Sulfate Donor Research Articles

B-350 The application of MALDI-TOF imaging to biomarker discovery in a mouse model of papillomavirus associated tumorigenesis

Abstract Background Human papillomavirus (HPV) can cause infection and dysplasia in cutaneous epithelium of immunodeficient patients. An animal model has been developed to study HPV-associated tumorigenesis using Nude mouse (Foxn1nu) and mouse papillomavirus (MmuPV1). This study applies MALDI-TOF imaging to compare the expression pattern of small molecules between viral-infected dysplastic mouse skin and mock-infected control tissue. Three sets of molecules showing distinct patterns are identified in correlation with the upregulation of an enzyme in related biosynthetic pathway. Methods Nude mice were wounded on tail and inoculated with MmPV1 suspension (1×109 viral DNA genomes per infected site) or PBS 24 hours later for treatment and control group, respectively. Tail skins were harvested 4 weeks post-infection and processed for cryosectioning. Tissue sections were analyzed using timsTOF flex (Bruker) under negative ion mode with 9-aminoacridine as the matrix. Viral infection (MmuPV1-L1 antigen) and enzyme expression (3'-phosphoadenosine 5'-phosphosulfate synthetase 1, PAPSS1) were assessed by immunofluorescence on adjacent sections. Results Three peaks demonstrated distinct distribution patterns in mouse skin. One peak at m/Z = 465 was identified as cholesterol sulfate (CS) by tandem MS and comparing with the standard. CS was enriched in keratinocytes, hair follicles, and blood vessels in normal skins, and it increased in dysplastic skins with enhanced angiogenesis. The combined peaks at m/Z = 327 & 329 were tentatively identified as docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA). These long chain polyunsaturated fatty acids (LC-PUFAs) were present in the dermis as foci partially co-localized with CS, and they also increased in virus-infected skins. The peak at m/Z = 426 may represent a combination of ADP and APS. Although these two isobars cannot be separated by tandem MS, APS was revealed by the presence of sulfur isotopes at expected natural abundance (34S and 32S). They were present at a low level in the epidermis of normal skins but significantly increased after virus infection. APS and ADP were closely associated with the proliferating cells, and the signal intensity also increased with the degree of tissue dysplasia. As a single factor, APS & ADP demonstrated a fair differential power for MmuPV1-induced dysplastic skin and normal control (AUC of ROC curve = 0.75). The bifunctional enzyme PAPSS1 catalyzes the conversion of ATP to 3’-phosphonato-5’-adenyl sulfate (PAPS) via APS as an intermediate. Interestingly, immunofluorescence showed the expression of PAPSS1 also increased significantly with the severity of tissue dysplasia and MmuPV1 infection. Conclusions MALDI-TOF imaging reveals three sets of molecules with distinct patterns in mouse skin, and their levels are increased during MmuPV1-induced epithelium dysplasia. As a precursor of the common sulfate donor (PAPS), increased APS and ADP in the epidermis of virus-infected skins indicate an upregulation of sulfation pathway and adenine metabolism. This finding is supported by enhanced expression of enzyme PAPSS1 and an increased sulfation reaction product, CS. In addition, DHA and DPA are also increased in the dermis of virus-infected skins, implying a disturbance in LC-PUFA metabolism. This study demonstrates combined use of MALDI-TOF imaging and immunostaining in the search of novel biomarkers of HPV-associated tumorigenesis.

Steroid sulfatase and sulfotransferases in the estrogen and androgen action of gynecological cancers: current status and perspectives.

Sulfatase (STS) and sulfotransferases (SULT) have important role in the biosynthesis and action of steroid hormones. STS catalyzes the hydrolysis of estrone-sulfate (E1-S) and dehydroepiandrosterone-sulfate (DHEA-S), while sulfotransferases catalyze the reverse reaction and require 3-phosphoadenosine-5-phosphosulfate as a sulfate donor. These enzymes control the concentration of active estrogens and androgens in peripheral tissues. Aberant expression of STS and SULT genes has been found in both, benign hormone-dependent diseases and hormone-dependent cancers. The aim of this review is to present the current knowledge on the role of STS and SULT in gynecological cancers, endometrial (EC) and ovarian cancer (OC). EC is the most common and OC the most lethal gynecological cancer. These cancers primarily affect postmenopausal women and therefore rely on the local production of steroid hormones from inactive precursors, either DHEA-S or E1-S. Following cellular uptake by organic anion transporting polypeptides (OATP) or organic anion transporters (OAT), STS and SULT regulate the formation of active estrogens and androgens, thus disturbed balance between STS and SULT can contribute to the onset and progression of cancer. The importance of these enzymes in peripheral estrogen biosynthesis has long been recognized, and this review provides new data on the important role of STS and SULT in the formation and action of androgens, their regulation and inhibition, and their potential as prognostic biomarkers.

Sulfate: a neglected (but potentially highly relevant) anion.

Sulfate is an important anion as sulfonation is essential in modulation of several compounds, such as exogens, polysaccharide chains of proteoglycans, cholesterol or cholesterol derivatives and tyrosine residues of several proteins. Sulfonation requires the presence of both the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and a sulfotransferase. Genetic disorders affecting sulfonation, associated with skeletal abnormalities, impaired neurological development and endocrinopathies, demonstrate the importance of sulfate. Yet sulfate is not measured in clinical practice. This review addresses sulfate metabolism and consequences of sulfonation defects, how to measure sulfate and why we should measure sulfate more often.

Crystal structure of activating sulfotransferase SgdX2 involved in biosynthesis of secondary metabolite sungeidine

Microorganisms synthesize a plethora of complex secondary metabolites, many of which are beneficial to human health, such as anticancer agents and antibiotics. Among these, the Sungeidines are a distinct class of secondary metabolites known for their bulky and intricate structures. They are produced by a specific biosynthetic gene cluster within the genome of the soil-dwelling actinomycete Micromonospora sp. MD118. A notable enzyme in the Sungeidine biosynthetic pathway is the activating sulfotransferase SgdX2. In this pathway, SgdX2 mediates a key sulfation step, after which the product undergoes spontaneous dehydration to yield a Sungeidine compound. To delineate the structural basis for SgdX2's substrate recognition and catalytic action, we have determined the crystal structure of SgdX2 in complex with its sulfate donor product, 3′-phosphoadenosine 5′-phosphate (PAP), at a resolution of 1.6 Å. Although SgdX2 presents a compact overall structure, its core elements are conserved among other activating sulfotransferases. Our structural analysis reveals a unique substrate-binding pocket that accommodates bulky, complex substrates, suggesting a specialized adaptation for Sungeidine synthesis. Moreover, we have constructed a substrate docking model that provides insights into the molecular interactions between SgdX2 and Sungeidine F, enhancing our understanding of the enzyme's specificity and catalytic mechanism. The model supports a general acid-base catalysis mechanism, akin to other sulfotransferases, and underscores the minor role of disordered regions in substrate recognition. This integrative study of crystallography and computational modeling advances our knowledge of microbial secondary metabolite biosynthesis and may facilitate the development of novel biotechnological applications.

Enhanced SLC35B2/SAV1 sulfation axis promotes tumor growth by inhibiting Hippo signaling in HCC.

Protein tyrosine sulfation (PTS) is a common posttranslational modification that regulates a variety of physiological and pathological processes. However, the role of PTS in cancer remains poorly understood. The goal of this study was to determine whether and how PTS plays a role in HCC progression. By mass spectrometry and bioinformatics analysis, we identified SAV1 as a novel substrate of PTS in HCC. Oxidative stress upregulates the transcription of SLC35B2, a Golgi-resident transporter of sulfate donor 3'-phosphoadenosine 5'-phosphosulfate, leading to increased sulfation of SAV1. Sulfation of SAV1 disrupts the formation of the SAV1-MST1 complex, resulting in a decrease of MST1 phosphorylation and subsequent inactivation of Hippo signaling. These molecular events ultimately foster the growth of HCC cells both in vivo and in vitro. Moreover, SLC35B2 is a novel transcription target gene of the Hippo pathway, constituting a positive feedback loop that facilitates HCC progression under oxidative stress. Our findings reveal a regulatory mechanism of the SLC35B2/SAV1 sulfation axis in response to oxidative stress, highlighting its potential as a promising therapeutic target for HCC.

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.

Abstract 6053: Sulfation is required for prostate cancer xenograft tumor formation but is dispensable for cell viabilityin vitro

Abstract Sulfation of proteins, carbohydrates, lipids, and xenobiotics is an essential post-translational modification (PTM) process thought to play critical roles in diverse biological processes ranging from detoxification, cell signaling, and extracellular matrix architecture, to immune modulation. Sulfation is accomplished by the universal sulfate donor, PAPS (3'-Phosphoadenosine-5'-phosphosulfate), which is synthesized by bifunctional enzymes PAPSS1 and PAPSS2 (PAPS synthases). The PAPSS2 gene situates near PTEN and is frequently deleted with PTEN across cancer types. Approximately 20% of prostate cancer patients exhibit loss of PTEN, and ~50% of these cases also sustain a loss of PAPSS2. However, the loss of PAPSS2 appears to be tolerated and possibly compensated by its functionally redundant paralogue, PAPSS1, located on chromosome 4q24. The functional redundancy between PAPSS1 and PAPSS2 suggests that these two genes may be collateral lethality pair provided that sulfation is essential for cancer cell viability. Thus, we hypothesize that targeting PAPSS1 in PTEN/PAPSS2-null prostate cancer can generate cancer-specific vulnerabilities while leaving normal cells undisturbed. To assess this possibility, knockdown and knockout of PAPSS1 in cell lines of PAPSS2-null and PAPSS2-wildtype background were generated to characterize cell viability in vitro and tumor formation in vivo. PAPS and APS, an intermediary product of the sulfation pathway, are measured to verify that no alternative pathways for sulfate donors exist and that the co-extinction of PAPSS1/2 eliminates all avenues of generating sulfate donors. Combined extinction of PAPSS1/2 across multiple cancer cell lines was shown to be tolerated in vitro, and recurrent changes in morphology were observed. Loss of sulfation verified by the disappearance of sulfotyrosine and mass spectrometry measurements of PAPS and APS are pending. Our In vitro results surprisingly indicate that a major PTM, like sulfation is entirely dispensable for cancer cell viability under normal culture conditions. However, PAPSS1/2-null cell lines demonstrated a profound delay in tumor formation and prolonged survival, suggesting that sulfation may be required for stromal and innate immune modulation. Citation Format: Ko-Chien Chen, Yonhong Liu, Chenchu Lin, Er-Yen (Nick) Yen, Francesca Citron, Xingdi Ma, Tan Lin, Philip Lorenzi, Florian Muller, Ronald DePinho. Sulfation is required for prostate cancer xenograft tumor formation but is dispensable for cell viabilityin vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6053.

What Controls the Sulfur Isotope Fractionation during Dissimilatory Sulfate Reduction?

Sulfate often behaves conservatively in the oxygenated environments but serves as an electron acceptor for microbial respiration in a wide range of natural and engineered systems where oxygen is depleted. As a ubiquitous anaerobic dissimilatory pathway, therefore, microbial reduction of sulfate to sulfide has been of continuing interest in the field of microbiology, ecology, biochemistry, and geochemistry. Stable isotopes of sulfur are an effective tool for tracking this catabolic process as microorganisms discriminate strongly against heavy isotopes when cleaving the sulfur-oxygen bond. Along with its high preservation potential in environmental archives, a wide variation in the sulfur isotope effects can provide insights into the physiology of sulfate reducing microorganisms across temporal and spatial barriers. A vast array of parameters, including phylogeny, temperature, respiration rate, and availability of sulfate, electron donor, and other essential nutrients, has been explored as a possible determinant of the magnitude of isotope fractionation, and there is now a broad consensus that the relative availability of sulfate and electron donors primarily controls the magnitude of fractionation. As the ratio shifts toward sulfate, the sulfur isotope fractionation increases. The results of conceptual models, centered on the reversibility of each enzymatic step in the dissimilatory sulfate reduction pathway, are in qualitative agreement with the observations, although the underlying intracellular mechanisms that translate the external stimuli into the isotopic phenotype remain largely unexplored experimentally. This minireview offers a snapshot of our current understanding of the sulfur isotope effects during dissimilatory sulfate reduction as well as their potential quantitative applications. It emphasizes the importance of sulfate respiration as a model system for the isotopic investigation of other respiratory pathways that utilize oxyanions as terminal electron acceptors.

Inhibition of the SLC35B2–TPST2 Axis of Tyrosine Sulfation Attenuates the Growth and Metastasis of Pancreatic Ductal Adenocarcinom

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the United States. Tyrosine sulfation, catalyzed by the tyrosylprotein sulfotransferase 2 (TPST2), is a post-translational modification essential for protein-protein interactions and cellular functions. Solute carrier family 35 member B (SLC35B2) is a key transporter that transports the universal sulfate donor 3'-phosphoadenosine 5'-phosphosulfate into the Golgi apparatus where the protein sulfation occurs. The goal of this study was to determine whether and how the SLC35B2-TPST2 axis of tyrosine sulfation plays a role in PDAC. Gene expression was analyzed in PDAC patients and mice. Human PDAC MIA PaCa-2 and PANC-1 cells were used for invitro studies. TPST2-deficient MIA PaCa-2 cells were generated to assess xenograft tumor growth invivo. Mouse PDAC cells derived from the KrasLSL-G12D/+;Tp53L/+;Pdx1-Cre (KPC) mice were used to generate Tpst2 knockout KPC cells to evaluate tumor growth and metastasis invivo. High expressions of SLC35B2 and TPST2 were correlated with poor PDAC patient survival. Knocking down SLC35B2 or TPST2, or pharmacologicically inhibiting sulfation, resulted in the inhibition of PDAC cell proliferation and migration invitro. TPST2-deficient MIA PaCa-2 cells showed inhibited xenograft tumor growth. Orthotopic inoculation of Tpst2 knockout KPC cells in mice showed inhibition of primary tumor growth, local invasion, and metastasis. Mechanistically, the integrin β4 was found to be a novel substrate of TPST2. Inhibition of sulfation destabilizes integrin β4 protein, which may have accounted for the suppression of metastasis. Targeting the SLC35B2-TPST2 axis of tyrosine sulfation may represent a novel approach for therapeutic intervention of PDAC.

Crystal structure of adenosine 5ʹ-phosphosulfate kinase isolated from Archaeoglobus fulgidus

The 3′-phosphoadenosine-5′-phosphosulfate (PAPS) molecule is essential during enzyme-catalyzed sulfation reactions as a sulfate donor and is an intermediate in the reduction of sulfate to sulfite in the sulfur assimilation pathway. PAPS is produced through a two-step reaction involving ATP sulfurylase and adenosine 5′-phosphosulfate (APS) kinase enzymes/domains. However, archaeal APS kinases have not yet been characterized and their mechanism of action remains unclear. Here, we first structurally characterized APS kinase from the hyperthermophilic archaeon Archaeoglobus fulgidus, (AfAPSK). We demonstrated the PAPS production activity of AfAPSK at the optimal growth temperature (83 °C). Furthermore, we determined the two crystal structures of AfAPSK: ADP complex and ATP analog adenylyl-imidodiphosphate (AMP-PNP)/Mg2+/APS complex. Structural and complementary mutational analyses revealed the catalytic and substrate recognition mechanisms of AfAPSK. This study also hints at the molecular basis behind the thermal stability of AfAPSK.

Remediation of high-concentration Cr(VI)-contaminated soils with FeSO4 combined with biostimulation: Cr(VI) transformation and stabilization

Efficient and sustainable technologies for cleaning up of hexavalent chromium (Cr(VI)) contaminated soil are under urgent demand. In this study, chemical reduction and biostimulation were combined to remediate contaminated soil with a high concentration of hexavalent Cr(VI) to compensate for the shortcomings of single-treatment technology. The Cr(VI) concentration, Cr speciation, and bacterial diversity of the contaminated soil were measured after combined remediation with ferrous sulfate (FeSO4) and different electron donors. The changes in various indexes in the soil after microbial death were also studied. The results indicated that the optimal reduction rate of Cr(VI) was as high as 95.20% when 10% FeSO4 was combined with lactic acid. The water-soluble fraction of Cr with the highest toxicity in the soil was almost completely converted into other stable fractions. Stabilization experiments proved that Cr(III) in the remediated soil did not revert to Cr(VI) after microorganism death, and had a good stabilization effect. The bacterial diversity results indicated that Acinetobacter spp. and Pseudomonas spp. were the dominant species during the biostimulation process and might be involved in the reduction of Cr(VI).

Phosphorylation and sulfation share a common biosynthetic pathway, but extend biochemical and evolutionary diversity of biological macromolecules in distinct ways.

Phosphate and sulfate groups are integral to energy metabolism and introduce negative charges into biological macromolecules. One purpose of such modifications is to elicit precise binding/activation of protein partners. The physico-chemical properties of the two groups, while superficially similar, differ in one important respect—the valency of the central (phosphorus or sulfur) atom. This dictates the distinct properties of their respective esters, di-esters and hence their charges, interactions with metal ions and their solubility. These, in turn, determine the contrasting roles for which each group has evolved in biological systems. Biosynthetic links exist between the two modifications; the sulfate donor 3′-phosphoadenosine-5′-phosphosulfate being formed from adenosine triphosphate (ATP) and adenosine phosphosulfate, while the latter is generated from sulfate anions and ATP. Furthermore, phosphorylation, by a xylosyl kinase (Fam20B, glycosaminoglycan xylosylkinase) of the xylose residue of the tetrasaccharide linker region that connects nascent glycosaminoglycan (GAG) chains to their parent proteoglycans, substantially accelerates their biosynthesis. Following observations that GAG chains can enter the cell nucleus, it is hypothesized that sulfated GAGs could influence events in the nucleus, which would complete a feedback loop uniting the complementary anionic modifications of phosphorylation and sulfation through complex, inter-connected signalling networks and warrants further exploration.

Bacterial Aryl Sulfotransferases in Selective and Sustainable Sulfation of Biologically Active Compounds using Novel Sulfate Donors.

Regioselective sulfation of bioactive compounds is a vital and scarcely studied topic in enzyme-catalyzed transformations and metabolomics. The major bottleneck of enzymatic sulfation consists in finding suitable sulfate donors. In this regard, 3'-phosphoadenosine 5'-phosphosulfate (PAPS)-independent aryl sulfotransferases using aromatic sulfate donors are a favored choice due to their cost-effectiveness. This work presents a unique study of five sulfate donors differing in their leaving group pKa values with a new His-tagged construct of aryl sulfotransferase from Desulfitobacterium hafniense (DhAST-tag). DhAST-tag was purified to homogeneity and biochemically characterized. Two new donors (3-nitrophenyl sulfate and 2-nitrophenyl sulfate) were synthesized. The kinetic parameters of these and other commercial sulfates (4-nitrophenyl, 4-methylumbelliferyl, and phenyl) revealed large differences with respect to the structure of the leaving group. These donors were screened for the sulfation of selected flavonoids (myricetin, chrysin) and phenolic acids (gallate, 3,4-dihydroxyphenylacetate). The donor impact on the sulfation regioselectivity and yield was assessed. The obtained regioselectively sulfated compounds are authentic human metabolites required as standards in clinical trials.

Heparan sulfate: In vitro and in vivo proof of efficacy of this new therapeutic strategy for halting Alzheimer disease‐related tauopathy development

AbstractBackgroundDeposition of amyloid plaques and neurofibrillary tangles composed of abnormally phosphorylated tau are the neuropathological hallmarks of Alzheimer's disease (AD). However, years before the accumulation of protein aggregates, the abnormal intraneuronal accumulation of heparan sulfates (HS) is observed. This phenomenon breaks a dogma, since HS are classically located on the cell surface and in the extracellular matrix, leading to a new cell autonomous concept in tauopathy, which is the conceptual core of the FET‐OPEN ArrestAD 737390 project. Previously, we provide evidence that inside the cell, HS act as molecular chaperones inducing in Tau conformational changes favoring its abnormal phosphorylation and aggregation (Sepulveda‐Diaz JE. Brain 2015, 138(Pt 5):1339‐54). Thus, molecules able to act on heparan sulfate pathway could represent a new class of compounds for the treatment of AD‐related tauopathies.MethodPAPS is the universal sulfate donor involved in HS biosynthesis and structurally related to ATP. Thus, we first screened kinase inhibitor libraries in a sulfotransferase enzymatic assay (Byrne et al., Biochem. J. 2018; 475:2417‐2433). This strategy, in addition to a HS mimetic, were evaluated for their ability to prevent tauopathy in cells. The best molecules were then evaluated in two transgenic mice model of AD. Thereafter, proof‐of‐concept of the efficacy of compounds selected with this strategy, as well as HS mimetic (Barritault D. Glycoconj J. 2017; 34(3):325‐338) for halting AD‐related tauopathies development was assessed first in vitro and then in vivo.ResultsThe screening of libraries allowed the identification of 3 hit compounds that, in addition to a HS mimetic, were evaluated for their ability to prevent tauopathy in cells. Two molecules, one HSSTs inhibitor and one heparan sulfate mimetic, were able to reduce tauopathy in cells and in 3xTg‐AD and rTg4510 mouse models of tauopathy. In the in vivo assessment in males and females 3xTg‐AD, the behavioral dose‐response of these molecules on the cognitive, neuropsychiatric‐like and motor AD‐phenotypes was determined.ConclusionOur results support the FET‐OPEN RIA ArrestAD cell autonomous hypothesis of tauopathy based in altered heparan sulfate biology and place HS and their biosynthetic enzymes as a potential therapeutic target for AD.

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.

Aqueous metabolome of tissue-specific conditional Pten-knockout mouse prostate cancer and TRAMP neuroendocrine carcinoma.

BackgroundMetabolic reprograming is now a recognized hallmark of cancer. The prostate‐specific phosphatase and tensin homolog deleted on chromosome 10 (Pten) gene‐conditional knockout (KO) mouse carcinogenesis model is highly desirable for studying prostate cancer biology and prevention due to its close resemblance of primary molecular defects and histopathological features of human prostate cancer. We have recently published macromolecular profiling of this model by proteomics and transcriptomics, denoting a preeminence of inflammation and myeloid suppressive immune cell features. Here, we performed metabolomic analyses of Pten‐KO prostate versus wild type (WT) counterpart for discernable changes in the aqueous metabolites and contrasted to those in the TRAMP neuroendocrine carcinoma (NECa).MethodsThree matched pairs of tissue‐specific conditional Pten‐KO mouse prostate and WT prostate of litter/cage‐mates at 20–22 weeks of age and three pairs of TRAMP NECa versus WT (28–31 weeks) were profiled for their global aqueous metabolite changes, using hydrophilic interaction liquid chromatography‐tandem mass spectrometry.ResultsThe Pten‐KO prostate increased purine nucleotide pools, cystathionine, and both reduced and oxidized glutathione (GSH, GSSG), and gluconate/glucuronate species in addition to cholesteryl sulfate and polyamine precursor ornithine. On the contrary, Pten‐KO prostate contained diminished pools of glycolytic intermediates and phosphorylcholine derivatives, select amino acids, and their metabolites. Bioinformatic integration revealed a significant shunting of glucose away from glycolysis‐citrate cycle and glycerol‐lipid genesis to pentose phosphate cycle for NADPH/GSH/GSSG redox and pentose moieties for purine and pyrimidine nucleotides, and glycosylation/glucuronidation. Implicit arginine catabolism to ornithine was consistent with immunosuppression in Pten‐KO model. While also increased in cystathionine‐GSH/GSSG, purine, and pyrimidine nucleotide pools and glucuronidation at the expense of glycolysis‐citrate cycle, the TRAMP NECa increased abundance of many amino acids, methyl donor S‐adenosyl‐methionine, and intermediates for phospholipids without increasing cholesteryl sulfate or ornithine.ConclusionsThe aqueous metabolomic patterns in Pten‐KO prostate and TRAMP NECa shared similarities in the greater pools of cystathionine, GSH/GSSG redox pair, and nucleotides and shunting away from glycolysis‐citrate cycle in both models. Remarkable metabolic distinctions between them included metabolisms of many amino acids (protein synthesis; arginine‐ornithine/immune suppression) and cholesteryl sulfate and methylation donor for epigenetic regulations.

The crystal structure of mouse SULT2A8 reveals the mechanism of 7α-hydroxyl, bile acid sulfation

Bile acids play essential roles in facilitating the intestinal absorption of lipophilic nutrients as well as regulation of glucose, lipid, and energy homeostasis via activation of some receptors. Bile acids are cytotoxic, and consequently their concentrations are tightly controlled. A critical pathway for bile acid elimination and detoxification is sulfation. The pattern of bile acid sulfation differs by species. Sulfation preferentially occurs at the 3α-OH of bile acids in humans, but at the 7α-OH in mice. A recent study identified mouse cytosolic sulfotransferase 2A8 (mSULT2A8) as the major hepatic 7α-hydroxyl bile acid-sulfating enzyme. To elucidate the 7α-OH specific sulfation mechanism of mSULT2A8, instead of 3α-OH specific sulfation in humans, we determined a crystal structure of mSULT2A8 in complex with cholic acid, a major bile acid, and 3′-phosphoadenosine-5′-phosphate, the sulfate donor product. Our study shows that bile acid-binding mode of mSULT2A8 and how the enzyme holds the 7α-OH group of bile acids at the catalytic center, revealing that the mechanism underlying 7α-OH specific sulfation. The structure shows the substrate binds to mSULT2A8 in an orientation perpendicular to that of human 3α-hydroxyl bile acid-sulfotransferase (hSULT2A1). The structure of the complex provides new insight into species different bile acid metabolism.

Complete biosynthesis of a sulfated chondroitin in Escherichia coli

Sulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production–chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 μg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.

Enhanced anticoagulant activity of hirudin-i analogue co-expressed with arylsulfotransferase in periplasm of E. coli BL21(DE3)

Hirudin, a blood anticoagulant, is the most potent natural thrombin inhibitor of leech origin. Its application is limited because it is difficult to obtain abundant natural hirudin directly from the leech. Although some bioengineering methods can significantly increase the production of hirudin, the reduced efficacy of recombinant hirudin (rH) remains a critical shortcoming. The lack of sulfation of tyrosine 63 in rH is an important cause of its inadequate performance. This article is the first report of periplasmic co-expression of an rH-I analogue with arylsulfotransferase (ASST) in E. coli BL21(DE3). Co-expressed rH-I analogue with sulfate donor substrate (p-nitrophenyl sulfate potassium) showed anticoagulant (rabbit and goat serum) activity twice more than rH-I analogue expressed without ASST, indicating its potential periplasmic sulfation. Moreover, purified rH-I analogue showed above 4.5 times higher anticoagulant activity compared to therapeutic anti-thrombotic heparin (HE). At the same time, pH-dependent differential solubility was employed to purify rH analogues from fermentation broth, which is a simple, fast and inexpensive purification technology, and can potentially be used for larger scale purification. This will also greatly improve the application of rH in clinical treatment.

The Role of Sulfotransferases in Liver Diseases.

The cytosolic sulfotransferases (SULTs) are phase II conjugating enzymes that catalyze the transfer of a sulfonate group from the universal sulfate donor 3'-phosphoadenosine-5'-phosphosulfate to a nucleophilic group of their substrates to generate hydrophilic products. Sulfation has a major effect on the chemical and functional homeostasis of substrate chemicals. SULTs are widely expressed in metabolically active or hormonally responsive tissues, including the liver and many extrahepatic tissues. The expression of SULTs exhibits isoform-, tissue-, sex-, and development-specific regulations. SULTs display a broad range of substrates including xenobiotics and endobiotics. The expression of SULTs has been shown to be transcriptionally regulated by members of the nuclear receptor superfamily, such as the peroxisome proliferator-activated receptors, pregnane X receptor, constitutive androstane receptor, vitamin D receptor, liver X receptors, farnesoid X receptor, retinoid-related orphan receptors, estrogen-related receptors, and hepatocyte nuclear factor 4α These nuclear receptors can be activated by numerous xenobiotics and endobiotics, such as fatty acids, bile acids, and oxysterols, many of which are substrates of SULTs. Due to their metabolism of xenobiotics and endobiotics, SULTs and their regulations are implicated in the pathogenesis of many diseases. This review is aimed to summarize the central role of major SULTs, including the SULT1 and SULT2 subfamilies, in the pathophysiology of liver and liver-related diseases. SIGNIFICANCE STATEMENT: Sulfotransferases (SULTs) are indispensable in the homeostasis of xenobiotics and endobiotics. Knowing SULTs and their regulations are implicated in human diseases, it is hoped that genetic or pharmacological manipulations of the expression and/or activity of SULTs can be used to affect the clinical outcome of diseases.