Peer review process
Revised: This Reviewed Preprint has been revised by the authors in response to the previous round of peer review; the eLife assessment and the public reviews have been updated where necessary by the editors and peer reviewers.
Read more about eLife’s peer review process.Editors
- Reviewing EditorRoel NusseStanford University, Stanford, United States of America
- Senior EditorUtpal BanerjeeUniversity of California, Los Angeles, Los Angeles, United States of America
Reviewer #1 (Public Review):
This manuscript presents a model in which combined action of the transporter-like protein DISP and the sheddases ADAM10/17 promote shedding of a mono-cholesteroylated Sonic Hedgehog (SHH) species following cleavage of palmitate from the dually lipidated precursor ligand. The authors propose that this leads to transfer of the cholesterol-modified SHH to HDL for solubilization. The minimal requirement for SHH release by this mechanism is proposed to be the covalently linked cholesterol modification because DISP could promote transfer of a cholesteroylated mCherry reporter protein to serum HDL. The authors used an in vitro system to demonstrate dependency on DISP/SCUBE2 for release of the cholesterol modified ligand. These results confirm previously published results from other groups (PMC3387659 and PMC3682496).
A strength of the work is the use of a bicistronic SHH-Hhat system to consistently generate dually-lipidated ligand to determine the quantity and lipidation status of SHH released into cell culture media.
Key shortcomings include the unusual normalization strategies used for many experiments and the lack of quantification/statistical analyses for several experiments. Due to these omissions, it is difficult to conclude that the data justify the conclusions. The significance of the data provided is overstated because many of the presented experiments confirm/support previously published work. The study provides a modest advance in understanding of the complex issue of SHH membrane extraction.
Reviewer #2 (Public Review):
Ehring et al. analyze contributions of Dispatched, Scube2, serum lipoproteins and Sonic Hedgehog lipid modifications to the generation of different Shh release forms. Hedgehog proteins are anchored in cellular membranes by N-terminal palmitate and C-terminal cholesterol modifications, yet spread through tissues and are released into the circulation. How Hedgehog proteins can be released, and in which form, remains controversial. The authors systematically dissect contributions of several previously identified factors, and present evidence that Disp, Scube2 and lipoproteins concertedly act to release a novel Shh variant that is cholesterol-modified but not palmitoylated. The results provide new insights into the function of Disp and Scube2 in Hedgehog release. The findings concerning the function of lipoproteins and cholesterol in Hedgehog release are largely confirmatory (PMID 23554573, 20685986). However, in light of the multitude of competing models for Hedgehog release, the present study is a valuable contribution that provides further insights into the relevance of lipoproteins in this process.
A novel and surprising finding of the present study is the differential removal of Shh N- or C-terminal lipid anchors depending on the presence of HDL and/or Disp. In particular, the identification of a non-palmitoylated but cholesterol-modified Shh variant that associates with lipoproteins is potentially important. The authors use RP-HPLC and defined controls to assess the properties of processed Shh forms, but their precise molecular identity remains to be defined. A caveat is the strong reliance on over-expression of Shh in a single cell line. The authors detect Shh variants that are released independently of Disp and Scube2 in secretion assays, which however are excluded from interpretation as experimental artifacts. Thus, it would be important to demonstrate key findings in cells that secrete Shh endogenously.