Recombinant Chicken WD repeat-containing protein 24 (WDR24), partial

What is WDR24 and what is its primary function in cellular systems?

WDR24 (WD repeat domain 24) is a protein-coding gene that produces a WD40 repeat-containing protein that serves as a critical component of the GATOR2 (GAP Activity Toward Rags) complex. The primary function of WDR24 is to act as a catalytic component within the GATOR2 complex, which plays an essential role in the amino acid-sensing branch of the mTORC1 signaling pathway. Specifically, WDR24 helps activate mTORC1 by indirectly inhibiting the GATOR1 subcomplex .

In cellular systems, WDR24 functions as an E3 ubiquitin-protein ligase targeting the GATOR1 complex. When amino acids are abundant, the WDR24-containing GATOR2 complex ubiquitinates the NPRL2 component of GATOR1, leading to GATOR1 inactivation and subsequent mTORC1 activation. This mechanism represents a crucial link between nutrient sensing and cellular growth regulation .

How does the GATOR2 complex function, and what is WDR24's specific role within it?

The GATOR2 complex functions as a positive regulator of mTORC1 signaling by inhibiting the GATOR1 complex, which itself acts as a negative regulator of mTORC1. Within this regulatory system, WDR24 serves as the catalytic subunit of GATOR2, mediating 'Lys-6'-linked ubiquitination of NPRL2 (a core component of GATOR1) when amino acids are abundant .

Studies in Drosophila have shown that WDR24 appears to be the core effector of GATOR2 activity, with other components like Mio and Seh1 functioning primarily as positive regulators of GATOR2 activity. This hierarchical organization suggests that WDR24 is essential for the GATOR2 complex to oppose GATOR1 activity in both germline and somatic cells .

What cellular pathways beyond mTORC1 signaling involve WDR24?

Beyond its well-established role in mTORC1 signaling, WDR24 plays significant roles in:

• Lysosomal function: WDR24 contributes to lysosome acidification, which is essential for the proper functioning of lysosomal enzymes and degradative processes .

• Autophagic flux: Research in both Drosophila and human cell lines demonstrates that WDR24 is necessary for efficient autophagic flux. In WDR24-deficient cells, there is notable accumulation of autolysosomes, LC3-II, and p62, indicating impaired autophagosomal degradation .

• Glucose sensing: Recent research has identified WDR24 as a component in glucose-responsive signaling. The AMPK-dependent phosphorylation of WDR24 represents a mechanistic link between glucose availability and mTORC1 activity regulation .

How does AMPK-dependent phosphorylation of WDR24 regulate the glucose-sensing capability of mTORC1?

AMPK-dependent phosphorylation of WDR24 represents a crucial mechanism linking glucose availability to mTORC1 activity. During glucose deprivation, increased AMPK activity leads to direct phosphorylation of WDR24 at the serine 155 residue (S155). This phosphorylation event disrupts the structural integrity of the GATOR2 complex, thereby relieving its inhibition of GATOR1, which subsequently suppresses mTORC1 activation .

The significance of this phosphorylation has been validated through in vivo studies with phospho-mutant mouse models. Phosphomimetic Wdr24 S155D knock-in mice exhibit early embryonic lethality and reduced mTORC1 activity, while phospho-deficient Wdr24 S155A knock-in mice show increased resistance to fasting conditions and elevated mTORC1 activity compared to wild-type littermates. These findings establish that AMPK-mediated phosphorylation of WDR24 serves as a molecular switch that modulates glucose-induced mTORC1 activation, providing a potential therapeutic target for diseases characterized by aberrant mTORC1 signaling .

What experimental models have been most effective for studying WDR24 function?

Several experimental models have proven valuable for investigating WDR24 function:

Drosophila models: The wdr24 mutant (CG7609) in Drosophila has been particularly informative. This model contains a 1.3 kb deletion removing 1163 bp of coding region including the start codon, resulting in a null allele. While wdr24 is not required for viability in Drosophila, mutants exhibit reduced egg chamber growth, female sterility, and accumulation of autolysosomes, making this model excellent for studying WDR24's role in growth and autophagy .

Mammalian cell culture: HeLa cell lines with WDR24 knockout (-/-) have successfully recapitulated the phenotypes observed in Drosophila mutants, including decreased TORC1 activity and accumulation of autolysosomes. These cell models allow for detailed biochemical analyses of WDR24's impact on lysosomal function and autophagic flux .

Mouse models: Genetically engineered mice with specific phosphorylation site mutations (S155D and S155A) have been instrumental in understanding WDR24's role in glucose sensing and mTORC1 regulation in vivo. These models demonstrate the physiological consequences of altered WDR24 phosphorylation states .

What is the relationship between WDR24 and lysosomal function/autophagic flux?

WDR24 plays a critical dual role in regulating both lysosomal function and autophagic flux:

Autophagic flux: Multiple experimental approaches have demonstrated that WDR24 deficiency leads to impaired autophagic flux. In WDR24 knockout HeLa cells, LC3-II levels are dramatically increased, while treatment with the lysosomal inhibitor chloroquine fails to further increase LC3-II levels (unlike in wild-type cells). This indicates a pre-existing block in autophagic flux independent of chloroquine treatment .

Autolysosome accumulation: Both Drosophila wdr24 mutants and human WDR24-deficient cells accumulate enlarged autolysosomes filled with undegraded material. In Drosophila models, these lysosomes fail to quench the GFP fluorescence of a GFP-mCherry-Atg8a fusion protein, providing further evidence of decreased lysosomal pH and degradative capacity .

p62 accumulation: WDR24-deficient cells show increased levels of p62, a ubiquitin-binding protein normally degraded through autophagy. This accumulation serves as another marker of reduced autophagic flux and confirms that WDR24 is required for efficient clearance of autophagy substrates .

What are the optimal expression systems for recombinant chicken WDR24 production?

Multiple expression systems have been developed for recombinant chicken WDR24 production, each with specific advantages depending on the research application:

What techniques are recommended for studying WDR24 phosphorylation dynamics?

To effectively study WDR24 phosphorylation dynamics, particularly at the critical S155 residue implicated in AMPK-dependent regulation, researchers should consider the following methodological approaches:

• Phospho-specific antibodies: Developing and utilizing antibodies that specifically recognize phosphorylated S155 on WDR24 enables direct detection of this modification in response to various stimuli like glucose deprivation.

• Phosphomimetic and phospho-deficient mutants: Creating S155D (phosphomimetic) and S155A (phospho-deficient) mutants allows researchers to assess the functional consequences of WDR24 phosphorylation in cellular contexts before moving to more complex in vivo models .

• In vitro kinase assays: Reconstituting the phosphorylation reaction using purified AMPK and recombinant WDR24 can confirm direct phosphorylation and enable quantitative measurements of phosphorylation kinetics.

• Mass spectrometry: Phosphoproteomic analysis provides unbiased detection of all phosphorylation sites on WDR24, potentially revealing additional regulatory modifications beyond S155.

• AMPK activators/inhibitors: Using pharmacological modulators of AMPK activity (e.g., AICAR as an activator, Compound C as an inhibitor) in conjunction with WDR24 phosphorylation detection can establish the AMPK-dependence of this modification in various physiological contexts .

What assays should be employed to measure WDR24's impact on autophagic flux?

To comprehensively assess WDR24's impact on autophagic flux, researchers should implement a combination of the following methodological approaches:

• LC3-II western blotting with and without lysosomal inhibitors: Measuring LC3-II levels in the presence and absence of chloroquine or bafilomycin A1 helps distinguish between increased autophagosome formation and decreased autophagosome clearance. In WDR24-deficient cells, LC3-II levels are already elevated and show minimal further increase with chloroquine treatment, indicating impaired autophagic flux .

• p62 accumulation: Monitoring p62 levels by western blotting or immunofluorescence provides insight into autophagic substrate clearance. Increased p62 in WDR24-deficient conditions indicates reduced autophagic degradation capacity .

• Tandem fluorescent-tagged LC3 (mRFP-GFP-LC3 or GFP-mCherry-Atg8a): This approach leverages the pH sensitivity of GFP to distinguish between autophagosomes (both GFP and RFP/mCherry positive) and autolysosomes (only RFP/mCherry positive due to GFP quenching in acidic environments). WDR24-deficient cells maintain GFP signal in presumptive autolysosomes, suggesting impaired lysosomal acidification .

• LysoTracker staining: This fluorescent dye accumulates in acidic organelles and can be used to assess lysosomal number and acidification status. WDR24 mutants typically show altered LysoTracker staining patterns .

• Transmission electron microscopy: Ultrastructural analysis of autophagic structures can reveal the accumulation of autolysosomes containing undegraded material in WDR24-deficient cells, providing direct visual evidence of impaired autophagic flux .

How might targeting the AMPK-WDR24 signaling axis offer therapeutic potential for mTORC1-related diseases?

The AMPK-WDR24 signaling axis represents a promising therapeutic target for diseases characterized by dysregulated mTORC1 activity. Research suggests that specifically modulating WDR24 phosphorylation at S155 could provide a more nuanced approach to controlling mTORC1 activity compared to direct mTORC1 inhibitors like rapamycin .

This approach may be particularly valuable for cancer treatment, where aberrant mTORC1 activation contributes to uncontrolled cell growth and proliferation. By targeting WDR24 phosphorylation, it may be possible to inhibit mTORC1 only under specific metabolic conditions (like glucose limitation), potentially reducing the side effects associated with blanket mTORC1 inhibition .

Emerging data from phospho-mutant mouse models supports this therapeutic potential. While complete WDR24 deficiency has severe developmental consequences, fine-tuning its activity through targeted modulation of its phosphorylation state offers a more subtle regulatory approach that could be therapeutically advantageous .

What is the relationship between WDR24 dysfunction and the diseases associated with the WDR24 gene?

The WDR24 gene has been associated with several diseases, primarily hematological disorders and neurological conditions. These include substance abuse, primary familial polycythemia, alpha thalassemia-intellectual disability syndrome type 1, and various hemoglobin-related disorders .

The mechanistic link between WDR24 dysfunction and these diseases likely involves its roles in:

• mTORC1 signaling regulation: Altered mTORC1 activity affects cell growth, proliferation, and protein synthesis, which may contribute to the developmental aspects of conditions like alpha thalassemia-intellectual disability syndrome.

• Autophagy and lysosomal function: Impaired protein and organelle turnover due to defective autophagy could contribute to neurological manifestations seen in some WDR24-associated conditions.

• Metabolic sensing: WDR24's role in coordinating nutrient availability with cellular growth may explain its association with metabolic conditions and substance abuse vulnerability.

What are the recommended reconstitution and storage protocols for recombinant WDR24?

For optimal handling of recombinant chicken WDR24 protein, researchers should follow these protocols:

Reconstitution procedure:

• Briefly centrifuge the vial containing lyophilized protein prior to opening

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 50% (or between 5-50% as needed for specific applications)

• Aliquot the reconstituted protein for long-term storage

Storage conditions:

• Store lyophilized powder at -20°C/-80°C upon receipt

• Store reconstituted protein aliquots at -20°C/-80°C

• Avoid repeated freeze-thaw cycles as this significantly reduces protein activity

• Working aliquots can be kept at 4°C for up to one week

The shelf life of the protein depends on several factors including storage temperature, buffer composition, and the intrinsic stability of the protein. Proper aliquoting and storage at -80°C generally ensures maximum retention of biological activity .

How can researchers effectively design knock-in models to study WDR24 phosphorylation?

Based on successful studies with WDR24 phospho-mutants, researchers should consider the following approach for designing knock-in models:

• Target residue selection: The serine 155 (S155) residue has been identified as a critical AMPK phosphorylation site in WDR24, making it an optimal target for phospho-mutant studies .

• Mutation design:

  • S155D mutation (replacing serine with aspartic acid) effectively mimics constitutive phosphorylation

  • S155A mutation (replacing serine with alanine) prevents phosphorylation at this site

• Genetic engineering techniques:

  • CRISPR/Cas9-mediated homology-directed repair allows for precise introduction of the desired mutations

  • Include silent mutations in the PAM site or guide RNA binding region to prevent re-cutting of successfully edited alleles

• Validation methods:

  • Genomic PCR and sequencing to confirm the presence of the desired mutation

  • Western blotting to verify WDR24 protein expression levels

  • Functional assays to assess mTORC1 activity (e.g., S6K phosphorylation) under various conditions

• Phenotypic analysis:

  • Embryonic development assessment (since S155D mutants exhibit embryonic lethality)

  • Metabolic stress response studies (particularly fasting resistance in S155A mutants)

  • Tissue-specific analyses of mTORC1 activity and autophagy markers

What controls are essential when studying WDR24's role in autophagic flux?

When investigating WDR24's impact on autophagic flux, the following controls are essential to ensure experimental validity and accurate interpretation of results:

• Genetic controls:

  • Include both heterozygous and wild-type controls alongside homozygous WDR24 mutants

  • Use genetic rescue experiments (expressing wild-type WDR24 in mutant backgrounds) to confirm phenotype specificity

• Double mutant controls:

  • Generate WDR24 and autophagy gene (e.g., Atg7) double mutants to distinguish between autophagy-dependent and autophagy-independent phenotypes

  • Example: LysoTracker-positive puncta persisting in WDR24/Atg7 double mutants indicates that these structures are lysosomes rather than autolysosomes

• Pharmacological controls:

  • Include appropriate controls for lysosomal inhibitors (chloroquine, bafilomycin A1)

  • Compare LC3-II accumulation patterns with and without inhibitors to distinguish between increased autophagosome formation and decreased clearance

• Nutrient status controls:

  • Perform experiments under both normal and nutrient-deprived conditions to assess basal versus induced autophagy

  • Control for consistent starvation protocols as variations in amino acid or glucose availability significantly impact results

• Reporters and markers:

  • Use multiple independent markers (LC3, p62, LAMP1) to corroborate findings

  • For tandem fluorescent reporters, include controls that artificially neutralize lysosomal pH to validate the reporter's pH sensitivity