The Structural–Functional Crosstalk of the Calsequestrin System: Insights and Pathological Implications

Round 1

Reviewer 1 Report

The review “The structural-functional crosstalk of the Calsequestrin system: insights and pathological implications” summarizes and analyzes data on the structural rearrangements of skeletal and heart isoforms of Calsequestrin (CASQ1 and 2) depending on the ionic microenvironment, mutations, and posttranslational modifications of certain residues, which, in turn, may change the Ca2+-binding properties in a way leading to cardiac pathologies. With all due respect to Prof. Silvia G Priori, who has made a great contribution to understanding the mechanisms of cardiac pathologies, we are forced to admit that the review in its present form is not very informative. Moreover, the complexity of the language does not match the simplicity of the reported ideas.

Major concerns

1.     Among the works cited by the authors, only 15 were published in the last 5 years. Is this due to the relatively slow progress in this area or due to the fact that the main data on the problem were obtained earlier? Is it possible to insert a paragraph summarizing most resent data on the problem?

2.     The authors very sparingly provide quantitative values of important characteristic parameters, which makes it difficult for the reader to assess the validity of their statements.

3.     In the present form, The Chapter 5 “Pathological implications of CASQs defects” rather touches (without detailed characteristics) on certain changes in the properties of proteins, but says practically nothing about “Pathological implications” of these changes. The same is true for the Table 1 (designated as Table 2).

4.     Authors state: “Two released crystal structures of a CASQ filament beautifully fit this hypothesis: in both cases, a solvent-accessible tunnel traverses the length of the linear polymer [28, 90] and is lined by several Ca2+-binding carboxylates. However, the very low pH conditions of the first one, published in 2020, may pose caution on its relevance as a Ca2+-bound state [28], whereas the detailed analysis of the second one (deposited with PDB ID: 7F05) must await its publication [90]”. It is not clear, what information is absent that precludes the detailed analysis?

 Minor points

1.     Some information seem missed: “In healthy cells, cardiac and 42 skeletal CASQ store about 50-75% of the SR Ca2+ content respectively [7]…”

2.     Stylistically questionable or controversial statements: “Still, dissecting the direct effect of CASQ conformational dynamics on the Ca2+-release channel versus the indirect effects in the striated muscle remains a technically challenging question”; “Structurally speaking, JNT and TRDN…”; “CRUs devoided of CASQ…”; “The Intrinsically Disordered portion is evidenced”; Phosphorylation at either T353 by Casein Kinase 2 [49, 58] or at S248 and S369 by Fam20C [19] amplifies the Ca2+-dependent properties…”; “the highly variable penetrance of inherited CASQ2 mutations [28,72], has recently led to the hypothesis…”; “The rich physico-chemical nature of CASQ”.

3.     Table 1 (designated as Table 2) seems not very convenient since columns related to CASQ1 and CASQ2 are mixed, which interferes with analysis.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

This is a review of the calsequestrin (CASQ) family of intralumenal Ca2+ buffers. It gives a detailed description of the molecular architecture and polymerisation of CASQ monomers and regulates Ca2+ release through ryanodine receptor (RyR) Ca2+ channels. The review then describes how post-translational modifications and mutations can alter CASQ architecture and function. Two novel aspects of this work are: i) categorisation of CASQ1 and CASQ2 mutations in terms of their effect on dimerisation and polymerisation; and ii) revisting the concept of CASQ polymers as resembling polyelectrolyte systems ("molecular wires"). Overall this review is written and presented to a very high standard. The language is clear and effective. The figures contribute to understanding of the concepts presented. It is likely to appeal to a broad readership, particularly to those interested in Ca2+ signalling and in macromolecular assemblies. Consequently, I thoroughly recommend publication of this work in MDPI Biomolecules. However, considering the following points should slightly increase the scope, reach and quality of this review:

MAJOR POINTS

1) It might be worth briefly mentioning the histidine rich calcium binding protein (HRC) as another structurally distinct, but functionally related SR intralumenal Ca2+ buffer? Like CASQs, this protein is regulated by phosphorylation by Fam20C and also interacts with triadin to regulate RyR-mediated Ca2+ release.

2) The role of protons in regulating CASQ architecture is described in Section 6.2. It might be useful to put this into physiological context. For example, does intralumenal pH alter during systole/diastole or in skeletal muscle excitation-contraction coupling? (I appreciate that there is lack of consensus on this (summarised in lines 607-608), but a little more detail might help a reader understand this point).

MINOR POINTS

1) Figure 1. The description of jSR curvature in the proximity of open RyRs (lines 59-61) is unnecessary and distracting.

2) Lines 187-189. It may not be clear to all readers that the HEK293 cell-line is a heterologous expression system (ie. it does not contain detectable levels of endogenous CASQ proteins).

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Authors addressed all my concerns.