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TLR-4 and downstream stuff does somebody work in this branch

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TLR-4 and downstream stuff does somebody work in this branch

Postby konstantin » Thu Nov 23, 2006 9:28 am

hello. I wouls like to ask, how can detect the activation or expression of TLR-4, MyD88, TRIF, TRAM, TRAF6 and other proteins in this pathway
any information will be valuable
thanks in advance.
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Postby Dr.Stein » Thu Nov 23, 2006 9:40 am

I guess you need their respective ligand with proper conjugate(s) as a marker to detect their presence :?
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Postby LilKim » Thu Nov 23, 2006 5:13 pm

wow, how ironic, I'm about to start writing a paper about that stuff.

Do you have access to journal subscriptions? You should read up and figure out what the TLR4 ligands are.

And how would you detect signalling? .. you'd have to do experiments involving biochemistry! It's not terribly difficult to do...

anyways, let me know what you're thinking and and how you'd go about figuring out signal detection... and i'll let you know what i'm thinking too.

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Postby konstantin » Fri Nov 24, 2006 2:51 am

yeah, may be it is not successful choice of words. but I really confused about this. I know that I can detect the interaction between MyD88 and TLR-4 or TRIF and TLR-4 through immunoprecipitation and western blot.
But, It is only interaction of two proteins.
my question is that with the help of what method I can investigate the expression or activation of TLR-4 or IRAK-1 and -4, MyD88, TRAF6 and other downstream proteins... I really confused about this... :( .
I read from articles that some researchers detect expression using RT-PCR on mRNA level. But I do not know, is it possible to investigate through western blot or other experiments?
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Postby Dr.Stein » Fri Nov 24, 2006 4:36 am

Immunohistochemistry is the basic method to detect any expression on membrane surface :)
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Postby LilKim » Fri Nov 24, 2006 6:22 pm

hey there

Ok... I think i see what you're asking now.

Personally, i don't think rtPCR would be an advantageous method to detect activity. It often used as a 'quick and dirty' technique that implies activity but all rtPCR really does is detect transcription of mRNA... rtPCR doesn't tell you if this mRNA is being translated into protein ... it doesn't tell you if the mRNA is unstable (therefore, quickly degraded before translation). Furthermore, just because you see an increasein mRNA levels doesn't necessarily mean that the TLR4 protien would be activiated.

surely.. rtPCR findings often 'imply' what you're trying to figure out... but the only way to tell is by following up with westerns to see if the protein is there.

So, i really don't think rtPCR is the best way to go to answer your questions.

So, i think you should think of your problem in these kinds of terms.... Ask yourself:

1. What happens to TLR when stimulated/activated with an agonist/ligand?

2. What happens to the adapter proteins upon receptor stimulation/activation? (ie myd88, tram, trif, mal;tirap)

3. What happens to irak, traf3/traf6 following actvation of the adapter protiens?

So essentially what i'm tryint to ask you is to investigate the physical interatction between the protiens along the pathway.

So... my crappy memory tells me that upon stimulation. TLR;s dimerize, resulting in subsequent dimerization of adaptor protiens ie. myd88, tram, trif, mal:tirap (depending on the ligand.. and i don't know TLR4 ligand off the top of my head)... following that dimerzation the 2 different IRAK protiens assosiciate... one phosphorlyates the other allowing association of traf3 or traf3.

Alright, with that said we know that activation results of ASSOCIATION of protiens and phosphorlation. Therefore, to determine activity along the pathway you'll need to devise and experiment that will test for association (ie. immunoprecipitation) or phosphorylation (ie. determine which residues are phosphorylated following activation and [wester] blot for these phosphorylation events using an antibody.

of course there are fancier ways to detect this .. such as mass spec.

if you would like, I can direct you to some excellent reviews and you can check them out.. they'll help you to understand.

but.. yeah, i'll stop now.

Tell me if this helps.
- KIM

p.s. Please double check what iv'e said for accuracy... I haven't started writing my paper yet, so i'm using my memory to explain this stuff (and sometimes my memory isn't soo accurate)
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thanks LiKim

Postby konstantin » Sat Nov 25, 2006 9:42 am

yes, you are right... that LPS is transferred to CD14 by a lipid transferase LPS-binding protein(LBP). LPS-CD-14 stimulates TLR-4-MD-2 on the cell surface. and this interaction causes oligomerization. Next, it trigger two pathways MyD88 dependent and independent one. MyD88 dependent is MyD88 recruits IRAK-4 and induces phosphorylation of IRAK-4 leading to the phosphorylation of IRAK-1. the p-IRAK-1 associates with TRAF-6 leading to the activation of the IKK complex resulting in the activation of NF-kB.
Independent one mediated through TIR domain-containing adapater indicing IFN-b (TRIF). TRIF-dependent pathway triggers the expression of INF-b andINF-inducible genes through TBK1 and IKKi. In addition, TRIF associates with RIP1 leading to the delayed NF-kB activation.
As you see, theoretically I understand what kinds of pathways are activated and what proteins take part in these pathways, but I want to investigate the effect of my drug on this pathways and it is very problematic for me.
some things confuse me such as, are oligomerization, dimerization and homodimerization of TLR-4 by LPS the same process? and can LPS induces the additional expression of TLR-4 or not? It is well known that LPS cause some modification in TLR-4 (dimerization or homodimerization) but can it cause expression of it ( I mean that after LPS induction, the number of TLR-4 is increased). I told you that I can identfy whether interaction of TLR-4-MyD88 or TLR-4-TRIF after treatment with my drug and stimulation with LPS. But how about down stream proteins....? for example may be my drug does not work at TLR-4-MyD88 and TLR-4-TRIF level, but lower. how can I check it? the effect of my drug on IRAK4, IRAK1, RIP1 or TRAF-6. I do not know this point. and the last thing what i do not know, how can I check the CD-14 and MD-2 proteins? this also confuses me.
yeah may be it is really reasonable to get rid off RT-PCR.
P.S. this is my first experience in forums, actually at the beginning of it , I did not hope that somebody will be interested in my problems. thanks a lot.
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Postby LilKim » Mon Nov 27, 2006 8:19 pm

hmm okay you have a ton of questions (and I don't think I know enough to answer most of them).

I know that TLR4 dimerizes upon LPS stimulation but I don't know if this is a homo./hetero./oligomerization process. I'm not sure if this is known but I'll see if I happen to come across anythin that addresses this question in the next week (while I read)

I also don't know if dimerization would stimulate TLR4 expression (.. that can be determined by rtPCR... you should also follow up with a western to see if the transcript is translated)

So, from what I gather, you're trying to find out what 'step' of the process your drug affects. That's a tough one... and it think that it would require alot of work. Off the top of my head the only thing that i could think is that you could mutagenize cells and look for a gain of function mutatants that are still able to signal via TLR4 (upon drug treatment) and then going BACK to determine which mutation made your cells resistant to the drug. (It is possible that the mutation in a protein in the pathway could make the protein/gene resistant to the function of the drug... but this is a 'chance' event) This is a HUGE undertaking because it is likely that many mutations will create a 'gain of function' situation. I cant' really think of an easier way to do it... cuz this could take a WHILE!!

lastly, I don't know enough about CD-14 aor MD-2 ... so I can't help you figure that part out ... but If I happen to come across somethine... i'll let you know

sorry that i can't help more

good luck anyways!
- KIM

p.s. let me know how things go
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Postby konstantin » Wed Nov 29, 2006 2:16 pm

hello LilKim. sorry that did not reply so long time ..... I searched information what could be useful to me and my project.
last friday, I had long discussion with my professor. so she agreed with me in some items and correct me in other ones...
anyway I started to grow my cells and may be at the end of this week I will begin my project. first of all, I would like to detect the expression of TLR-4 in time dependent manner using western blot. and secondly to investigate whether my drug has inhibitory effect on the expression of TLR-4 or not. If I get positive result, I'll continue. If not, I 'll rethink my project again :) . actually it is my plan for some time.
In addition: I am studying in South Korea. Here the mentor system is not so developed but I heard from some students that in USA, mentors are very usual thing. If you have some information about it, can you share with me?
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Postby LilKim » Fri Dec 01, 2006 2:26 am

Hey there!

Glad that you recieved some help with you project! I personaly don't know what i'd do WITHOUT my mentor... he is FANTASTIC! He helps me with everything and I feel very fortunate to have someone who is sooo supportive and helpful.

anyways ...

Have you devised a way to detect TLR4 signalling downstream of activation? AN easy way would the to use antibodies specific for phosphorylation of the Map kinasese (like Erk, p38, JNK..also you can look for degredation of IRAK4, IKBa)

of course you'd probably have to ask for antibodies... but I recommend you check out these papers:

http://www.ncbi.nlm.nih.gov/entrez/quer ... g+effector

(Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6. Hacker et al. Nature. 2006 Jan 12;439(7073):204-7. )

If you don't have access to this journal... please let me know, I will email you a copy of the paper.

Good luck with everything!
- KIM

p.s. don't worry about not being able to write back soon... i'll get your message whenver you send it
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Postby konstantin » Sun Dec 03, 2006 3:37 am

Hello Kim.
Ohhhh thanks a lot for that article. I just glimpsed through this article, if I have some question, I'll let you know. In my previous project, I have already checked out the phosphorylation of MAPKinases and activation of NF-kB as well as STAT-1. I and my professor got good results in that stuff, today we are going to investigate TLR signaling pathway and ....you got the point....
as I see, you are very familiar with mentor system and you have only good impressions about this. so can you recommend me some internet sites or links where I can find some mentor or something like this? I really want to have a mentor because I want to interact with other people in my major. I have the bunch of questions about foundation system, how to apply into conferences or how to choose apropriate conferences and so on. I heard that mentros can be very helpful in these questions. I am very glad to study in korea because in my homecountry science is completely stagnant. I can make here the modern experiments and it makes me happy. but on the other hand, here I understood that, except my skills in experiments and knowledge, I should be active and contact with many people in my branch and neighbors spheres in order to have a good career path.
Anyway, thanks very much I do not know how to appreciate you for your help. good bye.
By the way, just in case, my mail is konstantintsoy@yahoo.com
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