Formation of DNA Adducts by 1-Methoxy-3-indolylmethylalcohol, a Breakdown Product of a Glucosinolate, in the Mouse: Impact of the SULT1A1 Status-Wild-Type, Knockout or Humanised.

We previously found that feeding rats with broccoli or cauliflower leads to the formation of characteristic DNA adducts in the liver, intestine and various other tissues. We identified the critical substances in the plants as 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate and its degradation product 1-MIM-OH. DNA adduct formation and the mutagenicity of 1-MIM-OH in cell models were drastically enhanced when human sulfotransferase (SULT) 1A1 was expressed. The aim of this study was to clarify the role of SULT1A1 in DNA adduct formation by 1-MIM-OH in mouse tissues in vivo. Furthermore, we compared the endogenous mouse Sult1a1 and transgenic human SULT1A1 in the activation of 1-MIM-OH using genetically modified mouse strains. We orally treated male wild-type (wt) and Sult1a1-knockout (ko) mice, as well as corresponding lines carrying the human SULT1A1-SULT1A2 gene cluster (tg and ko-tg), with 1-MIM-OH. N2-(1-MIM)-dG and N6-(1-MIM)-dA adducts in DNA were analysed using isotope-dilution UPLC-MS/MS. In the liver, caecum and colon adducts were abundant in mice expressing mouse and/or human SULT1A1, but were drastically reduced in ko mice (1.2-10.6% of wt). In the kidney and small intestine, adduct levels were high in mice carrying human SULT1A1-SULT1A2 genes, but low in wt and ko mice (1.8-6.3% of tg-ko). In bone marrow, adduct levels were very low, independently of the SULT1A1 status. In the stomach, they were high in all four lines. Thus, adduct formation was primarily controlled by SULT1A1 in five out of seven tissues studied, with a strong impact of differences in the tissue distribution of mouse and human SULT1A1. The behaviour of 1-MIM-OH in these models (levels and tissue distribution of DNA adducts; impact of SULTs) was similar to that of methyleugenol, classified as "probably carcinogenic to humans". Thus, there is a need to test 1-MIM-OH for carcinogenicity in animal models and to study its adduct formation in humans consuming brassicaceous foodstuff.

Association of SULT1A2 rs1059491 with obesity and dyslipidaemia in southern Chinese adults.

In the sulfotransferase (SULT) superfamily, members of the SULT1 family mainly catalyse the sulfonation reaction of phenolic compounds, which is involved in the phase II metabolic detoxification process and plays a key role in endocrine homeostasis. A coding variant rs1059491 in the SULT1A2 gene has been reported to be associated with childhood obesity. This study aimed to investigate the association of rs1059491 with the risk of obesity and cardiometabolic abnormalities in adults. This case‒control study included 226 normal weight, 168 overweight and 72 obese adults who underwent a health examination in Taizhou, China. Genotyping of rs1059491 was performed by Sanger sequencing in exon 7 of the SULT1A2 coding region. Chi-squared tests, one-way ANOVA, and logistic regression models were applied. The minor allele frequencies of rs1059491 in the overweight combined with obesity and control groups were 0.0292 and 0.0686, respectively. No differences in weight and body mass index were detected between the TT genotype and GT + GG genotype under the dominant model, but the levels of serum triglycerides were significantly lower in G-allele carriers than in non-G-allele carriers (1.02 (0.74-1.32) vs. 1.35 (0.83-2.13) mmol/L, P = 0.011). The GT + GG genotype of rs1059491 versus the TT genotype reduced the risk of overweight and obesity by 54% (OR 0.46, 95% CI 0.22-0.96, P = 0.037) after adjusting for sex and age. Similar results were observed for hypertriglyceridaemia (OR 0.25, 95% CI 0.08-0.74, P = 0.013) and dyslipidaemia (OR 0.37, 95% CI 0.17-0.83, P = 0.015). However, these associations disappeared after correction for multiple tests. This study revealed that the coding variant rs1059491 is nominally associated with a decreased risk of obesity and dyslipidaemia in southern Chinese adults. The findings will be validated in larger studies including more detailed information on genetic background, lifestyle and weight change with age.

Association between Single Nucleotide Polymorphisms of SULT1A1, SULT1C4, ABCC2 and Phase II Flavanone Metabolites Excretion after Orange Juice Intake.

Citrus fruits and juices are a major source of dietary flavanones, and the regular consumption of these foods is inversely associated with the development of cardiometabolic diseases. However, the biological benefits depend on the bioavailability of these compounds, and previous studies have reported a large interindividual variability in the absorption and excretion of these compounds. Different factors, such as age, gender or genetic polymorphism of genes coding enzymes involved in the metabolism and transport of the flavanones, may explain this heterogeneity. This study aimed to assess the impact of single nucleotide polymorphism of sulfotransferases SULT1A1 and SULT1C4, and ABCC2 transporter genes on excretion of phase II flavanone metabolites in volunteers after 24 h of orange juice intake. Forty-six volunteers ingested a single dose of 500 mL of orange juice and 24-h urine was collected. The hesperetin and naringenin phase II metabolites were quantified in urine, and SNPs in SULT1A1, SULT1C4 and ABCC2 genes were genotyped. A significant (p < 0.05) relationship between the SNPs in these genes and the high excretion of phase II flavanone metabolites were observed. These results identified novel polymorphisms associated with higher absorption of flavanones, which may provide bases for future personalized nutritional guidelines for consuming flavanone-rich foods rich in these nutrients for better benefit from their health properties.

Sulfation of 12-hydroxy-nevirapine by human SULTs and the effects of genetic polymorphisms of SULT1A1 and SULT2A1.

Nevirapine (NVP) is an effective drug for the treatment of HIV infections, but its use is limited by a high incidence of severe skin rash and liver injury. 12-Hydroxynevirapine (12-OH-NVP) is the major metabolite of nevirapine. There is strong evidence that the sulfate of 12-OH-NVP is responsible for the skin rash. While several cytosolic sulfotransferases (SULTs) have been shown to be capable of sulfating 12-OH-NVP, the exact mechanism of sulfation in vivo is unclear. The current study aimed to clarify human SULT(s) and human organs that are capable of sulfating 12-OH-NVP and investigate the metabolic sulfation of 12-OH-NVP using cultured HepG2 human hepatoma cells. Enzymatic assays revealed that of the thirteen human SULTs, SULT1A1 and SULT2A1 displayed strong 12-OH-NVP-sulfating activity. 1-Phenyl-1-hexanol (PHHX), which applied topically prevents the skin rash in rats, inhibited 12-OH-NVP sulfation by SULT1A1 and SULT2A1, implying the involvement of these two enzymes in the sulfation of 12-OH-NVP in vivo. Among five human organ cytosols analyzed, liver cytosol displayed the strongest 12-OH-NVP-sulfating activity, while a low but significant activity was detected with skin cytosol. Cultured HepG2 cells were shown to be capable of sulfating 12-OH-NVP. The effects of genetic polymorphisms of SULT1A1 and SULT2A1 genes on the sulfation of 12-OH-NVP by SULT1A1 and SULT2A1 allozymes were investigated. Two SULT1A1 allozymes, Arg37Asp and Met223Val, showed no detectable 12-OH-NVP-sulfating activity, while a SULT2A1 allozyme, Met57Thr, displayed significantly higher 12-OH-NVP-sulfating activity compared with the wild-type enzyme. Collectively, these results contribute to a better understanding of the involvement of sulfation in NVP-induced skin rash and provide clues to the possible role of SULT genetic polymorphisms in the risk of this adverse reaction.

Aha1 Is an Autonomous Chaperone for SULT1A1.

The cochaperone Aha1 activates HSP90 ATPase to promote the folding of its client proteins; however, very few client proteins of Aha1 are known. With the use of an ascorbate peroxidase (APEX)-based proximity labeling method, we identified SULT1A1 as a proximity protein of HSP90 that is modulated by genetic depletion of Aha1. Immunoprecipitation followed by Western blot analysis showed the interaction of SULT1A1 with Aha1, but not HSP90. We also observed a reduced level of SULT1A1 protein upon genetic depletion of Aha1 but not upon pharmacological inhibition of HSP90, suggesting that the SULT1A1 protein level is regulated by Aha1 alone. Maturation-dependent interaction assay results showed that Aha1, but not HSP90, binds preferentially to newly synthesized SULT1A1. Reconstitution of Aha1-depleted cells with wild-type Aha1 and its E67K mutant, which is deficient in interacting with HSP90, restored SULT1A1 protein to the same level. Nonetheless, complementation of Aha1-depleted cells with an Aha1 mutant lacking the first 20 amino acids, which disrupts its autonomous chaperone function, was unable to rescue the SULT1A1 protein level. Together, our study revealed, for the first time, Aha1 as an autonomous chaperone in regulating SULT1A1. SULT1A1 is a phase-II metabolic enzyme, where it adds sulfate groups to hydroxyl functionalities in endogenous hormones and xenobiotic chemicals to improve their solubilities and promote their excretion. Thus, our work suggests the role of Aha1 cochaperone in modulating the detoxification of endogenous and environmental chemicals.

Impact of Pineapple Juice on Expression of CYP3A4, NAT2, SULT1A1 and OATP1B1 mRNA in HepG2 Cells.

<b>Background and Objective:</b> Pineapple (<i>Ananas comosus</i>) is a popular fruit worldwide with natural antioxidant properties. This study examined how pineapple modified the expression of drug-metabolizing enzymes (CYP1A2, CYP2C9, CYP3A4, UGT1A6, NAT2 and SULT1A1) and a drug transporter (OATP1B1) in human hepatocarcinoma (HepG2) cells. <b>Materials and Methods:</b> HepG2 cells (2.5×10⁵ cells/well in a 24-well plate) were incubated with pineapple juice extract (125-1,000 µg mL¹) for 48 hrs in phenol red-free medium. Resazurin reduction, ROS, AST and ALT assays were performed. The mRNA expression of target genes was determined by RT/qPCR. <b>Results:</b> Pineapple juice slightly reduced HepG2 cell viability to 80% of the control, while ROS, AST and ALT levels were not changed. Pineapple juice did not alter the expression of CYP1A2, CYP2C9 and UGT1A6 mRNA. All tested concentrations of pineapple juice suppressed CYP3A4, NAT2 and OATP1B1 expression, while SULT1A1 expression was induced. <b>Conclusion:</b> Though pineapple juice slightly decreased the viability of HepG2 cells, cell morphology and cell function remained normal. Pineapple juice disturbed the expression of phase I (CYP3A4) and phase II (NAT2 and SULT1A1) metabolizing genes and the drug transporter OATP1B1. Therefore, the consumption of excessive amounts of pineapple juice poses a risk for drug interactions.

SULT1A1 and SULT1A2 Associated Extensive Prolapse-Type Inflammatory Polyposis in Crohn's Colitis.

Mucosal prolapse syndrome most commonly involves the rectum and presents as solitary rectal ulcer syndrome and proctitis cystica profunda. Symptoms and endoscopic appearances are nonspecific. Histologically, mucosal prolapse is characterized by fibromuscular obliteration of the lamina propria, and displacement of crypts into submucosa and muscularis mucosae. Mucosal prolapse presenting as polyposis is rare and has only been reported involving the rectosigmoid colon. In this report, we describe a case of mucosal prolapse syndrome presenting as diffuse polyposis and colitis cystica profunda involving the hepatic, splenic flexures and descending colon in a teenage boy suffering from refractory fibrostenosing Crohn's disease. This patient was found to have possibly deleterious homozygous single nucleotide polymorphisms in both SULT1A1 and SULT1A2 genes within a unique polygenic variation of altered cell adhesion.

Genetic polymorphism of Arg213His variant in the SULT1A1 gene is associated with reduced susceptibility to lung cancer in North Indian population.

Sulfotransferases (SULTs) are phase II detoxification enzymes that is involved in the biotransformation of many compounds including tobacco carcinogens. A polymorphism in the SULT1A1 (Arg213His) gene results in reduced enzyme activity.We investigated the association between the SULT1A1 (Arg213/His) genotype and lung cancer (LC). This case-control study comprised of 550 cases and controls, matched on age, gender and smoking status.The variant genotype exhibited no association with LC risk, even after stratification on basis of histological subtypes. Male LC patients carrying the variant His213 allele (p = 0.02) did not exhibit an increased risk towards LC. Smokers harbouring the Arg/His genotype did demonstrate a reduced risk towards LC (AOR = 0.70; p = 0.019). Furthermore, the LC subjects who were heavy smokers and harbouring the Arg/His genotype (AOR = 0.28; p = 0.019) did not show a genetic predisposition towards LC susceptibility. The subjects who smoked pack years of above 40 and carrying the His/His (AOR = 0.28; p = 0.036) genotype were found to have a reduced risk for LC. Furthermore, 473 subjects were analysed in regards to overall survival, wherein the His/His genotype exhibited better OS than Arg/Arg genotype (11.30 vs. 8.07 months).This study provides evidence of no genetic predisposition towards LC risk associated with SULT1A1 Arg213His polymorphism in relation to tobacco smoking.

Physiologically relevant oxygen tensions differentially regulate hepatotoxic responses in HepG2 cells.

This study evaluates the impact of physiologically relevant oxygen tensions on the response of HepG2 cells to known inducers and hepatotoxic drugs. We compared transcriptional regulation and CYP1A activity after a 48 h exposure at atmospheric culture conditions (20% O2) with representative periportal (8% O2) and perivenous (3% O2) oxygen tensions. We evaluated cellular responses in 2D and 3D cultures at each oxygen tension in parallel, using monolayers and a paper-based culture platform that supports cells suspended in a collagen-rich environment. Our findings highlight that the toxicity, potency, and mechanism of action of drugs are dependent on both culture format and oxygen tension. HepG2 cells in 3D environments at physiologic oxygen tensions better matched primary human hepatocyte data than HepG2 cells cultured under standard conditions. Despite altered transcriptional regulation with decreasing oxygen tensions, we did not observe the zonation patterns of drug-metabolizing enzymes found in vivo. Our approach demonstrates that oxygen is an important regulator of liver function but it is not the sole regulator. It also highlights the utility of the 3D paper-based culture platform for continued mechanistic studies of microenvironmental influences on cellular responses.

Peculiarities of Immune Regulation in Men of Perm Region with Atherosclerosis Related to Polymorphism of Candidate Genes.

The study examined peculiarities of immune regulation and associated polymorphic variants of candidate genes in men with atherosclerosis in Perm region. The revealed deficiency of CD127 lymphocytes and Annexin V-FITC+7AAD- cells, as well as enhanced level of CD3+CD4+ lymphocytes against the background of variant alleles of candidate genes FAS (rs1159120), CPOX (rs1131857) and wild-type alleles SULT1A1 (rs9282861), MMP9 (rs17576) are responsible for peculiar features of hereditary determination and pathogenesis of atherosclerosis in examined sample (p<0.05). The genetically determined degradation of extracellular matrix in vascular wall and implication of regulated Fas/APO1 apoptosis in the development of progressive atherosclerotic lesions indicate important role of immune system in atherogenesis. The revealed immunological and genetic features are recommended as the markers for early diagnosis of atherosclerosis and its prevention in men of Perm region.

Suppressed Hepatic Production of Indoxyl Sulfate Attenuates Cisplatin-Induced Acute Kidney Injury in Sulfotransferase 1a1-Deficient Mice.

Endogenous factors involved in the progression of cisplatin nephropathy remain undetermined. Here, we demonstrate the toxico-pathological roles of indoxyl sulfate (IS), a sulfate-conjugated uremic toxin, and sulfotransferase 1A1 (SULT1A1), an enzyme involved in its synthesis, in cisplatin-induced acute kidney injury using Sult1a1-deficient (Sult1a1-/- KO) mice. With cisplatin administration, severe kidney dysfunction, tissue damage, and apoptosis were attenuated in Sult1a1-/- (KO) mice. Aryl hydrocarbon receptor (AhR) expression was increased by treatment with cisplatin in mouse kidney tissue. Moreover, the downregulation of antioxidant stress enzymes in wild-type (WT) mice was not observed in Sult1a1-/- (KO) mice. To investigate the effect of IS on the reactive oxygen species (ROS) levels, HK-2 cells were treated with cisplatin and IS. The ROS levels were significantly increased compared to cisplatin or IS treatment alone. IS-induced increases in ROS were reversed by downregulation of AhR, xanthine oxidase (XO), and NADPH oxidase 4 (NOX4). These findings suggest that SULT1A1 plays toxico-pathological roles in the progression of cisplatin-induced acute kidney injury, while the IS/AhR/ROS axis brings about oxidative stress.

Decreased phenol sulfotransferase activities associated with hyperserotonemia in autism spectrum disorders.

Hyperserotonemia is the most replicated biochemical abnormality associated with autism spectrum disorders (ASD). However, previous studies of serotonin synthesis, catabolism, and transport have not elucidated the mechanisms underlying this hyperserotonemia. Here we investigated serotonin sulfation by phenol sulfotransferases (PST) in blood samples from 97 individuals with ASD and their first-degree relatives (138 parents and 56 siblings), compared with 106 controls. We report a deficient activity of both PST isoforms (M and P) in platelets from individuals with ASD (35% and 78% of patients, respectively), confirmed in autoptic tissues (9 pineal gland samples from individuals with ASD-an important source of serotonin). Platelet PST-M deficiency was strongly associated with hyperserotonemia in individuals with ASD. We then explore genetic or pharmacologic modulation of PST activities in mice: variations of PST activities were associated with marked variations of blood serotonin, demonstrating the influence of the sulfation pathway on serotonemia. We also conducted in 1645 individuals an extensive study of SULT1A genes, encoding PST and mapping at highly polymorphic 16p11.2 locus, which did not reveal an association between copy number or single nucleotide variations and PST activity, blood serotonin or the risk of ASD. In contrast, our broader assessment of sulfation metabolism in ASD showed impairments of other sulfation-related markers, including inorganic sulfate, heparan-sulfate, and heparin sulfate-sulfotransferase. Our study proposes for the first time a compelling mechanism for hyperserotonemia, in a context of global impairment of sulfation metabolism in ASD.

Investigating the molecular mechanism of hydroxylated bromdiphenyl ethers to inhibit the thyroid hormone sulfotransferase SULT1A1.

Hydroxylated bromodiphenyl ethers (OH-BDEs) have raised great concern due to their potential endocrine disrupting effects on humans. In vitro experiments have indicated OH-BDEs can inhibit the activity of thyroid hormone (TH) sulfotransferases (SULTs); however, the molecular mechanism has not been investigated in depth. In this work, we employed 17 OH-BDEs with five or fewer Br atoms, and performed integrated computational simulations to unravel the possible inhibition mechanism of OH-BDEs on human SULT1A1. The molecular docking results demonstrate that OH-BDEs form hydrogen bonds with residues Lys106 and His108, and the neutral OH-BDEs show comparable binding energies with their anionic counterparts. The further hybrid quantum mechanical/molecular mechanical (QM/MM) calculations unravel a metabolic mechanism of OH-BDEs comprised by proton abstraction and sulfation steps. This mechanism is involved in the SULT1A1 inhibition by some OH-BDEs comprised of three or fewer Br atoms, while other OH-BDEs likely only form ternary complexes to competitively inhibit SULT1A1 activity. Moreover, the effect of the hydroxyl group of OH-BDEs on SULT1A1 inhibition potential follows the order of ortho-OH BDE > meta-OH BDE > para-OH BDE. These results provide an insight into the inhibition mechanism of OH-BDEs to SULT1A1 at the molecular level, which are beneficial in illuminating the molecular initiating events involved in the TH disruption of OH-BDEs.

Evaluation of a conserved tryptophanyl residue in donor substrate binding and catalysis by a phenol sulfotransferase (SULT1A1).

Structural determinations of members of the sulfotransferase (SULT) family suggest a direct interaction between a conserved tryptophanyl side chain and bound 3'-phosphoadenosine-5'-phosphate (PAP). We have prepared and purified mutants of the bovine SULT1A1, a very conserved homolog to the human SULT1A1, in which tryptophanyl-53 was sequentially trimmed to tyrosine, leucine, and alanine. Differential scanning fluorimetry indicated structural stabilities of the mutant proteins comparable to the wild type SULT1A1; however, less thermal stabilizations by PAP plus pentachlorophenol were observed with the mutants, suggesting weakened ligand binding. Protein fluorescence of the wild type enzyme decreased 6.5% upon binding PAP, whereas no changes occurred with the mutant enzymes. This reveals that W53, or its positional counterpart, has been the source of emission intensity changes used in previous investigations of other SULTs. Fluorescence resonance energy transfer from excited tryptophans to bound 7-hydroxycoumarin, as induced by PAP, indicated weakened binding of ligands to the mutant SULTs. This was also encountered and quantified in initial rate kinetic analyses. Ablation of the PAPS adenine-to-W53 ring interaction, shown by the W53A mutant enzyme, resulted in a 6.4-fold increase in KPAPS and a 92% decrease in kcat/KPAPS. Measured KPAPS values reveal the W53 indole ring contribution to PAPS binding to be 1.1 kcal/mol (4.6 kJ/mol). These results verify the structurally-inferred role for the π-π stacking interaction between PAP(S) and the conserved tryptophanyl residue in SULT1A1 and other members of the SULT family.

Expression of functional sulfotransferases (SULT) 1A1, 1A3, 1B1, 1C2, 1E1, and 2A1 in common marmosets.

Cytosolic sulfotransferases (SULTs), which mediate the conjugation of drugs with 3'-phosphoadenosine-5'-phosphosulfate, have been characterized in humans and cynomolgus monkeys. However, SULTs remain to be evaluated in common marmosets, a species of non-human primate often employed in drug metabolism and pharmacokinetic studies of endogenous and exogenous compounds. In this study, marmoset SULT1A1, 1A3, 1B1, 1C2, 1E1, and 2A1 cDNAs were isolated and characterized, based on genome data. The deduced amino acid sequences of these marmoset SULT cDNAs had high identities (90-95%) with their human orthologs, except for marmoset SULT2A1, which was only 81% identical to human SULT2A1. The amino acid sequences of the orthologs of these six SULTs in marmosets, monkeys, and humans were closely clustered in a phylogenetic tree. The structures and genomic organizations of marmoset SULT genes were similar to those of their human orthologs. Among the five marmoset tissues analyzed, SULT mRNAs showed typical expression patterns. The most abundant SULT mRNAs were SULT1B1 in liver, small intestine, and kidney; SULT1E1 in lung; and SULT1A3 in brain. Recombinant marmoset SULT1A1, 1A3, 1B1, 1C2, 1E1, and 2A1 proteins expressed in bacterial cytosolic fractions mediated sulfate conjugations with 3'-phosphoadenosine-5'-phosphosulfate of the following typical human SULT substrates: dopamine, 1-naphthol, p-nitrophenol, estradiol, and dehydroepiandrosterone. Taken together, these wide-ranging results suggest functional and molecular similarities of SULTs among marmosets, monkeys, and humans.

Comprehensive analysis of copy number variance and sensitivity to common targeted therapy in clear cell renal cell carcinoma: In silico analysis with in vitro validation.

BACKGROUND: Chromosomal rearrangements are common in clear cell renal cell carcinoma (ccRCC) and their roles in mediating sensitivity to tyrosine kinase inhibitors (TKIs) and mTOR inhibitors (mTORi) remain elusive. METHODS: We developed an in silico strategy by screening copy number variance (CNV) that was potentially related to TKI or mTORi sensitivity in ccRCC by reproducing the TCGA and GDSC datasets. Candidate genes should be both significantly prognostic and related to drug sensitivity or resistance, and were then validated in vitro. RESULTS: ADCYAP1 loss and GNAS gain were associated with sensitivity and resistance and to Cabozantinib, respectively. ACRBP gain and CTBP1 loss were associated with sensitivity and resistance and to Pazopanib, respectively. CDKN2A loss and SULT1A3 gain were associated with sensitivity and resistance and to Temsirolimus, respectively. CCNE1 gain was associated with resistance to Axitinib and LRP10 loss was associated with resistance to Sunitinib. Mutivariate analysis showed ADCYAP1, GNAS, and CCNE1 remained independently prognostic when adjusted for the rest. CONCLUSION: Here we show CNVs of several genes that are associated with sensitivity and resistance to commonly used TKIs and mTORi in ccRCC. Further validation and functional analyses are therefore needed.

Expression of enzymes for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis and their preparation for PAPS synthesis and regeneration.

The synthesis of sulfated polysaccharides involves the sulfation of simpler polysaccharide substrates, through the action sulfotransferases using the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Three enzymes are essential for the in vitro synthesis of PAPS, namely, pyrophosphatase (PPA), adenosine 5'-phosphosulfate kinase (APSK), and ATP sulfurylase (ATPS). The optimized enzyme expression ratio and effect on PAPS synthesis were evaluated using ePathBrick, a novel synthetic biology tool that assemble multiple genes in a single vector. The introduction of multiple promoters and stop codons at different location enable the bacterial system to fine tune expression level of the genes inserted. Recombinant vectors expressing PPA (U39393.1), ATPS (CP021243.1), and PPA (CP047127.1) were used for fermentations and resulted in volumetric yields of 400-1380 mg/L with accumulation of 34-66% in the soluble fraction. The enzymes from soluble fraction, without any further purification, were used for PAPS synthesis. The PAPS was used for the chemoenzymatic synthesis of a heparan sulfate polysaccharide and coupled with a PAPS-ASTIV regeneration system. ASTIV catalyzes the regeneration of PAPS. A recombinant vector expressing the enzyme ASTIV (from Rattus norvegicus) was used for fermentations and resulted in volumetric yield of 1153 mg/L enzyme with accumulation of 48% in the soluble fraction. In conclusion, we have successfully utilized a metabolic engineering approach to optimize the overall PAPS synthesis productivity. In addition, we have demonstrated that the ePathBrick system could be applied towards study and improvement of enzymatic synthesis conditions. In parallel, we have successfully demonstrated an autoinduction microbial fermentation towards the production of mammalian enzyme (ASTIV). KEY POINTS : • ePathBrick used to optimize expression levels of enzymes. • Protocols have been used for the production of recombinant enzymes. • High cell density fed-batch fermentations with high yields of soluble enzymes. • Robust fermentation protocol successfully transferred to contract manufacturing and research facilities.

Identification and targeting of selective vulnerability rendered by tamoxifen resistance.

BACKGROUND: The estrogen receptor (ER)-positive breast cancer represents over 80% of all breast cancer cases. Even though adjuvant hormone therapy with tamoxifen (TMX) is saving lives of patients with ER-positive breast cancer, the acquired resistance to TMX anti-estrogen therapy is the main hurdle for successful TMX therapy. Here we address the mechanism for TMX resistance and explore the ways to eradicate TMX-resistant breast cancer in both in vitro and ex vivo experiments. EXPERIMENTAL DESIGN: To identify compounds able to overcome TMX resistance, we used short-term and long-term viability assays in cancer cells in vitro and in patient samples in 3D ex vivo, analysis of gene expression profiles and cell line pharmacology database, shRNA screen, CRISPR-Cas9 genome editing, real-time PCR, immunofluorescent analysis, western blot, measurement of oxidative stress using flow cytometry, and thioredoxin reductase 1 enzymatic activity. RESULTS: Here, for the first time, we provide an ample evidence that a high level of the detoxifying enzyme SULT1A1 confers resistance to TMX therapy in both in vitro and ex vivo models and correlates with TMX resistance in metastatic samples in relapsed patients. Based on the data from different approaches, we identified three anticancer compounds, RITA (Reactivation of p53 and Induction of Tumor cell Apoptosis), aminoflavone (AF), and oncrasin-1 (ONC-1), whose tumor cell inhibition activity is dependent on SULT1A1. We discovered thioredoxin reductase 1 (TrxR1, encoded by TXNRD1) as a target of bio-activated RITA, AF, and ONC-1. SULT1A1 depletion prevented the inhibition of TrxR1, induction of oxidative stress, DNA damage signaling, and apoptosis triggered by the compounds. Notably, RITA efficiently suppressed TMX-unresponsive patient-derived breast cancer cells ex vivo. CONCLUSION: We have identified a mechanism of resistance to TMX via hyperactivated SULT1A1, which renders selective vulnerability to anticancer compounds RITA, AF, and ONC-1, and provide a rationale for a new combination therapy to overcome TMX resistance in breast cancer patients. Our novel findings may provide a strategy to circumvent TMX resistance and suggest that this approach could be developed further for the benefit of relapsed breast cancer patients.

Interaction of the Brain-Selective Sulfotransferase SULT4A1 with Other Cytosolic Sulfotransferases: Effects on Protein Expression and Function.

Sulfotransferase (SULT) 4A1 is a brain-selective sulfotransferase-like protein that has recently been shown to be essential for normal neuronal development in mice. In the present study, SULT4A1 was found to colocalize with SULT1A1/3 in human brain neurons. Using immunoprecipitation, SULT4A1 was shown to interact with both SULT1A1 and SULT1A3 when expressed in human cells. Mutation of the conserved dimerization motif located in the C terminus of the sulfotransferases prevented this interaction. Both ectopically expressed and endogenous SULT4A1 decreased SULT1A1/3 protein levels in neuronal cells, and this was also prevented by mutation of the dimerization motif. During differentiation of neuronal SH-SY5Y cells, there was a loss in SULT1A1/3 protein but an increase in SULT4A1 protein. This resulted in an increase in the toxicity of dopamine, a substrate for SULT1A3. Inhibition of SULT4A1 using small interference RNA abrogated the loss in SULT1A1/3 and reversed dopamine toxicity. These results show a reciprocal relationship between SULT4A1 and the other sulfotransferases, suggesting that it may act as a chaperone to control the expression of SULT1A1/3 in neuronal cells. SIGNIFICANCE STATEMENT: The catalytically inactive sulfotransferase (SULT) 4A1 may regulate the function of other SULTs by interacting with them via a conserved dimerization motif. In neuron-like cells, SULT4A1 is able to modulate dopamine toxicity by interacting with SULT1A3, potentially decreasing the metabolism of dopamine.

Paracetamol and Pain Modulation by TRPV1, UGT2B15, SULT1A1 Genotypes: A Randomized Clinical Trial in Healthy Volunteers.

BACKGROUND: The influence of the genetic polymorphism of enzymes and receptors involved in paracetamol metabolism and mechanism of action has not been investigated. This trial in healthy volunteers investigated the link between paracetamol pain relief and the genetic polymorphism of 23 enzymes and receptors. DESIGN: This randomized double-blind crossover controlled pilot study took place in the Clinical Pharmacology Department, University Hospital, Clermont-Ferrand, France. Forty-seven Caucasian volunteers were recruited. The trial consisted of two randomized sessions one week apart with oral paracetamol or placebo, and pain changes were evaluated with mechanical pain stimuli. The genetic polymorphism of 23 enzymes and receptors was studied, and correlations were made with pain relief. All tests are two-sided with a type I error at 0.05. RESULTS: Paracetamol was antinociceptive compared with placebo (222 ± 482 kPaxmin vs 23 ± 431 kPaxmin; P = 0.0047), and the study showed 30 paracetamol responders and 17 paracetamol nonresponders. Responders were characterized by TRPV1rs224534 A allele, UGT2B15rs1902023 TT genotype, and SULT1A1rs9282861 GG genotype (P < 0.05 for all). These findings confirm for the first time the involvement of a specific TRPV1 rs224534 variant in paracetamol antinociception. They also reveal a new antinociceptive role for specific variants of hepatic phase II enzymes associated with paracetamol metabolism. CONCLUSIONS: The study warrants larger clinical trials on these potential genomic markers of paracetamol analgesia in patients. Confirmation of the present findings would open the way to effective individualized pain treatment with paracetamol, the most commonly used analgesic worldwide.