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

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
In this study by Dong and colleagues, the role of endothelial Epsin-1 in the development of 
atherosclerosis was investigated. The authors identified an interaction of Epsin-1 with the 
endoplasmatic reticulum protein IP3R1, which regulates calcium release in the cytosol. Using 
isolated endothelial cells, the authors found that binding of Epsin-1 to IP3R1 led to a proteolytic 
degradation of IP3R1, which was mediated by the proteasome. Mapping studies using recombinant 
constructs with deleted domains pinpointed the interaction sites to the ubiquitin-interacting motif 
in Epsin-1 and the suppressor domain in IP3R1. Subsequent experiments revealed that 
ubiquitination of IP3R1 precedes Epsin-1 binding and degradation. The absence of Epsin-1 (and -2) 
maintained calcium homeostasis in endothelial cells treated with oxidized lipoproteins. In addition, 
whereas wild-type endothelial cells showed a strong upregulation of adhesion molecules after 
stimulation with TNFα, Epsin-deficiency showed a suppression of this upregulation after TNF-
stimulation. After this extensive in vitro characterization, mice with genetic deletions of endothelial 
Epsin-1 and -2 and of endothelial IP3R1 (or combinations thereof) were investigated in a diet-
induced model of atherosclerosis. Deletion or reduction of IP3R1 was found to accelerate 
atherosclerosis, while deletion of Epsins-1 and -2 led to a notable reduction in plaque formation. 
Deletion of a single IP3R1 allele on an Epsin-1 and -2 double endothelial knockout background 
partially restored atherosclerotic lesion formation to the wild-type situation. 
This is an interesting study, in which the role of the endothelium in the development of 
atherosclerosis is highlighted. Endothelium dysfunction, associated with disturbed intracellular 
calcium homeostasis, drives atherosclerotic plaque formation. This study links the Epsin-1-
mediated degradation of IP3R1 to endothelial dysfunction during plaque formation. Although of 
interest, a number of issues need to be addressed to increase impact and to resolve conceptual 
unclarities. 
 
1. Endothelial cells can be considered to be an integral part of the immune system, as they should 
be permissive to leukocyte (trans-)migration. Since the proteasomal targeting of IP3R1 by Epsin-1 
appear to be a physiologic function (of Epsin 1), how would general inflammation and the immune 
response be affected by (functional) Epsin-1/2 deficiency? This is a relevant question to this 
reviewer for 2 reasons: (i) The authors advocate the identified function of Epsin-1 to be a valuable 
drug target. (ii) The Epsin-1-mediated degradation of IP3R1 appears to occur during inflammation, 
irrespective of the trigger (oxidized lipoproteins, TNFα). A remark to the latter is that in animal 
models, it is unclear which is the trigger for IP3R1 ubiquitinylation, (diet-induced) hyperlipidemia, 
systemic inflammation, aberrant flow conditions, or all of those. 
The study would be strengthened if the authors could show and discuss whether: 
- Epsin-1 mediated endothelial dysfunction is a patho(physio)logic mechanism that relates more or 
less exclusively to vascular inflammation and atherogenesis. 
- Endothelial calcium homeostasis (e.g. artificially modulated by BAPTA-AM) closely relates to 
adhesion molecule expression and leukocyte trafficking, or whether a different downstream effect 
is responsible for this. 
- the increased influx of different subsets of inflammatory cells into the plaque is directly related to 
a supposed increased permissiveness of the endothelium when Epsins are present (can be tested 
by transmigration through different genotypes of cultured cells). 
 
2. A further question that arose during reading the manuscript is related to flow conditions. 
Although already touched down upon later in the manuscript (using cultured human endothelial 
cells under physiologic and pathologic flow conditions), it would be interesting to expand these 
experiments to those performed with the cultured mouse cells. The study would be conceptually 
coherent when aberrant flow conditions would lead to similar Epsin-1-directed degradation of 
IP3R1 as triggering with cytokines or oxidized lipoproteins. The authors might also relate IP3R1 
expression to the different regions of the aorta or carotid artery (e.g. inner vs outer curvature and 
abdominal, or carotid bifurcation). 

 
3. Endothelial IP3R1 is ubiquitinylated under "abnormal" conditions. Do the authors have any 
mechanistic explanation why IP3R1 expression and calcium homeostasis should be altered under 
these conditions? And how this occurs? 
 
Other remarks: 
As the study was done on a double Epsin-1/2 background, please discuss the implications for 
human disease more elaborately (where both epsins are present). Similar counts for the different 
IP3 receptors. 
Please more clearly indicate the statistical significance between groups in figure 6i. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
Dong, Chen and colleagues studied the role for Epsin 1 and 2 in atherosclerosis. They report that 
Epsin1 interacts with the inositol 1,4,5-trisphosphate receptor type I (IP3R1), only under 
atherogenic conditions (following stimulation with high doses of oxLDL, Cholesterol or 7-KC). They 
showed that the interaction is Ubiquitin-dependent and occurs between the UIM domain of Epsin 1 
and the suppressor domain of IP3R1. Some of the sites of Ub on IP3R1 were mapped to K126, 129 
and 143. They claimed that such interaction triggers proteasomal degradation of the channel. The 
rest of the manuscript (Fig. 4, 5 and 6) reports calcium levels and atherosclerosis phenotypes in 
Epsin or IP3R1 KO mice but is rather phenomenological and could just be circumstantial (affecting 
Ca2+ levels will have many pleiotropic effects). 
 
The interaction of Epsin1 and IP3R1 and the degradation of the latter under atherogenic conditions 
is new and interesting. However, the interpretation of the data for the molecular mechanism is 
very flawed and unlikely to be true. The data do not support the conclusion that such degradation 
is mediated by the proteasome. The authors need to do a rigorous investigation of the mechanism 
and provide direct evidence and they need to rule out alternative hypotheses. 
 
1) IP3R1 is a 6-pass transmembrane protein. How could the proteasome extract the 
transmembrane parts to degrade it? Transmembrane proteins are degraded in the lysosome 
(following endocytosis or autophagy), not by the proteasome. 
 
The only evidence the authors got to support proteasome degradation is the use of MG132. 
 
But this is not an evidence of proteasomal degradation. It is well known that blocking the 
proteasome by MG132 (or by any proteasomal drugs such as ALLN, Lactcystin, Bombesin etc..) 
induces several compensatory effects: it triggers unfolded protein response (UPR), which activates 
autophagy (reviewed 10 years ago in PubMed ID (PMID)20040365 and more recently in 
PMID30333975), and it decreases the availability of free Ub (thus indirectly slowing down 
endosomal sorting and lysosomal degradation). 
In addition some of the drugs may have side effects: MG132 was reported to affect transcription 
(PMID30647455 amongst others) and ALLN is also a Cathepsin inhibitor and thus will block 
Lysosomal degradation as well (PMID8087844). 
 
Compensatory autophagy following MG132 (or other proteasome inhibitors) is mediated by the 
IRE1 arm of the UPR (IRE1 etc..) and JNK1, which phosphorylates Bcl-2, thereby disrupting 
autophagy-inhibitory interaction with Beclin-1 (reviewed in PMID20040365). 
Interestingly, IP3R1 interacts with Bcl-2 (PMID15613488 and PMID19706527) and is known to be 
involved in autophagy (PMID30251688, PMID22082873, PMID23565295, PMID28254579). 
Moreover, Epsin is involved in autophagy in flies (PMID19305132). 
 
Furthermore, Ubiquitination of IP3R1 was found to be as much K48 (proteasome+autophagy) as 

K63 (Lysosomes), with K63 found to accumulates most rapidly least 40% of Ub was Mono-Ub 
(PMID18955483), which suggests an autophagic and/or lysosomal degradation, the proteasome 
needs poly-Ub) 
 
Finally, Lysosomal inhibition (NH4Cl and Chloroquine) blocked IP3R1 degradation (PMID9139693, 
despite what the authors of this paper said: Fig. 7 clearly showed that IP3R1 was at 100% of 
control levels upon AngII). 
 
Thus, the authors must rule out alternative hypotheses such as autophagy, ER-phagy and 
Lysosomal degradation. 
 
- The role for autophagy can be easily ruled out by depleting Atg5, Atg7 and p62, the use of 
autophagy inducers and inhibitors (serum starvation, rapamycin, resveratrol, 3MA and 
chloroquine). 
- Lysosomal degradation can be inhibited by Bafilomycin (more specific than NH4CL) and 
Leupeptides or other Cathepsin inhibitors. 
- UPR can be tested by GPR78 and phospho-eIF2alpha levels and inhibited by Salubrinal, IRE1a 
and ATF4 KD. 
Both the OxLDL and the MG132 effects should be tested upon autophagy, lysosomal and UPR 
inhibitions. 
 
 
The authors also ought to back up their claim that IP3R1 is degraded by the proteasome by 
providing direct evidence and mechanistic insights. How do Epsin actually link IP3R1 to the 
proteasome? Etc.. 
 
 
 
 
 
 
 
2) Fig. 1c, 1e, 1g, 3c, 3h and 3j: it is not clear that Ub is actually on IP3R1 (its Mw did not 
change). How did the authors rule out that the Ub is not on another protein being co-IP? I 
understand the K to R mutations, but this is on ectopically expressed proteins. 
 
3) Fig. 3j is apparent contradiction with Fig.3a: no Epsin1 and 2 increase upon oxLDL. Unless this 
is an issue with alignment Epsin1 and 2 blots on 3a? 
 
4) Reference 28 does not support line 98-100 (it is about Ca2+, not stress) 
 
 
 
Reviewer #3: 
Remarks to the Author: 
This is a study which makes the novel observation that the endocytic adaptor proteins epsin are 
linked to the ER Ca2+ channel IP3R1 in endothelial cells and that this attachment is related to 
IP3R1 degaradation by the Ub/proteasome pathway. The authors show that perturbation of this 
Ca2+ signaling pathway affects the susceptibility of endothelium to atherogenic stimuli. The 
expriments are a nice combination of cell culture and animal models. The results are well 
organized and the experiments are performed in a thorough manner. In my opinion, the basic 
manuscript is worthy of publication in Nature communications and should be of interest to a wide 
audience. Despite my generally positive view, I do have a number of specific concerns that the 
authors should be encouraged to address. These are detailed below: 
 

1) The authors provide data using mutant constructs that the site of interaction with IP3R1 is the 
suppressor domain and that interaction involves 3 lysines in this domain at K126,K129,K143. 
Wojcikiewicz and coworkers have characterized the Ub binding sites in all 3 IP3R isoforms and 
none of the sites were in the N-terminal portion of the IP3R. Although different experimental 
systems were used, the authors should reference these studies and provide an explanation for the 
discrepancies. Wojcikiewicz et al have also shown that the IP3Rs are ubiquitinated with both K48 
and K63 chains. Have the authors looked to see what kind of chains are being formed on the SD? 
 
2) Previous studies by the Wojcikiewicz lab have identified the adaptor proteins erlin1 &2 and the 
ring-finger E3 ligase RNF170 as being key to the ER associated degradation of IP3Rs. How are the 
epsin proteins integrated into this system? Are they mediating a completely separate degradation 
mechanism or are they alternative adaptors that plug in to the erlin/RNF170 complexes? 
 
3) As the authors state in their discussion the endothelial cells have all 3 IP3R isoforms which 
contribute to the Ca2+ signal and presumably all 3 isoforms are downregulated in response to 
oxLDL or 7-KC. Yet, the emphasis of the paper is on IP3R1 and loss of just the IP3R1 gene or 
restoration of one allele of IP3R1 is sufficient to increase/lower the number of plaques seen in the 
animal models fed a WD diet. Does this mean that IP3R2 and IP3R3 have no role in the endothelial 
cells and IP3R1 has some selective effect. How is the Ca2+ signal effected in MAECs derived from 
these animal models? 
 
4) Although the source of the animal models are mentioned in the supplementary table of 
materials, the origin of the IP3R1 floxed mice is not clear. 
 
5) Which specific endocytic proteins are being referred to on p6L91? Is this info in the 
supplementary Table? 
 
6) The authors state that there are SNPs in IP3R1 linked to increase risk of cardiovascular disease. 
They go on to suggest that these result in “gain or loss of function”. What is the evidence for this 
functional measurements and are the SNPs in the coding sequence of IP3R1? 
 
7) The panel (f) in Figure 2 is not well described in the text. If this panel uses just the receptor’s 
NTD and RD then presumably the cartoon hosing these constructs in (g) should precede (f). Things 
may be clearer if the construct # in the cartoon is also used in the labeling of (h). 
 
8) In supplemental Figure 7 the abbreviation “N-glyc” is not defined. What is the purpose of 
showing the PTMs and why is this shown for only aa1-53? It is unclear why the lysine 129 position 
is highlighted but not the other potential Ub sites. 
 
9) The series of experiments involving identifying interacting sites use HEK293T cells. The legend 
to Fig 3 includes a sentence to indicate that the experiments involved a 30min pretreatment with 
oxLDL to induce the Ub of the IP3R1 or constructs. The sentence should be included in the main 
text. It is not clear from the epxeriments that the treatment with oxLDL is absolutely necessary to 
see Ub of IP3R1 or to observe the interaction with the epsins since none of the experiments have a 
control from which the oxLDL treatment has been omitted. It should be noted that Wojcikiewicz 
and coworkers have proposed that the Ub/proteasome pathway may play a role in the basal 
turnover of IP3Rs.