Support information for this lesson:
1. Why are there different skin tones in humans?
Dark skin protects against the ultraviolet (UV; in particular UV-B) component of the sun light, which causes DNA damage and mutations in skin cells leading to skin cancer (Narayanan et al, 2010). Lighter skin does not provide this protection; therefore, people with this skin tone have a higher chance of skin dmage such as sunburn and are advised to use sun cream with a safe sun protection factor (SPF: a relative measure of how long a sunscreen will protect you from UV-B rays). On the other hand, sunlight promotes the synthesis of vitamin D, and in northern countries with less sunlight (hence reduced risk of skin cancer) it is therefore of benefit to have light skin. Vitamin D is important for calcium and phosphate absorption in the gut required for bone strength (Nair and Maseeh, 2012), and its deficiency can lead to rickets (a disease where bones become soft and malformed, often characterised by bow legs). Modern fortification of foods has reduced the number of people affected by rickets, but finding the right balance for sun exposure seems important.
2. What is skin pigmentation?
Pigments are molecules that absorb light; non-absorbed light is reflected and determines the pigments’ colour (see our light resource). The pigment primarily determining for our hair and skin colour is melanin. It is a polymer derived from the amino acid tyrosine (Fig. 2), but it is not a protein produced through ribosomal translation, but it is the product of a series of enzymatic reactions, referred to as a metabolic pathway.
3. Principles of metabolic pathways
Here we will briefly explain the principles of enzymes and metabolic pathways: (1) the “one gene-one enzyme” hypothesis, (2) the lock-and-key or induced-fit model, (3) co-factors, (4) feedback inhibition and (5) different forms of metabolic pathways.
Figure 3.Genes encode enzymes (green) or specific subunits of enzymes (not shown). Enzymes display pockets that precisely match the shapes of their substrates (orange). After the enzymatic reaction (here a binding reaction of the two molecules) the product is released.
(1) “One gene-one enzyme hypothesis” (Fig. 3): Enzymes are proteins encoded by genes which speed up (catalyse) chemical reactions. The “one gene-one-enzyme” hypothesis proposed by Beadle and Tatum (1941) stated that one gene codes for one enzyme and therefore each step in a metabolic pathway is controlled by a single gene. When it was recognised that enzymes can be complexes composed of different subunits, each encoded by a different gene, the hypothesis was renamed into the “one gene-one polypeptide” theory.
(2) “lock-and-key” or “induced-fit model” (Fig. 3) [LINK]: Substrates fit into the active site of an enzyme like “lock-and-key” which induces a shape change, so that they form a very tight enzyme-substrate complex (“induced-fit”) which lowers the activation energy for the enzymatic reaction. After this reaction, the product(s) is/are released and the enzyme is able to repeat the process.
(3) Co-factors: many enzymes use “helper molecules” that assist in biochemical transformations. Co-factors are either linked to the enzyme (they bind ‘covalently’) and are then referred to as ‘prosthetic group‘. If they bind only transiently during the catalytic process, they are referred to as ‘co-substrates‘. Co-factors are either metallic ions, or thy are organic molecules, such as vitamin derivatives, but not proteins; such organic co-factors are also called ‘co-enzymes‘. All have the same function of facilitating the enzymatic reaction: an inactive enzyme without the co-factor is called an ‘apoenzyme‘ and the complete enzyme with co-factor a ‘holoenzyme‘.
(4) Feedback inhibition (Fig. 4): It is important to have some control over reactions in the body, for example to prevent that all substrate is being used up when there is no actual need for the product. For this, enzymes can be activated or inhibited. For example, competitive inhibitors are molecules similar to the substrate which bind to and block the enzyme’s active site; in contrast, non-competitive/allosteric inhibitors bind somewhere else on an enzyme and change its shape so that the active site no longer binds the substrate. In feedback inhibition, the substrate itself acts as an inhibitor of the enzyme it produces, usually through allosteric inhibition. This regulation can be concentration-dependent: if the product is not used up and accumulates in the cell, it is more likely to bind and inhibit the enzyme, thus preventing further accumulation.
(5) Different forms of metabolic pathways (Fig. 5): Different metabolic pathways can be used to break a product down (catabolism) or to build up a new molecule (anabolism). They often involve several steps each catalysed by a different enzyme, modifying the molecule in a stepwise manner (Fig. 5). In nature, many metabolic pathways tend to be much more complicated involving many more enzymes, substrates and co-factors. Apart from having multiple steps, metabolic pathways can also be branched whereby one common intermediate substrate is used by different enzymes to produce different products (Fig. 6).
Figure 5. Exemplary metabolic pathway with several steps forming an anabolic pathway: Enzyme 1 catalyses the joining of the initial substrates to form a product which is the first intermediate substrate for Enzyme 2, the product of which forms the 2nd intermediate substrate for Enzyme 3 which, in turn, generates the final product. Below some exemplary intermediate steps of the Melanin pathway are given which is even more complex in reality.
Figure 6. In a branched metabolic pathway multiple products can produced from one common precursor. In this example Enzyme 1 produces a product which can be used as a substrate for 3 different enzymes to produce distinct products.
4. Studying metabolic pathways using genetics
How can we study metabolic pathways? The key strategy is to take random approaches to block single enzymes (without knowing which one!), and then to measure whether any substrates/products of interest either accumulate or vanish (Fig. 7). If the attempt worked and different enzymes within the same pathway were blocked in different experiments (Fig. 7), one can then deduce the sequence of enzymatic steps (i.e. which molecule are precursor and which are derivatives) and identify what enzymes act at what step in the process. Enzymes can be blocked using pharmacology or genetics.
The pharmacological approach uses so called drug screens using random chemical substances (referred to as ‘drugs’) which are derived for example from tropical plants or random synthesis in laboratories. These drugs are added one-by-one to cells, in the hope that they cause quantitative changes in substrate/products of the pathway in question; such drugs are then further scrutinised.
The genetic approach takes advantage of the fact that enzymes are encoded by genes which can be affected by mutations. Therefore, in analogy to drug screens, gene mutations are randomly induced (e.g. using radiation or chemical substances that induce mutations, hence also cancer!). If a mutation causes changes in substrate/product amounts, this mutation is then further scrutinised; if the gene affected by the mutation can be identified, it leads directly to the amino acid sequence of the enzyme!
Figure 7. When blocking enzymes, their substrates are likely to accumulate (unless used up by other enzymes in a branched metabolic pathway; Fig. 6), and their products are reduced or missing. Open arrows indicate that molecules come in from other pathways or sources.
5. An example of Drosophila eye pigments
The fruit fly Drosophila has red compound eyes, primarily determined by two types of pigments: brown ommochromes are found in primary and secondary pigment cells, and red pteridines are found in secondary pigment cells (Fig. 8; Shoup, 1966).
Figure 8. The Drosophila compound eye (shown on the left), is composed of single units called ommatidia (middle), each composed of 8 photoreceptor cells (only some shown) and support cells ensheathing the ommatidum and forming its lens system (cornea, pseudocone). The primary and secondary pigment cells contain specialised organelles, the lysosome-like pigment granules, which contain the enzymes to produce pigment: ommochromes are brown pigments in primary pigment cells and pteridines are red pigments in secondary pigment cells.
Both ommochromes and pteridines have their discrete metabolic pathways within the pigment granules, and the primary substrates (GTP for pteridines and tryptophan for ommochromes) are transferred into the granules via distinct transporter proteins composed of different subunits, one of which is common to both transporters (Fig. 9). Like enzymes, also the transporters are therefore essential for pigment production: affecting the GTP transporter causes absence of the red pteridins turning the eye colour into brown, affecting tryptophan transport affects the brown ommochromes resulting in red/scarlet eye colour, and affecting both transporters causes absence of eye colour, which is the famous white mutation discovered in 1910 (see the film; Mackenzie et al, 1999; Cagan and Ready, 1989).
Figure 9. Two distinct transporters sharing one subunit (2) import the Ommochrome (brown pigment) precursor Tryptophan and the Pteridine (scarlet pigment) precursor GTP into pigment granules. Metabolic pathways in these granules produce the respective pigments which add up to the brownish red eye colour of fruit flies.
Here we will focus on Pteridines which, themselves, represent a cocktail of pigments and provide a nice example of a branched metabolic pathway (Fig. 10). Amongst the Pteridines, a group of red pigments called Drosopterins are the essential fraction contributing to eye colour whereas Sepiapterin is visible as yellow in visible light, and Biopterin is only visible in UV light. This is still a simplified version of all molecules derived from this pathway, but sufficient to explain the principles of analysis.
As explained in section 4, metabolic pathway can be “deciphered” using mutations of genes encoding enzymes, in combination with measurements of their substrates/products. Two potential ways to measure substrates/products are the eye colour of flies (Fig. 12) and chromatography. There are many sophisticated ways of chromatography, such as thin layer chromatography, gas-liquid chromatography and ion exchange chromatography, but the simplest form that can be used, also in classrooms, is paper chromatography (Figs. 11 and 12).
Figure 11. The principle of paper chromatography. Chromatography paper is prepared by drawing a pencil line approx. 1 cm from the bottom of the paper; ~5 fly heads are crushed and transferred as a dot to the middle of the pencil line; the chromatography paper is placed in a sealed vial containing a solvent (a 1:1 mix of propan-2-ol and ammonium hydroxide) not reaching higher than 2-3 mm below the pencil line. The tube is covered with foil (to prevent light-induced break-down of the pigments) and left for 2.5 hrs (Ranmuson et al., 1960). The solvent climbs up the chromatography paper, taking with it and separating the eye pigments. The molecules separate out because they have different degrees to which they dissolve in the solvent (mobile phase) and/or interact with the paper (stationary phase). The retardation factor Rf for each pigment spot is calculated by dividing the furthest distance travelled by each pigment (measured from the pencil line) through the distance the solvent has travelled (i.e. distance from pencil line to solvent front). Once calculated this value can be compared to the reference list of Drosophila pigments (see the teacing resource documents) to identify the respective pigment.
Figure 12 shows some experimental results when removing the transporter genes brown, scarlet, white or the sepia gene coding for an important enzyme of the Drosopterin pathway. The combination of eye colour changes and chromatography data makes it possible to take apart the metabolic pathways step by step.
Figure 12. “Deciphering” the Drosophila metabolic pathway of Pteridines. A) The same pathway as shown in Fig. 10, but some genes are indicated (highlighted in yellow) which encode transporter proteins (brown, scarlet, white) or an enzyme for Drosopterin production (sepia, encoding “PDA synthase”). B) When using heads of normal flies (wt, wildtype) to perform chromatography as explained in Fig. 10, three pigments are visible under ultraviolet (UV) light, as indicated by numbers in green circles. If the sepia (se) gene is mutated, spot 2 is missing and eyes turn dark brown (C). Loss of the white gene (required for GTP and Tryptophan transport, A) causes a loss of all spots in the chromatography (B) and of all eye pigmentation (C). If abolishing only one transporter at a time by removing the scarlet (st) or brown (bw) gene, eyes turn scarlet (from the remaining Pteridins) or brown (from the remaining Ommochromes), respectively.
6. What mechanisms explain the north-south distribution in skin colour?
The skin is formed of an upper pigmented layer, the epidermis, and a lower layer, the dermis (see here an image of the skin). As mentioned in Section 2, the pigment in the epidermis is Melanin. Melanin is produced in pigment granules called melanosomes, produced in specialised skin cells called melanocytes in the lower layers of the epidermis. Melanosomes are ‘pinched off’ from melanocytes and ‘taken up’ by the keratinocytes (the actual cells forming the epidermis), and this process is referred to as phagocytosis. There are two types of Melanin in human skin, Eumelanin (brown or black) and Pheomelanin (yellow or red); these pigments are derived from a branched metabolic pathway using Tyrosine as the common precursor (Fig. 13). For more detail click here or read Yamaguchi et al. (2000).
Figure 13. The metabolic pathway producing Pheo- and Eumelanin. A) The common precursor is Tyrosine and its derivative DOPA from where the pathway branches. B) The difference in skin colour is determined by the amount of Eumelanin produced.
Skin colour is determined by the amount of Eumelanin: the higher the content the darker the skin. However, how can this be achieved? The Melanocortin receptor 1 (MC1R) is a protein in the membrane of melanocytes (Fig. 14). It is a so-called receptor molecule which can bind specific factors on the outside and, upon binding, trigger changes in the inside of the cell. As shown in Figure 13, MC1R is activated by melanocyte-stimulating hormone (MSH), but it is down-regulated by another hormone called Agouti signalling peptide (ASIP). Activated MC1R triggers the activity of factors in the cells that lead to the production of Eumelanin (Fig. 14); this may occur through enhancing the production of enzymes of the Eumelanin pathway (Fig. 13; e.g. by stimulating gene transcription) or through activating these enzymes themselves [see Section 3 (4)].
From the information above, we can conclude that a person with dark skin is likely to have high MSH levels (stimulating Eumelanin production), and a person with light skin to have low MSH and/or high ASIP levels (turning down Eumelanin production); a person with red hair and even lighter skin is likely to carry a mutation in the MC1R gene, thus having an even stronger balance shift from Eumelanin to Pheomelanin (Fig. 14). Some people have a condition called albinism in which Melanin is entirely absent, for example due to the absence of Tyrosinase or a Tyrosine importer (see Fig. 13). Accordingly, albinism is characterised by white skin and hair and often also red eyes (because lack of melanin pigment in the eye lets us see through to the highly innervated retina).
Figure 14. The MC1R receptor protein promotes Eumelanin production. It is hormonally regulated: activated by MSH and inhibited by its “antagonist” ADIP. Usually, these factors can explain different skin colours.
Humans are believed to have originated in Africa (Fig. 15; although this has recently been questioned: LINK). Therefore, high levels of MSH and darker skin were likely traits that were positively selected because they protect from skin cancer (Fig. 1). When humans migrated north, the selection criteria shifted towards lighter skin, especially since nutrition will not have been optimal to prevent Rickets (Fig. 1). Therefore, one can imagine that mild genetic mutations (referred to as genetic variations) were selected for which would reduce MSH expression, increase ASIP or reduce (but not abolish) MC1R activity, thus still leaving the flexible option of gaining tan in summer. In the grey and cloudy north even mutations in the MC1R gene were tolerated, in spite of the fact that the ability to tan is severely reduced.
There is one exception to this trend: indigenous peoples who live near the poles have darker skin yet live at very high latitudes where there is low UV. However, these populations do not suffer from the expected lack of vitamin D (Fig. 1) because they supplement it from their fish-rich diet. In recent years there has been a higher incidence of vitamin D deficiency due to changes in diet (Sharma et al, 2011).