Recombinant Mouse WD repeat-containing protein 37 (Wdr37)

What is WD repeat-containing protein 37 (Wdr37) and what are its structural characteristics?

Wdr37 belongs to the WD-repeat (WDR) protein superfamily, which are characterized by the presence of multiple WD40 domains. The WD motif is approximately 40 amino acids in length, typically ending with Tryptophan-Aspartic acid (Trp-Asp) residues, though amino acid sequence conservation is limited .

Mouse Wdr37 contains seven predicted WD40 domains that fold into a β-propeller structure . These domains form a series of four-stranded, antiparallel beta sheets that arrange into a higher-order cylindrical structure . This arrangement creates a stable platform that facilitates protein-protein interactions.

The protein is encoded by the Wdr37 gene located on mouse chromosome 10, and the full-length mouse Wdr37 protein consists of 496 amino acids .

What are the known biological functions of Wdr37 in mice?

Research has revealed several important functions of Wdr37 in mice:

• Complex formation with Pacs1: Wdr37 forms a mutually stabilizing complex with Pacs1, and these proteins require each other for optimal expression and stability .

• Calcium homeostasis regulation: The Pacs1-Wdr37 complex plays a critical role in endoplasmic reticulum (ER) Ca²⁺ handling in lymphocytes. Deletion of Wdr37 causes blunted Ca²⁺ release from the ER after antigen receptor stimulation .

• Lymphocyte population maintenance: Wdr37 is essential for maintaining lymphocyte quiescence. Wdr37⁻/⁻ mice demonstrate peripheral lymphopenia affecting both T and B cells .

• rRNA processing: Wdr37 has been implicated in maturation of 5.8S rRNA and maturation of LSU-rRNA, suggesting a role in ribosome biogenesis .

• T cell function: Wdr37 has been identified in a genome-scale gain-of-function CRISPR screen as a potential enhancer of CD8+ T cell cytotoxicity .

What are the recommended methods for detecting endogenous mouse Wdr37 protein?

Several methods have been validated for detecting endogenous mouse Wdr37:

• Western blotting: Using a specific anti-Wdr37 polyclonal antibody produced in rabbit is effective for detecting endogenous Wdr37 in mouse tissue lysates. Recommended dilutions should be empirically determined, but typically range between 1:500-1:2000 .

• Immunocytochemistry: Wdr37 can be detected by immunofluorescence microscopy using either unconjugated antibodies or FITC-conjugated Wdr37 antibodies. Wild-type Wdr37 is predominantly localized in the cytoplasm .

• ELISA: Commercial ELISA kits specific for mouse Wdr37 are available with a detection range of 0.156-10 ng/ml. These kits are optimized for tissue homogenates, cell lysates, and other biological fluids from mouse sources .

For all detection methods, appropriate controls should be included to ensure specificity, including samples from Wdr37-knockout mice where available.

How can recombinant mouse Wdr37 protein be effectively expressed and purified for research applications?

The following methodological approach is recommended based on published protocols:

• Expression system selection: Mammalian expression systems (HEK293 or CHO cells) are preferable for maintaining proper folding and post-translational modifications of Wdr37. For structural studies requiring higher yields, insect cell systems (Sf9/Sf21) have been successfully employed .

• Vector design: Use a pReceiver-M14 or similar plasmid with the full-length Wdr37 cDNA (1-496aa) inserted at appropriate restriction sites (e.g., HindIII and XbaI). Addition of an affinity tag (3xFLAG, 6xHis, or Myc) at either terminus facilitates purification .

• Purification strategy:

  • For FLAG-tagged constructs: Use anti-FLAG M2 affinity gel for immunoprecipitation

  • For His-tagged constructs: Use Ni-NTA agarose under native conditions

  • Apply a two-step purification with size-exclusion chromatography to enhance purity

• Protein stability considerations: Purified Wdr37 should be stored with 10% glycerol in an appropriate buffer (e.g., 50 mM HEPES pH 7.4, 150 mM NaCl) at -80°C. Avoid repeated freeze-thaw cycles as this significantly reduces protein activity .

• Quality control: Verify protein identity and integrity by SDS-PAGE, western blot, and mass spectrometry. Functional validation can be performed through binding assays with known interaction partners such as Pacs1 .

What are the known protein interaction partners of Wdr37 and how can these interactions be studied?

Key interaction partners of mouse Wdr37 include:

• Pacs1 and Pacs2: These phosphofurin acidic cluster sorting proteins form a complex with Wdr37. The interaction can be detected by co-immunoprecipitation and yeast two-hybrid assays, and confirmed by colocalization in immunocytochemistry .

• Self-association: Wdr37 has the ability to form homodimers, which can be studied through co-immunoprecipitation using differentially tagged versions of the protein .

Methodological approaches to study these interactions include:

• Co-immunoprecipitation (Co-IP):

  • Express FLAG-tagged Wdr37 and Myc-tagged potential interaction partners in cells

  • Lyse cells in 1% Triton X-100 buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EGTA, 10% glycerol)

  • Immunoprecipitate with anti-FLAG or anti-Myc antibodies

  • Analyze precipitated complexes by SDS-PAGE and western blotting

• Yeast two-hybrid assay:

  • Use the full-length coding sequence (1-494 aa) of Wdr37 as bait

  • Screen against a library (e.g., human fetal brain library)

  • Confirm positive interactions by reverse two-hybrid approach

• Immunocytochemistry colocalization:

  • Express fluorescently tagged versions of both proteins

  • Analyze subcellular localization using confocal microscopy

  • Quantify colocalization using Pearson's correlation coefficient or Manders' overlap coefficient

How does Wdr37 contribute to calcium flux regulation in lymphocytes?

Wdr37, in complex with Pacs1, plays an essential role in regulating calcium homeostasis in lymphocytes through the following mechanisms:

• Regulation of IP3 receptor expression: The Pacs1-Wdr37 complex maintains normal levels of inositol triphosphate receptors (IP3Rs), which are critical calcium release channels in the ER membrane. Wdr37-deficient cells show diminished IP3R expression .

• ER calcium store maintenance: The Pacs1-Wdr37 complex is required for normal ER Ca²⁺ handling. In Wdr37⁻/⁻ lymphocytes, there is blunted Ca²⁺ release from the ER following antigen receptor stimulation .

• Prevention of ER stress: Proper calcium homeostasis prevents ER stress. Loss of Wdr37 leads to increased ER stress, which can be measured by:

  • Elevated levels of ER stress markers (BiP, CHOP, XBP1 splicing)

  • Increased reactive oxygen species (ROS) production

  • Altered mitochondrial oxygen consumption

• Maintenance of cellular quiescence: The Pacs1-Wdr37 complex plays a critical role in maintaining lymphocyte quiescence through proper calcium signaling. Wdr37⁻/⁻ B cells show spontaneous loss of quiescence with increased proliferation and apoptosis in lymphocyte-replete environments in vivo .

To study these functions experimentally:

• Flow cytometry with calcium-sensitive fluorescent dyes (Fluo-4, Indo-1) can be used to measure Ca²⁺ flux in response to antigen receptor stimulation

• Comparison of responses in Ca²⁺-containing versus Ca²⁺-free media can distinguish between ER release and extracellular calcium entry

What developmental processes are affected by Wdr37 mutations in animal models?

Studies in zebrafish and mouse models have revealed several developmental processes affected by Wdr37 mutations:

• Zebrafish models: CRISPR-Cas9-mediated genome editing to generate zebrafish with missense variants in Wdr37 (p.Ser129Phe, p.Ser129Cys, p.Ser129Tyr) or frameshift alleles (p.Lys127Cysfs, p.Gln95Argfs) demonstrated:

  • Poor growth and larval lethality in heterozygotes with missense variants

  • Better survival in heterozygotes with frameshift alleles, suggesting a dominant-negative mechanism for the missense variants

  • Disruption of cholesterol biosynthesis pathways, as revealed by RNA-seq analysis

• Mouse models: Wdr37-knockout mice show:

  • Reduced peripheral blood T and B cell counts

  • Impaired calcium flux in response to B cell receptor (BCR) cross-linking

  • No reported gross neurological phenotypes, unlike human patients with WDR37 mutations

These animal models suggest that Wdr37 plays crucial roles in:

How do mutations in human WDR37 correlate with disease phenotypes and what can researchers learn from these correlations?

Human WDR37 mutations have been associated with a multisystemic syndrome with distinct mutation clusters corresponding to different phenotypic manifestations:

• N-terminal mutation cluster (amino acids 115-130):

  • Ocular anomalies: corneal opacity/Peters anomaly, coloboma, microcornea

  • Significant neurological impairment with structural brain defects and seizures

  • Dysmorphic facial features

  • Poor feeding and post-natal growth

  • Variable skeletal, cardiac, and genitourinary defects

  • Potential infant mortality

• Second mutation cluster (affecting the second WD40 motif and connecting region):

  • Located either within the second WD40 motif (c.659A>G p.(Asp220Gly)) or in a disordered protein region connecting the second and third WD40 motifs (c.778G>A p.(Asp260Asn) and c.770C>A p.(Pro257His))

  • Phenotypes overlap with the first cluster but show some distinctions

  • Mutations show normal cellular localization but lower expression levels

  • One variant (p.(Asp220Gly)) loses its ability to bind PACS1 and PACS2

Research insights from these human mutations:

This correlation between mutations and phenotypes provides researchers with valuable insights for designing experiments to understand WDR37's tissue-specific functions and molecular mechanisms.

How can CRISPR-based screening approaches be used to investigate Wdr37 function in immune cells?

CRISPR-based screening approaches offer powerful tools for investigating Wdr37 function in immune cells, as demonstrated by recent research:

• Gain-of-function screening approach:

  • A dgRNA (dead guide RNA) activation system can be used with active Cas9 in primary T cells isolated from Cas9 transgenic mice

  • Design a lentiviral T cell dgRNA activation (TdgA) vector system targeting Wdr37

  • Evaluate functional outcomes using assays such as CD107a expression in CD8+ T cell kill assays

• Loss-of-function screening:

  • Generate CRISPR knockout pools targeting Wdr37 in primary immune cells or immune cell lines

  • Assess effects on calcium flux, antigen receptor signaling, lymphocyte development, and homeostasis

  • Use competitive growth assays to identify fitness effects

• Domain-specific editing:

  • Apply precise editing to introduce specific mutations in WD40 domains or regions connecting them

  • Compare phenotypes to human disease-associated variants

  • Determine structure-function relationships

• Screening readouts for Wdr37 function:

  • Calcium flux measurements using flow cytometry and calcium-sensitive dyes

  • Expression of activation markers (CD69, CD25, CD107a)

  • Cell survival and proliferation assays

  • RNA-seq to identify dysregulated pathways (e.g., cholesterol biosynthesis)

The dgTKS (dgRNA T cell Kill assay activation Screen) approach identified Wdr37 as one of 26 significantly enriched genes that enhance CD8+ T cell function, suggesting potential applications in T cell engineering for immunotherapy .

What are the implications of Wdr37's interaction with PACS proteins for research on cellular trafficking and disease mechanisms?

The discovery that Wdr37 strongly binds and colocalizes with PACS1 and PACS2 (phosphofurin acidic cluster sorting proteins) has significant implications for research:

• Cellular trafficking research:

  • PACS proteins are involved in the sorting of membrane proteins between different cellular compartments

  • The Wdr37-PACS complex likely plays a role in protein trafficking pathways

  • Researchers can investigate how this complex regulates the localization of specific cargo proteins, particularly in the context of calcium signaling components (e.g., IP3Rs)

• Disease mechanism insights:

  • Mutations that disrupt Wdr37-PACS binding (e.g., p.Asp220Gly) may lead to missorting of critical proteins

  • PACS1 mutations are associated with a syndrome characterized by intellectual disability and distinctive facial features (Schuurs-Hoeijmakers syndrome)

  • The overlap between PACS1 syndrome and WDR37 syndrome phenotypes provides a mechanistic framework to understand both conditions

• Experimental approaches to investigate this interaction:

  • Structure determination of the Wdr37-PACS complex using cryo-EM or X-ray crystallography

  • Cargo identification through proximity labeling approaches (BioID, APEX)

  • Trafficking assays using fluorescently tagged reporter proteins

  • Measurement of protein half-lives with cycloheximide chase experiments

• Therapeutic implications:

  • The Pacs1-Wdr37 complex may represent a novel target for lymphoproliferative diseases

  • Disruption of this complex forces lymphocytes out of quiescence, potentially synergizing with existing therapies that target lymphocyte survival factors

  • Screens for small molecules that modulate this interaction could yield compounds with therapeutic potential

Data from cycloheximide pulse assays demonstrates that FLAG-Pacs1 and HA-Wdr37 are expressed at higher levels and decay more slowly when co-expressed compared to when each is expressed separately, confirming their mutual stabilization .

What are the critical considerations when designing site-directed mutagenesis experiments for Wdr37?

When performing site-directed mutagenesis of Wdr37, researchers should consider:

• Target selection based on functional domains:

  • WD40 domains are critical for protein function and interactions

  • The disordered regions connecting WD40 domains are also important for proper folding

  • Disease-associated variants provide high-value targets (e.g., p.Ser119Phe, p.Thr125Ile, p.Ser129Cys, p.Thr130Ile in N-terminal cluster; p.Asp220Gly, p.Asp260Asn, p.Pro257His in second cluster)

• Template selection and primer design:

  • Use a high-fidelity, sequence-verified plasmid containing full-length Wdr37 cDNA (e.g., pReceiver-M14 with Wdr37 inserted at HindIII and XbaI sites)

  • Design primers with the mutation centered and 15-20 nucleotides of matching sequence on each side

  • Ensure primers have similar melting temperatures and GC content

• Mutagenesis protocol optimization:

  • The QuikChange XL Site-Directed Mutagenesis Kit has been successfully used for Wdr37 mutations

  • Adjust extension time based on plasmid length (typically 1 minute/kb)

  • Verify all mutations by Sanger sequencing of the entire Wdr37 coding region

• Functional validation strategies:

  • Expression level assessment by western blot

  • Subcellular localization by immunocytochemistry

  • Protein-protein interaction testing (co-IP with known partners like PACS1/PACS2)

  • Appropriate functional assays (calcium flux in lymphocytes)

How can researchers address variability in detecting low-abundance Wdr37 protein in primary cells and tissues?

Detecting low-abundance Wdr37 in primary cells and tissues presents challenges that can be addressed through these approaches:

• Sample preparation optimization:

  • Use fresh samples whenever possible

  • Include protease inhibitors (complete protease inhibitor cocktail) in all extraction buffers

  • For tissue samples, optimize homogenization conditions to maximize protein extraction while minimizing degradation

  • Compare different lysis buffers (RIPA, NP-40, Triton X-100) to determine optimal extraction conditions

• Enrichment strategies:

  • Immunoprecipitation with validated anti-Wdr37 antibodies before western blot analysis

  • Subcellular fractionation to concentrate the cytoplasmic fraction where Wdr37 predominantly localizes

  • Use of specialized protein extraction kits designed for low-abundance proteins

• Detection method sensitivity enhancement:

  • For western blots: Use high-sensitivity ECL substrates (SuperSignal West Femto) and longer exposure times

  • For immunofluorescence: Use signal amplification systems (tyramide signal amplification)

  • For ELISA: Ensure sample concentrations are diluted to mid-range of the kit (0.156-10 ng/ml)

• Quantification approaches:

  • Use internal loading controls appropriate for the sample type

  • Consider normalization to total protein (Ponceau S staining) rather than single housekeeping proteins

  • Perform technical replicates (at least triplicate) and measure band intensity using image analysis software (e.g., ImageJ)

• Validation of signal specificity:

  • Include positive controls (recombinant Wdr37 protein)

  • Include negative controls (samples from Wdr37-knockout mice where available)

  • Confirm antibody specificity using multiple antibodies targeting different epitopes

The low baseline expression of Wdr37 in primary T cells compared to housekeeping genes like Gapdh has been documented, highlighting the importance of these optimization strategies .

What statistical analyses are most appropriate for interpreting Wdr37 expression or functional data across different experimental conditions?

When analyzing Wdr37 expression or functional data, researchers should consider these statistical approaches:

• For comparing expression levels across groups:

  • For normally distributed data: Two-tailed unpaired t-test (two groups) or one-way ANOVA with appropriate post-hoc tests (multiple groups)

  • For non-normally distributed data: Mann-Whitney U test (two groups) or Kruskal-Wallis with Dunn's post-hoc test (multiple groups)

  • Include multiple biological replicates (n≥3) to ensure statistical power

• For calcium flux experiments:

  • Area under the curve (AUC) analysis for comparing total calcium responses

  • Peak height analysis for maximum response magnitude

  • Time to peak for response kinetics

  • Two-way ANOVA with repeated measures to compare response curves over time

• For protein-protein interaction studies:

  • Quantify band intensities from co-IP experiments and normalize to input control

  • Use paired statistical tests when comparing wild-type and mutant interactions from the same experiment

  • Consider ANOVA with multiple comparisons when testing several mutations simultaneously

• For high-dimensional data (e.g., RNA-seq):

  • Appropriate normalization methods (e.g., DESeq2)

  • False discovery rate (FDR) correction for multiple testing

  • Pathway analysis (GSEA, IPA) to identify affected biological processes

  • Validation of key findings using orthogonal methods (qPCR, western blot)

• Data presentation best practices:

  • Show individual data points in addition to means and error bars

  • Clearly indicate sample sizes and definition of error bars (SEM vs. SD)

  • Include appropriate statistical test information and exact p-values

  • Consider violin or box plots for better visualization of data distribution

For example, when comparing expression levels of wild-type and mutant Wdr37, researchers have successfully used a two-tailed unpaired t-test after normalizing FLAG-tagged protein band intensity to the corresponding WDR37-Myc band intensity across triplicate experiments .

What are the most promising approaches for investigating Wdr37's role in cholesterol biosynthesis pathways?

Based on RNA-seq data showing significant upregulation of cholesterol biosynthesis pathways in zebrafish embryos carrying Wdr37 missense variants , several promising approaches can be pursued:

• Comprehensive lipid profiling:

  • Lipidomic analysis comparing wild-type and Wdr37-mutant/knockout tissues

  • Focus on sterol intermediates of the cholesterol biosynthesis pathway

  • Incorporate isotope labeling (C13-acetate) to measure de novo synthesis rates

  • Compare effects across different tissues (brain, liver, lymphocytes)

• Transcriptional regulation analysis:

  • ChIP-seq for SREBP transcription factors that regulate cholesterol biosynthesis

  • Luciferase reporter assays with cholesterol biosynthesis gene promoters

  • Investigation of potential direct interactions between Wdr37 and transcriptional regulators of lipid metabolism

• Functional metabolic studies:

  • Measure cellular cholesterol content using filipin staining or enzymatic assays

  • Assess responses to cholesterol depletion (statin treatment) or loading

  • Determine if cholesterol pathway modulation can rescue phenotypes in Wdr37-deficient models

  • Investigate membrane fluidity and lipid raft composition

• Structure-function analyses:

  • Map the domains of Wdr37 responsible for cholesterol pathway regulation

  • Determine if this function is independent of or connected to the Pacs1-Wdr37 interaction

  • Investigate potential binding to cholesterol or cholesterol biosynthesis enzymes

• Therapeutic implications:

  • Test whether statins or other cholesterol-lowering drugs mitigate phenotypes in animal models of Wdr37 deficiency

  • Explore connections between cholesterol metabolism and calcium signaling defects

  • Assess tissue-specific requirements for Wdr37 in cholesterol homeostasis

How might understanding Wdr37 function contribute to developing novel therapeutic approaches for lymphoproliferative diseases?

The discovery that the Pacs1-Wdr37 complex regulates lymphocyte quiescence offers promising therapeutic avenues:

• Target identification and validation:

  • Further characterize the structural interface between Pacs1 and Wdr37

  • Identify the minimal interaction domains required for complex formation

  • Develop high-throughput screening assays to identify small molecules that disrupt this interaction

• Preclinical disease models:

  • Test Wdr37 knockout or inhibition in mouse models of:

    • B-cell lymphomas with Bcl2 overexpression

    • T-cell lymphoproliferative disorders (lpr/Fas mutation models)

    • Leukemias driven by other oncogenic events (c-Myc, Bcr-Abl, constitutive Notch)

  • Assess combination therapies with existing agents (venetoclax, ibrutinib, idelalisib)

• Mechanism-based combination approaches:

  • Explore synergies between Pacs1-Wdr37 disruption and:

    • Direct apoptosis inducers (venetoclax/BCL2 inhibitors)

    • Kinase inhibitors (BTK, PI3K)

    • ER stress inducers

    • Calcium signaling modulators

• Therapeutic selectivity assessment:

  • Determine differential effects on malignant versus normal lymphocytes

  • Evaluate impacts on beneficial immune responses to vaccination or infection

  • Assess potential off-target effects in other tissues where Wdr37 functions

Research has shown that Pacs1 deletion does not impair normal humoral responses but strongly blocks lymphoproliferation resulting from Fas mutation and Bcl2 overexpression . This remarkable selectivity for pathological proliferation while preserving normal immune function makes the Pacs1-Wdr37 complex an attractive therapeutic target.

The table below summarizes potential therapeutic applications based on Wdr37 biology: