I would like to assay for the effect of Hectd2 activity on my protein of interest but the actual Hectd2 protein is too big. I would like to know if I can express the Hect domain only to test my hypothesis.
Question: why do you think it is too big? Too big for transfection? genecard says Hectd2 is 81kda, which would be fine for transfection. The HECT domain if active could still ubiquitin target proteins in transfected cells and in vitro. The problem is the that specificity (binding sites) is usually determined by interactions that occur outside the HECT domain. So you may get ubiquitination that is not specific or you may fail to get ubiquitination because you don't get the appropriate protein-protein interaction.
You should try different transfection reagents and systems. Efficient transfection of cDNAs for proteins that are hundreds of kda is possible and routinely done. I regularly co-transfect an E3 ligase that is 110 kda using Fugene Xtreme Gene HP.
I´m not sure but I think the HECT or RING domain refers only on the type of ubiquitin transfer from a donor E2. While the RING mediate "direct" transfer, the HECT E3 ligase type act as intermediate donor (Cell Mol Life Sci. 2004 Jul;61(13):1546-61). As this, I´m not sure if by simply increasing the HECT domain of your ligase is enough to increase your protein of interest polyubiquitination. Because is not the HECT/RING domain that defines substrate selectivity.
You sould give it a try and check if by increasing the HECT domain, you have a reduction in the putative substrate of this E3.
Hope this information is useful for you.
Hi, usually the HECT domain is active in absence of substrate. But, Is the substrate binding site of your protein in the fragment that you are expressing? If you overexpress your HECT ligase, is the degradation rate of your ligase higher? Sometime, the transfection works fine, but at the same time the cell degrades faster the overexpressed protein. And you only detect a fragment of it. That was reported for some HECT ligase.
As far as I understand, you want to test whether a protein is polyubiquitinated in a HECTd2-dependent fashion. As stated above by Virginia and Ivan the HECT domain is active on its own and isn't what defines the substrate specifity. Hence, overexpressing the HECT domain alone won't answer your question: it will ubiquitinate or polyubiquitinate in a promiscuous fashion (unspecifically). This will be the case in vivo but in vitro as well: throwing ATP, an E1 an E2 the HECT domain of HECTd2 alone in a tube will probably result in it being ubiquitinated. Then again, this will be true for a vast subsets of proteins here, as the HECT will ubiquitinate any protein.
If I wanted to assess the effect of HECTd2 on a protein, I'll use dominant negative mutants of this ligase and check whether your protein of interest is still ubiquitinated: if you know which part of HECTd2 does determine its specificity, try to overexpress it without the HECT domain. Theoritically, it should bind your protein of interest and sequesters it away from the wild type HECTd2 protein. Monitor the ubiquitination of your protein using one of the protocols given by Thorsten. In parallel, and I recommand you do that to ensure the validity of your result, you could try to knock HECTd2 down, which should also result in a lower (if any) ubiquitination of your protein.
That's what I would do if I couldn't overexpress the whole of the protein.
No problem, hope this helps. Nathaniel add the clue in his first answer.
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