Identification of ascorbate- and salicylate-responsive miRNAs and verification of the spectral control of miR395 in Arabidopsis.

We assumed that miRNAs might regulate the physiological and biochemical processes in plants through their effects on the redox system and phytohormones. To check this hypothesis, the transcriptome profile of wild-type Arabidopsis and lines with decreased ascorbate (Asc), glutathione (GSH), or salicylate (Sal) levels were compared. GSH deficiency did not influence the miRNA expression, whereas lower levels of Asc and Sal reduced the accumulation of 9 and 44 miRNAs, respectively, but only four miRNAs were upregulated. Bioinformatics analysis revealed that their over-represented target genes are associated with the synthesis of nitrogen-containing and aromatic compounds, nucleic acids, and sulphate assimilation. Among them, the sulphate reduction-related miR395 - ATP-sulfurylase couple was selected to check the assumed modulating role of the light spectrum. A greater induction of the Asc- and Sal-responsive miR395 was observed under sulphur starvation in far-red light compared to white and blue light in wild-type and GSH-deficient Arabidopsis lines. Sal deficiency inhibited the induction of miR395 by sulphur starvation in blue light, whereas Asc deficiency greatly reduced it independently of the spectrum. Interestingly, sulphur starvation decreased only the level of ATP sulfurylase 4 among the miR395 target genes in far-red light. The expression level of ATP sulfurylase 3 was higher in far-red light than in blue light in wild-type and Asc-deficient lines. The results indicate the coordinated control of miRNAs by the redox and hormonal system since 11 miRNAs were affected by both Asc and Sal deficiency. This process can be modulated by light spectrum, as shown for miR395.

ATP sulfurylase atypical leucine zipper interacts with Cys3 and calcineurin A in the regulation of sulfur amino acid biosynthesis in Cryptococcus neoformans.

Fungal pathogens are a major cause of death, especially among immunocompromised patients. Therapies against invasive fungal infections are restricted to a few antifungals; therefore, novel therapies are necessary. Nutritional signaling and regulation are important for pathogen establishment in the host. In Cryptococcus neoformans, the causal agent of fungal meningitis, amino acid uptake and biosynthesis are major aspects of nutritional adaptation. Disruptions in these pathways lead to virulence attenuation in an animal model of infection, especially for sulfur uptake and sulfur amino acid biosynthesis. Deletion of Cys3, the main transcription factor that controls these pathways, is the most deleterious gene knockout in vitro and in vivo, making it an important target for further application. Previously, we demonstrated that Cys3 is part of a protein complex, including calcineurin, which is necessary to maintain high Cys3 protein levels during sulfur uptake and sulfur amino acid biosynthesis. In the current study, other aspects of Cys3 regulation are explored. Two lines of evidence suggest that C. neoformans Cys3 does not interact with the F-box WD40 protein annotated as Met30, indicating another protein mediates Cys3 ubiquitin degradation. However, we found another level of Cys3 regulation, which involves protein interactions between Cys3 and ATP sulfurylase (MET3 gene). We show that an atypical leucine zipper at the N-terminus of ATP sulfurylase is essential for physical interaction with Cys3 and calcineurin. Our data suggests that Cys3 and ATP sulfurylase interact to regulate Cys3 transcriptional activity. This work evidences the complexity involved in the regulation of a transcription factor essential for the sulfur metabolism, which is a biological process important to nutritional adaptation, oxidative stress response, nucleic acid stability, and methylation. This information may be useful in designing novel therapies against fungal infections.

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.

Gene Identification, expression analysis and molecular docking of ATP sulfurylase in the selenization pathway of Cardamine hupingshanensis.

BACKGROUND: ATP sulfurylase (ATPS) is a crucial enzyme for the selenate assimilation pathway in plants. RESULTS: In this study, genome-wide and comparative analyses of ATPS in Cardamine hupingshanensis, including sequence and structural analyses, were performed. The expression of ChATPS gene family members in C. hupingshanensis under selenium (Se) stress was also investigated, and our results suggest that ChATPS1-2 play key roles in the response to Se stress. Nine ATPS genes were found from C. hupingshanensis, which share highly conserved sequences with ATPS from Arabidopsis thaliana. In addition, we performed molecular docking of ATP sulfurylase in complex with compounds ATP, selenate, selenite, sulfate, and sulfite. ChAPS3-1 was found to have stronger binding energies with all compounds tested. Among these complexes, amino acid residues Arg, Gly, Ser, Glu, and Asn were commonly present. CONCLUSION: Our study reveals the molecular mechanism of C. hupingshanensis ATP sulfurylase interacting with selenate, which is essential for understanding selenium assimilation. This information will guide further studies on the function of the ChATPS gene family in the selenium stress response and lay the foundation for the selenium metabolic pathway in higher plants.

A Reference Range for Plasma Levels of Inorganic Pyrophosphate in Children Using the ATP Sulfurylase Method.

PURPOSE: Generalized arterial calcification of infancy, pseudoxanthoma elasticum, autosomal recessive hypophosphatemic rickets type 2, and hypophosphatasia are rare inherited disorders associated with altered plasma levels of inorganic pyrophosphate (PPi). In this study, we aimed to establish a reference range for plasma PPi in the pediatric population, which would be essential to support its use as a biomarker in children with mineralization disorders. METHODS: Plasma samples were collected from 200 children aged 1 day to 18 years who underwent blood testing for medical conditions not affecting plasma PPi levels. PPi was measured in proband plasma utilizing a validated adenosine triphosphate (ATP) sulfurylase method. RESULTS: The analytical sensitivity of the ATP sulfurylase assay consisted of 0.15 to 10 µM PPi. Inter- and intra-assay coefficients of variability on identical samples were below 10%. The standard range of PPi in the blood plasma of children and adolescents aged 0 to 18 years was calculated as 2.36 to 4.44 µM, with a median of 3.17 µM, with no difference between male and female probands. PPi plasma levels did not differ significantly in different pediatric age groups. MAIN CONCLUSIONS: Our results yielded no noteworthy discrepancy to the reported standard range of plasma PPi in adults (2-5 µM). We propose the described ATP sulfurylase method as a diagnostic tool to measure PPi levels in plasma as a biomarker in the pediatric population.

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.

Changes in ATP Sulfurylase Activity in Response to Altered Cyanobacteria Growth Conditions.

We investigated variations in cell growth and ATP Sulfurylase (ATPS) activity when two cyanobacterial strains-Synechocystis sp. PCC6803 and Synechococcus sp. WH7803-were grown in conventional media, and media with low ammonium, low sulfate and a high CO2/low O2 atmosphere. In both organisms, a transition and adaptation to the reconstructed environmental media resulted in a decrease in ATPS activity. This variation appears to be decoupled from growth rate, suggesting the enzyme is not rate-limiting in S assimilation and raising questions about the role of ATPS redox regulation in cell physiology and throughout Earth history.

Intestinal Sulfation Is Essential to Protect Against Colitis and Colonic Carcinogenesis.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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-ß-muricholic acid, and deficiency in the formation of bile acid sulfates in the colon of Papss2ΔIE mice. CONCLUSIONS: 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.

Overexpression of ATP sulfurylase improves the sulfur amino acid content, enhances the accumulation of Bowman-Birk protease inhibitor and suppresses the accumulation of the ß-subunit of ß-conglycinin in soybean seeds.

ATP sulfurylase, an enzyme which catalyzes the conversion of sulfate to adenosine 5'-phosphosulfate (APS), plays a significant role in controlling sulfur metabolism in plants. In this study, we have expressed soybean plastid ATP sulfurylase isoform 1 in transgenic soybean without its transit peptide under the control of the 35S CaMV promoter. Subcellular fractionation and immunoblot analysis revealed that ATP sulfurylase isoform 1 was predominantly expressed in the cell cytoplasm. Compared with that of untransformed plants, the ATP sulfurylase activity was about 2.5-fold higher in developing seeds. High-resolution 2-D gel electrophoresis and immunoblot analyses revealed that transgenic soybean seeds overexpressing ATP sulfurylase accumulated very low levels of the ß-subunit of ß-conglycinin. In contrast, the accumulation of the cysteine-rich Bowman-Birk protease inhibitor was several fold higher in transgenic soybean plants when compared to the non-transgenic wild-type seeds. The overall protein content of the transgenic seeds was lowered by about 3% when compared to the wild-type seeds. Metabolite profiling by LC-MS and GC-MS quantified 124 seed metabolites out of which 84 were present in higher amounts and 40 were present in lower amounts in ATP sulfurylase overexpressing seeds compared to the wild-type seeds. Sulfate, cysteine, and some sulfur-containing secondary metabolites accumulated in higher amounts in ATP sulfurylase transgenic seeds. Additionally, ATP sulfurylase overexpressing seeds contained significantly higher amounts of phospholipids, lysophospholipids, diacylglycerols, sterols, and sulfolipids. Importantly, over expression of ATP sulfurylase resulted in 37-52% and 15-19% increases in the protein-bound cysteine and methionine content of transgenic seeds, respectively. Our results demonstrate that manipulating the expression levels of key sulfur assimilatory enzymes could be exploited to improve the nutritive value of soybean seeds.

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.

PoMet3 and PoMet14 associated with sulfate assimilation are essential for conidiogenesis and pathogenicity in Pyricularia oryzae.

Pyricularia oryzae is the causal agent of blast disease on staple gramineous crops. Sulphur is an essential element for the biosynthesis of cysteine and methionine in fungi. Here, we targeted the P. oryzae PoMET3 encoding the enzyme ATP sulfurylase, and PoMET14 encoding the APS (adenosine-5'-phosphosulphate) kinase that are involved in sulfate assimilation and sulphur-containing amino acids biosynthesis. In P. oryzae, deletion of PoMET3 or PoMET14 separately results in defects of conidiophore formation, significant impairments in conidiation, methionine and cysteine auxotrophy, limited invasive hypha extension, and remarkably reduced virulence on rice and barley. Furthermore, the defects of the null mutants could be restored by supplementing with exogenous cysteine or methionine. Our study explored the biological functions of sulfur assimilation and sulphur-containing amino acids biosynthesis in P. oryzae.

Sulfur application reduces cadmium uptake in edible parts of pakchoi (Brassica chinensis L.) by cadmium chelation and vacuolar sequestration.

Sulfur (S) application in pakchoi (Brassica chinensis L.) cultivation is vital for reducing cadmium (Cd) accumulation in the plants. However, the mechanism of S application on Cd uptake and translocation in pakchoi is unclear. In this study, a hydroponic experiment was performed to investigate the effects of S application on Cd accumulation in pakchoi at one Cd concentration (50 µM, in comparison to the control condition, 0 µM) and three S levels (0, 2, 4 mM). The results showed that excessive S application (4 mM) reduced Cd accumulation and alleviated pakchoi growth inhibition caused by Cd stress in shoots and roots. With increased S application, the proportion of Cd in the vacuolar fraction and the proportion of NaCl-extractable Cd increased in roots. Additionally, S application increased the content of glutathione (GSH) and phytochelatins (PCs). The reduced Cd uptake and accumulation in pakchoi shoots could have been due to increased Cd chelation and vacuolar sequestration in roots. In addition, sufficient S application (2 mM) increased the expression of γ-glutamylcysteine synthetase (GSH1) and nicotinamide synthase (NAS) in roots, and excessive S application upregulated the expression of ATP sulfurylase (ATPS) and phytochelatin synthase (PCs). This study provides evidence for the mechanism of mitigating Cd toxicity in pakchoi and will be helpful for developing strategies to reduce Cd accumulation in the edible parts of pakchoi through S fertilizer application.

The relationship between sulfur metabolism and tolerance of hexavalent chromium in Scenedesmus acutus (Spheropleales): Role of ATP sulfurylase.

Sulfur availability and the end products of its metabolism, cysteine, glutathione and phytochelatins, play an important role in heavy metal tolerance, chromium included. Sulfate and chromate not only compete for the transporters but also for assimilation enzymes and chromium tolerance in various organisms has been associated to differences in this pathway. We investigated the mechanisms of Cr(VI)-tolerance increase induced by S-starvation focusing on the role of ATP sulfurylase (ATS) in two strains of Scenedesmus acutus with different chromium sensitivity. S-starvation enhances the defence potential by increasing sulfate uptake/assimilation and decreasing chromium uptake, thus suggesting a change in the transport system. We isolated two isoforms of the enzyme, SaATS1 and SaATS2, with different sensitivity to sulfur availability, and analysed them in S-sufficient and S-replete condition both in standard and in chromium supplemented medium. SaATS2 expression is different in the two strains and presumably marks a different sulfur perception/exploitation in the Cr-tolerant. Its induction and silencing are compatible with a role in the transient tolerance increase induced by S-starvation. This enzyme can however hardly be responsible for the large cysteine production of the Cr-tolerant strain after starvation, suggesting that cytosolic rather than chloroplastic cysteine production is differently regulated in the two strains.

Genome-wide analyses of ATP sulfurylase (ATPS) genes in higher plants and expression profiles in sorghum (Sorghum bicolor) under cadmium and salinity stresses.

ATP sulfurylase (ATPS, EC: 2.7.7.4) is a crucial enzyme for sulfate assimilation pathway in both plastids and cytosol in plants. In this study, genome-wide and comparative analyses of ATPSs in 11 higher plant species, including sequence and structural analyses have been performed. Expression of ATPS genes in sorghum under cadmium (Cd) and salinity (NaCl) stresses were also investigated to provide a model experimental data for the regulation of ATPS genes under stress conditions. Thirty-one ATPS genes from 11 plant species were found. It showed that ATPSs from different species have high sequence divergences, which cause structural differences among them. Phylogenetic analysis has shown that there are two major types of ATPSs evolved in dicots while monocots were evolved to have one type of ATPs. Finally, expression analysis of ATPS genes revealed tissue and stress dependent expression pattern, which indicates expressions of ATPS genes are tightly regulated.

Enhanced PAPSS2/VCAN sulfation axis is essential for Snail-mediated breast cancer cell migration and metastasis.

The zinc finger protein Snail is a master regulator of epithelial-mesenchymal transition (EMT) and a strong inducer of tumor metastasis, yet the signal cascades triggered by Snail have not been completely revealed. Here, we report the discovery of the sulfation program that can be induced by Snail in breast cancer cells, and which plays an essential role in cell migration and metastasis. Specifically, Snail induces the expression of PAPSS2, a gene that encodes a rate-limiting enzyme in sulfation pathway, and VCAN, a gene that encodes the chondroitin sulfate proteoglycan Versican in multiple breast cancer cells. Depletion of PAPSS2 in MCF7 and MDA-MB-231 cells results in reduced cell migration, while overexpression of PAPSS2 promotes cell migration. Moreover, MDA-MB-231-shPAPSS2 cells display a significantly lower rate of lung metastasis and lower number of micrometastatic nodules in nude mice, and conversely, MDA-MB-231-PAPSS2 cells increase lung metastasis. Similarly, depletion of VCAN dampens the cell migration activity induced by Snail or PAPSS2 in MCF 10A cells. Moreover, PAPSS inhibitor sodium chlorate effectively decreases cell migration induced by Snail and PAPSS2. More importantly, the expression of Snail, PAPSS2, and VCAN is positively correlated in breast cancer tissues. Together, these findings are important for understanding the genetic programs that control tumor metastasis and may identify previously undetected therapeutic targets to treat metastatic disease.

Specific inhibitors of respiratory sulfate reduction: towards a mechanistic understanding.

Microbial sulfate reduction (SR) by sulfate-reducing micro-organisms (SRM) is a primary environmental mechanism of anaerobic organic matter mineralization, and as such influences carbon and sulfur cycling in many natural and engineered environments. In industrial systems, SR results in the generation of hydrogen sulfide, a toxic, corrosive gas with adverse human health effects and significant economic and environmental consequences. Therefore, there has been considerable interest in developing strategies for mitigating hydrogen sulfide production, and several specific inhibitors of SRM have been identified and characterized. Specific inhibitors are compounds that disrupt the metabolism of one group of organisms, with little or no effect on the rest of the community. Putative specific inhibitors of SRM have been used to control sulfidogenesis in industrial and engineered systems. Despite the value of these inhibitors, mechanistic and quantitative studies into the molecular mechanisms of their inhibition have been sparse and unsystematic. The insight garnered by such studies is essential if we are to have a more complete understanding of SR, including the past and current selective pressures acting upon it. Furthermore, the ability to reliably control sulfidogenesis - and potentially assimilatory sulfate pathways - relies on a thorough molecular understanding of inhibition. The scope of this review is to summarize the current state of the field: how we measure and understand inhibition, the targets of specific SR inhibitors and how SRM acclimatize and/or adapt to these stressors.

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.

ATP Sulfurylase is Essential for the Utilization of Sulfamate as a Sulfur Source in the Yeast Komagataella pastoris (syn. Pichia pastoris).

The methylotrophic yeast Komagataella pastoris (syn. Pichia pastoris) is one of the few known yeasts that can utilize sulfamate ([Formula: see text]) as a sulfur source. The biochemical pathway responsible for the catabolism of sulfamate has yet to be identified. The present study sought to investigate whether sulfamate catabolism proceeds through either of the inorganic sulfur intermediates sulfate ([Formula: see text]) or sulfite ([Formula: see text]) before its assimilation and subsequent incorporation into sulfur-containing amino acids and their derivatives. Two key genes in the K. pastoris inorganic sulfur assimilation pathway were deleted separately and the ability of each deletion mutant to utilize sulfamate and other selected sulfur sources was studied. Deletion of the MET3 gene (which encodes the enzyme ATP sulfurylase) did not affect growth on L-methionine, sulfite, methanesulfonate, or taurine but completely abolished growth on sulfate, methyl sulfate and sulfamate. Deletion of the MET5 gene (which encodes the ß subunit of the enzyme sulfite reductase) abolished growth on all tested sulfur sources except L-methionine. These results suggest that the catabolism of sulfamate proceeds through a sulfate intermediate before its assimilation.

High-Throughput Screening of Sulfated Proteins by Using a Genome-Wide Proteome Microarray and Protein Tyrosine Sulfation System.

Protein tyrosine sulfation (PTS) is a widespread posttranslational modification that induces intercellular and extracellular responses by regulating protein-protein interactions and enzymatic activity. Although PTS affects numerous physiological and pathological processes, only a small fraction of the total predicted sulfated proteins has been identified to date. Here, we localized the potential sulfation sites of Escherichia coli proteins on a proteome microarray by using a 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase-coupled tyrosylprotein sulfotransferase (TPST) catalysis system that involves in situ PAPS generation and TPST catalysis. Among the 4256 E. coli K12 proteins, 875 sulfated proteins were identified using antisulfotyrosine primary and Cy3-labeled antimouse secondary antibodies. Our findings add considerably to the list of potential proteins subjected to tyrosine sulfation. Similar procedures can be applied to identify sulfated proteins in yeast and human proteome microarrays, and we expect such approaches to contribute substantially to the understanding of important human diseases.

SOX9 protein is stabilized by TGF-ß and regulates PAPSS2 mRNA expression in chondrocytes.

OBJECTIVE: We previously identified 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2) as a transcriptional target of transforming growth factor ß (TGF-ß) in chondrocytes. PAPSS2 is required for proper sulfation of proteoglycans in cartilage. Defective sulfation in the matrix results in alterations in mechanical properties of the cartilage that would be expected to result in degeneration. The objective of this study was to identify factors that regulate PAPSS2 expression and compare to a known TGF-ß responsive gene, proteoglycan 4/lubricin (PRG4). In this study, TGF-ß-mediated regulation of SOX9 was characterized, and the involvement of SOX9 in regulation of PAPSS2 mRNA was investigated. DESIGN: Primary bovine articular chondrocytes grown in micromass culture and ATDC5 cells were used as the model system. Adenoviruses were used to express SOX9 and SMAD3. siRNA was used to knock-down Sox9 and Smad3. Western blot and real-time quantitative RT-PCR (qPCR) were used to measure changes in protein and mRNA levels in response to treatment. RESULTS: Over-expression of SOX9 was sufficient to up-regulate PAPSS2 mRNA. TGF-ß treatment of SOX9-expressing cells resulted in enhanced up-regulation of PAPSS2 mRNA, suggesting that SOX9 cooperates with TGF-ß signaling. Furthermore, Sox9 was required for full TGF-ß-mediated induction of Papss2. In contrast, PRG4 was regulated by SMAD3 but not SOX9. SOX9 protein levels were increased after treatment with TGF-ß, although SOX9 mRNA was not. SOX9 protein was post-translationally stabilized after treatment with TGF-ß. CONCLUSIONS: TGF-ß stabilizes SOX9 protein, and SOX9 is sufficient and necessary for TGF-ß-mediated regulation of PAPSS2 mRNA, providing a novel mechanism for TGF-ß-mediated gene regulation in chondrocytes.