Recombinant LEM protein 2 (lem-2)

What is LEM-2 protein and what structural domains does it contain?

LEM-2 is a novel inner nuclear membrane protein belonging to the LEM domain family. Structurally, it contains an N-terminal LEM (lamina-associated polypeptide–emerin–MAN1) domain that mediates binding to barrier-to-autointegration factor (BAF), a DNA-crosslinking protein. LEM-2 also features two predicted transmembrane domains and a MAN1-Src1p C-terminal (MSC) domain highly homologous to MAN1, though it lacks MAN1's C-terminal RNA-recognition motif . This structural organization places LEM-2 as a lamina-associated protein residing in the inner nuclear membrane where it contributes to nuclear structure organization .

What are the common synonyms and gene names for LEM-2 across different species?

LEM-2 nomenclature varies across species:

The protein is referenced by these various identifiers in scientific literature and databases, reflecting its conservation across species .

How does recombinant LEM-2 differ from native LEM-2 protein?

Recombinant LEM-2 protein is artificially produced in expression systems like E. coli, while maintaining the amino acid sequence of the native protein. The recombinant version typically includes affinity tags (such as His-tag) for purification purposes. For instance, the Caenorhabditis elegans full-length recombinant LEM-2 (Q9XTB5) contains all 500 amino acids (1-500aa) fused to an N-terminal His tag when expressed in E. coli . While these modifications facilitate protein isolation and detection, researchers should consider how tags might affect protein folding, function, or interaction studies. For most structural and biochemical analyses, recombinant LEM-2 closely resembles native protein behavior, particularly when expressed in eukaryotic systems that allow for proper post-translational modifications.

What expression systems are optimal for producing functional recombinant LEM-2 protein?

Multiple expression systems have been successfully used to produce recombinant LEM-2 protein, each with specific advantages:

What are the optimal storage and handling conditions for recombinant LEM-2 protein?

Recombinant LEM-2 protein requires careful handling to maintain stability and activity. For long-term storage, the following protocols are recommended:

• Store lyophilized protein at -20°C/-80°C upon receipt

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (optimal: 50%) for cryoprotection

• Aliquot to avoid repeated freeze-thaw cycles

• For working solutions, store at 4°C for no more than one week

The protein is typically supplied in a Tris/PBS-based buffer with 6% trehalose at pH 8.0 . Repeated freeze-thaw cycles significantly reduce protein activity, so creating single-use aliquots is strongly recommended. When working with the protein, maintaining proper temperature and pH conditions is crucial for preserving structural integrity and functional activity.

How can I design experiments to study LEM-2's role in nuclear envelope organization?

To investigate LEM-2's role in nuclear envelope organization, consider these methodological approaches:

• Localization studies: Use fluorescently tagged LEM-2 constructs to visualize its distribution in the nuclear envelope. Compare wild-type distribution with truncated variants lacking specific domains to determine which regions are necessary for proper localization.

• Protein-protein interaction assays: Implement co-immunoprecipitation or proximity ligation assays to study LEM-2's interactions with lamins, particularly lamin C, which has been shown to bind to LEM-2 in vitro .

• Overexpression studies: Controlled overexpression of LEM-2 can induce distinctive phenotypes, including accumulation in patches at the nuclear envelope and formation of membrane bridges between adjacent nuclei. These structures selectively recruit A-type lamins, emerin, MAN1, and BAF, while excluding lamin B and lamin B receptor .

• Knockout/knockdown approaches: Use CRISPR-Cas9 or RNAi to deplete LEM-2 and assess changes in nuclear morphology, lamin distribution, and nuclear envelope integrity.

• Super-resolution microscopy: Employ techniques like STORM or PALM to visualize the nanoscale organization of LEM-2 relative to other nuclear envelope components.

These approaches can be combined with computational modeling to better understand how LEM-2 contributes to the structural integrity and organization of the nuclear envelope.

What methods are effective for studying LEM-2's role in RNA surveillance mechanisms?

Based on recent research in S. pombe, LEM-2 plays a significant role in RNA surveillance, particularly in the degradation of non-coding RNAs and meiotic transcripts. To study this function, consider the following methodological approaches:

• RNA-seq analysis: Compare transcriptome profiles between wild-type and lem2Δ cells. Principal component analysis (PCA) has revealed similarities between transcriptome profiles of lem2Δ, rrp6Δ (nuclear exosome), and red1Δ (MTREC complex) mutants .

• RT-qPCR validation: Use RT-qPCR to quantify specific transcript levels, particularly meiotic genes (e.g., sme2, ssm4) and snoRNAs (e.g., sno20, snR42) in single and double mutants lacking LEM-2 and other exosome components .

• Co-immunoprecipitation: Investigate LEM-2's physical interactions with components of the MTREC complex, especially Red1, and the human homolog PAXT, which are implicated in exosome-mediated RNA degradation .

• RNA localization studies: Employ FISH (Fluorescence In Situ Hybridization) to visualize the localization of exosome substrates in relation to LEM-2 at the nuclear periphery.

• Nutrient response experiments: Test how different nutrient conditions affect LEM-2's regulation of meiotic transcripts, as the research suggests this pathway is environmentally responsive .

When designing these experiments, it's important to consider that LEM-2's function in RNA surveillance appears distinct from its role in heterochromatin silencing, suggesting multiple pathways may be involved.

How can I distinguish between LEM-2's direct and indirect effects on gene expression?

Distinguishing between direct and indirect effects of LEM-2 on gene expression requires sophisticated experimental approaches:

• ChIP-seq analysis: While LEM-2 is not directly DNA-binding, chromatin immunoprecipitation sequencing can identify genomic regions associated with LEM-2-containing complexes.

• CLIP-seq experiments: Since LEM-2 does not directly bind RNA but interacts with RNA-binding complexes, cross-linking immunoprecipitation sequencing can identify RNAs associated with LEM-2-containing ribonucleoprotein complexes.

• Tethering assays: Artificially tether LEM-2 to specific genomic loci using systems like LacO/LacI to determine if LEM-2 proximity is sufficient to affect expression of nearby genes.

• Rapid protein depletion: Use systems like the auxin-inducible degron to rapidly deplete LEM-2 and distinguish immediate (likely direct) from delayed (likely indirect) effects on gene expression.

• Domain-specific mutations: Create LEM-2 variants with mutations in specific domains to determine which interactions are necessary for observed effects on gene expression.

Research has shown that LEM-2 does not directly bind RNA but interacts with the exosome-targeting MTREC complex and its human homolog PAXT to promote RNA recruitment for degradation . This suggests that many of LEM-2's effects on gene expression are mediated through these protein-protein interactions rather than direct RNA binding.

How does LEM-2 function differ between model organisms?

LEM-2 exhibits both conserved and species-specific functions across different model organisms:

In S. pombe, LEM-2 has a well-characterized role in regulating nuclear-exosome-mediated RNA degradation, particularly affecting non-coding RNAs and meiotic transcripts . Analysis using the AnGeLi tool revealed 'ncRNA' as the group of genes most significantly altered in lem2∆ cells . This function appears to be modulated by nutrient availability, suggesting an environmentally responsive regulatory mechanism.

In contrast, studies in other organisms have focused more on LEM-2's structural roles in the nuclear envelope. When designing comparative studies, researchers should consider these functional variations and select appropriate readouts for their specific model system.

What are the methodological challenges in working with recombinant LEM-2 from different species?

Working with recombinant LEM-2 from different species presents several methodological challenges:

• Protein solubility and stability: As a membrane protein with transmembrane domains, LEM-2 can pose solubility challenges. Species-specific variations in hydrophobicity and folding may require different detergents or buffer conditions.

• Post-translational modifications: Eukaryotic LEM-2 proteins may require specific post-translational modifications for proper function that are not correctly added in prokaryotic expression systems.

• Protein-protein interactions: LEM-2's binding partners may vary between species, requiring careful selection of interaction assay components.

• Antibody cross-reactivity: Antibodies raised against LEM-2 from one species may not recognize orthologs from other species due to sequence divergence.

• Functional assays: Different species-specific readouts may be needed to assess functional activity (e.g., RNA surveillance in S. pombe versus nuclear envelope structure in mammalian cells).

To address these challenges, researchers often use a combination of expression systems matched to their experimental needs. For instance, structural studies might use E. coli-expressed protein with >90% purity , while functional studies might require expression in systems that better recapitulate the protein's native environment.

What are common pitfalls in recombinant LEM-2 purification and how can they be addressed?

Purifying recombinant LEM-2 protein presents several challenges due to its transmembrane domains and complex structure. Common pitfalls and their solutions include:

• Poor solubility:

  • Problem: Membrane proteins like LEM-2 often aggregate during expression and purification.

  • Solution: Use mild detergents (e.g., DDM, CHAPS) during extraction; consider fusion tags that enhance solubility (e.g., MBP, SUMO); optimize buffer conditions (pH, salt concentration).

• Low expression levels:

  • Problem: Transmembrane proteins often express poorly in heterologous systems.

  • Solution: Test multiple expression systems; consider using cell-free expression systems that have shown success with LEM-2 ; optimize codon usage for the expression host.

• Protein degradation:

  • Problem: Proteolytic degradation during expression or purification.

  • Solution: Add protease inhibitors; optimize purification temperature (typically 4°C); minimize time between cell lysis and purification steps.

• Tag interference:

  • Problem: Affinity tags may affect protein folding or function.

  • Solution: Compare N- and C-terminal tag placements; include a cleavable linker between the tag and protein; verify protein activity after tag removal.

• Protein aggregation during storage:

  • Problem: Purified protein forms aggregates upon storage.

  • Solution: Add glycerol (5-50%) as a cryoprotectant; store at -80°C in single-use aliquots; avoid repeated freeze-thaw cycles .

Commercial recombinant LEM-2 preparations typically achieve >85-90% purity as determined by SDS-PAGE , suggesting these challenges can be overcome with optimized protocols.

How can I validate the functional activity of purified recombinant LEM-2?

Validating the functional activity of purified recombinant LEM-2 requires assays that reflect its known biological functions:

• Protein-protein interaction assays:

  • Verify binding to known partners such as lamin C using pull-down assays or surface plasmon resonance

  • Test interaction with BAF (barrier-to-autointegration factor), a critical binding partner of the LEM domain

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm proper secondary structure

  • Size-exclusion chromatography to ensure the protein is not aggregated

  • Limited proteolysis to verify the protein is properly folded and resistant to non-specific degradation

• Membrane integration assays:

  • Liposome binding assays to verify the transmembrane domains can properly insert into lipid bilayers

  • Reconstitution into artificial membrane systems to test membrane integration capacity

• Functional reconstitution:

  • For S. pombe LEM-2, test interaction with components of the MTREC complex or PAXT

  • In vitro RNA degradation assays to assess if LEM-2 enhances exosome-mediated degradation of target RNAs

• Cell-based validation:

  • Complementation assays in lem2Δ cells to test if the recombinant protein can rescue mutant phenotypes

  • Localization studies with fluorescently labeled recombinant protein to verify proper targeting to the nuclear envelope

When validating recombinant LEM-2, it's important to include positive controls (e.g., commercially available validated protein) and negative controls (e.g., heat-denatured protein) to establish the specificity of your assays.

What are promising research directions for understanding LEM-2's role in disease mechanisms?

While the search results don't explicitly connect LEM-2 to disease mechanisms, several promising research directions emerge based on its functions:

• Neurodegenerative diseases: Given LEM-2's role in nuclear structure organization and RNA surveillance, investigating its potential involvement in neurodegenerative disorders characterized by nuclear envelope abnormalities (e.g., Huntington's disease, ALS) could yield valuable insights.

• Cancer biology: Exploring how LEM-2-mediated RNA degradation pathways might be dysregulated in cancer could reveal new therapeutic targets, particularly for cancers with aberrant non-coding RNA profiles.

• Aging-related nuclear dysfunction: As nuclear envelope integrity declines with age, studying how LEM-2 contributes to maintaining nuclear architecture throughout the lifespan could inform interventions for age-related cellular dysfunction.

• Developmental disorders: Investigating LEM-2's interactions with A-type lamins , mutations in which cause various laminopathies, might reveal contributory roles in these disorders.

• Stress response pathways: The environmentally responsive nature of LEM-2's regulation of meiotic transcripts suggests it might function in cellular stress adaptation, warranting investigation in models of cellular stress.

These directions would benefit from comparative studies across model organisms, leveraging the evolutionary conservation of LEM-2 while acknowledging species-specific functions.

How might advances in protein engineering impact future applications of recombinant LEM-2?

Emerging protein engineering technologies offer exciting possibilities for expanding recombinant LEM-2 applications:

• Designer LEM-2 variants: Creating chimeric proteins with modified domains could enable targeted manipulation of specific cellular pathways. For instance, engineered LEM-2 with enhanced RNA surveillance activity might be used to selectively degrade pathogenic RNAs.

• Optogenetic LEM-2 constructs: Light-controlled LEM-2 variants could allow temporal and spatial control of nuclear envelope reorganization or RNA degradation pathways in living cells.

• Biosensor applications: LEM-2 fusion proteins designed to report on nuclear envelope integrity or localized RNA processing events could serve as valuable research tools.

• CRISPR-based targeting: Fusion of catalytically inactive Cas proteins with LEM-2 domains could direct specific genomic loci to the nuclear periphery, enabling studies of how nuclear positioning affects gene expression.

• Therapeutic protein delivery: Engineered LEM-2 variants might facilitate nuclear targeting of therapeutic proteins in gene therapy applications.