OSSTMM

MHC class I molecules bind peptides of 8 - 10 amino acids length and p resent them to cytotoxic T cells. The source o f the peptides

are u su a lly cytosolic p ro tein s but th ere are also som e m in o r pathw ays by which antigens from other com partm ents or external sources can be presented (Carbone and Bevan, 1990; Kovacsovics- Bankowski and Rock, 1995).

The initial step in the generation o f antigenic epitopes for M H C class I m olecules is the proteolytic degradation o f proteins in the c y to s o l by la rg e m u l t i c a t a l y t i c p r o t e a s e s , r e f e r r e d to as p r o te a s o m e s . P ro te a s o m e s are e v o lu tio n a r y h ig h ly c o n s e r v e d p ro te a se s th at o c cu r in a v a rie ty o f d iffe re n t fo rm s w h ic h p re s u m a b ly re fle c t th e ir d iffe re n t c e llu la r tasks w h ich b e sid e antigen processing include degradation o f ubiq u itin y lated proteins by p ro teo ly sis (Belich and T row sdale, 1995; C iechanover, 1994; C ressw ell and Hughes, 1997). Five different proteolytic activities have been c h arac terise d so far: c h y m o try p sin -lik e , try p sin -lik e, p e p tid y lg lu tam y l-p ep tid e h y drolysing, bran ch ed ch ain am ino acid preferring and small neutral amino acid preferring (Eleuteri et al., 1997) T he p ro te a so m e core co m p lex is a 20S b a rr e l-s h a p e d m olecule that can be decorated with various associating proteins. The 20S core complex of both the archae bacterium T h e r m o p l a s m a a c i d o p h i l u m (Groll et al., 1997; Lowe et al., 1995) and yeast have b een c ry sta llise d rec en tly . The a n a ly sis o f the T . a c i d o p h i l u m

protein revealed that the 20S core com plex consisted of four rings o f either 7 identical a or p subunits. T he inner p su b u n its w e re found to be the catalytically active ones im plying that proteins have to enter the central tunnel of the barrel-shaped m olecule in o rd er to be degraded. The y east cry stal structure is far m o re co m p lex with 7 d istin c t a and p subunits. Only three o f the p subunits seem to be cataly tically active. Surprisingly, the in n er c a v ity o f the yeast p ro tea so m e seem s to be in a c c e s s ib le for proteins. It has been proposed that access to the inside of the 20S

proteasom e is regulated by additional subunits, such as the PA 28 regulator (Rubin and Finley, 1995).

In m a m m a lia n cells th ree o f the c o n s titu tiv e ' p r o te a s o m e p subunits can be replaced by interferon-y inducible subunits two of w h ich are encoded in the MHC. U p o n ex posure to in terfero n -y (IFNy) the two M HC class H-encoded subunits LM P2 and L M P7 re p la c e 6 and M B l (B elich et al., 1994), resp ectiv ely , w h e rea s M E C L -1, w hich is not M H C -en c o d ed but also I F N y - in d u c i b le replaces subunit Z (Groettrup et al., 1996; Hisam atsu et al., 1996; N a n d i et al., 1996). IFNy p r o d u c t i o n c an be i n d u c e d by in fla m m a tio n and virus infections. It seem s p lau sib le th at the replacem ent of the constitutive subunits by IFNy inducible ones in these situations has an effect on the peptides produced. Indeed, studies com paring the peptide repertoire of cells with those that h ad been stim u lated w ith IFNy d e m o n s tra te d th at the IFNy- inducible subunits seem ed to enhance cleavage after h y d ro p h o b ic and basic resid u es, w hile c leav ag e a fter a cid ic resid u es w as inhibited (Gaczynska et al., 1993; Gaczynska et al., 1994; Driscoll et al., 1993). These results are consistent with the idea that exchange o f the constitutive pro teaso m e subunits for IF N y -in d u cib le o n es le a d s to a m o re p r o n o u n c e d p r o d u c tio n o f p e p tid e s w ith h y d r o p h o b ic C -te rm in i c ap a b le o f b in d in g to M H C c la ss I m olecules. Alternatively, the replacem ent o f some subunits in only p a r t o f the p ro te a so m e p o o l m ig h t lead to a m o re v a r ie d p ro teo ly tic activity w hich w ould in crease the rep e rto ire o f the produced peptides.

IFNy no t only has an in flu e n ce on the p ro te a s o m e s u b u n it co m p o sitio n , it also upregulates the expression of PA28 w h ic h binds to the proteasom e core complex and increases its proteolytic

activity. The PA28 m olecule consists o f two subunits, a and (3 that form a hexam er ring that can bind to both ends o f the proteasom e core complex (Gray et al., 1994) In vitro peptide Cleavage studies sh o w e d th at p r o te a so m e s in the p re s e n c e o f PA 28 g e n e ra te peptides resulting from a double cleavage o f the substrate rath er than a single cleavage (Dick et al., 1996). By this mechanism the g e n e ra tio n o f c e rta in C T L e p ito p es was m a rk e d ly e n h an c ed .

Figure 1.6 Antigen presentation by MHC class I molecules

MHC class heavy chain and p 2 -m ic ro g lo b u lin are tr a n s la te d into the lumen of the rough ER where they associate. This process is aided by the chaperones calnexin and calreticulin (not shown in the graphic). The peptides that bind to MHC class I m olecules are g enerally deriv ed from the cytosol. C ytosolic proteins are degraded by the p ro teaso m e and the re s u ltin g p e p tid e s are tra n s p o rte d in to the E R by the h e t e r o d i m e r i c t r a n s p o r t e r a s s o c i a t e d w i t h a n t i g e n presentation (TAP). Here the peptides bind to M H C class I. m olecules form ing stable trim eric com plexes. T he relay o f peptides from TAP to the MHC class I molecules appears to be facilitated by the recen tly discovered m o lecu les tapasin which seems to bridge TAP and class I m olecules. Properly assembled MHC class I molecules leave the ER and reach the cells surface via the default secretory pathway. Cell surface MHC class I molecules present their peptidergic ligands to CD8+ cytotoxic T cells.

Figure 1.6 Antigen presentation by MHC class I molecules c y to s o l , ta p a sin P ro tea so m e

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O n ce pep tid es have b een produced by the p ro tea so m e in the cytosol they have to be transported into the ER in order to bind to M H C class I molecules. This transport process is m ediated by an A B C tra n s p o rte r m o le c u le in the ER m e m b ra n e c alle d T A P (transporter associated with antigen processing) consisting o f two subunits, T A PI and TA P2 (Neefjes et al., 1993). Both subunits are encoded in the MHC class II region and are IFN y-inducible (K elly et al., 1992). Each subunit has a C-terminal cytosolic ATP-binding d o m ain and m u ltip le p red icted transm em brane d o m ains (G ileadi and Higgins, 1997). T he detailed structure o f the m olecule and num ber o f the transm em brane domains has not been resolved yet. Peptide binding studies suggested that the peptide binding site is at the C-terminal end o f the molecules and is formed by regions of b oth T A P I and T A P2 (Nijenhuis and H am m erlin g , 1996). T he sp ecificity o f the pep tid e transport has been analysed using in vitro systems. In hum ans peptide transport by T A P seems to be fairly promiscuous, unlike in the mouse where a strong preference fo r peptides with hydrophobic C-termini is exhibited (Neefjes et al., 1995). It is unclear what biological significance these findings h a v e , sin ce c e rta in n a tu ra l CTL e p ito p e s are v e ry p o o r ly tran sp o rted in such in vitro assays (A ndrolew icz and C ressw ell,

1994).

In the ER the folding process of newly synthesised MHC class I m olecules is helped by a variety of general ER chaperones, like c a l n e x i n a n d c a l r e t i c u l i n , w h ic h are b o th s p e c i f i c f o r m onoglucosylated proteins (Hammond and Helenius, 1995; Jackson et al., 1994; S ad asiv an et al., 1996), and others, like E R 6 0 (Lindquist et al., 1998).

A bridging molecule betw een the MHC class I heavy chain-P2iîi - c a lreticu lin com plex and TAP has been iden tified recen tly and

te rm e d tap a sin (O rtm ann et al., 1997; Sadasivan et al., 1996). T a p a sin is another m em b er o f the im m u n o g lo b u lin su p e rfam ily w ith a m em brane p ro x im al dom ain h o m o lo g o u s to Ig c o n sta n t reg io n dom ains. C o-im m unoprecipitation studies show ed that fo u r tapasin molecules associate with a single T A P1/T A P2 heterodim er b u t th at each tapasin associates w ith only one M H C c lass I m olecule (Ortmann et al., 1997). This stoichiom etry m ight enhance the chances of a transported peptide to e n co u n ter at least one M H C class I molecule with an adequate peptide binding groove in a heterozygous individual.

M H C c la ss I heav y ch ain m o le c u le s that fa il to a s s o c ia te successfully with P2ni and/or peptide are retained in the ER and degraded. It has been shown recently that this process previously re fe rred to as E R -associated degradation, does not take place in the ER but is carried out in the cytosol by the proteasome. M H C class heavy chains destined for degradation are transported b ack into the cytosol via the Sec61 complex, where they are rap id ly deg ly co sy lated and degraded (Hughes et al., 1997; W iertz et al.,

1996).

In contrast, MHC class I molecules which have successfully bound a peptide assume a stable conformation and are transported to the cell surface where they serve as target antigens for cytotoxic, CD8- positive T cells.

1.8

A n tig en

p r e se n ta tio n

via

MHC

cla ss

II