Phosphoadenosine 5'-phosphosulfate Synthase Research Articles

Local Anesthetic Bupivacaine Inhibits Melanoma Proliferation and Metastasis by Targeting PAPSS2.

Melanoma is a highly invasive skin cancer with limited treatment strategies. Bupivacaine, a commonly used local anesthetic recognized for its safety, has shown promise in combating tumors. 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2) is a key enzyme in the sulfation process and is associated with the development and metastasis of various tumors. This study aimed to explore the mechanism by which bupivacaine inhibits melanoma proliferation and metastasis by targeting PAPSS2. The effects of bupivacaine on the proliferation of A375 and A2058 melanoma cells were evaluated using Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) labeling, and clonogenic assays. Cell migration, invasion, and PAPSS2 expression were evaluated using Transwell experiments and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) analysis. Additionally, an in vivo melanoma tumor model in nude mice was constructed to evaluate the impact of bupivacaine on melanoma growth and metastasis. Immunohistochemistry was used to assess tumor metastasis and PAPSS2 expression levels in the nude mouse model. Experimental results demonstrated that bupivacaine significantly inhibited melanoma proliferation and invasion compared to the control group. Notably, this inhibitory effect was partially reversed by PAPSS2 overexpression. In vivo experiments demonstrated that bupivacaine-treated nude mice exhibited reduced tumor volumes, weights, and fewer lung metastatic foci. Molecular analysis via qRT-PCR and immunohistochemistry analysis further indicated that bupivacaine significantly reduced PAPSS2 in tumor tissues. This study confirms that bupivacaine, a local anesthetic, can inhibit melanoma proliferation and metastasis by targeting the PAPSS2 signaling pathway. These findings suggest its potential as an anti-tumor medication and present new treatment strategies for melanoma.

Sulfation of chondroitin and bile acids converges to antagonize Wnt/β-catenin signaling and inhibit APC deficiency-induced gut tumorigenesis

Sulfation is a crucial and prevalent conjugation reaction involved in cellular processes and mammalian physiology. 3′-Phosphoadenosine 5′-phosphosulfate (PAPS) synthase 2 (PAPSS2) is the primary enzyme to generate the universal sulfonate donor PAPS. The involvement of PAPSS2-mediated sulfation in adenomatous polyposis coli (APC) mutation-promoted colonic carcinogenesis has not been reported. Here, we showed that the expression of PAPSS2 was decreased in human colon tumors along with cancer stages, and the lower expression of PAPSS2 was correlated with poor prognosis in advanced colon cancer. Gut epithelial-specific heterozygous Apc deficient and Papss2-knockout (ApcΔgut-HetPapss2Δgut) mice were created, and the phenotypes were compared to the spontaneous intestinal tumorigenesis of ApcΔgut-Het mice. ApcΔgut-HetPapss2Δgut mice were more sensitive to gut tumorigenesis, which was mechanistically accounted for by the activation of Wnt/β-catenin signaling pathway due to the suppression of chondroitin sulfation and inhibition of the farnesoid X receptor (FXR)-transducin-like enhancer of split 3 (TLE3) gene regulatory axis. Chondroitin sulfate supplementation in ApcΔgut-HetPapss2Δgut mice alleviated intestinal tumorigenesis. In summary, we have uncovered the protective role of PAPSS2-mediated chondroitin sulfation and bile acids–FXR–TLE3 activation in the prevention of gut carcinogenesis via the antagonization of Wnt/β-catenin signaling. Chondroitin sulfate may be explored as a therapeutic agent for Papss2 deficiency-associated colonic carcinogenesis.

A tale of two switches: Redox regulation of adenosine-5′-phosphosulfate kinase in humans and plants

The sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is a near-universal component of sulfur metabolism. In a report by Zhang etal. in this issue of Structure, X-ray crystal structures of the APS kinase domains from human PAPS synthase reveal dynamic substrate recognition and a regulatory "redox switch" analogous to that previously described only in plant APS kinases.

Redox switching mechanism of the adenosine 5′-phosphosulfate kinase domain (APSK2) of human PAPS synthase 2

Adenosine 5'-phosphosulfate kinase (APSK) catalyzes the rate-limiting biosynthetic step of the universal sulfuryl donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). In higher eukaryotes, the APSK and ATP sulfurylase (ATPS) domains are fused in a single chain. Humans have two bifunctional PAPS synthetase isoforms: PAPSS1 with the APSK1 domain and PAPSS2 containing the APSK2 domain. APSK2 displays a distinct higher activity for PAPSS2-mediated PAPS biosynthesis during tumorigenesis. How APSK2 achieves excess PAPS production has remained unclear. APSK1 and APSK2 lack the conventional redox-regulatory element present in plant PAPSS homologs. Here we elucidate the dynamic substrate recognition mechanism of APSK2. We discover that APSK1 contains a species-specific Cys-Cys redox-regulatory element that APSK2 lacks. The absence of this element in APSK2 enhances its enzymatic activity for excess PAPS production and promotes cancer development. Our results help to understand the roles of human PAPSSs during cell development and may facilitate PAPSS2-specific drug discovery.

Construction of a Protein Crystalline Inclusion-Based Enzyme Immobilization System for Biosynthesis of PAPS from ATP and Sulfate.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the bioactive form of sulfate and is involved in all biological sulfation reactions. The enzymatic transformation method for PAPS is promising, but the low efficiency and high cost of enzyme purification and storage restrict its practical applications. Here, we reported PAPS biosynthesis with a protein crystalline inclusion (PCI)-based enzyme immobilization system. First, the in vivo crystalline inclusion protein CipA was identified as an efficient auto-assembly tag for immobilizing the bifunctional PAPS synthase (ASAK). After characterizing the pyrophosphokinase activity of a polyphosphate exonuclease PaPPX from Pseudomonas aeruginosa, and optimizing the linker fragment, auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA were constructed. Then, the auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA with high stability were co-expressed and immobilized for constructing a transformation system. The highest transformation rate of PAPS from ATP and sulfate reached 90%, and the immobilized enzyme can be reused 10 times. The present work provided a convenient, efficient, and easy to be enlarged auto-immobilization system for PAPS biosynthesis from ATP and sulfate. The immobilization system also represented a new approach to reduce the production cost of PAPS by facilitating the purification, storage, and reuse of related enzymes, and it would boost the studies on biotechnological production of glycosaminoglycans and sulfur-containing natural compounds.

Clinical Diagnostic Values of Transfer RNA-Derived Fragment tRF-41-YDLBRY73W0K5KKOVD and its Effects on the Growth of Gastric Cancer Cells.

Gastric cancer (GC) is a serious disease with high mortality and poor prognosis. It is known that tRNA halves play key roles in the progression of cancer. This study explored the function of the tRNA half tRF-41-YDLBRY73W0K5KKOVD in GC. Quantitative real-time reverse transcription-polymerase chain reaction was used to measure RNA levels. The level of tRF-41-YDLBRY73W0K5KKOVD in GC cells was regulated by its mimics or inhibitor. Cell proliferation was evaluated by using a Cell Counting Kit-8 and EdU cell proliferation assay. A Transwell assay was used to detect cell migration. Flow cytometry was used to measure cell cycle and apoptosis. The results showed that tRF-41-YDLBRY73W0K5KKOVD expression was decreased in GC cells and tissues. Functionally, overexpression of tRF-41-YDLBRY73W0K5KKOVD inhibited cell proliferation, reduced migration, repressed the cell cycle, and promoted cell apoptosis in GC cells. Based on RNA sequencing results and luciferase reporter assays, 3'-phosphoadenosine-5'-phosphosulfate synthase 2 (PAPSS2) was identified as a target gene of tRF-41-YDLBRY73W0K5KKOVD. These findings indicated that tRF-41-YDLBRY73W0K5KKOVD inhibited GC progression, suggesting that tRF-41-YDLBRY73W0K5KKOVD might be a potential therapeutic target in GC.

DOP35 High-fat diet reduces gut microbiota-derived metabolite indole-acetic acid and aggravates colitis

Abstract Background It is widely accepted that high-fat diet (HFD) has been considered as one of the risk factors of inflammatory bowel disease(IBD), while the mechanism is little known. We aimed to examined whether dietary high fat promotes colonic inflammation through modulating gut microbial tryptophan metabolites. Methods The C57BL/6 female mice were fed with chow diet (12kcal% fat) or HFD (60kcal% fat) for 2 weeks. Fresh fecal samples were collected before administration of 2% DSS. LC-MS/MS analysis was performed to detect fecal microbial tryptophan metabolites. Next, 8-week-old mice were administrated 3-indoleacetic acid (IAA) for 7 days prior to 2.5% DSS treatment. The severity of colitis was assessed and the colonic sections were collected. RNA sequencing was performed to analyze the differential genes in intestinal tissue of mice fed with IAA or not. Real-time PCR and Western blotting were performed to detect the transcription and protein expression of mucin sulfation-related genes. High iron diamine-alcian blue (HID-AB) staining of colon tissues and LS174T cells was used to detect the abundance of sulfated mucin. Additionally, the Chromatin immunoprecipitation (ChIP) sequencing is performed to confirm the binding of aryl hydrocarbon receptor(AHR) to 3’-phosphoadenosine 5’-phosphosulfate (PAPS) synthase 2 (PAPSS2). Results Short-term HFD increased the susceptibility to colitis of mice. Mice fed with HFD have impaired tryptophan metabolism with remarkable lower level of IAA. IAA supplementation resulted in ameliorative colitis symptoms. Mechanistically, the transcriptome sequencing detected that IAA supplementation enriched the the cytosolic sulfonation pathway and significantly increased the expression of PAPSS2 of colon tissue, a rate-limiting enzyme on the mucin sulfation pathway. Next, in vivo and in vitro experiments verified IAA increased the expression of PAPSS2 and its downstream genes and promoted the mucin sulfation in golgi apparatus of colon through AHR. ChIP sequencing and ChIP-PCR verified IAA can enhance the binding of AHR to the promoter region of PAPSS2 , which further promoted the expression of PAPS transporter 2 (PAPST2 or Slc35b3) and ultimately promotes the mucin sulfation. Conclusion HFD altered the gut microbial tryptophan metabolites and disrupt the protective effect of IAA on the intestinal barrier. IAA could relief colitis through AHR-PAPSS2-PAPST2-mucin sulfation axis, which may present a potential approach for precise prevention and treatment of IBD.

Design of a biocatalytic cascade for the enzymatic sulfation of unsulfated chondroitin with in situ generation of PAPS.

Sulfation of molecules in living organisms is a process that plays a key role in their functionality. In mammals, the sulfation of polysaccharides (glycosaminoglycans) that form the proteoglycans present in the extracellular matrix is particularly important. These polysaccharides, through their degree and sulfation pattern, are involved in a variety of biological events as signal modulators in communication processes between the cell and its environment. Because of this great biological importance, there is a growing interest in the development of efficient and sustainable sulfation processes, such as those based on the use of sulfotransferase enzymes. These enzymes have the disadvantage of being 3'-phosphoadenosine 5'-phosphosulfate (PAPS) dependent, which is expensive and difficult to obtain. In the present study, a modular multienzyme system was developed to allow the in situ synthesis of PAPS and its coupling to a chondroitin sulfation system. For this purpose, the bifunctional enzyme PAPS synthase 1 (PAPSS1) from Homo sapiens, which contains the ATP sulfurylase and APS kinase activities in a single protein, and the enzyme chondroitin 4-O-sulfotransferase (C4ST-1) from Rattus norvegicus were overexpressed in E. coli. The product formed after coupling of the PAPS generation system and the chondroitin sulfation module was analyzed by NMR.

Markhor-derived Introgression of a Genomic Region Encompassing PAPSS2 Confers High-altitude Adaptability in Tibetan Goats.

Understanding the genetic mechanism of how animals adapt to extreme conditions is fundamental to determine the relationship between molecular evolution and changing environments. Goat is one of the first domesticated species and has evolved rapidly to adapt to diverse environments, including harsh high-altitude conditions with low temperature and poor oxygen supply but strong ultraviolet radiation. Here, we analyzed 331 genomes of domestic goats and wild caprid species living at varying altitudes (high > 3000 m above sea level and low < 1200 m), along with a reference-guided chromosome-scale assembly (contig-N50: 90.4 Mb) of a female Tibetan goat genome based on PacBio HiFi long reads, to dissect the genetic determinants underlying their adaptation to harsh conditions on the Qinghai-Tibetan Plateau (QTP). Population genomic analyses combined with genome-wide association studies (GWAS) revealed a genomic region harboring the 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2) gene showing strong association with high-altitude adaptability (PGWAS = 3.62 × 10-25) in Tibetan goats. Transcriptomic data from 13 tissues revealed that PAPSS2 was implicated in hypoxia-related pathways in Tibetan goats. We further verified potential functional role of PAPSS2 in response to hypoxia in PAPSS2-deficient cells. Introgression analyses suggested that the PAPSS2 haplotype conferring the high-altitude adaptability in Tibetan goats originated from a recent hybridization between goats and a wild caprid species, the markhor (Capra falconeri). In conclusion, our results uncover a hitherto unknown contribution of PAPSS2 to high-altitude adaptability in Tibetan goats on QTP, following interspecific introgression and natural selection.

Transcriptome responses in copepods Calanus finmarchicus, Calanus glacialis and Calanus hyperboreus exposed to phenanthrene and benzo[a]pyrene.

Arctic and sub-arctic pelagic organisms can be exposed to effluents and spills from offshore petroleum-related activities and thus it is important to understand how they respond to crude oil related contaminants such as polycyclic aromatic hydrocarbons (PAHs). The copepod species Calanus finmarchicus, Calanus glacialis and Calanus hyperboreus represent key links in the arctic marine food web. We performed a transcriptome analysis of the three species exposed to phenanthrene (Phe) and benzo[a]pyrene (BaP) representing low and high molecular weight PAHs, respectively. Differential expression of several genes involved in many cellular pathways was observed after 72h exposure to Phe (0.1μM) and BaP (0.1μM). In C. finmarchicus and C. glacialis, the exposure resulted in up-regulation of genes encoding enzymes in xenobiotic biotransformation, particularly the phase II cytosolic sulfonation system that include 3'-phosphoadenosine 5'-phosphosulfate synthase (PAPSS) and sulfotransferases (SULTs). The sulfonation pathway genes were more strongly induced by BaP than Phe in C. finmarchicus and C. glacialis but were not affected in C. hyperboreus. However, a larger number of genes and pathways were modulated in C. hyperboreus by the PAHs including genes encoding xenobiotic biotransformation and lipid metabolism enzymes, suggesting stronger responses in this species. The results suggest that the cytosolic sulfonation is a major phase II conjugation pathway for PAHs in C. finmarchicus and C. glacialis. Some of the biotransformation systems affected are known to be involved in metabolism of endogenous compounds such as ecdysteroids, which may suggest potential interference with physiological and developmental processes of the copepod species.

Differences in detoxification mechanism and gene expression changes of sulfur metabolism in coping with the air pollutant SO2 between the resistant and ordinary poplar variety

In this study, two poplar varieties with different resistance to sulfur dioxide were selected for a comparative experiment. SO2 fumigation to the poplars was carried out under controlled conditions to determine the variation in the activity of enzymes, the content of metabolites and the expression of enzymes genes in pathways of sulfur metabolism in plants. The results showed that the activity of enzymes and the content of sulfur metabolites were closely related to the response of the poplars to SO2 stress. Populus × euramericana cv. 'Purui' had two ways of detoxification: oxidation detoxification, oxidizing sulfite (SO32−) to sulfate SO42− by sulfite oxidase; reductive detoxification, SO32− being reduced to S2− by sulfite reductase (SiR). Moreover, OASTL and SAT activity, and levels of cysteine (CYS) and glutathione (GSH) also increased in P. × euramericana cv. 'Purui' in response to SO2 fumigation, and the gene expression encoding Glutathione S-transferases (GST), and some enzymes in cysteine and methionine metabolism was up-regulated. For Populus × euramericana cv. '74/76' with weaker resistance to sulfur dioxide, it only detoxified by increasing the activity of SiR, and but down-regulated the expression of gene encoding 3'-phosphoadenosine 5'-phosphosulfate synthase (PAPSS), which could affect the consumption of sulfite in the exposure to SO2. Thus, the SO2-resistant difference of the two poplar varieties is mainly attributed to variation in activity of the enzymes and content of their metabolites in pathways of sulfur metabolism, and gene expression of some enzymes in cysteine and methionine metabolism also plays a role in the resistant difference.

Inhibition of p53 Sulfoconjugation Prevents Oxidative Hepatotoxicity and Acute Liver Failure

Sulfoconjugation of small molecules or protein peptides is a key mechanism to ensure biochemical and functional homeostasis in mammals. The PAPS synthase 2 (PAPSS2) is the primary enzyme to synthesize the universal sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF), in which oxidative stress is a key pathogenic event, whereas sulfation of APAP contributes to its detoxification. The goal of this study was to determine whether and how PAPSS2 plays a role in APAP-induced ALF. Gene expression was analyzed in APAP-induced ALF in patients and mice. Liver-specific Papss2-knockout mice using Alb-Cre (Papss2ΔHC) or AAV8-TBG-Cre (Papss2iΔHC) were created and subjected to APAP-induced ALF. Primary human and mouse hepatocytes were used for invitro mechanistic analysis. The hepatic expression of PAPSS2 was decreased in APAP-induced ALF in patients and mice. Surprisingly, Papss2ΔHC mice were protected from APAP-induced hepatotoxicity despite having a decreased APAP sulfation, which was accompanied by increased hepatic antioxidative capacity through the activation of the p53-p2-Nrf2 axis. Treatment with a sulfation inhibitor also ameliorated APAP-induced hepatotoxicity. Gene knockdown experiments showed that the hepatoprotective effect of Papss2ΔHC was Nrf2, p53, and p21 dependent. Mechanistically, we identified p53 as a novel substrate of sulfation. Papss2 ablation led to p53 protein accumulation by preventing p53 sulfation, which disrupts p53-MDM2 interaction and p53 ubiquitination and increases p53 protein stability. We have uncovered a previously unrecognized and p53-mediated role of PAPSS2 in controlling oxidative response. Inhibition of p53 sulfation may be explored for the clinical management of APAP overdose.

Structural basis for the substrate recognition mechanism of ATP-sulfurylase domain of human PAPS synthase 2

Sulfation is an essential modification on biomolecules in living cells, and 3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is its unique and universal sulfate donor. Human PAPS synthases (PAPSS1 and 2) are the only enzymes that catalyze PAPS production from inorganic sulfate. Unexpectedly, PAPSS1 and PAPSS2 do not functional complement with each other, and abnormal function of PAPSS2 but not PAPSS1 leads to numerous human diseases including bone development diseases, hormone disorder and cancers. Here, we reported the crystal structures of ATP-sulfurylase domain of human PAPSS2 (ATPS2) and ATPS2 in complex with is product 5′-phosphosulfate (APS). We demonstrated that ATPS2 recognizes the substrates by using family conserved residues located on the HXXH and PP motifs, and achieves substrate binding and releasing by employing a non-conserved phenylalanine (Phe550) through a never observed flipping mechanism. Our discovery provides additional information to better understand the biological function of PAPSS2 especially in tumorigenesis, and may facilitate the drug discovery against this enzyme.

Role of aspartic acid residues D87 and D89 in APS kinase domain of human 3′-phosphoadenosine 5′-phosphosulfate synthase 1 and 2b: A commonality with phosphatases/kinases

3′-phosphoadenosine 5′-phosphosulfate (PAPS) is synthesized in two steps by PAPS synthase (PAPSS). PAPSS is comprised of ATP sulfurylase (ATPS) and APS kinase (APSK) domain activities. ATPS combines inorganic sulfate with α-phosphoryl of ATP to form adenosine 5′-phosphosulfate (APS) and PPi. In the second step APS is phosphorylated at 3′-OH using another mole of ATP to form PAPS and ADP catalyzed by APSK. The transfer of gamma-phosphoryl from ATP onto 3′-OH requires Mg2+ and purported to involve residues D87GD89N. We report that mutation of either aspartic residue to alanine completely abolishes APSK activity in PAPS formation. PAPSS is an, unique enzyme that binds to four different nucleotides: ATP and APS on both ATPS and APSK domains and ADP and PAPS exclusively on the APSK domain. The thermodynamic binding and the catalytic interplay must be very tightly controlled to form the end-product PAPS in the forward direction. Though APS binds to ATPS and APSK, in ATPS domain, the APS is a product and for APSK it is a substrate. DGDN motif is absent in ATPS and present in APSK. Mutation of D87 and D89 did not hamper ATPS activity however abolished APSK activity severely. Thus, D87GD89N region is required for stabilization of Mg2+-ATP, in the process of splitting the γ-phosphoryl from ATP and transfer of γ-phosphoryl onto 3′-OH of APS to form PAPS a process that cannot be achieved by ATPS domain. In addition, gamma32P-ATP, trapped phosphoryl enzyme intermediate more with PAPSS2 than with PAPSS1. This suggests inherent active site residues could control novel catalytic differences. Molecular docking studies of hPAPSS1with ATP + Mg2+ and APS of wild type and mutants supports the experimental results.

Intestinal Sulfation is Essential to Protect Against Colitis‐Associated Colonic Carcinogenesis

Sulfation is a conjugation reaction essential for numerous biochemical and cellular functions in mammals. The 3′-phosphoadenosine 5'-phosphosulfate (PAPS) synthase 2 (PAPSS2) is the key enzyme to generate PAPS, which is the universal sulfonate donor for all sulfation reactions. The goal of this study is to determine whether and how PAPSS2 plays a role in colitis and colitis-associated colonic carcinogenesis. The expression of PAPSS2 is decreased in the colon cancers of mice and humans. The lower expression of PAPSS2 in colon cancer patients is correlated with worse survival. In mice, intestinal ablation of the Papss2 gene (Papss2ΔIE) promoted colitis and associated colon cancer by damaging the intestinal mucosal barrier, increasing intestinal permeability and bacteria infiltration, and worsening the intestinal tumor microenvironment. Mechanistically, the Papss2ΔIE mice exhibited reduced intestinal sulfomucin content. Metabolomic analyses revealed the accumulation of bile acids including farnesoid X receptor (FXR) antagonist bile acid tauro-β-muricholic acid (T-β-MCA), as well as deficiency in the formation of bile acid-sulfates in the colon of Papss2ΔIE mice. In summary, we have uncovered the role of PAPSS2-mediated sulfation in colitis and associated colonic carcinogenesis. Intestinal sulfation may represent a potential diagnostic marker, and PAPSS2 may serve as a potential therapeutic target for inflammatory bowel disease and colon cancer.

Intestinal Sulfation Is Essential to Protect Against Colitis and Colonic Carcinogenesis

Sulfation is a conjugation reaction essential for numerous biochemical and cellular functions in mammals. The 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase 2 (PAPSS2) is the key enzyme to generate PAPS, which is the universal sulfonate donor for all sulfation reactions. The goal of this study was to determine whether and how PAPSS2 plays a role in colitis and colonic carcinogenesis. Tissue arrays of human colon cancer specimens, gene expression data, and clinical features of cancer patients were analyzed. Intestinal-specific Papss2 knockout mice (Papss2ΔIE) were created and subjected to dextran sodium sulfate-induced colitis and colonic carcinogenesis induced by a combined treatment of azoxymethane and dextran sodium sulfate or azoxymethane alone. The expression of PAPSS2 is decreased in the colon cancers of mice and humans. The lower expression of PAPSS2 in colon cancer patients is correlated with worse survival. Papss2ΔIE mice showed heightened sensitivity to colitis and colon cancer by damaging the intestinal mucosal barrier, increasing intestinal permeability and bacteria infiltration, and worsening the intestinal tumor microenvironment. Mechanistically, the Papss2ΔIE mice exhibited reduced intestinal sulfomucin content. Metabolomic analyses revealed the accumulation of bile acids, including the Farnesoid X receptor antagonist bile acid tauro-β-muricholic acid, and deficiency in the formation of bile acid sulfates in the colon of Papss2ΔIE mice. We have uncovered an important role of PAPSS2-mediated sulfation in colitis and colonic carcinogenesis. Intestinal sulfation may represent a potential diagnostic marker and PAPSS2 may serve as a potential therapeutic target for inflammatory bowel disease and colon cancer.

PAPSS2 ablation prevents acetaminophen‐induced hepatotoxicity in mice

Sulfate plays an essential role in numerous biochemical and cellular processes in mammalian physiology. The PAPS synthase 2 (PAPSS2) is the primary enzyme to synthesize 3'‐phosphoadenosine 5'‐phosphosulfate (PAPS), which is the universal sulfonate donor for all sulfonation reactions in mammals. Acetaminophen (APAP) overdose is the leading cause of acute liver failure in the United States, and sulfation is widely believed to contribute to the detoxification of many drugs including APAP. The goal of this study is to determine whether PAPSS2 plays a role in APAP‐induced hepatotoxicity. Surprisingly, we found the PAPSS2 knockout mice were protected from APAP‐induced hepatotoxicity, and this protection was accompanied by increased antioxidant ability through the activation Nrf2‐Keap1 and p53 pathways. The hepatoprotective effect of PAPSS2 ablation was abolished when p53 was knocked down. In summary, we have uncovered a unique role of PAPSS2 in APAP‐induced hepatotoxicity.Support or Funding InformationThis work was supported in part by National Institutes of Health Grants DK083952, ES023438

Melting Down Protein Stability: PAPS Synthase 2 in Patients and in a Cellular Environment.

Within the crowded and complex environment of the cell, a protein experiences stabilizing excluded-volume effects and destabilizing quinary interactions with other proteins. Which of these prevail, needs to be determined on a case-by-case basis. PAPS synthases are dimeric and bifunctional enzymes, providing activated sulfate in the form of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) for sulfation reactions. The human PAPS synthases PAPSS1 and PAPSS2 differ significantly in their protein stability as PAPSS2 is a naturally fragile protein. PAPS synthases bind a series of nucleotide ligands and some of them markedly stabilize these proteins. PAPS synthases are of biomedical relevance as destabilizing point mutations give rise to several pathologies. Genetic defects in PAPSS2 have been linked to bone and cartilage malformations as well as a steroid sulfation defect. All this makes PAPS synthases ideal to study protein unfolding, ligand binding, and the stabilizing and destabilizing factors in their cellular environment. This review provides an overview on current concepts of protein folding and stability and links this with our current understanding of the different disease mechanisms of PAPSS2-related pathologies with perspectives for future research and application.

Evolution of Catalysis: 3′‐Phospho adenosine 5′‐phosphosulfate synthase (PAPSS) and Methionine gamma Lyase deaminase (Mgld) as an Experimental and Computational Model System

Binding of substrate or ligand to a protein is the first common step between a receptor protein and an enzyme. However, the receptor protein usually bear low Kd, that is higher affinity towards the ligand. Whereas, many of the enzymes bear higher Km, that is relatively lower affinity. What is the criteria behind this distinction? The product, is the transformed substrate and it would have to leave the active site so that it can perform its function outside the binding pocket or it can be transformed further by a separate set (s) enzyme(s). Between this inter play, in a bifunctional enzyme, a product from one active site pocket is a substrate for the neighboring domain. Therefore, the product of domain [(1), ATP sulfurylase] is a substrate for the domain [(2) APS kinase], the new active site. This mandates that the active site 2 must have higher affinity (low Km) than that of the domain 1, so that it stays bound tighter and gets transformed into product 2. This would avoid futile cycle of substrate of domain 2 leaving back to be bound to domain 1 to become the substrate for the domain 1 backward reaction, or to occlude/inhibit. My data, from ATP sulfurylase (domain 1) and APS kinase (domain 2) of PAPSS kinetics proves the fundamentally perfected evolutionary concept. The computational data supports this concept as well. Afterall, nature rarely fails to achieve perfection and sophistication, especially after millions of years, as part of the evolutionary process. Certain enzymes like tryptophan synthetases have developed channeling as a means to perfect its overall efficiency of ultimate product formation. PAPSS is a good model system to address the binding energies, Km, Kcat. PAPSS is a standalone enzyme that binds to five different nucleotides ATP, AMP, ADP, APS, and PAPS. In addition, this enzyme binds to two different, a phospho and a sulfo nucleotides. Methionine gammalyase deaminase (Mgld) is a different example that use PLP as a coenzyme to assist catalysis. Mgld cleaves C‐S bond of methionine to form methylthiol and the PLP bound 2‐aminobutyrate is deaminated to form alpha‐keto butyrate. Here the active site is constrained and very little flexibility is observed. Thus, the evolution of enzyme active site catalysis will be discussed using PAPSS and Mgld systems.Support or Funding InformationN/AThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1

The high-energy sulfate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS), generated by human PAPS synthase isoforms PAPSS1 and PAPSS2, is required for all human sulfation pathways. Sulfotransferase SULT2A1 uses PAPS for sulfation of the androgen precursor dehydroepiandrosterone (DHEA), thereby reducing downstream activation of DHEA to active androgens. Human PAPSS2 mutations manifest with undetectable DHEA sulfate, androgen excess, and metabolic disease, suggesting that ubiquitous PAPSS1 cannot compensate for deficient PAPSS2 in supporting DHEA sulfation. In knockdown studies in human adrenocortical NCI-H295R1 cells, we found that PAPSS2, but not PAPSS1, is required for efficient DHEA sulfation. Specific APS kinase activity, the rate-limiting step in PAPS biosynthesis, did not differ between PAPSS1 and PAPSS2. Co-expression of cytoplasmic SULT2A1 with a cytoplasmic PAPSS2 variant supported DHEA sulfation more efficiently than co-expression with nuclear PAPSS2 or nuclear/cytosolic PAPSS1. Proximity ligation assays revealed protein–protein interactions between SULT2A1 and PAPSS2 and, to a lesser extent, PAPSS1. Molecular docking studies showed a putative binding site for SULT2A1 within the PAPSS2 APS kinase domain. Energy-dependent scoring of docking solutions identified the interaction as specific for the PAPSS2 and SULT2A1 isoforms. These findings elucidate the mechanistic basis for the selective requirement for PAPSS2 in human DHEA sulfation.