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  1. Evaluation Summary:

    Wayne Mitchell et al. report the study aimed to determine the structural features of cationic hydrophobic tetrapeptides in their cytoprotective efficacy. Detailed structural characterization of the peptides "free" in solution and bound to membranes is followed by their comparison in protecting cells from starvation-induced stress and the loss of viability. Overall, there are important and detailed observations regarding the peptide-membrane interactions, while their relevance to cytoprotection mechanisms in not demonstrated.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  2. Reviewer #2 (Public Review):

    Studies on improving mitochondrial function are clinically significant as many diseases and overall cellular metabolism is linked to mitochondrial health. C-terminal amidated synthetic tetrapeptides with an alternative cation and aromatic residues are being shown to improve mitochondrial health. These peptides can accumulate on the mitochondrial membrane and appear to improve electron transfer efficiency with a concomitant increase in ATP production and reduction in ROS production. The lead peptide SS-31( arg-Dmt-Lys-Phe-NH2 ) has been shown to improve mitochondrial health in disease conditions such as hypoxia, ischemia, and aging-related disorders. The unusual Dmt (2,6 dimethyltyrosine) at the second position of the tetrapeptide is shown to have a characteristic free radical quenching property. The peptide SS-31 appears to be a promising target for various mitochondrial disorders.

    In this study, Mitchell et al. tested the three best tetrapeptides with alternative basic/aromatic amino acid sequence setting SS-31 as a benchmark to understand the importance of side-chain and type of aromatic side chains role in the structure and binding to the membrane using extensive NMR, biophysical and MD simulation studies either in solution and in the membrane-bound state. Among the tested SS-20, SPN4 and SPN10 show similar or better features than known SS-31 peptides. The study depicts the importance of polar groups on aromatic side chains. In particular SPN10 decreases, the enthalpy of membrane interactions and ability reduce membrane surface potential. All the tested peptides were shown to be targeted to mitochondria, increasing the viability and ATP content in cell lines. In particular, SPN10 appears to have a more significant impact on the recovery from stress by improving mitochondrial health. These studies, in my view, would be important for the development of therapeutics based on SPN10.
    Broadly, the experimental design and the results support their conclusions. More discussion on how do these peptides improve mitochondrial health may be included.
    The idea of tetrapeptides binding to liposomes and calculating lipid to peptide binding ratios is quite good. However, it will make more sense to show their binding to the isolated mitochondria and compare the binding ratios of tetrapeptides to mitochondria.

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  3. Reviewer #1 (Public Review):

    Wayne Mitchell et al. report the study aimed to determine the structural features of cationic hydrophobic tetrapeptides in their cytoprotective efficacy. Detailed structural characterization of the peptides "free" in solution and bound to membranes is followed by their comparison in protecting cells from starvation-induced stress and the loss of viability. Overall, there are important and detailed observations regarding the peptide-membrane interactions, while their relevance to cytoprotection mechanisms in not demonstrated.

    Strengths:
    i. The authors performed and described detailed experimental and computational analyses of the structure of the peptides in solutions and in model membranes. The data obtained provide important insights into the nature of the interaction between the peptides and model membranes.
    ii. The use of isothermal titration calorimetry allowed the authors to investigate the equilibria of binding of the tetrapeptides to model membranes containing cardiolipin.
    iii. The observation of the reverse-turn conformation for all peptides except SS-31, when bound to membranes is interesting, even though its significance in the cytoprotection mechanisms is not clear.
    iv. An intriguing conclusion from the structural studies is the potential to improve membrane binding of the peptides by preparing the cyclized forms.
    Weaknesses:
    i. The manuscript mostly focuses on the mechanisms of interaction of the peptides with membranes and this is a strength of the paper. There is no clear link between hose data and cytoprotection. My recommendation is to modify the title to better reflect the major focus and strength of the paper.
    ii. Quantitative structure activity relationship requires a correlation between quantities describing structural feature(s) of the compound and its efficiency/potency. It is not clear what structural feature of the tetrapeptide is most influential of its potency and what efficiency/potency parameter would best reflect its action.
    iii. Determination of the structural effects on the compound function requires modulation of a single structural property and assessment of its effect on the compound's activity. Therefore, SS-31 may be compared to SPN4, as the only change is the replacement of Dmt residue with tyrosine. SS-20 may be compared with SPN10, as both phenyl rings are replaced by indole rings. However, comparison between SS-31 and SS-20 introduces more variables (replacement of Dmt by phenylalanine, and change in amino acid order in the peptide), which makes the interpretation difficult. It is not clear why testing the role of amino acid order was not performed without changing the identities of aromatic amino acids.
    iv. The cellular uptake of the peptides was determined only for selected biotinylated peptides (SS-31 and SPN10) and only in a qualitative manner (immunostaining, to confirm the presence of the peptides in cells). Comparison of the uptake requires quantitative analyses for all four peptides.
    v. Cell protection experiments are lacking an important control - non-stressed cells. Without such control, it is difficult to interpret the observed cytoprotection data.

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