Cuts by Caspase-14 Control the Proteolysis of Filaggrin

Although mutations in the filaggrin gene (FLG) have been shown to be associated with ichthyosis vulgaris and atopic dermatitis, the function and regulation of filaggrin remain incompletely understood. In this issue, Hoste et al. report that filaggrin is directly cleaved by caspase-14. Acting in concert with other proteases, caspase-14 controls the breakdown of filaggrin to free amino acids and amino acid derivatives that contribute to the hydration and UVB absorption capacity of the stratum corneum. These findings identify a new layer of complexity in the regulation of epidermal barrier function

Convergent evolution of cysteine-rich proteins in feathers and hair

Background Feathers and hair consist of cornified epidermal keratinocytes in which proteins are crosslinked via disulfide bonds between cysteine residues of structural proteins to establish mechanical resilience. Cysteine-rich keratin-associated proteins (KRTAPs) are important components of hair whereas the molecular components of feathers have remained incompletely known. Recently, we have identified a chicken gene, named epidermal differentiation cysteine-rich protein (EDCRP), that encodes a protein with a cysteine content of 36%. Here we have investigated the putative role of EDCRP in the molecular architecture and evolution of feathers. Results Comparative genomics showed that the presence of an EDCRP gene and the high cysteine content of the encoded proteins are conserved among birds. Avian EDCRPs contain a species-specific number of sequence repeats with the consensus sequence CCDPCQ(K/Q)(S/P)V, thus resembling mammalian cysteine-rich KRTAPs which also contain sequence repeats of similar sequence. However, differences in gene loci and exon-intron structures suggest that EDCRP and KRTAPs have not evolved from a common gene ancestor but represent the products of convergent sequence evolution. mRNA in situ hybridization demonstrated that chicken EDCRP is expressed in the subperiderm layer of the embryonic epidermis and in the barbule cells of growing feathers. This expression pattern supports the hypothesis that feathers are evolutionarily derived from the subperiderm. Conclusions The results of this study suggest that convergent sequence evolution of avian EDCRP and mammalian KRTAPs has contributed to independent evolution of feathers and hair, respectively.(VLID)486031

Evolutionary origin and diversification of epidermal barrier proteins in amniotes.

The evolution of amniotes has involved major molecular innovations in the epidermis. In particular, distinct structural proteins that undergo covalent cross-linking during cornification of keratinocytes facilitate the formation of mechanically resilient superficial cell layers and help to limit water loss to the environment. Special modes of cornification generate amniote-specific skin appendages such as claws, feathers, and hair. In mammals, many protein substrates of cornification are encoded by a cluster of genes, termed the epidermal differentiation complex (EDC). To provide a basis for hypotheses about the evolution of cornification proteins, we screened for homologs of the EDC in non-mammalian vertebrates. By comparative genomics, de novo gene prediction and gene expression analyses, we show that, in contrast to fish and amphibians, the chicken and the green anole lizard have EDC homologs comprising genes that are specifically expressed in the epidermis and in skin appendages. Our data suggest that an important component of the cornified protein envelope of mammalian keratinocytes, that is, loricrin, has originated in a common ancestor of modern amniotes, perhaps during the acquisition of a fully terrestrial lifestyle. Moreover, we provide evidence that the sauropsid-specific beta-keratins have evolved as a subclass of EDC genes. Based on the comprehensive characterization of the arrangement, exon-intron structures and conserved sequence elements of EDC genes, we propose new scenarios for the evolutionary origin of epidermal barrier proteins via fusion of neighboring S100A and peptidoglycan recognition protein genes, subsequent loss of exons and highly divergent sequence evolution

DNase1L2 Degrades Nuclear DNA during Corneocyte Formation

The removal of keratinocyte (KC) nuclear DNA by deoxyribonucleases (DNases) is an important step in the formation of normal stratum corneum (SC). However, the molecular identity of the DNA-degrading enzymes has so far remained elusive. Here we show that the endonuclease DNase1-like 2 (DNase1L2) is preferentially expressed in the epidermis and that its expression correlates with terminal differentiation of KC in vitro and in vivo. In biopsies of normal skin, DNase1L2 mRNA was regularly found in suprabasal KC and DNase1L2 protein was highly abundant in the stratum granulosum. In contrast to normal skin, DNase1L2 expression was downregulated in parakeratotic epidermis such as in psoriatic lesions. When DNase1L2 gene expression was knocked down by small interfering RNA in a human skin equivalent model, nuclei were maintained through all layers of the SC. Taken together, our data demonstrate that DNase1L2 plays an essential role in DNA degradation during terminal differentiation of epidermal KC

DNase 2 Is the Main DNA-Degrading Enzyme of the Stratum Corneum

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA

In situ labeling of DNA reveals interindividual variation in nuclear DNA breakdown in hair and may be useful to predict success of forensic genotyping of hair

Hair fibers are formed by keratinocytes of the hair follicle in a process that involves the breakdown of the nucleus including DNA. Accordingly, DNA can be isolated with high yield from the hair bulb which contains living keratinocytes, whereas it is difficult to prepare from the distal portions of hair fibers and from shed hair. Nevertheless, forensic investigations are successful in a fraction of shed hair samples found at crime scenes. Here, we report that interindividual differences in the completeness of DNA removal from hair corneocytes are major determinants of DNA content and success rates of forensic investigations of hair. Distal hair samples were permeabilized with ammonia and incubated with the DNA-specific dye Hoechst 33258 to label DNA in situ. Residual nuclear DNA was visualized under the fluorescence microscope. Hair from some donors did not contain any stainable nuclei, whereas hair of other donors contained a variable number of DNA-positive nuclear remnants. The number of DNA-containing nuclear remnants per millimeter of hair correlated with the amount of DNA that could be extracted and amplified by quantitative PCR. When individual hairs were investigated, only hairs in which DNA could be labeled in situ gave positive results in short tandem repeat typing. This study reveals that the completeness of DNA degradation during cornification of the hair is a polymorphic trait. Furthermore, our results suggest that in situ labeling of DNA in hair may be useful for predicting the probability of success of forensic analysis of nuclear DNA in shed hair

Assessing autophagy in archived tissue or how to capture autophagic flux from a tissue snapshot

Autophagy is a highly conserved degradation mechanism that is essential for maintaining cellular homeostasis. In human disease, autophagy pathways are frequently deregulated and there is immense interest in targeting autophagy for therapeutic approaches. Accordingly, there is a need to determine autophagic activity in human tissues, an endeavor that is hampered by the fact that autophagy is characterized by the flux of substrates whereas histology informs only about amounts and localization of substrates and regulators at a single timepoint. Despite this challenging task, considerable progress in establishing markers of autophagy has been made in recent years. The importance of establishing clear-cut autophagy markers that can be used for tissue analysis cannot be underestimated. In this review, we attempt to summarize known techniques to quantify autophagy in human tissue and their drawbacks. Furthermore, we provide some recommendations that should be taken into consideration to improve the reliability and the interpretation of autophagy biomarkers in human tissue samples.This work was supported by grants from the Bernese Cancer League, “Stiftung für klinisch-experimentelle Tumorforschung”, and the Werner and Hedy Berger-Janser Foundation for Cancer Research (to M.H.); by Institute of Health Carlos III (ISCIII) and FEDER funds from the EU (PI14/01085 and PI17/00093) and supported by Miguel Servet contract by ISCIII and FSE funds (CPII16/00023) (to M.M.); from the Spanish Ministry of Science, Innovation and Universities (RTI2018-096748-B-100 to N.A.); from the University Professor Training Fellowship, Ministry of Science, Innovation and University, Government of Spain (FPU17/00026) (to P.C.O); from the ISCIII (PI16/00090 and PI19/01266) and the Andalusian Government (Consejería de Igualdad, Salud y Políticas Sociales, PI-0198-2016) for their financial support, and from the Biomedical Research Network Center for Liver and Digestive Diseases (CIBERehd) founded by the ISCIII and co-financed by European Development Regional Fund (EDRF) “A way to achieve Europe” for their financial support (to J.M.), from Breakthrough Cancer Research, Ireland funding (to S.L.M); from the PI18/00442 grant integrated into the State Plan for R & D + I2013-2016 and funded by the ISCIII and the ERDF, a way to make Europe (to G.V.); from the Luxembourg National Research Fund (C18/BM/12670304/COMBATIC to B.J.); from the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, by the European Regional Development Fund (FEDER), through the Competitiveness Factors Operational Programme (COMPETE) (NORTE-01-0145-FEDER-000013) and from the projects POCI-01-0145-FEDER-028159 and POCI-01-0145-FEDER-030782 by FEDER, through the COMPETE (to P.L.); from National funds, through the Foundation for Science and Technology (FCT) (to P.L.); from ARRS—the Slovenian research agency, programme P1-0140: Proteolysis and its regulation (led by B. Turk) (to E.Ž.); from the Swiss Cancer Research (KFS-3360-02-2014) (to A.P, and M.P.T.) (KFS-3409-02-2014), and the Swiss National Science Foundation (31003A_173219) (to M.P.T.)

Multifaceted role of TREX2 in the skin defense against UV-induced skin carcinogenesis

TREX2 is a 3'-DNA exonuclease specifically expressed in keratinocytes. Here, we investigated the relevance and mechanisms of TREX2 in ultraviolet (UV)-induced skin carcinogenesis. TREX2 expression was up-regulated by chronic UV exposure whereas it was de-regulated or lost in human squamous cell carcinomas (SCCs). Moreover, we identified SNPs in the TREX2 gene that were more frequent in patients with head and neck SCCs than in healthy individuals. In mice, TREX2 deficiency led to enhanced susceptibility to UVB-induced skin carcinogenesis which was preceded by aberrant DNA damage removal and degradation as well as reduced inflammation. Specifically, TREX2 loss diminished the up-regulation of IL12 and IFNγ, key cytokines related to DNA repair and antitumor immunity. In UV-treated keratinocytes, TREX2 promoted DNA repair and passage to late apoptotic stages. Notably, TREX2 was recruited to low-density nuclear chromatin and micronuclei, where it interacted with phosphorylated H2AX histone, which is a critical player in both DNA repair and cell death. Altogether, our data provide new insights in the molecular mechanisms of TREX2 activity and establish cell autonomous and non-cell autonomous functions of TREX2 in the UVB-induced skin response