Cell signalling

(In this post I include a lot of diagrams. This blog layout allows you to click on the images and enlarge them, but you may find it more convenient to open them in new tabs, otherwise you have to hit 'back' and reload the page again, which can be a nuisance.)

This, then, is how the cells of the immune system effect an inflammatory response. I will now give a detailed discussion of what happens when a ligand such as peptidoglycan binds to TLR 1. In fact, I will take the case of TLR 1:TLR 2 signalling (remember TLR 1 can either act as a homodimer or a heterodimer with TLR 2). Again, this is not strictly necessary for understanding inflammation and can be read separately. I hope, however, that this will be a means of understanding cell signalling in general, as well as the specific processes by which a macrophage recognises a pathogen and participates in the inflammatory response (here, by releasing cytokines). Throughout, I will make use of my own fairly ugly and very simplistic diagrams. I have taken a lot of license with them and they are for purely illustrative purposes - mostly to help with keeping track of the proteins.

So, the story begins with a pathogen entering the body, perhaps through an open wound, perhaps through ingestion, perhaps by other means. Forming part of the cell wall of our pathogen is peptidoglycan:

Figure 1.3: A highly stylised representation of a bacterium. Obviously, the 'P's are for 'Peptidoglycan'

Our pathogen, once in the body, can be recognised by macrophages, which are found all over the body, ready to respond to pathogens, damaged cells and so on. Expressed on the surface of macrophages are many PRRs, including TLR 1 and TLR 2:

Figure 1.4

When a PAMP like peptidoglycan binds with TLR 1 and TLR 2, it initiates a signalling pathway called the MyD88-dependent pathway. The simultaneous binding of peptidoglycan to TLR 1 and (in our case) TLR 2, brings about a conformational change in these receptors which leads to a change in a specific domain of each receptor called the TIR domain (Toll/Interleukin-1R domain). The precise mechanisms by which this happens are not only very complicated, but not really relevant. In brief, intricate chemical interactions between the TLRs and peptidoglycan result in a physical change in the TLRs. The TIR domain is changed and can now interact with other proteins in the cell, which it couldn't have interacted with before:

Figure 1.5: I use cyan to indicate that the receptors are activated. Note, also, my simplistic representation of the change in the TIR domain (represented by triangles rather than semi-circles)

So, with the TIR domain now changed in both receptors, these receptors are able to associate with a protein in the cell called TIRAP (TIR Adaptor Protein). The TIR domain of TIRAP binds with the newly altered TIR domains of the receptors:

Figure 1.6

MyD88 is now recruited - it can now interact with the complex formed by the TIR domain of the receptors and TIRAP:

Figure 1.7

The proteins MyD88 and TIRAP are adaptor proteins. As we have just seen, the TLR 1 and TLR 2 undergo a conformational change when they bind with a PAMP. This changes the TIR domain of those receptors which attracts and binds with TIRAP, which leads to the recruitment of MyD88. The adaptor MyD88 has another domain called the death domain (DD). This serves as the adaptor. The TIR domain allows the MyD88 to interact with the TLRs, while the death domain interacts with the kinases¹ we discuss next. Once MyD88 has bound to the activated receptor it recruits another protein called IRAK4 (interleukin-1 receptor-associated kinase 4):

Figure 1.8: The squares on the side of MyD88 represent the death domain interactions. IRAK4 cannot bind directly to the TIR domains of TLR 1 or TLR 2 because it is not the right shape, with its square death domains. Note how the MyD88 serves as the adaptor, it can bind to both the TIR domains of TLR 1 and TLR 2 (in combination with TIRAP) and to IRAK4. This is just like a travel adaptor for an electrical appliance. I cannot, however, stress enough what a gross over-simplification this is. In reality, the domains of proteins are highly complex chemical structures which fit together in really rather astonishing ways

With this completed, the next stage is the recruitment of another protein - IRAK1 - which is also recruited through death domain interaction. IRAK4 undergoes phosphorylation, which is the process of transferring phosphate groups from one molecule to another. It is extremely important in the behaviour of proteins, for it transfers energy and also affects the structure of the protein. IRAK4 is phosphorylated and it phosphorylates IRAK1, which I believe then hyperautophosphorylates:²

Figure 1.9: Note my crude representation of the phosphorylation of IRAK1 by means of the ellipses. Phosphorylation is, as we've seen, the transfer of phosphate groups - PO₄^3- - which is the reason behind the 'PO₄'s

IRAK1 is now competent for binding with another protein called TRAF6 (TNF receptor associated factor 6). This it does and it is also ubiquitinated, which is where a chain of ubiquitin molecules are added to a protein (in this case IRAK1). Ubiquitin is a small regulatory protein involved in numerous processes such as these, which changes the behaviour of proteins. I believe TRAF6 may also be phosphorylated.

As I understand it, IRAK1 dissociates from the complex that's bound to the TLR - along with TRAF6 - and is transferred to a new complex:
 

Figure 1.10: The 'U's in circles obviously represent ubiquitin

The new complex in question is a complex of TAK1 (transforming growth factor beta-activated kinase 1), TAB1 (TAK1-Binding protein 1) and TAB2, which are associated to the membrane. TAB2 facilitates the binding of our complex of IRAK1 and TRAF6 to TAK1:

Figure 1.11: Don't worry too much about my phosphate groups, my diagrams are very rough and meant only to help in keeping track of things. I'm not much of an artist

TAK1, TAB1 and TAB2 are now phosphorylated. This causes dissociation, leaving IRAK1 bound to the membrane, while the complex of TAK1, TAB1, TAB2 and TRAF6 'breaks away.' This complex moves to the cytosol, which is a part of the cell, where TAK1 is activated. It is polyubiquitinated:
 

Figure 1.12

After all this, we begin to see the immensely beautiful and complex process of numerous chemical reactions which eventually transduces a signal and produces a response. Finally, we have the components which now activate MAP kinases and NF-κB. For the purposes of my example, I am going to consider precisely how NF-κB (which is involved in the production of cytokines, in this case) works.

As I have said, NF-κB is involved in the production of cytokines. When it is activated, it binds to particular DNA sequences (sc. response elements (RE)). More proteins and RNA polymerase are recruited and, by means of transcription and translation - which we will come to when we consider how the HIV virus works - cytokines are ultimately produced. This is the function of NF-κB here. When it is not activated, it is "stored" in the cytoplasm, where it is bound to proteins called IκBs (inhibitors of κB). There are four such proteins, but the best known is IκBα, which we may take as an example. Very simply, the IκB forms a complex with NF-κB, which keeps the NF-κB unactivated until such times as it is needed to bring about a change in the cell, so that it - for example - produces cytokines. Until it is needed, it is held in a kind of IκB storage:

Figure 1.13: The normal state of NFκB - it is unactivated and in a complex with IκBα

The IκBs can be induced to released NF-κB by IKK (IκB kinase). IKK is composed of two heterodimers - IKKα and IKKβ - and a regulatory protein IKKγ (or NEMO (NF-κB essential modulator)):

Figure 1.14: IKK

IKK ubiquitinates IκB:

Figure 1.15

Which causes structures called proteasomes to degrade (or proteolyze) the IκB. A proteasome is an enzyme which degrades proteins by breaking peptide bonds. With the IκB degraded, NF-κB is released and goes on to induce the production of cytokines, as I described above:

Figure 1.16: Note that the faded yellow of my IκBα is supposed to represent the fact that it has been degraded

So, to tie it all together. In our case - in the case of a macrophage with activated TLR 1 & TLR 2 receptors - the release of NF-κB is achieved by the TAK1 when it has been polyubiquitinated. At last we have all the pieces in place. The TAK1, which has been "prepared" by being polyubiquitinated, can now phosphorylate IKKβ. When this has happened, the IKKβ ubiquitinates IκB, releasing NF-κB by the mechanism described:

Figure 1.17

It is worth taking a moment just to appreciate a few things. Firstly, the NF-κB is held in what I have dubbed a kind of "storage," ready to be activated, but inactive when all is well. When PAMPs are recognised (and, therefore, when pathogens are present), the NF-κB is released, by means of the process I have outlined. This will lead to the production of cytokines, which should help to remove the pathogen. NF-κB is only released when pathogens are present and the more pathogens there are, the more PAMPs will bind with receptors on macrophages and the stronger the response will be. Once there are no pathogens left, there will be nothing to bind with the PRRs on the macrophages and so NF-κB will not be released and cytokines will not be produced.

The next thing to point out is this: When a PAMP binds with a receptor it brings about a change in the receptor which recruits adaptor proteins. These allow the signal (the recognition of the PAMP) to be transduced, as proteins already present in the cell can now be recruited and begin to interact. This couldn't have happened before, as the TIR domain of the TLRs cannot interact with the kinases such as IRAK4 which are involved in the signalling pathway. And so we see that the recognition of a pathogen is necessary for a response to take place, which is important as you would not want your immune system constantly activated - it would be bad for your own cells.

The process is also wonderfully intricate and this, too, is important as there are actually multiple pathways, and multiple responses which can be elicited, depending on the cell, the receptor and the signal. I have chosen one very specific event to walk you through, by way of example. However, these complex interactions allow complex responses to take place.

N.B.: Once again, the diagrams provided are for illustration only. I have tried to make them accurate but information is, understandably, scanty. The relative positions and interactions of the proteins shown in the diagrams should not be taken as necessarily representative. Whilst I don't wish to excuse myself of any shoddy research, I would also just like to emphasise that this discussion is purposed towards giving a deeper understanding of cell signalling, by means of considering a specific pathway, in a specific case. Through this, I hope to illuminate the discussion of inflammation. I may have unintentionally abbreviated or passed over some steps and various intricacies.

¹An enzyme which transfers phosphate groups to other molecules, i.e. an enzyme which phosphorylates
²What it says on the tin: IRAK1 phosphorylates itself. A lot