PAPSS1 Human
Description
Molecular Structure and Enzymatic Function
PAPSS1 comprises two functional domains:
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ATP sulfurylase domain (C-terminal): Converts ATP and inorganic sulfate to adenosine 5′-phosphosulfate (APS).
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APS kinase domain (N-terminal): Phosphorylates APS to generate PAPS .
The enzyme forms a homodimeric, asymmetric structure (PDB: 1X6V), where conformational changes in the kinase domain regulate substrate binding during catalysis . Key motifs include:
Tissue Expression and Isoforms
Humans express two PAPSS isoforms:
PAPSS1 is predominantly nuclear, while PAPSS2 is cytosolic, reflecting divergent regulatory roles .
Cancer Therapeutics
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Cisplatin Sensitivity: Low PAPSS1 expression correlates with enhanced cisplatin efficacy in non–small cell lung cancer (NSCLC) and ovarian cancer. Silencing PAPSS1 in A549 cells reduced cisplatin IC50 by 5-fold and impaired clonogenicity by 98.7% .
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Biomarker Potential: NSCLC and ovarian cancer patients with low PAPSS1 expression exhibit prolonged survival post-platinum chemotherapy .
Hepatocellular Carcinoma (HCC)
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A PAPSS1 haplotype (C-T-T at SNPs 29–31) increases HCC risk (OR = 3.41 for homozygotes) and associates with elevated AFP levels and poor survival in HBV-positive patients .
Genetic Disorders
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PAPSS2 mutations cause osteochondrodysplasias, but PAPSS1 deficiency is linked to impaired lymphocyte recruitment in high endothelial venules .
Sulfation Pathways and Cellular Roles
PAPS, synthesized by PAPSS1, drives sulfation of:
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Endogenous compounds: Steroids, glycosaminoglycans, neurotransmitters.
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Xenobiotics: Drugs (e.g., acetaminophen), environmental carcinogens .
Sulfation impacts extracellular matrix integrity, hormone inactivation, and drug metabolism. PAPSS1’s nuclear localization suggests additional roles in gene regulation or retroviral infection .
Therapeutic Implications
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Targeting PAPSS1: Inhibition could potentiate DNA-damaging agents (cisplatin, radiation) in tumors with high PAPSS1 expression .
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Preclinical Models: PAPSS1 knockdown reduced tumor growth in zebrafish and murine xenografts by 58% .
Genetic and Pharmacogenomic Considerations
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SNPs: Coding-region SNPs alter enzyme activity; rs3824987 (PAPSS1) links to sulfation capacity variability .
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Promoter Regions: Lack TATA boxes but contain GC-rich sequences, suggesting complex transcriptional regulation .
Evolutionary Context
Invertebrates (e.g., C. elegans) possess a single PAPSS2-like enzyme, indicating PAPSS2’s ancestral role. PAPSS1’s emergence in vertebrates可能与组织特异性硫化的需求有关 .
Product Specs
Q&A
What is PAPSS1 and what is its role in human biochemistry?
PAPSS1 is a protein-coding gene that produces a bifunctional enzyme with both ATP sulfurylase and APS kinase activity. This enzyme mediates two critical steps in sulfate activation: first transferring a sulfate group to ATP to yield adenosine 5'-phosphosulfate (APS), and then transferring a phosphate group from ATP to APS, yielding 3'-phosphoadenylylsulfate (PAPS). In mammals, PAPS is the sole source of sulfate for sulfotransferase enzymes, which catalyze the sulfate conjugation of numerous endogenous and exogenous compounds, including drugs and xenobiotics .
What is the genomic organization of human PAPSS1?
The human PAPSS1 gene consists of 12 exons spanning approximately 108 kb of genomic DNA. The gene maps to chromosome band 4q24 as determined by fluorescence in situ hybridization analysis. The 5'-flanking region of PAPSS1 does not include a TATA box sequence near the site of transcription initiation, which distinguishes it from PAPSS2. All splice junctions conform to the "GT-AG" rule, and the major PAPSS1 transcript is approximately 2.7 kb in length .
What diseases are associated with PAPSS1 dysfunction?
Diseases associated with PAPSS1 include Achondrogenesis, Type Ib and Spondyloepiphyseal Dysplasia With Congenital Joint Dislocations . These conditions highlight the importance of proper sulfation pathways in skeletal development and joint formation. Researchers investigating these conditions should focus on how disrupted sulfation affects extracellular matrix composition and structure.
What are recommended approaches for heterologous expression of PAPSS1?
For heterologous protein expression of human PAPSS1, researchers have successfully used modified pET-41 vectors with GST-His tags in E. coli BL21/Rosetta (DE3) cells. The purification workflow typically includes GSH-Sepharose purification, PreScission cleavage to remove tags, and gel filtration on Superdex 200 . When designing expression systems, researchers should consider that PAPSS1 functions as a dimer, which affects protein folding and stability.
What techniques can be used to study PAPSS1 stability?
To analyze PAPSS1 structural stability, thermal unfolding monitored by intrinsic tryptophan fluorescence and circular dichroism spectroscopy are effective approaches. These techniques have revealed important differences in protein stability between PAPSS1 and PAPSS2, with PAPSS1 showing markedly greater stability at physiological temperatures . When conducting these experiments, researchers should test the effects of natural ligands such as APS, which significantly stabilizes PAPS synthases.
How can researchers generate mutations in PAPSS1 for structure-function studies?
Point mutants of human PAPSS1 can be generated by site-directed mutagenesis with DpnI digestion of the parental DNA using commercial kits such as the QuikChange kit. This approach allows researchers to create specific amino acid substitutions to investigate the structural determinants of enzymatic activity, protein stability, and interactions with binding partners .
What are the key differences between PAPSS1 and PAPSS2?
Despite their similar enzymatic functions, PAPSS1 and PAPSS2 differ significantly in several aspects:
These differences explain why one isoform cannot complement for the other in biological systems .
How does APS affect the stability of PAPSS1 compared to PAPSS2?
APS (adenosine 5'-phosphosulfate), an intermediate in PAPS synthesis, significantly stabilizes both PAPS synthases, with a more pronounced effect on PAPSS2. For PAPSS2, APS binding more than doubles the half-life for unfolding at physiological temperatures and effectively prevents its aggregation on a time scale of days . This differential stabilization suggests that fluctuations in cellular APS concentration may have more dramatic effects on PAPSS2 activity than on PAPSS1.
What can evolutionary comparisons tell us about PAPSS1 function?
Invertebrates possess only one PAPS synthase enzyme that has been classified as PAPSS2-type by sequence-based machine learning techniques. The C. elegans PPS-1 protein behaves like human PAPSS2 in thermal unfolding experiments and stabilization by APS, suggesting that the less stable protein is evolutionarily older . This evolutionary perspective suggests that PAPSS1 may have evolved in vertebrates for specialized functions requiring greater protein stability.
How does PAPSS1 subcellular localization affect its function?
The subcellular distribution of PAPSS1 is more intricately regulated than previously thought, with access to both cytoplasm and nucleus . This dual localization suggests possible nuclear functions beyond the cytoplasmic role in general sulfation pathways. Researchers investigating this aspect should employ fluorescence microscopy with tagged PAPSS1 constructs under various cellular conditions and perform subcellular fractionation followed by enzymatic activity assays.
What is the significance of PAPSS1-PAPSS2 heterodimer formation?
Recent research has discovered that PAPSS1 and PAPSS2 can form heterodimers in addition to their respective homodimers. Interestingly, these heterodimers behave like an additive mixture of the respective homodimers without showing synergistic effects . To advance understanding in this area, researchers should investigate whether the ratio of heterodimers to homodimers changes under different physiological or pathological conditions, potentially representing a regulatory mechanism.
How does PAPSS1 contribute to cellular PAPS homeostasis?
PAPSS1 contributes to cellular PAPS homeostasis through its greater stability compared to PAPSS2. The enzymatic activity of PAPSS1 may provide a consistent baseline level of PAPS production, while the less stable but potentially more active PAPSS2 might respond more dynamically to changing cellular needs . Methodologically, this can be investigated using knockdown or overexpression of either isoform followed by metabolomic analysis of sulfation pathways.
What are effective techniques for measuring PAPSS1 enzymatic activity?
The table below outlines key techniques for assessing PAPSS1 enzymatic activity:
Researchers should consider the bifunctional nature of PAPSS1 when designing activity assays, as the ATP sulfurylase and APS kinase activities may have different kinetic properties.
How can protein-protein interactions of PAPSS1 be studied?
To investigate PAPSS1 protein-protein interactions, researchers can employ several complementary approaches: (1) Co-immunoprecipitation followed by mass spectrometry to identify interaction partners; (2) Yeast two-hybrid screening to discover novel interactions; (3) FRET or BRET assays to study dynamic interactions in living cells; and (4) Surface plasmon resonance or isothermal titration calorimetry for quantitative binding analysis. These techniques can reveal how PAPSS1 interacts with sulfotransferases or other cellular components to coordinate sulfation pathways.
What approaches are recommended for studying PAPSS1 in animal models?
When studying PAPSS1 in animal models, researchers should consider: (1) Knockout or conditional knockout models to assess developmental and tissue-specific functions; (2) Knockin models with tagged PAPSS1 for in vivo localization studies; (3) Humanized models expressing human PAPSS1 in place of the endogenous gene to study human-specific functions; and (4) C. elegans as a simpler model system to study basic aspects of PAPS synthase function, as it possesses only one PAPS synthase (PPS-1) that behaves similarly to human PAPSS2 .
What ethical and regulatory requirements apply to PAPSS1 research involving human subjects?
Research involving human subjects, including studies of PAPSS1 variants in patient populations, requires IRB approval prior to initiation, regardless of funding source. This applies to both biomedical and social-behavioral research projects . Researchers must submit a formal protocol for IRB review and cannot rely on informal determinations via email or phone conversations.
What are the considerations for student research projects involving PAPSS1?
Student academic research projects investigating PAPSS1 require IRB approval if they qualify as human subjects research, whether biomedical or social-behavioral in nature . Faculty advisors should guide students through the IRB submission process and ensure compliance with all research protections. This requirement applies regardless of whether the research is funded or unfunded.
Product Science Overview
3’-Phosphoadenosine 5’-Phosphosulfate Synthase 1 (PAPSS1) is a crucial enzyme in the biosynthesis of 3’-Phosphoadenosine 5’-Phosphosulfate (PAPS), a key molecule involved in sulfation reactions. Sulfation is an essential biochemical process that modifies various molecules, including hormones, drugs, and xenobiotics, enhancing their solubility and excretion.
PAPSS1 is a bifunctional enzyme that catalyzes two sequential reactions. It first converts adenosine 5’-phosphosulfate (APS) to PAPS using ATP as a phosphate donor. This enzyme is composed of two domains: an ATP sulfurylase domain and an APS kinase domain . The ATP sulfurylase domain catalyzes the formation of APS from ATP and sulfate, while the APS kinase domain phosphorylates APS to form PAPS .
PAPS is the most common coenzyme in sulfotransferase reactions, which are part of the sulfation pathways . These pathways are vital for the metabolism and detoxification of various endogenous and exogenous compounds. In humans, PAPSS1 and its isoform PAPSS2 are responsible for the endogenous synthesis of PAPS .
Recombinant PAPSS1 is produced using Escherichia coli (E. coli) expression systems. The recombinant protein is typically a non-glycosylated polypeptide chain containing 626 amino acids and has a molecular mass of approximately 70.9 kDa . It is purified using proprietary chromatographic techniques to ensure high purity and activity .
Recombinant PAPSS1 is typically stored at -20°C for long-term storage and at 4°C for short-term use. To maintain its stability, it is recommended to avoid multiple freeze-thaw cycles and to add a carrier protein, such as human serum albumin (HSA) or bovine serum albumin (BSA), for long-term storage .
