KC epidermis as well as wild type and Egfr17 KC epidermis at P10. r = pearson coefficient. Δ

Δ

Figure 3: Hierarchical Clustering. Hierarchical clustering of all replicates comparing wild type (WT) and Adam17 deficient (A17ΔKC) epidermis at P3 and P10 as well as all replicates comparing wild type (WT) and Egfr deficient (EgfrΔKC) epidermis at P3 and P10 using the Perseus software.

Figure 4: Proteins altered upon disruption of the ADAM17-EGFR signaling axis. Table of selected proteins displayed with their Fc in each replicate comparing wild type (WT) and Adam17ΔKC deficient (A17ΔKC) epidermis and comparing wild type (WT) and Egfr deficient (EgfrΔKC) epidermis at P3 and P10. Fc ≤ -0.58 or ≥ 0.58 corresponds to an alterations in protein abundance by more than 50 %. Background colors indicate magnitude of protein alteration (green: increase, red: decrease).

Figure 5: Overall abundance alterations comparing P3 and P10 and connectivity of all proteins altered in the quantitative proteome comparison of wild-type and Egfr

ΔKC

epidermis at P10. (A) Percentage of proteins that display no alteration, are increased by more than 50% (Fc ≥ 0.58), or decreased by more than 50% (Fc ≤ -0.58) comparing P3 and P10 of the proteome comparison of Adam17ΔKC deficient (A17ΔKC) and wild-type epidermis. Analysis was applied to average fold changes of both replicates for each time point using proteins altered 33 ACS Paragon Plus Environment


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according to our cutoff criteria and displayed in Supplementary table 9. (B) Percentage of proteins that display no alteration, are increased by more than 50% (Fc ≥ 0.58), or decreased by more than 50% (Fc ≤ -0.58) comparing P3 and P10 of the proteome comparison of EgfrΔKC deficient and wild-type epidermis. Analysis was applied to average fold changes of both replicates for each time point using proteins altered according to our cutoff criteria and displayed in Supplementary table 9. (C) The Search Tool for the Retrieval of Interacting Genes (STRING) was used to visualize connections between proteins altered in EgfrΔKC epidermis at P10 and pointed to the functional clusters “keratins and CE modifying enzymes” and “cysteine proteases and their inhibitors”. Different line colors represent the types of evidence for each association.

Figure 6: Western blot analysis for involucrin, protein S100-A9, and cathepsins B, D, E, and L. Western blot analysis of (A) wild type (WT) and Adam17ΔKC epidermis (A17ΔKC), (B) wild type (WT) and EgfrΔKC epidermis (EgfrΔKC), and (C) immortalized wild type (WT) and Adam17ΔKC keratinocytes. α-Tubulin served as loading control. Biological replicates of the proteomic samples were probed.

Figure 7: Cleavage site analysis by TAILS comparing wild type and Adam17ΔKC epidermis at P3. (A) Distribution of fold change values (log2) displayed as Box-Whisker plot. Dashed lines indicate alteration in abundance of more than 50 % (Fc < -0.58; Fc > 0.58). IQR = Interquartile Range (B) Distribution of dimethylated (naturally unmodified) N-termini binned by their relative position in the sequence of the corresponding protein for replicate 1. (C) Distribution of dimethylated (naturally unmodified) N-termini binned by their relative position in the sequence of the corresponding protein for replicate 2. (D) N-acetylation pattern upon Adam17ΔKC deletion. Sequence logos were generated with iceLogo

85

. (E) Acetylated N-termini identified in both

replicates, which do not reflect the native N-terminus according to the uniprot database. Non-prime sequences were derived by database search.

Figure 8: Proteolytic processing of filaggrin-2. (A) Dimethylated (naturally unmodified) Ntermini increased in protein abundance in both TAILS replicates comparing wild type (WT) and 34 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Adam17ΔKC (A17ΔKC) epidermis at P3. Non-prime sequences were derived by database search. Colors of arrows indicate position of cleavage site in filaggrin-2 in panel (B). (B) Schematic overview of the filaggrin-2 protein and the position of overrepresented cleavage sites indicated by colored arrows. Precise position and sequence of cleavage sites is displayed in panel (A).


Tholen et al., Fig. 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

B


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B

C

D

EgfrΔKC

Tholen et al., Fig. 2

IQR: 0.74 0.62 0.49 0.65 IQR: 0.59 0.54 0.70 0.73

A17ΔKC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

Page 37 of 44 Journal of Proteome Research


Tholen et al., Fig. 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



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Proteins affected affected at P3 different upon A17ΔKC in at least one genotype and EgfrΔKC

Proteins more affected at P10 in at least one genotype

Proteins similarly affected at P3 and P10 in at least one genotype

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fold change (log2 of A17ΔKC/WT ratio) P3 P3 P10 P10 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Protein name Cornifin-A -1.0 -1.4 -1.2 -0.5 Filaggrin -1.0 -0.8 x -1.4 Filaggrin-2 -1.0 -0.3 -1.6 -1.7 Involucrin -0.9 -1.5 x -0.8 Keratin, type I cytoskeletal 17 -0.6 -0.6 -0.4 -0.8 Keratin, type I cytoskeletal 27 -1.2 -1.2 -1.2 -2.8 Keratin, type I cytoskeletal 28 -1.2 x -0.5 -0.3 Keratin, type II cytoskeletal 1b -0.3 -0.4 -0.3 -1.6 Keratin, type II cytoskeletal 71 -0.7 0.7 -0.8 -1.0 Protein Crnn; Cornulin -0.8 -0.7 -1.2 -1.5 Trichohyalin-like protein 1 -2.6 -1.4 -1.0 -1.4 Protein S100-A3 -3.3 1.0 -0.7 -0.7 Protein S100-A9 2.8 3.7 -0.1 x 1.3 1.3 1.4 1.0 Alpha-1-antitrypsin 1-4; Serpin A1d Kininogen-1 0.1 0.8 0.4 0.7 Afadin -0.4 -0.5 -0.4 x Aurora kinase B -0.7 -0.8 0.7 x

Fold change (log2 of EgfrΔKC/WT ratio) P3 P3 P10 P10 Replicate 1 Replicate 2 Replicate 1 Replicate 2 -1.7 -1.6 -0.8 -0.6 -0.4 -0.5 -1.1 -1.6 -0.2 -0.6 -1.6 -0.7 -0.8 -0.4 -1.3 -1.2 -0.8 -1.1 -1.0 -1.2 -3.2 -2.0 -5.1 -3.2 -3.7 x -1.9 -0.9 -0.2 -0.8 -1.0 -1.2 -0.8 -2.5 -3.4 -2.6 -3.3 -1.2 -2.7 -2.5 -2.1 x -3.0 -2.4 -2.8 -3.8 -1.7 -1.7 1.5 1.9 1.3 0.4 -0.9 2.0 0.0 1.1 0.3 2.0 1.1 1.2 -0.4 -0.5 -0.6 -0.6 -0.5 -0.8 -1.0 -0.9

Hornerin Keratin, type I cytoskeletal 10 Keratin, type II cytoskeletal 1 Transglutaminase-3 Calpastatin Cathepsin B Cathepsin D Cathepsin L1 Cystatin-B; Stefin-B Perilipin-2 Phospholipase D3 Apolipoprotein E Apolipoprotein A-I Apolipoprotein A-II MCG129038; Protein Serpinb3a Kallikrein 6; Kallikrein 1; Neurosin Kallikrein related-peptidase 10 Cystatin E/M; Cystatin M/E

-0.2 0.7 0.2 -0.3 -0.4 -0.1 0.7 0.7 -1.0 -0.2 0.0 -1.0 0.4 0.2 -1.1 0.4 0.1 -0.1

-0.1 0.1 -0.4 0.0 -0.2 -0.1 -0.1 0.1 -0.3 -0.1 -0.3 -0.5 0.5 0.4 0.0 -0.5 -0.1 -0.4

-1.7 -1.1 -1.7 -0.5 -0.5 -0.6 -0.6 -0.7 -0.7 -0.9 -0.3 -2.3 0.7 0.7 0.1 1.3 0.9 0.9

x -0.5 x -0.3 -0.4 -0.7 -0.3 -0.8 -0.5 -0.9 -0.5 -1.6 0.8 0.8 x x 1.5 1.0

0.0 -0.3 0.0 0.2 -0.3 -0.7 -0.3 0.3 -0.3 0.2 -0.2 -1.3 -0.2 0.3 -0.6 -0.5 0.6 -0.7

-0.3 -0.3 -0.4 -0.3 0.0 -0.8 -0.4 -0.5 -0.5 -0.5 -0.5 -0.6 -0.2 1.1 -0.1 x -0.4 -0.6

-2.0 -1.1 -1.8 -0.7 -0.6 -1.1 -0.7 -1.0 -0.7 -0.9 -0.8 -2.6 0.6 0.7 1.2 2.2 0.9 0.2

-2.8 -2.1 -2.6 -0.9 -0.8 -0.7 -0.7 -0.9 -0.9 -0.5 -0.7 -2.3 0.5 0.5 0.9 2.0 1.2 0.4

Decorin Catenin alpha-1 Hemopexin Leucine-rich repeat flightlessinteracting protein 1

1.0 0.6 1.1

1.4 0.6 1.1

0.5 -0.1 0.6

-0.2 -0.2 0.3

x 0.8 0.8

0.8 0.8 0.9

-0.2 -0.4 0.1

-0.1 -0.4 0.3

-0.1

-0.8

-0.3

-0.2

-1.0

-1.0

-0.5

-0.6

0.3

0.0

0.0

0.1

x

0.4

0.7

0.9

-0.1 -0.4

-0.2 0.3

-0.2 1.3

-0.1 0.8

0.0 0.1

-0.2 -0.3

-1.4 0.4

-1.5 0.0

-1.3

-1.1

-1.0

-0.5

-0.2

-0.2

-0.4

0.3

1.3

0.7

0.0

0.3

0.8

-0.9

-0.9

-0.8

0.3

0.3

-0.7

-0.7

0.4

x

0.1

-0.2

Dihydropyrimidinase-related protein 2 Caspase-14 Death-associated protein-like 1 Eukaryotic translation initiation factor 3 subunit B Lactoylglutathione lyase Platelet-activating factor acetylhydrolase IB subunit alpha


Tholen et al., Fig. 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A


A17ΔKC: P3 à P10

B

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EgfrΔKC: P3 à P10

C


Page 41 of 44


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A


B

C



Tholen et al., Fig. 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

Page 42 of 44

B

IQR: 0.81 0.80

C

D

E Protein name tRNA (guanine-N(7)-)-methyltransferase ELAV-like protein 1 Potassium channel tetramerisation domain containing 18 Huntingtin-interacting protein K Exocyst complex component 6B

(Pseudo-) non-prime Prime sequence (experimentally sequence (from determined) database) MM AGAEAPQPQKR MSNGYEDHM AEDCRDDIGR MLKAELQASKGAM AGHEAEDVLDILR MRRRGEIEM ATEGDVELELETETSGPERPPEKPR MERAKM AEESLETAAEHER


Position of detected, acetylated N-termini 2 9 13 9 6

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A Fold change (log2 Fold change (log2 Non-prime sequence Prime sequence Cleavage site indicated by ↓ ΔKC ΔKC (from database) (experimentally determined) of A17 /WT ratio) of A17 /WT (at position) in replicate 1 ratio) in replicate 2 ↓ (976, 1124, 1202, 1286, RSPVHPESSE GEEHSVIPQR 1.3 1.3 1364, 1440, 1518, 1598, 1674, 1752, 1832, 1910, 1988) GQSNGFGENE SGPDQEGYQQR 1.0 0.9 ↓ (929) GRQQRMGSSH SSCCGPYGSGATQSSGCGQQR 1.3 1.3 ↓ (373) ↓ (1020, 1176, 1256, 1337, RPEVPTQDSS RQPQADQGQPSQSGSGR 0.6 6.8 1487, 1567, 1644, 1722, 1802, 1880) GNSTGFGEHG SSSHPLPSSGQNESSSGQSSR 0.9 1.5 ↓ (655) ↓ (967, 1197, 1277, 1358, QSSSGRSPRR SPVHPESSEGEEHSVVPQR 1.0 0.8 1431, 1509, 1589, 1665, 1743, 1823, 1901, 1979)

B



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For TOC only

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Skin barrier homeostasis ACS Paragon Plus Environment

Skin Barrier Defects Caused by Keratinocyte-Specific Deletion of

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