Closed EveCarter closed 1 year ago
mattodd
commented
1 year ago Hope @StructuralGenomicsConsortium/cnp5-plcz1 folks still OK for a short catch-up today. I think @AnnaGirtle is joining us - Anna is interested in helping out as James Murray Student Champion.
mattodd
commented
1 year ago Hi @AnnaGirtle !
Thanks for coming along today. So, not too many actions at the moment since we're in this period of trying different solutions for protein production. As mentioned, we're meeting with the Nuvisan folks tomorrow to discuss. @EveCarter could Anna be invited?
Today's meeting recording needs downloading from Zoom and uploading to Youtube. To make sure everything is findable we can use hashtags for the recording, but it helps also to have all the recordings in one channel. The instructions for doing this are here - Eve I can't remember if we have adequately tweaked these instructions to solve the technical SNAFU we had last time? It'd be a good tester to see if Anna could do it independently, obviously. It's easy within Zoom to give specific UCL people access to a recording.
Then the Youtube link gets added to this issue (above).
Then we need to start the next Issue, to plan for the meeting, whcih is meant to be on March 7th (first Tuesday of each month at 2pm UK time. However, that clashes with the next SGC Board meeting plus the 20-year SGC celebration of the SGC happening in Toronto. If it's all the same for everyone else could I suggest we schedule it for Monday, March 6th just as a one-off? The new meeting issue can be posted by copying the text from this one (click "edit" so you can see the underlying Markdown) and then tweaking it. It should end up looking very similar to this one!
In terms of things arising from today: It'd be really great to have a few tech slides from Madison if we don't have them, essentially on what's been tried recently. Just a copy of a recent deck. Nico was talking about non-human primates mating study, but it seemed a little early to be talking about this unless I'm missing the point. Karl says mouse better to work with than human, in terms of activity? A question arose that maybe we had the protein and it was too weak to see? Not sure how we check for that? Al asked about co-expression of something else alongside PLCz1 as being a potential key to getting happy protein, i.e. another protein with which it interacts. Mat has reached out to BMGF to see if they can put us in touch with Cyclica, who are working on some of the targets in this area, possibly PLCz1.
@EveCarter you've made a sensational job of the wiki for this project. If you could make a start on the "story so far" I reckon it'd be pretty short, because you can link out to so much detail in the other pages. All we need is a couple of paragraphs explaining to newbies what the project is, and where we're up to.
AnnaGirtle
commented
1 year ago Hi guys! It was great to meet you all today. I had a thought about the protein production issue, but I am new to the project so I am not sure if you have considered this yet.
PLCZ1 is pretty much exclusively expressed in the testis and is localized to the perinuclear cytosolic structure near the post-acrosomal sheath of the spermatozoon in mice (and in the cytosolic portion surrounding the acrosome in humans). These are both in very close proximity to post-acrosomal region of the sperm head, where CAPZA3 is expressed. The two genes are back-to-back in the genome and share a bidirectional promoter and there is some evidence that low fertility is associated with decreased expression of both genes. There is similar low expression in sperm lacking ACTL7A and ACTL9 (testis localized genes) as well. Is it possible that these actin-like proteins are stabilizing PLCZ1 if they are similarly colocalized?
EveCarter
commented
1 year ago Thanks all for coming to the meeting!
@AnnaGirtle, thanks for this suggestion - I will let one of the biologists reply as I am not sure. I will send you a message about uploading the meeting video and planning the next meeting.
I will also get to work on a "story so far" section on the github wiki.
March 6th works for me
mattodd
commented
1 year ago Great job uploading the recording @AnnaGirtle.
Date: Tue Feb 7th 2023 Time: 2pm UK time (other times) Place: https://ucl.zoom.us/j/94060098722 Previous Meeting: issue #5 Who can come?: Anyone. No need to say anything unless you'd like to. If you'd like to contribute, please join Github.
Attendees: Prof Matthew Todd (UCL), Dr Eve Carter (UCL), Prof Matilda Katan (UCL), Dr Madison Edwards (Uni Toronto), Prof Karl Swann (Uni Cardiff), Prof Al Edwards (SGC), Prof Opher Gileadi (SGC), Dr Nico Braeuer (Nuvisan), Anna Girtle (UCL)
Agenda:
1) Protein Production
Update from Madison Edwards and @EveCarter.
In meeting:
Madison shared that she has tried express PLCZ1 from different animals in E. coli but has not been able to get good yield. Constructs with loop deletions showed much better expression, but she believe they are insoluble and will run assays to confirm.
Opher discussed potentially using calmodulin to stabilize the protein and Matilda suggested IP3 given its success with PLCG1 and crystallography. Karl mentioned calmodulin may be inhibitory, but still worth trying given the difficulty we’ve had thus far. Aled suggested trying to incubate partially purified proteins in various additives in case we serendipitously find one that improves stability, and also highlighted the possibility that PLCZ1 may require an interacting partner to fold correctly (as proteins in a complex do).
Opher discussed the possibility of using constructs engineered with internal mutations in amino acids or alpha folds (Weizmann Institute has a search tool). May improve stability, but run the risk of diverging too far from the native protein with high mutation loads. Could take a low, medium, and high mutation load-construct and try to purify, then pare down to constructs with minimal mutations (ie. those sufficient for stable folding but not so many that function changes).
2) Any other business
L'esprit de l'escalier If you'd like to follow up after the meeting, please comment below. You can also email, but please be clear if anything in the email should not be public domain - the default is open.