New Role for LEKTI in Skin Barrier Formation: Label-Free

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New Role for LEKTI in Skin Barrier Formation: Label-Free Quantitative Proteomic Identification of Caspase 14 as a Novel Target for the Protease Inhibitor LEKTI Kate Bennett,† Robin Callard,† Wendy Heywood,† John Harper,† Arumugam Jayakumar,‡ Gary L.Clayman,‡ Wei-Li Di,† and Kevin Mills*,† Institute of Child Health & Great Ormond Street Hospital for Sick Children, University College London, 30 Guilford Street, London, WC1N 1EH, and Department of Head and Neck Surgery, UTMD Anderson Cancer Centre, 1515 Holcombe Blvd, Houston, Texas, 77030 Received April 15, 2010

Abstract: Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is recognized as a serine protease inhibitor and is thought to play a key role in skin barrier function through the inhibition of kallikrein (KLK) activities and regulation of skin desquamation. LEKTI has a total of 15 potential inhibitory domains, and we hypothesize that it has other potential targets in the skin. To identify candidate protease targets of LEKTI, a label-free quantitative proteomic approach was employed. This work describes a novel, rapid, and noninvasive method for the identification and quantitation of the major proteins present in the uppermost layers of the skin. By using cells scraped from the elbow, we were able to rapidly identify and quantitate 79 proteins. Caspase 14 and bleomycin hydrolase were identified as the proteases of highest abundance. Despite the fact that caspase 14 is a cysteine protease and LEKTI is described as a serine protease inhibitor, we demonstrate that caspase 14 is inhibited by full-length LEKTI and 5 recombinant fragments of LEKTI to varied extents. Details of the development of the methods used for the creation of the skin proteome and the inhibition of caspase 14 by LEKTI and implications for LEKTI as a multifunctional protease inhibitor are discussed. Keywords: skin proteome • caspase 14 • LEKTI • labelfree quantitative mass spectrometry • cysteine protease

Introduction LEKTI is a Kazal-type protease inhibitor expressed in the most differentiated layers of stratified epithelial tissues including the skin, tonsil, thymus, vagina and esophagus.1 In the epidermis, LEKTI expression has been localized to the uppermost layers, including the granular layer and stratum corneum.2,3 LEKTI is synthesized in its full-length form, which contains a total of 15 domains and is thought to be cleaved rapidly into a range of single or multidomain fragments that are secreted from the cell.2-5 Recessive mutations in SPINK5, * To whom correspondence should be addressed. Dr Kevin Mills, Tel +44207-905-2873e-mail: [email protected]. † University College London. ‡ UTMD Anderson Cancer Centre. 10.1021/pr1003467

 2010 American Chemical Society

the gene that encodes LEKTI, have been identified as the cause of Netherton syndrome,6 a chronic skin disease characterized by loss of skin barrier function.7-9 Polymorphisms in the SPINK5 gene have also been found to be associated with atopic dermatitis, a disease that shares several clinical features with Netherton syndrome including skin barrier dysfunction.10-12 LEKTI is recognized as a serine protease inhibitor by its ability to inhibit several serine proteases in vitro, including members of the KLK family.5,13-16 KLKs belong to a subgroup of serine proteases, many of which colocalize with LEKTI in the uppermost layers of the skin.2,3,17 KLKs play an essential role in skin desquamation through the degradation of corneodesmosomal proteins in the upper stratum corneum layer. In Netherton syndrome, where LEKTI expression is either absent or reduced, KLK5- and KLK7-like activities are increased, which leads to premature degradation of corneodesmosomes, overdesquamation and stratum corneum thinning.3,17,18 Overall, these observations have implicated an essential role for LEKTI in the regulation of skin desquamation and skin barrier function through the inhibition/control of KLK activities.5,16,19 The high number of potential inhibitory domains that LEKTI contains suggests that it may have multiple targets in the skin. The P1-Arg residues that reside within many inhibitory domains of LEKTI have been used to predict the interaction of LEKTI with trypsin-like KLKs.16 Interestingly, the P1 site of LEKTI domain 2 has been identified as an aspartic acid residue,16 which corresponds to the substrate specificity of proteases other than KLKs. This opens up the possibility that LEKTI also inhibits other families of proteases. In order to identify novel candidate targets of LEKTI we developed a mass spectrometrybased technique, which allowed the identification and quantitation of all major proteins in the epidermis. Several candidate proteases were identified, and the inhibitory activity of fulllength LEKTI and various LEKTI fragments were tested against these proteases. By this approach, caspase 14 was identified as a target for LEKTI.

Materials and Methods In Solution Digestion of Whole Skin Biopsy. Excess skin samples from 5 patients attending the plastic surgery unit for ear pinning procedures were snap frozen and stored at -80 °C. The dermis was removed and the epidermis washed with 3 × 1 mL of ice-cold phosphate buffered saline solution, to Journal of Proteome Research 2010, 9, 4289–4294 4289 Published on Web 06/09/2010

technical notes remove all contaminating blood proteins. Proteins were prepared for analysis by homogenizing the biopsy on ice, in 500 µL of 100 mM Tris-HCl, pH 7.2 containing 8 M urea and 5% ASB-14 detergent. The homogenate was then transferred to an Eppendorf vessel and shaken at 4 °C for 4 h. Residual and insoluble cellular debris were removed by centrifugation (10 000× g for 5 min) and protein levels determined in the supernatant using the bicinchoninic acid protein assay. An aliquot containing 400 µg equivalent of protein was taken and the protein precipitated by adding 3 volumes of ice-cold acetone and leaving for 12 h at -20 °C. The protein was recovered by centrifugation (10 000× g for 15 min), the supernatant discarded and the pellet lyophilized by freezedrying. Proteins were digested according the protocol developed for the skin scrapings below. In Solution Digestion of Skin Scrapings. One to 5 mg of skin cells (keratinocytes) were collected and combined from the elbow regions of 5 males and 5 female lab staff volunteers by gentle scraping and collection using Dermapak 3 skin kits (Dermaco Ltd., Bedfordshire, U.K.). Keratinocytes were transferred to 1.8 mL microcentrifuge vials and stored at -80 °C until analyzed. Keratinocytes were prepared for digestion by dissolving in 40 µL of 100 mM Tris, pH 7.8, containing 6 M urea, 5% ASB14 and shaking gently overnight at room temperature. Disulfide bridges were reduced by the addition of 3 µL of 100 mM Tris-HCL, pH 7.8 containing 5 M DTE and incubated at room temp for 60 min. Free thiol groups were carboamidomethylated followed by incubation with 6 µL of 100 mM TrisHCL, pH 7.8 containing 5 M iodoacetamide. The solution was then diluted with H2O to a final volume of 400 µL, vortexed and 2 µg of sequence grade trypsin added to the solution. Samples were incubated overnight at 37 °C in water bath. LC-MS/MS (ESI-QTOF MS). Prior to analysis each digest was spiked with 5 pmol of enolase (Saccharomyces cerevisiae). Automated quantitation of each protein was made using the label-free quantitation principle that the responses of the three biggest precursor peptide masses observed from the tryptic digest of any protein are proportional to the amount of protein present in the sample. Proteins amounts were quantitated by comparing the response of the largest peptides from each protein identified with those peptides of the enolase internal standard. Skin cell proteins were identified and quantitated by direct analysis of the reaction mixture described above. All analyses were performed using a nanoAcquity HPLC and QTOF Premier mass spectrometer (Waters Corporation, Manchester, U.K.). Peptides were trapped and desalted prior to reverse phase separation using a Symmetry C18 5 µm, 5 mm × 300 µm precolumn. Peptides were then separated prior to mass spectral analysis using a 15 cm × 75 µm C18 reverse phase analytical column. Peptides were loaded onto the precolumn at a flow rate of 4 µL/min in 0.1% formic acid for a total time of 4 min. Peptides were eluted off the precolumn and separated on the analytical column using a gradient of 3-40% acetonitrile [0.1% formic acid] over a period of 90 min and at a flow rate of 300 nL/min. The column was washed and regenerated at 300 nL/min for 10 min using a 99% acetonitrile [0.1%] rinse. After all nonpolar and non peptide material were removed the column was re-equilibrated at the initial starting conditions for 20 min. All column temp were maintained at 35 °C. Mass accuracy was maintained during the run using a lock spray of the peptide [glu1]-fibrinopeptide B delivered through the auxiliary pump of the nanoAcquity at a concentration of 300 fmol/L and at a flow rate of 300 nL/min. Peptides were 4290

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Bennett et al. analyzed in positive ion mode using a Q-Tof Premier mass spectrometer (Waters Corp., Manchester, U.K.) and was operated in v-mode with a typical resolving power of 10 000 fwhm. Prior to analyses, the TOF analyzer was calibrated using [glu1]fibrinopeptide B fragments obtained using a collision energy of 25 V and over the mass range 50-2000 m/z. Post calibration of data files were corrected using the doubly charged precursor ion of [glu1]-fibrinopeptide B (785.8426 m/z) with a sampling frequency of 30 s. Accurate mass LC-MS data were collected in a data independent and alternating, low and high collision energy mode. Each low/high acquisition was 1.5 s with 0.1 s interscan delay. Low energy data collections were performed at a constant collision energy of 4 V, high collision energy acquisitions were performed using a 15-40 V ramp over a 1.5 s time period and a complete low/high energy acquisition achieved every 3.2 s. Data Analysis of Skin Samples Analyzed by QTOF MS. ProteinLynx GlobalServer version 2.4 was used to process all data acquired. Protein identifications were obtained by searching UniProt/Swiss-Prot databases to which the sequence of yeast enolase was added manually. Protein identification from the low/high collision spectra for each sample was processed using a hierarchical approach where more than three fragment ions per peptide, seven fragment ions per protein and more than two peptides per protein had to be matched. Protein identification parameters used in the database searching included a 93% of the total proteins. The most abundant protein identified was keratin, type 1 cytoskeletal cytokeratin 10 and comprised approximately 13% of the total proteins. This corresponds to the fact that keratin 10 is one of the most predominant cytoskeletal intermediate filaments of epidermal cells during terminal differentiation20 and demonstrates the label-free quantitative proteomic method as an effective tool for skin proteomic analyses. The next most abundant class of molecules detected were the junction proteins including desmoglein (0.4%), desmoplakin (0.37%), plakoglobin (0.33%), desmocollin (0.22%) and corneodemosin (0.2%). Figure 2b shows a similar schematic representation of the proteins making up the uppermost layers of the epidermis but with the higher abundant keratin proteins removed from the analyses. Interestingly, no proteins were detected that are considered to be the major classes of proteins that play a role in differentiation of the epidermis i.e. filaggrin, LEKTI or any kallikreins. No serine proteases were detected and the only proteases identified were the cysteine proteases bleomycin hydrolase (0.14%) and caspase 14 (0.09%) (Figure 2c). Inhibition of Caspase 14 using LEKTI. The presence of high levels of caspase 14 in the epidermal biopsy material and skin

scrapings and the presence of a highly favorable aspartic acid residue in the P1 site of LEKTI domain 2, led us to hypothesize that LEKTI may have the potential to inhibit caspase 14. The ability of full-length rLEKTI and four rLEKTI fragments to inhibit caspase 14 was investigated. The rLEKTI fragments rLEKTI 1-6, rLEKTI 6-9′, rLEKTI 9-12 and rLEKTI 12-15 used in this study span all fifteen domains of LEKTI and are numbered according to the domains they contain. The amount of caspase 14 activity that was inhibited by each rLEKTI form was expressed using % decreases in A405 nm, relative to the uninhibited caspase 14 control, which was assumed to represent 0% inhibition (Figure 3). In the presence of an 8-fold molar excess of LEKTI, full-length rLEKTI demonstrated almost complete inhibition (93.4%) of caspase 14 activity. rLEKTI 1-6, rLEKTI 6-9′ and rLEKTI 9-12 all exerted strong inhibitory effects on caspase 14, inhibiting 63.6, 62 and 66.5% activity, respectively (Figure 3). Although rLEKTI 12-15 was found to be the least potent inhibitor of caspase 14, this rLEKTI form was still able to inhibit 42% activity. The plots of time versus A405 nm remained linear for each rLEKTI form, which suggested that the inhibition of caspase 14 by each rLEKTI form remained constant over the assay period. To confirm that the inhibition of caspase 14 by LEKTI was specific, the serine protease inhibitor alpha-1 antitrypsin, which was assumed not to inhibit caspase 14 in vitro, was used as a negative control (Figure 4). A405 nm values measured over a specific time period were similar to those observed for uninhibited caspase 14 and confirmed that alpha-1 antitrypsin did not inhibit caspase 14 activity. This suggested that the inhibition of caspase 14 by LEKTI represented specific and true inhibition and postulated caspase 14 as a novel target of LEKTI in the skin. Journal of Proteome Research • Vol. 9, No. 8, 2010 4291

technical notes

Bennett et al.

Figure 3. Inhibition of caspase 14 by rLEKTI 1-6, rLEKTI 6-9′, rLEKTI 9-12, rLEKTI 12-15 and full-length rLEKTI. A plot of time versus proportional increase of absorbance at 405 nm (A405 nm), representing the hydrolysis of a pNA substrate (Ac-WEHD-pNA) by caspase 14 in the presence of rLEKTI. rLEKTI forms are numbered according to the domains they contained. Uninhibited caspase 14 was used as a positive control.

Figure 4. Inhibition of caspase 14 by R1-antitrypsin (R1AT). The plots of time versus proportional increase of absorbance at 405 nm, representing the hydrolysis of a pNA substrate (Ac-WEHDpNA) by caspase 14 in the presence of R1AT.

Figure 2. Schematic representation of the percentage of proteins identified in skin scrapings (% of total fmol of protein). (a) Major proteins identified in the outermost layers of the skin. (b and c) Enlargement of the lesser abundant proteins in the regions marked in red and blue boxed/highlighted areas (a and b, respectively).

Discussion The label-free quantitative proteomic method developed in this study using LC-QTOF MS has proved to be an effective tool for the identification and quantitation of multiple proteins from epidermal biopsy material and skin scrapings. This has provided a much more rapid and sensitive alternative to conventional methods that require preseparation of proteins by gel electrophoresis. Traditional gel-based proteomics such as two-dimensional difference gel electrophoresis (2D-DIGE) are relatively ineffectual in the analysis of skin proteins because 4292

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of the extreme hydrophobic nature of the proteins and their extensive cross-linking to lipids and other proteins present in the epidermis. By using a mass spectrometry-based approach whereby all proteins were reduced to peptides for identification/quantitation, it was possible to overcome these technical difficulties. The proteins identified that provoked the most interest were the proteases, bleomycin hydrolase and caspase 14. Bleomycin hydrolase was discovered only in the skin scraping analyses whereas caspase 14 was identified in both skin scraping and the whole epidermal analyses. It was assumed that skin scrapings would only include the uppermost layers of the epidermis, which may suggest that bleomycin hydrolase is only present in these layers. The label-free quantitative method used in this study was slightly biased toward the identification of higher abundant proteins, which may explain why proteins such as filaggrin and involucrin, which are known to be present in the uppermost layers of the epidermis, were not identified. As a result, the detection of bleomycin hydrolase and caspase 14 and the absence of any kallikreins in these analyses indicate that cysteine proteases are present in higher abundance in the epidermis and may play a more important role in skin barrier function. They could also prove to be potential drug targets for therapy in Netherton syndrome and other skin disorders. These hypotheses are strengthened by the finding that caspase 14 can be inhibited by all forms of rLEKTI to various extents, with full-length rLEKTI demonstrating almost complete inhibition. Unfortunately, at the time of writing no commercial source of bleomycin hydrolase was available so we were unable to test the inhibitory capacity of LEKTI against this protease.

technical notes

New Role for LEKTI in Skin Barrier Formation The P1-site residue of an inhibitor can often be used to predict potential protease targets, based upon the P1 preference of the target. In 12 LEKTI domains the P1 residue has been identified as arginine (D3-14) and in one domain (D15) identified as lysine. This led to the assumption that trypsinlike proteases, such as the KLKs, which have strong preferences for P1-Arg or -Lys sites, were potential primary targets of LEKTI.16 Although caspase 14 has a strong preference for an aspartic acid at the P1 site, it was still found to be inhibited by LEKTI in this study. The P1 site of LEKTI domain 2 has been identified as an aspartic acid residue,16 which is consistent with rLEKTI 1-6 inhibition of caspase 14. However, rLEKTI 6-9′, rLEKTI 9-12 and rLEKTI 12-15 do not contain domain 2, but still inhibited caspase 14, suggesting that the presence of an aspartic acid in the P1 site of LEKTI was not a necessary prerequisite for effective inhibition. As this is the first study to report the inhibitory activity of LEKTI against a cysteine protease, it opens up the possibility that LEKTI acts not only as a serine protease inhibitor but also as a cysteine protease inhibitor, with LEKTI being a multifunctional protease inhibitor in the skin and elsewhere in the body. Caspase 14 is a member of the caspase family, which includes a group of cysteine proteases that cleave target proteins at specific aspartate residues.21 To date, 15 caspases have been reported (1-15), 9 of which have been detected in the human epidermis (caspase-1, -2, -3, -4, -6, -7, -9, -10 and -14).22 Caspase 14 is believed to be a nonapoptotic caspase, expressed mainly in the epidermis and is the only caspase that has been shown to be activated consistently during epidermal cornification22-24 suggesting role for caspase 14 in terminal keratinocyte differentiation.25 Caspase 14 has also been found to be involved in the processing and degradation of filaggrin fragments into free hygroscopic amino acids, which contribute to approximately 40% of the natural moisturising factors present in the stratum corneum.25,26 Therefore, the finding that LEKTI inhibits caspase 14 in vitro may implicate a novel regulatory role for LEKTI in epidermal hydration through the inhibition of caspase 14 activity. For future work, it would be interesting to investigate whether LEKTI is able to prevent the processing of filaggrin by caspase 14. Although the primary goal of this work was to identify potential targets of LEKTI, a more extensive study to create a skin proteome could prove beneficial in the study of the pathophysiology of disease and aging. A study of this format would need to consider such factors such as person-to-person variation, gender, ethnicity, changes in skin protein expression with age, body site variation and more importantly those changes associated with disease (atopic dermatitis, psoriasis etc.). This is possible by the use of skin scrapings for the identification of epidermal proteins. Conventional punch biopsies are highly invasive and unsuitable for visible areas of the body. The use of skin scrapings has provided an alternative noninvasive sampling method for proteomic analyses and will provide the opportunity to perform quantitative MS analyses from multiple sites on the body, to assess both intra- and interpersonal variation. In summary, caspase 14 has been implicated as a novel target of LEKTI. This work indicates a more significant role of cysteine proteases in the formation of the skin barrier than previously reported and also confirms that LEKTI has the potential to act as both a serine and a cysteine protease inhibitor. It also highlights the huge potential for mass spectrometry-based proteomics in the study of skin analyses.

Acknowledgment. We thank the National Eczema Society for the funding of this research. Thanks also go to Professor Bryan Winchester for his time, advice and knowledge regarding the inhibitory kinetics. We also thank Dr. Jonathon Fox and Dr. James Langridge for their advice and help in the label-free quantitative proteomic analyses. Supporting Information Available: The data has been tabulated to include the amount of sequence coverage obtained for each identified protein and the quantities of each protein detected in both nanograms and femtomoles. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Magert, H. J.; Standker, L.; Kreutzmann, P.; Zucht, H. D.; Reinecke, M.; Sommerhoff, C. P.; Fritz, H.; Forssmann, W. G. LEKTI, a novel 15-domain type of human serine proteinase inhibitor. J. Biol. Chem. 1999, 274 (31), 21499–21502. (2) Bitoun, E.; Micheloni, A.; Lamant, L.; Bonnart, C.; Tartaglia-Polcini, A.; Cobbold, C.; Al, S. T.; Mariotti, F.; Mazereeuw-Hautier, J.; Boralevi, F.; Hohl, D.; Harper, J.; Bodemer, C.; D’Alessio, M.; Hovnanian, A. LEKTI proteolytic processing in human primary keratinocytes, tissue distribution and defective expression in Netherton syndrome. Hum. Mol. Genet. 2003, 12 (19), 2417–2430. (3) Hachem, J. P.; Wagberg, F.; Schmuth, M.; Crumrine, D.; Lissens, W.; Jayakumar, A.; Houben, E.; Mauro, T. M.; Leonardsson, G.; Brattsand, M.; Egelrud, T.; Roseeuw, D.; Clayman, G. L.; Feingold, K. R.; Williams, M. L.; Elias, P. M. Serine protease activity and residual LEKTI expression determine phenotype in Netherton syndrome. J. Invest. Dermatol. 2006, 126 (7), 1609–1621. (4) Tartaglia-Polcini, A.; Bonnart, C.; Micheloni, A.; Cianfarani, F.; Andre, A.; Zambruno, G.; Hovnanian, A.; D’Alessio, M. SPINK5, the defective gene in netherton syndrome, encodes multiple LEKTI isoforms derived from alternative pre-mRNA processing. J. Invest. Dermatol. 2006, 126 (2), 315–324. (5) Deraison, C.; Bonnart, C.; Lopez, F.; Besson, C.; Robinson, R.; Jayakumar, A.; Wagberg, F.; Brattsand, M.; Hachem, J. P.; Leonardsson, G.; Hovnanian, A. LEKTI fragments specifically inhibit KLK5, KLK7, and KLK14 and control desquamation through a pHdependent interaction. Mol. Biol. Cell 2007, 18 (9), 3607–3619. (6) Chavanas, S.; Bodemer, C.; Rochat, A.; Hamel-Teillac, D.; Ali, M.; Irvine, A. D.; Bonafe, J. L.; Wilkinson, J.; Taieb, A.; Barrandon, Y.; Harper, J. I.; de, P. Y.; Hovnanian, A. Mutations in SPINK5, encoding a serine protease inhibitor, cause Netherton syndrome. Nat. Genet. 2000, 25 (2), 141–142. (7) Yang, T.; Liang, D.; Koch, P. J.; Hohl, D.; Kheradmand, F.; Overbeek, P. A. Epidermal detachment, desmosomal dissociation, and destabilization of corneodesmosin in Spink5-/-mice. Genes Dev. 2004, 18 (19), 2354–2358. (8) Descargues, P.; Deraison, C.; Bonnart, C.; Kreft, M.; Kishibe, M.; Ishida-Yamamoto, A.; Elias, P.; Barrandon, Y.; Zambruno, G.; Sonnenberg, A.; Hovnanian, A. Spink5-deficient mice mimic Netherton syndrome through degradation of desmoglein 1 by epidermal protease hyperactivity. Nat. Genet. 2005, 37 (1), 56–65. (9) Hewett, D. R.; Simons, A. L.; Mangan, N. E.; Jolin, H. E.; Green, S. M.; Fallon, P. G.; McKenzie, A. N. Lethal, neonatal ichthyosis with increased proteolytic processing of filaggrin in a mouse model of Netherton syndrome. Hum. Mol. Genet. 2005, 14 (2), 335–346. (10) Walley, A. J.; Chavanas, S.; Moffatt, M. F.; Esnouf, R. M.; Ubhi, B.; Lawrence, R.; Wong, K.; Abecasis, G. R.; Jones, E. Y.; Harper, J. I.; Hovnanian, A.; Cookson, W. O. Gene polymorphism in Netherton and common atopic disease. Nat. Genet. 2001, 29 (2), 175–178. (11) Kato, A.; Fukai, K.; Oiso, N.; Hosomi, N.; Murakami, T.; Ishii, M. Association of SPINK5 gene polymorphisms with atopic dermatitis in the Japanese population. Br. J. Dermatol. 2003, 148 (4), 665– 669. (12) Nishio, Y.; Noguchi, E.; Shibasaki, M.; Kamioka, M.; Ichikawa, E.; Ichikawa, K.; Umebayashi, Y.; Otsuka, F.; Arinami, T. Association between polymorphisms in the SPINK5 gene and atopic dermatitis in the Japanese. Genes Immun. 2003, 4 (7), 515–517. (13) Mitsudo, K.; Jayakumar, A.; Henderson, Y.; Frederick, M. J.; Kang, Y.; Wang, M.; El-Naggar, A. K.; Clayman, G. L. Inhibition of serine proteinases plasmin, trypsin, subtilisin A, cathepsin G, and elastase by LEKTI: a kinetic analysis. Biochemistry 2003, 42 (13), 3874–3881. (14) Egelrud, T.; Brattsand, M.; Kreutzmann, P.; Walden, M.; Vitzithum, K.; Marx, U. C.; Forssmann, W. G.; Magert, H. J. hK5 and hK7, two serine proteinases abundant in human skin, are inhibited by LEKTI domain 6. Br. J. Dermatol. 2005, 153 (6), 1200–1203.

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technical notes (15) Schechter, N. M.; Choi, E. J.; Wang, Z. M.; Hanakawa, Y.; Stanley, J. R.; Kang, Y.; Clayman, G. L.; Jayakumar, A. Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lymphoepithelial Kazal-type inhibitor (LEKTI). Biol. Chem. 2005, 386 (11), 1173–1184. (16) Borgono, C. A.; Michael, I. P.; Komatsu, N.; Jayakumar, A.; Kapadia, R.; Clayman, G. L.; Sotiropoulou, G.; Diamandis, E. P. A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation. J. Biol. Chem. 2007, 282 (6), 3640–3652. (17) Komatsu, N.; Takata, M.; Otsuki, N.; Ohka, R.; Amano, O.; Takehara, K.; Saijoh, K. Elevated stratum corneum hydrolytic activity in Netherton syndrome suggests an inhibitory regulation of desquamation by SPINK5-derived peptides. J. Invest. Dermatol. 2002, 118 (3), 436–443. (18) Descargues, P.; Deraison, C.; Prost, C.; Fraitag, S.; MazereeuwHautier, J.; D’Alessio, M.; Ishida-Yamamoto, A.; Bodemer, C.; Zambruno, G.; Hovnanian, A. Corneodesmosomal cadherins are preferential targets of stratum corneum trypsin- and chymotrypsin-like hyperactivity in Netherton syndrome. J. Invest. Dermatol. 2006, 126 (7), 1622–1632. (19) Caubet, C.; Jonca, N.; Brattsand, M.; Guerrin, M.; Bernard, D.; Schmidt, R.; Egelrud, T.; Simon, M.; Serre, G. Degradation of corneodesmosome proteins by two serine proteases of the kallikrein family, SCTE/KLK5/hK5 and SCCE/KLK7/hK7. J. Invest. Dermatol. 2004, 122 (5), 1235–1244. (20) Kartasova, T.; Roop, D. R.; Holbrook, K. A.; Yuspa, S. H. Mouse differentiation-specific keratins 1 and 10 require a preexisting keratin scaffold to form a filament network. J. Cell Biol. 1993, 120 (5), 1251–1261.

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Bennett et al. (21) Fischer, H.; Stichenwirth, M.; Dockal, M.; Ghannadan, M.; Buchberger, M.; Bach, J.; Kapetanopoulos, A.; Declercq, W.; Tschachler, E.; Eckhart, L. Stratum corneum-derived caspase-14 is catalytically active. FEBS Lett. 2004, 577 (3), 446–450. (22) Raymond, A. A.; Mechin, M. C.; Nachat, R.; Toulza, E.; Tazi-Ahnini, R.; Serre, G.; Simon, M. Nine procaspases are expressed in normal human epidermis, but only caspase-14 is fully processed. Br. J. Dermatol. 2007, 156 (3), 420–427. (23) Eckhart, L.; Declercq, W.; Ban, J.; Rendl, M.; Lengauer, B.; Mayer, C.; Lippens, S.; Vandenabeele, P.; Tschachler, E. Terminal differentiation of human keratinocytes and stratum corneum formation is associated with caspase-14 activation. J. Invest Dermatol. 2000, 115 (6), 1148–1151. (24) Lippens, S.; Kockx, M.; Knaapen, M.; Mortier, L.; Polakowska, R.; Verheyen, A.; Garmyn, M.; Zwijsen, A.; Formstecher, P.; Huylebroeck, D.; Vandenabeele, P.; Declercq, W. Epidermal differentiation does not involve the pro-apoptotic executioner caspases, but is associated with caspase-14 induction and processing. Cell Death. Differ. 2000, 7 (12), 1218–1224. (25) Denecker, G.; Ovaere, P.; Vandenabeele, P.; Declercq, W. Caspase14 reveals its secrets. J. Cell Biol. 2008, 180 (3), 451–458. (26) Denecker, G.; Hoste, E.; Gilbert, B.; Hochepied, T.; Ovaere, P.; Lippens, S.; Van den, B. C.; Van, D. P.; D’Herde, K.; Hachem, J. P.; Borgonie, G.; Presland, R. B.; Schoonjans, L.; Libert, C.; Vandekerckhove, J.; Gevaert, K.; Vandenabeele, P.; Declercq, W. Caspase14 protects against epidermal UVB photodamage and water loss. Nat. Cell Biol. 2007, 9 (6), 666–674.

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