Protein facilitates enzyme recruitment

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Protein facilitates enzyme recruitment

Postby nekameneka » Oct 04 2016 1:21 am

Hi everyone, it's me again.
Anyone has suggestion what is the best way to find out if my protein is important for recruitment of RNase?
Thanks! :)
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Re: Protein facilitates enzyme recruitment

Postby relaxin » Oct 04 2016 10:42 am

Use antisense RNA to block the expression of this protein.
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Re: Protein facilitates enzyme recruitment

Postby nekameneka » Oct 04 2016 11:24 am

Thank you for the suggestion. I would like to do that but is there some more simple way to check if my protein makes a complex with RNase? Is IP the choice to check it before I go further? Also, do you think endogenous levels of gene encoding this RNase would be enough? I know it's hard to guess but I would like to just check this possibility before I go to more extensive data if it would be worth to go that way.
Thank you :)
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Re: Protein facilitates enzyme recruitment

Postby relaxin » Oct 05 2016 12:41 pm

I do not know if the mechanism of enzyme recruitment involves direct binding. Binding could also be a mechanism of suppression, such as Rnase inhibitor:RNase complex. Of course, IP with antibody against the protein and Western blotting with antibody against RNase will prove the binding of these two proteins. Do you have any info of the protein of interest?
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Re: Protein facilitates enzyme recruitment

Postby nekameneka » Oct 06 2016 2:07 am

My protein is RNA binding helicase involved in immune response and the RNase I want to check is RNase L (degrading all RNAs in the cells upon viral infection).
I would like to know if these two "communicate" in some way and if they do how and when.
(Not sure if I can detect endogenous RNase L, tried to search but depends on cell types)
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Re: Protein facilitates enzyme recruitment

Postby relaxin » Oct 06 2016 9:55 am

I do not know much in this area of research. You may get some ideas by searching PubMed on papers using keywords: RNase L, MDA5, IFN-beta. It looks like RNAse L cleaves and identifies viral RNA and feeds it into MDA5 (a helicase) activation sites, enhancing the production of IFN-β. There is no stable binding between RNAse L and MDA5, and hence your IP experiment may not work. They demonstrate the interaction of these two molecules by the decrease of IFN-beta expression by siRNA tests silencing the expression of either RNAse L or MDA5.
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Re: Protein facilitates enzyme recruitment

Postby nekameneka » Oct 06 2016 10:19 am

Thanks so much for your reply but MDA5 is not my protein of interest, I might have idea about the other proteins involved in this. I connected some previous reports about RNase L behavior in different virus infections and its involvement in downregulation and upregulation of IFN and other mechanisms depending on type of virus, very interesting, I might check this IP and RNase siRNA, thanks for your advice.
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Re: Protein facilitates enzyme recruitment

Postby mchlbrmn » Oct 07 2016 12:56 pm

You can do an experiment transfecting the two interactors each tagged with a different epitope tag, FLAG and V5 for example. Reciprocal experiments can be done switching which is bait and prey.
If you want an idea of whether their is enough expression for the experiment to work using native protein, you might first check the native expression level by Western.
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Re: Protein facilitates enzyme recruitment

Postby nekameneka » Oct 07 2016 8:20 pm

Hi! :) Yes, I already expressed few proteins (which I think might be involved) and their mutants to see if I will get higher native level expressions of the target.
I plan to do fractionation with my lysate and if I really do get higher native expressions, in which cell compartment it would be. I also did IF with wt and mutants.
I know its a lot of work for just an assumption but it might be worthwhile because I can use lysates for other experiments.
Thank you for your suggestion, I will definitely do the bait and prey as you advise. (If I get anything interesting from this Western).
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