The Toll-like Receptor 4 and Signalling through Ubiquitylation
contribute to the establishment of the adaptive immune response, the dendritic cell must be regarded as the primary cellular bridge linking innate and adaptive immunity. As the major antigen-presenting cell, it signals the T-effector cells and thereby triggers the production of antigen-specific immunoglobulins.
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IFNis necessary for the maturation of antigen-presenting dendritic cells: increased expression of MHCII and costimulatory proteins including CD40, CD80 and CD86 (Chapter 17).
IFNand IL-12 are necessary for formation of Th1 cells, important in antigenspecific activation of B cells, leading to the production of specific antibodies. IL-6 stimulates the maturation of B cells towards high-level immunoglobulin-producing plasma cells.
TNFand IL-1 are essential for the increased expression of adhesion molecules on endothelial cells, leading to enhanced extravasation of leukocytes and dendritic cells at sites of infection (Chapter 16). Several chemokines (RANTES, MIP-1 , IL-8, CCL5, CXCL9, etc.) cause
activation of integrins on leukocytes, including dendritic cells. This leads to their enhanced extravasation and tissue infiltration in order to combat the infecting agent.
In Chapter 16 we show how the TLR4 response leads to recruitment of leukocytes at sites of infection. In Chapter 17 we explain how the innate immune response establishes an adaptive immune response, involving the antigen presenting molecule MHCII and signalling downstream of the T cell receptor (TCR).
Essay: ubiquitylation and SUMOylation
Ubiquitylation
Ubiquitylation is the process by which the peptide ubiquitin is coupled to the-amino groups of lysines present in the target protein (Figure 15.12). It is selfevident that it must specify its targets with great accuracy; if it did not, protein degradation would be uncontrolled. The reaction is reversible.71
The attachment of one or more ubiquitins does more than just marking proteins for destruction by the proteasome complex. It also serves as a signal for endocytosis of cell surface receptors and subsequent selection for lysosomal destruction. It is involved in the regulation of protein kinases, it regulates gene transcription and it acts as a protein–protein interaction motif involved in the recruitment of signalling complexes.72
Ubiquitylation: a process involving three activities (but not necessarily three proteins)
Ubiquitin conjugation is catalyzed in three steps. Similar to adenylation, ubiquitin is first ‘activated’, bound to E1-cysteine through a glycine–sulfydryl
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Signal Transduction
FIG 15.12 The ubiquitylation reaction.
(a) Structure of the Cbl E3-ligase complex. Cbl has three roles. It selects substrate, in this example the phosphorylated kinase ZAP-70 bound by the SH2 domain of Cbl. It recruits the E2-conjugating enzyme (UbcH7, loaded with ubiquitin) through its RING domain and contributes to the transfer of the glycine-76 of ubiquitin to a lysine in the substrate (not shown). Zn2 ions indicated as red spheres. (b) The ubiquitylation reaction occurs in three steps. Following adenylation (‘activation’), ubiquitin is bound to E1-cysteine through a thioester bond. This allows transfer of the ubiquitin to E2-cysteine (residue 86 in this example). With substrate bound to E3, the ubiquitin transfers to a lysine -amino group to create a stable isopeptide bond. This reaction can be repeated by addition of further ubiquitins. Only polyubiquitylated proteins are recognized for destruction. (1aar70 and 1fbv73).
thioester link. It is then transiently coupled to a cysteine on the E2conjugating enzyme. The substrate (target protein) and E2-ubiquitin are brought in close proximity through a third component, E3-ligase, which determines the specificity of the conjugation reaction (Figure 15.12). Ubiquitin detaches from E2 to form an isopeptide bond linking the -carboxyl group of its terminal glycine with an -amino group of a lysine residue in the substrate.
63K or 48K conjugation
Repeated conjugation results in the formation of polyubiquitin chains. Ubiquitin itself possesses six lysines, all of which can make the link to the N-terminal glycine, but two of these, Lys48 and Lys63, predominate (48K or 63K type). Recognition of target proteins by the proteasome complex requires four or more linked 48K-type ubiquitins (Figure 15.13). The 63K-type polyubiquitin chain, though not excluded, is not necessarily involved in
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The Toll-like Receptor 4 and Signalling through Ubiquitylation
FIG 15.13 K48or K63-linked ubiquitin chains.
Ubiquitins are coupled to each other through isopeptide linkages between either Lys48 or Lys63 and the C-terminal Gly76. K48-type polyubiquitin chains are recognized by ubiquitin binding proteins that communicate with the proteasome.
K63-linked ubiquitins play a role in the recruitment of signalling complexes (1aar70).
recognition by the proteasome, serving more readily as a docking site for other ubiquitin-binding proteins. It also plays a role in cell signalling,
importantly in TNF- -mediated activation of NF- B.74 Two examples of K63type polyubiquitin-binding proteins relevant for this discussion are NEMO and TAB2 but how these make the distinction between 48Kand 63K-type ubiquitylation is not known.
Two classes of E3-ubiquitin ligases
There are two classes of E3-ubiquitin ligases; RINGand HECT-type (Figure 15.14).
1.The RING type are abundant. The RING domain comprises a series of zinc finger motifs that act as a protein–protein interaction domain. Some RINGdomain proteins interact with E2-ubiquitin conjugating enzymes. This class of E3-ubiquitin ligases comes in two flavours:
•Single protein. Here the E3-ligase acts both as a binding site for E2 and as a receptor for substrates. An example is Mdm2 involved in the ubiquitylation of p53, Cbl, involved in the ubiquitylation of the EGFR (Figure 21, page 350) and the kinase ZAP-70 (that acts downstream of the T cell receptor) (Figure 17.3, page 518).
•Multiprotein complex. The RING domain protein binds the E2-ubiquitin conjugating enzyme, but the E3-ligase and the receptor are kept apart by scaffold proteins. Examples are SCF and APC/C, both involved in destruction of components of the cell cycle machinery (anaphase promoting complex/cyclosome).
2.The HECT type E3-ligase harbours both E2and E3-activity.75 An example is Nedd4 which operates in the regulation of the activity and plasma membrane expression of ENaC, an epithelial cell-surface Na channel. We return to HECT type E3-ligases in relation to signalling by TGF (Chapter 20). A variant, Nedd4-2, plays a role in the regulation of destruction of PTEN (page 668 onwards).
The ENaC Na channel is involved in fluid resorption in kidney, lung, and colon. Mutated ENaC, resistant to the attention of Nedd4, results in its enhanced expression at the cell surface. This leads to excessive re-uptake of Na in nephrons, giving rise to hypertension (Liddle’s syndrome).76
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FIG 15.14 Examples of ubiquitinand sumo-ligase complexes.
The human genome contains five genes that code for E1-ubiquitin activating proteins. These transfer ubiquitin to a much wider range of E2-conjugating enzymes (37 genes). Relevant examples are UbcH5, UbcH7, UbcH4, and Ubc13 (other Ubcs can replace Ubc4). Ubc13 is only active when bound to MMS2. In the case of the RING domain-containing ligase complexes, the E2-conjugating enzyme transfers the ubiquitin chain to substrate which is bound to a separate E3-ligase. With Mdm2, Cbl, and TRAF6, the E3-ligase constitutes a single protein; in other cases it forms a multiprotein complex comprising a RING domain protein (Rbx1), scaffold proteins (Cullin and Skp1), and a receptor ( -Tcrp-1 or Skp2). In the case of HECT-type E3-ligases, both activities (E3 and E2) are harboured within one protein (NEDD4, for instance). Proteins can also be modified by ubiquitin-like SUMO proteins. Specialized E1-SUMO activating proteins are SAE1 and -2. These transfer the SUMO to Ubc9, which, in the presence of PIAS, is transferred to substrate.
Ubiquitin-binding proteins
The first entity that binds ubiquitin with high specificity to be identified was the regulatory proteasome particle S5a (RPN10).77 However, since deletion of the homologous gene in yeast has somewhat modest phenotypic effects, a search for additional proteins was instigated. We have already mentioned NEMO and TAB2/3 but there are many others, of which a large proportion is involved in targeting proteins to the proteasome.78 Representative examples and their domains are shown in Figure 15.15.
SUMO and sumoylation
Overall, SUMO (small ubiquitin-like modifier) and ubiquitin share only
18% sequence identity, but the folded structures of their C-termini are almost superimposable. In vertebrates, there are three SUMO-related peptides. SUMO is implicated in a number of regulatory events such as enhancement of protein stability, protein–protein interactions, subcellular translocation, and transcriptional control.80–82 Because ubiquitin and SUMO compete for the
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