Recombinant Rat IQ and AAA domain-containing protein 1-like (Iqca1p1), partial

What is Recombinant Rat IQ and AAA domain-containing protein 1-like (Iqca1p1)?

Recombinant Rat Iqca1p1 is a laboratory-produced version of the naturally occurring Iqca1 protein found in rats. It belongs to the family of proteins containing both IQ motifs (calmodulin-binding domains) and AAA domains (ATPases Associated with diverse cellular Activities). This recombinant protein is typically expressed using bacterial, yeast, or mammalian expression systems and purified for experimental use. The "partial" designation indicates that the recombinant protein represents a fragment rather than the full-length protein, which allows researchers to study specific domains or regions of interest .

How does Iqca1p1 relate to other IQ domain-containing proteins?

Iqca1p1 shares structural similarity with the IQGAP family of scaffold proteins, particularly in its IQ domains. While Iqca1p1 remains less characterized, the IQGAP family proteins (IQGAP1, IQGAP2, and IQGAP3) function as multi-domain scaffold proteins that integrate multiple signaling pathways. These proteins contain calponin homology domains, IQ motifs, RasGAP-related domains, and C-terminal regions that interact with various binding partners . Proteomics studies have identified Iqca (IQ and AAA domain-containing protein 1) as differentially expressed in traumatic brain injury models, suggesting potential roles in cellular stress responses .

What are the primary structural features of Iqca1p1?

Iqca1p1 contains IQ motifs, which are approximately 25-amino acid sequences that bind calmodulin in a calcium-independent manner. The AAA domains are highly conserved regions containing Walker A and Walker B motifs involved in nucleotide binding and hydrolysis. Unlike the better-characterized IQGAP1 (which contains a calponin homology domain, WW domain, IQ domains, GRD domain, and RasGAP C-terminus), Iqca1p1's structural organization features a combination of IQ and AAA domains, suggesting potentially distinct functional roles in cellular processes .

What are the common experimental applications for recombinant Iqca1p1?

Recombinant Iqca1p1 can be utilized in:

• Protein-protein interaction studies to identify binding partners

• Immunization for antibody production

• In vitro kinase assays to examine potential phosphorylation

• Pull-down assays to validate interactions with suspected partners

• Structural studies using X-ray crystallography or NMR

• Cell-based assays examining the effects of exogenous protein addition
  Similar to IQGAP1, which serves as a scaffold for multiple signaling pathways including MAPK and PI3K/Akt pathways, Iqca1p1 might function in coordinating protein complexes, making it valuable for studying cellular signaling mechanisms .

How can recombinant Iqca1p1 be used to study cellular signaling pathways?

Based on knowledge of related proteins, researchers can employ recombinant Iqca1p1 to:

• Identify interaction partners using affinity purification followed by mass spectrometry

• Disrupt endogenous protein interactions through competitive binding

• Examine effects on MAPK and PI3K/Akt pathways, similar to IQGAP proteins

• Investigate potential roles in cytoskeletal organization

• Study responses to cellular stressors or stimuli
  For example, IQGAP1 has been shown to modulate Rac1 activity and enhance the accumulation of actin filaments, E-cadherin, and β-catenin at cell-cell contacts. Similar methodological approaches could reveal whether Iqca1p1 participates in analogous cellular processes .

What expression systems are optimal for producing recombinant Iqca1p1?

What purification strategies work best for recombinant Iqca1p1?

For optimal purification of recombinant Iqca1p1:

• Tag selection: Histidine tags are commonly used for initial affinity purification, though GST tags may improve solubility for challenging constructs. Similar approaches have been used for IQGAP1 purification, as noted in research utilizing GST-IQGAP1 fusion proteins .

• Multi-step purification protocol:

  • Initial capture: Affinity chromatography using nickel or glutathione resins

  • Intermediate purification: Ion exchange chromatography based on theoretical pI

  • Polishing: Size exclusion chromatography to achieve high purity

  • Optional: Tag removal using specific proteases if the tag interferes with function

• Quality control assessments:

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

  • Mass spectrometry for precise molecular weight determination

  • Circular dichroism to assess secondary structure

  • Dynamic light scattering to evaluate homogeneity

How can recombinant Iqca1p1 be used to investigate its role in traumatic brain injury models?

Proteomics analysis using iTRAQ has identified Iqca as differentially expressed following traumatic brain injury (TBI), with an observed fold change of 1.26 (p=0.0043) . To investigate its functional role:

• In vitro models:

  • Primary neuronal or glial cultures subjected to mechanical injury or oxygen-glucose deprivation

  • Addition of recombinant Iqca1p1 to evaluate protective or detrimental effects

  • Examination of downstream signaling using phospho-specific antibodies for MAPK pathways

• In vivo approaches:

  • Intracerebroventricular injection of recombinant protein following experimental TBI

  • Assessment of neurological outcomes using behavioral tests

  • Histological and biochemical analyses of brain tissue

• Molecular approaches:

  • Protein-protein interaction studies under TBI-like conditions

  • Phosphoproteomic analysis to identify post-translational modifications

  • Comparison with known TBI-responsive proteins like transthyretin (Ttr)

What techniques can be used to study Iqca1p1 interactions with small GTPases and cytoskeletal components?

Drawing from knowledge of IQGAP1, which interacts with Rac1 and participates in actin filament organization:

• Co-immunoprecipitation assays:

  • Using recombinant Iqca1p1 as bait to pull down interacting proteins

  • Reciprocal co-IPs with suspected partners

  • Validation with specific antibodies for small GTPases (Rac1, Cdc42)

• In vitro binding assays:

  • GST pull-down assays with purified GTPases (GDP-bound vs. GTP-γS-bound)

  • Surface plasmon resonance to determine binding kinetics

  • Fluorescence polarization assays to measure direct interactions

• Cellular localization studies:

  • Co-localization of fluorescently tagged proteins

  • Live-cell imaging to track dynamics during cellular processes

  • FRET/BRET approaches to measure protein proximity in living cells

• Functional assays:

  • Actin polymerization assays in the presence of recombinant protein

  • GTPase activity assays to assess effects on nucleotide hydrolysis

  • Cell spreading and migration assays to evaluate cytoskeletal effects

How should researchers interpret contradictory findings when studying Iqca1p1 function?

When facing contradictory results in Iqca1p1 research:

• Experimental context evaluation:

  • Cell types may express different binding partners

  • Environmental conditions (serum levels, confluency) affect signaling

  • Differentiation state influences protein function

• Technical considerations:

  • Tag position might interfere with specific interactions

  • Expression levels could cause non-physiological interactions

  • Post-translational modifications may be absent in certain systems

• Resolution strategies:

  • Employ multiple complementary techniques (genetic knockdown, overexpression, recombinant protein addition)

  • Use domain mapping to identify functional regions

  • Test in multiple cell types and under various conditions

  • Consider temporal dynamics of interactions
    For example, studies of IQGAP1's role in B cell development demonstrated its importance for marginal zone B cell formation while also showing its participation in both T-dependent and T-independent antibody responses, highlighting the need to examine protein function across multiple cellular contexts .

What controls are essential when performing interaction studies with recombinant Iqca1p1?

For rigorous interaction studies:

• Negative controls:

  • Non-related proteins of similar size and properties

  • Heat-denatured recombinant protein

  • Empty vector/tag-only preparations

  • Competitive inhibition with excess untagged protein

• Positive controls:

  • Known interaction partners from related proteins

  • Artificially engineered high-affinity interactions

  • Domains with well-characterized binding properties

• Validation controls:

  • Reciprocal pull-downs with suspected partners

  • Domain deletion/mutation to confirm specific binding regions

  • Dose-dependency experiments

  • Competition with synthetic peptides corresponding to binding sites

How might Iqca1p1 be involved in signaling pathways relevant to neurological disorders?

Based on proteomics data showing Iqca upregulation after traumatic brain injury (fold change 1.26, p=0.0043), potential research directions include:

• Signaling pathway analysis:

  • Investigation of MAPK pathway involvement, similar to IQGAP1's role in scaffolding MEK/ERK signaling

  • Examination of PI3K/Akt pathway interactions, which have neuroprotective functions

  • Assessment of calcium-dependent signaling through IQ domain interactions

• Experimental approaches:

  • Co-immunoprecipitation followed by phosphoproteomic analysis

  • Pharmacological inhibition of suspected pathways

  • RNAi or CRISPR-based knockdown/knockout studies

  • Overexpression of wild-type vs. mutant forms in neuronal models

• Potential functional implications:

  • Cytoskeletal reorganization during neuronal injury response

  • Regulation of cell survival pathways following traumatic insult

  • Modulation of inflammatory responses in glial cells

What experimental designs could evaluate Iqca1p1's potential role in receptor tyrosine kinase signaling?

Drawing from IQGAP1's established roles in receptor tyrosine kinase (RTK) signaling:

• In vitro kinase assays:

  • Recombinant Iqca1p1 as potential substrate for RTKs

  • Phosphorylation site mapping using mass spectrometry

  • Effects of phosphorylation on protein-protein interactions

• Cell-based approaches:

  • EGF or insulin stimulation in cells expressing tagged Iqca1p1

  • Time-course analysis of complex formation

  • Phosphoproteomic analysis to identify signaling nodes

  • Comparison of wild-type vs. phospho-mutant effects

• Proximity-based methods:

  • BioID or APEX2 fusion proteins to identify neighbors upon receptor activation

  • PLA (proximity ligation assay) to visualize interactions with RTK components

  • FRET sensors to detect conformational changes following stimulation