LIMD2 Human

What is the molecular structure of human LIMD2?

Human LIMD2 is a small protein (127 amino acids) characterized by the presence of LIM domains, which are defined by two adjacent cysteine-histidine-rich zinc fingers separated by a hydrophobic linker . The protein sequence includes specific motifs that enable protein-protein interactions and downstream signaling. The full-length human protein sequence is:

MGSMFQAAGAAQATPSHDAKGGGSSTV QRSKSFSLERAQVKETCAACQKTVYPMER LVADKLIFHNSCFCCKHCHTKLSLGSYAAL HGEFYCKPHFQQLFKSKGNYDEGFGRKQHK ELWAHKEVDPGTKTA

This structural arrangement enables LIMD2 to interact with various binding partners and participate in critical cellular functions. The protein's relatively small size combined with its interaction domains makes it an efficient mediator in signaling cascades.

What are the primary cellular functions of LIMD2?

LIMD2 primarily acts as an activator of the protein-kinase ILK (Integrin-linked kinase), thereby regulating cell motility and migration . The molecular function of LIMD2 depends on its binding to target proteins, through which it influences various cellular processes including:

• Control of gene transcription

• Cytoskeleton organization to regulate cell growth

• Cell motility and division

• Cell lineage specification

• Organ development

These functions explain why LIMD2 dysregulation is frequently associated with pathological conditions, particularly cancer progression and metastasis. Its ability to interface with multiple binding partners makes it a pivotal protein in cellular signaling networks.

What techniques are most effective for detecting LIMD2 expression in human samples?

Several methodologies have proven effective for detecting LIMD2 in human samples, each with specific applications:

• RT-qPCR (Reverse Transcription-Quantitative PCR): For mRNA expression analysis, using specific primers targeting LIMD2 transcripts . This method can quantify relative expression levels across different tissue samples.

• Western Blotting: For protein detection, using validated antibodies such as:

  • The antibody developed by Peng et al. (now commercialized by Merck as MABC1168) which recognizes full-length human LIMD2

  • Ab221110 which recognizes amino acid residues 105-126 (C-terminal region)

• Copy Number Analysis by qPCR: For determining gene amplification or deletion, using primers targeting LIMD2 genomic regions compared to reference genes like ACTB .

• Immunohistochemistry: For tissue localization in clinical samples, using specific antibodies visualized through appropriate detection systems.

When analyzing LIMD2 expression, researchers should consider using multiple detection methods for cross-validation, as each technique provides complementary information about expression at different biological levels.

How can researchers accurately quantify LIMD2 expression levels?

Accurate quantification of LIMD2 requires rigorous methodology and appropriate controls:

• For protein quantification: Western blotting followed by densitometry analysis using software such as ImageJ, normalizing to housekeeping proteins . Signals can be visualized using systems like Immobilon Western and captured with imaging equipment such as ImageQuant LAS 4000.

• For mRNA quantification: RT-qPCR using the 2^(-ΔΔCt) method with validated reference genes. In studies examining LIMD2 in cancer, GAPDH has been used successfully as a reference gene .

• For copy number analysis: qPCR-based approaches comparing amplification of LIMD2 to reference genes, with copy numbers below 0.5 defined as deleted and above 1.5 as amplified .

• For normalization: When comparing across samples, researchers should use appropriate normal tissues as controls. For example, peripheral blood mononuclear cells (PBMCs) have been used as controls in copy number studies .

What is the prognostic significance of LIMD2 in different cancer types?

Intriguingly, LIMD2 demonstrates cancer type-specific prognostic implications:

These contrasting roles suggest that LIMD2's function may be context-dependent and influenced by tissue-specific factors, tumor microenvironment, or different binding partners across cancer types.

How does LIMD2 affect cancer cell phenotypes at the cellular level?

LIMD2 influences multiple cancer-associated cellular behaviors:

• Cell Proliferation: Knockdown of LIMD2 in NSCLC cells causes remarkable decreases in cancer cell proliferation .

• Cell Migration: LIMD2 affects cell motility through its role as an activator of protein-kinase ILK . In vitro studies using wound-healing assays have demonstrated that LIMD2 knockdown reduces migration capacity in cancer cells .

• Cell Invasion: Transwell assays have shown that LIMD2 knockdown significantly decreases invasion potential in cancer cells .

• Metastasis Cascade: In papillary thyroid cancer, LIMD2 regulates key steps of the metastasis cascade, including processes involved in epithelial-mesenchymal transition (EMT) and genomic instability .

These findings collectively indicate that LIMD2 promotes an aggressive cancer phenotype, particularly in NSCLC and PTC, by enhancing cellular processes associated with tumor progression and metastasis.

What pathways and molecular mechanisms are influenced by LIMD2?

LIMD2 interacts with several key signaling pathways:

• MAPK Signaling: CRISPR/Cas9-mediated knockout studies of LIMD2 in PTC cell lines have explored the phosphorylation state of multiple kinases associated with the three major families of MAPK that are linked to:

  • Cell proliferation and differentiation

  • Cell survival

  • Cell migration and invasion

• T cell receptor signaling pathway: Gene set enrichment analysis (GSEA) has shown that high LIMD2 expression in ECA correlates with enrichment of the T cell receptor signaling pathway .

• DNA Replication: High LIMD2 expression in ECA shows enrichment in DNA replication pathways .

• Autophagy-related signaling: LIMD2 shows positive relationships with several autophagy-related genes, including BCL-2 .

Understanding these pathway interactions helps explain how LIMD2 influences various cellular processes and contributes to cancer progression through multiple mechanisms.

How is LIMD2 expression regulated at the genetic and epigenetic levels?

LIMD2 expression is regulated through several mechanisms:

• MicroRNA Regulation: LIMD2 has been identified as a direct target of microRNA-124 (miR-124) in NSCLC. Bioinformatics analysis and luciferase reporter gene assays confirmed that miR-124, a known tumor suppressor in NSCLC, negatively regulates LIMD2 expression . An inverse correlation between miR-124 and LIMD2 expression has been observed in NSCLC tissues.

• Copy Number Variation: LIMD2 copy number can influence its expression levels, with amplification potentially leading to overexpression. The copy number state can be determined using qPCR-based approaches .

• Transcriptional Regulation: Though not fully elucidated in the provided search results, transcriptional regulation likely plays a role in the tissue-specific and context-dependent expression of LIMD2.

These regulatory mechanisms provide potential therapeutic targets for modulating LIMD2 expression in pathological conditions, particularly in cancers where it promotes disease progression.

What genetic modification approaches are effective for studying LIMD2 function?

Several genetic modification approaches have been successfully implemented to study LIMD2:

• CRISPR/Cas9-mediated knockout: This approach has been used to create LIMD2 knockout in PTC cell lines to study its role in regulating key steps of the metastasis cascade . Complete knockout enables clear assessment of loss-of-function effects.

• RNA interference (RNAi): Knockdown of LIMD2 using siRNA or shRNA has been employed to study its role in NSCLC cell proliferation, migration, and invasion . This approach is valuable for partial and transient reduction of expression.

• miRNA modulation: Since LIMD2 is regulated by miR-124, modulating this miRNA's expression provides an indirect method to study LIMD2 regulation and function in cancers like NSCLC .

When designing genetic modification experiments, researchers should consider appropriate controls, verification of knockout/knockdown efficiency, and potential off-target effects. Experimental validation of modification success can be performed using a combination of RT-qPCR, Western blotting, and functional assays.

What is the relationship between LIMD2 and tumor immunity?

LIMD2 demonstrates significant associations with immune components in the tumor microenvironment:

• Immune Cell Infiltration: Based on TCGA database analyses using CIBERSORT, LIMD2 expression has been correlated with the infiltration of various immune cell populations in tumor tissues .

• T Cell Function: Gene set enrichment analysis has shown that high LIMD2 expression in ECA correlates with enrichment of the T cell receptor signaling pathway . This suggests a potential role in modulating T cell responses within the tumor microenvironment.

• Memory activated CD4 T cells: High LIMD2 expression correlates with greater proportions of memory activated CD4 T cells, which have been associated with poor prognosis in some cancer types .

These findings suggest that LIMD2 may influence tumor progression not only through direct effects on cancer cells but also by modulating the immune microenvironment. This connection offers potential for investigating LIMD2 in the context of immunotherapeutic approaches.

What are the key unanswered questions about LIMD2 in human health and disease?

Several critical knowledge gaps remain in our understanding of LIMD2:

• Tissue-specific functions: Why does LIMD2 appear to have opposing prognostic implications in different cancer types (e.g., favorable in ECA but unfavorable in NSCLC)?

• Binding partners: What are the complete set of LIMD2 binding partners across different tissue types and disease states?

• Regulation mechanisms: Beyond miR-124, what other regulatory mechanisms control LIMD2 expression in normal and pathological conditions?

• Normal physiological role: What is the full spectrum of LIMD2's functions in normal human physiology and development?

• Therapeutic potential: Can LIMD2 be effectively targeted for cancer therapy, and would such approaches need to be cancer-type specific?

Addressing these questions will require integrated approaches combining genomics, proteomics, and functional studies across multiple experimental systems.

What novel methodologies are emerging for LIMD2 research?

Emerging methodologies with potential applications in LIMD2 research include:

• Single-cell analysis: Single-cell RNA sequencing and proteomics could reveal cell-type specific expression patterns and functions of LIMD2 within heterogeneous tissues and tumor microenvironments.

• Protein interaction mapping: Techniques like BioID, proximity labeling, or protein microarrays could help identify the complete interactome of LIMD2 across different cellular contexts.

• In vivo imaging: Development of LIMD2-specific probes could enable real-time monitoring of its expression and activity in animal models of disease.

• Structure-function studies: Detailed structural analysis through crystallography or cryo-EM could inform rational design of LIMD2 inhibitors or modulators.

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data could provide a more comprehensive understanding of LIMD2's role in cellular networks.

These approaches could overcome current limitations in LIMD2 research and accelerate the translation of basic findings into clinical applications.