I want to deliver an improperly folded protein (may / may not be improperly folded) into the animal cell. Does it has any chance to get properly folded inside the cell before it get delivered to specified destination ie, nucleus or mitochondria???
It is important to mention that I am using cell penetrating peptide as the delivery methodology into the cells. Secondly, the proteins to be delivered are nuclear transcription factors. Thirdly, what if I would give cells the heat shock?? This would activate the cellular chaperon cascade and may allow proper folding of the delivered protein.
Kindly quote any reference if available
Don't think so.. even if the protein enters the cell.. if the protein is not very essential for the cell to survive or it lacks such type of proteins.. why a cell will waste energy to fold such a unnecessary protein? Though you can always label the protein and see its fate internally.. Hope you succeed.
Interesting question... Can be monitored using single molecule studies in a reconstituted system..
Hi Tarun, I have my doubts. Cells produce chaperones that aid in properly folding newly synthesized proteins. Proteins that are not properly folded are tagged with ubiquitin and degraded by proteasomes.
It might work. Some studies actually use denatured proteins and probe refolding in differently reconstituted chaperone systems.
Further, the hsp70/hsp40 system seems promising. Very recently it could be shown that denatured rhodanese is actually expanded by an ATP driven mechanism, indicating that this might help to process and prepare it for downstream chaperone systems. doi: 10.1073/pnas.1407086111.
Still, it is hard to predict whether your target protein will be recognized. In accordance with the previous posts I suggest that you perform a careful labeling of the protein. Further, you should definetely check whether your protein is getting degraded.
Cheers
You might like to take a look at
TAT-mediated protein transduction into mammalian cells.
Becker-Hapak M, McAllister SS, Dowdy SF.
Methods. 2001 Jul;24(3):247-56.
PMID: 11403574
The authors intentionally denature their TAT-fused proteins by solubilisation in urea followed by rapid removal of the urea. In some cases they found this increased the biological effects of the proteins on cells - they speculate this facilitates entry into the cells. These findings suggest that at least these proteins, once in the cells, can refold to an active conformation.
Definitely can. As stated above by Rajeev and Michael, many bacterial toxins are assisted in folding by chaperones after their cross the membrane. This might depend on the way you deliver the protein of interest though.
If your goal is to re-purify and get more folded protein I think it is not a good idea. I would try adding glycerol to the cells producing the protein to help folding, or something like that. By contrast, if you plan to investigate this, there are some systems to detect by FACS the amount of enzymatic activity of a protein delivered to the cyosol (look for cross presentation assays). By tagging the protein you should be able to know the total amount of protein and at the same time the enzymatic activity (a measure of total and folded, respectively) at single cells.
Hi there,
I do not know what would be your delivery method of a misfoldedc protein into any cells. You may first let people know what is your delivery method. However, any external delivery done which goes through the cell membrane, would cause your protein to end up in the clatherin or non-clatherin coated pits for delivery to the lysosomes. There in lysosomes, the pH is 5 and your protein becomes more denatured than ever and hence more non-functional than ever.
Try to explain your problem more extensively.
Regards,
GCK
Hi everyone, thank you for the valuable replies.
The question has been explained in much deeper context now. kindly go through it and suggest the plausible condition.
Thank You
Hi Tarun, thanks for the added information.
Some questions: Does your misfolded protein have either a nuclear or mitochondrial translocation signal?
There are cytosolic chaperons, e.g. MSF and hsp70, that aid in importing proteins into mitochondria, but as far as I know, most chaperones are in the ER.
Does your cell penetrating peptide target endocytic pathways and is it delivered to endosomes?
Further clarifying the concept:
The protein which i m trying to deliver has a tat peptide (act as CPP) and a nuclear localizing signal.
What I feel once the cells adhered on a surface are given the treatment of protein the CPP will hep out the protein to enter inside the cells though endocytosis. However the CPP have the property to escape from the endosomes. So the protein of interest will also escape. Now, we assume that major proportion of the proteins exist in the cytoplasm and there are present the cytosolic chaperones hsp70, hsp40 and hsp90. Now if we give a heat stress to the cells, i presume it is most likely the stress response system will get activated and will try to correctly fold the misfolded protein. and then it will be transported to nucleus.
Is my presumption correct?????
Kindly also suggest are there any nuclear chaperon????
Hi Tarun, your procedure looks good on paper.
Can you put a biotin on your peptide to see what happens to it in the cell?
Hi Tarun,
Keep in mind that even with the TAT-tag, most of your protein will stay in endocytic vesicles and end up going to lysosomes for degradataion. Expect that only a very small fraction of your protein will reach the cytoplasm and the nucleus.
Good luck.
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