There was a closed meeting of a group interested in PLCz1 on Oct 4th 2022. The notes below were taken during the meeting and have been filtered so as to be non-confidential, but if anyone would like anything changed, please let @mattodd or @EveCarter know. Similarly, please correct any errors.
Present: @mattodd , @EveCarter (UCL), Al Edwards, Madison Edwards (SGC Toronto), Dan Goldberg and Stephen Ward (BMGF), Anke Mueller-Fahrnow, Charlotte Kopitz (Nuvisan), Karl Swann (Cardiff), Claudia Tredup (SGC Frankfurt).
PLCz1 protein stability was highlighted as a key challenge, particularly where the protein lacks tags. The whole protein is needed for activity. Domain swapping with PLCd1 has been done to generate chimera proteins. EFEF swaps maintain activity, but XY and C2 domain swaps stops the activity. These findings tally with locations on the protein where mutations have been found to destroy activity e.g. I489F (published mutation, but about 20 are known). There is no effect on protein activity in vitro, but changes the %binding of something else. C2 domain mutations maintain catalytic activity, but still don't get the calcium oscillations, so that region is involved in something as yet not understood. The XY region is critical for binding PIP2. The C2 domain binds PI3P.
The other PLCs are 50-100 times less potent in their calcium oscillation effects and only zeta works in eggs. If PLCz1 is over-expressed in regular mammalian cells there is no effect on calcium oscillation, but if you take that protein and put it in eggs it causes the oscillations, so it is highly specific in its effects. PLCdelta1 does not cause these oscillations at the same concentrations.
In mouse eggs the protein does not bind to the plasma membrane, but is instead located in the cytoplasm associated with vesicles where pip2 is located.
Al Edwards asked whether antibody reagents for PLCz1 would be useful, specifically:
1) Have human knockout cells been made? Answer: mouse KO has been made, the colony is frozen down and mothballed. There is the same in a group in Japan. What about actual sperm from someone with the knockout - apparently many people who are infertile have PLCz1 knockout. (Interesting comment). Commercial antibodies have not performed well.
2) Would purified PLCz1 be a useful reagent that could make fertilisation more effective? Answer: yes. Has been tried, and the roadblock is the stability of the isolated protein, and dose calibration. There is a patent on PLCz1 held by Cardiff that runs out about now.
Madison Edwards gave a protein update. Trying to secure slides. Existing approaches with NusA and His6-MBP tags not working well. Alternative needed. Karl tried a lot with bacteria, not baculoviruses.
If we can find a promiscuous inhibitor, we can set up a nanoBRET assay for in-cell target engagement. Works on lysates.
Assays: (now at #1 ) it was suggested that we reach out to John Sondek at UNC. Avanti lipids sell a fluorogenic probe. Already used in screen of inhibitors vs other PLCs. Karl's current assay in eggs (endpoint assay) can do 100 compounds a year, which will be v useful.
Did not discuss:
1) where should we look for the best focussed libraries of relevance, given the nearest neighbour proteins are dissimilar.
2) How does the PLCz1 get into the egg?
There was a closed meeting of a group interested in PLCz1 on Oct 4th 2022. The notes below were taken during the meeting and have been filtered so as to be non-confidential, but if anyone would like anything changed, please let @mattodd or @EveCarter know. Similarly, please correct any errors.
Present: @mattodd , @EveCarter (UCL), Al Edwards, Madison Edwards (SGC Toronto), Dan Goldberg and Stephen Ward (BMGF), Anke Mueller-Fahrnow, Charlotte Kopitz (Nuvisan), Karl Swann (Cardiff), Claudia Tredup (SGC Frankfurt).
PLCz1 protein stability was highlighted as a key challenge, particularly where the protein lacks tags. The whole protein is needed for activity. Domain swapping with PLCd1 has been done to generate chimera proteins. EFEF swaps maintain activity, but XY and C2 domain swaps stops the activity. These findings tally with locations on the protein where mutations have been found to destroy activity e.g. I489F (published mutation, but about 20 are known). There is no effect on protein activity in vitro, but changes the %binding of something else. C2 domain mutations maintain catalytic activity, but still don't get the calcium oscillations, so that region is involved in something as yet not understood. The XY region is critical for binding PIP2. The C2 domain binds PI3P.
The other PLCs are 50-100 times less potent in their calcium oscillation effects and only zeta works in eggs. If PLCz1 is over-expressed in regular mammalian cells there is no effect on calcium oscillation, but if you take that protein and put it in eggs it causes the oscillations, so it is highly specific in its effects. PLCdelta1 does not cause these oscillations at the same concentrations.
In mouse eggs the protein does not bind to the plasma membrane, but is instead located in the cytoplasm associated with vesicles where pip2 is located.
Al Edwards asked whether antibody reagents for PLCz1 would be useful, specifically: 1) Have human knockout cells been made? Answer: mouse KO has been made, the colony is frozen down and mothballed. There is the same in a group in Japan. What about actual sperm from someone with the knockout - apparently many people who are infertile have PLCz1 knockout. (Interesting comment). Commercial antibodies have not performed well. 2) Would purified PLCz1 be a useful reagent that could make fertilisation more effective? Answer: yes. Has been tried, and the roadblock is the stability of the isolated protein, and dose calibration. There is a patent on PLCz1 held by Cardiff that runs out about now.
Madison Edwards gave a protein update. Trying to secure slides. Existing approaches with NusA and His6-MBP tags not working well. Alternative needed. Karl tried a lot with bacteria, not baculoviruses.
If we can find a promiscuous inhibitor, we can set up a nanoBRET assay for in-cell target engagement. Works on lysates.
Assays: (now at #1 ) it was suggested that we reach out to John Sondek at UNC. Avanti lipids sell a fluorogenic probe. Already used in screen of inhibitors vs other PLCs. Karl's current assay in eggs (endpoint assay) can do 100 compounds a year, which will be v useful.
Did not discuss: 1) where should we look for the best focussed libraries of relevance, given the nearest neighbour proteins are dissimilar. 2) How does the PLCz1 get into the egg?
Nuvisan gave an update on their current thinking about this target. They have an interest in this area.