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Netherton syndrome (NS) is a severe skin disease caused by the loss of protease inhibitor LEKTI, which leads to the dysregulation of epidermal proteases and severe skin-barrier defects. KLK5 was proposed as a major protease in NS pathology, however its inactivation is not sufficient to rescue the lethal phenotype of LEKTI-deficient mice. In this study, we further elucidated the in vivo roles of the epidermal proteases in NS using a set of mouse models individually or simultaneously deficient for KLK5 and KLK7 on the genetic background of a novel NS-mouse model. We show that although the ablation of KLK5 or KLK7 is not sufficient to rescue the lethal effect of LEKTI-deficiency simultaneous deficiency of both KLKs completely rescues the epidermal barrier and the postnatal lethality allowing mice to reach adulthood with fully functional skin and normal hair growth. We report that not only KLK5 but also KLK7 plays an important role in the inflammation and defective differentiation in NS and KLK7 activity is not solely dependent on activation by KLK5. Altogether, these findings show that unregulated activities of KLK5 and KLK7 are responsible for NS development and both proteases should become targets for NS therapy.

The human kallikrein-related peptidase 4 (KLK4) and the transcribed pseudogene KLKP1 are reported to be highly expressed in the prostate. When trying to clone transcripts of KLKP1, we partly failed. Instead, we identified an androgen-regulated transcript, KLK4T2, which appeared to be a splice variant of KLK4 that also contained exons of KLKP1. Expression analysis of KLK4, KLK4T2, and KLKP1 transcripts in prostate cancer cell lines showed high levels of KLKP1 transcripts in the nucleus and in unfractionated cell extract, whereas it was almost completely absent in the cytoplasmatic fraction. This was in contrast to KLK4 and KLK4T2, which displayed high to moderate levels in the cytoplasm. In patient cohorts we found significantly higher expression of both KLK4T2 and KLK4 in benign prostatic hyperplasia compared to both primary prostate cancer and bone metastasis. Analysis of tissue panels demonstrated the highest expression of KLK4T2 in the prostate, but in contrast to the classical KLK4, relatively high levels were also found in placenta. So far, the function of KLK4T2 is still to be explored, but the structure of the translation product indicated that it generates a 17.4 kDa intracellular protein with possible regulatory function.

Proteinase-activated receptor-2 (PAR2) is a seven transmembrane spanning, G-protein-coupled receptor, present on the membrane of many cell types including keratinocytes. In skin, PAR2 is suggested to play a regulatory role during inflammation, epidermal barrier function, and pruritus. PAR2 is activated by trypsin-like proteases by a unique mechanism where cleavage of the receptor leads to the release of a small peptide, which activates the receptor as a tethered ligand. The endogenous activators of PAR2 on keratinocytes have not been identified as of yet. Potential candidates are kallikrein-related peptidases (KLKs) expressed by epidermal cells. Therefore, the ability of four human skin-derived KLKs was examined with regard to their capacity to activate PAR2in vitro. PAR2 cleavage was followed by immunofluorescence analysis and functional activation by measurements of changes in intracellular calcium levels. We found that KLK5 and KLK14, but neither KLK7 nor KLK8, induced PAR2 signalling. We conclude that certain, but not all, epidermal KLKs are capable of activating PAR2. We could also show the coexpression of KLK14 and PAR2 receptor in inflammatory skin disorders. These in vitro results suggest that KLKs may take part in PAR2 activation in the epidermis and thereby in PAR2-mediated inflammatory responses, including epidermal barrier repair and pruritus. The role of KLKs in PAR2 activation in vivo remains to be elucidated.

Corneodesmosin (CDSN), desmoglein 1 (DSG1), and desmocollin 1 (DSC1) are adhesive proteins of the extracellular part of the corneodesmosomes, the junctional structures that mediate corneocyte cohesion. The degradation of these proteins at the epidermis surface is necessary for desquamation. Two serine proteases of the kallikrein family synthesized as inactive precursors have been implicated in this process: the stratum corneum chymotryptic enzyme (SCCE/KLK7/hK7) and the stratum corneum tryptic enzyme (SCTE/KLK5/hK5). Here, we analyzed the capacity of these enzymes to cleave DSG1, DSC1, and epidermal or recombinant forms of CDSN, at an acidic pH close to that of the stratum corneum. SCCE directly cleaved CDSN and DSC1 but was unable to degrade DSG1. But incubation with SCTE induced degradation of the three corneodesmosomal components. Using the recombinant form of CDSN, either with its N-glycan chain or enzymatically deglycosylated, we also demonstrated that oligosaccharide residues do not protect CDSN against proteolysis by SCCE. Moreover, our results suggest that SCTE is able to activate the proform of SCCE. These results strongly suggest that the two kalikreins are involved in desquamation. A model is proposed for desquamation that could be regulated by a precisely controlled protease–protease inhibitor balance.

Engagement of an extended β-sheet is a common substrate/inhibitor interaction at the active site of serine proteases and is an important feature of Laskowski mechanism inhibitors that present a substrate-like loop to a target protease. This loop is cleaved but subsequently relegated forming a stable inhibitor/protease complex. Laskowski inhibitors are ubiquitous in nature and are used extensively in serine protease inhibitor design. However, most studies concentrate on introducing new sidechain interactions rather than the direct contributions of the substrate-like β-sheet to enzyme inhibition. Here we report the crystal structure of an simplified β-sheet inhibitory motif within the Sunflower Trypsin Inhibitor (SFTI) in complex with trypsin. We show that the intramolecular hydrogen bond network of this SFTI variant (SFTI-TCTR) engages the inhibitor sidechains that would normally interact with a target protease, giving mainchain interactions a more prominent role in complex formation. Despite having reduced sidechain interactions, this SFTI variant is remarkably potent and inhibits a diverse range of serine proteases. Crystal structural analysis and molecular modelling of SFTI-TCTR complexes again indicates an interface dominated by β-sheet interactions, highlighting the importance of this motif and the adaptability of SFTI as a scaffold for inhibitor design.

A previously unreported Kazal-type serine protease inhibitor, serine protease inhibitor Kazal type 9 (SPINK9), was identified in human skin. SPINK9 expression was strong in palmar epidermis, but not detectable or very low in non palmoplantar skin. Analysis of a human cDNA panel showed intermediate expression in thymus, pancreas, liver, and brain, and low or undetectable expression in other tissues. Using kallikrein-related peptidases (KLKs) 5, 7, 8, and 14, thrombin, trypsin, and chymotrypsin, inhibition with recombinant SPINK9 was seen only for KLK5 using low molecular weight substrates, with an apparent Ki of 65nM. Also KLK5 degradation of fibrinogen was totally inhibited by SPINK9. Slight inhibition of KLK8 using fibrinogen substrate could be observed using high concentrations of SPINK9. Analyses by surface plasmon resonance showed heterogeneous binding to SPINK9 of KLK5 and KLK8, but no binding of KLK7 or KLK14. KLK5 has been suggested to play a central role in skin desquamation as an initiating activating enzyme in proteolytic cascades formed by KLKs. An apparently KLK5-specific inhibitor, such as SPINK9, may play a significant regulatory role in such cascades. We suggest a possible role for SPINK9 in the site-specific epidermal differentiation of palms and soles.

Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor’s stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.

Serine proteases belonging to the kallikrein group may play a central role in desquamation. We have identified human kallikreins 5, 7, and 14 (hK5, hK7, hK14) in catalytically active form in stratum corneum. All three enzymes are produced as inactive precursors. In this work, we prepared recombinant enzymes and enzyme precursors and characterized the catalytic properties of hK5 and hK14. With peptide substrates hK5 and hK14 both showed trypsin-like specificity and alkaline pH-optima. For the substrates tested, hK14 was superior to hK5 as regards maximum catalytic rate as well as catalytic efficiency. hK5, but not hK14, could activate pro-hK7 in a reaction which was optimal at pH 5–7. hK5 could activate its own precursor as well as pro-hK14. This was in contrast to hK14, which could activate pro-hK5 but not its own precursor. The activation of pro-hK5 either by auto-activation or by hK14 occurred at maximum rate at neutral or weakly alkaline pH, whereas activation of pro-hK14 by hK5 was optimal at pH 6–7. We conclude that the enzymes studied may be part of a protease cascade in the stratum corneum, and that the observed pH effects may have physiological relevance.

Background Patients with metastatic prostate cancer (PC) are treated with androgen deprivation therapy (ADT) that initially reduces metastasis growth, but after some time lethal castration-resistant PC (CRPC) develops. A better understanding of the tumor biology in bone metastases is needed to guide further treatment developments. Subgroups of PC bone metastases based on transcriptome profiling have been previously identified by our research team, and specifically, heterogeneities related to androgen receptor (AR) activity have been described. Epigenetic alterations during PC progression remain elusive and this study aims to explore promoter gene methylation signatures in relation to gene expression and tumor AR activity. Materials and methods Genome-wide promoter-associated CpG methylation signatures of a total of 94 tumor samples, including paired non-malignant and malignant primary tumor areas originating from radical prostatectomy samples ( n  = 12), and bone metastasis samples of separate patients with hormone-naive ( n  = 14), short-term castrated ( n  = 4) or CRPC ( n  = 52) disease were analyzed using the Infinium Methylation EPIC arrays, along with gene expression analysis by Illumina Bead Chip arrays ( n  = 90). AR activity was defined from expression levels of genes associated with canonical AR activity. Results Integrated epigenome and transcriptome analysis identified pronounced hypermethylation in malignant compared to non-malignant areas of localized prostate tumors. Metastases showed an overall hypomethylation in relation to primary PC, including CpGs in the AR promoter accompanied with induction of AR mRNA levels. We identified a Methylation Classifier for Androgen receptor activity (MCA) signature, which separated metastases into two clusters (MCA positive/negative) related to tumor characteristics and patient prognosis. The MCA positive metastases showed low methylation levels of genes associated with canonical AR signaling and patients had a more favorable prognosis after ADT. In contrast, MCA negative patients had low AR activity associated with hypermethylation of AR-associated genes, and a worse prognosis after ADT. Conclusions A promoter methylation signature classifies PC bone metastases into two groups and predicts tumor AR activity and patient prognosis after ADT. The explanation for the methylation diversities observed during PC progression and their biological and clinical relevance need further exploration.