Recombinant Pig Importin subunit alpha-8 (KPNA7), partial

What is KPNA7 and how does it function within the karyopherin alpha family?

KPNA7 is a member of the karyopherin alpha (KPNA) family of nuclear import receptors. In mammals, the KPNA family is encoded by seven genes that generate isoforms with 42–86% sequence identity . While all KPNA isoforms share the same general protein architecture and recognize nuclear localization signals (NLS), KPNA7 stands out as a highly divergent member, sharing only 54.7% identity with its closest relative, KPNA2 .

KPNA7 functions as an adaptor protein that recognizes NLS-containing cargo proteins and facilitates their transport into the nucleus in conjunction with Importin-β (Imp-β). Unlike other family members, KPNA7 can bind to Imp-β in the absence of NLS cargo, suggesting a unique regulatory mechanism . This distinct property may be crucial for its specialized functions during early development.

How is KPNA7 expression regulated in different tissues and developmental stages?

KPNA7 exhibits a highly specialized expression pattern. It is expressed at high levels during early embryonic development but becomes reduced to very low or absent levels in most adult tissues . This restricted expression pattern suggests a critical role in early developmental processes. Interestingly, KPNA7 can be re-expressed in certain cancer cells, particularly in pancreatic and breast cancer cells, suggesting its potential involvement in cancer pathogenesis .

The developmental regulation of KPNA7 expression likely involves tissue-specific transcription factors and epigenetic mechanisms, though the exact regulatory pathways remain to be fully elucidated. Understanding these regulatory mechanisms represents an important area for future research.

What are the key structural domains of KPNA7 and how do they compare to other KPNA family members?

KPNA7 maintains the canonical domain architecture of karyopherin alpha proteins but with significant sequence divergence. The protein contains:

• An N-terminal Importin-β binding (IBB) domain that interacts with Importin-β

• A central region composed of armadillo (ARM) repeats that form the NLS-binding groove

• A C-terminal region involved in cargo release and recycling

The IBB domain of KPNA7 is particularly divergent, sharing only 48% identity with the KPNA2 IBB domain and merely 24% identity with the KPNA5 IBB domain . This divergence likely contributes to KPNA7's unique binding properties, including its ability to bind Importin-β in the absence of NLS cargo .

The ARM repeats in KPNA7 also show variations that may affect its cargo specificity and binding affinity. These structural differences highlight KPNA7's specialized function compared to other KPNA family members.

How does the IBB domain of KPNA7 differ functionally from other KPNA proteins?

The IBB domain of KPNA7 exhibits functional properties distinct from other KPNA family members. While typically the IBB domain serves to occlude the NLS binding groove and maintain an auto-inhibited "closed" state prior to NLS cargo binding, KPNA7's IBB appears to function differently .

This unique property may be particularly important during early embryonic development when rapid nuclear import of specific factors is required.

What role does KPNA7 play in pig reproductive traits and early embryonic development?

KPNA7 is essential for early embryogenesis and normal fertility in pigs . Research has demonstrated significant associations between single nucleotide polymorphisms (SNPs) in the KPNA7 gene and important reproductive traits in French Large White pigs .

Specifically, certain KPNA7 genotypes are associated with:

These findings suggest that KPNA7 genetic variants influence reproductive performance in pigs, likely through effects on early embryonic development and fertility .

How can researchers study KPNA7's function in early embryogenesis?

To investigate KPNA7's role in early embryogenesis, researchers can employ several methodological approaches:

• Gene knockdown/knockout studies: Using RNA interference (siRNA/shRNA) or CRISPR-Cas9 technology to reduce or eliminate KPNA7 expression in early embryos.

• Transgenic animal models: Generating KPNA7-deficient or KPNA7-overexpressing pig models to observe developmental outcomes.

• In vitro fertilization and embryo culture: Studying the effects of KPNA7 manipulation on embryonic development in vitro.

• Immunofluorescence and live imaging: Tracking KPNA7 localization and dynamics during early embryonic divisions.

• Identification of KPNA7 cargo proteins: Using pull-down assays and mass spectrometry to identify NLS-containing proteins transported by KPNA7 during early development.

• SNP genotyping and association studies: Correlating KPNA7 genetic variants with reproductive outcomes in different pig breeds .

These approaches can provide insights into the specific mechanisms by which KPNA7 influences early embryonic development and reproductive traits in pigs.

What techniques can be used to study KPNA7's interactions with importin-β and cargo proteins?

Several techniques can be employed to investigate KPNA7's interactions with importin-β and cargo proteins:

• GST pull-down assays: This approach has been successfully used to demonstrate KPNA7's binding to importin-β and to identify interacting proteins . Recombinant GST-tagged importin-β can be immobilized on glutathione beads and incubated with KPNA7 to assess direct binding .

• Co-immunoprecipitation (Co-IP): This technique can be used to capture protein complexes from cell lysates to verify interactions in a cellular context.

• Yeast two-hybrid screening: This can identify novel KPNA7-interacting proteins from a cDNA library.

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC): These biophysical techniques can quantitatively measure binding affinities and kinetics between KPNA7 and its partners.

• Liquid chromatography with tandem mass spectrometry (LC-MS/MS): This approach has been used to identify KPNA1 and KPNA7 interacting proteins in porcine fibroblast cells .

• Fluorescence resonance energy transfer (FRET): This can be used to detect protein-protein interactions in living cells.

These methodologies can reveal the specific binding partners of KPNA7 and how these interactions contribute to its function in nuclear import and early embryonic development.

How do KPNA7-interacting proteins differ from those of other KPNA family members?

Research utilizing GST pull-down assays coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS) has revealed that KPNA7-interacting proteins generally share common characteristics with those binding to other KPNA family members, but with some important distinctions .

KPNA7-interacting proteins:

• Are predominantly nuclear proteins that possess nuclear localization signals

• Include chromatin remodeling enzymes and transcription factors that need nuclear access to exert their functions

• May include proteins specifically involved in early embryonic development

The differential expression patterns of KPNA isoforms across tissues and developmental stages suggest that subtle differences in their cargo preferences might be important in specific biological contexts . KPNA7's unique binding properties, including its ability to bind importin-β in the absence of NLS cargo, suggest it may have specialized cargo recognition capabilities compared to other KPNA family members .

Further comparative proteomic studies are needed to fully characterize the unique interactome of KPNA7 versus other KPNA proteins.

What are the optimal systems and conditions for expressing recombinant pig KPNA7?

For successful expression of recombinant pig KPNA7, researchers should consider the following expression systems and conditions:

• E. coli expression system:

  • Recommended strains: BL21(DE3), Rosetta(DE3), or Arctic Express for proteins with rare codons

  • Expression vector: pGEX for GST-tagged protein or pET for His-tagged protein

  • Induction: 0.1-0.5 mM IPTG at lower temperatures (16-25°C) to enhance solubility

  • Culture conditions: Growth at 37°C until OD600 reaches 0.6-0.8, then temperature reduction for induction

• Insect cell expression system (for better folding):

  • Baculovirus expression vector system using Sf9 or Hi5 cells

  • Vectors containing polyhedrin or p10 promoters

  • Culture temperature: 27°C with proper aeration

• Mammalian cell expression system (for authentic post-translational modifications):

  • HEK293 or CHO cells

  • Vectors with strong promoters (CMV or EF1α)

  • Transient or stable transfection methods

• Fusion tags to consider:

  • N-terminal GST tag (facilitates solubility and affinity purification)

  • His-tag (for IMAC purification)

  • MBP tag (enhances solubility)

• Co-expression with importin-β may enhance stability and solubility due to their natural interaction.

The choice of expression system should be guided by the intended experimental use of the recombinant protein, required purity, and functional assays planned.

What purification strategies are most effective for obtaining active recombinant KPNA7?

To obtain pure, active recombinant KPNA7, a multi-step purification strategy is recommended:

• Initial capture:

  • For GST-tagged KPNA7: Glutathione affinity chromatography

  • For His-tagged KPNA7: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins

  • Buffer conditions: pH 7.5-8.0, 300-500 mM NaCl, with protease inhibitors

• Tag removal (if necessary):

  • Specific protease treatment (e.g., TEV protease for TEV cleavage sites)

  • Optimization of cleavage conditions (temperature, time, buffer)

• Intermediate purification:

  • Ion exchange chromatography (IEX)

  • For KPNA7 (theoretical pI ~4.8-5.2): Cation exchange at pH 6.0 or anion exchange at pH 8.0

• Polishing step:

  • Size exclusion chromatography to remove aggregates and provide buffer exchange

  • Recommended buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol

• Quality control assessments:

  • SDS-PAGE and Western blot to confirm purity and identity

  • Dynamic light scattering to assess homogeneity

  • Circular dichroism to verify proper folding

  • Activity assays: Binding studies with importin-β and NLS-containing cargo proteins

• Storage considerations:

  • Store at -80°C in small aliquots

  • Include 10-20% glycerol or sucrose as cryoprotectants

  • Avoid repeated freeze-thaw cycles

This purification workflow ensures high purity recombinant KPNA7 suitable for structural studies, binding assays, and functional characterization.

How does KPNA7 expression affect cancer cell growth and nuclear morphology?

KPNA7, though minimally expressed in most adult tissues, can be re-expressed in certain cancer cells with significant functional consequences . Research has demonstrated that:

• Silencing of KPNA7 using siRNA-based knockdown results in dramatic reduction in both pancreatic and breast cancer cell growth, regardless of the endogenous KPNA7 expression level .

• This growth inhibition is accompanied by:

  • A decrease in the fraction of S-phase cells

  • Aberrant number of centrosomes

  • Severe distortion of mitotic spindles

• KPNA7 depletion profoundly affects nuclear morphology:

  • Leads to reorganization of nuclear lamina proteins (lamin A/C and B1)

  • Causes drastic alterations in nuclear shape, resulting in lobulated and elongated nuclei

These findings suggest that KPNA7 plays a crucial role in cancer cell proliferation by regulating proper mitosis and maintaining nuclear envelope integrity. The re-expression of KPNA7 in cancer cells may represent an oncogenic mechanism that supports cancer cell growth and division.

What experimental approaches can be used to study KPNA7's role in disease models?

Researchers can employ various experimental approaches to investigate KPNA7's role in disease models:

• RNA interference and gene editing:

  • siRNA or shRNA-mediated knockdown of KPNA7 in cancer cell lines

  • CRISPR-Cas9-mediated knockout or mutation of KPNA7

  • Rescue experiments with wild-type or mutant KPNA7

• Cell proliferation and cell cycle analysis:

  • MTT/XTT assays or cell counting to assess growth inhibition

  • Flow cytometry with propidium iodide or EdU incorporation to analyze cell cycle distribution

  • BrdU incorporation assays to measure DNA synthesis

• Microscopy techniques:

  • Immunofluorescent staining to visualize nuclear morphology and centrosome numbers

  • Live-cell imaging to track mitotic progression

  • Super-resolution microscopy to examine nuclear lamina organization

• Animal models:

  • Xenograft models using KPNA7-manipulated cancer cells

  • Genetically engineered mouse models with tissue-specific KPNA7 alterations

  • Patient-derived xenografts to study KPNA7 in human tumors

• Multi-omics approaches:

  • RNA-seq to identify transcriptional changes following KPNA7 manipulation

  • ChIP-seq to examine changes in chromatin binding of transcription factors

  • Proteomics to identify altered nuclear import substrates

• Clinical correlation studies:

  • Analysis of KPNA7 expression in human tumor samples

  • Correlation with clinical parameters and patient outcomes

These approaches can provide comprehensive insights into KPNA7's role in cancer pathogenesis and potential as a therapeutic target.

How can researchers analyze the subcellular localization and dynamics of KPNA7?

To investigate the subcellular localization and dynamics of KPNA7, researchers can utilize several advanced techniques:

• Immunofluorescence microscopy:

  • Fixed-cell immunostaining with specific anti-KPNA7 antibodies

  • Co-staining with markers for nuclear envelope, nuclear pore complexes, and other cellular compartments

  • Confocal microscopy for high-resolution localization analysis

• Live-cell imaging:

  • Fusion of KPNA7 with fluorescent proteins (GFP, mCherry, etc.)

  • Time-lapse microscopy to track KPNA7 movement between cytoplasm and nucleus

  • Photobleaching techniques (FRAP, FLIP) to measure transport kinetics

• Subcellular fractionation:

  • Biochemical separation of nuclear and cytoplasmic fractions

  • Western blot analysis of KPNA7 distribution across fractions

  • Density gradient centrifugation for more detailed compartment analysis

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins to identify proteins in close proximity to KPNA7

  • Spatial mapping of KPNA7 interactome in different cellular compartments

• Super-resolution microscopy:

  • STORM, PALM, or SIM imaging for nanoscale localization

  • Visualization of KPNA7 in relation to nuclear pore complexes

Research has already demonstrated that KPNA7 shows predominantly nuclear localization in HeLa cells, which differs from other importin α family members . This distinct localization pattern suggests unique functional properties that can be further explored using these techniques.

What methods can be used to measure KPNA7 binding affinity to importin-β and NLS-containing cargoes?

Several quantitative methods can be employed to measure binding affinities between KPNA7 and its interaction partners:

• Surface Plasmon Resonance (SPR):

  • Real-time measurement of binding kinetics (kon and koff)

  • Determination of equilibrium dissociation constants (KD)

  • Requires immobilization of purified KPNA7 or its binding partners on sensor chips

• Isothermal Titration Calorimetry (ITC):

  • Direct measurement of binding thermodynamics (ΔH, ΔS, ΔG)

  • No protein labeling or immobilization required

  • Provides stoichiometry information

• Microscale Thermophoresis (MST):

  • Measures changes in thermophoretic mobility upon binding

  • Requires minimal protein amounts

  • Works well with fluorescently labeled proteins

• Fluorescence Anisotropy/Polarization:

  • Measures changes in rotational diffusion upon binding

  • Useful for studying interactions with fluorescently labeled NLS peptides

  • Allows for competition assays

• Bio-Layer Interferometry (BLI):

  • Real-time measurement of binding similar to SPR

  • No microfluidics required

  • Good for kinetic screening of multiple interactions

• Analytical Ultracentrifugation (AUC):

  • Monitors complex formation in solution

  • No labeling required

  • Provides information on stoichiometry and shape

• AlphaScreen/AlphaLISA assays:

  • Bead-based proximity assay

  • High-throughput compatible

  • Good for screening interactions or inhibitors

These methods can reveal the unique binding properties of KPNA7, such as its reported higher affinity for importin-β compared to other KPNA family members , and help elucidate how these properties contribute to KPNA7's specialized functions in nuclear import.

How do SNPs in the porcine KPNA7 gene affect protein function and reproductive traits?

Single nucleotide polymorphisms (SNPs) in the porcine KPNA7 gene have been associated with significant effects on reproductive traits . Six new SNPs were identified in the KPNA7 gene in French Large White pigs, with several showing statistically significant associations with reproductive performance .

The functional effects of these SNPs may include:

• Altered protein structure or stability: SNPs in coding regions may affect the three-dimensional structure of KPNA7, potentially altering its binding properties with importin-β or NLS-containing cargo proteins.

• Modified expression levels: SNPs in regulatory regions may affect transcription factor binding sites, enhancers, or promoter elements, leading to changes in KPNA7 expression levels during critical developmental stages.

• Altered splicing patterns: SNPs near splice junctions may affect the efficiency of mRNA splicing, potentially leading to alternative KPNA7 isoforms with different functional properties.

• Post-translational modifications: SNPs that alter amino acid sequences may create or eliminate sites for post-translational modifications, affecting KPNA7 regulation.

The specific mechanisms by which these genetic variations influence reproductive traits require further investigation through functional genomics approaches and protein structure-function analyses.

What genomic techniques can be used to identify and characterize KPNA7 variants in different pig breeds?

Researchers can employ several genomic techniques to identify and characterize KPNA7 variants across pig breeds:

• Targeted sequencing:

  • PCR amplification and Sanger sequencing of KPNA7 exons and regulatory regions

  • Targeted next-generation sequencing panels including KPNA7

  • Useful for focused analysis of specific regions in many individuals

• Whole-genome sequencing (WGS):

  • Comprehensive approach to identify all variants in and around KPNA7

  • Enables discovery of structural variants and distant regulatory elements

  • Provides context of genomic architecture

• RNA sequencing (RNA-seq):

  • Analysis of KPNA7 expression levels across tissues and developmental stages

  • Identification of alternative splicing variants

  • Detection of allele-specific expression

• SNP genotyping arrays:

  • High-throughput screening of known KPNA7 variants in large populations

  • Cost-effective for association studies with reproductive traits

  • Commercial porcine SNP chips can be supplemented with custom KPNA7 markers

• Genome-wide association studies (GWAS):

  • Identification of associations between KPNA7 variants and reproductive phenotypes

  • Detection of quantitative trait loci (QTLs) in the KPNA7 region

• Comparative genomics:

  • Analysis of KPNA7 conservation and variation across pig breeds and related species

  • Identification of functionally important regions based on evolutionary conservation

• Epigenomic profiling:

  • Analysis of DNA methylation, histone modifications, and chromatin accessibility

  • Understanding regulatory mechanisms affecting KPNA7 expression

These approaches can provide comprehensive insights into KPNA7 genetic diversity and its implications for reproductive performance across different pig breeds and populations.

What are the most promising research areas for understanding KPNA7's specialized functions?

Several research directions hold particular promise for advancing our understanding of KPNA7's specialized functions:

• Developmental regulation of KPNA7 expression:

  • Elucidation of transcriptional and epigenetic mechanisms controlling KPNA7's restricted expression pattern

  • Identification of signaling pathways regulating KPNA7 during embryogenesis

• KPNA7-specific cargo identification:

  • Comprehensive proteomic profiling of KPNA7-interacting proteins during early development

  • Comparative analysis with other KPNA family members to identify unique cargo specificities

• Structural biology approaches:

  • Crystal or cryo-EM structures of KPNA7 alone and in complex with importin-β and/or cargo

  • Molecular dynamics simulations to understand the unique properties of KPNA7's IBB domain

• KPNA7 in nuclear envelope biology:

  • Investigation of KPNA7's role in nuclear lamina organization and nuclear morphology

  • Analysis of KPNA7's interactions with nuclear envelope proteins

• Translational applications in reproductive technology:

  • Development of KPNA7-based markers for embryo quality assessment

  • Exploration of KPNA7 as a target for improving reproductive efficiency in livestock

• KPNA7 in cellular reprogramming:

  • Investigation of KPNA7's potential role in cellular reprogramming and pluripotency

  • Analysis of KPNA7 in the context of induced pluripotent stem cells (iPSCs)

These research directions could significantly advance our understanding of the specialized functions of KPNA7 in early development and reproduction, potentially leading to applications in both basic science and agricultural biotechnology.

How might KPNA7 research contribute to advances in reproductive technologies and cancer therapeutics?

KPNA7 research has significant potential to contribute to advances in both reproductive technologies and cancer therapeutics:

Reproductive Technologies:

• Embryo selection markers: KPNA7 expression levels or localization patterns could serve as biomarkers for embryo quality assessment in assisted reproductive technologies.

• Genetic improvement strategies: Selection for beneficial KPNA7 variants could enhance reproductive traits in breeding programs, as evidenced by the association between KPNA7 SNPs and reproductive performance in pigs .

• In vitro maturation optimization: Understanding KPNA7's role in oocyte maturation and early embryogenesis could inform improved protocols for in vitro embryo production.

• Embryo culture system development: Knowledge of KPNA7-dependent nuclear import pathways could guide the development of culture media supplements that support optimal early embryonic development.

• Gene editing applications: KPNA7 could be a target for genetic modification to enhance fertility or reproductive efficiency in livestock.

Cancer Therapeutics:

• Novel therapeutic targets: The re-expression of KPNA7 in cancer cells and its critical role in cancer cell growth suggest it could be a promising therapeutic target.

• Diagnostic and prognostic markers: KPNA7 expression patterns could serve as biomarkers for certain cancer types or stages.

• Drug development strategies: Small molecule inhibitors targeting the KPNA7-importin-β interaction or KPNA7-cargo interactions could be developed as anti-cancer agents.

• Combination therapy approaches: Understanding how KPNA7 depletion affects cell cycle progression and nuclear morphology could inform combination therapies that synergize with these effects.

• Personalized medicine applications: KPNA7 expression profiles could potentially guide treatment selection for individual cancer patients.