Keratins are the major structural protein of vertebrate epidermis, constituting up to 85% of a fully differentiated keratinocyte (Fuchs 1995). Keratins belong to a superfamily of intermediate filament (IF) proteins that form alpha-helical coiled-coil dimers, which associate laterally and end-to-end to form approximately 10 nm diameter filaments. Keratin filaments are heteropolymeric, formed from equal amounts of acidic type I and basic /neutral type 2 keratins. Humans have 54 keratin genes (Schweitzer et al. 2006). They have highly specific expression patterns, related to the epithelial type and stage of differentiation. Roughly half of human keratins are specific to hair follicles (Langbein & Schweizer 2005). Keratin filaments bundle into tonofilaments that span the cytoplasm and bind to desmosomes and other cell membrane structures (Waschke 2008). This reflects their primary function, maintaining the mechanical stability of individual cells and epithelial tissues (Moll et al. 2008).
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Lipases are enzymes that hydrolyse dietary lipids such as fats, oils and triglycerides. The majority of human lipases are secreted by the pancreas and function mainly in the digestive system. Lipase members K, M and N (LIPK, M and N), however, all appear to play a role in the last step of keratinocyte differentiation where they are proposed to hydrolyse triglycerides to free fatty acids and glycerol which is essential to stratum corneum hydration (Toulza et al. 2007).
The first step in keratin assembly is the formation of coiled-coil heterodimers consisting of an acidic type I keratin and a basic or neutral type II keratin (Coulombe & Fuchs 1990, Hatzfeld & Webber 1990, Steinert 1990). In humans, the type I keratins are K9-24, K25-28, which are specific to the inner root sheath of hair, and the hair-specific keratins K31-K38. The type II keratins are K1-8, K71-80 and K81-86 (Bragula & Homberger 2009). Binding between dimer pairs is remarkably strong and can form even in 9M urea (Coulombe & Fuchs 1990). The ~50 nm long middle rod region of keratin protein aligns with its partner in a parallel orientation (Pauling & Corey 1953, Hanukoglu & Fuchs 1983, Parry et al. 1985, Steinert et al. 1994). The rod region is sufficient to form a heterodimer and subsequent tetramers, but the assembly of keratin filaments requires the non-helical head and tail regions (Wilson et al. 1992). The assembly of rod domain heterodimers has asymmetric salt bridges, hydrogen bonds and hydrophobic contacts, and surface of the heterodimer interface exhibits a notable charge polarization (Lee et al. 2012).
In vitro, virtually any type I keratin can dimerize with any type II keratin, leading to the formation of 10-nm long filaments (Franke et al. 1983, Hatzfeld et al. 1987). In vivo, the composition of keratin heterodimers is probably determined by expression. Differing keratin combinations are not characteristic of entire tissues, but probably confer particular functional properties to cells and tissue regions (Bragulla & Homberger 2009). Certain combinations are characteristic of a cell type, e.g. K18/K8 in simple epithelia. At least some keratins can be replaced with no loss of functionality of the keratin filament, e.g. K1/K10, K1/K9, K2/K9, K2/K10 in epithelia (Coulombe & Omary 2002). Suprabasal cells of stratified epithelia express different keratin pairs in different tissues, e.g. skin epidermis predominantly expresses K1/K10, the anterior corneal epithelium produces K3/K12, esophageal epithelium produces K4/K13 (Eichner & Kahn 1990) while hyperproliferative suprabasal cells are characterized by K6/K16 (Sun 2006).
Keratin dimers associate in antiparallel orientation to form tetramers (Wood & Inglis 1984, Quinlan et al. 1984). The rod regions of heterodimers align, but alignment of the head and tail regions differs between keratin types. Soft-keratinizing-cornifying cell keratins are slightly out of phase, by 7-8 amino acids, while keratin heterodimers of hard-keratinizing-cornifying cells are in register and consequently there is no overlap between the head and tail domains when a tetramer is formed (Jones et al. 1997). Protofilament tetramers have a diameter of about 2 nm (Aebi et al. 1983, Eicher & Kahn 1990). Heterodimers and tetramers represent the stable building blocks of larger octamers (Herrmann & Abei 2004) and Unit Length Filaments (ULFs), which have a diameter of 20 nm (Parry et al. 2001, Hermann et al. 2007). The tetramers are stabilized by a hydrophobic stripe exposed at the surface of coiled-coil keratin heterodimers (Bernot et al. 2005).
Mammalian keratin filaments are produced by the lateral and longitudinal aggregation of subunits, such as tetrameric protofilaments and octameric protofibrils (Aebi et al. 1983). The extent of aggregation depends on the pH and osmolarity of the surrounding cytoplasm (Yamada et al. 2002, Magin et al. 2007). Filaments have a cross-section of 32 keratin molecules (Jones et al. 1997).
Mammalian keratins form soluble short full-width filaments called unit length full-width particles (Parry et al. 2007), unit length filaments (ULFs) (Herrmann et al. 2002) or intermediate filament-like particles (Steinert 1991). These are formed by the lateral association of tetramers. ULFs are ~ 70 nm long, with a diameter of ~20 nm. The diameter shrinks during formation of filaments (Parry et al. 2001). X-ray diffraction suggests that ULFs are tube-like structures formed from eight tetramers in non-cornified cells (Parry et al. 2007). In cornified hair cells, the tetramers are thought to be arranged in a seven-member ring, with an eighth in the centre (Parry et al. 2007).
Keratin filaments can rapidly disassemble and reassemble, allowing flexibility for the cytoskeleton. Keratin building blocks accumulate at the cell periphery near focal adhesions. Polymerization is regulated by signaling molecules, e.g. heat shock proteins, 14-3-3 proteins, kinases and phosphatases (Magin et al. 2007, Kayser et al. 2003).
Keratin filaments bind cell-cell adhesion complexes such as desmosomes and hemidesmosomes, transferring mechanical forces between cells and maintaining cytoskeletal integrity (Hanakawa et al. 2002). The stability of the tonofilament-desmosome interaction depends, in part, on the type of keratin present in the cell (Loschke et al. 2016).
At the ultrastructural level, desmosomes appear as electron dense discs approximately 0.2-0.5 μm in diameter, which assemble in a mirror-image arrangement at cell-cell interfaces (North et al. 1999, Al-Amoudi et al. 2011, Kowalczyk & Green 2013). Large bundles of filaments extend from the nuclear surface and cell interior out towards the plasma membrane, where they attach to desmosomes by interweaving with the cytoplasmic plaque of the adhesive complex. The head domains of keratins bind the tail domains of desmosomal cadherin molecules such as plakoglobin (Dusek et al. 2007), plectin, periplakin, envoplakin and desmoplakin (Bornslaeger et al. 1996, Kazerounian et al. 2002), thereby anchoring the cytoskeleton to the cell membrane.
The five major desmosomal components are the desmosomal cadherins, represented by desmogleins (DSG1-4) and desmocollins (DSC1-3), the armadillo family members, plakoglobin (PG) and the plakophilins (PKP1-3), and the plakin linker protein desmoplakin (DSP), which anchors the intermediate keratin filaments.
Certain adhesion complex proteins are expressed only when cornification commences. These include desmoglein-1, desmocollin-1, envoplakin, periplakin, plakophilin-1 and corneodesmosin (Candi et al. 2005). This expression is associated with changes in desmosome mophology whereby the cytoplasmic plaque integrates with the cornified envelope (Serre et al. 1991, Simon et al. 2001). Deregulation of desmosome formation can lead to degenerative cutaneous diseases (Brooke et al. 2012, Cirillo 2014).
Hair consists of three major structural components: the cuticle, the cortex and the central medulla. Approximately 90% of cortical cells contain longitudinally arrayed keratin filaments. These filaments have a surrounding matrix that contains keratin-associated proteins (KAPs) that are involved in the formation of cornified, resilient hair shafts (Shimomura & Ito 2005, Lee et al. 2006, Harland et al., 2010, Gong et al. 2016). KAPs forming extensive disulfide cross-links with keratin filaments (Marshall et al. 1991).
The proliferative cells that give rise to hair fibres are located in the bulb at the base of the hair follicle. As they leave the germinative compartment, trichocytic differentiation begins and in matrix, cuticular, and cortical cells, the genes for keratins and KAPs (KRTAPs) are expressed. In the lower and middle cortex, keratin filaments are embedded in a matrix that consists of KAPs. Based on amino acid composition, three classes of KAPs have been described, the high sulfur KAPs (<30 mol % cysteine content), the ultrahigh sulfur KAPs (>30 mol % cysteine content), and the high tyrosine/glycine KAPs (Rogers et al. 2001). KAPs can be divided into subfamilies based on amino acid composition and phylogenetic relationships (Wu et al. 2008). Humans have approximately 100 KAP genes (Wu et al. 2008). Compared to the conserved structure and modality of keratins within mammals, KAP genes differ significantly between species and are likely to explain the variety of characteristics seen in hard keratin appendages such as feathers, claws, scales and hair (Wu et al. 2008, Khan et al. 2014). KAPs are crucial for the assembly of keratin intermediate filaments into arrays and likely to affect attributes of hair such as strength, rigidity and chemical inertness (Parry & Steinert 1999, Koster et al. 2015).
During cornification a network of keratin intermediate filaments (KIF) and filaggrin (FLG) becomes crosslinked to the cornified envelope (CE). This facilitates the collapse and flattening of cells in the outermost stratum corneum to produce squames (Dale et al. 1978, Mack et al. 1993, Candi et al. 2005, Gruber et al. 2011).
Envoplakin (EVPL) is insoluble under physiological conditions but soluble as a heterodimer with periplakin (PPL) (Kalinin et al. 2004). The heterodimers provide a firm but flexible structures (Al-Jassar et al. 2013). EVPL and PPL deposition and crosslinking are amongst the earliest events of cornification (Kalinin et al. 2002, Candi et al. 2005). They partially colocalize with desmosomal proteins and keratin intermediate filaments (Ruhrberg et al. 1996, DiColandrea et al. 2000), linking the cornified envelope to desmosomes and keratin filaments (Ruhrberg et al. 1996, 1997). PPL also associates with cortical actin at the interdesmosomal plasma membrane (DiColandrea et al. 2000, Groot et al. 2004).
The current model of cornified envelope (CE) formation suggests that crosslinking between envoplakin (EVPL), periplakin (PPL), involucrin (IVL) and small proline-rich proteins (SPRs) results in the formation of a layer along the entire inner surface of the plasma membrane, including desmosomes, forming a scaffold to which other precursors are added to form the mature CE (Steinert & Marekov 1999, Kalinin et al. 2002, Candi et al. 2001).
Transglutaminases (TGs) are believed to mediate the intramolecular bonds involved in CE formation. They catalyze inter-protein bond formation by forming a thiolester acyl-enzyme intermediate and subsequently transferring the acyl residue to a primary amine (Folk & Finlayson 1977, Folk 1980). The amine acceptor is generally provided by the epsilon-amino group of a protein-bound lysine and the link formed is an N6-(gamma-glutamyl)lysine isopeptide bond.
CE assembly is thought to be initiated on the inner face of the plasma membrane between desmosomes by the cross-linking of involucrin to itself, to envoplakin and perhaps to periplakin (Steinert & Marekov 1999). The extent of homo- and heterologous cross-linking varies as the CE matures. In the immature CE, EVPL, IVL, SPR1, and SPR2 are largely cross-linked to themselves; EVPL-IVL and IVL-SPR crosslinks are common while cross-links between desmoplakin (DSP) and IVL or DSP and EVPL are not. Later there are many more cross-links between DSP and IVL, DSP and EVL, or IVL and type II keratins. Loricrin (LOR) cross-linking to other protein partners appears later.
Transglutaminase-1 (TGM1) can crosslink IVL (Simon & Green 1988, Nemes et al. 1999), LOR (Candi et al. 2001), SPR3 (Steinert et al. 1999) and is thought to be responsible for EVPL crosslinking to itself and to IVL (Steinert & Marekov 1999). TGM5 can catalyse homo-crosslinking in LOR, SPR1, SPR2, and IVL, and hetero-crosslinks between LOR-SPR3 (Candi et al. 2001).
Transglutaminase-1 (TGM1) and involucrin (IVL) are expressed shortly after envoplakin and periplakin. TGM1 associates with the plasma membrane via C14-16 fatty-acid adducts on its C-terminus (Steinert et al. 1996). IVL deposition precedes that of most other cornified envelope proteins (Nemes & Steinert 1999). It can bind the plasma membrane in a calcium and phosphatidyl-serine dependent manner, where it becomes a substrate for membrane-bound TGM1 and TGM5 (Nemes et al. 1999, Candi et al. 2001).
Lamellar bodies (LBs) ) are lipid-rich organelles produced by keratinocytes and secreted to form an impermeable water barrier (Feingold & Elias 2014). The lipids in LBs contain phospholipids, glucosylceramides, sphingomyelin and cholesterol (Feingold 2007). These lipids, some of which are keratinization specific, are synthesized and accumulate in the trans Golgi apparatus, budding off as LBs that accumulate in the granular layer (Wertz & van den Bergh 1998). ). LB lipids also organize into characteristic intercellular lamellae (Kalinin et al. 2002). LBs fuse with the plasma membrane (Schmitz & Muller 1991, Chattopadhyay et al. 2003) delivering lipids which become ester-linked to involucrin and probably other cornified envelope proteins by TG1 (Nemes et al. 1991) and possibly TG5 (Candi et al. 2005), forming a monomolecular layer termed the lipid envelope. Eventually these lipids replace the plasma membrane lipid bilayer, which is reabsorbed. Extracellularly, the LB lipids are further metabolized to have a unique composition and are 50% ceramides, 25% cholesterol, and 15% free fatty acids (Feingold 2007).
The inital scaffold of the cornified envelope (CE) is reinforced by the inclusion of loricrin (LOR) and small proline-rich proteins (SPRs), which together comprise about 75% of the total mass of the CE. Other proteins include filaggrin (FLG) (8%), elafin (6%), cystatin A (5%), involucrin (IVL) and keratin intermediate filaments (KIFs) (about 2% each) (Steinert & Marekov 1995). Other minor proteins include repetin (RPTN), trichohyalin (TCHH) and elafin (PI3) (Steinert & Marekov 1997, Steinert et al. 1998).
LOR is poorly soluble in vivo, while SPRs are very soluble. Both are preferred substrates of cytosolic transglutaminase-3 (TGM3) (Candi et al. 1999, Steinert et al. 1999, Tarcsa et al. 1999), which suggests that TGM3 may cross-link LOR and SPRs to create soluble complexes that are more easily translocated to the cell periphery (Kalinin et al. 2002). These cross-linked oligomers are good substrates for TGM1 (Candi et al. 1999, Steinert et al. 1999) which may link the LOR-SPR complexes to the CE scaffold. LOR can also be crosslinked by TGM5 (Candi et al. 2001). SPR content varies in epithelia from different body sites and increasing SPR content correlates with mechanical requirements of the tissue (Steinert et al. 1998). In humans LOR is initially deposited in the granular layer of the epidermis in keratohyalin granules, intermixed with profilaggrin (Yoneda & Steinert 1993). These are encoded in a linked 'Epidermal Differentiation Complex.' (Kypriotou et al. 2012, Niehues et al. 2016).
As the main component of the CE (Steinert & Marekov 1995), LOR is thought to function as the main reinforcement protein. LOR proteins are extensively crosslinked through isopeptide bonds but also crosslinked to SPRs, which may function as bridging proteins between LOR molecules (Candi et al. 2005). LOR can also form crosslinks with keratin and filaggrin (Steinert & Marekov 1995). CE crosslinking involves TGM1, TGM3 and TGM5 (Lorand & Graham 2003). The type-II keratin chains (K1, K2e and K5) are crosslinked at a specific Lys residue that is located in a conserved region of the V1 subdomain of the head domain (Steinert & Marekov 1995). IVL can be crosslinked by TGM1, which preferentially crosslinks Gln495 and Gln496 (Simon & Green 1998). In vitro, LOR is a substrate for TGM1-3 and 5 (Candi et al. 1995). In the epidermis, TGM1, TGM5 and TGM3 are believed to crosslink LOR sequentially; an initial attachment by TGM1 and 5 forms interchain crosslinks followed by a compaction process that involves TGM3 (Candi et al. 2005). SPRs are also TGM substrates, particularly TGM3 (Candi et al. 1999, Tarcsa et al. 1998, Steinert et al. 1999).
FLG binds KIFs, aggregating them into tight bundles. As a component of the CE, FLG 'glues' KIFs to the CE and coordinates the structure of cornifying cells (Steinert & Marekov 1995, Candi et al. 2005).
Envoplakin:periplakin heterodimers (EVPL:PPL) bind the plasma membrane. In vitro, EVPL:PPL can bind lipid vesicles in response to increasing Ca2+, suggesting that translocation and binding to the plasma membrane is regulated by elevation of intracellular Ca2+ (Kalinin et al. 2004).
Late envelope proteins or late cornified envelope proteins (LCEs) are a family of 18 proteins that are expressed after assembly of the cornified envelope (CE) is advanced (Marshall et al. 2000, Kypriotou et al. 2012). They are incorporated into the CE late in the process of envelope maturation during epidermal differentiation. They are probable substrates for epidermal transglutaminases and proposed to link CE proteins and mediate differences in barrier quality, perhaps through interaction with cytoplasmic components of the cornified cell (Marshall et al. 2001).
Human LCEs fall into distinct structural groups, encoded by genes which form clusters on the genome at 1q21 (Marshall et al. 2001, Niehues et al. 2016). Group 1 are expressed predominantly in epidermis. Group 4 (LEP 13-17) have highest expression in internal epithelia (Wang et al. 2001, Marshall et al. 2001).
Kazrin (KAZN) is an evolutionarily-conserved cytoplasmic and nuclear protein that was identified as a binding partner of periplakin (PPL), a component of epidermal desmosomes (DS) and the cornified envelope (CE) (Groot et al. 2004).
Kazrin has at least 5 different isoforms. Overexpression of the short isoform kazrinE stimulates the terminal differentiation of cultured human keratinocytes and is associated with a reduction in F-actin content, disruption of DS assembly, and changes in cell shape. Overexpression of activated RhoA rescues the effects on cell shape and adhesion. Conversely, knockdown of the longest isoform kazrinA impairs terminal differentiation, independently of RhoA activity (Sevilla et al. 2008a). KazrinE colocalizes with stabilized microtubules in differentiating keratinocytes (Nachat et al. 2009). All KAZN isoforms can form complexes with one another (Nachat et al. 2009), suggesting that like periplakin and envoplakin, it may form part of the cortical scaffold that integrates the actin cytoskeleton with DS (Ruhrberg et al. 1997, Kalinin et al. 2001, Groot et al. 2004). In Xenopus embryos, depletion of endogenous KAZN results in striking defects in axial elongation, muscle and notochord differentiation, and epidermal morphogenesis. These effects are believed to be due to disruption of cell-cell junctions (Sevilla et al. 2008b, Cho et al. 2010). However, mice with a knockout that removes exons 5-15 of KAZN had normal epidermal morphogenesis and homeostasis (Chhatriwala et al. 2012).
In fully cornified cells, filaggrin is degraded into free amino acids. This high concentration of hydrophillic amino acids is essential for water retention and contributes to the osmolarity, and consequently the flexibility of the cornified layer (Candi et al. 2005). Filaggrin monomers are a direct target for cleavage by the aspartate-specific protease caspase 14 (Denecker et al. 2007, 2008, Hoste et al. 2011, Eckhart & Tschachler 2011). Proteases able to process profilaggrin into fillagrin in vitro include microbial ST14 (Profillagrin endopeptidase 1, PEP1), CAPN1 (mu-calpain), furin, PACE4 and matriptase MT-SP1 (Reising et al. 1995, Yamazaki et al. 1997, Pearton et al. 2001, List et al. 2003, Candi et al. 2005), but these proteases do not appear to have a role in the degradation of filaggrin that occurs at a late stage in keratinization.
PERP (p53 effector related to PMP-22) is a p53/p63 target gene involved in DNA damage-induced apoptosis (Flores et al. 2002). It is a tetraspan membrane protein,distantly related to members of the claudin/PMP-22/EMP family of four-pass membrane proteins (Attardi et al. 2000). It has an epithelial-specific expression pattern during embryogenesis and localizes to desmosomes. Perp -/- knockout mice exhibit numerous desmosomal structural defects, suggesting a role for Perp in promoting the stable assembly of desmosomal adhesive complexes (Ihrie et al. 2005).
Corneodesmosomes (CDS) are an ultrastructurally modified form of desmosomes (DS) (Chapman & Walsh 1990). When DS are transformed into CDS between the stratum granulosum and the stratum corneum, the desmoglea loses its trilamellar structure and becomes homogeneously electron dense. On the cytoplasmic side, the attachment plaque (desmosomal plaque) becomes incorporated into the cornified cell envelope (CE). Keratin filaments are connected to the attachment plaque in DS; this association is no longer visible in CDS.
Like DS, desmoglein and desmocollin constitute the extracellular parts of CDS (Simon et al. 1997), but there is an additional unique extracellular component known as corneodesmosin (CDSN). CDSN is a 52- to 56-kDa glycoprotein produced by keratinocytes that is incorporated into the desmoglea of DS shortly before their transformation into CDS during cornification (Serre et al. 1991). It is stored and secreted by Lamellar bodies. After secretion CDSN localizes to the extracellular structures of CDS and covalently cross-links to the CE. This step coincides with the morphological transformation of DS into CDS. In vitro studies suggest that CDSN mediates homophilic binding to counterparts on adjacent corneocytes (Ishida-Yamamoto & Kishibe 2011). Cleavage of desmoglein, desmocollin and CDSN is a key step in desquamation.
In differentiating keratinocytes, fusion of lamellar body (LB) membranes with the plasma membrane enriches the plasma membrane with lipids including omega-OH-ceramides. Their fatty acid chains are long enough to span the lipid bilayer, so that the omega-OH projects into the cell. In vitro data have shown that the membrane-anchored transglutaminase 1 enzyme can covalently esterify these ceramides onto glutamine residues of cornified envelope scaffold proteins (Nemes et al. 1999). Eventually, the ceramides replace the bilayer plasma membrane and are thought to serve to interdigitate with and organize the extracellular lipids into characteristic lamellae (Kalinin et al. 2001).
At low pH, the complex of the D8D9 fragment of SPINK5 (Serine protease inhibitor Kazal-type 5, also known as LEKTI, Lympho-epithelial Kazal-type-related inhibitor), consisting of residues 490 - 624 of the full-length protein, and KLK5 (Kallikrein-5) dissociates, releasing active KLK5 protease. In normal skin, this event occurs extracellularly in upper layers of the skin (Deraison et al. 2007).
The D8D9 fragment of SPINK5 (Serine protease inhibitor Kazal-type 5, also known as LEKTI, Lympho-epithelial Kazal-type-related inhibitor), consisting of residues 490 - 624 of the full-length protein, binds to KLK5 (Kallikrein-5), inactivating the latter. At neutral pH, complex formation is effectively irreversible. In normal skin, this event occurs extracellularly in the stratum corneum of the skin. As the complex is carried into layers nearer the surface of the skin, falling pH triggers its dissociation and release of active KLK5. Mutations that inactivate SPINK5 are associated with a severe skin disorder, Netherton syndrome (NS, MIM 256500), whose symptoms include premature desquamation (Deraison et al, 2007; Fortugno et al. 2011). Consistent with the hypothesis that SPINK5-mediated inhibition of KLK5 activity is a key feature of regulating normal desquamation, the NS-like phenotype of mice whose SPINK5-homologous gene has been knocked out is reversed in mice missing both SPINK5 and KLK5 activitiies (Furio et al. 2015).
Filaggrin is initially synthesized as a large, insoluble, highly phosphorylated precursor containing many tandem copies of 324 residues. This precursor is dephosphorylated and proteolytically cleaved by several proteases, including the undefined protease PEP1 (Resing et al. 1996), mu-calpain (Yamazaki et al. 1997), furin, PCSK6 (PACE4) (Pearton et al. 2001), PRSS8 (cap1) (Leyvraz et al. 2005), ST14 (matriptase) (List et al. 2003), CELA2 (Bonnart et al. 2010), CASP14 (Denecker et al. 2007) and Kallikrein-related peptidase 5 (KLK5) (Sakabe et al. 2013). Filaggrin is further processed and proteolytically degraded by CASP14 (Eckhart & Tschachler 2011).
ST14 (Suppressor of tumorigenicity 14, also known as matriptase) associated with the plasma membrane catalyzes the hydrolytic cleavage of proKLK5 (pro-Kallekrein-5) to yield active KLK5 enzyme. The activity of human ST14 enzyme is inferred from that of its well-characterized mouse homolog (Sales et al. 2010). Consistent with this inference, deficiencies of the human enzyme are associated with an ichthyosis syndrome (MIM 602400) as is a knockout mutation of mouse St14 (Alef et al. 2009).
In the low-pH environment of the upper layers of the stratum corneum, KLK5 (Kallikrein 5) dissociates from its complex with SPINK5 (Serine protease inhibitor kazal-type 5) (Deraison et al. 2007) and is free to cleave proCELA2 (Elastase 2), activating it (Bonnart et al. 2010).
Extracellular SPINK6 (Serine protease inhibitor Kazal-type 6) binds KLK14 (kallekrein-related peptidase 14), inactivating the latter. KLK14 activity contributes to the process of desquamation, and SPINK6 binding may play a role in the regulation of that process (Meyer-Hoffert et al. 2010).
Extracellular SPINK6 (Serine protease inhibitor Kazal-type 6) binds KLK5 (kallekrein-related peptidase 5), inactivating the latter. KLK5 activity contributes to the process of desquamation, and SPINK6 binding may play a role in the regulation of that process (Meyer-Hoffert et al. 2010).
Extracellular SPINK9 (Serine protease inhibitor Kazal-type 6) binds KLK5 (kallekrein-related peptidase 5), inactivating the latter. KLK5 activity contributes to the process of desquamation, and SPINK9 binding may play a role in the regulation of that process (Brattsand et al. 2009; Meyer-Hoffert et al. 2009).
In vitro, KLK5 (Kallikrein 5) catalyzes the slow cleavage of a four-residue aminoterminal propeptide from proKLK8 (pro-Kallekrein 8), to generate active KLK8. The abundance of KLK8 in the stratum corneum and its serine endopeptidase activity are consistent with a role for KLK8 in formation of the stratum corneum, desquamation, or both. Physiological substrates for KLK8 remains to be identified, however, as do possible additional activators of it (Eissa et al. 2011). A possible pathogenic role for KLK8 is suggested by the recent demonstration that it can mediate the extracellular cleavage of the L1 capsid protein of human papilloma viruses, facilitating their infection of human host cells (Cerqueira et al. 2015).
Filaggrin is initially synthesized as a large, insoluble, highly phosphorylated precursor containing many tandem copies of 324 residues. This precursor is dephosphorylated and proteolytically cleaved by several proteases, including the undefined protease PEP1 (Resing et al. 1996), mu-calpain (Yamazaki et al. 1997), furin, PCSK6 (PACE4) (Pearton et al. 2001), PRSS8 (cap1) (Leyvraz et al. 2005), ST14 (matriptase) (List et al. 2003), CELA2 (Bonnart et al. 2010), CASP14 (Denecker et al. 2007) and Kallikrein-related peptidase 5 (KLK5) (Sakabe et al. 2013). Filaggrin is further processed and proteolytically degraded by CASP14 (Eckhart & Tschachler 2011).
During cornification, a network of keratin intermediate filaments (KIF) and filaggrin (FLG) becomes crosslinked to the cornified envelope (CE). The FLG-KIF linkage occurs primarily through a specific lysine residue on the head domain of type II keratin chains, which can crosslink with several CE proteins including loricrin, SPRs, envoplakin and involucrin (Dale et al. 1978, Manabe et al. 1991, Mack et al. 1993, Steinert et al. 1995, Candi et al. 1998). This FLG-KIF crosslinking is believed to organise intermediate filaments into bundles, which stabilize the keratin network (Steinert et al. 1981, Candi et al. 2005) and facilitate the collapse and flattening of cells in the outermost stratum corneum to produce squames. Cell flattening can occur in the absence of FLG, but at the ultrastructural level loss-of-function mutations in FLG are associated with disorganized keratin filaments, impaired lamellar body loading and abnormal architecture of the lamellar bilayer (Gruber et al. 2011).
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profilaggrin processing
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proteasesIn vitro, virtually any type I keratin can dimerize with any type II keratin, leading to the formation of 10-nm long filaments (Franke et al. 1983, Hatzfeld et al. 1987). In vivo, the composition of keratin heterodimers is probably determined by expression. Differing keratin combinations are not characteristic of entire tissues, but probably confer particular functional properties to cells and tissue regions (Bragulla & Homberger 2009). Certain combinations are characteristic of a cell type, e.g. K18/K8 in simple epithelia. At least some keratins can be replaced with no loss of functionality of the keratin filament, e.g. K1/K10, K1/K9, K2/K9, K2/K10 in epithelia (Coulombe & Omary 2002). Suprabasal cells of stratified epithelia express different keratin pairs in different tissues, e.g. skin epidermis predominantly expresses K1/K10, the anterior corneal epithelium produces K3/K12, esophageal epithelium produces K4/K13 (Eichner & Kahn 1990) while hyperproliferative suprabasal cells are characterized by K6/K16 (Sun 2006).
Keratin filaments can rapidly disassemble and reassemble, allowing flexibility for the cytoskeleton. Keratin building blocks accumulate at the cell periphery near focal adhesions. Polymerization is regulated by signaling molecules, e.g. heat shock proteins, 14-3-3 proteins, kinases and phosphatases (Magin et al. 2007, Kayser et al. 2003).
At the ultrastructural level, desmosomes appear as electron dense discs approximately 0.2-0.5 μm in diameter, which assemble in a mirror-image arrangement at cell-cell interfaces (North et al. 1999, Al-Amoudi et al. 2011, Kowalczyk & Green 2013). Large bundles of filaments extend from the nuclear surface and cell interior out towards the plasma membrane, where they attach to desmosomes by interweaving with the cytoplasmic plaque of the adhesive complex. The head domains of keratins bind the tail domains of desmosomal cadherin molecules such as plakoglobin (Dusek et al. 2007), plectin, periplakin, envoplakin and desmoplakin (Bornslaeger et al. 1996, Kazerounian et al. 2002), thereby anchoring the cytoskeleton to the cell membrane.
The five major desmosomal components are the desmosomal cadherins, represented by desmogleins (DSG1-4) and desmocollins (DSC1-3), the armadillo family members, plakoglobin (PG) and the plakophilins (PKP1-3), and the plakin linker protein desmoplakin (DSP), which anchors the intermediate keratin filaments.
Certain adhesion complex proteins are expressed only when cornification commences. These include desmoglein-1, desmocollin-1, envoplakin, periplakin, plakophilin-1 and corneodesmosin (Candi et al. 2005). This expression is associated with changes in desmosome mophology whereby the cytoplasmic plaque integrates with the cornified envelope (Serre et al. 1991, Simon et al. 2001). Deregulation of desmosome formation can lead to degenerative cutaneous diseases (Brooke et al. 2012, Cirillo 2014).
The proliferative cells that give rise to hair fibres are located in the bulb at the base of the hair follicle. As they leave the germinative compartment, trichocytic differentiation begins and in matrix, cuticular, and cortical cells, the genes for keratins and KAPs (KRTAPs) are expressed. In the lower and middle cortex, keratin filaments are embedded in a matrix that consists of KAPs. Based on amino acid composition, three classes of KAPs have been described, the high sulfur KAPs (<30 mol % cysteine content), the ultrahigh sulfur KAPs (>30 mol % cysteine content), and the high tyrosine/glycine KAPs (Rogers et al. 2001). KAPs can be divided into subfamilies based on amino acid composition and phylogenetic relationships (Wu et al. 2008). Humans have approximately 100 KAP genes (Wu et al. 2008). Compared to the conserved structure and modality of keratins within mammals, KAP genes differ significantly between species and are likely to explain the variety of characteristics seen in hard keratin appendages such as feathers, claws, scales and hair (Wu et al. 2008, Khan et al. 2014). KAPs are crucial for the assembly of keratin intermediate filaments into arrays and likely to affect attributes of hair such as strength, rigidity and chemical inertness (Parry & Steinert 1999, Koster et al. 2015).
Transglutaminases (TGs) are believed to mediate the intramolecular bonds involved in CE formation. They catalyze inter-protein bond formation by forming a thiolester acyl-enzyme intermediate and subsequently transferring the acyl residue to a primary amine (Folk & Finlayson 1977, Folk 1980). The amine acceptor is generally provided by the epsilon-amino group of a protein-bound lysine and the link formed is an N6-(gamma-glutamyl)lysine isopeptide bond.
CE assembly is thought to be initiated on the inner face of the plasma membrane between desmosomes by the cross-linking of involucrin to itself, to envoplakin and perhaps to periplakin (Steinert & Marekov 1999). The extent of homo- and heterologous cross-linking varies as the CE matures. In the immature CE, EVPL, IVL, SPR1, and SPR2 are largely cross-linked to themselves; EVPL-IVL and IVL-SPR crosslinks are common while cross-links between desmoplakin (DSP) and IVL or DSP and EVPL are not. Later there are many more cross-links between DSP and IVL, DSP and EVL, or IVL and type II keratins. Loricrin (LOR) cross-linking to other protein partners appears later.
Transglutaminase-1 (TGM1) can crosslink IVL (Simon & Green 1988, Nemes et al. 1999), LOR (Candi et al. 2001), SPR3 (Steinert et al. 1999) and is thought to be responsible for EVPL crosslinking to itself and to IVL (Steinert & Marekov 1999). TGM5 can catalyse homo-crosslinking in LOR, SPR1, SPR2, and IVL, and hetero-crosslinks between LOR-SPR3 (Candi et al. 2001).
LOR is poorly soluble in vivo, while SPRs are very soluble. Both are preferred substrates of cytosolic transglutaminase-3 (TGM3) (Candi et al. 1999, Steinert et al. 1999, Tarcsa et al. 1999), which suggests that TGM3 may cross-link LOR and SPRs to create soluble complexes that are more easily translocated to the cell periphery (Kalinin et al. 2002). These cross-linked oligomers are good substrates for TGM1 (Candi et al. 1999, Steinert et al. 1999) which may link the LOR-SPR complexes to the CE scaffold. LOR can also be crosslinked by TGM5 (Candi et al. 2001). SPR content varies in epithelia from different body sites and increasing SPR content correlates with mechanical requirements of the tissue (Steinert et al. 1998). In humans LOR is initially deposited in the granular layer of the epidermis in keratohyalin granules, intermixed with profilaggrin (Yoneda & Steinert 1993). These are encoded in a linked 'Epidermal Differentiation Complex.' (Kypriotou et al. 2012, Niehues et al. 2016).
As the main component of the CE (Steinert & Marekov 1995), LOR is thought to function as the main reinforcement protein. LOR proteins are extensively crosslinked through isopeptide bonds but also crosslinked to SPRs, which may function as bridging proteins between LOR molecules (Candi et al. 2005). LOR can also form crosslinks with keratin and filaggrin (Steinert & Marekov 1995). CE crosslinking involves TGM1, TGM3 and TGM5 (Lorand & Graham 2003). The type-II keratin chains (K1, K2e and K5) are crosslinked at a specific Lys residue that is located in a conserved region of the V1 subdomain of the head domain (Steinert & Marekov 1995). IVL can be crosslinked by TGM1, which preferentially crosslinks Gln495 and Gln496 (Simon & Green 1998). In vitro, LOR is a substrate for TGM1-3 and 5 (Candi et al. 1995). In the epidermis, TGM1, TGM5 and TGM3 are believed to crosslink LOR sequentially; an initial attachment by TGM1 and 5 forms interchain crosslinks followed by a compaction process that involves TGM3 (Candi et al. 2005). SPRs are also TGM substrates, particularly TGM3 (Candi et al. 1999, Tarcsa et al. 1998, Steinert et al. 1999).
FLG binds KIFs, aggregating them into tight bundles. As a component of the CE, FLG 'glues' KIFs to the CE and coordinates the structure of cornifying cells (Steinert & Marekov 1995, Candi et al. 2005).
Human LCEs fall into distinct structural groups, encoded by genes which form clusters on the genome at 1q21 (Marshall et al. 2001, Niehues et al. 2016). Group 1 are expressed predominantly in epidermis. Group 4 (LEP 13-17) have highest expression in internal epithelia (Wang et al. 2001, Marshall et al. 2001).
Kazrin has at least 5 different isoforms. Overexpression of the short isoform kazrinE stimulates the terminal differentiation of cultured human keratinocytes and is associated with a reduction in F-actin content, disruption of DS assembly, and changes in cell shape. Overexpression of activated RhoA rescues the effects on cell shape and adhesion. Conversely, knockdown of the longest isoform kazrinA impairs terminal differentiation, independently of RhoA activity (Sevilla et al. 2008a). KazrinE colocalizes with stabilized microtubules in differentiating keratinocytes (Nachat et al. 2009). All KAZN isoforms can form complexes with one another (Nachat et al. 2009), suggesting that like periplakin and envoplakin, it may form part of the cortical scaffold that integrates the actin cytoskeleton with DS (Ruhrberg et al. 1997, Kalinin et al. 2001, Groot et al. 2004). In Xenopus embryos, depletion of endogenous KAZN results in striking defects in axial elongation, muscle and notochord differentiation, and epidermal morphogenesis. These effects are believed to be due to disruption of cell-cell junctions (Sevilla et al. 2008b, Cho et al. 2010). However, mice with a knockout that removes exons 5-15 of KAZN had normal epidermal morphogenesis and homeostasis (Chhatriwala et al. 2012).
Like DS, desmoglein and desmocollin constitute the extracellular parts of CDS (Simon et al. 1997), but there is an additional unique extracellular component known as corneodesmosin (CDSN). CDSN is a 52- to 56-kDa glycoprotein produced by keratinocytes that is incorporated into the desmoglea of DS shortly before their transformation into CDS during cornification (Serre et al. 1991). It is stored and secreted by Lamellar bodies. After secretion CDSN localizes to the extracellular structures of CDS and covalently cross-links to the CE. This step coincides with the morphological transformation of DS into CDS. In vitro studies suggest that CDSN mediates homophilic binding to counterparts on adjacent corneocytes (Ishida-Yamamoto & Kishibe 2011). Cleavage of desmoglein, desmocollin and CDSN is a key step in desquamation.