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2004). KLK5 degrades DSG1, causing When Activity Requires Breaking Up: abnormal detach- ment, leading to a severe skin barrier LEKTI Proteolytic Activation Cascade defect that favors increased allergen penetration in the skin. In parallel, for Specific Proteinase Inhibition unrestrained KLK5 activity triggers an Laetitia Furio1,2 and Alain Hovnanian1,2,3 autonomous inflammatory cascade by activating PAR-2 signaling, resulting Lymphoepithelial Kazal-type related inhibitor (LEKTI) is a multidomain in the production of major pro-inflam- proteinase inhibitor whose defective expression causes matory molecules and pro–T helper (NS). LEKTI is encoded by SPINK5, which is also a susceptibility for atopic type 2 (Th2) cytokines such as thymic disease. In this issue, Fortugno et al. report an elegant and thorough study of the stromal lymphopoietin (TSLP). This LEKTI proteolytic activation process in which they identify the precise nature of specific KLK5–PAR2–TSLP pathway, in the cleavage sites used and the bioactive fragments generated. They propose addition to producing thymus and acti- a proteolytic activation model in human skin and confirm differential inhib­ vation-regulated chemokine and mac- ition of (KLK) 5, 7, and 14 by the major physiological LEKTI fragments. rophage-derived chemokine, IL-1, and They show that these bioactive fragments inhibit KLK-mediated proteolysis of tumor necrosis factor-α, contributes to desmoglein 1 (DSG1) and suggest a fine-tuned inhibition process controlling the creation of a pro-inflammatory and target serine proteinase (SP) activity. pro-allergic Th2 microenvironment Journal of Investigative Dermatology (2011) 131, 2169–2173. doi:10.1038/jid.2011.295 that predisposes NS patients to severe skin allergy, asthma, and food allergy (Bonnart et al., 2010; Briot et al., 2009, 2010; Fontao et al., 2011). Proteases and their inhibitors as in the control of protease activity. In essential actors in skin desquamation the , they include LEKTI LEKTI isoform structure, transport, and inflammation: loss of control in NS (or LEKTI-1), LEKTI-2, SPINK6, skin-­ and secretion Proteinases and regulation of their activ- derived anti­leukoprotease (SKALP/ LEKTI cDNA was cloned by Magert et ity play important roles in many aspects ), secre­tory leukocyte protease al. from D1 and D6 fragments isolated of skin biology and function, such inhibitor, bikunin, hurpin, other SP from blood ultrafiltrates (Magert et al., as epidermal differentiation, barrier inhibitors (), and cystatins 1999). LEKTI is encoded by SPINK5, formation and shedding, inflamma- (Brattsand et al., 2009; Magert et al., which maps to 5q33.1 tion, immune responses, host defense, 2005; Meyer-Hoffert, 2009; Zeeuwen, within a gene family cluster includ- wound healing, and hair formation 2004). ing SPINK6 and SPINK9, encoding (Meyer-Hoffert, 2009; Ovaere et al., The importance of a tight control of the single-domain inhibitors SPINK6 2009). Human tissue (KLKs) protease activity has been dramatical- and LEKTI-2, respectively (Brattsand are secreted SPs with (chymo)- ly illustrated in NS, a rare and severe et al., 2009; Meyer-Hoffert, 2009; like specificity that comprise 15 mem- genetic skin disease in which loss of Meyer-Hoffert et al., 2010). SPINK5 bers, several of which are expressed balance between LEKTI, a SP inhibitor, consists of 33 exons spread over 61 in the skin (Borgoño et al., 2007). In and its target proteinases leads to a pro- kb, encoding 15 domains preceded particular, KLK5, KLK7, and KLK14 are found skin barrier defect with severe by a peptide signal and interspaced by involved in the desquamation of super- inflammatory and allergic manifesta- linker regions. The LEKTI pri- ficial layers of corneocytes by degrad- tions (Chavanas et al., 2000; Bitoun mary structure shows that each domain ing corneodesmosomal components. et al., 2002; Deraison et al., 2007; shares a Kazal-type related motif found They also activate protease-activated Descargues et al., 2006). Studies in NS in SP inhibitors. In fact, only domains 2 receptor-2 (PAR-2), resulting in the patients and in murine models have and 15 contain the classic Kazal motif production of inflammatory cyto- shown that LEKTI deficiency results in defined by the presence of 6-cysteine kines (Brattsand et al., 2005; Hachem unopposed KLK5 activity, leading to residues with precise spacing; other et al., 2006; Komatsu et al., 2005). a dual mechanism (Descargues et al., domains exhibit only a 4-cysteine Protease inhibitors are essential actors 2005; Hewett et al., 2005; Yang et al., pattern. This allows the formation of two or three intramolecular bonds, resulting in a rigid reactive cen- ter loop that mimics the substrate and 1INSERM U781, Paris, France; 2Université Paris V René Descartes, Paris, France and 3Department of Genetics and Department of Dermatology, Necker Hospital, Paris, France traps the target protease. Each LEKTI Correspondence: Alain Hovnanian, Hôpital Necker-Enfants Malades, Service de Génétique, Tour Lavoisier, domain, except for D1, D2, and D15, 3ème étage, 149 rue de Sèvres, 75743 Paris Cedex 15, France. E-mail: [email protected] shares an arginine residue at position

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LEKTI proteolytic activation cascade generates a wide variety of bioactive fragments In this issue, Fortugno et al. report the results of a careful and exhaustive study of LEKTI proteolytic processing aimed at identifying the precise nature of the generated LEKTI polypeptides. The investigators combined antibody map- ping, N-terminal sequencing, and spe- cific mutagenesis of predicted cleavage sites to identify precisely the cleavage sites used and the corresponding LEKTI fragments generated. Pro-LEKTI proteo- lytic processing was first studied in nor- mal human epidermis, in differentiated normal human keratinocytes (NHKs), and in human embryonic kidney cells transfected with an expression plas- mid containing the cDNA encoding for Figure 1. model of LEKTI proteolytic activation cascade in human keratinocytes and inhibitory LEKTI-FL, LEKTI-L, and LEKTI-Sh. The properties of physiological fragments. The three LEKTI isoforms—LEKTI-FL, LEKTI-L, and LEKTI-Sh—are resulting proteolytic patterns showed represented (see text for their description). The four arginine residues used for cleavage by are bands of 65/68, 42, 37, 30, and 23 indicated. The three isoforms are cleaved at Arg 355 to generate D6–D15-FL (102 kDa), D6–D15-L (105 kDa), or D10–D13-Sh (79 kDa) fragments, which are rapidly cleaved at Arg625 to produce fragments kDa, comparable to those reported D10–D15-FL (65 kDa), D10–D15-L (68 kDa), and D10–D13-Sh (42 kDa), as well as fragment D6–D9 by Deraison et al. (2007). The 68-kDa (37 kDa), which is common to the three isoforms. D6–D9 is subsequently cleaved into D6 (7 kDa) and band seen with LEKTI-L corresponds to D7–D9 (30 kDa) following cleavage at Arg 425. D7–D9 is further cleaved at Arg 489 to generate D7 (7 the extra 30-amino-acid residues of this kDa) and D8–D9 (23 kDa). Proteolytic processing of fragment D1–D5 was not included in this study isoform in linker region 13. Fragments and is not yet fully understood. D1 and D5 are represented as single domains because they, like D6, of 102, 105, and 79 kDa were also were identified from human blood ultrafiltrates (Magert et al., 1999). Additional proteolytic fragments detected in human embryonic kidney derived from D2–D4 are not represented. However, there is evidence that fragments of 20, 15, 13, 11, and 10 kDa are generated from this fragment, the identity of which remains to be established (Deraison cells transfected with LEKTI-FL, LEKTI-L, et al., 2007). Domains are color coded according to their inhibitory properties found in Fortugno et and LEKTI-Sh cDNA, respectively, and al. (this issue, 2011) for C-terminal fragments (D6–D9, D10–D13, D10–D15, and derived fragments). in keratinocytes. These fragments corre- For N-terminal fragments (D1–D5), D1–D6 was previously shown to inhibit strongly KLK5 and KLK14 spond to an intermediate form not seen (Borgoño et al., 2007), D5 inhibits KLK5 and KLK14 (Deraison et al., 2007), and D1 does not inhibit any in human epidermis, possibly attribut- of these KLKs. Note that, except for D6–D9, the physiological proteolytic fragments do not match able to rapid processing. LEKTI fragments previously investigated for inhibition studies. Pink, preferential and strong inhibition Fortugno et al. (2011) subsequently of KLK5 and KLK14; blue, preferential but weaker inhibition of KLK7; yellow, unknown or no inhibition of KLK5 or KLK7. LEKTI domains are represented as cylinders; linker regions are shown as curved black used in silico analysis to identify poten- lines between domains. The peptide signal sequence is indicated in green at the N-terminal end of the tial furin cleavage sites. Numerous molecules. K, classic Kazal-type domain (D2 and D15). potential sites were identified, but four arginine residues with the highest scores were selected for functional studies P1, which predicts activity against isoform is longer (LEKTI-L), carry- (Arg355, Arg425, Arg489, and Arg625). trypsin-like SPs (Goettig et al., 2010). ing a 30-amino-acid residue insertion Each of these sites was subsequently Linkers are arginine- and lysine-rich between the thirteenth and fourteenth mutated to inactivate them, and the sequences that represent putative domains. All three SPINK5 alternative effect on Pro-LEKTI proteolytic process- cleavage sites for convertin/- transcripts are translated into protein ing was analyzed in human embryonic like such as furin. in differentiated keratinocytes, with kidney cells transfected with the mutated Tartaglia-Polcini et al. have shown that LEKTI-FL being the most abundant. cDNA for LEKTI-FL. The 42-kDa LEKTI LEKTI is transcribed into three different LEKTI pro-protein is rapidly cleaved fragment was used as a prototype to vali- transcripts that differ through their car- intracellularly, generating a number of date this strategy. N-terminal sequencing boxy-terminal end by alternative pro- potentially bioactive fragments (Bitoun of the purified 42-kDa band showed that cessing (Tartaglia-Polcini et al., 2006). et al., 2003; Tartaglia-Polcini et al., it is generated from cleavage at Arg625 In addition to the 15-domain isoform 2006). LEKTI is transported in lamel- in linker region 9. Site-directed muta­ (LEKTI-FL), SPINK5 encodes a shorter lar granules, separated from KLK5 and genesis of Arg625 was subsequently LEKTI isoform (LEKTI-Sh), containing KLK7, and secreted at the granular used to confirm that the 65- and 68-kDa only the first 13 domains resulting from and stratum corneum interface (Ishida- fragments were generated from cleavage differential poly­adenylation. The third Yamamoto et al., 2005). at the same site. These results identified

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unambiguously the 65- and 68-kDa identified LEKTI fragments of 20, 15, of four overlapping rLEKTI multidomain fragments as being D10–D15 derived 13, 11, and 10 kDa, recognized by D1– fragments covering the entire molecule from LEKTI-FL and LEKTI-L, respectively, D6 antibodies from human epidermal against multiple KLKs involved in epi- and the 42-kDa band as corresponding extract and conditioned medium of cul- dermal desquamation. They found that to D10–D13 from LEKTI-Sh. tured NHK (Deraison et al., 2007). The rLEKTI D1–D6, rLEKTI D6–D9, and The identity of the other 37-, 30-, 11- and 10-kDa bands were proposed rLEKTI D9–D12 were potent inhibitors and 23-kDa fragments was determined to correspond to D1, D5, and/or D6 pre- of KLK5 and KLK14 and that they also by mutating the other three potential viously isolated from the serum, but the inhibited KLK6 and KLK13 to a lesser furin cleavage sites with the highest identity of the other bands remains to be extent. By contrast, rLEKTI D12–D15 scores (Arg355, Arg425, and Arg489 in determined. inhibited KLK5 only. This work expanded linker regions 5, 6, and 7, respectively) the spectrum of KLKs inhibited by LEKTI in the LEKTI-FL sequence. This strategy KLK5 and KLK14 as major target SPs fragments to KLK6 and KLK13, and KLK6 allowed the investigators to confirm the of LEKTI physiological fragments was shown to degrade DSG1. use of these cleavage sites for proteolytic Initial functional studies aimed at assess- processing and assigned the 37-, 30-, ing LEKTI’s inhibitory capacity were and 23-kDa bands to D6–D9, D7–D9, performed with a LEKTI full-length mol- Inhibitors of kallikrein- and D8–D9, respectively. As stated by ecule (Mitsudo et al., 2003). Protease- mediated proteolysis Fortugno et al. (2011), differences in the inhibition assays showed inhibition suggest fine-tuned composition of the 31-kDa LEKTI frag- of the SPs trypsin, , subtilisin inhibition and a potential ments proposed in Deraison et al. (2007) A, G, and human neutro- | are attributable to overestimation of the phil . The cysteine proteinases for new therapies. molecular weight of the D8–D9 frag- papain or cathepsin K, L, or S were not ment by Ahmed et al. (2001). inhibited. However, subsequent studies This study identified the furin-sensitive of rLEKTI fragments reported a differ- Fortugno et al. (2011) assessed the sites used during pro-LEKTI proteolytic ent spectrum of inhibition. Specifically, inhibitory properties of the LEKTI physi- processing, allowing the identification rLEKTI D6-D9 was shown to inhibit ological fragments that they identified of the LEKTI bioactive fragments gen- trypsin, subtilisin A, KLK5, and KLK7 against KLK5, KLK7, and KLK14. They erated. Fortugno et al. (2011) propose (Jayakumar et al., 2004; Schechter et al., found D6–D9 and derived multido- the following succession of cleavages 2005); rLEKTI D6 inhibits trypsin, KLK5, main fragments D7–D9 and D8–D9 to during the activation cascade in NHK and KLK7, but not subtilisin A (Egelrud be highly efficient inhibitors of KLK5 (Figure 1). The full-length protein (145 et al., 2005; Kreutzmann et al., 2004); and KLK14 but weak inhibitors of KLK7. kDa) is cleaved into a D6–D15 frag- rLEKTI D9–D12 inhibits KLK5, but not Conversely, they found D10–D15 to be a ment (102 kDa), which is rapidly pro- KLK7 (Schechter et al., 2005); and rLEKTI weak inhibitor of KLK5 and KLK14, but cessed to generate a D6–D9 fragment D15 inhibits trypsin and plasmin, but not that it inhibited KLK7 more efficiently. (37 kDa), a D10–D15 fragment (65 kDa), KLK5 or KLK7 (Egelrud et al., 2005). The Single domains (D6 and D7) showed and smaller amino-terminal fragments LEKTI inhibitory spectrum thus seems to weaker inhibition than corresponding not reported in this study. The D10–D15 be complex and highly dependent on multidomain fragments. fragment shows no evidence of subse- the domains considered. These results confirm the differential quent cleavage. By contrast, the D6–D9 Deraison et al. (2007) examined the inhibitory properties of LEKTI domains. fragment undergoes further proteolysis inhibition profiles of proposed physi- They also support the notion that KLK5 to release single domains (D6 and then ological LEKTI fragments against a and KLK14 are major targets of physi- D7), as well as the corresponding three- wide range of human SPs. All fragments ological LEKTI fragments, with D6–D9 (D7–D9) and two-domain (D6–D9) tested (D5, D6, D8–D11, and D9–D15) and its derived multidomain fragments fragments. The authors conclude that in inhibited KLK5, KL7, and KLK14 only. being much more potent KLK5 and normal keratinocytes and human epi- No inhibition was observed with the KLK14 inhibitors than D10–D15. These dermis, pro-LEKTI proteolytic processing human SPs KLK8, , , results are consistent with previous work results in the generation of a wide variety elastase, plasmin, KLK3, and . that identified D8–D11 as the most effec- of LEKTI polypeptides, which comprise D8–D11 showed the strongest inhibi- tive LEKTI fragment, suggesting that at least D6–D9, D7–D9, D8–D9, D6, tion properties toward KLK5 and KLK14, D8–D9 could account for this activity D7, D10–D15, and D10–D13 frag- whereas KLK7 was less efficiently inhib- (Deraison et al., 2007). Fortugno et al. ments, with the latter being generated ited. D5, D6, and D9–D15 were weaker (2011) also show that KLK7 is inhibited from LEKTI-Sh, which lacks D14 and inhibitors of KLK5, KLK7, and KLK14. to a lesser extent by D10–D15. However, D15 (Figure 1). From these studies, it appeared that the the fact that LEKTI is degraded by KLK7 Fortugno and colleagues mainly main target proteases of these LEKTI frag- suggests that LEKTI is not a major KLK7 investigated the carboxy-terminal region ments were KLK5 and KLK14 and, to a inhibitor. In addition, none of the LEKTI of the protein, from D6 to D15. Previous lesser extent, KLK7. domains contains a large hydrophobic work on physiological proteolytic pro- An exhaustive study by Borgoño et Phe, Try, or Tyr residue at position P1, as cessing of the amino-terminal region al. (2007) tested the inhibition potential are found in -like inhibitors

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(Goettig et al., 2010). Nevertheless, enous target proteinases. Likewise, the expression in Netherton syndrome. Hum Mol LEKTI could regulate KLK7 activity large number of LEKTI domains would Genet 12:2417–30 through KLK5-mediated KLK7 activation. contribute to quickly release a signifi- Bonnart C, Deraison C, Lacroix M et al. (2010) From these data, it appears that the cant number of bioactive fragments at Elastase 2 is expressed in human and mouse epidermis and impairs skin barrier function in inhibitory capabilities of LEKTI fragments the site of injury. LEKTI would thus act Netherton syndrome through filaggrin and lipid and LEKTI domains do not necessarily as a “cluster bomb”—able to rapidly misprocessing. J Clin Invest 120:871–82 correlate with the number of domains mobilize a significant number of bioac- Borgoño CA, Michael IP, Komatsu N et al. (2007) contained in each fragment. Although tive fragments from a single molecule. A potential role for multiple tissue kallikrein unique domains (D6, D7) are usually serine proteases in epidermal desquamation. J Biol Chem 282:3640–52 weaker inhibitors than multidomain frag- Impact on the development of new Brattsand M, Stefansson K, Lundh C et al. (2005) A ments, two-domain fragments (D8–D9) treatments for NS and allergy proteolytic cascade of kallikreins in the stratum can be as potent as four-domain frag- Understanding the physiological LEKTI corneum. J Invest Dermatol 124:198–203 ments (D6–D9). This suggests that the fragments and their inhibitory properties Brattsand M, Stefansson K, Hubiche T et al. (2009) conformation of multidomain fragments has the potential to influence the design SPINK9: a selective, skin-specific Kazal-type could modify the accessibility of inhibi- of new treatments for NS. In topical pro- inhibitor. J Invest Dermatol 129:1656–65 tory reactive loops and/or the binding to tein-replacement therapy, penetration of Briot A, Deraison C, Lacroix M et al. (2009) putative allosteric sites, thus influencing selected LEKTI fragments would be ham- Kallikrein 5 induces -like the inhibitory capacity of the fragment pered by their large molecular weight lesions through PAR2-mediated thymic stromal (Jayakumar et al., 2004). and would require skin delivery systems. lymphopoietin expression in Netherton Nevertheless, the profound skin barrier syndrome. J Exp Med 206:1135–47 What do these results tell us about LEKTI defect seen in NS could facilitate diffu- Briot A, Lacroix M, Robin A et al. (2010) Par2 biological function? inactivation inhibits early production of TSLP, sion through a disrupted stratum cor- but not cutaneous inflammation, in Netherton These studies identify the nature of physio­ neum. Alternatively, and perhaps more syndrome adult mouse model. J Invest Dermatol logical LEKTI fragments derived from D6 importantly, these results confirm that 130:2736–42 to D15, crucial information for assessing KLK5, KLK14, and KLK7 are potential Chavanas S, Bodemer C, Rochat A et al. (2000) their biological functions. They confirm therapeutic targets whose specific inhi- Mutations in SPINK5, encoding a serine that KLK5 and KLK14 are major target bition by small lead compounds might protease inhibitor, cause Netherton syndrome. Nat Genet 25:141–2 proteases of D6–D9 and derived domains, block unrestrained protease activity, Deraison C, Bonnart C, Lopez F et al. (2007) LEKTI whereas fragments D10–D15 are more leading to pharmacological inhibition of fragments specifically inhibit KLK5, KLK7, and potent KLK7 inhibitors. KLK5, KLK14, the pathogenic cascades. KLK14 and control desquamation through and KLK7 play a central role in skin des- Finally, in light of the work by Fortugno a pH-dependent interaction. Mol Biol Cell quamation. In fact, Fortugno et al. (2011) et al. (2011), spontaneous mutations 18:3607–19 showed that LEKTI fragments derived abolishing or creating furin cleavage sites Descargues P, Deraison C, Bonnart C et al. (2005) Spink5-deficient mice mimic Netherton from D6–D9 had a differential capability in linker regions may impact LEKTI pro- syndrome through degradation of desmoglein 1 to inhibit KLK14- versus KLK5-mediated cessing and proteolytic activation. These by epidermal protease hyperactivity. Nat Genet DSG1 degradation in vitro. Further stud- mutations could underlie more subtle SP 37:56–65 ies may explore the inhibitory capacities deregulation in the epidermis, which may Descargues P, Deraison C, Prost C et al. (2006) of these physio­logical fragments toward have a clinical impact and contribute to a Corneodesmosomal cadherins are preferential targets of stratum corneum trypsin- and other epidermal KLKs and SPs. genetic predisposition to eczema (Walley chymotrypsin-like hyperactivity in Netherton These results are consistent with the et al., 2001). syndrome. J Invest Dermatol 126:1622–32 biological cascades described in NS Egelrud T, Brattsand M, Kreutzmann P et al. (2005) CONFLICT OF INTEREST hK5 and hK7, two serine proteinases abundant patients and in mouse models of NS The authors state no conflict of interest. (Briot et al., 2009, 2010; Descargues et in human skin, are inhibited by LEKTI domain 6. Br J Dermatol 153:1200–3 al., 2005). In these models, unopposed ACKNOWLEDGMENTS Fontao L, Laffitte E, Briot A et al. (2011) Infliximab KLK5 activity initiates an autonomous The authors’ work is supported in part by the Agence Nationale de la Recherche (ANR-08- infusions for Netherton syndrome: sustained cascade, leading to stratum corneum GENO-033) and the Fondation pour la Recherche clinical improvement correlates with a reduction detachment and skin barrier defect, Médicale (FRM-DAL20051205066). of thymic stromal lymphopoietin levels in the inflammation, allergy, filaggrin degrada- skin. J Invest Dermatol 131:1947–50 tion, and lipid abnormalities. Notably, References Fortugno P, Bresciani A, Paolini C et al. (2011) Proteolytic activation cascade of the Netherton proteases not inhibited by LEKTI directly, Ahmed A, Kandola P, Ziada G et al. (2001) Purification and partial amino acid sequence syndrome–defective protein, LEKTI, in the such as elastase 2, also play important of from human epidermal keratinocyte epidermis: implications for skin . roles in NS, as shown by a transgenic conditioned medium. J Protein Chem 20:273–8 J Invest Dermatol 131:2223–32 mouse model that overexpresses elastase Bitoun E, Chavanas S, Irvine A et al. (2002) Goettig P, Magdolen V, Brandstetter H (2010) Natural 2 (Bonnart et al., 2010). Netherton syndrome: disease expression and and synthetic inhibitors of kallikrein-related The different inhibition properties spectrum of SPINK5 mutations in 21 families. peptidases (KLKs). Biochimie 92:1546–67 J Invest Dermatol 118:352–61 displayed by LEKTI fragments probably Hachem JP, Houben E, Crumrine D et al. (2006) Bitoun E, Micheloni A, Lamant L et al. (2003) Serine protease signaling of epidermal represent an advantage in addressing LEKTI proteolytic processing in human primary permeability barrier homeostasis. J Invest the diversity of exogenous and/or endog- keratinocytes, tissue distribution and defective Dermatol 126:2074–86

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Hewett DR, Simons AL, Mangan NE et al. (2005) See related article on pg 2233 Lethal, neonatal ichthyosis with increased proteolytic processing of filaggrin in a mouse model of Netherton syndrome. Hum Mol Genet Cuts by Caspase-14 Control the 14:335–46 Ishida-Yamamoto A, Deraison C et al. (2005) LEKTI is localized in lamellar granules, separated Proteolysis of Filaggrin from KLK5 and KLK7, and is secreted in the Leopold Eckhart1 and Erwin Tschachler1,2 extracellular spaces of the superficial stratum granulosum. J Invest Dermatol 124:360–6 Although mutations in the filaggrin gene (FLG) have been shown to be associated Jayakumar A, Kang Y, Mitsudo K et al. (2004) with ichthyosis vulgaris and atopic dermatitis, the function and regulation of Expression of LEKTI domains 6–9’ in the baculovirus expression system: recombinant filaggrin remain incompletely understood. In this issue, Hoste et al. report that LEKTI domains 6–9’ inhibit trypsin and subtilisin filaggrin is directly cleaved by caspase-14. Acting in concert with other proteases, A. Protein Expr Purif 35:93–101 caspase-14 controls the breakdown of filaggrin to free amino acids and amino Komatsu N, Saijoh K, Toyama T et al. (2005) acid derivatives that contribute to the hydration and UVB absorption capacity Multiple tissue kallikrein mRNA and protein of the stratum corneum. These findings identify a new layer of complexity in the expression in normal skin and skin diseases. Br J Dermatol 153:274–81 regulation of epidermal barrier function. Kreutzmann P, Schulz A, Standker L et al. (2004) Journal of Investigative Dermatology (2011) 131, 2173–2175. doi:10.1038/jid.2011.282 Recombinant production, purification and biochemical characterization of domain 6 of LEKTI: a temporary Kazal-type-related serine proteinase inhibitor. J Chromatogr B Analyt Processing of filaggrin in the epidermis underlying layers of the skin against UVB Technol Biomed Life Sci 803:75–81 Filaggrin became a focus of dermato­ radiation (Barresi et al., 2011). In addition Magert HJ, Standker L, Kreutzmann P et al. (1999) LEKTI, a novel 15-domain type of human serine logical research when mutations in its to proteolysis, protein phosphorylation, proteinase inhibitor. J Biol Chem 274:21499–502 gene (FLG) were found to be associ- dephosphorylation, and deimination Magert HJ, Drogemuller K, Raghunath M (2005) ated with ichthyosis vulgaris and atopic control the progressive conversion of Serine proteinase inhibitors in the skin: role in dermatitis (Irvine and McLean, 2006). profilaggrin to its amino acid constituents homeostasis and disease. Curr Protein Pept Sci Because filaggrin is expressed only in (Sandilands et al., 2009). The report by 6:241–54 keratinocytes, defects in the epidermal Hoste et al. (2011, this issue) uncovers a Meyer-Hoffert U (2009) Reddish, scaly, and itchy: barrier are now considered likely to pre- novel part of the complex processing of how proteases and their inhibitors contribute to inflammatory skin diseases. Arch Immunol Ther cede immune activation and allergies in filaggrin and highlights the experimental Exp (Warsz) 57:345–54 patients with atopic dermatitis (Irvine and challenges faced by investigators studying Meyer-Hoffert U, Wu Z, Kantyka T et al. (2010) McLean, 2006). Although the mechanism biochemical processes in the outermost Isolation of SPINK6 in human skin: selective of FLG-associated barrier defects has not layers of the skin. inhibitor of kallikrein-related peptidases. J Biol yet been fully elucidated, recent stud- Chem 285:32174–81 ies have revealed multiple interactions Caspase-14 is a filaggrin-processing Mitsudo K, Jayakumar A, Henderson Y et al. (2003) Inhibition of serine proteinases plasmin, trypsin, between filaggrin and other proteins that The cysteine protease caspase-14 was subtilisin A, cathepsin G, and elastase by LEKTI: are critical for its physiological function. linked to the breakdown of filaggrin a kinetic analysis. Biochemistry 42:3874–81 Like other members of the S100-fused- in 2007 by Declercq and colleagues, Ovaere P, Lippens S, Vandenabeele P (2009) The type , filaggrin is expressed who generated and characterized a emerging roles of serine protease cascades in as a precursor protein consisting of an caspase-14 knockout mouse model the epidermis. Trends Biochem Sci 34:453–63 N-terminal S100 domain and a series (Denecker et al., 2007). They found that Schechter NM, Choi EJ, Wang ZM et al. (2005) of filaggrin repeats. Proteolytic process- caspase-14 deficiency was associated Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lympho-epithelial ing yields filaggrin monomers that are with the accumulation of incompletely Kazal-type inhibitor (LEKTI). Biol Chem important in the aggregation of interme- degraded filaggrin fragments within the 386:1173–84 diate filaments during cornification of stratum corneum, a decrease in stratum Tartaglia-Polcini A, Bonnart C, Micheloni A et al. keratinocytes (Steinert et al., 1981). In the corneum hydration, increased trans­ (2006) SPINK5, the defective gene in Netherton stratum corneum, complete proteolysis of epidermal water loss, and sensitivity syndrome, encodes multiple LEKTI isoforms filaggrin leads to the release of free amino to UVB photodamage. In an extension derived from alternative pre-mRNA processing. J Invest Dermatol 126:315–24 acids that function as components of the of this work, Hoste et al. (2011) now Walley AJ, Chavanas S, Moffatt MF et al. (2001) natural moisturizing factor (NMF) (Scott provide evidence for the molecular Gene polymorphism in Netherton and common and Harding, 1986) (Figure 1). Moreover, mechanisms underlying the phenotype atopic disease. Nat Genet 29:175–8 filaggrin-derived histidine is converted of caspase-14 knockout mice. The Yang T, Liang D, Koch PJ et al. (2004) Epidermal to urocanic acid, which protects the investigators identify direct cleavage sites detachment, desmosomal dissociation, and destabilization of in Spink5-/- mice. Dev 18:2354–8 1Department of Dermatology, Medical University of Vienna, Vienna, Austria; 2Centre de Recherches et Zeeuwen PL (2004) Epidermal differentiation: the d’Investigations Epidermiques et Sensorielles, Neuilly, France role of proteases and their inhibitors. Eur J Cell Correspondence: Leopold Eckhart, Department of Dermatology, Medical University of Vienna, 1090 Biol 83:761–3 Vienna, Austria. E-mail: [email protected]

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