Biochemistry of skin

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Biochemistry of skin

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

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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 m2 in 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.

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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.

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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, Langerhans cells
  
• Stratum basale maturing/aging keratinocytes,
  melanocytes, Merkel cells (receptor cells)

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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 helical structure
• 2 types
• type I acidic keratins
• type II basic keratins
• heterodimer type I forming a coild coil with
  type II

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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 dimeric colid 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/Protein20Pa
pers/Voet26Voet20chapter6.pdf
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Composition and Structure of Keratin

• Intra- and intermolecular hydrogen bonds, disufid
  bridges 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

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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.

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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

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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.

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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.

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Epidermal Cell Differentiation and Turnover

• Importan factors for keratocyte differentiation
  are Ca2-dependent integrins the receptors for
  the extracellular matrix fibronectin binding.
• Laminin and collagen IV 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, lamellar granules.
• Lipids from lamelar granules form the sheets of
  the lipid permeability of the lipid permeability
  barrier of the epidermis.

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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

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Melanocytes

• Cover pictureThe 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.

Hume et al. JCB 2001152 (4) 795
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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.

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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)

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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 wavelength of 280 -
  320 nm
• Both eumelanin and pheomelanin play important
  protective role in binding to cations, anions,
  drugs, chemicals, etc.

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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.
• Quick responds 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.

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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
• a-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).

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Other Epidermal Cells

• Langerhans cells - dendritic cells - arise from
  bone 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 a 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.

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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.

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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

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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

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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

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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.

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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)

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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

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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.

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Mineral Composition of Sweat
sodium 0.9 g/l
potassium 0.2 g/l
calcium 15 mg/l
magnesium 1.3 mg/l

zinc 0.4 mg/l
copper 0.3 0.8 mg/l
iron 1 mg/l
chromium 0.1 mg/l
nickel 0.05 mg/l
lead 0.05 mg/l
Microelements
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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.

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Skin Metabolism

• Primary source for energy production in epidermis
  is glucose from 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).

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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).

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Skin Metabolism

• Lipid metabolism - components
• a) membranes,
• b) major constituents of permeability barrier,
• c) energy supply
• Synthesis from 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 - generally with lipases (yields in
  FA for neutral lipids TG, sterol esters) in
  outermost layers of epidermis (e.g. formation of
  prostaglandins)

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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)