In Silico Analysis of Protein–Protein Interactions of Putative Endoplasmic Reticulum Metallopeptidase 1 in Schizosaccharomyces pombe

Round 1

Reviewer 1 Report

Authors can further clearly establish the linkage of the erm1/ERMP1 in S. pombe to humans and how it’s an appropriate inference to pathogenesis in humans. That is still very unclear in the present state of the manuscript.

Was there a prior in vitro study that led to the identification of the erm1 gene in S. pombe? and emphasized its importance to disease development in humans?

Was there any other method that was used to improve the structure of the model giving it fell below the acceptable threshold on a Ramachandran plot for a quality model? e.g. structural minimisation? Also, authors should show other results from the structural validation as assertions made from a poor-quality model structure may be incorrect.  z-DOPE score is also an important criterion to assess the energy minima of the modeled conformation. Moreover, was the quality of the AlphaFold structure validated using the same tools used to validate other models? Additionally, MODELLER can be used to obtain good quality ‘structurally minimized’ models and to fill in the missing cleavage loop of interest instead of using models from different sources and algorithms that may not be comparable.

Are there any similarities or identities or conservations between the Amk2, Ypt5, Pex12, Oca8, Fis1, and Pmc1 predicted for S. pombe and in humans or how else did authors justify the inference of this PPI in humans? Do they correspond to any of the proteins in the human PPI network predicted in Figure 1?

Also, the authors should clarify how the template search was done. Perhaps databases like HHPred could help identify template structures with higher % identities.

For the docking section, authors should clarify how this was carried out. Was the docking site in the protein specifically defined in the docking parameters or the peptides were allowed to randomly bind to the Ermp1 M28 domain?

Authors can use LigPlot software to provide more details on the interaction mechanisms of the protein-peptide complexes, especially for residues involved in the formation of the complex.

Although there seem to be minor corrections to the English language, the article needs to be reworded to make it clearer and to convey the message more articulately making it less confusing.

Author Response

Introduction and discussion sections were written and revised. In particular, it has been demonstrated by functional complementation of mutants of S. pombe with human genes that genetic conservation between both species is very high. This model organism has been useful for the functional analysis of eukaryotic genes related to the cell cycle and the regulation of gene expression, as well as the study of biochemical aspects of genetic products such as the identification of domains related to catalytic activity and interaction with other proteins. Fortunately, S. pombe conserves the UPR signaling pathway, so in this microorganism proteotoxic stress in ER and its relationship with Ermp1 can be studied. In such a way that the most essential function of this type of proteases that has been conserved throughout millions of years of evolution can be identified. Because there is no precedent for the proteolytic characterization of human ERMP1 and other Fxna peptidases, the study of Ermp1 in a microorganism that is easily manipulated in the laboratory such as S. pombe could lay the foundations for characterization at the level of gene expression and the importance of functional conservation of the human-yeast M28 domain.

Reviewer 2 Report

I have a major concern: why only Biogrid?

this issue is not clarified...and given the different scenario in the different data bases of interactomics it should be at least discussed

No major problem

Author Response

Introduction and results sections were written and revised. There are databases that compile the PPIs reported in publications based on experimental evidence such as BIND, DIP, INTACT, MINT and BioGRID. BioGRID is an open-access database resource hosting gene and protein interactions from multiple species, including yeast, worm, fly, and human. All content is selected from experimental evidence reported in scientific publications, being the most complete repository. About 1.93 million of the reported interactions can be used to build interaction networks that facilitate biomedical research, particularly related to human health and diseases.

Reviewer 3 Report

Although the attempt to elucidate the possible mechanism of Metallopeptidase 1 could be interesting, unfortunately the paper does not convincingly explore this kind of aspect, and should be rigorously revised before its possible acceptance. Below my main concerns:

-Unfortunately performing this kind of study without study the dynamics of the proteins and of interactions has very limited sense. Authors should consider to perform additional computational studies for providing an overall in silico assessment. In particular the complexes should be subjected to MD simulation to analyze the dynamics of interactions found by docking. This is necessary considering that protein-protein docking is a challenging task, and providing only docking results without validating them with MD is not acceptable. Furthermore, from the dynamics, it will be possible to evaluate the binding energy/affinity for highlighting the different affinity of the selected protein with possible client proteins, considering that probably the Metallopeptidase 1 could have preferred client proteins.

Without this assessment the work is not complete and cannot be accepted. If the authors will provide further in silico studies as suggested I will be happy to reconsider my decision.

Moderate changes are requested

Author Response

Molecular dynamics studies were performed in Desmond to assess the stability of the interaction complexes: Ermp1-Pex12, Ermp1-Amk2 and Ermp1-Ypt5. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) was reported. the RMSD values for all systems exhibited reasonable fluctuations, ranging from 0.8 to 2.6 Å throughout the simulations. The interaction contacts within the complexes consistently exhibited a stable behavior, indicating that the binding stability of the analyzed proteins can be confidently regarded.

Round 2

Reviewer 1 Report

All comments raised have been responded to by the authors.

Author Response

All comments raised have been responded to by the authors.

Ready

Reviewer 2 Report

The subject of this paper is marginal. the bioinformatics analysis is routinary

Author Response

The authors addressed my main concerns related to MD simulation. Unfortunately, the authors did not provide a comprehensive analysis of the trajectory, considering that only the stability of the systems was evaluated. To establish and understand the affinity and mechanism, the authors should calculate the affinity of different client proteins using the entire MD trajectory, to provide a possible preferred binding of Emp1 with the selected client proteins. This calculation will add additional information regarding the topic discussed by the authors.

Answer

The objective of the study and therefore its conclusions were more clearly defined.-

Dynamic studies have been carried out focused on stability given the scope of the project, this manuscript presents results from the model, selection of possible target proteins of ERPM1, prediction of cutting sites and correlation with docking studies and analysis of the interaction site, were added dynamic studies in order to observe the stability of the protein-protein complex and thereby predict whether the time is sufficient to promote cleavage, the trajectory and mechanism analysis studies are interesting, but we consider that they are beyond the scope of this manuscript, given that what would be the next step with the selection of proteins would be an in vitro study, to first observe the proteolytic activity of ERPM1 and with the correlation with what was predicted, according to that continue with studies where we can explain in detail the activity and the mechanism, but in another phase of study.

Reviewer 3 Report

Authors addressed my main concerns related to the MD simulation. Unfortunately, authors did not provide a comprehensive analysis of the trajectory, considering that only the stability of the systems were evaluated. In order to establish and understand the affinity and the mechanism, authors should calculate the affinity of different client proteins using the entire MD trajectory, to provide a potential preferred binding of Emp1 with the selected client proteins. This calculation will add additional insight with respect to the topic treated by the authors.

minor editing should be performed

Author Response

The objective of the study and therefore its conclusions were more clearly defined.

The paper is an initial study to investigate interactions of Ermp1 with potential target proteins. Remembering that it is a putative protein and we do not have any background on its biological function or structural characterization. This is explained in the introduction of the article. Therefore, in this study the first in silico predictions of Ermp1 in S. pombe are reported.

The authors addressed my main concerns related to MD simulation. Unfortunately, the authors did not provide a comprehensive analysis of the trajectory, considering that only the stability of the systems was evaluated. To establish and understand the affinity and mechanism, the authors should calculate the affinity of different client proteins using the entire MD trajectory, to provide a possible preferred binding of Emp1 with the selected client proteins. This calculation will add additional information regarding the topic discussed by the authors.

Answer

Dynamic studies have been carried out focused on stability given the scope of the project, this manuscript presents results from the model, selection of possible target proteins of ERPM1, prediction of cutting sites and correlation with docking studies and analysis of the interaction site, were added dynamic studies in order to observe the stability of the protein-protein complex and thereby predict whether the time is sufficient to promote cleavage, the trajectory and mechanism analysis studies are interesting, but we consider that they are beyond the scope of this manuscript, given that what would be the next step with the selection of proteins would be an in vitro study, to first observe the proteolytic activity of ERPM1 and with the correlation with what was predicted, according to that continue with studies where we can explain in detail the activity and the mechanism, but in another phase of study.

Round 3

Reviewer 3 Report

Authors addressed the concerns. In my opinion after the revision done the manuscript can be published.

minor