ALDOC Human, His

Basic Research Questions

• What is the molecular structure and biochemical function of human ALDOC?

  Human ALDOC is a member of the fructose-bisphosphate aldolase class I gene family. It consists of 364 amino acids with a molecular mass of approximately 40.5 kDa . ALDOC is primarily expressed in the hippocampus and Purkinje cells of the brain .

  Functionally, ALDOC catalyzes the reversible conversion of fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This reaction is a critical step in both glycolysis and gluconeogenesis. ALDOC is involved in two major pathways: glucose metabolism and glycosaminoglycan metabolism .

  Recent research has also identified ALDOC's involvement in cancer progression, particularly in non-small cell lung cancer where it affects MYC-mediated transcription and regulates the Wnt/β-catenin pathway .

• How is recombinant His-tagged ALDOC typically produced and purified?

  Recombinant human ALDOC protein with a His-tag can be produced using several expression systems, with insect cell and mammalian expression systems being most common for maintaining proper folding and post-translational modifications.

  According to available research products, His-tagged ALDOC protein is typically produced as follows:

  Expression System	Tag Position	Protein Length	Purification Method
  Insect Cells	C-terminal His	1-364aa	Affinity chromatography
  Human Cells	C-terminal 6His	Phe2-Tyr364	Affinity chromatography

  The expressed protein undergoes affinity purification using nickel or cobalt resin columns that bind the His-tag. Further purification may employ size exclusion chromatography to achieve >90% purity as determined by SDS-PAGE .

• What are the optimal storage and reconstitution conditions for His-tagged ALDOC?

  For optimal stability and activity of His-tagged ALDOC:

  Storage conditions:

  • Store at -20°C to -80°C upon receipt

  • Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

  • Long-term storage should be at -80°C for maximum stability

  Reconstitution protocol:

  • For lyophilized protein, it is recommended to add sterile water to prepare a stock solution of 0.2 µg/µl

  • Centrifuge the vial at 4°C before opening to recover the entire contents

  • After reconstitution, the protein solution can be stored in Tris/PBS-based buffer with 5-50% glycerol

  Working solution stability:

  • Minimize freeze-thaw cycles as they can lead to protein denaturation

  • For experiments, maintain the protein on ice when not in use

  • Stability studies indicate that aliquoted protein maintains >95% activity for 6 months when stored properly

Advanced Research Questions

• How can researchers design experiments to study ALDOC's role in cancer progression?

  To investigate ALDOC's role in cancer progression, researchers can implement the following experimental design approach:

  1. Expression Analysis:

  • Immunohistochemistry for comparing ALDOC protein expression in tumor tissues versus normal tissues

  • qRT-PCR and Western blot analysis to quantify ALDOC expression levels

  2. Functional Studies:

  • Gene knockdown using siRNA or CRISPR-Cas9 to assess the effect of ALDOC depletion on cancer cell proliferation, migration, and invasion

  • Overexpression studies using vector-based ALDOC expression systems

  3. Pathway Analysis:

  • Investigation of MYC-mediated transcriptional effects, as ALDOC has been shown to affect MYC-mediated UBE2N transcription

  • Assessment of Wnt/β-catenin pathway activation using reporter assays such as TOP/FOP flash

  4. In vivo Models:

  • Xenograft models with ALDOC-modulated cancer cells to evaluate tumor growth and metastasis

  • Patient-derived xenografts to maintain tumor heterogeneity

  5. Multi-omics Integration:

  • Combine proteomics, transcriptomics, and metabolomics data to understand the comprehensive role of ALDOC in cancer metabolism

  Recent studies have demonstrated that ALDOC promotes non-small cell lung cancer through affecting MYC-mediated UBE2N transcription and regulating the Wnt/β-catenin pathway , providing a methodological template for similar studies in other cancer types.

• What experimental approaches can elucidate ALDOC's role in immune infiltration and the tumor microenvironment?

  Research has shown that ALDOC is significantly associated with immune infiltration in gastric cancer and regulates macrophage differentiation . To investigate this aspect of ALDOC function, researchers can employ:

  1. Immune Cell Profiling:

  • Flow cytometry to quantify immune cell populations in models with altered ALDOC expression

  • Multiplex immunohistochemistry to visualize the spatial distribution of immune cells relative to ALDOC-expressing cells

  2. Co-culture Systems:

  • Design co-culture experiments with cancer cells (ALDOC-modulated) and immune cells (macrophages, T cells)

  • Assess changes in immune cell function, polarization, and cytokine production

  3. Cytokine/Chemokine Analysis:

  • Multiplex ELISA or cytokine arrays to profile secreted factors

  • qRT-PCR to measure changes in cytokine/chemokine gene expression

  4. Transcriptomic Analysis:

  • RNA-seq of immune cells exposed to conditioned media from ALDOC-overexpressing or ALDOC-silenced cancer cells

  • Bioinformatic analysis to identify enriched immune-related pathways

  5. In vivo Immune Monitoring:

  • Use of immunocompetent mouse models to study how ALDOC modulation affects immune infiltration

  • Single-cell RNA-seq of tumor-infiltrating immune cells

  This comprehensive approach can help elucidate how ALDOC influences the tumor microenvironment and immune response, expanding on findings that ALDOC regulates macrophage differentiation in gastric cancer .

• How to design Taguchi experimental methods to optimize ALDOC protein production and purification?

  Taguchi's experimental design is a statistical method that can efficiently optimize ALDOC protein production by identifying the most influential factors while minimizing the number of experiments . For ALDOC-His production optimization:

  1. Factor Identification and Level Definition:

  Factor	Low Level	High Level
  Temperature	25°C	37°C
  Induction time	4 hours	24 hours
  IPTG concentration	0.1 mM	1.0 mM
  Media composition	Minimal	Rich
  Cell density at induction	OD600 0.6	OD600 1.2
  pH	6.8	7.5

  2. Orthogonal Array Selection:

  • For 6 factors at 2 levels, use an L8 orthogonal array (8 experiments instead of 64 with full factorial)

  3. Response Variables:

  • Protein yield (mg/L culture)

  • Protein purity (% by SDS-PAGE)

  • Functional activity (enzymatic assay)

  4. Analysis Methodology:

  • Calculate signal-to-noise ratios for each factor level

  • Perform analysis of variance (ANOVA) to determine statistical significance

  • Identify optimal factor settings for maximum ALDOC production

  5. Confirmation Experiment:

  • Conduct validation runs using the predicted optimal conditions

  • Compare actual results with predicted results

  This approach allows systematic optimization of ALDOC production conditions while maintaining robust process performance against external variations, ensuring consistently high-quality protein for research applications .

• What analytical techniques are most effective for studying ALDOC interactions with transcription factors like MYC?

  Given ALDOC's involvement in affecting MYC-mediated transcription , several techniques can be employed to study this interaction:

  1. Protein-Protein Interaction Assays:

  • Co-immunoprecipitation (Co-IP) with antibodies against ALDOC and MYC

  • Proximity ligation assay (PLA) to visualize interactions in situ

  • FRET or BRET assays using fluorescently tagged proteins

  • Yeast two-hybrid screening to identify interaction domains

  2. Chromatin-Associated Studies:

  • Chromatin immunoprecipitation (ChIP) to assess MYC binding to target genes in the presence/absence of ALDOC

  • ChIP-seq to analyze genome-wide binding patterns

  • Re-ChIP (sequential ChIP) to confirm co-occupancy of ALDOC and MYC at specific genomic loci

  3. Functional Transcription Assays:

  • Luciferase reporter assays with MYC-responsive promoters

  • Gene expression analysis following ALDOC modulation, focusing on known MYC target genes

  4. Domain Mapping:

  • Expression of truncated versions of ALDOC to identify domains required for MYC interaction

  • Site-directed mutagenesis of key residues to pinpoint specific interaction sites

  5. Structural Analysis:

  • X-ray crystallography or cryo-EM of ALDOC-MYC complexes

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  6. Kinetic and Thermodynamic Measurements:

  • Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to determine binding affinities and kinetics

  These approaches provide complementary information about how ALDOC affects MYC-mediated transcription, which has been implicated in cancer progression .

• How can researchers distinguish between the metabolic and non-metabolic functions of ALDOC in experimental settings?

  ALDOC has both canonical metabolic functions in glycolysis and non-canonical roles in cancer progression and immune regulation . To distinguish between these functions:

  1. Catalytic Mutant Studies:

  • Generate catalytically inactive ALDOC mutants (targeting active site residues)

  • Compare phenotypes between wildtype ALDOC and catalytic mutants to separate enzymatic from non-enzymatic functions

  2. Subcellular Localization Analysis:

  • Fluorescent tagging and confocal microscopy to track ALDOC localization

  • Subcellular fractionation followed by Western blotting

  • Correlation of non-glycolytic functions with nuclear or membrane localization versus cytoplasmic (glycolytic) functions

  3. Metabolic Flux Analysis:

  • Use of isotope-labeled glucose (13C) followed by mass spectrometry to measure glycolytic flux

  • Seahorse XF analyzer to measure extracellular acidification rate (ECAR) and oxygen consumption rate (OCR)

  • Comparing metabolic changes with non-metabolic phenotypes after ALDOC modulation

  4. Rescue Experiments:

  • After ALDOC knockdown, attempt to rescue phenotypes by:
    a) Adding glycolytic metabolites (bypassing ALDOC's enzymatic function)
    b) Re-expressing wildtype or mutant ALDOC

  • Different rescue patterns can distinguish metabolic from non-metabolic roles

  5. Proteomics Approach:

  • Identify ALDOC interaction partners using mass spectrometry

  • Network analysis to separate metabolic partners from signaling partners

  These methods allow researchers to deconvolute the multifaceted roles of ALDOC, particularly important when studying its contributions to cancer progression that extend beyond glycolysis .