Recombinant Human Keratin-associated protein 5-10 (KRTAP5-10)

What is KRTAP5-10 and how does it differ from other keratin-associated proteins?

KRTAP5-10 (also known as KAP5.10) belongs to the ultrahigh sulfur subgroup of the keratin-associated protein (KRTAP/KAP) superfamily. This superfamily comprises over 100 genes unique to mammals that are primarily involved in hair formation. The KRTAP5 subfamily is characterized by particularly high cysteine content (>30% of amino acid composition), which facilitates crosslinking of keratin intermediate filaments through disulfide bonds. KRTAP5-10 specifically consists of 202 amino acids in humans, with a distinctive sequence rich in cysteine residues that contribute to its structural functions .

While KRTAP5-10 shares structural similarities with other KRTAP5 subfamily members like KRTAP5-5, each has unique expression patterns and potentially distinct regulatory functions in different tissues. Research on related family members suggests these proteins may have roles beyond mere structural components in hair, including potential involvement in cellular processes like cytoskeletal organization .

What expression systems are most effective for producing recombinant KRTAP5-10?

Cell-free protein synthesis (CFPS) systems have demonstrated particularly high efficiency for expressing recombinant KRTAP5-10. The ALiCE® system, which utilizes lysate from Nicotiana tabacum c.v., has been successfully employed to produce soluble KRTAP5-10 protein with functional post-translational modifications . This approach circumvents challenges often encountered with traditional cell-based expression methods for cysteine-rich proteins.

For effective expression and purification, consider these methodological steps:

• Optimize codon usage for the expression system

• Include appropriate affinity tags (e.g., Strep-Tag) for one-step purification

• Maintain reducing conditions during purification to prevent premature disulfide bond formation

• Validate protein identity through mass spectrometry and functional assays

• Store purified protein with stabilizing agents to prevent aggregation

Plant-based expression systems offer advantages for KRTAP5-10 production due to their ability to handle complex post-translational modifications while avoiding endotoxin contamination inherent to bacterial systems .

What are the recommended methods for detecting KRTAP5-10 in experimental systems?

For reliable detection of KRTAP5-10 in research applications, multiple complementary approaches should be employed:

Protein-level detection:

• Western blotting using antibodies against KRTAP5-10 or epitope tags (e.g., Strep-Tag)

• ELISA for quantitative assessment

• Immunofluorescence microscopy for localization studies

• SDS-PAGE analysis for molecular weight confirmation

mRNA-level detection:

• RT-PCR for presence/absence determination

• qRT-PCR for quantitative expression analysis

• RNA-seq for comprehensive transcriptome profiling

What experimental approaches can differentiate between the structural and signaling functions of KRTAP5-10?

To distinguish between structural roles (keratin crosslinking) and potential signaling functions of KRTAP5-10, implement these methodological approaches:

For structural function assessment:

• Electron microscopy to visualize cytoskeletal architecture

• Atomic force microscopy to measure changes in cellular stiffness

• Biochemical crosslinking assays to identify direct keratin binding partners

• Domain mutation studies to identify regions critical for structural integrity

For signaling function assessment:

• Proximity labeling techniques (BioID, APEX) to identify non-cytoskeletal interaction partners

• Phosphoproteomics to detect changes in signaling pathway activation

• Reporter assays for relevant transcription factors

• Time-course studies following KRTAP5-10 manipulation to distinguish immediate versus delayed effects

Importantly, structure-function studies should utilize both gain-of-function (overexpression) and loss-of-function (knockdown/knockout) approaches. This bidirectional manipulation helps determine whether observed phenotypes result from direct KRTAP5-10 activity versus indirect compensatory mechanisms.

How does post-translational modification affect KRTAP5-10 function and interactions?

The high cysteine content of KRTAP5-10 suggests redox regulation plays a significant role in its function. Researchers investigating post-translational modifications (PTMs) of KRTAP5-10 should consider:

• Disulfide bond mapping: Using non-reducing versus reducing conditions in combination with mass spectrometry to identify specific disulfide linkages that form under different cellular conditions

• Oxidative modification detection: Employing proteomic approaches to identify other oxidative modifications of cysteine residues (e.g., S-glutathionylation, S-nitrosylation) that may regulate protein function

• Phosphorylation analysis: Investigating potential serine/threonine/tyrosine phosphorylation events that could modulate protein-protein interactions

• Experimental methodology for PTM-function relationships:

  • Site-directed mutagenesis of specific cysteine residues to determine their contribution to protein function

  • Treatment with redox-modulating compounds to assess functional changes

  • Creation of phosphomimetic mutants to simulate constitutive phosphorylation

The PTM status of KRTAP5-10 likely varies between different cellular compartments and in response to environmental stressors, potentially serving as a mechanism for context-dependent regulation of its function.

What controls are essential when studying KRTAP5-10 function in cellular systems?

Rigorous experimental design for KRTAP5-10 research requires these critical controls:

For knockdown/knockout studies:

• Multiple independent siRNA/shRNA sequences or CRISPR guide RNAs to minimize off-target effects

• Rescue experiments with shRNA-resistant KRTAP5-10 to confirm phenotype specificity

• Assessment of other KRTAP family member expression to identify compensatory mechanisms

• Non-targeting siRNA/shRNA or non-targeting guide RNA controls

For overexpression studies:

• Empty vector controls

• Expression of unrelated proteins of similar size to control for non-specific effects

• Expression of related KRTAP family members to assess specificity

• Titration of expression levels to avoid artifacts from excessive overexpression

For all studies:

• Verification of manipulation at both mRNA and protein levels

• Time-course experiments to distinguish immediate from adaptive responses

• Assessment in multiple cell types to determine context-dependency

• Parallel analysis of related KRTAP family members (particularly KRTAP5-5) for comparative insights

How should researchers design experiments to study potential KRTAP5-10 involvement in cancer progression?

Based on findings with the related protein KRTAP5-5, researchers investigating KRTAP5-10's potential role in cancer should implement these methodological approaches:

• Expression profiling:

  • Analyze KRTAP5-10 expression across cancer types using public databases

  • Validate findings with qRT-PCR and immunohistochemistry in patient samples

  • Correlate expression with clinical parameters and survival outcomes

• Functional assessment in cancer models:

  • Generate stable KRTAP5-10 knockdown and overexpression cell lines

  • Evaluate effects on:

    • Proliferation and apoptosis

    • Cell motility (scratch wound assays)

    • Invasion through extracellular matrix

    • Endothelial monolayer disruption (vascular invasion models)

• Cytoskeletal and adhesion analysis:

  • Examine keratin and vimentin intermediate filament expression and organization

  • Assess F-actin cytoskeletal architecture

  • Quantify expression of integrins, particularly α6/β4-integrins

  • Measure cell-matrix and cell-cell adhesion properties

• In vivo models:

  • Implant KRTAP5-10-modified cancer cells in appropriate animal models

  • Assess tumor growth, local invasion, and metastatic spread

  • Analyze vascular invasion through zebrafish or mouse models

  • Evaluate circulating tumor cell isolation and extravasation capacity

The experimental design should include appropriate controls as outlined in the previous question and incorporate both gain- and loss-of-function approaches to comprehensively assess KRTAP5-10's contribution to cancer-relevant phenotypes.

How should researchers interpret contradictory findings between KRTAP5-10 and other KRTAP family members?

The KRTAP superfamily comprises over 100 genes with potential functional redundancy and context-dependent activities. When facing contradictory results between KRTAP5-10 and other family members (particularly KRTAP5-5), consider these analytical approaches:

• Sequence comparison analysis:

  • Align protein sequences to identify conserved versus divergent domains

  • Map differences to functional domains that might explain phenotypic variations

  • Examine species conservation patterns for evolutionary insights

• Expression pattern comparison:

  • Analyze co-expression versus differential expression across tissues

  • Determine if contradictory findings correlate with expression differences

  • Consider potential competitive or cooperative interactions between family members

• Functional domain swap experiments:

  • Create chimeric proteins by exchanging domains between KRTAP5-10 and other members

  • Identify which regions confer specific functional properties

  • Determine if contradictions arise from domain-specific activities

• Methodological considerations:

  • Assess antibody cross-reactivity between family members

  • Evaluate specificity of knockdown/knockout approaches

  • Consider different cellular contexts that might explain contradictory results

Remember that apparent contradictions may reflect genuine biological complexity rather than experimental error, as paralogous proteins often evolve distinct functions while maintaining structural similarity.

What bioinformatic approaches are most useful for predicting KRTAP5-10 function and interaction partners?

Given the limited published research specifically on KRTAP5-10, computational approaches provide valuable guidance for experimental design:

• Structural prediction and analysis:

  • Use homology modeling based on related proteins with known structures

  • Employ cysteine clustering analysis to predict disulfide bonding patterns

  • Implement molecular dynamics simulations to assess conformational flexibility

• Interaction partner prediction:

  • Apply co-expression analysis across tissue and single-cell datasets

  • Utilize protein-protein interaction predictors based on sequence features

  • Examine shared promoter elements with genes of known function

  • Consider STRING database analysis with appropriate confidence thresholds

• Functional annotation transfer:

  • Identify functions of other KRTAP family members, particularly KRTAP5-5

  • Assess GO term enrichment for statistically significant patterns

  • Use interactome analysis to place KRTAP5-10 in functional networks

• Evolutionary analysis:

  • Conduct phylogenetic analysis across species to identify conserved functions

  • Examine selection pressure on different regions of the protein

  • Compare tissue-specific expression patterns across species

The knowledge value of KRTAP5-10 in various categories (cell line: 0.82, tissue: 0.35, microRNA: 0.33, cellular component: 0.25) suggests targeted approaches focusing first on cell line-based validation of predicted interactions .

Beyond hair biology, what novel research areas might benefit from investigating KRTAP5-10?

While traditionally associated with hair formation, emerging evidence from related keratin-associated proteins suggests KRTAP5-10 may have broader biological significance. Researchers should consider these promising investigative directions:

• Cancer biology:

  • Investigate KRTAP5-10's potential role in cytoskeletal regulation during epithelial-mesenchymal transition

  • Assess its contribution to cancer cell invasion and metastasis

  • Examine correlations between expression and patient outcomes across cancer types

• Cellular stress response:

  • Explore how the cysteine-rich nature of KRTAP5-10 might function in cellular redox sensing

  • Investigate its potential protective role against oxidative damage

  • Examine expression changes during various cellular stress conditions

• Tissue mechanical properties:

  • Study KRTAP5-10's contribution to cellular and tissue stiffness

  • Investigate its role in mechanotransduction pathways

  • Assess potential involvement in diseases characterized by altered tissue mechanics

• Developmental biology:

  • Characterize expression patterns during embryonic and postnatal development

  • Investigate potential roles in epithelial morphogenesis

  • Explore contributions to stem cell maintenance in epithelial tissues

The limited current knowledge about KRTAP5-10 (PubMed score: 0.16) suggests these areas represent fertile ground for novel discoveries that may expand our understanding of keratin-associated protein functions beyond their classical structural roles .

What methodological advances would most benefit KRTAP5-10 research?

Advancement in KRTAP5-10 research would be accelerated by these methodological innovations:

• Improved protein production systems:

  • Development of specialized expression systems for cysteine-rich proteins

  • Optimization of folding conditions to ensure native disulfide bond formation

  • Creation of isotopically labeled KRTAP5-10 for structural studies

• Enhanced detection tools:

  • Generation of highly specific antibodies that differentiate between KRTAP family members

  • Development of proximity labeling approaches for in vivo interaction studies

  • Creation of fluorescent protein fusions that maintain native function

• Advanced imaging techniques:

  • Implementation of super-resolution microscopy to visualize cytoskeletal interactions

  • Application of correlative light and electron microscopy to connect function with ultrastructure

  • Development of live-cell imaging approaches to track dynamic interactions

• Physiologically relevant model systems:

  • Creation of organoid models expressing fluorescently tagged KRTAP5-10

  • Development of tissue-specific knockout mouse models

  • Implementation of human-derived ex vivo tissue models

These methodological advances would facilitate more comprehensive understanding of KRTAP5-10's biological functions and potential relevance to human health and disease.