**2. Ubiquitin proteasome system**

The degradation and processing of cellular proteins is critical for cell survival, growth, and cell division. Proteolysis via the proteasome pathway plays an important role in a variety of basic cellular processes. These processes are regulation of cell cycle and division, modulation of the immune and inflammatory responses, intracellular signaling, and development and differentiation [Goldberg, 2003].

Cellular proteins are mainly degraded in two ways: lysosomal degradation and ubiquitinmediated degradation. Proteolysis in lysosomes is a non-specific process. In higher

Proteasome Targeted Therapies in Rheumatoid Arthritis 135

Ubiquitin is a 76 amino acid protein conserved across eukaryotic cells. The covalent attachment of ubiquitin to a substrate protein is a highly regulated process and can be controlled at multiple points. Ubiquitin is first activated by an activating enzyme, E1. This step requires ATP to generate a high-energy thioester intermediate, E1-S~ubiquitin. The thioester attachment induces a conformational change in the E1 that promotes association with an ubiquitin carrier protein, E2. Next, activated ubiquitin is transferred to the E2 via formation of an additional high-energy thiol intermediate, E2-S~ubiquitin, leading to dissociation from the E1 [Huang et al, 2007]. In the third step, a substrate-specific ubiquitin E3 ligase interacts with the target protein-E2 ubiquitin complex to transfer ubiquitin to the target protein (Fig 2). Additional ubiquitin proteins are attached to the initial ubiquitin via a lysine linkage forming polyubiquitin chains that may be linear or branched [Kim et al, 2007; Pickart et al, 2004]. The protein must be polyubiquitinated for Ub-dependent protein

Fig. 2. Polyubiquitination of substrate protein. Ubiquitin (Ub) is activated by enzyme E1 and translocated to enzyme E2. In the last stage, E3 ligase conjugates Ub to the substrate protein. In mammals, there are only two E1 ligases [Jin et al, 2007], but dozens of E2 ligases, and hundreds of E3 ligases. Ubiquitination specificity is determined principally by this large variety of E3 ligases, which generate the vast number of E2/E3 combinations that each

The proteasome is a cylindrical shaped structure with a molecular weight of 1,500 to 2,000 kD, located both in the cytoplasm and in nucleus in eukaryotes. It consists of two 19S regulatory complex and a core 20S catalytic complex (Fig. 3). It is also denoted the 26S

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

**2.1 Enzymatic cascade** 

degradation by the proteasome.

target specific groups of protein substrates.

proteasome [Orlowski, 1990; Ciechanover, 1998].

**2.2.1 The 19S regulatory complex** 

**2.2 Proteasome structure** 

eukaryotes, membrane-associated and extracellular proteins captured during endocytosis (e.g. viral, bacterial) are destroyed in lysosomes. Degradation of the vast majority (80-90%) of intracellular proteins is proteasome mediated [Ciechanover, 2005]. The ubiquitin proteasome system (UPS) controls the degradion of proteins in the cytosol, nucleus as well as in the luminal endoplasmic reticulum in eukaryotic cells (Goldberg, 2003).

Ubiquitin-mediated degradation of a protein involves two discrete and successive steps: first, the conjugation of multiple moieties of ubiquitin (Ub) to the protein substrate; multiple copies of ubiquitin covalently bind to available lysine residues on target proteins in a threestep process. Second, recognition of polyubiquitinated proteins by the 19S proteasome complex; Ub chain is cleaved by deubiquitinated enzymes (DUB), the substrate protein is unfolded and enters the 20S core for degradation. Then the substrate protein is cleaved into smaller peptide chains (5-20 amino acids), which are further degraded into constituent amino acids and are recycled by the cell [Goldberg, 2003]. The polyubiquitin chain is also broken down by the hydrolase enzymes and free Ub molecules are recycled by the cell [Kisselev et al., 1999] (Fig. 1).

Fig. 1. The ubiquitin-dependent degradation of protein

This process has been named the "ubiquitin-dependent degradation of protein" and was first discovered by A. Ciechanover, A. Hershko, and I. Rose who were later awarded the Nobel Prize in 2004 [Sorokin et al., 2009].

### **2.1 Enzymatic cascade**

134 Rheumatoid Arthritis – Treatment

eukaryotes, membrane-associated and extracellular proteins captured during endocytosis (e.g. viral, bacterial) are destroyed in lysosomes. Degradation of the vast majority (80-90%) of intracellular proteins is proteasome mediated [Ciechanover, 2005]. The ubiquitin proteasome system (UPS) controls the degradion of proteins in the cytosol, nucleus as well

Ubiquitin-mediated degradation of a protein involves two discrete and successive steps: first, the conjugation of multiple moieties of ubiquitin (Ub) to the protein substrate; multiple copies of ubiquitin covalently bind to available lysine residues on target proteins in a threestep process. Second, recognition of polyubiquitinated proteins by the 19S proteasome complex; Ub chain is cleaved by deubiquitinated enzymes (DUB), the substrate protein is unfolded and enters the 20S core for degradation. Then the substrate protein is cleaved into smaller peptide chains (5-20 amino acids), which are further degraded into constituent amino acids and are recycled by the cell [Goldberg, 2003]. The polyubiquitin chain is also broken down by the hydrolase enzymes and free Ub molecules are recycled by the cell

as in the luminal endoplasmic reticulum in eukaryotic cells (Goldberg, 2003).

[Kisselev et al., 1999] (Fig. 1).

Fig. 1. The ubiquitin-dependent degradation of protein

Nobel Prize in 2004 [Sorokin et al., 2009].

This process has been named the "ubiquitin-dependent degradation of protein" and was first discovered by A. Ciechanover, A. Hershko, and I. Rose who were later awarded the Ubiquitin is a 76 amino acid protein conserved across eukaryotic cells. The covalent attachment of ubiquitin to a substrate protein is a highly regulated process and can be controlled at multiple points. Ubiquitin is first activated by an activating enzyme, E1. This step requires ATP to generate a high-energy thioester intermediate, E1-S~ubiquitin. The thioester attachment induces a conformational change in the E1 that promotes association with an ubiquitin carrier protein, E2. Next, activated ubiquitin is transferred to the E2 via formation of an additional high-energy thiol intermediate, E2-S~ubiquitin, leading to dissociation from the E1 [Huang et al, 2007]. In the third step, a substrate-specific ubiquitin E3 ligase interacts with the target protein-E2 ubiquitin complex to transfer ubiquitin to the target protein (Fig 2). Additional ubiquitin proteins are attached to the initial ubiquitin via a lysine linkage forming polyubiquitin chains that may be linear or branched [Kim et al, 2007; Pickart et al, 2004]. The protein must be polyubiquitinated for Ub-dependent protein degradation by the proteasome.

Fig. 2. Polyubiquitination of substrate protein. Ubiquitin (Ub) is activated by enzyme E1 and translocated to enzyme E2. In the last stage, E3 ligase conjugates Ub to the substrate protein.

In mammals, there are only two E1 ligases [Jin et al, 2007], but dozens of E2 ligases, and hundreds of E3 ligases. Ubiquitination specificity is determined principally by this large variety of E3 ligases, which generate the vast number of E2/E3 combinations that each target specific groups of protein substrates.

#### **2.2 Proteasome structure**

The proteasome is a cylindrical shaped structure with a molecular weight of 1,500 to 2,000 kD, located both in the cytoplasm and in nucleus in eukaryotes. It consists of two 19S regulatory complex and a core 20S catalytic complex (Fig. 3). It is also denoted the 26S proteasome [Orlowski, 1990; Ciechanover, 1998].
