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.

Chemical, Physical, and Toxicological Properties of V-Agents.

V-agents are exceedingly toxic organophosphate nerve agents. The most widely known V-agents are the phosphonylated thiocholines VX and VR. Nonetheless, other V-subclasses have been synthesized. Here, a holistic overview of V-agents is provided, where these compounds have been categorized based on their structures to facilitate their study. A total of seven subclasses of V-agents have been identified, including phospho(n/r)ylated selenocholines and non-sulfur-containing agents, such as VP and EA-1576 (EA: Edgewood Arsenal). Certain V-agents have been designed through the conversion of phosphorylated pesticides to their respective phosphonylated analogs, such as EA-1576 derived from mevinphos. Further, this review provides a description of their production, physical properties, toxicity, and stability during storage. Importantly, V-agents constitute a percutaneous hazard, while their high stability ensures the contamination of the exposed area for weeks. The danger of V-agents was highlighted in the 1968 VX accident in Utah. Until now, VX has been used in limited cases of terrorist attacks and assassinations, but there is an increased concern about potential terrorist production and use. For this reason, studying the chemistry of VX and other less-studied V-agents is important to understand their properties and develop potential countermeasures.

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.

Improvements, Variations and Biomedical Applications of the Michaelis-Arbuzov Reaction.

Compounds bearing the phosphorus-carbon (P-C) bond have important pharmacological, biochemical, and toxicological properties. Historically, the most notable reaction for the formation of the P-C bond is the Michaelis-Arbuzov reaction, first described in 1898. The classical Michaelis-Arbuzov reaction entails a reaction between an alkyl halide and a trialkyl phosphite to yield a dialkylalkylphosphonate. Nonetheless, deviations from the classical mechanisms and new modifications have appeared that allowed the expansion of the library of reactants and consequently the chemical space of the yielded products. These involve the use of Lewis acid catalysts, green methods, ultrasound, microwave, photochemically-assisted reactions, aryne-based reactions, etc. Here, a detailed presentation of the Michaelis-Arbuzov reaction and its developments and applications in the synthesis of biomedically important agents is provided. Certain examples of such applications include the development of alkylphosphonofluoridates as serine hydrolase inhibitors and activity-based probes, and the P-C containing antiviral and anticancer agents.

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.

New molecular diagnostic trends and biomarkers for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rare and fatal neurodegenerative disorder. Two forms are recognized, familial (FALS) that accounts for 5-10% of ALS cases, and sporadic (SALS) that accounts for the rest. Early diagnosis of ALS is important because it improves their therapeutic efficacy. Current diagnosis is based on clinical assessment and requires approximately 12 months, leading to a significant delay in drug administration. Therefore, new methods are required for the earlier diagnosis of ALS. Screening for pathogenic variants in known ALS-associated genes is already exploited as a diagnostic tool in ALS but cannot be applied for population-based screening. New circulating biomarkers (proteins or small molecules) are needed for initial screening, whereas specific diagnostic methods can be applied to confirm the presence of pathogenic variants in the selected population subgroup. Lipids appear as promising biomarkers for population-based screening and for monitoring disease progression. Genetic analysis can also assist in the prediction of disease progression by analyzing disease-modifying genes, for example, EPHA4 and CHGB. Furthermore, molecular diagnosis will aid the stratification of ALS patients for improved pharmacological approaches. Here, we discuss current and novel diagnostic strategies and how they can be applied to revolutionize the field of ALS molecular diagnosis.

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.

A Closed Chemobrionic System as a Biochemical Delivery Platform.

Inorganic cells bearing calcium silicate membranes were prepared and resembled closed chemical gardens. It was demonstrated that these inorganic cells can successfully be loaded with natural products, proteins and plasmid DNA, and their cargo can be released in a controlled manner. These cells demonstrated the ability of chemical gardens to act as platforms for the sustained delivery of biomolecules and are expected to introduce chemical gardens in the field of biosciences.

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.

The Generation of an Organic Inverted Chemical Garden.

A chemical garden based on iron salt that grows in organic solvents and ions is demonstrated for the first time. This prototype chemical garden develops in an inverted orientation, thus providing evidence that downward growth is feasible.

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.

Underestimations in the In Silico-Predicted Toxicities of V-Agents.

V-agents are exceedingly toxic nerve agents. Recently, it was highlighted that V-agents constitute a diverse subclass of compounds with most of them not extensively studied. Although chemical weapons have been banned under the Chemical Weapons Convention (CWC), there is an increased concern for chemical terrorism. Thus, it is important to understand their properties and toxicities, especially since some of these agents are not included in the CWC list. Nonetheless, to achieve this goal, the testing of a huge number of compounds is needed. Alternatively, in silico toxicology offers a great advantage for the rapid assessment of toxic compounds. Here, various in silico tools (TEST, VEGA, pkCSM ProTox-II) were used to estimate the acute oral toxicity (LD50) of different V-agents and compare them with experimental values. These programs underestimated the toxicity of V-agents, and certain V-agents were estimated to be relatively non-toxic. TEST was also used to estimate the physical properties and found to provide good approximations for densities, surface tensions and vapor pressures but not for viscosities. Thus, attention should be paid when interpreting and estimating the toxicities of V-agents in silico, and it is necessary to conduct future detailed experiments to understand their properties and develop effective countermeasures.

Growth of chemical gardens in gaseous acidic atmospheres.

The generation of chemobrionic architectures through slow injection of aqueous silicate solution in gaseous TiCl4 is demonstrated. The tubes were characterized by XRD, SEM and wet chemistry control experiments, and their mechanism of formation was unraveled. These structures serve as laboratory models for calthemites or soda straws.

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.