Recombinant Rat WD repeat-containing protein 89 (Wdr89)

What is Wdr89 and what is its general function in cellular processes?

Wdr89 (WD repeat domain 89) is a member of the WD repeat family of proteins. These proteins are characterized by the presence of multiple WD repeats, which are conserved sequences of approximately 40 amino acids that typically end with tryptophan-aspartic acid (WD). In human WDR89, six WD repeats have been identified . These repeats form β-propeller structures that serve as platforms for protein-protein interactions, allowing WD repeat proteins to function as adaptor proteins that facilitate the formation of multiprotein complexes .

While the specific cellular function of Wdr89 has not been fully characterized, WD repeat proteins generally participate in diverse cellular processes including signal transduction, cell cycle regulation, vesicular trafficking, cytoskeletal assembly, and transcriptional regulation. The β-propeller structure created by the WD repeats provides multiple protein interaction surfaces that can allow these proteins to function as molecular scaffolds.

What expression systems are most suitable for producing recombinant Wdr89?

Several expression systems can be used to produce recombinant Wdr89, each with distinct advantages:

For initial structural studies where post-translational modifications may not be critical, E. coli or yeast expression systems provide the best yields and shorter turnaround times . For functional studies requiring proper protein folding and activity, insect cells with baculovirus or mammalian cell expression systems are recommended as they provide necessary post-translational modifications . The selection should be based on the specific research objectives and the properties of Wdr89 being investigated.

How can I verify the identity and purity of recombinant Rat Wdr89?

Verification of identity and purity of recombinant Rat Wdr89 should follow a multi-method approach:

• SDS-PAGE analysis: To confirm molecular weight and initial purity assessment

• Western blotting: Using anti-Wdr89 antibodies or anti-tag antibodies if the recombinant protein contains fusion tags

• Mass spectrometry: For precise molecular weight determination and peptide mapping

• Size exclusion chromatography: To assess oligomeric state and homogeneity

• Circular dichroism: To evaluate secondary structure integrity

For tagged recombinant Wdr89 (such as His-tagged variants similar to the UAF1/WDR48 approach), specific validation can include tag-based affinity purification followed by tag detection methods . When using antibody-based detection, it may be advisable to perform blocking experiments with protein fragments, similar to the approach described for WDR89 control fragments, using a 100x molar excess of the protein fragment based on concentration and molecular weight .

What are the optimal buffer conditions for maintaining Wdr89 stability during purification and storage?

Optimal buffer conditions for Wdr89 stability should be determined empirically, but general recommendations based on WD repeat proteins include:

For long-term storage, recombinant Wdr89 can be kept in buffer containing 10% glycerol at -80°C, similar to the storage conditions used for WDR89 adenovirus preparations . Avoid multiple freeze-thaw cycles by preparing single-use aliquots. For functional studies, buffer optimization may be required to ensure proper protein activity, particularly if Wdr89 is involved in enzymatic reactions or protein-protein interactions within multiprotein complexes.

How do post-translational modifications affect Wdr89 function, and which expression system best preserves these modifications?

Post-translational modifications (PTMs) potentially critical for Wdr89 function may include phosphorylation, ubiquitination, and glycosylation. These modifications can affect protein folding, stability, localization, and protein-protein interactions.

Based on studies with other WD repeat proteins like UAF1/WDR48, PTMs can significantly impact function. For instance, UAF1/WDR48 participates in deubiquitination processes and interacts with other proteins in a manner that may be regulated by PTMs .

For preserving native PTMs:

• Mammalian expression systems provide the most authentic mammalian PTMs and are recommended for functional studies requiring native modification patterns.

• Insect cell expression with baculovirus offers a compromise between yield and PTM fidelity and is suitable for many applications requiring proper protein folding and basic PTMs .

• Yeast expression systems provide some PTMs but may differ from mammalian patterns.

• E. coli systems lack most PTMs and are unsuitable when modifications are critical for function.

If specific PTMs are known to be essential for Wdr89 function, targeted approaches such as site-specific ubiquitylation or SUMOylation using genetic-code expansion techniques (as referenced for UAF1 ) could be employed to generate properly modified recombinant protein.

What are the known interaction partners of Wdr89, and how can these interactions be studied in vitro?

While specific interaction partners of rat Wdr89 are not extensively documented in the provided search results, WD repeat proteins typically function in multiprotein complexes. Based on the structural characteristics of WD repeat domains serving as protein-protein interaction platforms , several approaches can be used to identify and study Wdr89 interactions:

To study these interactions in vitro, recombinant Wdr89 could be expressed with affinity tags (such as His-tag as used with UAF1/WDR48 ) to facilitate purification and interaction studies. For studying the stoichiometry of interactions, analytical techniques such as size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) would be appropriate.

What functional assays can be used to evaluate Wdr89 activity in vitro?

As a WD repeat protein likely involved in protein-protein interactions and potentially in multiprotein complexes, several functional assays can be employed to evaluate Wdr89 activity:

• Protein-protein interaction assays:

  • ELISA-based binding assays with potential partners

  • Fluorescence resonance energy transfer (FRET) with labeled interaction partners

  • Isothermal titration calorimetry (ITC) for thermodynamic binding parameters

• Complex formation analysis:

  • Size exclusion chromatography to detect complex formation

  • Analytical ultracentrifugation to determine complex stoichiometry

  • Native gel electrophoresis to visualize intact complexes

• If Wdr89 is involved in enzymatic complexes (by analogy with UAF1/WDR48 involvement in deubiquitination ):

  • Specific enzymatic assays with reconstituted complexes

  • For example, if Wdr89 interacts with deubiquitinating enzymes, deubiquitination assays using substrates like Ubiquitin-AMC could be adapted, with optimization of reaction conditions and stoichiometry as recommended for UAF1

Each assay should include appropriate controls and standardization to ensure reproducibility. Additionally, correlation of in vitro findings with cellular functions would provide validation of the physiological relevance of the observed activities.

How does Wdr89 expression change in different rat tissues and developmental stages?

While the provided search results don't contain specific information about Wdr89 expression patterns across rat tissues and developmental stages, a comprehensive expression analysis would typically include:

• Tissue-specific expression analysis:

  • qRT-PCR analysis of Wdr89 mRNA across major rat tissues

  • Western blot analysis of protein levels in tissue lysates

  • Immunohistochemical staining to visualize tissue distribution

• Developmental expression profiling:

  • Analysis of expression at different embryonic stages

  • Postnatal development expression changes

  • Comparison between juvenile and adult expression patterns

• Cell type-specific expression:

  • Single-cell RNA sequencing data analysis

  • Immunofluorescence microscopy with cell type-specific markers

This information would be valuable for understanding the physiological context of Wdr89 function and could guide experimental design for functional studies in specific tissues or developmental stages of interest.

What is the subcellular localization of Wdr89, and how does this relate to its function?

Determination of Wdr89 subcellular localization is crucial for understanding its functional context. Several complementary approaches can be used:

• Immunofluorescence microscopy:

  • Using specific anti-Wdr89 antibodies

  • Co-staining with organelle markers

• Subcellular fractionation:

  • Biochemical separation of cellular compartments

  • Western blotting of fractions to detect Wdr89

• Live-cell imaging:

  • Expression of fluorescently tagged Wdr89

  • Time-lapse microscopy to monitor dynamics

• Proximity labeling:

  • APEX or BioID fusion to identify proteins in the same subcellular location

The subcellular localization of Wdr89 would provide insights into its potential functions. For instance, nuclear localization might suggest roles in transcriptional regulation or DNA repair, while cytoplasmic localization could indicate involvement in signaling pathways or vesicular transport. If Wdr89 shows similar functions to other WD repeat proteins, it may be involved in multiprotein complexes with specific subcellular distributions related to their functions.

How can CRISPR-Cas9 gene editing be optimized for studying Wdr89 function in rat models?

CRISPR-Cas9 gene editing provides powerful approaches for studying Wdr89 function in rat models. Optimization strategies include:

• gRNA design considerations:

  • Target early exons to ensure functional disruption

  • Select gRNAs with high on-target and low off-target scores

  • Design multiple gRNAs to increase editing efficiency

• Verification of editing efficiency:

  • T7 endonuclease I assay or Surveyor assay for initial screening

  • Sanger sequencing of PCR amplicons spanning the target site

  • Next-generation sequencing for comprehensive mutation analysis

• Experimental design options:

  • Complete knockout for loss-of-function studies

  • Knock-in of point mutations to study specific domains

  • Introduction of epitope tags for protein detection

  • Conditional knockout using Cre-loxP systems

• Phenotypic analysis approaches:

  • Molecular characterization (RNA-seq, proteomics)

  • Cellular phenotyping (proliferation, morphology)

  • Physiological assessment based on tissues of interest

When introducing Wdr89 modifications, considerations should be given to potential compensatory mechanisms by other WD repeat proteins. Additionally, since WD repeat proteins often function in multiprotein complexes, disruption of Wdr89 might affect the assembly or function of larger complexes, potentially resulting in pleiotropic effects that require careful interpretation.

What roles might Wdr89 play in rat hippocampal function and memory formation?

Given that WD repeat proteins function in diverse cellular processes and the hippocampus is crucial for memory formation, potential roles of Wdr89 in rat hippocampal function could be explored through several experimental approaches:

• Expression analysis in hippocampal regions:

  • Quantification of Wdr89 expression in different hippocampal subregions (CA1, CA3, dentate gyrus)

  • Analysis of expression changes following memory-related tasks

• Functional studies through selective manipulation:

  • Targeted knockdown using viral delivery of shRNA to specific hippocampal regions

  • Overexpression studies to assess gain-of-function effects

  • Behavioral testing using paradigms like those described in the hippocampal lesion studies

• Molecular interaction studies:

  • Identification of hippocampus-specific interaction partners

  • Analysis of activity-dependent changes in protein interactions

While specific information on Wdr89's role in hippocampal function is not provided in the search results, the detailed methodologies described for studying recognition and recency memory in rats with hippocampal lesions provide robust experimental paradigms that could be adapted for investigating Wdr89 function. These include the bow-tie maze protocol for object recognition testing and the recency memory protocol, which could reveal whether Wdr89 manipulation affects specific aspects of memory formation or retrieval.

How do mutations in Wdr89 affect protein stability and function in rat models?

Mutations in Wdr89 could significantly impact protein stability and function through several mechanisms:

• Structural stability effects:

  • Mutations in core WD repeat residues may disrupt the β-propeller structure

  • Alterations in conserved residues could affect protein folding

  • Changes in surface residues might impact interaction partner binding

• Methodological approaches to assess mutation effects:

  • Thermal shift assays to measure changes in protein stability

  • Circular dichroism to detect alterations in secondary structure

  • Limited proteolysis to identify regions of altered conformation

  • In silico modeling to predict structural changes

• Functional consequences evaluation:

  • Protein-protein interaction assays with known partners

  • Subcellular localization analysis of mutant proteins

  • Rescue experiments in knockout backgrounds

  • Phenotypic analysis in rat models carrying specific mutations

When generating rat models with Wdr89 mutations, consideration should be given to both null mutations that eliminate protein expression and missense mutations that might affect specific functions while preserving others. This would allow dissection of domain-specific functions and potentially reveal separation-of-function phenotypes that could provide insights into the diverse roles of Wdr89 in different cellular processes or tissues.

What are common pitfalls in the expression and purification of recombinant Wdr89, and how can they be overcome?

Several challenges may arise during expression and purification of recombinant Wdr89:

Expression in insect cells with baculovirus or mammalian cells can provide necessary post-translational modifications for proper protein folding and maintain activity , as demonstrated with other WD repeat-containing proteins. For E. coli expression, specialized strains designed for expression of eukaryotic proteins with rare codons may improve yield. If protein solubility remains problematic, structural biology approaches such as limited proteolysis may identify stable domains suitable for expression as individual constructs.

How can the specificity and sensitivity of antibodies against rat Wdr89 be validated for research applications?

Rigorous validation of antibodies against rat Wdr89 is essential for reliable research outcomes. A comprehensive validation approach includes:

• Western blot validation:

  • Testing against recombinant Wdr89 as positive control

  • Testing against tissue lysates from multiple rat tissues

  • Competition experiments with blocking peptides

  • Comparison of signal in wildtype vs. Wdr89-depleted samples

• Immunoprecipitation validation:

  • Pulldown of endogenous Wdr89 from rat tissue lysates

  • Mass spectrometry confirmation of precipitated protein

  • Co-immunoprecipitation of known interaction partners

• Immunohistochemistry/immunofluorescence validation:

  • Comparison of staining patterns with multiple antibodies

  • Blocking experiments with antigen excess (similar to the approach described for WDR89 control fragments )

  • Controls using tissues from knockout or knockdown models

• Cross-reactivity assessment:

  • Testing against closely related WD repeat proteins

  • Species cross-reactivity testing if working with multiple models

For blocking experiments, a 100x molar excess of the protein fragment control based on concentration and molecular weight is recommended, with pre-incubation of the antibody-protein control fragment mixture for 30 minutes at room temperature .

What are the best strategies for delivering recombinant Wdr89 or Wdr89-expressing constructs into rat primary neurons?

Delivering recombinant proteins or expression constructs to primary neurons presents unique challenges due to their post-mitotic nature and sensitivity. Several approaches can be considered:

• For recombinant protein delivery:

  • Protein transduction domains (cell-penetrating peptides)

  • Liposome-based delivery systems

  • Microinjection for targeted delivery to individual neurons

• For genetic constructs:

  • Viral vectors:

    • Adenovirus systems (similar to the WDR89 adenovirus ) with titers >1×10^6 pfu/mL

    • Lentiviral systems for stable integration and long-term expression

    • AAV vectors for high-efficiency neuronal transduction

  • Non-viral approaches:

    • Lipofection (though typically lower efficiency in neurons)

    • Nucleofection for higher efficiency

    • Calcium phosphate precipitation for cultured neurons

• Considerations for experimental design:

  • Expression level control using inducible promoters

  • Cell-type specificity using neuron-specific promoters

  • Temporal control using optogenetic or chemogenetic regulators

For viral delivery, adenoviral systems like those described for human WDR89 can be adapted for rat Wdr89, with appropriate species-specific modifications to the construct. When using viral vectors, verification of transduction efficiency through reporter gene expression is essential before proceeding with functional experiments.

How is high-throughput screening being used to identify small molecule modulators of Wdr89 function?

High-throughput screening (HTS) approaches for identifying modulators of Wdr89 function could include:

• Biochemical screening approaches:

  • Protein-protein interaction disruption assays

  • Activity-based assays if Wdr89 is part of enzyme complexes

  • Thermal shift assays to identify stabilizing compounds

• Cell-based screening strategies:

  • Reporter gene assays linked to Wdr89-dependent pathways

  • Phenotypic screens in Wdr89-manipulated cell lines

  • High-content imaging to detect changes in subcellular localization

• In silico screening methods:

  • Structure-based virtual screening if crystal structure is available

  • Pharmacophore modeling based on known interaction interfaces

  • Fragment-based approaches to identify chemical starting points

• Validation of screening hits:

  • Dose-response relationships

  • Target engagement in cellular contexts

  • Selectivity profiling against other WD repeat proteins

While specific small molecule modulators of Wdr89 are not described in the provided search results, approaches similar to those used for other WD repeat proteins could be applied. Development of tool compounds through HTS would provide valuable reagents for dissecting Wdr89 function in complex biological systems and potentially reveal therapeutic opportunities if Wdr89 is implicated in disease processes.

What emerging techniques are advancing our understanding of Wdr89's role in protein-protein interaction networks?

Several cutting-edge techniques are enhancing our understanding of protein-protein interaction networks for WD repeat proteins like Wdr89:

• Proximity-based labeling approaches:

  • BioID, TurboID, or APEX2 fusion proteins to identify proximal interactors

  • Split-BioID for mapping interactions with specific partners

  • Spatially resolved proximity labeling for subcellular interaction mapping

• Advanced structural biology methods:

  • Cryo-electron microscopy for complex structure determination

  • Hydrogen-deuterium exchange mass spectrometry for mapping interaction surfaces

  • Cross-linking mass spectrometry to capture transient interactions

• Single-molecule techniques:

  • Single-molecule FRET to study conformational dynamics

  • Optical tweezers to measure interaction forces

  • Super-resolution microscopy to visualize protein complexes in situ

• Computational advances:

  • Machine learning approaches to predict interaction networks

  • Molecular dynamics simulations to model complex formation

  • Network analysis tools to identify functional modules

These techniques could be applied to understand how Wdr89, like other WD repeat proteins, functions as a scaffold in multiprotein complexes. For instance, techniques used to study UAF1/WDR48 interactions with deubiquitinating enzymes could be adapted for Wdr89 to determine if it participates in similar regulatory complexes.

How can systems biology approaches integrate Wdr89 function into broader cellular pathways?

Systems biology approaches offer powerful frameworks for integrating Wdr89 function into broader cellular contexts:

• Multi-omics integration strategies:

  • Combining transcriptomics, proteomics, and interactomics data

  • Correlation of Wdr89 expression with global gene expression patterns

  • Network analysis to position Wdr89 within cellular pathways

• Perturbation biology approaches:

  • Systematic Wdr89 perturbation (knockout, knockdown, overexpression)

  • Measurement of multi-parametric cellular responses

  • Computational modeling of response networks

• Comparative systems analysis:

  • Cross-species comparison of WD repeat protein functions

  • Evolutionary analysis of conserved interaction networks

  • Identification of species-specific adaptations

• Mathematical modeling applications:

  • Ordinary differential equation models of Wdr89-containing pathways

  • Stochastic modeling of protein complex assembly

  • Agent-based models of spatial organization

By integrating multiple data types and experimental approaches, systems biology can help position Wdr89 within the broader cellular context, revealing how it contributes to emergent cellular properties and organismal physiology. This integrative approach would be particularly valuable for understanding the function of WD repeat proteins like Wdr89, which likely participate in multiple protein complexes and cellular processes.