UPRT Human
Description
Biochemical Overview of UPRT Human
Uracil phosphoribosyltransferase (UPRT) is a key enzyme in the pyrimidine salvage pathway, catalyzing the conversion of uracil and 5-phosphoribosyl-1-R-diphosphate (PRPP) to uridine monophosphate (UMP) . This reaction enables cells to recycle uracil into nucleotides, bypassing de novo synthesis. While UPRT activity is well-documented in lower eukaryotes, its role in humans remains debated due to conflicting experimental evidence .
Key Biochemical Properties
Gene and Protein Structure
The UPRT gene (ENSG00000094841) encodes a 36.2 kDa protein with a conserved catalytic domain. Key structural features include:
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N-terminal His-tag: Facilitates purification in recombinant expression systems .
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Amino Acid Sequence: Includes motifs critical for PRPP and uracil binding, though two residues in the uracil-binding region are absent compared to active bacterial homologs .
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Paralog: Shares sequence similarity with UCKL1, though functional divergence exists .
Table: UPRT Gene and Protein Attributes
Functional Roles in Metabolism and Disease
UPRT Human plays dual roles in nucleotide metabolism and disease pathobiology:
Disease Associations
UPRT is implicated in:
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Coccidiosis: Linked via homology to parasitic UPRT targets .
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Cancer: Potential therapeutic target for 5-fluorouracil (5-FU) activation in gene therapy .
Table: UPRT-Associated Diseases and Pathways
| Disease/Pathway | Mechanism | Source |
|---|---|---|
| Coccidiosis | Homology to Toxoplasma gondii UPRT, a drug target | |
| Cancer Therapy | Converts 5-FU to toxic 5-fluorouridine monophosphate in UPRT-expressing cells |
Clinical and Therapeutic Applications
UPRT Human serves as a cornerstone in suicide gene therapy systems:
CD::UPRT/5-FC System
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Mechanism: Co-expressing cytosine deaminase (CD) and UPRT enables sequential conversion of 5-fluorocytosine (5-FC) to 5-FU, then to toxic 5-fluorouridine monophosphate (5-FUMP) .
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Bystander Effect: Kills adjacent non-transduced cells via gap junctions or secreted metabolites .
Preclinical Insights
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In Vivo Monitoring: 19F-Magnetic Resonance Spectroscopy (19F-MRS) tracks 5-FU accumulation, validating UPRT functionality .
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Viral Complementation: Recombinant cytomegalovirus expressing UPRT enables selective RNA labeling in latently infected cells .
Catalytic Activity Debates
Recombinant human UPRT shows minimal enzymatic activity in vitro, raising questions about its physiological relevance . Proposed explanations include:
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Missing Co-factors: Requires ancillary proteins absent in heterologous systems.
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Alternative Functions: May regulate nucleotide pools via non-catalytic roles .
Gene Expression Patterns
RT-PCR data reveal tissue-specific expression, with highest levels in hematopoietic organs . This aligns with UPRT’s proposed role in immune cell nucleotide metabolism.
Recombinant Protein Production
UPRT Human is produced in E. coli as a His-tagged fusion protein (ENZ-742) for research use .
Product Specs
Q&A
What is human UPRT and what is its primary biological role?
Human Uracil phosphoribosyltransferase (UPRT) is an enzyme involved in the pyrimidine salvage pathway, theoretically catalyzing the conversion of uracil and 5-phosphoribosyl-1-R-diphosphate to uridine monophosphate. This pathway is critical for recycling nucleosides generated by DNA or RNA degradation. While the enzyme is highly conserved from prokaryotes to humans, there has been debate about its functional activity in higher eukaryotes including humans.
What is the molecular structure of human UPRT?
Human UPRTase is a 309 amino acid protein containing a putative uracil phosphoribosyltransferase domain. Structural analysis reveals that human UPRT differs from microbial versions in the uracil-binding region, specifically lacking two amino acids that are present in prokaryotic orthologs. This structural difference may explain the apparent differences in enzymatic activity between human and microbial UPRT enzymes.
Where is human UPRT localized within cells?
Subcellular localization studies using UPRTase-EGFP fusion protein revealed that human UPRTase is distributed in both the nucleus and cytoplasm of cells. This dual localization suggests potential roles in both compartments, possibly related to different aspects of pyrimidine metabolism or potentially other functions yet to be fully characterized.
What is the tissue distribution pattern of human UPRT?
RT-PCR analysis has demonstrated that human UPRTase exhibits tissue-specific expression patterns. The enzyme is strongly expressed in blood leukocytes, liver, spleen, and thymus. Lower levels of expression have been detected in the prostate, heart, brain, lung, and skeletal muscle. This differential expression pattern suggests tissue-specific roles for the enzyme.
How does UPRT expression vary across developmental stages?
While comprehensive developmental expression data is limited in the current literature, studies in model organisms like Drosophila suggest that UPRT activity is required for larval growth, pre-pupal/pupal viability, and long-term adult lifespan. This indicates that the enzyme likely plays important roles during various developmental stages in higher eukaryotes, though human-specific developmental expression patterns require further investigation.
How can researchers accurately measure UPRT activity in human cells?
Methodology recommendation: Due to challenges in measuring direct enzymatic activity, researchers should employ multiple complementary approaches:
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Recombinant protein assays with varied buffer conditions
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Cell-based functional assays measuring uracil incorporation
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Genetic complementation studies in UPRT-deficient organisms
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Metabolic labeling using isotope-labeled uracil
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Mass spectrometry to track conversion of uracil to UMP in cell extracts
What factors might explain the discrepancy between expected and observed human UPRT activity?
Several hypotheses have been proposed to explain why human UPRT activity is difficult to detect despite conservation of the enzyme:
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Human UPRT may require additional cofactors or protein partners not present in standard in vitro assays
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The enzyme may have acquired altered substrate specificity in higher eukaryotes
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Post-translational modifications may regulate activity in vivo
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The structural differences in the uracil-binding region (lacking two critical amino acids) may significantly alter catalytic properties
What can studies in model organisms tell us about human UPRT function?
Research in Drosophila provides valuable insights into potential human UPRT functions. The Drosophila UPRT homologue (krishah) is required for larval growth, pre-pupal/pupal viability, and long-term adult lifespan. These findings challenge the previous notion that UPRT is non-essential in higher eukaryotes and suggest that human UPRT likely plays important physiological roles that have been underappreciated.
How should researchers approach the purification of human UPRT protein?
Methodology recommendation:
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Clone the complete ORF of human UPRTase into an appropriate expression vector (pQE30 has been successfully used)
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Express in E. coli M15 or equivalent expression system
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Purify using Ni-NTA affinity chromatography if using His-tagged constructs
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Verify protein integrity by SDS-PAGE and Western blotting
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Consider additional purification steps (ion exchange, size exclusion) to achieve highest purity
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Test activity under various buffer conditions with appropriate controls
How might we reconcile conflicting data on human UPRT activity?
The apparent contradiction between evolutionary conservation of UPRT and lack of detectable activity presents a fascinating research challenge. Advanced researchers should consider:
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Developing more sensitive assays that might detect low-level UPRT activity
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Identifying potential protein partners or cofactors required for human UPRT function
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Investigating alternative substrates or reaction conditions
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Exploring whether human UPRT might have evolved new functions while retaining structural similarity to ancestral enzymes
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Using CRISPR/Cas9 to create UPRT-knockout human cell lines to assess metabolic consequences
What unknown factors might be necessary for human UPRT function in vivo?
Current research suggests that human UPRT might require additional, currently unknown factors to function in vivo. These could include:
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Specific post-translational modifications
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Protein-protein interactions with cofactors
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Subcellular compartmentalization effects
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Allosteric regulators
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Alternative substrates or reaction pathways
Investigating these possibilities represents an important frontier in UPRT research and could resolve longstanding questions about the enzyme's function in humans.
Product Science Overview
The human recombinant form of uracil phosphoribosyltransferase was identified and characterized through various studies. Initially, this enzyme was reported in plants like Arabidopsis thaliana, but not in mammals . However, a novel family of uracil phosphoribosyltransferase was later discovered in humans. The gene encoding this enzyme was isolated from a human fetal brain library and named human UPRTase .
The open reading frame (ORF) of the human UPRTase gene was cloned into the pQE30 vector and expressed in Escherichia coli M15 cells. The protein was then purified using Ni-NTA affinity chromatography . Despite successful purification, the enzymatic activity of UPRTase could not be detected through spectrophotometry .
Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that human UPRTase is expressed in various tissues, including the prostate, heart, brain, lung, and skeletal muscle . Subcellular localization studies using UPRTase-EGFP fusion protein showed that the enzyme is distributed in both the nucleus and cytoplasm of AD293 cells .
Human recombinant UPRTase has potential applications in the rational design of drugs for treating parasitic infections and cancer . The enzyme’s role in the pyrimidine salvage pathway makes it a target for developing antimetabolite drugs, which can inhibit nucleotide synthesis in rapidly dividing cells, such as cancer cells .
