LEM-Domain-Containing Inner Nuclear Membrane Proteins: Emerging Regulators of Intranuclear Signaling
_Fu.png)
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis review summarized the functions of representative LEM-D proteins, including LAP2, emerin, and MAN1, on their regulations of intranuclear signaling pathways, and they discussed how these proteins regulate the activity of transcription factors involved in Hedgehog, Wnt/β-catenin, STAT3, Notch, and transforming growth factor-β (TGF-β) signaling at the inner nuclear membrane and controlling their access to chromatin. It is interesting and fit the journals’ requirements. But there are several comments need to be answered.
Comments:
- Most of the data of the transcription factors controlled by the LEM-D proteins are from studies of muscle cells or connected to muscle diseases. Are there other studies on different tissues or different disease model?
- Are there any structure studies on the LEM-D proteins or their specific domains, please summarize.
- As different regulators such as LAP2, emerin, or MAN1 can control different transcription factors, and the signaling pathways such as Hedgehog, Wnt, etc. are different in different cell types, please summarize the regulation network in a temporal or spatial regulation especially maybe different in different tissues.
English writing need to be improved.
Author Response
Manuscript ID: ijms-4081893
Manuscript title: LEM-Domain–Containing Inner Nuclear Membrane Proteins: Emerging Regulators of Intranuclear signaling
We sincerely thank the reviewers for their careful evaluation of our manuscript for the previous title
“The nuclear inner membrane proteins shared LAP2–emerin–MAN1-domain (LEM-D); newly discovered regulators of intranuclear signaling”
and for their constructive and insightful comments.
We have carefully addressed all points raised and revised the manuscript accordingly.
All changes are reflected in the revised version, and the corresponding sections are indicated below.
Reviewer 1
Comment 1: Most of the data of the transcription factors controlled by the LEM-D proteins are from studies of muscle cells or connected to muscle diseases. Are there other studies on different tissues or different disease model?
Response: We appreciate this important point. We agree that earlier studies on LEM-D proteins have been strongly enriched in muscle-related systems, largely due to the pronounced phenotypes observed in mechanically active tissues. To address this concern, we have revised the manuscript to explicitly acknowledge this bias and to highlight evidence from non-muscle contexts where available.
We added a paragraph in the Introduction summarizing non-muscle contexts (e.g., cancer-related dysregulation and neural differentiation contexts for LAP2; cardiac involvement in emerin-linked disease; and developmental/vascular roles of MAN1 and BMP/TGF-β outputs). We also highlight that pathway consequences can differ by tissue due to differences in mechanical environment and signaling networks.
Specifically, we have expanded the discussion to emphasize that LEM-D proteins are ubiquitously expressed and that emerging studies suggest broader, tissue-dependent regulatory roles beyond muscle. We also clarified that the predominance of muscle-related data reflects current research emphasis rather than biological exclusivity.
Revisions Made
- Section 1 (Introduction) – expanded tissue-context paragraph;
Added / revised sentences:
“Although a substantial portion of mechanistic evidence linking LEM-D proteins to transcriptional regulation has been generated in skeletal muscle models, accumulating studies support broader, tissue-dependent roles of INM proteins. In particular, LAP2 isoforms have been implicated in regulatory programs beyond muscle, including neural differentiation and cancer-associated dysregulation. Likewise, emerin-linked diseases frequently display prominent cardiac involvement, and MAN1 has been linked to developmental and vascular contexts through BMP/TGF-β–dependent signaling outputs. These examples underscore that downstream consequences of INM-mediated transcription-factor gating can diverge across tissues depending on lineage-specific transcriptional programs, mechanical environment, and available signaling networks.”
-Section 6 (Conclusion , paragraphs 3–4) – reinforced cross-tissue relevance.
Added / revised sentences:
“These insights also provide a mechanistic framework for understanding how defects in nuclear envelope proteins can lead to tissue-specific pathologies despite their ubiquitous expression.”
Comment 2: Are there any structure studies on the LEM-D proteins or their specific domains, please summarize.
Response: We thank the reviewer for this valuable suggestion. In response, we strengthened a concise structural biology summary focusing on domain-level evidence that supports functional interactions. Because full-length INM proteins are challenging to resolve structurally, we summarize available structures/biophysical studies that define key interaction modules, including the conserved LEM motif, LAP2α dimerization, the lamin A/C–BAF–emerin interface, and the MAN1 C-terminal RRM-like/UHM region that mediates SMAD recruitment.
Revisions Made
-Section 1 (Introduction) – added/expanded structural-summary paragraph;
Added / revised sentences:
“At the molecular level, the LEM motif itself has been structurally characterized, supporting its role as a conserved interaction module shared among multiple INM proteins. For LAP2, the structural basis of dimerization has been resolved, providing insight into how LAP2 may assemble and scaffold regulatory complexes within the nucleoplasm. In emerin-associated assemblies, structural analysis of the lamin A/C–BAF–emerin ternary complex has identified specific interfaces, disruption of which has been linked to autosomal recessive progeroid disease. In the case of MAN1, detailed structural and biophysical studies have defined the Smad–MAN1 interaction at the INM. Nuclear magnetic resonance analyses indicate that the C-terminal RRM-like region characterized as a U2AF homology motif, which is adapted for protein–protein interactions through a hydrophobic pocket that recruits SMAD proteins. Collectively, these structural data are consistent with the concept that LEM-D proteins act as organized platforms integrating nuclear architecture with signal-dependent transcriptional control.”
-Figure 1. – context retained as domain-architecture overview.
-Section 4 (MAN1 and Regulators of Intranuclear Signaling) –
Expanded discussion of MAN1–Smad structural interactions and their functional implications.
Comment 3: As different regulators such as LAP2, emerin, or MAN1 can control different transcription factors, and the signaling pathways such as Hedgehog, Wnt, etc. are different in different cell types, please summarize the regulation network in a temporal or spatial regulation especially maybe different in different tissues.
Response: We agree and have strengthened the spatiotemporal and tissue-context framework. We now explicitly describe LEM-D proteins as spatial gatekeepers that retain signaling regulators at the inner nuclear membrane (INM) and, in a signal- and PTM-dependent manner, permit their release and chromatin engagement. We added/updated Figure 2 as an integrative schematic summarizing LAP2-, emerin-, and MAN1-centered nodes (Hedgehog/GLI1, Wnt/β-catenin, STAT3, Notch, TGF-β/Smads) and we expanded the Conclusion to emphasize that tissue-specific pathway utilization and mechanical context shape distinct outputs.
Revisions Made
- Section 5.4 and Section 6 (Conclusion) – reinforced spatiotemporal/tissue-dependent integration language.
Added / revised sentences:
Section 5.4 – “Collectively, these findings support a unifying concept in which LEM-domain proteins act as central organizers that couple nuclear architecture to intranuclear signaling pathways governing muscle development, regeneration, and disease. Rather than acting through single linear pathways, LAP2, emerin, and MAN1 integrate multiple signaling inputs at the nuclear envelope, shaping transcriptional outputs in a tissue- and context-dependent manner. Understanding how LEM-domain proteins coordinate intranuclear signaling networks provides a conceptual basis for future mechanistic and translational studies in muscle-wasting diseases. Targeting nuclear envelope–associated signaling regulators, including STAT3-, Wnt-, Notch-, and Smad-dependent pathways, may offer new strategies to restore balanced transcriptional control and improve muscle function.”
Conclusion (paragraphs 2–3) – “While many studies have focused on muscle-related phenotypes, the regulatory principles governed by LEM-domain proteins are not restricted to a single tissue. Instead, the downstream consequences of LEM-domain–mediated signaling diverge across tissues depending on pathway utilization, transcription factor availability, and mechanical context. In skeletal muscle, emerin-dependent coordination of Notch and Wnt signaling contributes to myogenic progression, whereas STAT3 exemplifies context-dependent signaling by promoting muscle atrophy in disease settings while functioning as an oncogene in non-muscular cells. These examples underscore the broader relevance of spatiotemporal gating at the inner nuclear membrane. Mechanistically, individual LEM-domain proteins regulate distinct but convergent signaling pathways: LAP2 modulates transcriptional activity through dynamic interactions with factors such as GLI1 and β-catenin, emerin spatially restrains signaling mediators including STAT3 and the Notch intracellular domain, and MAN1 fine-tunes TGF–Smad signaling at the nuclear periphery.”
Comment on the Quality of English Language : The English writing needs to be improved.
Response: We appreciate this comment. The entire manuscript has undergone careful language revision to improve clarity, conciseness, and readability. Redundant descriptions were condensed, and overly repetitive explanations—particularly in the LAP2 section—were streamlined.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis review article addresses an important topic by positioning LEM-domain proteins as regulators of intranuclear signaling. One clear strength appears in the Introduction, where the authors define LEM-domain proteins and link nuclear architecture to transcriptional control. This section sets a solid foundation and uses appropriate references to establish context.
The section on LAP2 is informative but overly long. For example, the discussion of GLI1 regulation in Section 2 repeats the same core idea across several paragraphs, namely that LAP2 retains GLI1 at the inner nuclear membrane in an acetylation-dependent manner. This mechanism is clear early on, and later descriptions add limited new insight. Condensing this material would improve focus and readability.
In the emerin section, the discussion of STAT3 signaling is one of the strongest parts of the manuscript. The authors clearly describe how emerin restricts STAT3 at the nuclear membrane and link this mechanism to muscle homeostasis. However, when the text connects this regulation to disease onset, especially in Emery–Dreifuss muscular dystrophy, the language sometimes implies direct causation. The cited studies support association rather than proof of causality, and this distinction should be stated more clearly.
The MAN1 section provides a solid overview of TGF-β and Smad signaling. The description of Smad2 and Smad3 sequestration at the nuclear envelope is clear, but the role of the RRM domain is mentioned without enough explanation. A brief clarification of its functional relevance would strengthen this section.
The reference list is comprehensive and current, which is a strong point. The manuscript is scientifically sound, but it would benefit from reduced repetition, clearer limits on disease claims, and tighter language throughout.
Author Response
Journal: International Journal of Molecular Sciences (IJMS)
Manuscript ID: ijms-4081893
Manuscript title: LEM-Domain–Containing Inner Nuclear Membrane Proteins: Emerging Regulators of Intranuclear signaling
Response to Reviewers
We sincerely thank the reviewers for their careful evaluation of our manuscript for the previous title
“The nuclear inner membrane proteins shared LAP2–emerin–MAN1-domain (LEM-D); newly discovered regulators of intranuclear signaling”
and for their constructive and insightful comments.
We have carefully addressed all points raised and revised the manuscript accordingly.
All changes are reflected in the revised version, and the corresponding sections are indicated below.
Reviewer 2
Comment 1: The section on LAP2 is informative but overly long. Condensing this material would improve focus and readability.
Response: We agree with this assessment. The LAP2 section was carefully condensed by removing repetitive descriptions of the GLI1 acetylation-dependent retention mechanism while retaining the core mechanistic insights.
Revisions Made
-Section 2 (LAP2 Isoforms…) – streamlined the GLI1/Hedgehog discussion. Repetitive descriptions were merged, and the GLI1 regulatory mechanism is now described more succinctly without loss of conceptual clarity.
Revised sentences:
“LAP2β associates with GLI1 and HDAC1. Acetylated GLI1 is preferentially retained at the INM, whereas HDAC1-mediated deacetylation promotes GLI1 release from the INM and facilitates Hedgehog target-gene activation. This acetylation-dependent gating mechanism underscores how LAP2 can spatially regulate intranuclear signaling outputs”.
Comment 2: In the emerin section, the language sometimes implies direct causation when connecting regulation to disease onset; the cited studies support association rather than proof of causality. Please state this distinction more clearly.
Response: We appreciate this important clarification. We have revised the language throughout the emerin section to avoid overstatement of causality and to clearly distinguish mechanistic regulation from disease association. The revised wording now explicitly notes that current evidence supports an association between altered emerin–STAT3 regulation and EDMD-related phenotypes, while a direct causal link to disease onset is not yet established.
Revisions Made
-Section 3 (Emerin…) and Section 5.2 (Emerin and Nuclear Envelope …)– Language has been carefully revised to emphasize association and mechanistic contribution rather than direct causation.
Revised sentences:
Section 3 – “Experimental evidence indicates that emerin attenuates STAT3 signaling by retaining STAT3 at the INM, thereby delaying its access to chromatin and transcriptional activation. This regulatory mechanism has been implicated in skeletal muscle homeostasis and suggests a potential point of intervention in conditions associated with aberrant STAT3 activity. Importantly, available data supports an association between altered emerin–STAT3 regulation and EDMD-related phenotypes, while a direct causal link to disease onset has not yet been established.”
Section 5.2– “These regulatory effects are particularly relevant during myogenesis where the temporal balance between Notch and Wnt signaling orchestrates the progression of muscle precursor cells along the myogenic lineage. Altered STAT3 activity associated with emerin perturbation may correlate with changes in Pax7 expression and enhanced proliferation during myogenic differentiation. These observations suggest that emerin-dependent modulation of STAT3 signaling may influence muscle stem cell behavior, without constituting a direct causal mechanism for disease onset”
Comment 3: The MAN1 section provides a solid overview of TGF-β and Smad signaling, but the role of the RRM domain is mentioned without enough explanation. Please clarify its functional relevance.
Response: We thank the reviewer for highlighting this point. We have expanded the explanation of the MAN1 C-terminal “RRM-like” region. We now clarify that this region is more accurately described as a U2AF homology motif (UHM), which mediates protein–protein interactions rather than canonical RNA binding. We also explain how its hydrophobic pocket recruits SMAD proteins to support sequestration at the nuclear periphery and thereby antagonize TGF-β/Smad signaling.
Revisions Made
-Section 4 (MAN1…) – expanded the RRM/UHM paragraph to clarify functional relevance for SMAD recruitment and spatial sequestration.
Added / revised sentences:
“The protein architecture further includes a carboxyl-terminal RNA recognition motif (RRM), underscoring the structural complexity of MAN1 and its capacity to interact with multiple nuclear factors. Structural studies using nuclear magnetic resonance have provided critical insight into the molecular basis of MAN1–Smad interactions. These analyses revealed that the C-terminal RRM of MAN1 is more accurately classified as a U2AF homology motif (UHM), rather than a classical RNA-binding RRM. This UHM is specifically optimized for protein–protein interactions by forming a hydrophobic pocket that recruits SMAD proteins, thereby antagonizing TGF-β signaling at the nuclear periphery. This structural specialization enables MAN1 to function as a spatial regulator rather than a simple transcriptional repressor.”
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors answered all the questions and comments, the revised version is good to publish.
