Recombinant Mouse Kelch domain-containing protein 7A (Klhdc7a)

What is Klhdc7a and how is it classified within protein families?

Klhdc7a (Kelch Domain Containing 7A) is a member of the evolutionarily conserved Kelch protein superfamily. This superfamily contains 63 alternate protein coding members split into three subfamilies: Kelch like (KLHL), Kelch-repeat and bric-a-bracs (BTB) domain containing (KBTBD), and Kelch domain containing protein (KLHDC). Klhdc7a belongs to the KLHDC subfamily, which is one of the smallest within the Kelch superfamily, containing only 10 primary members .

What is currently known about the structure of mouse Klhdc7a protein?

While specific structural information about mouse Klhdc7a is limited, the full-length mouse Klhdc7a protein consists of 773 amino acids (AA 1-773). The protein shares 76% sequence identity with its human ortholog . Recent structural and biochemical advances for KLHDC2, another member of the KLHDC subfamily, have provided insights that aid our understanding of other KLHDC family members, including Klhdc7a . The KLHDC proteins typically contain Kelch domain repeats that form a β-propeller structure involved in protein-protein interactions.

What is known about the genomic organization of mouse Klhdc7a?

The human ortholog KLHDC7A gene is located at chromosome 1p36.13, and contains a single exon . This single-exon structure is unusual and suggests the gene may have evolved through retrotransposition. For the mouse Klhdc7a gene, limited genomic organization data is available in the search results, but based on its human ortholog structure, it likely has similar characteristics.

What expression systems are most effective for producing recombinant mouse Klhdc7a?

Recombinant mouse Klhdc7a protein has been successfully expressed in HEK-293 cells, a human embryonic kidney cell line commonly used for recombinant mammalian protein expression . This mammalian expression system offers advantages for proper folding and post-translational modifications of mouse proteins. Alternative expression systems might include cell-free protein synthesis (CFPS), which has been used for other KLHDC family proteins .

What purification tags and methods are recommended for recombinant mouse Klhdc7a?

Commercially available recombinant mouse Klhdc7a is typically produced with a His-tag, which facilitates purification through immobilized metal affinity chromatography (IMAC) . Alternative tags such as Strep-tag have also been used for KLHDC family proteins . Purification quality can be assessed using techniques such as SDS-PAGE, Western blot, anti-tag ELISA, and analytical SEC (HPLC), with purity levels of >90% achievable for well-optimized protocols.

How should recombinant mouse Klhdc7a be stored to maintain stability?

For optimal stability, recombinant mouse Klhdc7a should be stored at -80°C . The protein is typically formulated as a liquid, though specific buffer compositions may vary. It's advisable to avoid repeated freeze-thaw cycles as this can lead to protein degradation and loss of activity. For carrier-free recombinant proteins, reconstitution in PBS at concentrations around 100 μg/mL is often recommended, following protocols similar to those used for related proteins .

What is known about the subcellular localization of Klhdc7a?

While specific information about mouse Klhdc7a localization is limited, studies of KLHDC7B (a related protein) have shown that it can localize to both cytoplasmic and nuclear compartments . In FLAG-tagged KLHDC7B-transfected HeLa cells, KLHDC7B was found in both cytosolic and nuclear fractions, and this localization pattern was maintained even under ER stress conditions . By analogy, mouse Klhdc7a might exhibit similar localization patterns, though direct experimental verification is needed.

What functional assays can be used to study Klhdc7a activity?

Based on what is known about related KLHDC proteins, several functional assays might be applicable to study mouse Klhdc7a:

• Reporter gene assays: These can be used to study transcriptional regulation effects, similar to those used to demonstrate that the risk T-allele of rs2992756 significantly lowers KLHDC7A promoter activity .

• Apoptosis assays: Given the role of KLHDC7B in apoptosis, assays measuring PARP cleavage, cell viability, and caspase activation could be relevant for investigating potential apoptotic functions of Klhdc7a .

• Protein interaction studies: Co-immunoprecipitation assays to identify binding partners, which could provide insights into the cellular pathways involving Klhdc7a.

• Subcellular localization studies: Immunofluorescence or cell fractionation experiments using tagged recombinant Klhdc7a to determine its distribution within cells .

What is the evidence linking Klhdc7a to human diseases?

While mouse Klhdc7a itself has not been directly linked to diseases, its human ortholog KLHDC7A has been implicated in breast cancer risk through genome-wide association studies (GWAS). Specifically, a variant (rs2992756) located 84bp from the transcription start site of KLHDC7A has been strongly associated with breast cancer risk (P=1.6x10^-15) . This variant is situated in the proximal promoter of KLHDC7A and influences its expression level .

How do genetic variants in KLHDC7A affect its function?

Reporter assays have demonstrated that the risk T-allele of rs2992756 in the KLHDC7A promoter significantly lowers its activity compared to the reference construct . This suggests that decreased expression of KLHDC7A might contribute to increased breast cancer risk, though the precise mechanism remains to be elucidated. This functional evidence strengthens the case for KLHDC7A as the target gene at the 1p36 breast cancer risk locus .

The following table summarizes key genetic variants associated with KLHDC7A:

What methodological approaches have been used to establish KLHDC7A as a breast cancer risk gene?

Several complementary approaches have been used to identify and validate KLHDC7A as a breast cancer risk gene:

• Genome-wide association studies (GWAS): Identified variants near KLHDC7A that are associated with breast cancer risk .

• Transcriptome-wide association studies (TWAS): Identified KLHDC7A as a target gene at the 1p36 breast cancer risk locus .

• Reporter assays: Demonstrated that the risk variant affects KLHDC7A promoter activity .

• Integrative bioinformatic approaches: Tools like DSNetwork were used to visualize and integrate multiple functional annotations for variants, helping to identify rs2992756 as the best candidate causal variant at the 1p36 locus .

How can CRISPR/Cas9 be utilized to study Klhdc7a function in mouse models?

CRISPR/Cas9 technology offers powerful approaches to study Klhdc7a function:

• Knockout studies: Complete elimination of Klhdc7a expression to determine its essential functions.

• Knockin of specific variants: Introduction of variants corresponding to human disease-associated polymorphisms to study their effects in vivo.

• Tagging of endogenous protein: Insertion of epitope or fluorescent tags to track endogenous Klhdc7a localization and interactions.

For Klhdc7a specifically, the single-exon structure (if similar to its human ortholog) may simplify CRISPR targeting strategies . The CRISPR/Cas9 system could be used to generate mice with point mutations by microinjecting guide RNA, hCas9 mRNA, and single-stranded donor oligonucleotides into mouse zygotes, creating specific genomic modifications in Klhdc7a with lower cost and shorter timeframes compared to traditional methods .

What are the challenges in studying the evolutionary conservation of Klhdc7a?

Studying evolutionary conservation of Klhdc7a presents several challenges:

• Limited functional characterization: The lack of detailed functional information about Klhdc7a makes it difficult to assess functional conservation across species.

• Single-exon structure: If mouse Klhdc7a has a single-exon structure like its human ortholog, this unusual genomic organization may complicate evolutionary analyses.

• Sequence divergence: Mouse Klhdc7a shares 76% sequence identity with human KLHDC7A, indicating significant divergence that may reflect species-specific functions .

• Subfamily complexity: The Kelch superfamily contains 63 members across three subfamilies, making it challenging to disentangle the evolutionary relationships and functional divergence among these proteins .

How can comparative studies between different KLHDC family members inform our understanding of Klhdc7a?

Comparative studies between KLHDC family members can provide valuable insights:

• Structural comparisons: Recent structural advances in understanding KLHDC2 can inform predictions about Klhdc7a structure and function .

• Functional parallels: Studies showing KLHDC7B's role in ER stress and apoptosis suggest potential similar roles for Klhdc7a .

• Expression pattern analysis: Comparing tissue-specific expression patterns across KLHDC family members may reveal functional specialization.

• Protein interaction networks: Identifying common and distinct interaction partners among KLHDC family members can illuminate their cellular functions.

A systematic approach comparing all ten KLHDC subfamily members could help position Klhdc7a within this family's functional landscape and generate testable hypotheses about its specific roles.

What are the critical controls needed when performing functional studies with recombinant mouse Klhdc7a?

When designing experiments with recombinant mouse Klhdc7a, the following controls are essential:

• Empty vector controls: For overexpression studies, cells transfected with empty vector provide baseline comparison.

• Inactive mutant controls: Generating a mutant version of Klhdc7a with disrupted Kelch domains can help confirm specificity of observed effects.

• Antibody validation: For recombinant protein detection, pre-incubation of antibodies with a protein control fragment (such as human KLHDC7A aa 705-776) can be used for blocking experiments to demonstrate specificity .

• siRNA controls: When using RNA interference, non-targeting siRNA controls and validation of knockdown efficiency through RT-qPCR are critical .

• Species-specific controls: When extrapolating between mouse and human systems, species-specific differences should be considered, as mouse Klhdc7a shares only 76% sequence identity with its human ortholog .

How can researchers address the current gaps in understanding Klhdc7a function?

To advance understanding of mouse Klhdc7a function, researchers could:

• Generate comprehensive expression profiles: Determine the tissue-specific expression patterns of Klhdc7a in mouse development and adulthood.

• Identify interaction partners: Use techniques such as yeast two-hybrid screening, co-immunoprecipitation followed by mass spectrometry, or proximity labeling approaches to identify proteins that interact with Klhdc7a.

• Develop knockout mouse models: Generate and characterize Klhdc7a knockout mice to determine the phenotypic consequences of Klhdc7a deficiency.

• Investigate potential role in cancer: Given the human KLHDC7A association with breast cancer risk, explore whether mouse Klhdc7a plays similar roles in mammary tumor models.

• Examine stress response functions: Based on KLHDC7B's role in ER stress, investigate whether Klhdc7a is similarly involved in cellular stress response pathways.

These approaches would help fill the significant knowledge gaps that currently exist regarding the biological functions of Klhdc7a.