Peer review of: Epsin-mediated degradation of IP3R1 fuels atherosclerosis (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The authors have sufficiently revised the manuscript and I have no further comments. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
My main concern was the absence of direct evidence for the molecular mechanism mediating the 
degradation of a multi-pass channel (IP3R1) by the proteasome. The only evidence linking the 
degradation to the receptor was the use of proteasome inhibitors, but these have many pleiotropic 
effects, including Ub availability and indirect effects on autophagy and lysosomal degradation. 
 
I appreciate the effort by the authors to address some of my concerns. Unfortunately, the new 
experiments on Figure S3 are not rigorous as they lack basic controls to check that their 
treatments (Leupeptin and salubrinal) actually worked, and so are inconclusive at this stage. 
 
Likewise, the ULK KO only blocks general autophagy (although here too, no controls were done), 
not selective ER-phagy, see PMID31100386 for a recent review. So this experiment did not rule 
out ER-phagy and the concern still remains. 
 
The new experiment finding that is Epins bind to the Erlin1-2 and RNF170 complex is a good 
addition to the paper but is not a proof that IP3R1 is degraded by the proteasome. It is just the 
identification of an E3 ligase that may ubiquitinate it (the importance of RNF170 was only inferred, 
was not tested in the present manuscript). 
 
The argument by the authors that the there is a precedent of the IP3R1 to be degraded by the 
proteasome during the ERAD response is very weak for 2 reasons: 
1. the ERAD response handles misfolded or damages protein within the lumen of the ER, followed 
by retro-translocation into the cytosol and then ubiquitin-dependent degradation by the 
proteasome. How this applies to a mature, fully folded and membrane embedded receptor (IP3R1) 
upon recognition by cytosolic proteins (Epsins), is totally unclear to me. 
 
2. the papers cited by the authors (Lu JP et al JBC 2011) and the rest of the literature on ERAD-
mediated degradation of IP3R1 (PMID12421829, 16103111, 10839985, 19751772 etc.) suffers 
from exactly the same flaws than the present study as the only evidence ever linking the 
degradation of IP3R1 to the proteasome was the use of MG132, ALLN etc.. thus the concern this is 
an indirect effect blocking another mechanism is still valid. 
 
In conclusion, I am sorry to say that the authors have not convinced me much more. I appreciate 
their effort to address my comments but their experiments either did not address some of my 
comments or were not rigorous enough to rule out other explanations or provide direct evidence 
for the molecular mechanism. 
 
However, my disagreement concerns only a small part of the study and it should not distract us 
from the quality and novelty of the rest of the manuscript. I do not wish to block the publication of 
the study but, in absence of absence of direct evidence or clear molecular mechanism, I encourage 
the authors to consider revising their claim about IP3R1 being degraded by the proteasome in this 
set up. Perhaps being more nuanced about the possibility of alternative mechanisms (ER-phagy 
etc.) would be wise at this stage. 
 
 
 

Reviewer #3: 
Remarks to the Author: 
The authors have addressed my comments with regard to a) K48/63 sites of Ub linkage; b) role of 
RNF170; c) role of other IP3R isoforms and d) role of other IP3R isoforms. They have also cleared 
up a number of minor points that I raised regarding the text. 
 
I do not the authors have adequately dealt with my point that the SD is the site of Ub being 
different from what is known from proteomic analysis of Ub attachment sites in IP3R1. The authors 
response to reviewers makes the valid point that this could just be due to the use of a different 
cell type. This should be insetted as a sentence in the paper. 
 
Some other comments 
 
1) Wojcikiewicz has proposed that the Ub/proteasomal degradation of IP3Rs can be viewed as a 
response of the cell to chronic elevation of IP3. Do the atherogenic stimuli like oxLDL increase IP3? 
 
2) It would be useful to show the coomassie gel lane for the IgG control used in the mass-spec 
studies. Similarly, are the list of proteins included for the controls in the supplementary table 2? 
 
3) It is made clear at the end of the dicussion that the epsin is in the PM and the IP3R is in the ER. 
However, the consequences of this different localization of the proteins is not discussed in relation 
to the data or the model. Are the events being studied by the authors occurring at contact points 
between the PM and ER. 
 
4) The discussion on p16 is repetitive and can be shortened.