PRPS2 Human

What is PRPS2 and what is its primary cellular function?

PRPS2 (Phosphoribosyl-pyrophosphate synthetase 2) is a rate-limiting enzyme that plays a crucial role in purine and pyrimidine nucleotide synthesis. Located on the X chromosome in the p22 region, PRPS2 belongs to the family of phosphoribosylpyrophosphate synthetases (PRS) and shares approximately 95% homology with PRPS1 at the amino acid sequence level . The enzyme catalyzes the conversion of ribose 5-phosphate to phosphoribosyl pyrophosphate (PRPP), which serves as a substrate for both de novo and salvage pathways of nucleotide synthesis. PRPS2 is highly expressed in several tissues, including the thymus, adipose tissue, and testes . Unlike many other metabolic enzymes, PRPS2 has been identified as having tissue-specific functions that extend beyond its catalytic role in nucleotide biosynthesis, particularly in contexts such as cancer metabolism and reproductive biology .

How does PRPS2 differ from PRPS1 in structure and function?

Despite sharing 95% amino acid sequence homology with PRPS1, PRPS2 exhibits distinct functional properties that set it apart . The key structural difference lies in a 3-amino acid insertion (V103-G104-E105) present in PRPS2 but absent in PRPS1 . This insertion causes significant steric clash at the interface of the PRPS hexamer, which explains PRPS2's lower enzyme activity compared to PRPS1 .

Functionally, PRPS2 differs from PRPS1 in several important ways:

What mechanisms regulate PRPS2 activity in human cells?

PRPS2 activity is regulated through multiple sophisticated mechanisms:

• Allosteric regulation: PRPS2 activity is subject to nucleotide feedback inhibition. Studies have shown that ADP and GDP significantly reduce PRPP production in cells with functional PRPS2, indicating a direct allosteric regulatory mechanism .

• Complex formation: PRPS2 forms a hexameric complex with PRPS1 and other PRPS-associated proteins. The stability of this hexamer directly influences enzymatic activity, with mutations affecting the interface regions resulting in altered enzyme function .

• Transcriptional regulation: In contexts such as male fertility, PRPS2 expression is regulated by transcription factors including E2F1, as confirmed through luciferase reporter assays .

• Post-translational modifications: Though not extensively detailed in the provided research, enzymatic activity can be regulated through phosphorylation and other post-translational modifications common to metabolic enzymes.

How do mutations in PRPS2 contribute to cancer pathogenesis and relapse?

PRPS2 mutations have been identified as drivers of cancer pathogenesis and relapse, particularly in childhood acute lymphoblastic leukemia (ALL). Through comprehensive analysis of 210 matched diagnosis-relapse ALL samples across two independent cohorts, researchers identified therapy-induced and recurrent relapse-specific mutations in the PRPS2 gene with a frequency of approximately 2.8-2.9% . These mutations were exclusively found in relapsed childhood ALL patients who had undergone thiopurine therapy.

The molecular mechanism by which PRPS2 mutations drive relapse involves altered PRPS1/2 hexamer stability. Functional PRPS2 mutations influence purine metabolism specifically during thiopurine treatment by:

• Destabilizing the PRPS1/2 hexamer structure

• Reducing nucleotide feedback inhibition of PRPS activity

• Enhancing thiopurine resistance

For example, the PRPS2 P173R mutation demonstrated increased resistance to thiopurines in xenograft models . This mutation and others affect the structural integrity of the PRPS hexamer, leading to altered enzymatic properties that ultimately confer drug resistance.

Importantly, patients with PRPS2 mutations did not concurrently harbor PRPS1 mutations, suggesting that these represent distinct pathological mechanisms. These findings establish functional PRPS2 mutations as significant prognostic factors for ALL relapse and potential biomarkers for treatment response .

What is the role of PRPS2 in male fertility and reproductive biology?

PRPS2 plays a critical role in male fertility through its influence on spermatogenesis. Research has established a direct relationship between PRPS2 expression and hypospermatogenesis, which is characterized by low production of spermatozoa and is a prevalent condition in azoospermic patients .

The mechanism by which PRPS2 affects male fertility involves regulation of spermatogenic cell apoptosis. Studies show that:

• PRPS2 depletion significantly increases the number of apoptotic spermatogenic cells both in vitro and in vivo

• PRPS2 expression is downregulated in mouse models of hypospermatogenesis

• Experimental knockdown of PRPS2 in mouse testes results in hypospermatogenesis and accelerated apoptosis of spermatogenic cells

At the molecular level, PRPS2 regulates spermatogenic cell apoptosis through the E2F1/P53/Bcl-xl/Bcl-2/Caspase 6/Caspase 9 pathway. E2F transcription factor 1 (E2F1) has been confirmed as a direct target gene of PRPS2 . When PRPS2 expression is downregulated in spermatogenic cells, there is:

• Decreased expression of E2F1, Bcl-2, and Bcl-xl

• Elevated expression of P53, Caspase 6, and Caspase 9

• Increased rates of apoptosis

This pathway differs from the mechanism by which PRPS2 influences Sertoli cell apoptosis in Sertoli Cell-Only Syndrome (SCOS), highlighting the cell-specific functions of PRPS2 in the male reproductive system .

How does PRPS2 mediate drug resistance in cancer therapy?

PRPS2 mediates drug resistance in cancer therapy, particularly to thiopurines, through alterations in purine metabolism. Thiopurines are first-line drugs in ALL chemotherapy, and their efficacy depends on proper purine metabolic pathways . PRPS2 mutations acquired during therapy can confer resistance through several mechanisms:

In knockout studies, deletion of either PRPS1 or PRPS2 markedly reduced dNTP pools, but with different effects on feedback inhibition: PRPS2 knockout cells maintained sensitivity to ADP/GDP inhibition, while PRPS1 knockout cells did not . This demonstrates the distinct roles of these enzymes in drug metabolism.

The table below summarizes the effects of PRPS2 status on thiopurine sensitivity and PRPS regulation:

These findings highlight PRPS2 as a potential therapeutic target and biomarker for predicting treatment response in ALL and possibly other cancers treated with thiopurines .

What are the established methods for measuring PRPS2 enzymatic activity?

• In vitro PRPS enzyme activity assay: This involves measuring the conversion of ribose 5-phosphate to PRPP. In research contexts, this can be done by:

  • Isolating cytosolic fractions from cells or tissues

  • Incubating with substrates (ribose 5-phosphate and ATP)

  • Measuring PRPP formation through coupled enzyme assays or chromatographic techniques

  • Comparing activity with and without nucleotide inhibitors (ADP/GDP) to assess feedback regulation

• Metabolite flux analysis: Researchers can trace the incorporation of labeled precursors into downstream metabolites using techniques such as ultra-performance liquid-chromatography tandem mass spectrometry (UPLC-MS/MS). This approach was utilized to measure dNTP pools in PRPS2 knockout cells compared to controls .

• Luciferase reporter assays: To evaluate the correlation between PRPS2 and its target genes (such as E2F1), luciferase reporter assays can be employed. Research has shown that the luciferase activity of E2F1 was significantly increased in cells overexpressing PRPS2 compared to controls .

• Erythrocyte PRPS activity measurement: In clinical settings, PRPS activity can be measured in erythrocytes, as demonstrated in patients with PRPS1 mutations. This involves a similar enzymatic assay but uses red blood cells as the source material .

These methods can be complemented with protein expression analysis through Western blotting and gene expression analysis through quantitative real-time PCR to provide a comprehensive assessment of PRPS2 function in experimental systems.

What genetic manipulation techniques are effective for studying PRPS2 function?

Several genetic manipulation techniques have proven effective for studying PRPS2 function in research settings:

• CRISPR/Cas9 gene editing: This has been successfully employed to establish PRPS2 knockout cell lines. The technique allows for precise deletion or modification of the PRPS2 gene to study its function. In comparative studies, both PRPS1 and PRPS2 knockout lines were generated using CRISPR/Cas9 technology to assess their distinct roles in nucleotide metabolism and drug sensitivity .

• RNA interference (RNAi): Small-hairpin RNA (shRNA) targeting PRPS2 has been used to down-regulate its expression in cell culture and animal models. In studies of spermatogenesis, PRPS2 expression was knocked down in mouse testes using shRNA constructs, resulting in hypospermatogenesis and accelerated apoptosis of spermatogenic cells .

• Overexpression systems: Plasmid vectors containing the PRPS2 gene have been utilized to overexpress PRPS2 in various cell types. This approach allows researchers to study the effects of increased PRPS2 activity. Experimental data shows that PRPS2 overexpression suppressed the expression of Caspase 6 and Caspase 9 in spermatogenic cells .

• Site-directed mutagenesis: To study specific mutations identified in clinical samples, site-directed mutagenesis can be employed to introduce these mutations into wild-type PRPS2 expression constructs. This approach was used to characterize the functional consequences of PRPS2 mutations found in relapsed ALL patients .

• Xenograft models: For in vivo studies, cells with manipulated PRPS2 expression or mutations can be transplanted into immunodeficient mice. This technique was used to demonstrate that the PRPS2 P173R mutation increased thiopurine resistance in xenograft models .

Each of these approaches offers specific advantages for investigating different aspects of PRPS2 function, from basic enzymatic properties to complex roles in disease pathogenesis.

What experimental models best represent human PRPS2 function and regulation?

The selection of appropriate experimental models is crucial for accurately representing human PRPS2 function and regulation. Based on current research methodologies, the following models have proven valuable:

• Human cell lines:

  • ALL cell lines (e.g., Reh cells): Particularly useful for studying PRPS2's role in leukemia and drug resistance. These have been employed to investigate the effects of PRPS2 mutations on thiopurine sensitivity .

  • Spermatogenic cell lines (e.g., GC1 and GC2 cells): Effective for studying PRPS2's role in male fertility and reproduction .

• Mouse models:

  • Hypospermatogenesis mouse models: These have been used to study the correlation between PRPS2 expression and male fertility. Researchers observed a significant decrease in PRPS2 expression in these models compared to normal controls .

  • Xenograft models: Human leukemia cells with PRPS2 mutations can be implanted into immunodeficient mice to study drug resistance in vivo .

• Patient-derived samples:

  • Primary ALL samples: Matched diagnosis-relapse samples from ALL patients have been crucial for identifying therapy-induced PRPS2 mutations .

  • Testicular biopsies: Samples from patients with hypospermatogenesis can provide insights into PRPS2's role in human fertility .

• Biochemical reconstitution systems:

  • Purified recombinant proteins: For detailed biochemical studies of enzyme kinetics and structure-function relationships, purified recombinant PRPS2 protein can be used, though this was not specifically detailed in the provided research.

When selecting a model system, researchers should consider:

• The specific aspect of PRPS2 biology being studied

• The need to differentiate PRPS1 and PRPS2 activities

• The relevance to human disease pathology

• Technical limitations of each model

How does PRPS2 integrate with broader metabolic networks in human cells?

PRPS2 functions as a critical node in cellular metabolic networks, connecting multiple pathways essential for cell growth, division, and survival. Its integration within these networks can be understood through several key interactions:

• Purine and pyrimidine biosynthesis: As a rate-limiting enzyme in nucleotide synthesis, PRPS2 catalyzes the formation of PRPP, which serves as a substrate for both de novo and salvage pathways of nucleotide synthesis . This positions PRPS2 at the intersection of glucose metabolism (which provides ribose-5-phosphate via the pentose phosphate pathway) and nucleotide production.

• Cell cycle regulation: PRPS2 interacts with cell cycle regulators, particularly through its relationship with E2F1, a key transcription factor involved in cell proliferation and apoptosis . This connection links metabolic status to cell cycle progression and cellular fate decisions.

• Apoptotic pathways: Research demonstrates that PRPS2 influences the expression of multiple components of the apoptotic machinery, including Bcl-2, Bcl-xl, P53, Caspase 6, and Caspase 9 . This creates a direct link between nucleotide metabolism and cell survival pathways.

• Feedback regulation networks: PRPS2 is subject to nucleotide feedback inhibition, with ADP and GDP serving as key regulators . This creates a self-regulating loop that balances nucleotide production with cellular demand.

• Stress response pathways: While not explicitly detailed in the provided research, PRPS2's role in cancer metabolism suggests its involvement in cellular responses to stress conditions such as hypoxia or nutrient deprivation, which are common in tumor microenvironments .

The integrated nature of these pathways is evidenced by the downstream effects of PRPS2 manipulation. For instance, knockout studies show that deletion of PRPS2 significantly alters dNTP pools, affecting not just nucleotide levels but potentially genomic stability and cellular response to DNA-damaging agents .

What is the relationship between PRPS2 activity and dNTP pool regulation?

The relationship between PRPS2 activity and dNTP pool regulation is fundamental to understanding cellular metabolism and disease processes, particularly in contexts of drug resistance and cancer progression:

• Direct contribution to dNTP synthesis: PRPS2 catalyzes the formation of PRPP, which is an essential substrate for both de novo and salvage pathways of nucleotide synthesis. This directly influences the availability of nucleotides for DNA and RNA synthesis .

• Experimental evidence: Metabolite flux analysis has demonstrated that deletion of either PRPS1 or PRPS2 markedly reduces dNTP pools in cellular models. Specifically, knockout studies in Reh cells showed significant reductions in dNTP levels when either PRPS1 or PRPS2 was deleted, though there was no significant difference between PRPS1 KO and PRPS2 KO in terms of the magnitude of reduction .

• Feedback regulation: PRPS2 is critical for nucleotide feedback inhibition of PRPS activity. Experiments have shown that ADP and GDP treatment significantly reduces PRPP production in control and PRPS2 KO cells but not in PRPS1 KO cells, indicating different roles in feedback regulation .

• Implications for disease: Altered dNTP pools affect genomic stability and can influence cellular responses to chemotherapeutic agents, particularly those that target nucleotide metabolism like thiopurines. PRPS2 mutations found in relapsed ALL patients lead to reduced nucleotide feedback inhibition, potentially allowing for continued nucleotide synthesis even in the presence of inhibitory signals .

The table below summarizes experimental findings on PRPS status and dNTP pools:

These findings highlight the critical role of PRPS2 in maintaining dNTP homeostasis and its potential as a therapeutic target in diseases characterized by dysregulated nucleotide metabolism .

How does PRPS2 function differ across tissue types and developmental stages?

PRPS2 exhibits notable tissue-specific functions and developmental regulation, though the provided research materials offer limited direct comparison across multiple tissues. Based on available information and inferences from the research:

These tissue-specific and developmental differences have significant implications for understanding disease processes and developing targeted therapeutic approaches. For instance, targeting PRPS2 in leukemia treatment would need to consider potential effects on reproductive and other tissues where the enzyme plays important physiological roles .

How can PRPS2 mutations serve as biomarkers in cancer diagnostics and treatment?

PRPS2 mutations have emerging potential as clinically valuable biomarkers in cancer diagnostics and treatment, particularly in the context of childhood acute lymphoblastic leukemia (ALL):

• Prediction of relapse risk: Research has identified PRPS2 mutations in approximately 2.8-2.9% of relapsed childhood ALL cases across multiple cohorts . These mutations were specifically found in patients who had received thiopurine therapy, suggesting their value as predictive biomarkers for treatment failure and relapse risk. The exclusive presence of these mutations in relapsed samples (not at initial diagnosis) indicates their acquisition during treatment, making them potential early markers of developing resistance.

• Therapy selection guidance: Detection of PRPS2 mutations could inform treatment decisions:

  • Patients with emerging PRPS2 mutations might benefit from alternative therapeutic strategies that bypass the affected metabolic pathways

  • The functional characterization of specific mutations could help predict which drugs might remain effective despite the mutation

• Monitoring treatment response: Serial testing for PRPS2 mutations during treatment could provide valuable information about:

  • The emergence of resistant clones

  • The need for treatment adjustment

  • The likelihood of long-term remission

• Methodological approaches for detection:

  • Ultra-deep sequencing has been successfully employed to identify PRPS2 mutations in ALL samples

  • This approach allows for detection of subclonal mutations that might be missed by standard sequencing methods

  • Liquid biopsy approaches could potentially enable less invasive monitoring for PRPS2 mutations

• Combined biomarker panels: The greatest clinical utility may come from combining PRPS2 mutation analysis with other markers:

  • PRPS1 mutations (found at higher frequencies of 3.9-13% in relapsed ALL)

  • Other mutations affecting purine metabolism

  • Standard prognostic markers like minimal residual disease

The specificity of PRPS2 mutations to thiopurine-treated relapsed cases suggests they may be particularly valuable as biomarkers of acquired drug resistance, potentially enabling earlier intervention before clinical relapse becomes evident .

What therapeutic approaches target PRPS2 or its downstream pathways?

Several therapeutic approaches targeting PRPS2 or its downstream pathways are under investigation or show potential for development:

• Direct PRPS2 inhibition:

  • While specific PRPS2 inhibitors are not detailed in the provided research, selective targeting of PRPS2 over PRPS1 could potentially overcome thiopurine resistance while minimizing side effects associated with broader inhibition of purine synthesis

  • The structural differences between PRPS1 and PRPS2, particularly the 3-amino acid insertion (V103-G104-E105) in PRPS2, provide a potential basis for developing selective inhibitors

• Modulation of PRPS2 regulation:

  • Approaches targeting the allosteric regulation of PRPS2 could restore sensitivity to feedback inhibition in mutated enzymes

  • Compounds that stabilize the PRPS1/PRPS2 hexamer might counteract the effects of mutations that disrupt this structure

• Targeting downstream metabolic pathways:

  • Alternative inhibitors of purine synthesis that act downstream of PRPS2 could bypass resistance mechanisms

  • Combination therapies targeting multiple points in the nucleotide synthesis pathway might prevent the emergence of resistance

• Apoptotic pathway modulation:

  • In contexts where PRPS2 affects apoptotic pathways (as in spermatogenic cells), therapies targeting the E2F1/P53/Bcl-xl/Bcl-2/Caspase pathway could potentially compensate for altered PRPS2 function

  • Experimental evidence shows that when E2F1 and shPRPS2 vectors were cotransfected in spermatogenic cell lines, the percentage of apoptotic cells was significantly reduced

• Nucleotide analog approaches:

  • Novel nucleotide analogs that are less dependent on the normal feedback mechanisms might remain effective against cells with PRPS2 mutations

  • Design of such analogs would need to consider the altered enzyme kinetics resulting from PRPS2 mutations

The development of these therapeutic approaches requires careful consideration of tissue-specific PRPS2 functions to minimize off-target effects, particularly on reproductive tissues where PRPS2 plays important physiological roles .

What are the most pressing unanswered questions in PRPS2 research?

Despite significant advances in understanding PRPS2 function, several critical questions remain unanswered, representing important directions for future research:

• Structural and enzymatic properties:

  • What is the complete three-dimensional structure of the PRPS1/PRPS2 hexamer, and how do mutations affect this structure?

  • How does the 3-amino acid insertion in PRPS2 (V103-G104-E105) precisely influence enzymatic activity and regulation?

  • What are the tissue-specific differences in PRPS2 complex formation and activity?

• Metabolic integration:

  • How does PRPS2 function integrate with broader cellular metabolic networks beyond nucleotide synthesis?

  • What is the relationship between PRPS2 activity and cellular energy status or nutrient availability?

  • How does PRPS2 function change under conditions of metabolic stress common in disease states?

• Disease mechanisms:

  • What is the full spectrum of diseases influenced by PRPS2 dysfunction beyond leukemia and male infertility?

  • Are there PRPS2 mutations or alterations associated with other cancer types or metabolic disorders?

  • How do epigenetic changes affect PRPS2 expression and function in different pathological contexts?

• Therapeutic development:

  • Can PRPS2-specific inhibitors be developed that don't affect PRPS1 function?

  • What combination therapies might prevent or overcome PRPS2-mediated drug resistance?

  • Could PRPS2 modulation have applications in treating male infertility?

• Clinical applications:

  • What is the optimal method for monitoring PRPS2 mutations in clinical samples?

  • Can early detection of emerging PRPS2 mutations improve outcomes in leukemia treatment?

  • Are there patient subgroups particularly susceptible to developing PRPS2 mutations during therapy?

• Evolutionary and comparative biology:

  • Why has PRPS2 evolved as a separate enzyme from PRPS1 despite their high sequence homology?

  • What can comparative studies across species tell us about the specialized functions of PRPS2?

  • Are there species-specific differences in PRPS2 regulation that could inform human disease research?

Addressing these questions will require multidisciplinary approaches integrating structural biology, biochemistry, cell biology, clinical research, and computational methods. The findings would advance both basic understanding of cellular metabolism and the development of novel therapeutic strategies for PRPS2-related disorders .