**2.2.1 The 19S regulatory complex**

Ubiquitin-tagged proteins are recognized by the 19S regulatory complex, where the ubiquitin tags are removed. ATPases with chaperone-like activity at the base of the 19S regulatory complex then unfold the protein substrates and feed them into the inner catalytic compartments of the 20S proteasome cylinder [Ciechanover, 2005]. The opening into the 20S catalytic chamber is small (approximately 1.3 nm), and significant unfolding of the substrate

Proteasome Targeted Therapies in Rheumatoid Arthritis 137

The 20S proteasome complex has chymotryptic, tryptic, and peptidylglutamyl-like activities [Ciechanover, 2005; Orlowski, 1990]. It is conformationally flexible with active catalytic sites located on the inner surface of the cylinder where protein substrates bind. Proteins unfolded and without Ub tag, enter the inner chamber, where they are hydrolyzed by six active proteolytic sites on the - β subunits (two sites each on the β1-, β2-, and β5-subunits) into small polypeptides ranging from three to 22 amino acids in length. Proteins cannot enter the inner cylinder through the outer walls of the 20S proteasome because the gaps between the rings are tight [Lowe et al., 1995; Stein et al., 1996]. In eukaryotic cells, 26S proteasome are localized both in the cytoplasm and in the nucleus. This distribution is tissue-specific [Lowe

A role for UPS in the pathogenesis of human diseases was first suggested some two decades ago. With the broad spectrum of protein substrates and the complex enzymatic machinery involved in targeting them and practically all intracellular processes being controlled by the UPS, it is not surprising that the proteasome pathway is involved in the pathogenesis of

The UPS plays significant role in immune and inflammatory processes. It has been shown that UPS takes part in the antigen processing in antigen presenting cells, regulates the transmission of signals from T-cell antigen receptors and the co-stimulatory CD28 molecule and is involved in activation of transcription factor-ĸB (NF-κB). NF-κB is the key regulator of the activity of genes of many inflammatory cytokines, chemokines and cell adhesion molecules [Sorokin, 2009]. The function of UPS in the activation of NF-kB is the most

NF-kB is a family of dimeric transcription factors. The NF-kB family consists of five members: p50, p52, p65/RelA, c-rel, and RelB [Neumann & M. Neumann, 2007]. p50 and p52 are formed as a result of processing from precursors p105 and p100, respectively. The processing of p105 can be performed both by the Ub-dependent pathway by the 26S proteasome [Coux & Goldberg, 1998] and by the ATP-/Ub-independent pathway by the 20S proteasome [Moorthy, 2006]. NF-ĸB activation promotes the expression of variety of target genes involved in the immune response, reparation reactions, and apoptosis. These include the pro-inammatory cytokines IL-1β and TNF-α, extracellular matrix metalloproteinase (MMPs), prostaglandins and nitric oxide. IL-1β and TNF-α, in particular, have been shown to play pivotal roles in the pathogenesis of RA both in preclinical [Han et al., 1998] and clinical studies using biological agents such as etanercept and iniximab [Carteron, 2000;

The UPS activate NF-κB in two stages. At first, the proteasome performs ubiquitin dependent processing of phosphorylated precursors p105 and p100 with the formation of active subunits of transcription factors p50 (NF-κB1) and p52 (NF-κB2). NF-κB is composed of p50 and p65 subunits, and in non-stimulated cells it is retained in the cytoplasm in a latent form associated with inhibitory protein IĸB. Following exposure of the cell to a variety of extracellular stimuli such as cytokines, viral and bacterial products and stress, IκB is phosphorylated, poly-ubiquitinated (which is recognized by the 19S regulatory subunit of Proteasome) and is finally rapidly degraded by the 26S proteasome. The released active heterodimer is translocated into the nucleus where it activates the transcription of

et al., 1995].

Cunnane, 2001].

**3. UPS in immune and inflammatory response** 

malignant, autoimmune, and neurodegenerative diseases.

important and will be discussed here in details.

corresponding genes [Van Waes et al., 2007] (Fig 5).

Fig. 3. The 26S proteasome, composed of two regulatory 19S and one catalytic 20S subunits.

is required for successful entering into the 20S subunit [Pickart, 2000]. A molecular gate (Nterminal tail of the α3-subunit) also guards the opening, but it is constitutively open when the 19S regulatory units are bound to the 20S proteasome [Groll, et al., 2000]. There are also multiple different 11S regulatory complexes that can replace the 19S regulator [Hill et al, 2002]. These alternate regulators do not have ATPase function and do not bind polyubiquitin chains. Proteasomes with 11S substitutions for 19S regulators have higher levels of proteolytic activity [Cascio et al, 2002; Fruh et al, 1994].
