RAB24 Human
Description
RAB24 is fused to a 24 amino acid His-tag at N-terminus & purified by proprietary chromatographic techniques.
Product Specs
Q&A
What are the primary molecular functions of RAB24 in human cells?
RAB24 operates as an atypical small GTPase with roles in autophagic compartment clearance, cell division, and cancer cell survival. Unlike canonical RAB proteins, it exhibits unique biochemical properties, including weaker GTPase activity and preferential localization to autophagic vacuoles under nutrient-rich conditions . Key methodologies to study its functions include:
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siRNA silencing paired with transmission electron microscopy to quantify autophagic vacuoles .
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Immunofluorescence colocalization studies with LC3 (autophagosome marker) and tubulin (microtubule marker) .
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Subcellular fractionation combined with proteinase protection assays to map membrane association .
Table 1: Experimental Models for RAB24 Functional Studies
How does RAB24 contribute to autophagic flux under basal vs. starvation conditions?
RAB24 is critical for basal autophagy termination but dispensable during acute nutrient deprivation. Under nutrient-rich conditions, RAB24 facilitates autolysosome clearance, as shown by:
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mRFP-GFP-LC3 reporter assays: RAB24 silencing causes accumulation of acidic autolysosomes (GFP-negative/RFP-positive puncta) .
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Long-lived protein degradation assays: ~20% reduction in degradation efficiency in RAB24-depleted cells .
In contrast, during 2-hour amino acid starvation (e.g., EBSS treatment), RAB24 silencing does not impair autophagosome formation or clearance .
What disease associations have been identified for RAB24 dysregulation?
RAB24 perturbations are linked to:
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Neurodegeneration: Mutations cause ataxia in dogs; reduced clearance of polyglutamine aggregates in human cell models .
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Cancer: Overexpression in hepatocellular carcinoma enhances cell motility and survival .
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Metabolic disorders: RAB24 overexpression correlates with hepatic steatosis .
Key evidence includes transcriptomic analyses of carotid atherosclerosis plaques showing reduced RAB24 mRNA in symptomatic patients .
How can contradictory findings about RAB24’s role in autophagy be resolved?
Discrepancies arise from context-dependent roles (e.g., basal vs. induced autophagy) and model system variability. To address this:
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Conditional knockout models: Use tissue-specific RAB24 deletions to isolate physiological roles.
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Autophagic flux assays: Combine lysosomal inhibitors (bafilomycin A1) with LC3-II immunoblotting to distinguish formation vs. clearance defects .
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Single-cell imaging: Track RAB24 dynamics in real-time using HaloTag fusion proteins.
Table 2: Contradictory Findings in RAB24 Studies
What are the implications of RAB24’s atypical post-translational modifications?
RAB24 exhibits divergent prenylation requirements and tyrosine phosphorylation:
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Prenylation mutants (CCΔ/CC→SS): Fail to localize to autophagic compartments, impairing clearance .
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Phosphorylation mutants (Y17F/Y172F): Retain partial function, suggesting phosphorylation fine-tunes activity .
Methodological recommendations: -
Metabolic labeling: Use ³H-mevalonate to assess prenylation efficiency.
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Phosphoproteomics: Combine anti-phosphotyrosine IP with mass spectrometry in stress conditions.
How does RAB24 interact with microtubules during mitosis?
RAB24 localizes to the mitotic spindle and midbody, regulating chromosome segregation and cytokinesis. Key approaches:
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Live-cell imaging of GFP-RAB24: Reveals dynamic association with microtubules during metaphase-to-telophase transitions .
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Co-sedimentation assays: Confirm direct binding to taxol-stabilized microtubules .
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Phenotypic analysis: RAB24 depletion causes chromatin bridges (90% of HeLa cells) and multinucleation .
What technical challenges exist in studying RAB24’s GTPase activity?
Challenges stem from its low intrinsic GTP hydrolysis rate and predominant GTP-bound state. Solutions include:
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Fluorescent GTP analogs: Use BODIPY-FL-GTPγS to monitor nucleotide binding in vitro.
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Dominant-negative mutants: Overexpress RAB24-S67N (GDP-locked) to block effector interactions .
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Structural studies: Cryo-EM of RAB24-GTP complexes to identify unique effector domains.
How could RAB24 be targeted therapeutically in cancer?
Strategy 1: Inhibit oncogenic RAB24 in hepatocellular carcinoma via:
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Autophagy-dependent approach: Small molecules blocking RAB24-LC3 interaction (e.g., virtual screening of C-terminal domain binders).
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Autophagy-independent approach: CRISPRi to suppress RAB24-driven motility pathways.
Strategy 2: Enhance RAB24 in neurodegenerative diseases using AAV-mediated overexpression in neuronal models .
Methodological Recommendations
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For autophagy studies: Use tandem mRFP-GFP-LC3 reporters + lysosomal inhibitors to isolate RAB24’s role in autophagic termination .
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For mitotic roles: Combine siRNA silencing with time-lapse microscopy (≥48-hour imaging) to capture multinucleation events .
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For disease modeling: Prioritize patient-derived xenografts (PDXs) over immortalized lines to capture endogenous RAB24 expression dynamics .
Product Science Overview
The RAB24 gene is a protein-coding gene associated with several biological pathways, including the innate immune system and protein metabolism . The gene is located on chromosome 5 and has several aliases, including Ras-Related Protein Rab-24 . The protein encoded by this gene is involved in autophagy-related processes, which are essential for cellular homeostasis and the degradation of unnecessary or dysfunctional cellular components .
RAB24, like other Rab proteins, functions as a molecular switch that cycles between an active GTP-bound state and an inactive GDP-bound state. This cycling is crucial for its role in regulating intracellular trafficking. The protein’s GTPase activity allows it to hydrolyze GTP to GDP, thereby switching from an active to an inactive state .
RAB24 is expressed in various tissues, including lymphoid tissue, bone marrow, testis, skeletal muscle, and several others . Its expression is particularly notable in cells involved in the immune response and protein ubiquitination processes . The protein’s localization within cells is essential for its function in autophagy and intracellular trafficking.
Mutations or dysregulation of the RAB24 gene have been associated with certain diseases, including Bartter Syndrome, Type 2, Antenatal, and Migraine With or Without Aura 1 . Understanding the role of RAB24 in these conditions can provide insights into potential therapeutic targets for these diseases.
Research on RAB24 continues to uncover its various roles in cellular processes and its potential implications in disease. The recombinant form of the human RAB24 protein is used in various research applications to study its function and interactions with other proteins.
