Review of Comparative proximity biotinylation produces an inventory of RAB18-interactions and implicates RAB18 in cholesterol mobilization
Creators: name: Anonymous
Publisher: name: Zenodo
Year: 2020

description: Summary As a possible path to better understand and develop treatments for Warburg Micro Syndrome (WMS), the authors have investigated the networks of protein-protein interactions involving genes mutated in this rare genetic disease. The goals of the work are to identify new proteins involved in the pathophysiology of the disease and to better understand the molecular and cellular effects of disease-causing mutations. The data will likely be of interest to researchers studying WMS and RAB18, the protein focused on here, but reviewers expressed some concerns about the validation and interpretation of the presented protein interaction data. Reviewer #1 This manuscript used proximity biotinylation to discriminate functional RAB18 interactions. The authors provide some evidence for several of the interactors and some functional data supporting a role for RAB18 in modulating cholesterol mobilization. <strong>Major concerns and questions:</strong> 1) Based on the spectral counts, the author calculated a mutant:WT ratios as a readout to identify nucleotide-binding-dependent effectors. But it is important to show that WT protein and mutant protein have similar expression level to begin with. And the intracellular localization of the mutation and WT should also be determined. Do they show the similar intracellular localization? 2) The ratio of mutation:WT is useful to remove some background. But this may omit some very highly interacting proteins just because their fold change is low. The converse is true for rare proteins. It would be better to have a list of candidate effectors based on the absolute counts. 3) Sec22A knockdown will change the morphology of lipid droplets. A knockdown efficiency test and some representative fluorescence images here would make this data more compelling. 4) Same comment for the cholesterol mobilization experiment. Expression level of the protein is needed. Figure 7A is rather confusing, as the Gln67Leu mutation already has higher CE before loading HDL. Why is this this? Better uptake or reduced efflux? What is the de novo cholesterol synthesis activity in this cell line? Reviewer #2 This study used WT and mutant RAB18 to look for interacting proteins in normal and GEF-deficient cells. A catalog of interactions that are regulated by nucleotide binding and/or GEF activity were uncovered. Among identified proteins, there are known/established ones and there are some new ones. Initial validation was carried out for some newly identified effectors such as TMCO4 and Sec22A. <strong>Major concerns and questions:</strong> 1) While the addition of new RAB18 effectors is useful to researchers who are interested in RAB18, the overall conclusion that RAB18 may regulate membrane contacts and lipid metabolism is not new. 2) Figure 7: the effect of RAB18 on cholesterol esterification and efflux may arise from multiple causes. This set of experiments do not provide any real insights into RAB18's role in cholesterol metabolism. 3) Given RAB18's interaction with ORP2, TMEM24 and OCRL, perhaps the authors may examine plasma membrane PIP2. The results would be more specific and novel. Reviewer #3 This study by Kiss and colleagues reports the findings of proximity biotinylation experiments for the discovery of novel RAB18 effectors. The authors perform careful proteomic analysis that appears well-controlled and successful in recapitulating known interactions. That small GTPase interactions can be identified with this approach has been previously demonstrated, though the application of this approach to RAB18 is novel and of interest to the field. A number of intriguing findings with potentially important implications are reported. However, this manuscript has several weaknesses. <strong>Major concerns and questions:</strong> 1) As the authors report, proximity biotinylation may not reflect direct protein-protein interactions but simply colocalization of bait and prey proteins. A true protein-protein interaction ideally would be further supported by ancillary experiments such as in vitro binding or co-immunoprecipitation, including an assessment of whether the interaction is affected by the GTP- or GDP-bound state. While co-IP in WT and GEF-deficient cells was performed for 1 candidate interactor (TMC04, Figure 6C), protein-protein interactions were not tested for the other 2, with the latter relying on either repeat BioID (SPG20, Figure 3A) or reciprocal BioID (SEC22A, Figure 5B). 2) Putative RAB18 interactions may be affected by the BioID fusion itself or by heterologous expression. While it is reassuring that known interactors were detected with this approach, the conclusions would be better supported by testing the localization of the fusion protein in comparison to endogenous RAB18, and/or by rescue of a phenotype associated with RAB18-deficiency. 3) Conclusions about the dependence of RAB18 interactions on its GTP or GDP-bound state rely on differences observed in cells with deficiency of RAB18 GEFs. It is certainly possible however that RAB3GAP may serve as a GEF for other GTPases, or have other functions, that cause the observed differences in labeling. The conclusions would be strengthened by additional experiments showing a direct effect - e.g. reproducing the disrupted labeling of candidate effectors with a GDP-locked RAB18 point mutant, or showing that RAB3GAP deficiency reduces binding of a candidate effector to RAB18. 4) The putative role of SEC22A in regulating lipid droplet morphology relies on siRNA perturbations that are prone to off-target effects. This is especially concerning given the high degree of sequence similarity between SEC22A and SEC22B, the latter of which has a known role in regulating LD morphology. Rescue of this phenotype with a siRNA-resistant SEC22A cDNA would rule out this possibility. 5) The finding of SPG20 protein abundance being affected by RAB18-deficiency relies on immunofluorescence with an antibody exhibiting cross-reactivity. While the authors do attempt to adjust for this non-specific background fluorescence, this conclusion would be strengthened by immunoblotting for a change in abundance of the specific band corresponding to SPG20. If confirmed, measurement of SPG20 transcripts levels would also help clarify the level of regulation for the altered protein abundance. 6) The influence of stable expression of a RAB18 GTP-locked point mutant on cholesterol metabolism is intriguing but the experimentation appears perfunctory. For 14C-CE cellular levels in 14C-oleate-loaded cells (Figure 7A), the most striking difference is the greatly enhanced synthesis level of CE at t=0. Is the subsequent drop due to an effect of RAB18 on efflux, or simply a consequence of the higher starting level at t=0? For efflux assays on 3H-cholesterol-loaded cells (Figure 7B), the data is only presented as a ratio of 3H activity in media relative to lysates after a 5 hr incubation with HDL. Interpretation of these results would be aided by a more detailed analysis. How does 3H-cholesterol uptake compare after 24 hr incubation but prior to addition of HDL (t=0)? After the 5 hr HDL chase, are the differences in the ratio driven by an increase in extracellular activity, a decrease in intracellular activity, or both? Ultimately these conclusions would be better supported by a more detailed analysis. Does disruption of the candidate effectors phenocopy the effect of RAB18 disruption? Are any known mediators of cholesterol efflux affected by RAB18 disruption? While a comprehensive mechanism may be reasonably considered beyond the scope of this paper, some additional descriptive analysis would be useful in interpreting these findings.