The family of TNF proteins and receptors

TNFand - are members of a large family of cytokines (Figure 16.3).29 They are initially generated in precursor form, as transmembrane proteins arranged as stable homotrimers. From these, soluble homotrimeric cytokines are released by the action membrane bound metalloproteases (so-called furins). With one or two exceptions, these ligands bind their receptors both in a membrane bound and soluble state. One example of these exceptions is FASL (CD95L). As a membrane protein this causes apoptosis of cells bearing the FAS (CD95), whereas when soluble, it prevents apoptosis.30 Another example is TNF- , which only effectively activates the TNFR2 in its membrane form (memTNF- ) but not in its soluble form (sTNF- ).31

The TNF ligands signal through 29 different receptors, members of the TNFR family (Figure 16.3). These receptors can be divided into two groups: those having a death domain (DD), e.g. the ubiquitously expressed TNFR1, and those without, e.g. TNFR2, which is typically found in cells of the immune system and whose expression level is context dependent. Members of the first group interact with DD-possessing proteins and couple to a caspase-activating pathway leading to cell death. The second group has the capacity to interact with the effector TRAF. The possession of a DD, however, does not restrict the receptor from signalling to pathways that have little to do with cell death. In fact, one of these pathways mediates activation of NF- B and constitutes an important cell survival signal.32 Conversely, death can be induced by receptors of the TNFR2 family that do not contain death domains.

Although some TNF-receptor signalling components have macabre names, TNFshould be considered an inflammatory cytokine with an optional ability to cause cell death.33 Generally, TNFR1 is the key mediator of TNFsignalling.

TNFand regulation of adhesion molecule expression in endothelial cells

Receptor activation

In the absence of ligand, the N-terminal cysteine-rich domains (PLAD) of TNFR-1 and -2 interact with each other, maintaining the silent state (Figures 16.4 and 16.5). Binding to the trimeric ligand TNFeither induces an activating conformational change or allows the formation of higher-order complexes that bestow signal competence.33 Non-engaged receptors interact with a signal silencing protein, SODD (silencer of DD). When over-expressed, this blocks the NF- B pathway as described below.34

In endothelial cells, binding of TNFcauses enhanced expression of VCAM-1 and the ICAMs.36 It causes release of chemokines (IL-8, MCP-3, MIP-1a) and cytokines (IL-1 , IL-6, TNF- , GM-CSF) and the expression of the enzymes

TNFwas initially named lymphotoxin- .

Furin is also exploited by a number of pathogenic microorganisms. Thus, the envelope proteins of viruses such as influenza, HIV, and dengue fever must be processed by furin in order to become functional. Likewise,

the toxins from anthrax and Pseudomonas must be cleaved in order to become membrane permeant. Compounds capable of acting

as inhibitors of furin are currently being

considered as therapeutic agents for the treatment of anthrax infection.

Fig 16.3  Domain architecture of the ligands and receptors of the TNF superfamily.

All ligands, except TNF-βand VEGI, are transmembrane proteins and have local effects. Under pathological conditions they can be liberated by a

variety of proteases, some of which are indicated: furin, operating in the Golgi, and ADAM matrilysin at the plasma membrane. The C-terminal segments, carrying the TNF homology domain, have 20–30% sequence identity and bind the receptor. The receptors are transmembrane proteins, except for DCRs 1 and 3 and OPG (which possess inositol-lipid anchors). NGFR is a low affinity nerve growth factor receptor that is structurally related to the TNF receptors but has quite distinct actions. Figure adapted from Aggarwal.29

NF- B: nuclear factorB, a protein complex that attaches to the immunoglobulin lightchain gene.38

iNOS, cyclo-oxygenase-2, and cytosolic PLA2. As a result of all this, the adherence of leukocytes is greatly enhanced.37

All these effects of TNFand of inflammatory stimuli are mediated through the transcription factors NF- B (Figure 16.6) and AP-1(ATF2, C-Jun).

Signalling downstream of TNFR1

Activation of the TNF1R (receptor for TNF- ) causes SODD to dissociate and initiates the formation of two complexes on the basis of homophilic

In the Prosite database, the TRAF domains are referred to as MATH domains because of their resemblance to Meprin (meprin and traf homology).

DD interactions. The DD was first identified as a stretch of 80 amino acids in FAS and TNFR1 necessary for the induction of programmed cell death (apoptosis).39 Through the DD domain the TNFR1 recruits:

•TRADD, an adaptor that is complexed with TRAF2.40 This multifunctional protein comprises two interaction domains with which, besides TRADD, it binds to non-DD-containing TNF receptors (such as TNFR2) and to

downstream signalling components such as RIP or the MAP3kinases, ASK1 and MEKK1. It is also contains a RING zinc finger and multiple TRAF zinc finger motifs, so qualifying it as an E3-ubiquitin ligase (see page 467).

It complexes with the E2-conjugating enzyme complex Ubc13/MMS2 responsible for a K63-type polyubiquitylation reaction (see Figure 16.6, also page 468).

•RIP1. This has a DD at the C-terminus, an intermediate region which contains an important ubiquitylation site (K377), and an N-terminal serine/threonine kinase domain that has no apparent function in TNFsignalling.41 Binding of TRADD and RIP1 is mutually exclusive so that they associate with different receptors (Figures 16.4 and 16.5).