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Home , Argyria, Elejalde syndrome, Griscelli syndrome

1, Introduction Introduction

History of research on human pigmentation although can be traced back to 2200 BC, the quest for understanding the mechanism of various aspects of pigmentation still continues. There is no doubt that the visual impressions of body form and color are important in the interactions within and between human communities. Further a number of or malformations are known to be associated with various pigmentory disorders. Thus this area of research is of tremendous importance.

In biology, a pigment is any material resulting in color of plant or animal cells, which is the result of selective color absorption. Many biological structures, such as , eyes, fur and hair contain pigments (such as ) in specialized cells called chromatophores. Study of pigmentation patterns provides a model system to study genetic as well as phenotypic variations as it provides a manifestation of the complex interplay between environmental cues, signal transduction pathways, several modifications at transcriptional- protein level. Thus pigment genes were "pioneers" for the exploration of mouse genetics leading to the identification of 127 different loci of which 63 different genes have been charaterised so far (Silvers 1979; Bennett and Lamourex, 2003; Getting 2005). A summary of the location and function of important genes in melanogenesis identified till date is presented in table 1. Introduction

Mouse Coat Human Human Mutation/ Protein Function Colour Locus Chromosome Phenotype proteins Oxidation of tyrosine, Albino(c) TYR llq-q21 Tyrosinase OCAl DOPA DHICA oxidation, Brown (b) TYRPl 9p23 Gp75/TYRP1 0CA3 TYR stabilization Dopachrome Slaty (sit) DCT 13q32 TYRP2 7 tautomerase DHICA (si) SILV 12pl3-ql4 GplOO/pMell7/silver 7 polymerizadon/stabling Pink eyed 0CA2 15qll.2-ql2 P-protein 0CA2 PH of melanosome dilute (p) Underwhite Homology of sugar LOC51151 5pl4.3-ql2.3 AIM-1 OCA4 (uw) transporters Signal proteins Agouti (s) ASIP 20qll.2-ql2 Agouti signal protein 7 MCIR antagonist G-protein coupled Extension (e) MCIR 16q24.3 MSH receptor Red Hair receptor Pomc(l) POMC 2p23.3 POMC,MSH,ACTH OA MCIR antagonist Wardenburg G-protein coupled Oal OAl Xp22.3 OAl protein syndrome type receptor 2 Micropthalmia MITF 3pl2.3-14.1 MITF Transcription factor (mi) Melanosome transport/uptake by keratinocyte Griscelli Dilute (d) MY05A 15q21 Va Motor protein syndrome Griscelli Ashen (ash) RAB27A 15q21 Rab27a RAS family protein syndrome G-protein coupled F2rll F2RL1 5ql3 PAR2 7 receptor

Table.l Important genes in melanogenesis, protein product and function (Adaptedfrom Sturm etal., 2001) Introduction

1.1 Biochemistry of Pigmentation

1.1.1 , epidermal -melanin unit,

Melanin synthesis takes place in specialized cells termed as (Odland and Reed, 1967). The majority of melanocytes are found in the epidermis of the skin, stria vascularis of inner ear, pigmented retinal epithelium and choroid layer of the eye. These melanocytes comprise a very small proportion of cells present in the epidermis (<1%) and virtually account for all the visible pigmentation in skin, hair and eyes (Jimbow et al., 1993). One melanocyte provides melanin pigment to approximately 36 keratinocytes, forming what is known as an epidermal melanin unit (Fig. IB).

Melanin is synthesized through a multistep biochemical pathway in specialized organelles known as the melanosomes. (Fig. lA) Melanosomes are the specialized members of the lysosomal lineage and are formed from the trans-Golgi network (TGN) (Orlow, 1995). Stage I, originally termed a "premelanosome," is a relatively spherical organelle with an amorphous matrix. In stage II, the organelle is ovoid and contains a fibrillar internal matrix. In stage III, the deposition of melanin on the melanosomal matrix becomes evident; and in stage IV, the organelle is completely filled with melanin (reviewed in Nordlund et al., 1998). However, the sequence in which melanosomal proteins are sorted to the organelle and the role(s) they play in its maturation remain largely unknown. Melanosomes are related to , and both types of organelles evolve initially via the same pathway (Diment et al 1995; Dell'Angelica et al., 2000 ). Raposo et al. (2001) recently reported that the intracellular processing of GPlOO follows a unique route to form stage I melanosomes that diverges from conventional lysosomes just past the early endosome stage. Introduction

R -_ •- SER \, RER v.^-*.- Y • % - ;-... /. .• • • ^=^ '^ Golgi Pre-meionosome ' - /• • * • .•** ,',. • . t Mclonosomc v^ .^ i E^ melanosome keratinocvtes Melanin synthesis' melanocyte

tyroi DOPA DOPAquinonr ^^ CysteinjiDOPA

Tyros inaje Glutathiont e I cjreteme i 5,6 Dihydroxyindok DOPAthromf Fheo-melanin \ Indole 5,6- quinoiiF ^ $,6~dihydToxyindole 2caiboxylic acid 1 TYRPl Eu-melanin Indole 5,6 quinone caitioxvljc acid

Fig. 1 |A| Formation of melanosome. |B| Epidermal melanin unit |C| melanin synthesis pathway (adaptedfrom Aiuh et ai, 2007)

1.1.2 Melanin synthesis pathway

Melanin synthesis is a highly cooperative process carried out by tyrosinase family proteins (Fig. Ic). It starts with hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) ( Furumura et al., 1996). This is the most important reaction in the pathway since the spontaneous rate of tyrosine hydroxylation is negligible. The subsequent step i.e., oxidation of dopa to dopaquinone is also catalysed by tyrosinase. Dopaquinone can give rise to dihydroxyindole spontaneously (DHI) or in presence of TRP2/ Dopachrome Introduction

tautomerase gives rise to dihydroxyindole carboxylic acid (DHICA). The DHICA so formed is further oxidised to indole quinones by TRPl. The indole quinone and the carboxylic acid derivatives of the indole quinines, polymerize in a poorly understood reaction to form the eumelanin. The pathway for synthesis of pheomelanin is less well understood. Although both eumelanin and pheomelanins are present to various degrees in human skin and hair (Liu et al, 2005), eumelanin remains the most predominant type of the melanin pigment in humans ( Wielgus and Sama, 2005)

1.1.3 Types of melanin

Melanins, the end products of complex multistep transformations of L- tyrosine, are polyacetylene, polyaniline, and polypyrrole polymorphous and multifunctional biopolymers (reviewed in Slominski et al, 2004), represented by eumelanin, pheomelanin, neuromelanin, and mixed melanin pigment, respectively (Prota, 1992,1995). The most common form of biological melanin is a polymer of either or both of two monomer molecules: indolequinone, and dihydroxyindole carboxylic acid. Melanin exists in the plant, animal and protista kingdoms, where it serves as a pigment.

Eumelanin polymers have long been thought to comprise numerous cross-linked 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (structure dipicted in Fig. 2) polymers and are brown black in colour (Sato e/o/., 1985; Hearing, 1987; Thody e?a/., 1991; Prota e? o/., 1992; Norlund et al, 1998; Meredith and Sama, 2006). Introduction

"^-'l^ DHI (HO) SO 10 yx^^ :x)H 01 DHICA

Fig. 2. The basic monomeric building blocks of eumelanin (DHI and DHICA) and their redox forms. (Adapted from Meredith and Sarna, 2006).

Tyrosine can be converted to eumelanin through the following sequential intermediates,

Tyrosine —> DOPA —• dopaquinone —> leucodopachrome —> dopachrome —• 5,6- dihydroxyindoIe-2-carboxylic acid —• quinone —+ eumelanin

Tyrosine —> DOPA —> dopaquinone —+ leucodopachrome -^ dopachrome —> 5,6- dihydroxyindole —• quinone —> eumelanin

Pheomelanins range from yellow to reddish brown pigments. Biosynthesis of pheomelanins differs from that of eumelanins chemically, in that its oligomer structure incorporates the amino acid L-cysteine, as well as DHI and DHICA units. Pheomelanins are mainly concentrated in lips, nipples, glans of the penis and vagina.

Tyrosine can be converted to pheomelanin through the following sequential intermediates.

Tyrosine —> DOPA -^ dopaquinone + cysteine - 5-S-cysteinyldopa - benzothiazine intermediate —»pheomelanin

Tyrosine —> DOPA —> dopaquinone + cysteine ^ 2-S- cysteinyldopa ^ benzothiazine intermediate -^ pheomelanin Introduction

Neuromelanin is the dark pigment present in pigment bearing neurons of four deep brain nuclei: the substantia nigra - Pars Compacta part, the locus ceruleus , the dorsal motor nucleus of the vagus nerve (cranial nerve X), and the median raphe nucleus of the pons. Neuromelanins are macropolymers composed of aminochromes and noradrenalinochromes (Stepien et al, 1989; Cartsmen et o/.,1992; Odh et al, 1994; Double et al, 2000). Similar to other , neuromelanins are brown/black pigment with stable paramagnetic properties, insoluble in organic solvents, bleached by hydrogen peroxide, and labeled by silver stain (Zecca et al, 2001). Neuromelanins have mixed properties of both eu- and pheomelanins; they chelate and interact with several inorganic and organic compounds (Aime et al, 1994; Double et al, 2000).

1.1.4 Importance of pigmentaion

Cutaneous melanin pigment plays a critical role in camouflage, mimicry, social communication, and protection against harmful effects of solar radiation. Melanin is believed to be a photoprotective pigment. The protective action of melanin is related to its high efficiency to absorb and scatter photons, particularly the higher energy photons from the UV and blue part of the solar spectrum. As a result of ultrafast photodynamics, energy of the absorbed photons is rapidly and efficiently converted into heat (Ye and Simon, 2003). Melanin is important for skin homeostasis and tanning itself represents a distress signal (Gilchrest and Eller,1999). Introduction

1,1.5 Pigment disorders

A variety of diseases and abnormal conditions that involve pigmentation arise, either from absence of or loss of pigmentation or pigment cells, or from the excess production of pigment. Few pigmenation disorders are listed below. Becker Congenital Patterned Leukodermas Drug-Induced Pigmentation Hermansky-Pudlak Syndrome Hypomelanosis of Ito Idiopathic Guttate Hypomelanosis Laugier-Hunziker Syndrome Mongolian Spot Post-inflammatory

http://www.emedicine.com/derni/DISEASES OF PIGMENTATION.htm

Oculocutaneous albinism type 1 (OCAl) (Mendelian Inheritance in Man (OMIM) 203100) is the type with (usually) the least amount of pigment. People with this type of abnormality generally have very pale skin, "white" (actually translucent) hair and light blue eyes. OCAl is caused by an alteration of the tyrosinase gene, and can occur in two variations. The first is OCAla, in Introduction

which the organism cannot develop pigment at all. Vision usually ranges from 20/200 to 20/400. The second is OCA lb and it has several subtypes itself Some individuals with OCA lb can tan and develop pigment. One subtype of OCA lb is called OCA lb TS (temperature sensitive), where the tyrosinase can only function below a certain temperature, which causes the body hair in cooler body regions to develop pigment (i.e. get darker). Another variant of OCA lb, called Albinism, yellow mutant type (OMIM: 606952) is more common among the Amish than in other populations, and results in blonde hair and the eventual development of skin pigmentation during infancy, though at birth is difficult to distinguish from other types. About 1 in 40,000 people have some form of OCAl(Boissy and Nordlund, 1997; Peracha 2005, http://www.emedicine.com/oph/topic260.htm).

Oculocutaneous albinism type 2 (OCA2) (OMIM: 203200), the most common type of albinism, is caused by mutation of the P gene. People with 0CA2 generally have more pigment and better vision than those with OCAl, but cannot tan like some with OCAlb. A little pigment can develop in or moles (King et al, 2003). People with 0CA2 usually have fair skin but not as pale as OCAl, and pale blonde to golden or reddish-blonde hair, and most commonly blue eyes. Affected people of African descent usually have a different phenotype (appearance): yellow hair, pale skin, and blue, gray or hazel eyes. About 1 in 15,000 people have 0CA2. (Boissy and Nordlund, 1997; Peracha, 2005; http://www.emedicine.com/oph/topic260.htm).

Oculocutaneous albinism type 3 (OCA3, or rufous albinism) (OMIM: 203290) has only been partially researched and documented. It is caused by mutation of the tyrosinase-related protein-1 (TRPl) gene. Cases have been reported in Africa and New Guinea. Affected individuals typically have red hair, reddish-brown skin and blue or gray eyes. Variants may be the recently- identified minimal pigment type albinism (OMIM: 203280) and rufous

10 Introduction

oculocutaneous albinism (ROCA or xanthism) (OMIM: 278400). The incidence rate of 0CA3 is unknown (Boissy and Nordlund, 1997).

Oculocutaneous albinism type 4 (OCA4) (OMIM: 606574) is very rare outside of Japan, where 0CA4 accounts for 24% of albinism cases. 0CA4 can only be distinguished from 0CA2 through genetic testing, and is caused by mutation of the membrane-associated transporter protein (MATP) gene (Boissy and Nordlund, 1997; Peracha, 2005; http://www. emedicine. com/oph/topic260. htm).

Hermansky-Pudlak syndrome (HPS) (OMIM: 203300) is not a type of OCA, technically, but has similar features. HPS has a great range of degrees of pigmentation, from OCA la-like to almost-normal coloring. Vision usually ranges from 20/60 to 20/200. Apart from the and impaired vision, people with HPS lack dense bodies in their blood platelets which are responsible for releasing clotting factors. For this reason, HPS patients bruise easily and have a hard time stopping bleeding once it begins (bleeding diathesis, similarly to hemophilia). HPS has seven known forms (HPS-1 through HPS-7), each caused by a different autosomal recessive gene mutation. HPS-1 and HPS-4 may also include pulmonary fibrosis, or scarring of lung tissue that prevents the necessary expansion and contraction during breathing. It is believed that this is due to a buildup of fatty ceroid in the lungs. Colitis, or inflammation in the large intestine, is another symptom of most types of HPS, which may cause diarrhea, nausea, and blood in the stool. HPS is rare generally, but affects 1 in 1800 Puerto Ricans, and is typically fatal by middle age (Boissy and Nordlund, 1997; Peracha, 2005; http://www.emedicine.com/oph/topic260.htm).

Chediak-Higashi syndrome (CHS) (OMIM: 214500), like HPS, is not technically a form of OCA, but produces similar results. CHS, caused by mutation of the LYST gene, is very rare, and is associated with other medical

11 Introduction

problems, such as immune system dysfunction that leads to a high infant mortality rate, HPS-like hemophilia, and neurological problems, among many others, in 85% of sufferers. (Boissy and Nordlund, 1997; Peracha, 2005; http://www. emedicine. com/oph/topic260. htm).

Griscelli syndrome (GS) is similar to CHS in symptoms (and also very rare). It is divided into three types, GSl (OMIM: 214450), GS2 (OMIM: 607624) and GS3 (OMIM: 609227). Each type is due to a different autosomal recessive gene mutation. Type 1 produces mainly neurological problems in addition to albinism, while type 2 produces mainly immunological issues as well as the hypopigmentation, and type 3 only evidences hypomelanosis without either of the other sorts of problems. People affected by GS differ in appearance from those with OCA, having silvery-grey hair. A fourth and even rarer variant, partial albinism and syndrome (PAID) (OMIM: 604228), has been identified and requires further study. An additional type called Elejalde syndrome (OMIM: 256710) may exist, but some researchers believe it is actually simply GSl. GS2, because of its immune system effects, results in a very high mortality rate among children and young adults that have it (Boissy and Nordlund, 1997; Peracha,2005; http://www. emedicine. com/oph/topic260. htm).

Vitiligo (or leukoderma) is a in which melanocytes (the cells that make pigment) in the skin are destroyed. As a result, white patches appear on the skin in different parts of the body. Similar patches also appear on both the mucous membranes (tissues that line the inside of the mouth and nose), and the retina (inner layer of the eyeball). The hair that grows on areas affected by vitiligo sometimes turns white.Vitiligo is a chronic skin condition that causes loss of pigment, resulting in irregular pale patches of skin. (http://www.niams.nih.gov/hi/topics/vitiligo/vitiligo.pdf.) The precise cause of vitiligo is complex and not ftilly understood. There is some evidence suggesting it is caused by a combination of auto-immune, genetic, and

12 Introduction

environmental factors. VitiUgo is associated with autoimmune and inflammatory diseases, commonly thyroid overexpression and underexpression (Jin et al., 2007). Vitiligo generally appears in one of the three following patterns.

Focal pattern - the is limited to one or only a few areas.

Segmental pattern - depigmented patches develop on only one side of the body.

Generalized pattern - the most common pattern, depigmentation occurs symmetrically on both sides of the body.

In addition to white patches on the skin, people with vitiligo may have premature graying of the scalp hair, eyelashes, eyebrows, and beard. People with dark skin may notice a loss of color inside their mouths.

Treatment: Topical psoralen plus UV-A phototherapy (PUVA therapy) is a well-established treatment for nonsegmental vitiligo (reviewed in Huggins et al, 2004). Narrow-band UV-B (NB-UVB) phototherapy is a treatment of choice for patients with active, generalized vitiligo with an effectiveness of 63% against this form of the (Njoo et al, 1999). Topical therapeutics are frequently used in the treatment of vitiligo. Melagenine, a topical agent derived from placenta, has been used successfully in treating some childhood cases of vitiligo (Xu and Wej, 2004). In addition to this, surgical therapies like autologus skin grafting, skin grafts using blisters, autologous melanocyte transplants, micropigmentation can offer improvement in appearance to the patients who have vitiligo that has been stable for at least 3 years. For people who have vitiligo on more than 50 percent of their bodies, depigmentation may be the best treatment option. This treatment involves fading the rest of the skin on the body to match the areas that are already white.

13 Introduction

Melanoma is a form of skin cancer that occurs in the melanocytes. The appearance and growth of melanoma will differ depending on the morphologic type:

Superficial spreading melanoma Superficial spreading melanoma is the most common type of melanoma, representing approximately 70% of all melanomas. This melanoma goes through a prolonged (years) horizontal growth pattern on the skin before becoming invasive. Superficial spreading melanomas are flat or slightly elevated brown lesions with black, blue or pink discoloration which are typically greater than 6 mm in diameter and have irregular asymmetric borders. These melanomas may be found on any body surface, especially the head, neck, and trunk of males and the lower extremities of females.

Nodular melanoma Nodular melanoma represents about 15% of all melanomas and becomes invasive soon after first appearing. A nodular melanoma typically looks similar to a blood vessel growth, presenting as a dark brown-to-black papule or dome-shaped nodule, however 5% of nodular melanomas are amelanotic (see below). These melanomas are commonly found on all body surfaces, especially the trunk of males.

Acral-lentiginous melanoma represents approximately 8% of all melanomas and is the most common melanoma in dark-skinned people. Acral-lentiginous melanomas represent up to 70% of melanomas in blacks and up to 46% in Asians. This type can occur on the palms, soles and nail beds. Like nodular melanoma, acral-lentiginous melanoma is extremely aggressive, with rapid progression from the horizontal to vertical growth phase.

Lentigo maligna melanoma accounts for approximately 5% of melanomas. melanomas are typica 'y found on sun-exposed areas of the Introduction

skin in adults and are clearly linked to exposure to the sun. Often many years pass between the first appearance of this melanoma and when it becomes invasive. The precursor lesion is typically greater than 3 cm in diameter, and upon becoming invasive develops a dark brown-to-black color or raised blue- black nodule.

Desmoplastic melanoma is rare, representing approximately 1.7% of all melanomas. This type of melanoma is locally aggressive and is difficult to diagnose both clinically and microscopically. The majority of these tumors occur on the head and neck of elderly patients and one half are amelanotic.

Amelanotic melanoma is rare and is challenging to diagnose because there is an absence of pigmentation (color). However, hallmark traits of melanoma, such as changes in size, borders, and symmetry, are present in this melanoma.

15 Introduction

1.2 Major factors in melanogenic pathway

1.2.1Tyrosinase (ECl.14.18.1) The gene The mouse tyrosinase gene maps to the albino (c) locus on chromosome 7 and to human chromosome 11 ( Hearing and Tsukamoto, 1991). This gene is made up 5 exons and 4 introns and spans about 60kb in mouse.The cis-regulatory elements of the TYR gene are many and complex (Ferguson et a!.,1997). Several studies have been done to identify these regulatory elements. Transgenic expression studies using 270 bp of the mouse TYR 5% promoter region have exhibited both cell type specificity, allowing expression to occur in neural crest-derived melanocytes and in the melanocytes in the pigmented epithelium of the retina (which are derived from the optic cup), as well as temporal regulation (Kluppel et al., 1991; Beermann et al., 1992). Within this 270-bp region in the mouse, both positive and negative regulatory sequences have been identified (Ganss et al., 1994). Many of the same regulatory sequences have been identified in the human TYR promoter located in a region between _270 and _80 bp from the start codon (Fig. 3) (Kikuchi et al., 1989; Ponnazhagan et al., 1994; Ferguson et al., 1997). An important regulatory element, an 11-bp motif, AGTCATGTGCT, was found at_104 bp upstream of the transcriptional start site and has been labeled the 'M'-box (for melanocyte) or the TYR proximal element (TPE). The CANNTG motif has been found to bind a family of transcription factors, characterized by the presence of a conserved helix-loop-helix structure required for DNA binding and activation (Hodgkinson et al., 1993).

One trans-acting binding protein product is the human homologue of the mouse microphthalmia (mi) gene, which encodes a melanocyte specific factor called the microphthalmia-associated transcription factor (MITF). This factor binds to both the M-box and the TDE, a cis-acting element 1.86 kb upstream of the transcriptional start site (Yasumoto et al., 1994; Hemesath et Introduction

'ai a. o > »3_

I (GA)„ repeat KXltf ^ T r T I r Exon 1 -2000 -1500 -1000 -500

Fig. 3. Potential cis-acting regulatory sites in the 5' promoter of the TYR gene, (adaptedfror, Oetiing. 2000). al. 1994). The human MITF gene was found to direct transcription preferentially in melanin-producing cells for both the mouse TYR and TRP 1 genes (Yasumoto et ai, 1995). A second factor, termed an 'ubiquitous transcription factor', was also found to bind the Mbox, but its role in activating melanocyte-specific transcription is unknown (Lowings et al, 1992). Other transcription factors, N-Oct-3 and N-Oct-5, are expressed at high levels in melanoma cells and may be involved in mechanisms controlling the transcription of pigment-specific genes (Sturm et al, 1994). There are several other regulatory sequences found in the proximal TYR promoter that may be important in the correct expression of the TYR gene in melanocytes (Fig. 3). A cis-acting regulatory element 12-15 kb from the initiation codon has also been identified in the mouse (Porter et al, 1991; Porter and Meyer, 1994). This region contains two DNase I hypersensitive sites (HS site) within a 200 bp region embedded within a scaffold:matrix attachment region (S:MAR) (Ganss et al, 1994, Porter et al, 1999) and was found to be reartanged in the hypopigmented chinchilla-mottled (Tyrc-m) mouse revealing the importance of these sites for the proper expression of TYR (Porter et al, 1991). A human homologue to this site has yet to be identified, but if it exists, could play an important role in OCAl.

17 Introduction

The protein Human tyrosinase is a type 1 membrane glycoprotein that contains 529 amino acid including an 18 amino acid N-terminal signal sequence (Fig. 4). The human (NP_000363) and mouse (NP_035791) tyrosinase proteins are highly conserved, possessing 85% sequence identity (Kwon et al., 1988a,b). The mature protein can be divided into three domains: (i) the N-terminal 455

GlycosjH ation sites

1 III ft T TTTT N-terminal OoOlOO (D(DI) 1 00 00 0 1 0 j C-termina 1 1 Signal Copper-binding Transmembrane sequence sites domain

Fig. 4. Structure of tyrosinase (adaptedfrom Wang and Hebert. 2006) amino acid lumenal ectodomain; (ii) a single transmembrane domain; and (iii) a C-terminal cytoplasmic tail. The ectodomain of human tyrosinase possesses the enzymatic activity. It has seven consensus N-linked glycosylation sites and 17 Cys residues. One Cys residue is located in its cleaved signal sequence and another in the cytoplasmic tail, leaving 15 Cys available for disulfide bonding under the oxidizing conditions of the endoplasmic reticulum (ER) lumen. Six of the seven glycosylation sites are conserved in mouse tyrosinase while all the lumenal Cys are conserved. The N-linked glycosylation sites are spaced throughout the lumenal domain, while the Cys residues are concentrated in three Cys-rich clusters. Both human and mouse tyrosinase have a single 26 amino acid hydrophobic transmembrane domain which is approximately 70% conserved. The transmembrane domain anchors tyrosinase in the melanosome membrane with the N-terminal ectodomain facing the lumen.

18 Introduction

Mouse tyrosinase is four amino acid longer than human tyrosinase, because of an extension of the C-terminal cytoplasmic tail. The C-terminal tails of both human and mouse tyrosinase possess two well-studied intracellular targeting signals. These signals include a di-leucine (LL) motif and a tyrosine-based motif (YXXB, where B is a hydrophobic residue) that will be discussed in more detail below (Blagoveshchenskaya et al., 1999; Calvo et al, 1999; Honing et al, 1998; Simmen et al, 1999). Newly synthesized tyrosinase has a molecular weight of- 55 kDa and is glycosylated en route to the melanosome to a final molecular weight of -75-80 kDa (Jimemez et al, 1988; Hearing and Tsukamoto, 1991) Two additional TRPs stimulate eumelanin synthetic rate: TRPl, product of TRPl (human) or b (mouse) locus, and TRP2 product of TRP2/DCT (human) and slaty locus (mice).

1.2.2 Tyrosinase Related Protein 1 (TRPl)

The gene The TRPl gene also called as gp75, has been mapped to the brown locus (b) chromosome 4 in mouse (Jackson, 1988; Bennett et al, 1990) and in humans on chromosome 9 (Murty et al, 1992). The TRPl gene encompasses 8 exons separated by seven introns including the first non-translated, exon (Budd and Jackson, 1995; Sturm et al, 1995). Mutations in the brown locus lead to synthesis of brown melanins rather than black melanin indicating it to be an important step in eumelanogenesis. Halaban and Moellmann (1990) showed that the b locus protein is a catalase and is identical to a known human melanosomal protein, gp75. Mutations in TRPl also lead to structural changes in melanosomes (Prota et al, 1995); melanosomes produced in brown melanosomes are round and relatively disorganized as compared to that in eumelanosomes. A recent study suggests that TRPl protein is essential for cell division of melanocyte in culture (Sarangrajan et al, 2000) Introduction

The protein TRPl has the 5,6- dihydroxyindole-2-carboxylic acid oxidase activity that catalyses an intermediate step in the melanin synthesis pathway (Kobayashi et al., 1994). Newly synthesized protein is ~ 53 kDa and matures to around 75 kDa (Halaban and Moellman, 1990; Hearing and Tsukamoto, 1991). This protein is present in a higher quantity compared to tyrosinase in both murine and human melanocytes (Setaluri et al., 1990). It has been proposed that the TRPs might interact in a multienzyme complex and the presence of high molecular weight aggregates have been shown (Orlow et al., 1994). TRPl has recently been shown to be actively involved in stabilizing the tyrosinase protein and allowing its efficient activity (Kobayashi et al., 1998)

1.2.3 Tyrosinase related protein 2 (TRP2/Dct) (EC 5.3.2.3)

The gene The TRP2 gene has been mapped to the slaty locus on the mouse chromosome 14 (Jackson et al., 1992) and human chromosome 13 (Yokoyama et al, 1994). Mutations in the slaty locus result in the dilution of coat color and slightly yellowish ears.

The protein The molecular weight of fully processed protein is 75-80 kDa. Multiple forms of TRP2 generated posttranslationaly by alternative poly (A) usage or by alternative splicing of mRNA. It catalyzes the conversion of dopachrome to DHICA (Aroca et al., 1990). Although the specific function of TRPl has been in dispute, mutations in its structural gene result in the formation of brown rather than black melanin. (Kobayashi et al, 1994). The melanocyte-specific proteins TRPl and dopachrome tautomerase (DCT or TRP2) stabilize tyrosinase in the early secretory pathway (Kobayashi et al, 1998). These proteins appear to constitute a melanogenic complex that helps

20 Introduction

to increase the activity of tyrosinase (Manga et al, 2000). It appears that dimerization of which expressed a defective TRPl protein (C86Y) (Francis et al.,2003).

1.2.4 Tyrosinase secretory pathway For proteins that traverse the secretory pathway, the ER provides a protective folding environment by coupling a series of foldases, molecular chaperones, folding sensors, and covalent modifiers, which work in concert to assist with the proper maturation of nascent proteins. (Petrescu et al., 1997, 2000; Ujvari et al., 2001; Francis et al., 2003). The co-translational and co- translocational maturation events include signal sequence cleavage, glycosylation, disulfide bond formation, and chaperone binding (Chen et a I., 1995; Hebert et al., 1997; Molinari and Helenius, 2000; Kowarik et al., 2002; Daniels et al., 2003; Schnell and Hebert, 2003). The maturation process continues post-translationaly in the ER until a ftiUy folded and assembled protein is packaged into vesicles that exit the ER for the Golgi. The translocation of tyrosinase into the ER lumen is guided by its N- terminal cleavable signal sequence. This study found that the signal sequence of tyrosinase was first cleaved when the cleavage site was located 142 amino acid away from the ribosomal P-site (Wang et al., 2005). N-linked glycosylation is important for the folding of some of eukaryotic proteins. Human tyrosinase is heterogenously glycosylated with either six or all seven of its glycosylation sites being occupied (Ujvari et al., 2001). Mouse tyrosinase lacks the hypoglycosylated site at Asn272. Glycosylation is essential for tyrosinase activity, as inhibition of glycosylation with tunicamycin abolishes proper tyrosinase maturation and its subsequent enzymatic activity (Imokawa and Mishima, 1981; Mishima and Imokawa, 1983; Takahashi and Parsons, 1992). Selective sites of glycosylation on tyrosinase are required for its proper maturation and stability, while others are

21 Introduction

Tyrosinase dimer V

ER

Golgi

Endosome I Melanosome

Stage IV Stage IH

Fig. 5. Tyrosinase maturation and trafficking through the secretory pathway. (1) Tyrosinase folds in the ER co-translationaly and dimerizes. The quality control system in the ER ensures the folding and assembly are correct. (2) The export-competent tyrosinase is transported to the cis-Golgi network in COPII vesicles. (3) In the trans-Golgi network (TGN), the N-linked glycans are modified further to complex sugars and copper is loaded. (4) Tyrosinase is transported out of the TGN to the melanosomes. (5) The melanogenic complex is formed in melanosomes, which mature through the various stages. {Adapted from Wang and Hehert, 2006) dispensable (.Gershoni-Baruch et ai, 1994; Halaban et al., 2000; Branza- Nichita et al., 2000). ER Hsp70 family member BiP is the first chaperone that associates with tyrosinase during its maturation. It binds tyrosinase co- translationaly at an early stage of translocation, when the N terminus of tyrosinase is only 142-170 residues away from the P-site of the ribosome (Wang et al., 2005). As the hydrophilicity of the nascent chains increases with the addition of the glycans, and these glycans are subsequently trimmed to their monoglucosylated state, the Hsp70 chaperone system hands tyrosinase

22 Introduction

off to the lectin chaperone system to assist with the later stages of tyrosinase folding until it reaches its native conformation. It seems that the two general chaperone systems calnexin/calreticulin and BiP complex can exchange substrates to optimize the folding reaction for tyrosinase (Petrescu et al., 1997;Branza-Nichita et al., 2000; Meunier et al., 2002; Deprez et al, 2005). Tyrosinase forms homodimers after properly folding in the ER, prior to being transported to the Golgi (Halaban et al, 2002a; Francis et al., 2003). Once tyrosinase has properly matured in the ER, it is packaged into COPII- coated vesicles that bud from the smooth ER membrane. Tyrosinase then travels by way of the traditional anterograde pathway to the ER-Golgi intermediate compartment where it is subsequently sorted to the cis-Golgi. In the Golgi, additional mannose residues are removed by the Golgi mannosidases I and II (Helenius and Aebi, 2001). Reports suggest that tyrosinase receives its copper prior to its exit from the TGN. Therefore, tyrosinase likely becomes enzymatically active in the TGN unless additional downstream factors are required for its activation (Hebert, 2006). The sorting of membrane proteins from the TGN to lysosomal-related organelles such as melanosomes are generally mediated by signals localized to the C-terminal cytosolic tail of the cargo protein. Tyrosinase, as well as the other melanosomal proteins including TRPl, p- protein and Pmell7possess dileucine motifs in their cytoplasmic tails (Calvo et al., 1999; Vijayasaradhi et al., 1995). The putative melanocyte membrane transporters p-protein and MATP appear to be critical for the proper maturation, processing and trafficking of tyrosinase to post-Golgi melanosomes. This is the mode in which tyrosinase traverses the secretory pathway. However reports indicate that only 50% wild-type tyrosinase reaches its mature form under optimal conditions (Halaban, 1997). Although the endogenous degradation of tyrosinase was first observed several decades ago, little had been clarified about the specific mechanism(s) that regulates

23 Introduction

tyrosinase degradation until proteasomes were found to be involved in that process (Halaban et ai, 1997).

1.3 Proteasomes Proteasomes are multicatalytic non-lysosomal proteases mainly involved in intracellular protein degradation (Hilt et al, 1993; Scherrer and Bey, 1994; Coux et al, 1996; Voges et al, 1999; Ciechanover et al, 2000). Proteasomes belong to the family of N-terminal nucleophile hydrolases, with their active sites being formed by the N-terminal threonine residues of their P- subunits. The proteasome is a modular structure. One or two regulatory particles (RP) attach to the outer surface of the core particle (CP).(Figure 8) Based on the regulatory particles attached, proteasomes are classified based on these regulatory particles attached (Glickman and Ciechanover, 2001). 26S proteasomes are responsible for cytosolic protein degradation in eukaryotes (Chandu and Nandi 2004).

24 Introduction

19SRegulatur,''^'"^y^, J particle i[^f^

VT~^ } ( '•'. Vi nsy \TP

f'•-—i >-6 'Li" ,L;•'•, 20S Proteasome A 1 1_ -'^••'

p/ ,) i'J j''~) P131

Fig. 6. Various proteasome modulating activating complexes (adapted from www.biomol.com/ubiquitons&zomes)

1.3.1 26S proteasome The proteasome holoenzyme (also known as the 26S proteasome) is a -2.5 MDa complex made up of two copies each of at least 32 different subunits that are highly conserved among all eukaryotes (Fig. 6). The overall structure can be divided into two major sub-complexes: the 20S CP that contains the protease subunits and the 19S regulatory particle (RP) that regulates the function of the former (Fig. 6). The 20S CP is a barrel-shaped structure made up of four rings of seven subunits each. The two inner (3-rings contain the proteolytic active sites facing inward into a sequestered proteolytic chamber (GroU et al, 1997; Loewe et al, 1997). One or two regulatory particles attach to the surface of the outer a-rings of the 20S CP to form the 26S proteasome holoenzyme (Fig. 6). The 19S RP itself can be ftirther dissected into two multisubunit substructures (Table 2), a lid and a base (Glickmane/o/., 1998).

25 Introduction

1.3.2 20S proteasome 20S proteasome or the core particle is a barrel shaped structure that contains the protease subunits are composed of four heptameric stacked rings ( a7 p7 p7 a7) and the outer rings are made up of a-type subunits whereas the inner two rings are made up of P-type subunits. The quaternary structure of 20S proteasomes is conserved from bacteria, including archaea, to mammals and the active sites are present inside a central "chamber for degradation" (Voges et al 1999; Pickart and Cohen 2004). All the subunits in a ring are identical in the 20S proteasome from T. acidophilum. In eukaryotes, each ring is composed of as many as 7 different a-type or 7 different P-type subunits (Table 2). Studies of human 20S proteasomes on peptide libraries demonstrated that proteasomes can cleave peptide bonds at the PI position (the amino acid immediately proximal to the peptide bond that is cleaved) of most amino acids, with a preference for leucine and alanine. Also, amino acids proximal to the Pi position, i.e. at P3 and P4 positions, glutamine, valine, isoleucine, leucine and asparagine influence peptide cleavage by 20S proteasomes (Harris et al., 2001).

26 Introduction

20S a-type subunits Nomenclature Human Seq. length MW Baumeister 'Old' Coux Groll Miscellaneous UniProtKB4 (amino (Da) etal. human etal. etal. acids) al iota Pro-a6 alsc Pros27, p27k, C7, a6 PSMA6 246 27399 Prs2, Y8, Prs2, Sell a2 C3 Pro-a2 a2_sc Pre8, Prs4, Y7 a2 PSMA2 233 25767 a3 C9 Pro-a4 a3_sc Pre9, Prs5,Y13 a4 PSMA4 261 29484 a4 C6 Pro-a3 a4_sc XAPC-7, Pre6 a7 PSMA7 248 27887 a5 zeta Pro-al a5_sc Pup2, Doa5 a5 PSMA5 241 26411 a6 C2 Pro-a5 a6_sc nu, Pros30, p30k, al PSMAl 263 29556 Pre5 a? C8 Pro-a7 a7_sc ' PrelO, Prsl,Cl, a3 PSMA3 254 28302 Prcl

27 Introcfuction

20S P-type subunits Pl Y Pro-P3pl_sc delta, Lmp9, Pre3 P6 PSMB6 P39/205125358/1 21904 Pli Lmp2 Pro-p3 Ring 12 p9 PSMB9 1219/199123264/1 21276 P2 Pro-p2p2_sc Lmpl9, MC14, Pupl P7 PSMB7 |2 77/234129965/1 25218 P2i MECL-lPro-p2 LmplO P10|PSMB10(273/234J28936/ 24648 P3 CIO Pro-p6p3_sc theta, Pup3 P3 PSMB3 205 22949 P4 C7 Pro-p4p4_sc Prel.Cll P2 PSMB2 201 22836 P5 Pro-pi P5_sc epsilon, Lmpl7, MBl,Pre2, Doa3, Prgl P5 PSMB5 &08/204I22897/1 22458 P5i Lmp7 Pro-pi RinglO, Y2,C13 P8 PSMB8 [276/204130354/1 22660 p6 C5 Pro-p5p6_sc gamma, Pre7, Prs3,C5, Ptsl Pl PSMBl 241 26489 P7 N3 Pro-p7p7_sc beta, Pros26, Pre4 p4 PSMB4 1264/219129192/1 24380

28 Introcfuction

19S Proteasome

Finley Dubiel Miscellaneous UniProtKB4 Human Seq. length MW etal. etal. (amino acids) (Da) 19S (PA700) regulator ATPase subunits Rptl S7 p48, Mssl, Yta3,Cim5 Subunit 7 PSMC2 432 48503 Rpt2 S4 p56, Yhs4. Yta5, Mts2 Subunit 4 PSMCl 440 49185 Rpt3 S6b S6, p48, Tbp7, Yta2, Subunit 6b PSMC4 418 47336 Yntl,MS73 Rpt4 SlOb p42, Sug2, Pcsl, Subunit 10b PSMC6 389 44173 Crll3,CADp44 Rpt5 S6a S6\p50, Tbpl, Ytal Subunit 6a PSMC3 439 49204 Rpt6 S8 p45,Tripl,Sugl,Cim3, Subunit 8 PSMC5 406 45626 Crl3,Tbyl,TbplO, m56 19S (PA700) regulator non-ATPase subunits Rpnl S2 p97, Trap2,Nasl,Hrd2, Subunit 2 PSMD2 908 100200 Rpdl,Mts4 Rpn2 SI pll2, Sen3 Subunit 1 PSMDl 953 105836 Rpn3 S3 p58, Sun2 Subunit 3 PSMD3 534 60978 Rpn4 Sonl,Ufd5 531 60153 Rpn5 p55, Nas5 Subunit 12 PSMDl 2 455 52773 Rpn6 S9 p44.5, Nas4/6? Subunit 11 PSMDU 421 47333 Rpn8 S12 p40, Mov-34h, Nas3 Subunit 7 PSMD7 324 37025 Rpn9 Sll p40.5, Lesl,Nas7 Subunt 13 PSMDl 3 376 42918 RpnlO S5a p54, ASFl,Sunl, Subunit 4 PSMD4 377 40736 McbUMbpl Rpnll S13 Pohl,Mprl,Padlh Subunit 14 PSMDl 4 310 34577 Rpnl 2 S14 p31,Ninl,Mts3 Subunit 8 PSMD8 257 30005 Rpnl 3 YLR421C 156 17902 S5b p50.5 Subunit 5 PSMD5 503 56065 S15 p27-L Subunit 9 PSMD9 223 24654 p28, Gankyrin, Nas6 Subunit 10 PSMDIO 226 24428

29 Introduction

lis Activator Nomenclature GENE Seq. length MW Dubiel et al. Ma et al. Realini et al. Kandil et al. UniProtKB4 (human) (amino acids) (Da)

llSa PA28a REGa Subunit 1 PSMEl 249 28723

lisp PA28P REGP Subunit 2 PSME2 238 27230 llSy PA28y REG7 Ki antigen Subunit 3 PSME3 254 29506

Table. 2. Proteasome nomenclature {http://www.biomol.com/SiteData/docs/Ubiqmtons&Zomes%20Fmal/86568ecl0c5cf64a00405a85c445 a40d/Ubiquitons&Zomes%20Final.pdJ)

30 Introduction

1.3.3 Ubiquitin proteasome System Proteins targeted for destruction are distinguished as "intracellular" and "extracellular". Extracellular proteins such as the blood coagulation factors, immunoglobulins, albumin, cargo-carrying proteins and peptide hormones (such as insulin) are taken up via pinocytosis or receptor-mediated endocytosis. They are then carried via a series of vesicles (endosomes) that fuse with primary lysosomes where they are degraded. During this process, the

Degradation Deubiquitylation

ATAP AD P 26S proteasome X^ -

Ub Conjugation -k Fig. 7. Ubiquitin proteasome pathway extracellular proteins are never exposed to the intracellular environment (the cytosol) and remain "extracellular" (topologically) throughout. Degradation of proteins in lysosomes is not specific, and all engulfed proteins exposed to lysosomal proteases are degraded at approximately the same rate. Whereas, intracellular proteins are degraded by a highly specific process and different proteins have half-life times that vary from a few minutes (e.g., the tumor suppressor p53) to several days (e.g., the muscle proteins and myosin)

31 Introduction

and up to a few years (crystalline). As seen in figure 7, ubiquitin is activated by the ubiquitin-activating enzyme El, a ubiquitin-carrier protein, E2 (ubiquitin-conjugating enzyme, UBC), and ATP. The product of this reaction is a high-energy E2_ubiquitin thiol ester intermediate. The protein substrate is bound to a specific ubiquitin-protein ligase via a defined recognition motif, E3. the protein is multiubiquitinated via multiple (n) cycles of conjugation of ubiquitin to the target substrate and synthesis of a polyubiquitin chain. E2 transfers the first activated ubiquitin moiety directly to the E3-bound substrate, and in following cycles, to previously conjugated ubiquitin moiety. Direct transfer of activated ubiquitin from E2 to the E3-bound substrate occurs in substrates targeted by RING finger E3s, the activated ubiquitin moiety is transferred from E2 to a high-energy thiol intermediate on E3, before its conjugation to the E3-bound substrate or to the previously conjugated ubiquitin moiety. This reaction is catalyzed by HECT domain E3s. Ubiquitin- tagged substrate is degraded by the 26S proteasome complex and short peptides are released. Ubiquitin is recycled via the activity of deubiquitinating enzymes (DUBs).

32 Introduction

1.3.4 Functions of the Proteasome

PriilfiM >s OIK- \nii/\mc ®f * Ubiquitin -^ . IQ pathway ^ ^ • '

Q, "»e» ® Untoldi-d protein

I >^*"'*« Proecswd

'".;- (D Amtao- \ I »| t P--"'"'" ill T.pir.«pomr >r Mnc-i Short Antigen peptides Peptides presentation

Fig. 8. Functions of the proteasome. 1. covalent conjugation of the protein to multiubiquitin chain 2. Ubiquitin-protein conjugates are specifically targeted to the proteasome. 3. Deubiquitination 4. Deubiquitinated substrates 5. Ubiquitin-independent degradation 6. Chaperon mediated degradation of unfolded proteins 7. Proteolysis of proteins to shorter length peptides 8. Further degradation of peptides to amino acids 9. Binding of peptides to the ER Tap transporter for presentation on MHC class 1 molecules. 10. Limited processing of substrates. 11. Refolding and rescue of the proteins from proteolysis. 12. DUB-mediated processing of polyubiquitin chains (adapted from Glickman and Ciechanover, 2002)

As reviwed by Nandi et al., (2006) the UPS play major roles in several biological processes (Fig. 8). The key ones are listed below,

Regulation of the cell cycle: The levels of regulatory proteins (e.g. cyclin B, CDK inhibitor p27kipl) are modulated at different phases of the cell cycle and the UPS is essential for cells to exit mitosis. The two major classes of E3 ligases that are involved in this process are the SCF complexes and the anaphase promoting complex/ cyclosome. In general, SCF regulate entry into S phase and recognizes substrates post-phosphorylation. The anaphase promoting complex is important for sister chromatid separation, exit from

33 Introduction

mitosis and degrades cell cycle regulators containing a nine amino acid motif known as the destruction box (Murray 2004).

Cancer and cell survival: The tumor suppressor p53 plays a key role in regulating cell cycle arrest, DNA repair and apoptosis. Under normal conditions, p53 levels are low due to binding to Mdm2, an E3 ubiquitin ligase. After DNA damage, p53 is phosphorylated resulting in reduced interaction with Mdm2, and induction of cell death. Not surprisingly, mutations in p53 are often associated with different human cancers. Interestingly, the human papilloma virus encodes a E3 ligase (E6-AP) which degrades p53 and is involved in generation of cervical tumors (Ciechanover and Iwai 2004).

Inflammatory responses: NF- KB is a key transcription factor involved in the inflammatory response. NF- KB is bound to inhibitor- KB and is found in the cytosol. On appropriate stimulation, inhibitor- KB is phosphorylated and degraded by the UPS. Free NF- KB enters into the nucleus and induces the expression of several genes involved in the inflammatory response (Karin and Ben-Neriah 2000).

Immune response: MHC class I molecules present peptides to CD8+ T cells. This process involves the digestion of self or microbial proteins into peptides by the UPS that are presented on MHC class I. Inhibition of UPS leads to the impairment of the biogenesis of MHC class I molecules (Kloetzel 2004).

Protein misfolding: The UPS interacts with members of the heat shock family and cofactors to eliminate misfolded proteins. A direct relation between protein unfolding and degradation is via CHIP, an E3 ligase and a Hsc70 interacting protein (McDonough and Patterson 2003).

34 Introduction

ER associated degradation: The UPS is also involved in the degradation of misfolded proteins in the ER which involves retro-translocation of misfolded proteins from the ER to cytoplasm via the Sec61 translocon. Other components of this pathway also include a cytosolic N-glycanase, ATPases, e.g. Cdc48p/p97/valosin-containing protein, and associated cofactors (Kostova and Wolf 2003). A clinical manifestation of this is observed in patients suffering from cystic fibrosis where the mutant F508 CFTR protein is retained exclusively in the ER and degraded by the UPS (Ward et al 1995). The role of chaperones and factors involved in ER-associated degradation is an active area of investigation.

Disease progression: Angelman syndrome is characterized by severe phenotypic defects including mental retardation, seizures and abnormal gait. Mutations in the E3 ligase, E6-AP cause Angelman syndrome, the first human disorder to be identified with a defect in the UPS (Kishino et al 1997). Another E3 ligase that contains the HECT domain, NEDD4, regulates the number of sodium channels on the cell surface. Mutations in NEDD4 cause hypertension associated with hypokalemic metabolic alkalosis, low plasma renin activity, and suppressed aldosterone secretion, together termed as the Liddle syndrome (Staub et al 1997).

35 Introduction

1.4 Link between proteasomes and tyrosinase The synthesis and the degradation of tyrosinase are tightly coupled to its function, and are influential parameters that regulate melanin synthesis. In instances where a melanogenic inhibitor decreases tyrosinase protein levels but has little effect on its mRNA levels (reviewed in Ando et al., 2007) it is like that the degradation of tyrosinase was accelerated by that agent. Early studies on the stability of tyrosinase revealed that tyrosinase is degraded endogenously in melanoma cells (Saeki and Oikawa, 1980; Jimenez et al., 1988; Martynez- Liarte et al., 1988). The rate of tyrosinase degradation was found to be increased by acidification of the culture medium (Saeki and Oikawa, 1980), indicating that the degradation of tyrosinase could be altered by environmental factors surrounding melanocytic cells. Although the endogenous degradation of tyrosinase was first observed several decades ago, little had been clarified about the specific mechanism(s) that regulates tyrosinase degradation until proteasomes were found to be involved in that process (Halaban et al., 1997). Studies on carbohydrate modifications of tyrosinase have revealed that tyrosinase destined for degradation in the ER is proteolyzed by proteasomes via ER-associated protein degradation (ERAD) (Wang and Androlewicz, 2000; Svedine et al., 2004). ERAD is a mechanism for quality-control which involves retention in the ER and retro-translocation into the cytosol of misfolded or unassembled secretory proteins followed by their deglycosylation, ubiquitylation, and subsequent proteolysis by proteasomes (Bonifacino and Weissman, 1998; Plemper and Wolf, 1999; Ellgaard and Helenius, 2003).

1.5 Scope and organization of tlie tliesis Post translational regulation of tyrosinase seems to be a major mechanism in regulation of melanogenesis. Extensive study is being carried out to understand the same. It has been indicated that tyrosinase destined for degradation in the ER is proteolyzed by proteasomes via ER-associated

36 Introduction

protein degradation (ERAD). More recently however, certain observations indicated that degradation of immature form of tyrosinase due to retention in ER might not be always true (Hall and Orlow, 2005 and Ando et ai, 2007). Thus, these observations put together indicate an ambiguity in the mechanism of amelanogenesis in melanoma cells. Besides this in most of the studies level of melanogenesis is studied between different cell types or regulation of mutant tyrosinase is studied. Here in the present study we are using B16 mouse melanoma cells as a model system, the salient features being; I) Inspite of being transformed the cells continue to synthesize melanin. II) the cells spontaneously fluctuate between melanotic and amelanotic phenotypes and retain the capacity to remelanize. Ill) Are easy to maintain in culture as compared to normal melanocytes. These factors facilitate the study of degradation pattern of tyrosinase in melanoma cells without any external manipulation.. Thus the work in present thesis is aimed at elucidating the role of proteasomes and regulation of melanin biosynthesis in different phenotypes of mouse melanoma cells. With this the study has been divided in three parts as,

3.1 Degradation of tyrosinase and role of proteasomes in the degradation of tyrosinase and tyrosinase related proteins in melanoma cells

3.2 Tyrosinase in Amelanotic and Melanotic Melanoma Cells: Study of Degradation Pattern.

3.3 Phenotype-specific protein pattern of B16 melanoma cells and puriflcation of 20S and 26S proteasomes from 816 melanoma cells

37