PRPS1 Human

What is PRPS1 and what is its primary function in human cells?

PRPS1 is the gene encoding phosphoribosyl pyrophosphate synthetase 1 (PRS-I), a key enzyme that catalyzes the first step of nucleotide synthesis necessary for both the de novo and salvage pathways of purine and pyrimidine biosynthesis . The enzyme converts ribose 5-phosphate to phosphoribosyl pyrophosphate (PRPP), which serves as a critical precursor for nucleotide synthesis. This reaction requires ATP and is regulated by various allosteric mechanisms. PRPS1 is expressed in multiple tissues and plays a fundamental role in cellular metabolism by providing the building blocks for DNA and RNA synthesis, as well as contributing to energy metabolism through nucleotide-dependent pathways .

What is the genomic location of PRPS1 and how is the gene structured?

PRPS1 is located on the X chromosome at position Xq22.3 . The gene consists of multiple exons encoding a protein of approximately 318 amino acids. Being X-linked, PRPS1-related disorders typically show X-linked inheritance patterns, affecting males more severely than females who carry one functional copy of the gene. The gene structure includes regulatory regions that control its expression in different tissues, though tissue-specific regulation of PRPS1 remains an area requiring further investigation .

What spectrum of disorders is associated with PRPS1 mutations?

PRPS1 mutations are associated with a spectrum of clinical conditions, ranging from non-syndromic to complex syndromic disorders. The main PRPS1-related disorders include:

• X-linked non-syndromic sensorineural hearing loss (DFNX1): Characterized primarily by progressive hearing impairment without other major symptoms

• Charcot-Marie-Tooth neuropathy type X-5 (CMTX5): Features peripheral neuropathy and hearing loss

• Arts syndrome: A more severe condition presenting with intellectual disability, early-onset hypotonia, ataxia, delayed motor development, hearing impairment, and optic atrophy

• Novel severe phenotypes: Recent research has identified even more severe presentations including prenatal growth restriction, retinal dystrophy, diabetes insipidus, intellectual disability, and spastic quadriparesis

The severity of these conditions appears to correlate with the degree of reduction in PRS-I enzymatic activity, with milder reductions associated with non-syndromic hearing loss and more severe reductions linked to complex syndromes .

How does PRPS1 enzyme activity correlate with disease phenotypes?

The correlation between PRPS1 enzyme activity and disease phenotypes follows a relatively consistent pattern, with the severity of clinical manifestations generally corresponding to the degree of enzyme dysfunction. Studies measuring PRS-I activity in patient erythrocytes have demonstrated that:

• Mild reductions (approximately 60-70% of normal activity) are typically associated with non-syndromic hearing loss (DFNX1)

• More substantial reductions (30-50% of normal activity) correspond to conditions like CMTX5 with peripheral neuropathy and hearing impairment

• Severe reductions (<30% of normal activity) are seen in complex syndromes like Arts syndrome and the most recently described severe phenotypes

This progressive spectrum suggests a threshold effect, where different tissues have varying sensitivity to reduced PRPS1 activity, with the auditory system being particularly vulnerable to even mild reductions in enzyme function .

How do different missense mutations in PRPS1 affect protein structure and enzyme function?

Different missense mutations in PRPS1 can affect protein structure and enzyme function through various mechanisms:

• Structural Alterations: Mutations may disrupt the tertiary structure of the protein, affecting catalytic sites or regulatory domains. For example, the novel missense mutation c.586C>T (p.Arg196Trp) causes a significant reduction in PRS-I activity, likely by altering a critical structural region of the enzyme .

• Filament Assembly Disruption: Recent structural studies have revealed that PRPS1 forms functional filaments that regulate its activity. Some disease-causing mutations disrupt these assembly contacts, reducing enzymatic efficiency. The conserved assembly interface stabilizes the binding site for the essential activator phosphate, thereby increasing enzyme activity in the filament state .

• Allosteric Regulation Interference: Mutations can affect the binding of allosteric regulators such as ADP or phosphate. The 2023 structural studies by Hvorecny et al. demonstrated that PRPS1 filaments stabilize allosteric sites, providing an additional layer of activity regulation .

• Catalytic Coupling Disruption: Active PRPS1 filaments exhibit coupling between catalysis in one active site with product release in an adjacent site, a mechanism that can be disrupted by certain mutations .

These diverse effects underscore the importance of both functional enzymatic assays and structural studies in understanding the pathogenic mechanisms of PRPS1 mutations.

What is the role of PRPS1 in cancer development and progression?

PRPS1 plays a significant role in cancer development and progression, particularly in colorectal cancer:

• Upregulated Enzymatic Activity: Despite only minor changes in mRNA and protein expression levels, PRPS1 enzymatic activity is significantly upregulated in colorectal cancer tissues compared to adjacent normal tissues .

• Correlation with Disease Progression: Higher PRPS1 enzymatic activity levels correlate with more advanced colorectal cancer cases. Analysis of 184 patients with complete follow-up information revealed that PRPS1 enzymatic activity strongly correlates with colorectal cancer recurrence .

• Survival Prediction: Patients with colorectal cancer exhibiting low PRPS1 enzymatic activity demonstrated higher survival rates compared to those with high enzymatic activity, suggesting PRPS1 activity as a potential prognostic marker .

• Cell Cycle Regulation: Research indicates that PRPS1 activity is regulated in a cell-cycle-dependent manner, with phosphorylation events modulating its function during different phases of the cell cycle to support the increased nucleotide demand during S phase .

These findings suggest that targeting PRPS1 activity could represent a novel therapeutic strategy for cancer treatment, particularly for colorectal cancers with high PRPS1 activity.

How does PRPS1 filament formation contribute to enzyme regulation?

PRPS1 filament formation represents a sophisticated mechanism of enzyme regulation that has been conserved throughout evolution:

• Conformational Stabilization: PRPS1 assembles into filaments with fixed assembly contacts that can accommodate both active and inhibited conformations of the enzyme. This assembly stabilizes the binding site for the essential activator phosphate, thereby increasing enzymatic activity in the filament state .

• Allosteric Control: The filamentous structure provides an additional layer of allosteric control over enzyme activity. Some disease-causing mutations alter this assembly, supporting a direct link between filament stability and catalytic activity .

• Catalytic Coupling: Structural studies of active PRPS1 filaments turning over substrate have revealed coupling between catalysis in one active site with product release in an adjacent site, demonstrating cooperative behavior within the filament .

• Metabolic Homeostasis: The ability of PRPS1 to form regulated filaments likely has significant impacts on metabolic homeostasis, allowing for coordinated control of nucleotide synthesis across multiple enzyme molecules .

This filament-based regulatory mechanism adds to our understanding of assembly-based enzyme control strategies and provides new insights into how mutations might disrupt enzyme function through effects on quaternary structure.

What are the established methods for measuring PRPS1 enzymatic activity?

Several validated approaches exist for measuring PRPS1 enzymatic activity, with the most common methods including:

• HPLC-Based End-Point Assay:

  • Purify PRPS1 from cell lysates (using antibody-based or tag-based approaches)

  • Incubate purified enzyme in reaction buffer containing:

    • 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

    • 0.25 mmol/L P¹P⁵-diadenosine pentaphosphate (Ap5A)

  • Terminate reaction with equal volume of 0.1 mol/L EDTA

  • Measure AMP concentration by HPLC

  • Calculate AMP/PRPS1 ratio to determine enzymatic activity

• Erythrocyte Assay for Clinical Samples:

  • Isolate erythrocytes from patient blood samples

  • Lyse cells and purify endogenous PRPS1

  • Measure enzymatic activity using similar reaction conditions as above

  • Compare activity levels to control samples

• Recombinant Protein Analysis:

  • Express wild-type or mutant PRPS1 in bacterial or mammalian expression systems

  • Purify using affinity tags (commonly FLAG or His-tags)

  • Perform in vitro activity assays under various conditions to assess kinetic parameters

  • Compare parameters between wild-type and mutant proteins

These methodologies enable researchers to quantitatively assess how mutations, inhibitors, or cellular conditions affect PRPS1 function.

What structural biology techniques have been used to study PRPS1?

Multiple structural biology techniques have been employed to elucidate PRPS1 structure and function:

• X-ray Crystallography:

  • Has provided high-resolution structures of human PRPS1 in different conformational states

  • Revealed the detailed architecture of the active site and allosteric regulatory sites

  • The structure with PDB ID 8DBC represents human PRPS1 with phosphate in a hexameric arrangement

• Cryo-Electron Microscopy (Cryo-EM):

  • Recently used to determine the structures of human PRPS1 filaments in both active and inhibited states

  • Revealed how the conserved assembly interface stabilizes the binding site for the essential activator phosphate

  • Demonstrated the coupling of catalysis between adjacent active sites in the filament

• Small-Angle X-ray Scattering (SAXS):

  • Used to study the solution behavior of PRPS1 and confirm oligomerization states

  • Helps bridge the gap between high-resolution structural studies and functional analyses

• Molecular Dynamics Simulations:

  • Complementary computational approach to understand conformational changes and allosteric regulation

  • Used to predict the effects of mutations on protein stability and function

These structural studies have been instrumental in understanding how PRPS1 mutations lead to disease by affecting protein folding, oligomerization, allosteric regulation, or catalytic activity.

What genetic approaches are most effective for studying PRPS1 variants?

Several genetic approaches have proven effective for studying PRPS1 variants:

• Next-Generation Sequencing (NGS) for Variant Discovery:

  • Whole-exome sequencing has been particularly successful in identifying novel PRPS1 variants in patients with suspected X-linked hearing loss or neurological disorders

  • Targeted gene panels including PRPS1 and other hearing loss genes can be cost-effective for clinical diagnosis

  • Segregation analysis within families helps confirm pathogenicity of identified variants

• CRISPR-Cas9 Gene Editing for Functional Studies:

  • Generation of cell lines carrying patient-specific PRPS1 mutations

  • Creation of isogenic control lines for comparative studies

  • Introduction of reporter tags to study protein localization and dynamics

• Patient-Derived Cell Models:

  • Fibroblasts or lymphoblasts from patients carrying PRPS1 mutations provide valuable tools for studying endogenous mutant protein

  • Can be reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to relevant cell types (neurons, inner ear hair cells, etc.)

• Animal Models:

  • Mouse models with targeted mutations in Prps1 help understand tissue-specific effects

  • Zebrafish models offer advantages for studying hearing and neurological phenotypes

• Transcriptomic Analysis:

  • RNA-seq to identify downstream effects of PRPS1 mutations on gene expression

  • Analysis of alternative splicing or expression of compensatory genes

These genetic approaches, often used in combination, provide comprehensive insights into the pathogenic mechanisms of PRPS1 variants and potential therapeutic strategies.

How can PRPS1 activity serve as a biomarker in cancer prognosis?

PRPS1 activity has emerging potential as a cancer biomarker, particularly in colorectal cancer:

• Prognostic Value:

  • Studies of colorectal cancer patients reveal that PRPS1 enzymatic activity strongly correlates with disease recurrence

  • Kruskal-Wallis statistical analysis demonstrated that patients with low PRPS1 enzymatic activity had significantly higher survival rates compared to those with high activity

  • This correlation remained significant even when accounting for other clinical parameters

• Disease Progression Correlation:

  • Analysis of 184 patients with complete follow-up information showed that higher PRPS1 enzymatic activity levels were observed in more advanced colorectal cancer cases

  • The activity levels appear to increase progressively with disease stage

• Measurement Methodology for Clinical Application:

  • For tumor tissues, specimens are homogenized in lysis buffer and centrifuged to remove debris

  • PRPS1 is antibody-purified from the supernatant

  • Enzymatic activity is measured using standardized assays

  • The AMP/PRPS1 calculation provides a quantitative measure of enzymatic activity

• Advantages Over Expression-Based Markers:

  • Despite only minor differences in mRNA and protein expression levels between normal and cancer tissues, the enzymatic activity shows much more significant and consistent differences

  • This suggests that post-translational modifications or cofactor availability may be more important in regulating PRPS1 function in cancer

These findings indicate that PRPS1 enzymatic activity could serve as a valuable biomarker for colorectal cancer prognosis, potentially guiding treatment decisions and follow-up protocols.

What therapeutic strategies targeting PRPS1 are being investigated?

Several therapeutic strategies targeting PRPS1 are under investigation, with potential applications in both cancer and genetic disorders:

• Direct Enzyme Inhibitors:

  • Small molecule inhibitors of PRPS1 enzymatic activity are being developed

  • ADP is a natural inhibitor of PRPS1, and analogs with improved pharmacological properties are being explored

  • Structure-based drug design using the recently determined PRPS1 structures (including PDB ID 8DBC) is facilitating the development of specific inhibitors

• Filament Disruption Approaches:

  • Compounds that specifically disrupt PRPS1 filament formation could provide a novel regulatory mechanism

  • The recently characterized filament assembly interface offers a potential target for such interventions

• Substrate Supplementation for Loss-of-Function Disorders:

  • S-adenosylmethionine (SAM) supplementation has shown benefits in some patients with Arts syndrome

  • Nucleotide supplementation strategies aim to bypass the metabolic block caused by PRPS1 deficiency

• Gene Therapy Approaches:

  • For X-linked disorders caused by PRPS1 loss-of-function mutations, gene replacement strategies are being investigated

  • AAV-mediated delivery of functional PRPS1 to affected tissues like the inner ear could potentially prevent or treat hearing loss

• Targeting Post-Translational Modifications:

  • Cell-cycle-dependent phosphorylation of PRPS1 regulates its activity in cancer cells

  • Identifying and targeting the kinases responsible for these modifications represents a potential therapeutic strategy

These diverse approaches demonstrate the therapeutic potential of targeting PRPS1 in multiple disease contexts, although most remain in preclinical development stages.

What are the major unresolved questions in PRPS1 research?

Despite significant advances, several critical questions about PRPS1 remain unresolved:

• Tissue-Specific Effects:

  • Why are certain tissues (particularly the auditory system) more sensitive to PRPS1 dysfunction than others?

  • What compensatory mechanisms exist in different cell types that might explain the variable manifestations of PRPS1 mutations?

• Regulatory Networks:

  • How is PRPS1 integrated into broader cellular metabolic networks?

  • What transcription factors and signaling pathways regulate PRPS1 expression in different contexts?

  • How do the recently discovered filamentous structures of PRPS1 interact with other cellular components?

• Function of PRPS1 Paralogs:

  • What are the functional differences between PRPS1 and its paralogs PRPS2 and PRPSL1?

  • Could these paralogs be targeted for compensatory upregulation as a therapeutic strategy?

• Cancer Metabolism:

  • How precisely does cell-cycle-dependent phosphorylation regulate PRPS1 activity?

  • Which kinases and phosphatases are responsible for PRPS1 regulation in cancer cells?

  • How does PRPS1 activity interact with other metabolic adaptations in cancer?

• Pharmacological Challenges:

  • How can PRPS1 inhibitors be designed to target cancer cells without affecting normal tissues?

  • What delivery strategies could effectively target PRPS1 therapeutics to the inner ear or other affected tissues in genetic disorders?

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, genetics, and clinical research.

What methodological challenges exist in studying PRPS1 and how might they be overcome?

Researchers face several methodological challenges when studying PRPS1:

• Measuring Native Enzyme Activity:

  • Challenge: Current methods typically require protein purification, which may not reflect in vivo activity

  • Solution: Development of cell-based assays using metabolic labeling or biosensors that can detect PRPS1 activity in intact cells

• Studying Tissue-Specific Effects:

  • Challenge: Many affected tissues (like inner ear hair cells) are difficult to access or culture

  • Solution: Development of organoid models or differentiation protocols for iPSCs to generate relevant cell types

  • Alternative: Conditional knockout animal models with tissue-specific Prps1 inactivation

• Visualizing Filament Formation in Cells:

  • Challenge: PRPS1 filaments have primarily been studied in vitro

  • Solution: Advanced imaging techniques like super-resolution microscopy or correlative light and electron microscopy to visualize filaments in cellular contexts

  • Alternative: Development of biosensors that report on PRPS1 oligomerization state

• Translating Structural Insights to Functional Understanding:

  • Challenge: Connecting structural features to cellular phenotypes

  • Solution: Integrated approaches combining structural characterization with cellular, biochemical, and genetic studies

  • Example: Using the recently determined structures (such as PDB ID 8DBC) to guide mutagenesis studies in cellular models

• Therapeutic Delivery:

  • Challenge: Delivering therapeutics to affected tissues like the central nervous system or inner ear

  • Solution: Exploring novel delivery vehicles such as exosomes, nanoparticles, or viral vectors with enhanced tissue tropism

Overcoming these challenges requires collaborative efforts across disciplines and continued technological innovation in both basic research and translational applications.