The primary article for this week makes use of Y2H approaches only. Based on what we know about PDZ domains, do you think they'd obtain different or additional results if they had used affinity purification/mass spectrometry? According to the reviews, AP/MS can be more useful for obtaining protein complex data, so what I'm wondering is if PDZ domains facilitate the formation of protein complexes, or just binary interactions.
The researchers found in their study that 51% of proteins that interact with PDZ domains of c elegans do not contain the classical C-terminal consensus sequence they predicted. Do the target ligands outside of the c-terminus for these proteins have a specific common factor making them suitable for PDZ binding? Or is this a question that must be answered with future studies into these unexpected protein locations?
I'm going to piggyback what Victoria said because I was wondering about the same thing and she stole the words right out of my mouth...is there any pattern to the GO terms of the PDZ domains that interact with their ligands without using the C-terminal?
Why in the primary research article did they choose to transform the 93 PDZ domains found in C. elegans to yeast for identifying protein interactions? What makes yeast a good organisms to study protein interaction of PDZ domains as opposed to studying the protein directly in C. elegans?
I might be completely missing the point, but what I got out of the paper was just proof of principle that a) Y2H works for studying the interaction between proteins and PDZ domains and b) that essentially PDZ domains are way more complicated than they thought and need to be studied more? I was also a bit confused about the c-terminal stuff too; is the c-terminal important for other protein-domain interactions or is it specific to PDZ?
My question is similar to Gina's -- The authors mention that Y2H can uncover physiologically relevant binding events, and so they sought to confirm their results using co-IP. Y2H can also uncover indirect interactors, which could be physiologically relevant (in complexes, maybe?). I'm wondering to what extent you believe their experimental logic, protocol, and findings reflect this possibility, and if you believe they adequately incorporated this possibility in to their interactome map.
In one of the reviews, it was mentioned that the Y2H method only detects binary interactions between proteins. Are their currently methods to detect interactions between more than two proteins at once (perhaps I missed the answer to this in the review)? Furthermore, (as many great, more specific questions have already been asked in regards to the primary paper, I will ask a more abstract one) what do you think are the broader implications of the research conducted in the primary paper, as I did not feel they addressed this well in the paper?
In the primary paper, they note that 81% of the PDZ domains gave at least one interaction partner. Were the other 20% just missed by the Y2H approach? Are they functionally redundant and no longer serve a distinct purpose? They listed multiple times that Y2H screens miss potential protein-protein interactions due to "cDNA representation in non-normalized libraries." What does this mean?
Do you suspect that the other target ligands outside the C-terminus could help develop the definition of PDZ domains? By tagging and following the other target ligands, maybe the importance of the C-terminus binding channels could be better understood?
Why did the authors fail to identify a clear internal motif that could be uniquely responsible for PDZ domain binding? How can they overcome this in future research?
The authors initially state the goal of the paper is to identify the protein partners of the PDZ domains in C. elegans by conducting a “comprehensive search.” While I find their argument on the merits of domain-centered interactome studies fairly convincing, I feel there are shortfalls to the methods. Would environmental stressors, not accounted for in the methods of this study, lead to differing results in the PDZ interactome than those reported in the study?
The authors first identified many different PDZ domains and the protein-protein interactions involved using a cDNA library but then only tested a small sample en vivo. Do you think there were any bias in which ones they selected to further research? Do you think if they used all of interactions found in the cDNA library, they could have found more definitive results or do you think their sample was sufficient?
PDZ domains bind proteins that participate in many types of cellular processes. Are any human diseases known to be caused by mutations which disrupt the binding between PDZ domains and their binding partners?
I was curious about the fact that only 44% of the PDZ containing proteins were associated with gene ontology terms. This seems like a very low number considering the amount of PDZ containing proteins they were analyzing and the kinds of interactions they posed. What was the reason behind this?
The article talked about the frequent use of non-consensus binding and I was a little bit confused regarding what these binding sites actually were and how they differed from the other domains that were discussed. What I am understanding is that the authors had thoughts about the binding sites and domains that were occuring but instead found these non-consensus binding sites which were much more complicated than they originally predicted?
I guess I was just wondering what the difference is between canonical and non-canonical PDZ domain proteins are? What is the reasoning behind the categorization?
The Lenfant paper states "It is clear that global Y2H screens only reveal a fraction of potential protein-protein interactions". The Bruckner review paper mentions using mass spec to elucidate protein protein interactions. Would using MS be more useful than Y2H for finding the other protein-protein interactions if time/money/equipment was not an issue?
Do the researchers propose a mechanism for the non-canonical binding of certain proteins to PDZ domains? Is it possible for certain proteins to use multiple binding domains to form the same connections?