EVALUACIÓN DEL SISTEMA

Cull WD P-TRCP

SCFP-TRCP

I

B Binding and Ubiquitination

F igu re 1-12: Schem atic R epresen tation o f th e U b iq u itin ation o f P hosp horylated iK B a by the C om plex.

P-1'RCP is the substrate reeognition subunit o f the SCF E3 ligase system. It does not have eatalytie aetivity itself, but

binds to p - k B a via its W D40 domain and promotes substrate ubiquitination through the reeruitment o f the E2 enzym e.

UBC 5. Skpl is thought to be the adapter m olecule that binds to the F-box m otif in E3RS*''®, forming a link between

E3RS'"^ and the other components o f the SCF com plex, such as Cul-1 and the E l and E2 enzym es.

W in sto n et a i, (1 9 9 9 ) sh o w e d that d e p le tio n o f S k p l from c e ll ly sa te s r esu lts in c o d e p le tio n o f an d s u b seq u e n t in h ib itio n o f p -lK B a u b iq u itin a tio n , in d ic a tin g that th e F -b o x o f E3RS'"® in tera cts w ith S k p l

(W in sto n et al., 1 9 9 9 ). It is a lso th o u g h t that S k p l and ESRS'*^ form a p r e e x istin g c o m p le x in c e lls , a s th e tw o p r o tein s can b e c o im m u n o p r e c ip ita ted e v e n in u n stim u la ted c e lls (S p en ce r et a i , 1 9 9 9 ). It is lik e ly th er efo re, that S k p l a c ts a s an adapter m o le c u le r e s p o n s ib le for lin k in g th e ESRS'*"® b o u n d su b stra te to th e

1.12.2 Degradation o f Ubiquitin-Protein Conjugates

Polyubiquitination marks proteins for degradation by the 26S proteasome (Jentsch and Schlenker, 1995). The proteasome is a large, 268 multicatalytic protease, which is composed of two subcomplexes (Figure 1- 13). The proteolytic activity of the proteasome is found within its core particle (CP)/20S proteasome, which is able to hydrolyse small peptides and some unfolded proteins. This CP however, cannot degrade multi-ubiquitinated proteins, therefore substrates are prepared for degradation by regulatory factors (198 proteasome) that attach to the two outer faces of the CP (Glickman et al., 1998). The 208 complex is barrel shaped, consisting of four stacked rings. The two outer rings consist of seven genetically related a- subunits, while the two inner rings are composed of P-subunits, which are less conserved and contain the peptidase active sites (BochtlCT et al., 2000). The 198 complex, which caps the CP, has been further resolved into two subcomplexes, referred to as the base (consisting of the six non-redundant ATPases, Rpnl-6 and the non-ATPases ^ n 2 (si), Rpnl(82) and RpnlO(85A)) and the lid (consisting of 8 non- ATPase subunits). The base is thought to create a gate in the a ring of the 208 complex, through which substrates can be inserted into the proteolytic chamber. The base is also responsible for unfolding denatured proteins, before translocating them into the chamber. It is assumed that the lid is essential for polyubiquitin substrate processing prior to degradation.

In most cases the proteasome cleaves protein substrates into small peptides of varying length (3-23 amino acids) (Kisselev et al., 1999; Nussbaum et al., 1998). These peptide products do not accumulate in the cell and are rapidly hydrolysed by a series of downstream proteases, or transported through the ER to be presented to the immune system by the MHC class I (Ben-8hahar et a l, 1999; Michalek et a l, 1993). In certain cases however, the proteasome does not degrade proteins to completion and acts as a regulator to control cellular activities. For example, during the processing of the p i 05 precursor of p50, \s4iich acts as an IkB protein, the C-terminal region of pi 05 that becomes ubiquitinated, is proteolysed by the proteasome and the 50kDa N-tominal region is released as a stable and active protein (Palombella et a l, 1994). The precursor of p52, p i 00 is also processed by the proteasome in a similar fashion (Heusch et a l, 1999). As previously described, the other IkB proteins are also ubiquitinated and this targets them for degradation by the proteasome and results in free NFkB dimCTS translocating to the nucleus to activate target genes.

19s R P X LID B A SE 20s C P N o n -A T P a ses A T P a ses

F igu re 1-13: T h e 26S P roteasom e. ITie proteasome is eom posed o f tw o subcom plexes; the core particle (20s CP) and

the regulatory particle (19s RP), which itself consists o f a lid and a base, l l i e CP is eom posed o f four heptagonal rings,

the tw o inner rings com prising hom ologous p-subunits which contain functional active peptidase sites and two outer

rings contianing a-subunits. The 20s CP forms a chamber in w hich small peptidase and unfolded proteins are

hydrolysed, fh e base o f the RP is an ATPase ring, which serves multiple roles such as selecting substrates, preparing

them for degradation. The lid is required for multi-ubiquitin protein degradation.

1.12.3 The Regulatory Role Played by Ubiquitin in IKK Activation

Polyubiquitin chains that are recognised by the proteasome are typically formed through linkage of lysine- 48 (K48) of one ubiquitin with the C-terminus of the next (Chau et a i, 1989). Polyubiquitin chains linked through other lysine residues of ubiquitin have been detected in cells, but the function of these chains remains elusive (Pickart, 1997). As mentioned earlier, recent studies have identified the IkB-E2 as a member of the UBC4/5 family and IkB-E3 as a multiprotein complex consisting of Skpl, Cul-1, Roc-1 and an F-box/WD40 protein called PXRCP/Slimb which binds specifically to phosphorylated IkB (Maniatis,

1999). Analysis of cells from TRAF6-deficient mice has revealed that TRAF6 is essential for IL-ip, CD40 and EPS-mediated IKK activation (Lomaga et a i, 1999; Naito et a i, 1999).

Interestingly, Deng et al, (2000) have just demonstrated that TRAF6 acts as an E3 Ubiquitin-ligase (Deng

et al, 2000). By purification of the complex that links TRAF6 to IKK, they were able to show that the RING finger domain of TRAF6 facilitates the assembly of K63-linked polyubiquitin chains by a dimeric ubiquitin-conjugating enzyme (E2) complex composed of UbcI3 and UevIA. Additionally, they demonstrated that assembly of this ubiquitin chain was essential for IKK activation in response to IL-ip stimulation and that IKK activation did not involve proteasomal degradation (Deng et a l, 2000). It therefore seems likely that ubiquitination, rather than degradation plays a regulatory role in IKK activation.

1.13 Aims

The aims of my thesis were to:

• Assess the effects of a series of IRAK-1 variants on IL-ip and TNFa-driven NExB-dependent reporter gene activation.

• To study the biochemical properties of endogenous and transfected IRAKI-containing complexes in resting cells and during the signal transduction process following IL-ip stimulation.