Reactive Sterol Electrophiles: Mechanisms of Formation and Reactions with Proteins and Amino Acid Nucleophiles^†

Round 1

Reviewer 1 Report

This is an excellent review!

I found numerous typo errors that may be due to the converision of the word format into pdf

I found some minor errors 

Line 129, “-substituted” something is missing;

Line 171, what does BDEs mean?

Line 205,”-fragmentation” something is missing?

Line 214: “epoxide ratio” alpha: beta?

Line 226,227: “-epoxide” something is missing?

Figure 10a: “7-DHC 5,6-epoxide” greek characters are missing;

Figure 10b: “-tocopherol”, alpha-tocopherol?

Line 364: “-alkyl” Number is missing

Line 392: “-epoxide”. Number is missing

Line 399: “microsomal”. It is not a microsomal enzyme but a soluble one.

Line 401: “5,6-epoxide”. Only the 5,6alpha isomer of 5,6epoxide of cholesterol was shown to be glutathionylated. The 5,6beta isomer was unreactive. see Watabe T et al, BBRC , 1979 whether 7DHCep are substrate of this enzyme is currently unknown.

Line 428: “-6 fatty”. Number is missing

Line 464,465, “A peptide.” is something missing?

Figure 14: Could you separate a little bit more CH2 from the double bond?

Line 544: what does ALS mean?

line 399 the rat GST-B is not a microsomal enzyme but a cytosolic enzyme

line 401: only the 5,6alpha isomer of 5,6 epoxide of cholesterol was shown to be glutathionylated. The 5,6beta isomer was unreactive. This was done on purified enzymes from rat liver. see Watabe T et al, BBRC , 1979. Whether or not the human GSTA1 catalyzes the same reaction than the rat GST-B is currently unknown. It is not known either that the 5alpha,6alpha -epoxycholesterol product of glutathionylation exists as a metabolite in mammalian tissues. The steroselectivity of the reaction of glutathionylation suggests that this is not a general mechanism of epoxysterols conjugation.

Author Response

We thank the reviewer for the thorough examination of the manuscript.  All corrections suggested were made.  They were indeed caused by the conversion of the original manuscript to the editorial copy.

Reviewer 2 Report

In the abstract, the statement "The single lipid present in the highest concentration in most membranes is cholesterol..." needs to be documented with details and references in the text. Furthermore, it is misleading because cholesterol is not the focus of the review. Rather, it is the precursor, 7-DHC, that is the primary biologically relevant molecule.

line 84: "The     -unsaturated" should be "The α,β-unsaturated"

line 91: remove inappropriate carriage return and provide literature citation for the claim "highest concentrations"

line 115: delete "yet"

p.3, Figure 1: show AY994 inhibition of DHCR7

line 123: insert Δ

line 129: insert greek symbol

lines 186-187: force M^- to next line

lines 226, 227, 230: insert α

line 231: give reference after "mammalian tissues"

line 264: "6" should be "7"

line 274: insert "(Figure 7B) " after "A and B"

line 277: "7" should be "7-"

line 287: "7" should be "7-"

line 293: "was" should be "is" 3x

line 309: "(k )" should be "(k_p)"

Figure 10, panel A: should have "7-DHC 5β,6β-epoxide"

Figure 10, panel B: 2 alphas missing

Figure 10, legend: "ectamgle" should be "rectangle"

line 364: should be "β-alkyl"

line 377: "but large" should be "but a large"

line 392: should be "from α- or β-epoxide"

line 428: should be "ω-fatty"

lines 464 and 465: should be "the Abeta peptide"

Figure 15 legend: "alkynyllLipid" should be "alkynyl lipid"

lines 521 and 529: units are corrupt, e.g., "100  M" and "20.  M"

line 550: "structure and" should b "structure, and"

Figure 16, legend: should be "beads. Beads"

Figure 17, legend: should be "beads. Beads"

I have a major problem with the statement "imine reduction with borohydride or cyanoborohydride has been used to stabilize the adducts", e.g., on page 15. Referring to figure 12 it is a concern to me that adduct formation, that may be insignificant at equilibrium, could be driven to "significant levels" by hydride reduction driving the equilibrium to produce adduct. At the very least this confounding factor should be acknowledged. Preferably the possibility should be ruled out or shown to be minimal and insignificant. Otherwise, the results of such experiments should not be trusted.

Author Response

We thank the reviewer for his thorough reading of the manuscript.  We have made the minor changes to the text as suggested. We note that many of these formatting errors were introduced in the editorial process. Two recommendations required more substantial editing.  The first concerned comments about cholesterol being the single compound present in the highest concentration in most cells.  We take the reviewers point and we've re-written that sentence to mention specific tissues, ie. brain, liver, CNS.  The second comment is a thoughtful one.  It concerns the question of whether we're looking at adducts under thermodynamic or kinetic control based on the reduction of imines by borohydride.  We also take this point and we've added a caveat to the text.