PRPS1 Antibody

What is PRPS1 and why is it significant in biological research?

PRPS1 (Phosphoribosyl pyrophosphate synthetase 1) is a key enzyme that catalyzes the synthesis of phosphoribosylpyrophosphate (PRPP), which is essential for nucleotide synthesis. This enzyme plays a critical role in controlling the pentose phosphate pathway (PPP) by catalyzing the phosphoribosylation of D-ribose 5-phosphate (R-5P) to 5-phosphoribosyl-1-pyrophosphate. PRPS1 is significant in biological research because it:

• Controls purine biosynthesis and supplies precursors for DNA and RNA synthesis

• Regulates the PPP through a feedback loop of the PRPS1 substrate R-5P

• Has implications in diverse developmental processes

• Is involved in multiple disease states including cancer and genetic disorders

In humans, PRPS1 is one of three isoforms (PRPS1, PRPS2, and PRPS1L1) that have been identified, with PRPS1 being the most ubiquitously expressed gene of the three .

What are the typical applications for PRPS1 antibodies in research settings?

PRPS1 antibodies are utilized across multiple research applications including:

• Western blotting (WB) for protein expression level detection

• Immunohistochemistry (IHC-P) for tissue localization studies

• Immunocytochemistry (ICC) and immunofluorescence (IF) for cellular localization

• Immunoprecipitation (IP) for protein-protein interaction studies

• ChIP assays for studying transcriptional regulation

These applications enable researchers to investigate PRPS1 expression patterns, subcellular localization, protein interactions, and functional roles in various experimental contexts .

What are the optimal conditions for using PRPS1 antibodies in Western blotting?

For optimal Western blotting results with PRPS1 antibodies, researchers should consider:

• Sample preparation: Using freshly prepared whole cell lysates (30-50 μg of protein)

• Gel concentration: 7.5-10% SDS-PAGE gels work well for PRPS1 detection

• Dilution ratios: Antibody dilutions between 1:1000 to 1:10000 have been reported effective, depending on the specific antibody

• Predicted band size: Approximately 34 kDa for human PRPS1

• Compatible samples: Human (cell lines like HeLa, 293T, HCT116), mouse, and rat tissues have been successfully tested

It is recommended to include appropriate positive controls and optimize blocking conditions to minimize background signal. When investigating specific phosphorylation states (such as S103 phosphorylation), specialized phospho-specific antibodies may be required .

How is PRPS1 enzyme activity measured in research settings?

The measurement of PRPS1 enzymatic activity involves several key steps:

• Protein isolation and purification:

  • Immunoprecipitation using specific antibodies (common for tissue samples)

  • Affinity purification with tagged PRPS1 (e.g., FLAG-tagged PRPS1)

• Activity assay conditions:

  • Reaction buffer composition: 50 mmol/L Tris-HCl (pH 7.4), 5 mmol/L MgCl₂, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 32 mmol/L Na₃PO₄, 0.5 mmol/L ATP, 0.15 mmol/L ribose 5'-phosphate, and 0.25 mmol/L P1P5-diadenosine pentaphosphate Ap5A

  • Incubation: 37°C for 15 minutes

  • Reaction termination: Equal volume of 0.1 mol/L EDTA

• For tissue samples:

  • Homogenization and centrifugation to remove debris

  • Antibody purification of PRPS1 from the supernatant

  • Activity measurement using standardized protocols

This methodology allows for quantitative comparison of PRPS1 activity between experimental conditions or between normal and pathological states.

What sample preparation techniques enhance the specificity of PRPS1 immunodetection?

To enhance specificity in PRPS1 immunodetection, researchers should consider:

• Fixation methods:

  • For IHC-P: Heat-mediated antigen retrieval using 10mM citrate buffer (pH 6.0) is recommended prior to staining

  • For ICC/IF: Methanol fixation has demonstrated good results with PRPS1 antibodies

• Blocking optimization:

  • Use appropriate blocking agents to reduce non-specific binding

  • Optimize antibody concentration through titration experiments

• Controls:

  • Include tissues or cells with known PRPS1 expression levels as positive controls

  • Include PRPS1 knockdown or knockout samples as negative controls when available

  • Consider testing different antibody clones targeting distinct epitopes for validation

• For phosphorylation-specific detection:

  • Include phosphatase treatment controls

  • Use phospho-specific antibodies (such as those that detect S103 phosphorylation)

These techniques help minimize background and ensure reliable, reproducible results across different experimental conditions.

How does PRPS1 contribute to cancer progression?

PRPS1 plays multiple roles in cancer progression through several mechanisms:

• Cell cycle regulation:

  • PRPS1 activity peaks during S phase of the cell cycle

  • CDK1 upregulates PRPS1 activity through phosphorylation at S103

  • This phosphorylation enhances nucleotide synthesis necessary for DNA replication

  • Loss of S103 phosphorylation delays cell cycle progression and decreases proliferation

• In melanoma:

  • PRPS1 is significantly upregulated in melanoma tissues compared to normal nevi

  • Higher expression is observed in metastatic melanoma compared to primary melanoma

  • PRPS1 promotes melanoma cell proliferation by:

    • Enhancing expression of cell cycle proteins (cyclin E1, CDK2)

    • Decreasing expression of cell cycle inhibitors (P16)

    • Increasing the proportion of cells in S and G2 phases

• In colorectal cancer:

  • PRPS1 activity is higher in colorectal cancer samples than in adjacent tissues

  • S103 phosphorylation serves as a potential diagnostic indicator

These findings suggest that PRPS1 and particularly its phosphorylated form could serve as both a diagnostic biomarker and therapeutic target in certain cancers.

What mutations in PRPS1 are associated with human diseases?

PRPS1 mutations result in four distinct disease syndromes, each with varying clinical manifestations:

• PRS-I superactivity:

  • Caused by regulatory defects from point mutations in the PRPS1 open reading frame

  • Characterized by uric acid overproduction, mental retardation, ataxia, hypotonia, and hearing impairment

  • Results from increased enzyme activity leading to accelerated nucleotide degradation to uric acid

• X-linked Charcot-Marie-Tooth disease-5 (CMTX5):

  • Characterized by peripheral neuropathy, hearing impairment, and optic atrophy

  • Results from reduced PRPS1 activity

• Arts syndrome:

  • More severe manifestation including central neuropathy and impaired immune system

  • Caused by loss-of-function mutations (e.g., c.455T>C/p.L152P and c.398A>C/p.Q133P)

  • Characterized by undetectable urine hypoxanthine and reduced plasma uric acid levels

  • Sural nerve biopsy shows mild paranodal demyelination indicating peripheral neuropathy

• X-linked nonsyndromic sensorineural deafness (DFN2):

  • Presents as postlingual progressive hearing loss without other symptoms

  • Results from mild reduction in PRPS1 activity

These diverse disease phenotypes highlight the critical importance of properly regulated PRPS1 activity in multiple physiological systems.

What is the role of PRPS1 in immune responses?

Research has revealed several important aspects of PRPS1's role in immune function:

• In yellow drum fish (Nibea albiflora):

  • NaPRPS1 expression is significantly upregulated following Vibrio harveyi infection

  • Expression increases in head-kidney, liver, and brain tissues, peaking at 72 hours post-infection

  • Immunohistochemistry confirms increased NaPRPS1 protein in head-kidney and brain tissues after pathogen exposure

• Interaction with immune signaling pathways:

  • PRPS1 physically interacts with MyD88 (myeloid differentiation factor 88), as demonstrated by pull-down assays and co-immunoprecipitation

  • MyD88 is a key adaptor protein in innate immune signaling pathways

  • This interaction suggests PRPS1 may coordinate with MyD88 to regulate immune responses to pathogens

• Clinical implications:

  • Mutations causing reduced PRPS1 activity (as in Arts syndrome) are associated with impaired immune system function

  • This further supports PRPS1's critical role in normal immune responses

These findings suggest PRPS1 may have broader functions beyond nucleotide synthesis, particularly in coordinating cellular responses to pathogens through interaction with key immune signaling molecules.

How can researchers investigate the phosphorylation status of PRPS1?

Investigating PRPS1 phosphorylation requires specialized techniques:

• Mass spectrometry-based phosphorylation site mapping:

  • Immunoprecipitate PRPS1 (using tagged versions like PRPS1-FLAG)

  • Subject samples to SDS-PAGE separation

  • Perform in-gel trypsin digestion

  • Analyze peptides by LC/MS-MS (Obitrap-XL mass spectrometer)

  • Identify phosphopeptides using database search tools (Mascot, Sequest)

  • Confirm with phosphopeptide analysis software (PhosphoRS)

• In vitro kinase assays:

  • Express and purify recombinant PRPS1 (e.g., GST-PRPS1)

  • Incubate with purified kinases (such as His-CDK1)

  • Use kinase buffer containing ATP

  • Detect phosphorylation by western blotting with phospho-specific antibodies

• Phospho-specific antibodies:

  • Use antibodies that specifically detect phosphorylated residues (e.g., S103)

  • Validate specificity using phosphatase-treated samples as controls

  • Apply in western blotting, IHC, or IF applications to detect phosphorylated PRPS1

These approaches allow researchers to investigate the regulatory mechanisms controlling PRPS1 activity and its role in various cellular processes.

What are the best approaches for studying protein-protein interactions involving PRPS1?

Several complementary approaches can be used to study PRPS1 protein interactions:

• GST pull-down assays:

  • Express and purify GST-tagged PRPS1 from E. coli

  • Immobilize on glutathione resin

  • Incubate with cell or tissue lysates

  • Elute bound proteins and analyze by SDS-PAGE

  • Identify interaction partners by mass spectrometry

  • This approach successfully identified MyD88 as a PRPS1-interacting protein

• Co-immunoprecipitation (Co-IP):

  • Use antibodies against endogenous PRPS1 or tagged versions

  • Perform reciprocal Co-IPs to confirm interactions

  • Western blot to detect specific interacting proteins

  • This method validated the PRPS1-MyD88 interaction

• Proximity-based labeling:

  • Express PRPS1 fused to enzymes like BioID or APEX

  • Allow biotinylation of proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

• Fluorescence techniques:

  • Fluorescence resonance energy transfer (FRET)

  • Bimolecular fluorescence complementation (BiFC)

  • These provide spatial information about interactions in living cells

These approaches provide complementary information about PRPS1's interactome and help elucidate its role in various cellular pathways beyond its enzymatic function.

What experimental models are most suitable for studying PRPS1 function?

Based on the research literature, several experimental models have proven valuable for PRPS1 research:

• Cell line models:

  • Human cancer cell lines: HCT116, LoVo (colorectal cancer), A875, SK-MEL-110 (melanoma), HeLa, and 293T cells

  • These models are suitable for:

    • Overexpression and knockdown studies

    • Cell proliferation and cell cycle analyses

    • Protein interaction studies

    • Subcellular localization studies

• Animal models:

  • Fish models: Yellow drum (Nibea albiflora) for studying immune functions

  • Rodent models: Mouse and rat tissues show cross-reactivity with many PRPS1 antibodies

  • These models enable in vivo studies of PRPS1 function in complex physiological contexts

• Patient-derived samples:

  • Erythrocytes and fibroblasts from patients with PRPS1 mutations

  • Tissue samples from patients with various PRPS1-related disorders

  • These provide clinically relevant insights into PRPS1 dysfunction

• Genetic manipulation approaches:

  • CRISPR/Cas9 for generating knockout or knock-in models

  • Site-directed mutagenesis to study specific mutations (e.g., phosphorylation sites)

  • Inducible expression systems to control PRPS1 levels temporally

Selection of the appropriate model should be guided by the specific research question, considering factors such as tissue-specific expression patterns and the availability of relevant disease models.

How can researchers address specificity issues with PRPS1 antibodies?

When facing specificity challenges with PRPS1 antibodies, researchers should consider:

• Validation strategies:

  • Test antibodies on PRPS1 knockout or knockdown samples

  • Compare results from multiple antibodies targeting different epitopes

  • Perform peptide competition assays to confirm specificity

  • Validate using recombinant PRPS1 protein as a positive control

• Cross-reactivity considerations:

  • Be aware of potential cross-reactivity with other PRPS isoforms (PRPS2, PRPS1L1)

  • Select antibodies raised against unique regions of PRPS1

  • Confirm specificity in tissues expressing multiple PRPS isoforms

• Application-specific optimization:

  • For Western blotting: Optimize blocking conditions and antibody dilutions

  • For IHC/ICC: Test multiple antigen retrieval methods

  • For IP: Compare different lysis conditions and buffer compositions

• Consider using tagged PRPS1 constructs:

  • When possible, use epitope-tagged versions (FLAG, HA, etc.) for which highly specific antibodies are available

  • This strategy was successfully employed in several PRPS1 studies

These approaches help ensure that experimental observations are truly attributable to PRPS1 rather than to non-specific binding or cross-reactivity.

What controls should be included when measuring PRPS1 enzyme activity?

To ensure reliable PRPS1 activity measurements, researchers should include:

• Positive controls:

  • Samples with known high PRPS1 activity (e.g., proliferating cancer cells)

  • Recombinant PRPS1 protein with confirmed activity

  • Cells overexpressing wild-type PRPS1

• Negative controls:

  • Samples treated with PRPS inhibitors

  • Heat-inactivated enzyme preparations

  • PRPS1 knockdown or knockout samples when available

• Validation controls:

  • Include substrate-free reactions to account for background

  • Test multiple protein concentrations to ensure linearity

  • Include time-course measurements to confirm enzyme stability

• Physiological controls:

  • Compare activity in different cell cycle phases

  • Include both normal and disease-state samples

  • For phosphorylation studies, include phosphatase-treated samples

• Specificity controls:

  • Compare activity in samples with different PRPS isoform expression profiles

  • Include isoform-specific inhibition or depletion controls

These controls help ensure that measured activities accurately reflect PRPS1 function and provide context for interpreting experimental results.

How can researchers distinguish between effects on PRPS1 expression versus activity?

Distinguishing between changes in PRPS1 expression and activity requires parallel analytical approaches:

• Expression analysis techniques:

  • RT-qPCR for mRNA expression levels

  • Western blotting for protein expression levels

  • Immunohistochemistry or immunofluorescence for tissue/cellular distribution

  • These methods quantify the amount of PRPS1 present

• Activity measurement approaches:

  • Enzymatic assays measuring PRPP production

  • Metabolic analyses of downstream products

  • Assessment of phosphorylation state (particularly S103)

  • These methods determine functional capacity

• Comparative analysis:

  • Calculate specific activity (activity per unit of protein)

  • Plot correlation between expression and activity

  • Identify conditions where expression and activity are discordant

• Mechanistic investigations:

  • For post-translational modifications: Use phospho-specific antibodies

  • For protein-protein interactions: Perform co-IP studies

  • For allosteric regulation: Analyze metabolite levels

This integrated approach allows researchers to determine whether changes in PRPS1 function result from altered expression levels, post-translational modifications, protein interactions, or other regulatory mechanisms. The literature demonstrates cases where PRPS1 activity changes independently of expression levels, particularly in cell cycle regulation where phosphorylation at S103 enhances activity without affecting expression .

What is known about the relationship between PRPS1 and the NRF2 pathway in cancer?

Recent research has uncovered an important relationship between PRPS1 and the NRF2 pathway in cancer:

• Regulatory mechanism:

  • NRF2 (Nuclear factor erythroid 2-related factor 2) can direct PRPS1 upregulation

  • This relationship has been documented in melanoma progression

  • ChIP assays using anti-NRF2 antibodies have confirmed this interaction

• Functional significance:

  • NRF2-directed PRPS1 upregulation promotes malignant progression

  • This mechanism influences cell proliferation by affecting cell cycle regulation

  • PRPS1 upregulation increases the proportion of cells in S and G2 phases

  • The pathway enhances expression of cell cycle proteins like cyclin E1 and CDK2

• Translational implications:

  • This pathway represents a potential therapeutic target

  • Dual targeting of NRF2 and PRPS1 might offer synergistic anti-cancer effects

  • PRPS1 expression levels could serve as biomarkers for NRF2 pathway activation

This emerging understanding of the NRF2-PRPS1 axis provides new insights into the complex regulatory networks controlling nucleotide metabolism in cancer cells and offers novel opportunities for therapeutic intervention.

How does PRPS1 intersect with immune signaling pathways?

Research has revealed several important intersections between PRPS1 and immune signaling:

• Direct interaction with immune signaling components:

  • PRPS1 physically interacts with MyD88, a critical adaptor in innate immune signaling

  • This interaction has been confirmed through multiple methods including:

    • Semi-in vivo pull-down assays

    • Mass spectrometry identification

    • Reciprocal pull-down assays

    • Co-immunoprecipitation

• Coordinated expression patterns:

  • Both PRPS1 and MyD88 show inducible expression following pathogen exposure

  • This coordinated response suggests functional cooperation during immune responses

  • In yellow drum fish, both proteins are upregulated following Vibrio harveyi infection

• Potential mechanistic implications:

  • PRPS1 may influence nucleotide availability for immune signaling

  • The interaction with MyD88 suggests PRPS1 might directly modulate immune signal transduction

  • This connection potentially links metabolic regulation with immune function

• Clinical relevance:

  • PRPS1 mutations causing reduced activity (as in Arts syndrome) are associated with impaired immune function

  • This suggests PRPS1's role in immunity extends beyond model organisms to humans

These findings point to a previously unappreciated role for PRPS1 in immune regulation, potentially through both metabolic and non-metabolic mechanisms.

What is the current understanding of PRPS1 in neurodevelopmental disorders?

PRPS1 plays significant roles in neurodevelopmental contexts, as evidenced by several disease associations:

• Neurological manifestations of PRPS1 mutations:

  • Arts syndrome: Central neuropathy, peripheral neuropathy, hearing impairment, optic atrophy

  • CMTX5: Peripheral neuropathy, hearing impairment, optic atrophy

  • PRS-I superactivity: Mental retardation, ataxia, hypotonia, hearing impairment

  • DFN2: Isolated postlingual progressive hearing loss

• Pathophysiological mechanisms:

  • Loss-of-function mutations (e.g., p.L152P, p.Q133P) result in:

    • Reduced PRPS1 enzyme activity

    • Mild paranodal demyelination observed in sural nerve biopsies

    • Altered nucleotide metabolism affecting neural development and function

• Neuroanatomical significance:

  • High expression of PRPS1 in brain tissue under normal conditions

  • Tissue-specific expression patterns suggest specialized roles in neural tissues

  • Particular importance in auditory pathways, as evidenced by hearing loss in multiple PRPS1-related disorders

• Research approaches:

  • Patient-derived samples (erythrocytes, fibroblasts) allow for enzymatic and molecular analysis

  • Clinical characterization through specialized assessments (e.g., sleep studies, neuroimaging)

  • Genetic testing to identify and characterize specific mutations

Understanding these neurological manifestations provides insights into both the normal roles of PRPS1 in neurodevelopment and the consequences of its dysfunction, potentially guiding therapeutic approaches for affected individuals.