This review resulted from the graduate-level course "How to Read and Evaluate Scientific Papers and Preprints" from the University of São Paulo, which aimed to provide students with the opportunity to review scientific articles, develop critical and constructive discussions on the endless frontiers of knowledge, and understand the peer review process.
Reviewer Team
Lucas Athayde https://orcid.org/0000-0003-4033-2686
Rosangela Silva Santos https://orcid.org/0000-0003-3546-2873
Sara Ventura https://orcid.org/0000-0003-4238-4471
Summary
Employing a variety of methodological approaches including structural modeling, phylogenetics, gene silencing, and mass spectrometry, the authors investigate 5 HEDH activity in A549 cells, an enzyme associated with lipid dehydrogenation that leads to 5-KETE formation. After confirming 5-HEDH activity through detection of its reversible oxidation products, 5-KETE and 5-HETE, the study proceeds with a gene-level investigation and screening for potential microsomal dehydrogenases involved in this lipid peroxidation. Additionally, the authors provide insights that may extend to other types of cells, as evidenced by functional studies in zebrafish.
The authors uncover the role of DHRS7 in redox signaling, demonstrating its NADPH-dependent regulation of 5-KETE production. Unlike classical redox signaling based on thiol oxidation, they present DHRS7 as a redox-regulated enzyme that acts in 5-KETE biosynthesis, which is a highly novel and interesting mechanism. Under stress conditions, DHRS7 facilitates rapid inflammatory signaling and leukocyte recruitment through the 5-KETE pathway.
Major Comments
Identification of DHRS7 as a 5-HEDH Candidate
The screening successfully identified DHRS7 as a candidate enzyme with 5 HEDH activity. LC-MS profiling of wild-type cell lines revealed increased 5-KETE levels in cells exposed to 5-HEDE and vice versa, with a stronger effect observed in 5-KETE accumulation. Surprisingly, 5-KETE production appeared to be favored under non-stress conditions. We would like to see a description of the data normalization to exclude the possibility that these differences arise from variations in cell viability, abundance, or any other experimental conditions.
Alternative Pathways and Metabolic Compensation
Another observation is the reduction in 5-KETE and 5-HETE levels in DHRS7 knockout cells, corroborating the proposed DHRS7 role. But the persistence of these metabolites in silenced cells even at lower concentrations is curious, which leads to a few hypotheses such as the activation of endogenous compensatory mechanisms and exogenous uptake of these metabolites from the media. To further investigate the origin of these residual metabolite levels, it is essential to quantify 5-KETE and 5-HETE in the culture medium to test if the cells' knockouts exposure to these lipids could explain these residual levels.
To evaluate if these residual metabolites could have originated from endogenous mechanisms, a transcriptome analysis could be used to identify other redox-active enzymes differentially expressed that could exhibit potential 5-HEDH activity thereby compensating DHRS7 silencing.
Subcellular Localization of DHRS7
The authors employed GFP-tagged DHRS7 constructs for subcellular localization and genetic complementation assays in DHRS7 KO A549 cells and zebrafish Dhrs7 KO models. This strategy successfully restored the wild-type phenotype, as evidenced by increased 5-HETE and 5-KETE levels upon treatment with precursors, corroborating the initial hypothesis of DRS7 as a 5-HEDH. However, the conclusion that DHRS7 localizes to microsomes lacks experimental validation, as no microsomal markers were used in confocal imaging. Indeed, cellular fractionation and Western blot assays indicate DHRS7 also localized in the nucleus, granules, and microsomes, but GFP fluorescence was absent in the nucleus. Given that the antibody used for Western blotting was available, it’s not clear why the authors did not employ an anti-DHRS7 antibody for immunofluorescence to precisely determine its intracellular distribution rather than relying on GFP-based localization assays.
Kinetic Characterization of DHRS7 Activity
The authors conducted enzymatic kinetics assays for DHRS7 and applied a Michaelis-Menten model for data fitting. However, the fitting shows clear deviations, with a notably low coefficient of determination (R²), suggesting that this model may not adequately describe DHRS7’s catalytic properties. In fact, a typical Michaelian model does not appear to fit the data distribution. To ensure that these discrepancies are not due to experimental artifacts, we recommend repeat the experiments performing additional replicates. Furthermore, considering the probable reversibility of 5-KETE conversion, alternative kinetic models that account for this factor should be explored.
Minor Comments
● Lines 13-15: The authors state, "As the DHRS7 (but not DHRS3) knockdown effect was consistent with 5-HEDH activity, we tested whether DHRS7 also promoted 5-KETE reduction. This was the case." However, Figure S1D shows a reduction in 5-HETE, not 5-KETE. More clarification is needed.
● Cell Line Justification: The authors discuss DHRS7 expression in various contexts but do not justify their choice of A549 cells. Since none of the cited references mention DHRS7 expression in this line, a description of the rationale for model choice is necessary.
● Figure 1E: We recommend a dose-time response curve for DOX treatment to monitor DHRS7 expression levels. This would determine if DHRS7 expression remains at physiological levels or becomes supraphysiological, thereby ensuring that subsequent treatments also occur under physiological conditions.
● Figure 1F: We recommend a more clear description that these are cells lacking the DHRS7 construct to enhance reader understanding.
● Figure 2B: H₂O₂ is not a direct intermediate in the 5-HETE/5-KETE pathway, though it may indirectly influence it by activating 5-LOX, altering redox balance (NADP⁺/NADPH), and contributing to ferroptosis. The statement "Lipid peroxidation stimulates 5-KETE production through DHRS7" would be more robust if 5-LOX activity was assessed. We suggest a validation experiment: Treat DOX-induced DHRS7-expressing cells with H₂O₂ and measure markers of lipid peroxidation, such as malondialdehyde (MDA), isoprostanes, or 4-hydroxy-2-nonenal (4-HNE). This would confirm lipid peroxidation’s role in 5-KETE production.
● Figure 3D: Overexpression methodology in HEK cells is unclear. The knockout was achieved using CRISPR/Cas9, not shRNA. More clarification is needed on whether overexpression was introduced via a separate construct post-knockout, whether a CRISPR activation (CRISPRa) or DOX-inducible system was used, or if overexpression was performed in a different cell line.
● Figure 4A: The heatmap visualization of metabolite concentrations was clear, but cluster analysis would provide more information related to the significance of observed alterations.
The authors declare that they have no competing interests.
The authors declare that they used generative AI to come up with new ideas for their review.
Correction
The authors that the use of Artificial Inteligence was done solely for corrections in the english language but not for idea generation.
The author of this comment declares that they have no competing interests.