There is a paper, which made me very upset and angry!
Huang W., Ghisletti S., Saijo K., Gandhi M., Aouadi M., Tesz G.J., Zhang D.X., Yao J., Czech M.P., Goode B.L., Rosenfeld M.G., Glass C.K.
Coronin 2A mediates actin-dependent de-repression of inflammatory response genes.
Nature, 2011 Feb 17;470(7334):414-8
Toll-like receptors (TLRs) function as initiators of inflammation through their ability to sense pathogen-associated molecular patterns and products of tissue damage. Transcriptional activation of many TLR-responsive genes requires an initial de-repression step in which nuclear receptor co-repressor (NCoR) complexes are actively removed from the promoters of target genes to relieve basal repression. Ligand-dependent SUMOylation of liver X receptors (LXRs) has been found to suppress TLR4-induced transcription potently by preventing the NCoR clearance step, but the underlying mechanisms remain enigmatic. Here we provide evidence that coronin 2A (CORO2A), a component of the NCoR complex of previously unknown function, mediates TLR-induced NCoR turnover by a mechanism involving interaction with oligomeric nuclear actin. SUMOylated LXRs block NCoR turnover by binding to a conserved SUMO2/SUMO3-interaction motif in CORO2A and preventing actin recruitment. Intriguingly, the LXR transrepression pathway can itself be inactivated by inflammatory signals that induce calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ)-dependent phosphorylation of LXRs, leading to their deSUMOylation by the SUMO protease SENP3 and release from CORO2A. These findings uncover a CORO2A-actin-dependent mechanism for the de-repression of inflammatory response genes that can be differentially regulated by phosphorylation and by nuclear receptor signalling pathways that control immunity and homeostasis. [PMID: 21331046]
Below are my comments to some of their data:
- Fig.1b: no control for cytoplasmic staining - I could get similar image for any protein by just moving up the focus plane
- No mutant protein stability estimate - I bet that their delta N mutant is not stable. Not sure about SIMmut, K11A/R13A; which might be fine.
- Fig.5b: use IP Actin to show the actin binding activity - Never seen it in the literature! In the buffer with EGTA, all actin filaments are disassembled. If it is such a robust G-actin binding protein, why is it not in cytoplasmic?
- Why actin is recruited to the promoters? Again, based on their experimental details, it is G-actin binds to the promoters. But they claim a mechanism involving interaction with oligomeric nuclear actin.
- Why using 10 uM NEM in their buffer for co-IP and ChIP? NEM, a blocker of vesicular transport, reacts with cysteine containing proteins? Since all vesicles were broken in the buffer with NP-40, NEM will reacting with all Cysteine containing proteins in the lysate? Is the result reliable?
- No comments on NCoR clearance, SUMO, LXR, CaMKII, etc. I am not an expert of those experiments.
Because of the limitation of 140 characters in a tweet, the actually posted comments are:
PMID: 21331046 @NatureNews: No comments on NCoR clearance, SUMO, LXR, CaMKII, etc. I am not an expert of those experiments.
PMID: 21331046 @NatureNews: ... not block vesicular transport. NEM will react with all Cysteines on proteins in lysate! Are results reliable?
PMID: 21331046 @NatureNews: Why using 10 uM NEM in the buffer for co-IP and ChIP? Since all vesicles were broken in the buffer with NP-40...
PMID: 21331046 @NatureNews: Based on exp details, it is G-actin binds to promoters. But the claim is a mechanism involving oligomeric actin
PMID: 21331046 @NatureNews: In buffer with EGTA, all actin filaments are disassembled. Such robust G-actin binding, why not in cytoplasmic?
PMID: 21331046 @NatureNews: Fig.5b - use IP Actin to show actin binding activity - Never seen in literatures! With EGTA, filaments all gone.
PMID: 21331046 @NatureNews: No protein stability estimate - I bet their delta N mutant is not stable. Not sure about SIMmut, K11A/R13A
PMID: 21331046 @NatureNews: Fig.1b no control for cytoplasmic staining - I could get the image for any protein by moving up the focus plane
New in 'Experiment'
- Haploid stem cells fertilize oocytes to generate live mice
- How to use ImageJ to quantify fluorescent distribution?
- Two fluorescence protein from Atsushi Miyawaki group
- Use ImageJ macro to facilitate and automate image processing
- Spot detection from noise images: averaging, filtering, fitting
- Batch extract PubMed ID from EndNote database, and else...