The MAL Protein, an Integral Component of Specialized Membranes, in Normal Cells and Cancer
Round 1
Reviewer 1 Report
In this review article Rubio-Ramos, Labat-de-Hoz, Correas & Alonso present a comprehensive overview of the current knowledge of the trans-membrane protein MAL. The review is well organised. The role of MAL in organisation of high-density packing of lipids in membranes is well described and linked to the known functional roles of MAL in membrane trafficking in specialized cells, such as kidney epithelial cells and oligodendrocytes. The discussion of the role of MAL in the uptake of clostridium epsilon toxin via caveolae is interesting and may be applicable to other toxins. The deregulation of MAL expression in various types of cancer is interesting and may potentially be harnessed as a prognostic or diagnostic tool in the future. The review will be of interest to readers of both the membrane trafficking field as well as for researchers working with clinical data sets. The manuscript is written in an excellent manner with informative figures and tables included.
Minor comment:
Line 817: there is one typo ‘sinceeg’ instead of ‘since eg.’
Author Response
Page 25, line 866: the typo has been corrected.
Reviewer 2 Report
Rubio-Ramos and colleagues have written a comprehensive review about the MAL protein, a four transmembrane protein expressed in human T cells, polarized epithelial cells and myelin forming cells. They describe MAL as belonging to the proteolipid proteins which partitions exclusively to detergent-insoluble membranes. They suggest that this protein has functions in specialized membranes in the cells where it is expressed. They provide extensive evidence that MAL is active in organization of condensed membranes. Moreover, they point out that the MAL gene is hypermethylated in several cancers which results in loss of MAL expression and indicates a potential role of MAL as a tumor suppressor. The authors have extensively studied this protein and describe their results in numerous, which makes them highly qualified to give an overview about the biology of MAL which is complex and incompletely understood.
There are several points that should be addressed:
Major points:
- The authors mention their own work which indicates that in human T cells MAL colocalizes with Lck an non-receptor tyrosine kinase which has an essential role in T cell receptor signaling and T cell activation. This work implicated MAL as a transporter of Lck and they reported that MAL is required for the correct assembly of the immunological synapse (IS). A recent study has demonstrated that MAL is downregulated during T cell activation and differentiation. Importantly the authors of this study found that MAL was dispensable for membrane localization of Lck in a T cell line and that knockout of MAL did neither significantly impair T cell activation nor the localization of Lck in the immunological synapse (Leitner et al. Eur. J. Immunol. 2021; DOI: 10.1002/eji.202048603). The results of this study which suggest a more subtle role of MAL in T cell function should be discussed in light of their data. A non-essential role of MAL in Lck transport and T cell activation is also supported by the fact that MAL is not expressed in mouse T cells and that CRISPR-screen for genes which play and important role in T cell activation yielded high rankings for Lck but not MAL.
- The authors should carefully check the legend of figure 2 - something seems to be wrong in the assignment of the panels.
Minor points:
check line 194 – something seems to be missing there.
Line 298: The authors write the CD28 and ICOS have different ligands – this is not completely accurate since human (but not mouse) ICOSL also weakly binds to CD28
Check lines 446-447: This sentence needs attention.
There seems to be a lot of wrong spacings e.g lines 311, 436, 467, 526
Author Response
Point 1. Response. We have added in page 10, lines 336-341, the following sentence: “In a recent study based on the use of a new mAb (MT3), generated to CD3-coprecipitated complexes, that stains the T-cell surface only when MAL is expressed, it was suggested that MAL is involved in T cell differentiation but not in Lck transport [100]. However, since biochemical evidence showing that mAb MT3 recognizes MAL are lacking, the possibility cannot discounted that mAb MT3 recognizes a protein whose transport to the cell surface is dependent on MAL, or a CD3 complex-associated epitope that is unmasked only in the presence of MAL.”
Point 2. Response: We have corrected the order of the panels in the legend of figure 2.
Point 3. Response. We have completed the sentence in page 6, line 205, by adding “Ib”.
Point 4. Response: We have modified the sentence (now in page 10, line 314) by removing “CD28 and ICOS have different ligands”.
Point 5. Response: We have rewritten the entire sentence. The modification, which is on page 14, lines 474-477, of the revised manuscript now reads: : “In mice, influenza virus infection impairs the development of the fetal immune system and causes downregulation of the expression of the MAL gene in developing fetal thymus [138]. However, since MAL is not expressed either in adult thymus or peripheral T cells in mice [45], it is not clear that this effect is related to the observed defects in immune system development.”
Point 6: Response: We have removed the incorrect spacings throughout the manuscript.
Reviewer 3 Report
In this review manuscript, authors discuss the roles of MAL gene in specialized membrane trafficking and signaling pathways in polarized epithelial cell,s T lymphocytes, myelin-forming cells and cancer cells. Furthermore, epigenetic regulation of MAL genes was discussed.
The authors provided comprehensive explanation on the function of MAL gene that would help scientists working in this field.
Some suggestions
In table 2, answer, yes, in the column of immuno-histochemical analysis of MAL is not clear. It would helpful if the levels of MAL are increased or not.
The same goes for the column of 'effect of exogenous expression of MAL on tumor cell functions'. It would be helpful if clearer explanation on the effects are described.
In table 1, offering references in the table would be helpful.
Author Response
Point 1. Response: Page 23, line 798: The top row of Table 2 has been modified to make clearer the changes observed in in the levels of MAL in cancer cells and the effect of exogenous MAL expression on characteristic tumor cell functions.
Point 2. Response: The references are now indicated in the text.
Reviewer 4 Report
This manuscript is a very well-written, very interesting, and updated review on MAL protein, from a pioneering group devoted for many years to the research of the MAL protein and cellular mechanism the protein is involved with.
There are only two minor comments:
Figure 2 legend (or figure 2 panels): change C for D (and D for C)
Lines 509-510 and related figure 3B (this comment is just a suggestion): although the molecular mechanism of action of botulinum and tetanus toxin are different from those of epsilon toxin, the suggestion both kinds of clostridial toxins target SNARE and MAL proteins respectively, is appropriate. However, it should be emphasized that both kinds of clostridial toxins (neurotoxins and epsilon toxin) have a different cellular mechanism of action and are involved in different membrane traffic compartments (for instance, there is no evidence that MAL, as the putative receptor of epsilon toxin, is located at the nerve terminals, the main target of botulinum and tetanus toxin).
Author Response
Point 1. Response: We have corrected the order of the panels in the legend of figure 2.
Point 2. Response: We have added the following sentence on page 16, lines 555-557: “Despite this parallelism, it should be noted that the botulinum and tetanus neurotoxins, which act on nerve terminals, have a different mechanism of action and are involved in different membrane trafficking compartments from those of ETX”.
