Biochemistry of skin

1. Biochemistry of skin Jana Novotná Department of Med. Biochemistry 2nd Faculty of Medicine Charles University

2. Skin • it provides barrier against a range of noxious stress (UV irradiation, mechanical, chemical and biological insults); • acts as the periphery„sensing“ system; • system which maintaining body homeostasis. • 2 m2in area • ~ 2.5 mm thick on average • constitutes 6% of our total body weight (5 – 6 kg) • barrier to prevent a desiccation and temperature balance • protection to the UV radiation – absorbing pigmentation system • complex immuno-regulatory network protection • normal skin pH is somewhat acidic - the range of 4.2. to 5.6.

3. Human skin layers • Mammalian skin is composed of two primary layers: • the epidermis, which provides waterproofing and serves as a barrier to infection; • the dermis is responsible for the tensile strength of skin. Its main functions are to regulate temperature and to supply the epidermis with nutrient-saturated blood. Much of the body's water supply is stored within the dermis.

4. Epidermis • An external stratified, non-vasularized epithelium (75 – 150 mm thick), continually keratinizing • Stratum corneum – 15 – 30 sheet of non-viable, but biochemicaly active corneocytes • Stratum granulosum – 3 – 5 sheet of non-dividing keratinocytes, producing keratino-hyalin • Stratum spinosum – 8 – 10 sheet of keratinocytes with limited dividing capacity, Langerhan´s cells • Stratum basale – maturing/aging keratinocytes, melanocytes, Merkelcells (receptor cells)

5. Keratins • keratinocytes contain filaments of the keratin intermediate filament (KIF) family (cyto-skeleton) • hair, nails, horny layers of the skin – are formed from keratin cytoskeleton of dead cells. • two primary groups of keratins, the a-keratins and the b-keratins • a-keratins occur in mammals, b-keratins in birds, reptiles • both form are right handed helicalstructure • 2 types • type I – acidic keratins • type II – basic keratins • heterodimer – type I forming a coild coil with type II

6. Composition and Structure of Keratin • Human skin contains ~ 20 genetically different keratins • Long stretches a-helix is interrupted by short non-helical segments • The most abundant amino acid are glycine and alanine, cysteine can account for up to 24% • Contact between 2 a-helices are formed by hydrophobic amino acid side chain on 1 edge of each helix • two polypeptides form a dimericcolid coil • protofilaments are formed from two staggered rows of head-to-tail associated coils • protofilaments dimerize to form a protofibril and four of which form a microfibril http://biochem118.stanford.edu/Papers/Protein%20Papers/Voet%26Voet%20chapter6.pdf

7. Composition and Structure of Keratin • Intra- and intermolecular hydrogenbonds, disufidbridges occure at all keratins. • In cells, keratin type I forms pair with keratine type II • Different keratin types are expressed in different cell types and different layers of epidermis: • cytoskeleton of epithelial cells - K14 (type I) & K5 (type II), K18 (type I) & K8 (type II) • Basal layer – K13 (type I) & K4 (type II) • Spinus and granular layer – K10 (type I) & K1 (type II) • Stratum corneum – K3 (type I) & K12 (type II) • Hairs and nails – various other keratin pairs

8. The Epidermal Permeability Barrier • Barrier function in human epidermis depends on transglutaminase-mediated cross-linking of structural proteins and lipids („biological glues“) • post-translation modification of proteins – formation of covalent bond between a e-amino group of lysine and the free amine group of glutamine. • Bonds formed by transglutaminase exhibit high resistance to proteolytic degradation. • Proteins are than highly resistent to mechanical perturbation and proteolysis. • The quality of the S. corneum barrier depends on the presence of equimolar concentration of ceramides, cholesterol and fatty acids. • Changes in the concentration of any of these can affect barrier quality.

9. Fatty Acids in Epidermis • Arachidonic acid and 20-carbon PUFA can be metabolized by either cyclooxygenase or lipoxygenase pathways → prostaglandins, hydroxyeicosatetraenoic acids. • phospholipids are starting point for the arachidonic acid pathway during inflammation (allergic reaction) • Some of these metabolites can interact with signaling system in proliferating and differentiating epidermal cells → modulation of protein kinase C, nuclear MAP-kinase

10. Epidermal Cell Differentiation and Turnover • Basal keratinocytes → transformation ~ 30 days to corneocytes. • Damage cells are removed by normal squamation. • Genetic damage - (UV-R) → trigger apoptosis (within hours) – „sunburn“ cells. • Skin protection against UV-R – concentrating transferred melanin over vulnerable keratocyte nucleus. • Other insults can induce keratnocyte apoptosis – chemical, mechanical, immunological. • The principal marker for keratinocyte/epidermal differentiation is expression of particular keratin pairs.

11. Epidermal Cell Differentiation and Turnover • Majority of over 30 keratins currently known. • Proliferative basal keratinocytes express K5 and K14; • keratinocytes in the early stages of maturation/differentiation switch to K1 and K10. • The „pluri-potent“ stem cells for keratinocytes sebaceous gland and epidermis rised from hair folicules. • Ca2+ plays pivotal role in epidermal differentiation - 4-fold increase of extracellular Ca2+ in S. corneum. • Keratinocyte differentiation is regulated by hormones and vitamins - D3 and retinol from diet, thyroid hormones and steroid hormones. • The skin has nucleas receptors for glucocorticoids, estrogen, androgen and progesterone.

12. Epidermal Cell Differentiation and Turnover • Importan factors for keratocyte differentiation are Ca2+-dependent integrins – the receptors for the extracellular matrix fibronectin binding. • Laminin and collagenIV and VII (basemen membrane components) – regulation of keratinocytes migration on basement membrane (very important during wound healing). • Migrating keratinocytes produce many matrix metalloproteinases. • Mature keratinocytes (in S. graulosum) contain protein-rich, keratohyalin granules and lipid-rich, lamellargranules. • Lipids from lamelar granules form the sheets of the lipid permeability of the lipid permeability barrier of the epidermis.

13. Melanocytes • Melanocytes are melanin-producing cells located in S. basale • Precursor - melanoblast • Melanin is stored in the melanosomes. • „Epidermal melanin unit“ - the anatomical relationship between keratinocytes and melanocytes. • 1 melanocyte is in contact with ~ 40 keratinocytes • Melanocytes extend arms to transfer melanosomes into the keratinocytes

14. Cover picture:The Rab27a GTPase associates with melanosomes and regulates their transport to, and retention in, the peripheral cytoplasm in skin melanocytes. Melanosome transport also requires the microtubule and actin cytoskeleton. Staining of a cultured murine melanocyte for filamentous actin (red) and microtubules (blue) reveals a close relationship between Rab27a-labelled melanosomes (green) and these cytoskeletal elements. Melanocytes Hume et al. JCB 2001;152 (4): 795

15. Formation of melanosomes • Melanosomes - elliptic membrane-bound organelles (melanin synthesis). • Synthesis of matrix proteins and tyrosinase(TYR) on the rough endoplasmic reticulum. • TYR undergoes post translational modification in the form of glycosylation in the Golgi apparatus. • Fusion of premelanosomes with coated vesicles containing tyrosinase - formation of the melanosome. • Melanosome migrates into one of the dendrites of the melanocyte → transfer to a neighboring keratinocyte.

16. Production of Melanin • Three enzymes in melanosomes whih absolutely required for different melanin type synthesis • tyrosinase (TYR) – responsible for critical step of melanogenesis (tyrosine hydroxylation) • tyrosinase-related protein 1 (TYR1) and DOPAchrome tautomerase (DHI = 5,6-dihydroxyindole; DHICA = 5,6-dihydroxyindole-2-carboxylic acid)

17. Melanins • Melanins are polymorphous and multifunctional polymers of eumelanin, pheomelanin, mixed melanins (a combination of the two); and neuromelanin • Mammalian cells produce black-brown eumelanin and yellow-redish pheomelanin • Eumelanin - highly heterogenous polymer consisting of DHI and DHICA units in reduced or oxidized states. • Pheomelanin - mainly sulfur-containing benzothiazine derivatives. • Neuromelanin is produces in dopaminergic neurons of substantia nigra. • Melanin absorbs UV light at a wavelengthof280 - 320 nm • Both eumelanin and pheomelanin play important protective role in binding to cations, anions, drugs, chemicals, etc.

18. Factors Involve in Melanin Production • The melanin granules accumulate above the nuclei of keratinocytes and absorb harmful UV-R before it can reach the nucleus and damage the DNA. • Quickresponds of the melanocyte-keratinocyte complex to a wide range of environmental stimuli (paracrine and/or autocrine) - to UV-R, melanocyte-stimulating hormone (MSH), endothelins, growth factors, cytokines, etc. • UV-R exposure→ melanocytes increase their expression of proopiomelanocortin (POMC, the precursor of MSH) and its receptor melanocortin 1 receptor (MC1-R), TYR and TYRP1, protein kinase C (PKC), and other signaling factors.

19. Factors Involve in Melanin Production • Fibroblasts (possibly other cells in skin) - produce cytokines, growth factors, and inflammatory mediators that can increase melanin production and/or stimulate melanin transfer to keratinocytes by melanocytes. • Other factors derived from keratinocytes which can regulate proliferation and/or differentiation of melanocytes: • α-MSH, ACTH, basic fibroblast growth factor (b-FGF), nerve growth factor (NGF), endothelins, granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), and hepatocyte growth factor (HGF).

20. Other Epidermal Cells • Langerhans cells - dendritic cells - arise frombone marrow early in embryonic development, occupie 2 - 8% of epidermis • important element of the immune system, interacting with T-cells • resided in suprabasal layer - attracted to keranocytes by E- cadherin receptor • their motion is regulated by specific integrin receptor and by α –TNF • in the stratum germinativum interacts with the allergen and migrates to the lymphoid gland - then “teache” the T cells about the allergen • interact specifically with T-lymphocytes and keratinocytes to initiate host response to antigens (allergens) • UV B stimulates synthesis and release of TNF- by keratinocytes which in turn modifies the behavior and morphology of Langerhans cells, decreases their total number.

21. Langehans cell Allergen cytokine T cell Activated T cell

22. Other Epidermal Cells • Merkel cells – location in S. germinativum • have synaptic contacts with sensory nerve endings • associated with the sense of „light touch“ discrimination of shapes and textures.

23. Dermis • responsible for the tensile strength of skin • main functions – regulation of temperature and to supply the epidermis with nutrient • much of the body's water supply is stored within the dermis • components: • connective tissue • hair follicles • sweat glands • sebaceous or oil glands • apocrine glands • lymph vessels • blood vessels • The main cell type - fibroblast

24. Dermal Proteins and Extracellular Matrix • Collagen – about 90% of total dermal proteins • predominatly type I (85 – 90%), • type III (8 -11%), • minor type V (2 – 4%), (papillary dermis, matrix around vessels, nerves), • type VI – associated with fibrils and interfibrillar spaces (responsible for fine structure in early prenatal development of skin). • Elastin, proteoglycans, glycoproteins, water and hyaluronic acid Collagen structure - refer to lecture on Collagens Elastin, proteoglycans, glycoproteins – refer to lecture on Extracellular matrix

25. Skin Appendages • Skin plays in the body homeostasis, therefore is well-equiped with secretory (release of chemicals from cells for physiological function) and excretory (elimination of weste products of metabolism) capacity. • sweat glands[can be sweat secreted with strong odour (apocrine) or with a faint odour (eccrine)]. • sebaceous glands (secrete sebum onto hair follicle to oil the hair). • hair follicle

26. Sweat Glands • 3 – 4 million eccrine sweat glands are in our skin – each producing water perspiration (serves mainly to cool us) and maintain core temperature at 37.5oC. • At maximum output the eccrine sweat glands can excrete as much as 3 l/hour, and heat loss is more than 18 kcal min-1. • Humans utilize eccrine sweat glands as primary form of cooling. • Apocrine sweat glands are larger, have different mechanism of secretion, and are limited to axila and perianal area.

27. Sweat Secretion • Eccrine gland activity is regulated via neural stimulation using sympathetic nerve fibers distributed around the gland. • Neurotransmitter is acelylcholine • Sweating is controlled from hypothalamus (a center in the preoptic and anterior regions), where thermosensitive neurons are located. • The stimulus for perspiration: • direct heating alone (39 to 46oC) • physiological sweating due to nerve reflex arise from sweat centers in brain cortex (emotional), hypothalamus (thermoregulation)

28. Eccrine Sweat • contains mainly water (99.0 – 99.5%). It also contains electrolytes NaCl, K+ and HCO3-, and other simpl molecules - lactate, urea, ammonia, amino acids (serin ornithin, citrulin, aspartic acid)and minerals. • Mineral composition varies with the individual: • their acclimatisation to heat, • exercise and sweating, • the particular stress source (exercise, sauna, etc.), • the duration of sweating, and the composition of minerals in the body

29. Apocrine Sweat • In lower mammals – secretion of pheromones (trigger sexual and territorial response) • In humans – the significance of apocrine secretion of pheromones is not completely understood. • Apocrine gland begin secreting at puberty • Apocrine duct exit to the surface via he hair follicle. • Apocrine sweat – more viscous, with milky consistency due to high content of fatty acids, cholesterol, squalene, triglycerides, androgens, ammonia, sugars.

30. Mineral Composition of Sweat Microelements

31. Sebaceous Glands • Glands secrete an oily/waxy matter, called sebum, to lubricate the skin and hair • Composition – 25% wax monoesters, 41% triglycerides, 16% free fatty acids, 1% squalene, small amount cholesterol esters and cholesterol.

32. Skin Metabolism • Primary source for energy production in epidermis is glucosefrom circulation – diffuses into keratinocytes without effect of insulin. Large proportion of glucose is catabolized up lactate (even in presence of oxygen) • citric acid cycle does operate in epidermis – explanation why this cycle is inefficient is due to wide fluctuation of temperature and blood flow in skin. • 20% of glucose is metabolized by pentose-phosphate pathway (PPP) – production of NADPH and pentose for both FA synthesis and nucleic acids. • Secondary source of energy - fatty acids derived from both epidermal stores and exogenous sources (when glucose flow is limited, then FA are metabolized).

33. Skin Metabolism • Glycogen– small amount under physiological conditions, however, elevation in all manner of injury of epidermis or during hair growth in follicle – explanation – energy when skin needs to be repaired or to use glucose immediately, most probably – disequilibrilium in metabolic processes. • Furthermore, glucose is substrate also for synthesis of lipids, polysaccharides, glycoproteins and nucleic acids. • GAG and proteoglycans – highly charged and attract water – forming gels (see also lecture about ECM).

34. Skin Metabolism Lipid metabolism - components: a) membranes, b) major constituents of permeability barrier, c) energy supply • Synthesisfrom both glucose catabolism,from AA and circulating FA - lipogenesis is going on in all layers of epidermis - sebum synthesis → in sebaceous glands (higher synthesis of sebum is after sexual maturation). • Degradation - generallywithlipases (yields in FA for neutral lipids – TG, sterol esters) – inoutermost layers of epidermis(e.g. formation of prostaglandins)

35. Skin Immune System • Skin not only provides immune protection for itself, but also for the whole body. • Cell types containing battery of mediators of immune response • Langerhanse cells, monocytes, macrophages, mast cells (cooperation with T-cells) • Cell types producing free radicals, anti-bacterial peptides, cytokines chemokines, pro- and anti-inflammatory mediators • Neutrophils, eosinophils, basophils. • B-cells secrete immunoglobulins (antibodies)