The Cornified Envelope: A Versatile Contributor to the Epidermal Barrier

“Long considered only as a resistant protective carapace around corneocyte, the cornified envelope and its many protein and lipid components are now known as multipurpose actors of the epidermal barrier with lipid-organizing, antioxidant, and antimicrobial properties.”Michel SimonView Large Image Figure ViewerDownload Hi-res image Download (PPT) “Long considered only as a resistant protective carapace around corneocyte, the cornified envelope and its many protein and lipid components are now known as multipurpose actors of the epidermal barrier with lipid-organizing, antioxidant, and antimicrobial properties.” The cornified envelope (CE) is a lipid-protein cross-linked structure that replaces the plasma membrane at the corneocyte periphery. For a long time, it has been considered only as a mechanical protection of the epidermis against physical aggressions from the external environment. Recent works have shown that several components of CE, including the Late Cornified Envelope (LCE) proteins, hornerin and filaggrin-2, possess broad-spectrum antimicrobial activity. In their recent article in the Journal of Investigative Dermatology, Niehues et al., 2023Niehues H. Rikken G. Kersten F.F.J. Eeftens J.M. van Vlijmen-Willems I.M.J.J. Rodijk-Olthuis D. et al.CYSRT1: an antimicrobial epidermal protein that can interact with late cornified envelope proteins.J Invest Dermatol. 2023; 143: 1498-1508.e7Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar, using yeast two-hybrid screens and co-immunoprecipitation assay, identify an interacting partner of most LCE proteins, a protein of previously unknown function named cysteine-rich tail 1 protein (CYSRT1). The authors show that CYSRT1 gene and protein sequences are conserved among vertebrates, and that CYSRT1 protein is mainly expressed in the stratified squamous epithelia, including the epidermis and proximal digestive tract epithelia, where it is located at the cell periphery in CE. Their data reveals that CYSRT1 expression is up-regulated by epidermal barrier disruption, and, importantly, that CYSRT1 displays anti-microbial activity against Pseudomonas aeruginosa (a major cause of mortality when the epidermal barrier is compromised, e.g., in burn patients). Thus, this study not only proposes a function for CYSRT1 for the first time, but it enlarges the spectrum of bactericidal activities constitutively present in the upper epidermis: a) antibacterial proteins located at the periphery of corneocytes and classically recognized as CE components; b) antimicrobial peptides, such as cathelicidin and beta-defensins, secreted by granular keratinocytes in the extracellular spaces; and c) antimicrobial lipids, including free fatty acids and sphingosine, also present in the intercorneocyte spaces. Moreover, the study highlights the fact that CE should be considered as a multipurpose protective structure conferring mechanical resistance to the epidermis, as a scaffold for intercorneocyte lipid lamellae organization and as the first-line of defense against skin oxidative stress and bacteria invasion (Figure 1). The CE is a 15-nm-thick, insoluble, and cross-linked protein network that results from the formation of ε-(γ-glutamyl)lysine isopeptide bonds catalyzed by the calcium-dependent transglutaminases (EC 2.3.2.13) between numerous precursors, including involucrin, loricrin, desmosomal proteins, and FLG. The CE, together with corneodesmosomes, provides structural integrity to the stratum corneum and is responsible for the physical epidermal barrier function (Candi et al., 2005Candi E. Schmidt R. Melino G. The cornified envelope: a model of cell death in the skin.Nat Rev Mol Cell Biol. 2005; 6: 328-340Crossref PubMed Scopus (1328) Google Scholar). The complexity observed in the CE protein composition is justified by the importance of its role in the skin homeostasis and the necessity to adapt to the skin environment. A monolayer of ω-hydroxyglucosylceramides, ω-hydroxyceramides, and ω-hydroxyacids is covalently attached to the CE outer surface and forms the cornified lipid envelope. This CE part is involved in the structural organization of lipids (cholesterol, ceramides, and free fatty acids) secreted by granular keratinocytes (KCs) in the extracellular space and in the formation of intercellular lamellae. In patients with some forms of autosomal recessive congenital ichthyosis, in canine analogs, and in Alox12b and Pnpla1 knockout mice, the absence of a cornified lipid envelope is paralleled by disorganized extracellular lipid lamellae. The cornified lipid envelope is therefore of importance for the permeability skin barrier function (Wertz, 2021Wertz P.W. Lipid metabolic events underlying the formation of the corneocyte lipid envelope.Skin Pharmacol Physiol. 2021; 34: 38-50Crossref PubMed Scopus (8) Google Scholar). In direct contact with the air and exposed to various environmental aggressions, including UVR and exposure to xenobiotics, the epidermis must deal with a high level of ROS. Besides a rich enzymatic equipment of detoxifying enzymes such as catalase, superoxide dismutases, and peroxidases in the stratum corneum, CE proteins may also play a role in protecting the epidermis from the toxicity of these highly reactive molecules. Indeed, Vermeij et al., 2011Vermeij W.P. Alia A. Backendorf C. ROS quenching potential of the epidermal cornified cell envelope.J Invest Dermatol. 2011; 131: 1435-1441Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar showed that purified CEs, either intact or fragmented by sonication, decrease singlet oxygen lifetime. The researchers then identified SPRR proteins, especially SPRR4, loricrin, FLG-2, and KPRP, all CE proteins, as quenchers of ROS through oxidation of their numerous cysteine residues; this leads to the formation of intramolecular and intermolecular disulfide bridges. In line with this, SPRR4 expression is induced by UV exposure of KCs suggesting an adaptive response to increasing oxidative stress. Accordingly, SPRR2 proteins are induced after activation of NRF2, a transcription factor known to regulate the expression of enzymes that protect the cell against oxidative stress. Recent data indicate that SPRR family proteins, when soluble, may have additional functions at very high ROS levels, localizing to the nucleus where they protect the DNA from chromosomal damage (Zabini et al., 2023Zabini A. Zimmer Y. Medová M. Beyond keratinocyte differentiation: emerging new biology of small proline-rich proteins.Trends Cell Biol. 2023; 33: 5-8Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). In 1994, N. Katunuma and his team were the first to identify cystatin-alpha as a component of CEs that inhibits the growth of a bacterium, namely Staphylococcus aureus. This suggested that CE proteins may have antimicrobial properties (Takahashi et al., 1994Takahashi M. Tezuka T. Katunuma N. Inhibition of growth and cysteine proteinase activity of Staphylococcus aureus V8 by phosphorylated cystatin alpha in skin cornified envelope.FEBS Lett. 1994; 355: 275-278Crossref PubMed Scopus (51) Google Scholar). More than 20 years later, this hypothesis was confirmed by two groups in Nijmegen (The Netherlands) and Kiel (Germany). Niehues and collaborators showed first that LCE3A-C proteins inhibit the growth of a variety of gram-positive and gram-negative bacteria such as S. epidermidis and Cutibacterium acnes. LCE3B and LCE3C proteins are upregulated in psoriasis like antimicrobial peptides, such as beta-defensins. This indicates that LCE3 proteins function like other antimicrobial peptides, which probably explains why deletion of the LCE3B and LCE3C genes is a risk factor for psoriasis. The same authors showed that all LCE proteins possess similar antimicrobial activity, but with differences in killing efficiency and specificity, with LCE6A having the most broad-spectrum activity (Niehues et al., 2022Niehues H. van der Krieken D.A. Ederveen T.H.A. Jansen P.A.M. van Niftrik L. Mesman R. et al.Antimicrobial late cornified envelope proteins: the psoriasis risk factor deletion of LCE3B/C genes affects microbiota composition.J Invest Dermatol. 2022; 142: 1947-1955.e6Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). In parallel, Latendorf et al., 2019Latendorf T. Gerstel U. Wu Z. Bartels J. Becker A. Tholey A. et al.Cationic intrinsically disordered antimicrobial peptides (CIDAMPs) represent a new paradigm of innate defense with a potential for novel anti-infectives.Sci Rep. 2019; 9: 3331Crossref PubMed Scopus (37) Google Scholar revealed that peptides derived from LCE2B, LCE3B, and LCE3C but also from hornerin exhibit at pH 5.5, the pH of skin surface, microbicidal activities directed against various pathogens, such as P. aeruginosa, S. aureus, Corynebacterium simulans, and Candida albicans. In 2022, a group from the University of Texas Southwestern Medical Center (Dallas, TX) proved that both mouse and human SPRR proteins (SPRR1B, SPRR2A, and Sprr1a) displayed potent in vitro antibacterial activity against methicillin-resistant S. aureus, P. aeruginosa, and S. epidermidis but not against the gram-negative bacteria Escherichia coli. Convincingly, these authors showed that mice lacking both Sprr1a and Sprr2a are more susceptible to P. aeruginosa and methicillin-resistant S. aureus skin infections than wild-type mice (Zhang et al., 2022Zhang C. Hu Z. Lone A.G. Artami M. Edwards M. Zouboulis C.C. et al.Small proline-rich proteins (SPRRs) are epidermally produced antimicrobial proteins that defend the cutaneous barrier by direct bacterial membrane disruption.eLife. 2022; 11e76729Crossref Scopus (12) Google Scholar). A limitation of these studies, except that of Takahashi et al., 1994Takahashi M. Tezuka T. Katunuma N. Inhibition of growth and cysteine proteinase activity of Staphylococcus aureus V8 by phosphorylated cystatin alpha in skin cornified envelope.FEBS Lett. 1994; 355: 275-278Crossref PubMed Scopus (51) Google Scholar, is the fact that we do not know whether the CE proteins display their antimicrobial activities only before cross-linking when they are soluble in the granular KC cytoplasm or when they are cross-linked to the envelope. We can imagine that proteases produced by the bacteria themselves could cleave the CE components from the exterior and release active peptides. Accordingly, Latendorf et al. (2019) isolated many hornerin-derived peptides from stratum corneum extracts with antibacterial properties at the nanomolar to micromolar range. The next question is, how do SPRR, LCE, CYSRT1, and other proteins inhibit the growth of and/or kill the bacteria? Mechanistic studies showed that SPRR proteins, particularly human SPRR1B and mouse Sprr1a, exert their bactericidal activity through disruption of the bacterial membrane after binding to the negatively charged part of phospholipids (Zhang et al., 2022Zhang C. Hu Z. Lone A.G. Artami M. Edwards M. Zouboulis C.C. et al.Small proline-rich proteins (SPRRs) are epidermally produced antimicrobial proteins that defend the cutaneous barrier by direct bacterial membrane disruption.eLife. 2022; 11e76729Crossref Scopus (12) Google Scholar), as is known for many antimicrobial peptides, including cathelicidins and beta-defensins. In addition, Latendorf et al., 2019Latendorf T. Gerstel U. Wu Z. Bartels J. Becker A. Tholey A. et al.Cationic intrinsically disordered antimicrobial peptides (CIDAMPs) represent a new paradigm of innate defense with a potential for novel anti-infectives.Sci Rep. 2019; 9: 3331Crossref PubMed Scopus (37) Google Scholar observed that the hornerin-derived peptides were hydrophilic, positively charged at pH 5.5, and contained a high proportion of disorder-promoting amino acids (Gly, His, Lys, SER, Arg, Gln, Asp, Glu, and Pro) and a very low proportion of order-promoting hydrophobic amino acids (Leu, Val, Met, Iso, Tyr, Trp, and Phe). The bactericidal activity seemed to be independent of the exact amino acid sequence by itself. Similar peptide properties are shared by other CE proteins with antimicrobial properties, namely FLG, FLG-2, and LCE proteins. These peptides were collectively called cationic intrinsically disordered antimicrobial peptides. Do additional CE proteins with such an amino acid content bias, for example, involucrin, also display antibacterial activity? This merits further investigation. Findings of the same group show that the HRNR-derived peptides pass through the E. coli membrane and exert bactericidal activity by targeting the ribosomes (Gerstel et al., 2018Gerstel U. Latendorf T. Bartels J. Becker A. Tholey A. Schröder J.M. Hornerin contains a Linked Series of Ribosome-Targeting Peptide antibiotics.Sci Rep. 2018; 8: 16158Crossref PubMed Scopus (19) Google Scholar). In their publication in the Journal of Investigative Dermatology, Niehues et al., 2023Niehues H. Rikken G. Kersten F.F.J. Eeftens J.M. van Vlijmen-Willems I.M.J.J. Rodijk-Olthuis D. et al.CYSRT1: an antimicrobial epidermal protein that can interact with late cornified envelope proteins.J Invest Dermatol. 2023; 143: 1498-1508.e7Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar show that CYSRT1 is a very cationic protein containing 55.6% of disorder-promoting amino acids and only 18.1% of order-promoting amino acids. Whether CYSRT1 and HRNR-derived peptides share a similar antibacterial mode of action or whether CYSRT1 binds metals, including Zn2+, like other antimicrobial peptides, should be tested. How CYSRT1 expression is controlled is poorly understood. The expression of SPRR2 proteins is upregulated in lung and intestinal allergic inflammation through Th2-type cytokines. Apparently, this is not the case in atopic dermatitis. This discrepancy has to be confirmed and explained. Evidence was also published to suggest that covalently bound ω-hydroxyceramides, the lipid part of CEs, serve as a reservoir for free sphingosine after ceramidase hydrolysis. Because sphingosine is a potent broad-acting antimicrobial molecule (Wertz, 2021Wertz P.W. Lipid metabolic events underlying the formation of the corneocyte lipid envelope.Skin Pharmacol Physiol. 2021; 34: 38-50Crossref PubMed Scopus (8) Google Scholar), this may further enlarge the antimicrobial repertoire at the skin surface used to defend the host against infection. The contribution of each component of the CE to the antimicrobial activities should be studied in vivo. It would be also important to know to what extent one can compensate for the absence of the other. Studies with animal models and human skin substitutes would be useful. A putative connection between the detoxifying properties and the antimicrobial capacity of CE would probably be the subject of studies in the future. Because there is a tight interplay between the skin surface microbiota and the cutaneous immune system, these observations may have implications for our understanding of and proposed treatment strategies for skin inflammatory diseases; therefore, these warrant detailed investigation. Nathalie Jonca: http://orcid.org/0000-0003-3602-0102 Michel Simon: http://orcid.org/0000-0003-3655-6329 The authors state no conflict of interest. The authors are supported by the Toulouse University, Institut national de la santé et de la recherche médicale, and Centre national de la recherche scientifique. CYSRT1: An Antimicrobial Epidermal Protein that Can Interact with Late Cornified Envelope ProteinsJournal of Investigative DermatologyVol. 143Issue 8PreviewLate cornified envelope (LCE) proteins are small cationic epidermal proteins with antimicrobial properties, and the combined deletion of LCE3B and LCE3C genes is a risk factor for psoriasis that affects skin microbiome composition. In a yeast two-hybrid screen, we identified CYSRT1 as an interacting partner of members of all LCE groups except LCE6. These interactions were confirmed in a mammalian cell system by coimmunoprecipitation. CYSRT1 is a protein of unknown function that is specifically expressed in cutaneous and oral epithelia and spatially colocalizes with LCE proteins in the upper layers of the suprabasal epidermis. Full-Text PDF Open Access