Ljubljana, Slovenia 2006/Background
From 2006.igem.org
Although in the past years all participating iGEM teams focused on bacteria and yeasts, we decided to modify signalling pathways in higher eukaryotes, t.i. in human cells. This is far more complicated system to understand but we feel that the recently accumulated knowledge enables rational planning of cells with novel properties.
Background
When bacteria invade the human body, it has to respond quickly by activation of the innate branch of the immune system, which recognizes the broad spectrum of molecules, specifical for pathogenic microorganisms. Cells which represent the first line of defence are macrophages and the way they act is rather complicated. On the surface of these cells (actually inserted into cell membrane) are several types of receptor molecules, commonly abbreviated as TLR (toll-like receptors). These can specifically bind various components of bacteria (e.g. lipopolysaccharides which are part of bacterial outer membranes, flagellin from flegella, peptidoglycans, lipopeptides, and also nonmethylated oligonucleotides containing CpG motif...) and after binding, they activate the cell signalling pathway that, after a complex phosphorylation cascade, ends in the cell nucleus and activates the transcription of immune response molecules, such as cytokines and chemokines. This rather complicated pathway is schematically depicted here, although many additional connections are being added constantly. This kind of cell signalling is the basis of our efficient innate immune response (i.e. the fast-acting, not mediated by antibodies) against microbes. In some instances, however, extensive signalling mediated by TLR is not beneficial. Under some not yet fully understood circumstances, bacteria can trigger this same signalling pathway just that it results in excessive inflammatory response. As result, sepsis occurs, which can lead to severe organ failure and in about 20% cases cosequences are fatal. Response to bacterial stimulus is a double-edged sword - too strong response may lead to sepsis, while the too weak response does not contain the bacterial infection, therefor both extremes have to be in the balance.
komentarji:
Signalling pathway of bacterial recognition of the human innate immune system Toll like receptors (TLRs) are key regulators of innate immunity, sensing and responding to invading microorganisms. So far we know eleven different receptors that recognize specific molecular patterns that are present in microbial components, e.g. lipopeptides, double and single stranded RNA of viruses, CpG containing DNA, glycolipids, and variety of others. We have focused on TLR4 and TLR5 that respond to presence of LPS and flagellin respectively, so both sense the presence of bacteria.
After binding of bacterial constituents to TLR, its intracellular TIR-domain interacts with either MyD88 (MyD88 dependent way) or TRIF (MyD88 independent way), two signaling molecules in the cytoplasm that transmit signal to other components of the cascade, as shown on picture below. TLR4 can induce response through both ways, TLR5 only through MyD88 depended.
MyD88 dependent pathway When receptors sense the presence of microbial components, stimulation of TLRs occurs and triggers the association of MyD88 (myeloid differentiation primary-response protein 88), which in turn recruits IRAK4, thereby allowing association with IRAK1. IRAK4 phosphorylates IRAK1 and with phosphorylated IRAK1, TRAF6 (tumour-necrosis-factor receptor- associated factor 6) associates. The complex of phosphorylated IRAK1 and TRAF6 dissociats from the receptor and form a complex with TAK1, TAB1 and TAB2, which induces the phosphorylation of TAB2 and TAK1. This leads to the ubiquitylation of TRAF6, which induces the activation of TAK1. TAK1 phosphorylates IKK complex (inhibitor of nuclear factor-?B (I?B)-kinase complex), which consists of IKK-?, IKK-ß and IKK-?. The IKK complex then phosphorylates I?B, which leads to its ubiquitylation and subsequent degradation. This allows NF-?B to translocate to the nucleus and to induce the expression of its target genes, such as cytokines and chemokines. Inflammatory cytokines, chemokines and interferons attract cells of immune system (lymphocytes, macrophages) to infected area. This seems to be beneficial to organism, but it can also lead to severe inflammation and sepsis. Some receptors mediate response through MyD88-independent pathway. A number of genes known to be interferon(IFN)-inducible genes was identified.
komentarji:
Project proposal In the beginning we have discussed following project ideas:
1. New mechanism of tolerance - use of inhibitors that interfere with NF-kappaB (transcription factor mentioned above) - inhibition with dominant negative proteins involved in signaling pathway, this proteins could be labeled with degradation tags (PEST sequence) and inhibition would be temporal (negative feedback loop)
2. Cell response to pathogen, that cells usualy can not recognise - for example response to beta glucans of fungi
3. Find a connection/shortening of signaling pathways to make it more efficient and include more responses
Selected project proposal
The basic idea of our project was to introduce the feedback loop,which would decrease the response to the persistent or repeated stimulus. However completly shutting down the response at bacterial stimulation is not a good solution. Ideally the feedback loop should decrease the response when it is too high but recover the responsiveness of the system after some time. Inhibition of the response could be achieved by activating the expression of the dominant-negative adapter protein, that inactivates the signaling pathway. Decreasing the lifetime of the dominant-negative inhibitior by the addition of rapid degradation tag (PEST sequence) should inactivate the inhibition and reset (restore) the normal responsiveness of the immune system.
This idea is similar to the natural mechanism of tolerance, which is already present in living cells and which decrease the response to repeated bacterial stimulation. This natural tolerance is activated slowly, on the order of days and operates through several different mechanisms (Figure). Our feedback mechanism (i.e. artificial tolerance) should decrease the response within hours and thus "attack" the signaling pathway at the point, which has not been used in the natural system.
komentarji:
Approach Mathematical model of signaling More to come
Parts design
At first we had to design primers to replicate a desired DNA fragment. In primers we included restriction sites - on left site XbaI and on the right site SpeI, NcoI and PstI. We cloned that part in to BioBrick plasmids with ccdB domain to get all restriction sites needed for BioBrick assembly. We had to design all parts de novo, since no parts like promoters, terminators, desired proteins for signaling pathway modification, degradation flags and reporters had been designed so far - neither to work in mammalian cells. List of desired constructs is shown below. For our use we designed a special vector (BBa_J52017) with terminator to simplify constructs assembly. All our composite parts (promoter plus part) were then cloned in this vector.
We also needed fusion proteins e.g. dnMyd88-rLuc-PEST (BBa_J52013) - that is our dominant negative protein linked with reporter and degradation flag. This parts are designed like basic parts, because only 2 amino acid long linker formed during biobrick assembly could affect on tertiary structure of proteins. That is why we have made this parts using PCR Overlap Extension. With primers we introduced a six amino acids long linker in between protein - reporter and reporter - degradation flag. These parts were then combined with promoter (NF-kappaB) in biobrick assembly technique. Part was then inserted in vector with terminator and ready to use in human cells (HEK 293).
|**reg. number**|**part**|**vektor**| |BBa_J52008|rluc|pSB1AK3| |BBa_J52012|rluc-link-pest191|pSB1AK3| |BBa_J52011|MyD88-link-rluc-verija2|pSB1AK3| |BBa_J52013|MyD88-link-rluc-link-PEST191|pSB1AK3| |BBa_J52023|NFkB+rluc-link-PEST191|pSB1AK3+TER| |BBa_J52010|NFkB|pSB1AC3| |BBa_J52014|NFkB+MyD-link-Rluc|pSB1AK3+TER| |BBa_J52018|NFkB+rluc|pSB1AK3+TER| |BBa_J52036|NFkB+MyD|pSB1AK3+TER| |BBa_J52024|NFkB+MyD-Rluc-PEST191|pSB1AK3+TER| |BBa_J52034|CMV|pSB1A2| |BBa_J52038|CMV+rluc|pSB1AK3+TER| |BBa_J52039|CMV+rluc-PEST191|pSB1AK3+TER| |BBa_J52642|GFP|pSB1AK3| |BBa_J52648|CMV+GFP|pSB1AK3+TER| |BBa_J52040|CMV+GFP-PEST191|pSB1AK3+TER| |BBa_J52035|MyD|pSB1AK3| |BBa_J52026|MyD-GFP|pSB1AK3| |BBa_J52028|GFP-PEST191|pSB1AK3| |BBa_J52029|NFkB+GFP-PEST191|pSB1AK3+TER| |BBa_J52027|NFkB+MyD-GFP|pSB1AK3+TER| |BBa_J52019|TRAF6|pSB1AK3| |BBa_J52021|TRAF6-GFP|pSB1AK3| |BBa_J52022|NFkB+TRAF6-GFP|pSB1AK3+TER| |BBa_J52016|terminator|pSB1AK3| |BBa_J52017|pSB1AK3+TER||
Transfection In September and October we started to work on transfection of our constructs (some parts were still in progress) into human embrional kidney cells (HEK293) and three detection system mentioned below. At first we had to optimize the methods (read articles, test negative and positive controls) and learn how to work with human cells. Then we transfected cells with our construct. Experiments are still in progress. Despite transfection with our construct, we also have to transfect cellc with TLRs, because strain HEK293 expresses only TLR3 and TLR6. Sepsis is usually response to pathogenic Gramm negative bacteria. Their outer membrane contains LPS (lipoproteins) that is recognized by TLR4 and accessory molecule MD2. At the beginning we transformed all cells with our constructs and additional plasmids, one coding TLR4 receptor and another MD2. Very soon we found out that our plasmids were contaminated with LPS of E. coli (strain DH5alpha used for transformation), since there was no difference in response between stimulated (with LPS) and unstimulated cells. To overcome this problem, now we are using TLR5. This receptor detects bacterial flagelin. Signal transfer through this receptor does not depend on presence of LPS.
Detection systems Requests for the optimal detection system are: -velocity; -sensitivity; -paralelization; -Optical sygnal; -on living cells; -price.
On the basis of those requests we have decided for the following detection systems: flow cytometry, luminometry and ELISA. Members of those subgroups are Jernej and Ota, Alja, Moni and Rok and Jelka and Matej respectively.
Which constuct are used in individual detection system and short description of system itself:
Flow cytometry More to come.
Luminometry Used composite parts: NFkB-MyD-luc, NFkB-MyD-luc-PEST, NFkB-lucPEST, CMV-luc, CMV-luc-PEST With parts that are under NFkB promoter we want to prove inhibitory effect of dnMyD88 on signal pathway (on the basis of luminiscence decrease). Parts with PEST tag should be decomposed and therefor luminiscence should increase. We found out that it takes 4 hours for any response. We also want to calculate half-life of rLuc and rLuc-PEST, so we used those two constructs that are expressed under constituitive promoter CMV. Synthesis was inhibited by adding cycloheximide in intervals
ELISA: Used Domposite parts: NFkB – dnMyD88-terminator (name), NF-kappaB – TRAF6 – GFP – terminator (name)
Komentarji:
Anticipated results and their significance for understanding the mechanism of immune response
Transfected cells have now TLR receptor (4 or 5) and our construct under NF-kappaB promoter. We expect that after stimulation (with LPS or flagellin) cytoplasmic NF-kappaB become active, migrate into nucleus and induce expression of our construct. Accumulating of dominant negative protein should block downstream signalling pathway. Concentration of active transcription factor should decrease and no more product should be synthesized. The rate of transcription and translation should be detected with detection systems - decrease of luminescence, free NF-kapaB and::: (depends on detection system). In case that our construct is followed by PEST (degradation) sequence, it is expected that product should degrade. If stimulus is still present, NF-kappaB will become active. Our product will be synthesized again.
Komentarji
Troubleshooting Luminometry: Luminiscence cannot be measured in vivo since celentrazine is decays too quickly, many parallells and controls are needed and therefor few 396-well plates are used in which pattern of transfection stimulation is very complicated.
Terms Dominant negative protein – protein that is mutated in way that has only one domain, that interacts only with one protein in signaling pathway (in our example) upstream but not downstream. The result is blockade of pathway.
MyD88 – protein at the beginning of our signaling pathway that transfer signal from TLR receptor to downstream proteins (IRAK4) resulting in NF-kappaB activation.
TRAF6 – protein that functions as a signalling mediator which binds to IRAK1 and transfer signal downstream and also resulting in NF-kappaB activation.
NF-kappaB – transcription factor that function as heterodimer NF-?B dimers are usually sequestered in the cytoplasm in an inactive form by molecules of the inhibitor of NF-kappaB (I?B). Activation of NF-?B involves the phosphorylation and proteolysis of the I?B proteins, release and nuclear translocation of the NF-kappaB, resulting in activation of NF-kappaB-dependent transcription.
References
Financial support
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Komntarji: - Terms bi se pojavljal kot 'floating bar' (predlagal Marko)
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