Reconstructing the epidermal proteolytic cascades in health and disease.

The epidermis is the outer stratified epithelium of the skin, forming the physical barrier that is indispensable for homeostasis. Epidermal proteolysis, mainly but not exclusively executed by kallikrein-related peptidases (KLKs), is tightly regulated to ensure maintenance of physiological skin renewal and an intact skin barrier. Perturbation of epidermal proteolytic networks is implicated in a wide array of rare and common skin pathologies of diverse genetic backgrounds. Recent studies of monogenic human skin diseases and newly developed animal models have revealed new mechanisms of regulation of proteolytic pathways in epidermal physiology and in disease states. These new data have challenged some accepted views, for example the role of matriptase in epidermal desquamation, which turned out to be restricted to mouse skin. The significance of PAR2 signaling in skin inflammation should also be reconsidered in the face of recent findings. Cumulatively, recent studies necessitate a sophisticated redefinition of the proteolytic and signaling pathways that operate in human skin. We elaborate how epidermal proteolysis is finely regulated at multiple levels, and in a spatial manner that has not been taken into consideration so far, in which specific proteases are confined to distinct epidermal sublayers. Of interest, transglutaminases have emerged as regulators of epidermal proteolysis and desquamation by spatially fixing endogenous protease inhibitors, constituting regulatory factors that were not recognized before. Furthermore, new evidence suggests a link between proteolysis and lipid metabolism. By synthesis of established notions and recent discoveries, we provide an up-to-date critical evaluation and synthesis of current knowledge and the extended complexity of proteolysis regulation and signaling pathways in skin. © 2022 The Pathological Society of Great Britain and Ireland.

Cocktails of KLK5 Protease Inhibitors and Anti-TNFα Therapeutics: an Effective Treatment for Netherton Syndrome.

Netherton syndrome (NS) is a rare, severe type of ichthyosis, often lethal in neonates, for which there is no therapy. Spink5-/- mice recapitulate major NS hallmarks and die homogeneously within 5 h from birth due to severe epidermal barrier defect leading to dehydration. Spink5-/-Klk5-/- mice survive neonatal lethality, indicating that KLK5 could be a drug target for NS. Nevertheless, after a week, these mice developed epidermal inflammation and signs of barrier defect leading to lethality. Here we tested whether anti-TNFα strategy in combination with anti-KLK5 could provide a long-term effective therapy for NS. Deletion of Tnfa in Spink5-/- suppressed the inflammatory phenotype but did not rescue neonatal lethality of Spink5-/- indicating that anti-TNFα therapy alone would not be sufficient to treat NS. Interestingly, in Spink5-/-Klk5-/-Tnfa-/- mice, NS features were rescued, and mice lived normally for 16-18 months. For the first time, evidence is provided that a combination of anti-TNFα and anti-KLK5 therapeutics represents an effective therapeutic strategy for NS. Notably, anti-TNFα factors are marketed and used widely, while LMW KLK5 inhibitors are being developed.

Kallikreins emerge as new regulators of viral infections.

Kallikrein-related peptidases (KLKs) or kallikreins have been linked to diverse (patho) physiological processes, such as the epidermal desquamation and inflammation, seminal clot liquefaction, neurodegeneration, and cancer. Recent mounting evidence suggests that KLKs also represent important regulators of viral infections. It is well-established that certain enveloped viruses, including influenza and coronaviruses, require proteolytic processing of their hemagglutinin or spike proteins, respectively, to infect host cells. Similarly, the capsid protein of the non-enveloped papillomavirus L1 should be proteolytically cleaved for viral uncoating. Consequently, extracellular or membrane-bound proteases of the host cells are instrumental for viral infections and represent potential targets for drug development. Here, we summarize how extracellular proteolysis mediated by the kallikreins is implicated in the process of influenza (and potentially coronavirus and papillomavirus) entry into host cells. Besides direct proteolytic activation of viruses, KLK5 and 12 promote viral entry indirectly through proteolytic cascade events, like the activation of thrombolytic enzymes that also can process hemagglutinin, while additional functions of KLKs in infection cannot be excluded. In the light of recent evidence, KLKs represent potential host targets for the development of new antivirals. Humanized animal models to validate their key functions in viral infections will be valuable.

Deficiency of the serine peptidase Kallikrein 6 does not affect the levels and the pathological accumulation of a-synuclein in mouse brain.

Several lines of evidence indicate that the propagation of misfolded α-synuclein (α-syn) plays a central role in the progression and manifestation of Parkinson's disease. Pathogenic α-syn species can be present in the extracellular space. Thus, the identification and modulation of the key enzymes implicated in extracellular α-syn turnover becomes vital. Kallikrein peptidase 6 has been identified as one of the major α-syn degrading enzymes and has been implicated in the clearance of extracellular α-syn. However, the physiological role of this enzyme in regulating α-syn, in vivo, still remains elusive. Here, by utilizing Klk6 knock-out (Klk6-/- ) mice as our experimental model, we provide insight into the physiologic relevance of endogenous KLK6 expression on α-syn processing. Behavioral phenotyping showed that Klk6-/- mice display no gross behavioral abnormalities. Further in vivo characterization of this mouse model, in the context of α-syn accumulation, showed that KLK6 deletion had no impact on the protein levels of intracellular or extracellular α-syn. Upon in vivo administration of α-syn pre-formed fibrils (PFF), α-syn pathologic accumulations were evident both in the brains of Klk6-/- mice and wt mice without significant differences. Intrastriatal delivery of active KLK6, did not affect secreted α-syn levels observed in the A53T α-syn over-expressing mice. These findings suggest that in the in vivo setting of PFF pathology induction, KLK6 alone is not able to modulate pathology transmission. Our study raises implications for the use of recombinant α-syn fibrils in α-syn turnover studies.

Redirecting drug repositioning to discover innovative cosmeceuticals.

Skin appearance is essential for self-esteem and quality of life; consequently, skin care products represent a huge market. In particular, cosmeceuticals constitute a hybrid category of skin care formulations, at the interphase of cosmetics and pharmaceuticals, rationally designed to target (patho) physiological mechanisms aiming to enhance skin health and appearance. Cosmeceuticals are marketed as anti-ageing, anti-wrinkle, hair regrowth, skin whitening and wound healing agents with special emphasis on scar-free healing. An overview on recent cutting-edge advances concerning the discovery and development of enhanced performance cosmeceuticals by drug repositioning approaches is presented here. In this context, we propose "target repositioning," a new term, to highlight that druggable protein targets implicated in multiple diseases (hubs in the diseasome) can be exploited to accelerate the discovery of molecularly targeted cosmeceuticals that can promote skin health as an added benefit, which is a novel concept not described before. In this direction, emphasis is placed on the role of mouse models, for often untreatable skin diseases, as well as recent breakthroughs on monogenic rare skin syndromes, in promoting compound repositioning to innovative cosmeceuticals.

Plausible Emergence of Biochemistry in Enceladus Based on Chemobrionics.

Saturn's satellite Enceladus is proposed to have a soda-type subsurface ocean with temperature able to support life and an iron ore-based core. Here, it was demonstrated that ocean chemistry related to Enceladus can support the development of Fe-based hydrothermal vents, one of the places suggested to be the cradle of life. The Fe-based chemical gardens were characterized with Fourier-transform (FT)IR spectroscopy and XRD. The developed chemobrionic structures catalyzed the condensation polymerization of simple organic prebiotic molecules to kerogens. Further, they could passively catalyze the condensation of the prebiotic molecule formamide to larger polymers, suggesting that elementary biochemical precursors could have emerged in Enceladus.

Generation of a quenched phosphonate activity-based probe for labelling the active KLK7 protease.

Kallikrein 7 (KLK7) is a chymotrypsin-like serine protease with established roles in skin diseases like the rare Netherton syndrome, an overdesquamating and inflammatory condition, but also common atopic dermatitis, and a potential drug target for these and possibly other diseases. Nevertheless, tools to determine the active KLK7 enzyme are not available. Here, a mixed alkyl aryl phosphonate quenched activity-based probe that detects the active KLK7 was developed and evaluated in vitro. This KLK7-qABP can potentially be used to monitor KLK7 activity in vivo.

Research in practice: Towards deciphering the role of epidermal proteases in recessive dystrophic epidermolysis bullosa progression.

Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable severe skin disease caused by loss of collagen VII, an extracellular protein that ensures skin cohesion. It manifests in skin blistering and unresolved cycles of wounding and healing that progressively lead to dermal stiffening and early development of aggressive cutaneous squamous cell carcinomas. Inflammation and subsequent tissue fibrosis highly contribute to RDEB pathogenicity and targeting them could provide new therapeutic options. Kallikreins (KLKs) are epidermal secreted proteases, which contribute to skin desquamation and inflammation. Kallikreins are involved in the pathogenesis of several inflammatory skin disorders, but interestingly also in the initiation and progression of different cancers. Our project aims at deciphering the role of KLKs in inflammation, fibrosis, and tumor development in RDEB.

Activography reveals aberrant proteolysis in desquamating diseases of differing backgrounds.

The role of epidermal proteolysis in overdesquamation was revealed in Netherton syndrome, a rare ichthyosis due to genetic deficiency of the LEKTI inhibitor of serine proteases. Recently, we developed activography, a new histochemical method, to spatially localize and semiquantitatively assess proteolytic activities using activity-based probes. Activography provides specificity and versatility compared to in situ zymography, the only available method to determine enzymatic activities in tissue biopsies. Here, activography was validated in skin biopsies obtained from an array of distinct disorders and compared with in situ zymography. Activography provides a methodological advancement due to its simplicity and specificity and can be readily adapted as a routine diagnostic assay. Interestingly, the levels of epidermal proteolysis correlated with the degree of desquamation independent of skin pathology. Thus, deregulated epidermal proteolysis likely represents a universal mechanism underlying aberrant desquamation.

KLK6 protease accelerates skin tumor formation and progression.

Kallikrein-related peptidase 6 (KLK6) is a serine protease that is aberrantly altered in various types of cancer, but its role in non-melanoma skin cancer has not been investigated. KLK6 is active in epidermis and has been linked to normal skin differentiation. Thus, we investigated whether it could be implicated in skin tumorigenesis in vivo. Carcinogenesis was induced in Klk6-/- mice by epidermal application of 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol 13-acetate (DMBA/TPA), and multistage skin tumor development and progression was monitored closely until squamous cell carcinomas (SCCs) and invasive tumors formed. Klk6-/- (but also Klk6+/-) mice were highly resistant to tumor growth/development manifested by their highly diminished numbers and delayed onset of tumors compared with wild-type (wt) mice. Histological analyses of the few tumors that developed in Klk6-/- after prolonged (>1 year) chemical challenge revealed that these were mainly benign papillomas, whereas in wt mice tumors progressed to SCCs. Inflammation was attenuated in Klk6-/- skin following chronic exposure to TPA, indicated by markedly low expression of proinflammatory cytokines, in direct contrast to wt. Further, in Klk6-/- mice, the ability of implanted nascent PDVC57 skin cancer cells to form tumors was highly diminished. Our study identified KLK6 as a new tumor-promoting factor of early skin cancer and suggested that KLK6 is an important molecular link in the development of skin inflammation and in tumor-promoting inflammatory processes.

KLK5, a novel potential suppressor of vaginal carcinogenesis.

Vaginal cancer is rare and largely unexplored. We found here that kallikrein-related peptidase 5 (KLK5) is coordinately expressed along with other KLKs in all stratified epithelia, including vagina, pointing to potential role(s) in differentiation. Further, we propose that KLK5 could be implicated in vaginal cancer development based on the fact that Klk5-/- mice are prone to develop vaginal tumors when exposed to 7,12-dimethylbenz[a]anthracene. Nf-κb activation is markedly enhanced in Klk5-/-, leading to increased resistance to apoptosis of mutated vaginal cells. This explains the higher tumor numbers observed in Klk5-/- compared to wildtype. Thus, KLK5 may represent a putative suppressor of vaginal cancer.

KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome.

Netherton Syndrome (NS) is a rare and severe autosomal recessive skin disease which can be life-threatening in infants. The disease is characterized by extensive skin desquamation, inflammation, allergic manifestations and hair shaft defects. NS is caused by loss-of-function mutations in SPINK5 encoding the LEKTI serine protease inhibitor. LEKTI deficiency results in unopposed activities of kallikrein-related peptidases (KLKs) and aberrantly increased proteolysis in the epidermis. Spink5⁻/⁻ mice recapitulate the NS phenotype, display enhanced epidermal Klk5 and Klk7 protease activities and die within a few hours after birth because of a severe skin barrier defect. However the contribution of these various proteases in the physiopathology remains to be determined. In this study, we developed a new murine model in which Klk5 and Spink5 were both knocked out to assess whether Klk5 deletion is sufficient to reverse the NS phenotype in Spink5⁻/⁻ mice. By repeated intercrossing between Klk5⁻/⁻ mice with Spink5⁻/⁻ mice, we generated Spink5⁻/⁻Klk5⁻/⁻ animals. We showed that Klk5 knock-out in Lekti-deficient newborn mice rescues neonatal lethality, reverses the severe skin barrier defect, restores epidermal structure and prevents skin inflammation. Specifically, using in situ zymography and specific protease substrates, we showed that Klk5 knockout reduced epidermal proteolytic activity, particularly its downstream targets proteases KLK7, KLK14 and ELA2. By immunostaining, western blot, histology and electron microscopy analyses, we provide evidence that desmosomes and corneodesmosomes remain intact and that epidermal differentiation is restored in Spink5⁻/⁻Klk5⁻/⁻. Quantitative RT-PCR analyses and immunostainings revealed absence of inflammation and allergy in Spink5⁻/⁻Klk5⁻/⁻ skin. Notably, Il-1ß, Il17A and Tslp levels were normalized. Our results provide in vivo evidence that KLK5 knockout is sufficient to reverse NS-like symptoms manifested in Spink5⁻/⁻ skin. These findings illustrate the crucial role of protease regulation in skin homeostasis and inflammation, and establish KLK5 inhibition as a major therapeutic target for NS.

Resistance of naturally secreted α-synuclein to proteolysis.

Recent evidence suggests that specific extracellular α-synuclein (α-syn) strains are implicated in the progression of Parkinson's disease (PD) pathology. It is plausible that deregulation in the normal processing of secreted α-syn may be a causative risk factor for PD. To date, the degradation mechanisms involved have received very little attention. Here, we sought to investigate factors that regulate extracellular α-syn levels. We show, for the first time, that cell-secreted α-syn forms are resistant to direct proteolysis by kallikrein-related peptidase 6 (KLK6), an extracellular enzyme known to cleave recombinant α-syn. This differential susceptibility appears to be partially due to the association of secreted α-syn with lipids. We further provide evidence that secreted α-syn can be cleaved by KLK6 indirectly through activation of a secreted metalloprotease, suggestive of the involvement of a proteolytic cascade in the catabolism of secreted α-syn. Our results clearly suggest that physiological modifications affect the biochemical behavior of secreted α-syn and provide novel insights into mechanisms and potential targets for therapeutic interventions.-Ximerakis, M., Pampalakis, G., Roumeliotis, T. I., Sykioti, V.-S., Garbis, S. D., Stefanis, L., Sotiropoulou, G., Vekrellis, K. Resistance of naturally secreted α-synuclein to proteolysis.

Unraveling ice multiplication in winter orographic clouds via in-situ observations, remote sensing and modeling.

Recent years have shown that secondary ice production (SIP) is ubiquitous, affecting all clouds from polar to tropical regions. SIP is not described well in models and may explain biases in warm mixed-phase cloud ice content and structure. Through modeling constrained by in-situ observations and its synergy with radar we show that SIP in orographic clouds exert a profound impact on the vertical distribution of hydrometeors and precipitation, especially in seeder-feeder cloud configurations. The mesoscale model simulations coupled with a radar simulator strongly support that enhanced aggregation and SIP through ice-ice collisions contribute to observed spectral bimodalities, skewing the Doppler spectra toward the slower-falling side at temperatures within the dendritic growth layer, ranging from -20 °C to -10 °C. This unique signature provides an opportunity to infer long-term SIP occurrences from the global cloud radar data archive, particularly for this underexplored temperature regime.

Towards the elucidation of the regulatory network guiding the insulin producing cells' differentiation.

This study pertains to the regulatory network of neurogenin3 (NGN3, approved symbol: NEUROG3), the main regulator of insulin producing cells' formation. In silico regulatory region analyses of known and novel targets of NGN3 revealed the presence of two variants of a regulatory module that appeared conserved at the most phylogenetically distant species with pancreas. Both variants of this module contained binding sites of six transcription factors implicated in pancreas development. Nevertheless, an additional factor was found only into the module of the down-regulated by NGN3 genes. Whole genome analyses confirmed the statistical significance of these regulatory modules. Investigation of protein-protein interactions among the factors bound into these sequences indicated the formation of alternative protein complexes resulting into the up- or down-regulation of the respective genes. Subsequently, an NGN3-guided regulatory network, was modeled, describing the interactions among the analyzed genes with their transcriptional regulators, leading into the differentiation of cells capable of producing insulin.

One round of SELEX for the generation of DNA aptamers directed against KLK6.

Kallikrein-related peptidase 6 (KLK6) is an active serine protease that has been implicated in common pathologies, including neurodegenerative disorders such as Parkinson and Alzheimer disease and certain types of cancer. Antibodies, either polyclonal or monoclonal, that exhibit specificity for distinct members of the extended kallikrein family, including KLK6, were developed. With the exception of KLK3/PSA, the identification and generation of aptamers, as potential new tools with improved characteristics demanded for therapeutic and diagnostic applications, has not been explored for KLKs. Here, we report for the first time the identification of novel DNA aptamers against KLK6 that were isolated using a modified systemic evolution of ligands by exponential enrichment technique. The identified aptamers were characterized using fluorescence spectroscopy, competition ELISA, and quartz crystal microbalance, and two aptamers (008 and 022) were found to exhibit high affinity (K(d) in the low nanomolar range) for KLK6. Aptamers were tested for their ability to bind to serum albumin, to demonstrate their specificity for their target, and the possible involvement of such proteins in the transport of aptamers into the bloodstream. The developed aptamers are expected to assist the development of novel diagnostic, biosensing, and therapeutic strategies.

Activity-Based Probes for Proteases Pave the Way to Theranostic Applications.

Proteases are important enzymes in health and disease. Their activities are regulated at multiple levels. In fact, proteases are synthesized as inactive proenzymes (zymogens) that are activated by proteolytic removal of their pro-peptide sequence and can remain active or their activity can be attenuated by complex formation with specific endogenous inhibitors or by limited proteolysis or degradation. Consequently, quite often, only a fraction of the protease molecules is in the active/functional form, thus, the abundance of a protease is not always linearly proportional to the (patho)physiological function(s). Therefore, assays to determine the active forms of proteases are needed, not only in research but also in molecular diagnosis and therapy. Activity-based probes (ABPs) are chemical entities that bind covalently to the active enzyme/protease. ABPs carry a detection tag to enable localization and quantification of specific enzymatic/proteolytic activities with applications in molecular imaging and diagnosis. Moreover, ABPs act as suicide inhibitors of proteases, which can be exploited for delineation of the functional role(s) of a given protease in (patho) biological context and as potential therapeutics. In this sense, ABPs represent new theranostic agents. We outline recent developments pertaining to ABPs for proteases with potential therapeutic applications, with the aim to highlight their importance in theranostics.

Keratinocyte differentiation and proteolytic pathways in skin (patho) physiology.

The epidermis is a stratified epithelium that forms the barrier between the organism and its environment. It is mainly composed of keratinocytes at various stages of differentiation. The stratum corneum is the outermost layer of the epidermis and is formed of multiple layers of anucleated keratinocytes called corneocytes. We aim to highlight the roles of epidermal differentiation and proteolysis in skin diseases. Skin biopsies isolated from Spink5-/- mice, the established model of Netherton syndrome (NS), and from patients with NS, seborrheic dermatitis (SD) and psoriasis, as well as healthy controls, were analyzed by histology and immunohistochemistry. Our results showed that NS, SD, and psoriasis are all characterized by abnormal epidermal differentiation, manifested by hyperplasia, hyperkeratosis, and parakeratosis. At the molecular level, abnormal differentiation is accompanied by increased expression of involucrin and decreased expression of loricrin in NS and psoriasis. Increased epidermal proteolysis associated with increased kallikrein-related peptidases (KLKs) expression is also observed in both NS and psoriatic epidermis. Furthermore, reduced expression of desmosomal proteins is observed in NS, but increased in psoriasis. Since desmosomal proteins are proteolytic substrates and control keratinocyte differentiation, their altered expression directly links epidermal proteolysis to differentiation. In conclusion, abnormal cellular differentiation and proteolysis are interconnected and underlie the pathology of NS, SD and psoriasis.

A chemogenomic approach is required for effective treatment of amyotrophic lateral sclerosis.

ALS is a fatal untreatable disease involving degeneration of motor neurons. Μultiple causative genes encoding proteins with versatile functions have been identified indicating that diverse biological pathways lead to ALS. Chemical entities still represent a promising choice to delay ALS progression, attenuate symptoms and/or increase life expectancy, but also gene-based and stem cell-based therapies are in the process of development, and some are tested in clinical trials. Various compounds proved effective in transgenic models overexpressing distinct ALS causative genes unfortunately though, they showed no efficacy in clinical trials. Notably, while animal models provide a uniform genetic background for preclinical testing, ALS patients are not stratified, and the distinct genetic forms of ALS are treated as one group, which could explain the observed discrepancies between treating genetically homogeneous mice and quite heterogeneous patient cohorts. We suggest that chemical entity-genotype correlation should be exploited to guide patient stratification for pharmacotherapy, that is administered drugs should be selected based on the ALS genetic background.