1.1. The ubiquitin-proteasome pathway
Ubiquitin is a highly conserved, 76-amino acid protein that is ubiquitously expressed in eukaryotic cells 1. This small protein controls almost all aspects of a cell's life and death, through its covalent modification of other cellular proteins in a process known as ubiquitination 2,3. The enzymatic cascade of ubiquitination begins with ubiquitin activation by an E1 (ubiquitin-activating enzyme), followed by transfer of the activated ubiquitin to an E2 (ubiquitin-conjugating enzyme, also known as Ubc), and ends with conjugation of ubiquitin to a target protein through the formation of an isopeptide bond between the carboxyl terminus of ubiquitin and an ε-amino group of a lysine residue on the protein substrate. The last step requires a member of a very large family of ubiquitin-protein ligases (E3), which, together with E2s, determine substrate specificity. E3s can be divided into two categories, depending on whether they contain a HECT (homology to E6AP C-terminus) or RING (really interesting new gene) domain. The HECT domain E3s contain an active-site cysteine, which can accept ubiquitin from an E2 and transfer the ubiquitin to a target protein. In contrast, the RING domain E3s do not contain a conventional enzyme active site, but they promote ubiquitination by binding to both protein substrates and E2s, facilitating the conjugation of ubiquitin to specific protein targets. Ubiquitination reactions are reversed by members of a large family of deubiquitination enzymes (DUBs, also known as isopeptidases)4,5. Thus, ubiquitination is a reversible covalent modification, similar to phosphorylation.
Ubiquitin has seven lysines, each of which can be conjugated by another ubiquitin to form a polyubiquitin chain 6. The topology of polyubiquitin chains can influence the fate of target proteins. For example, polyubiquitin chains linked through lysine 48 (K48) of ubiquitin normally target a protein for degradation by the proteasome, whereas K63 polyubiquitin chains have functions independent of proteolysis, including protein kinase activation, DNA repair and membrane trafficking. Monoubiquitination usually does not lead to proteasomal degradation; instead, it regulates important cellular functions such as chromatin remodeling and vesicle trafficking.
1.2. The NF-κB pathway
Both the proteolytic and non-proteolytic functions of ubiquitin are critically important for the regulation of nuclear factor kappa B (NF-κB), a family of transcription factors actively involved in the regulation of immunity, inflammation, and cell survival 7. The NF-κB /Rel family includes RelA (p65), c-Rel, RelB, p50 and p52. They share an N-terminal Rel homology domain (RHD), which mediates dimerization, nuclear translocation, DNA binding and association with the inhibitory proteins IκBs. p50 and p52 are generated from their precursors p105 and p100, respectively, through proteasomal degradation of the C-terminal IκB-like ankyrin repeats.
The NF-κB activation pathways are classified into canonical and non-canonical pathways; the canonical pathway leads to the degradation of IκB, whereas the non-canonical pathway involves the processing of p100 to the mature subunit p52 8. The canonical pathway is activated by most NF-κB stimulatory ligands, including proinflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β), and microbial ligands such as bacterial lipopolysaccharides (LPS) and viral nucleic acids. These ligands bind to their receptors and trigger distinct signaling pathways that converge on a large kinase complex consisting of the catalytic subunits IKKα and IKKβ (IκB kinase α and β), and an essential regulatory subunit NEMO (NF-κB essential modulator, also known as IKKγ or IKKAP). The IKK complex phosphorylates IκBs, and targets these inhibitors for polyubiquitination and subsequent degradation by the proteasome. The liberated NF-κB enters the nucleus to turn on the transcription of target genes. The non-canonical pathway is activated by a subset of receptors in B cells, such as CD40 and B cell activating factor receptor (BAFF-R). These receptors initiate a signaling cascade leading to activation of IKKα, which phosphorylates p100. Phosphorylated p100 is polyubiquitinated and then its C-terminus is selectively degraded by the proteasome, sparing the N-terminal Rel homology domain to generate the mature p52 subunit. p52 forms a dimer with RelB and the dimeric complex enters the nucleus to turn on the expression of genes that are important for B cell maturation and activation.
In both canonical and non-canonical pathways, IKK is key to NF-κB activation. Mounting evidence shows that ubiquitination and deubiquitination play a central role in IKK regulation by diverse NF-κB signaling pathways 9,10. In particular, K63 polyubiquitination mediates the activation of IKK and mitogen-activated protein kinases (MAPKs) through a proteasome-independent mechanism. In this chapter, we will discuss recent progress in understanding the roles of ubiquitin in three steps of the NF-κB pathway: IκB degradation, processing of NF-κB precursors and activation of IKK and other kinases. In addition, we will discuss how deubiquitination enzymes negatively regulate the NF-κB pathway, and how dysfunction of these enzymes may lead to human diseases.