[The intestinal microbiota: A new player in depression?] / Le microbiote intestinal : un nouvel acteur de la dépression ?

Depression is the leading cause of disability in the world according to the World Health Organization. The effectiveness of the available antidepressant therapies is limited. Data from the literature suggest that some subtypes of depression may be associated with chronic low grade inflammation. The uncovering of the role of intestinal microbiota in the development of the immune system and its bidirectional communication with the brain have led to growing interest on reciprocal interactions between inflammation, microbiota and depression. Our purpose is to review the state of knowledge on these interactions. METHODS: We carried out a literature search on Pubmed, Go pubmed, psyC info, Elsevier, Embase until August 13, 2016 using the keywords "depression", "microbiota" and "inflammation". RESULTS: Dysbiosis reported in patients suffering from depression seems to contribute to low grade systemic inflammation which in turn feeds back depression. The hypothetical mechanisms behind these interactions are multiple: leaky gut, hyperreactivity of the corticotropic axis, disturbed neurotransmission. Abnormal microbial exposure during childhood and perinatal stress are reported to influence both the maturation of the immune system and the microbiota hence contributing to the ethiopathogeny of depression. There is no evidence in the literature to support a role for diet. CONCLUSION: The evidence supporting a causal relationship between dysbiosis and depression through low grade inflammation is limited and precludes us from drawing firm conclusions. Further studies are needed to improve our knowledge.

Biochemical and molecular characterization of flavonoid 7-sulfotransferase from Arabidopsis thaliana.

Flavonoid compounds play important roles as flower pigments, stress metabolites formed in response to UV, during pollen germination and for polar auxin transport (Trends Plant Sci. 1 (1996) 377). Flavonoid sulfate esters are common in plants, especially the Asteraceae; however, due to the lack of information regarding the factors that regulate their accumulation, their exact role remains to be elucidated. The biosynthesis of flavonol sulfate esters is catalyzed by a number of position specific flavonol sulfotransferases (STs). An Arabidopsis thaliana database search has allowed us to identify and classify 18 putative ST coding sequences. We report here the cloning and characterization of the AtST3a member of this family that is expressed at early stages of seedling development and in the inflorescence stem and siliques of mature plants. The recombinant AtST3a protein exhibits strict specificity for position 7 of flavonoids. In contrast to previously characterized flavonol 7-ST from Flaveria bidentis that sulfonates only flavonol disulfates, AtST3a was found to accept as substrates a number of flavonols and flavone aglycones, as well as their monosulfate esters. The discovery of a flavonol ST from A. thaliana suggests that flavonol sulfates are more widely distributed than originally believed and this model plant could be used to study their biological significance.

Inactivation of brassinosteroid biological activity by a salicylate-inducible steroid sulfotransferase from Brassica napus.

Recent discoveries from brassinosteroid-deficient mutants led to the recognition that plants, like animals, use steroids to regulate their growth and development. We describe the characterization of one member of a Brassica napus sulfotransferase gene family coding for an enzyme that catalyzes the O-sulfonation of brassinosteroids and of mammalian estrogenic steroids. The enzyme is specific for the hydroxyl group at position 22 of brassinosteroids with a preference for 24-epicathasterone, an intermediate in the biosynthesis of 24-epibrassinolide. Enzymatic sulfonation of 24-epibrassinolide abolishes its biological activity in the bean second internode bioassay. This mechanism of hormone inactivation by sulfonation is similar to the modulation of estrogen biological activity observed in mammals. Furthermore, the expression of the B. napus steroid sulfotransferase genes was found to be induced by salicylic acid, a signal molecule in the plant defense response. This pattern of expression suggests that, in addition to an increased synthesis of proteins having antimicrobial properties, plants respond to pathogen infection by modulating steroid-dependent growth and developmental processes.

3'-Phosphoadenosine 5'-phosphosulfate binding site of flavonol 3-sulfotransferase studied by affinity chromatography and 31P NMR.

The function of Lys-59, Arg-141, and Arg-277 in PAPS binding and catalysis of the flavonol 3-sulfotransferase was investigated. Affinity chromatography of conservative mutants with PAPS analogues allowed us to determine that Lys-59 interacts with the 5' portion of the nucleotide, while Arg-141 interacts with the 3' portion, confirming assignments deduced from the crystal structure of mouse estrogen sulfotransferase [Kakuta, Y., Pedersen, L. G., Carter, C. W. , Negishi, M., and Pedersen, L. C. (1997) Nat. Struct. Biol. 4, 904-908]. The affinity chromatography method could be used to characterize site-directed mutants for other types of enzymes that bind nucleoside 3',5'- or 2',5'-diphosphates. 31P NMR spectra of enzyme-PAP complexes were recorded for the wild-type enzyme and K59R and K59A mutants. The results of these experiments suggest that Lys-59 is involved in the determination of the proper orientation of the phosphosulfate group for catalysis.

Recent developments in the study of the structure-function relationship of flavonol sulfotransferases.

With the rapid proliferation of sulfotransferase (ST) cDNA sequences in the last 5 years, consensus sequences were identified in four conserved regions. The association of these regions with substrate binding or catalysis was tested in several site-directed mutagenesis studies. Due to their strict substrate and position specificities, the flavonol 3- and 4'-STs represent an advantageous model system for the study of the structure-function relationship of cytosolic STs. Using a combination of chimeric and site-directed mutant proteins, a domain was identified containing all the determinants responsible for the substrate specificity of these enzymes, and characterized amino acid residues conserved in all cloned STs that are involved in substrate binding and catalysis. This paper summarizes the results of these studies.

Mutational analysis of domain II of flavonol 3-sulfotransferase.

The flavonol 3- and 4'-sulfotransferases (ST) from Flaveria chloraefolia catalyze the transfer of the sulfonate group from 3'-phosphoadenosine 5'-phosphosulfate (PAdoPS) to position 3 of flavonol aglycones and position 4' of flavonol 3-sulfates. We identified previously a protein segment, designated domain II, that contains all the determinants responsible for the specificity of these enzymes. Within domain II, at least five amino acids specific to the 4'-ST that could bind the sulfate group of quercetin 3-sulfate were identified. In this study, these amino acid residues were introduced at equivalent positions in the flavonol 3-ST sequence by site-directed mutagenesis of the cloned cDNA. No reversal of the substrate specificity was observed after the individual mutations. However, mutation of Leu95 to Tyr had different effects on the kinetic constants depending on the substitution pattern of the flavonoid B ring, suggesting that the tyrosine side chain may be in direct contact with this part of the molecule. The function of conserved amino acids present in domain II was also investigated. Unconservative mutations at Lys134, Tyr137 and Tyr150 resulted in protein instability in solution, suggesting that these residues might be important for the structural stability of the enzyme. Replacement of Arg140 with Lys or Ser had no effect on protein stability, but resulted in a strong reduction in specific activity. The results of photoaffinity-labeling experiments with PAdoP[35S]S suggest that this residue is required to bind the cosubstrate. In addition, the reduced affinity of [Ser140]ST for 3'-phosphoadenosine 5'-phosphate (PAdoP)-agarose indicates that Arg140 is also involved in binding the coproduct. Replacement of His118 with Glu or Ala resulted in a strong reduction in catalytic activity. However, [Lys118]ST retained a significant amount of catalytic activity. The results of photoaffinity-labeling experiments with PAdoP[35S]S and affinity chromatography on PAdoP-agarose suggest that His118 might be involved in catalysis in the flavonol 3-ST.

Sulfation and sulfotransferases 6: Biochemistry and molecular biology of plant sulfotransferases.

It is now well established that, in mammals, sulfate conjugation constitutes an important reaction in the transformation of xenobiotics and in the modulation of the biological activity of steroid hormones and neurotransmitter. The presence of a sulfate group on some molecules can also be a prerequisite for their biological function. For example, it is well known that the sulfate groups are directly involved in the molecular interaction between heparin and antithrombin III. In plants, sulfation also seems to play an important role in the intermolecular recognition and signaling processes, as indicated by the requirement of a sulfate moiety for the biological activity of gallic acid glucoside sulfate in the seismonastic and gravitropic movements of plants, and of Nod RM1 in the cortical cell division during early nodule initiation in Rhizobium meliloti-alfalfa interaction. In addition, recent studies indicate that flavonoid conjugates, including the sulfate esters, may play a role in the regulation of plant growth by strongly binding the naphthylphthalamic acid receptor, thus blocking the quercetin-stimulated accumulation of the auxin phytohormone. Although several sulfated metabolites are known to accumulate in a variety of plant species, the study of enzymes that catalyze the sulfation reaction in plants lagged considerably compared to those conducted with their mammalian homologs. This apparent lack of interest may have been because the function of plant-sulfated metabolites is difficult to predict, since their accumulation is often restricted to a limited number of species. Despite this limitation, several plant sulfotransferases (STs) have been characterized at the biochemical level, and the cDNA clones encoding six plant STs have been isolated. Based on sequence homology, the plant ST coding sequences are grouped under the SULT3 family, also known as the flavonol ST family. This review summarizes our current knowledge of the plant STs and focuses on the functional significance of the sulfate conjugation in plant growth, development, and adaptation to stress.

The enzyme involved in sulfation of the turgorin, gallic acid 4-O-(beta-D-glucopyranosyl-6'-sulfate) is pulvini-localized in Mimosa pudica.

A sulfotransferase (ST) which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to gallic acid glucoside was characterized from microsomal preparations of Mimosa pudica. The product of the reaction was found to co-elute on HPLC with the periodic leaf movement factor 1 (PLMF-1)(gallic acid beta-D-gluco-pyranosyl-6'-sulfate). The distribution of the enzyme activity was restricted to plasma membrane preparations from primary, secondary and tertiary pulvini. The M. pudica ST activity was inhibited in a dose-dependent manner in the presence of an antibody raised against the flavonol 3-sulfotransferase of Flaveria chloraefolia, suggesting structural similarities between the two proteins. Western blot analysis of M. pudica protein extracts using these antibodies indicated the presence of a cross-reactive polypeptide with an apparent molecular mass of 42,000 Da whose distribution correlates with the presence of the gallic acid glucoside ST activity. Indirect immunogold labeling of resin-embedded sections from tertiary pulvini showed a specific localization of gold particles on the sieve-tube plasma membranes. The label distribution was uniform and other cellular organelles and membrane systems displayed little or no labeling. The results of the Western blot and immunocytochemical studies are consistent with the detection of the gallic acid glucoside ST activity in plasma membrane preparations of M. pudica pulvini cells. The specific tissue distribution of the ST in motor organ phloem cells suggests that this is the site of synthesis and/or accumulation of PLMF-1 and supports the proposed hypothesis that PLMF-1 may be acting as a chemical signal during the seismonastic response of M. pudica.

Identification of amino acid residues critical for catalysis and cosubstrate binding in the flavonol 3-sulfotransferase.

The comparison of the deduced amino acid sequences of plant and animal sulfotransferases (ST) has allowed the identification of four well conserved regions, and previous experimental evidence suggested that regions I and IV might be involved in the binding of the cosubstrate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Moreover, region IV is homologous to the glycine-rich phosphate binding loop (P-loop) motif known to be involved in nucleotide phosphate binding in several protein families. In this study, the function of amino acid residues within these two regions was investigated by site-directed mutagenesis of the plant flavonol 3-ST. In region I, our results identify Lys59 as critical for catalysis, since replacement of this residue with alanine resulted in a 300-fold decrease in specific activity, while a 15-fold reduction was observed after the conservative replacement with arginine. Photoaffinity labeling of K59R and K59A with [35S]PAPS revealed that Lys59 is not required for cosubstrate binding. However, the K59A mutant had a reduced affinity for 3'-phosphoadenosine 5'-phosphate (PAP)-agarose, suggesting that Lys59 may participate in the stabilization of an intermediate during the reaction. In region IV, all substitutions of Arg276 resulted in a marked decrease in specific activity. Conservative and unconservative replacements of Arg276 resulted in weak photoaffinity labeling with [35S]PAPS and the R276A/T73A and R276E enzymes displayed reduced affinities for PAP-agarose, suggesting that the Arg276 side chain is required to bind the cosubstrate. The analysis of the kinetic constants of mutant enzymes at residues Lys277, Gly281, and Lys284 allowed to confirm that region IV is involved in cosubstrate binding.

Chimeric flavonol sulfotransferases define a domain responsible for substrate and position specificities.

The pFST3 and pFST4' cDNAs encode flavonol sulfotransferases (ST) that are 69% identical in amino acid sequence yet exhibit strict substrate and position specificities. To determine the domain responsible for the properties of the flavonol STs, several chimeric flavonol STs were constructed by the reciprocal exchange of DNA fragments derived from the plasmids pFST3 and pFST4' and by the expression of the corresponding chimeric proteins in Escherichia coli. The chimeric enzymes were enzymatically active even though their activities were reduced compared to the parent enzymes. The specificity of the resulting hybrid proteins indicates that an interval of the flavonol STs spanning amino acids 92-194 of the flavonol 3-ST sequence contains the determinant of the substrate and position preferences. From the comparison of the amino acid sequences between plant and animal STs, this interval can be subdivided into a highly conserved region corresponding to positions 134-152 of the flavonol 3-ST, flanked by two regions of high divergence from 98 to 110 and 153 to 170. In view of the similarities in length and hydropathic profiles as well as the presence of four conserved regions between plant and animal STs, the results of these experiments suggest that this interval is involved in the recognition of substrates and/or catalysis in all STs.

Cloning and regulation of flavonol 3-sulfotransferase in cell-suspension cultures of Flaveria bidentis.

Flaveria spp. accumulate flavonol sulfate esters whose biosynthesis is catalyzed by a number of position-specific flavonol sulfotransferases. Although the accumulation of sulfated flavonols appears to be tissue specific and developmentally regulated and to vary among related species, little is known about the mechanism of regulation controlling the synthesis of these metabolites. In the present work, we report the isolation of a cDNA clone from Flaveria bidentis (pBFST3) encoding flavonol 3-sulfotransferase (F3-ST), which catalyzes the first step in the biosynthesis of flavonol polysulfates. This clone (pBFST3) was expressed in Escherichia coli and produced an F3-ST with high affinity for the flavonol aglycones, quercetin, and its 7-methyl derivative, rhamnetin. In addition, the synthetic auxin 2,4-dichlorophenoxyacetic acid was shown to induce F3-ST enzyme activity and F3-ST mRNA transcript levels in cell cultures of F. bidentis. The F3-ST mRNA levels increased within the first 3 h, reaching a maximum after 24 h of treatment, and remained elevated for up to 48 h. Treatments with either quercetin 3-sulfate or quercetin 3,7,4'-trisulfate reduced F3-ST enzyme activity in cell cultures but had no effect on the transcript levels. These results are discussed in relation to the putative role of flavonoid conjugates in the regulation of auxin transport.

Molecular characterization of two plant flavonol sulfotransferases.

cDNA clones coding for flavonol 3- and 4'-sulfotransferases (STs) were isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA extracted from terminal buds of Flaveria chloraefolia. Sequence analysis revealed full-length cDNA clones with open reading frames of 933 and 960 base pairs, which encode polypeptides containing 311 and 320 amino acids, respectively. This corresponds to a molecular mass of 36,442 Da for the 3-ST and 37,212 Da for the 4'-ST. Expression of these clones in Escherichia coli led to the synthesis of beta-galactosidase-ST fusion proteins having the same substrate and position specificities as those for the 3- and 4'-flavonol ST enzymes isolated from the plant. Comparison of the deduced amino acid sequence of the two clones revealed an overall identity of 69% in 311 amino acid residues. The two flavonol STs of F. chloraefolia also shared significant sequence similarities with steroid and aryl STs found in animal tissues and with the senescence marker protein 2 isolated from rat liver, suggesting an evolutionary link between plant and animal STs.

Novel flavonol 3-sulfotransferase. Purification, kinetic properties, and partial amino acid sequence.

A flavonol sulfotransferase (EC 2.8.2.-), which catalyzes the transfer of the sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the 3-hydroxyl group of flavonol aglycones, has been purified to apparent homogeneity from Flaveria chloraefolia. The specific activity of flavonol 3-sulfotransferase was enriched 2000-fold, as compared with the homogenate, with a recovery of 9%. The molecular mass of the native and denatured enzyme was found to be 34.5 kDa, suggesting that the active from of the enzyme is a monomer. The enzyme exhibited expressed specificity for position 3 of flavonol aglycones, showed two activity optima at pH 6.0 and 8.5, did not require divalent cations, and was not inhibited by either EDTA or sulfhydryl group reagents. The results of substrate interaction kinetics and product inhibition are consistent with an Ordered Bi Bi mechanism where 3'-phosphoadenosine 5'-phosphosulfate is the first substrate to bind to the enzyme and 3'-phosphoadenosine 5'-phosphate is the final product to be released. The amino acid sequence of two peptides representing 17 and 33 amino acids showed no significant sequence similarity with the amino acid sequences reported for animal sulfotransferases. Antibodies raised against F. chloraefolia 3-sulfotransferase were found to cross-react with the 3'- and 4'-sulfotransferase activities of the same plant, suggesting that the three enzymes are structurally related.

Spatial Distribution of Flavonoid Conjugates in Relation to Glucosyltransferase and Sulfotransferase Activities in Flaveria bidentis.

The spatial distribution of sulfated and glucosylated flavonols as well as of the enzymes involved in the later steps of their biosynthesis, sulfotransferase and glucosyltransferase, were investigated in the shoots of Flaveria bidentis. The highest amounts of both types of flavonoid conjugates (as micromole per gram fresh weight) and the highest activities of their enzymes (as picokat per milligram) were detected in the terminal bud and the first pair of leaves. Sulfotransferase activity was also highest in the upper stem segments and in the basal section of the leaves. Western blot analysis of protein extracts showed that variations in sulfotransferase activity in different tissues correlate well with the amounts of immunodetected enzyme protein. These results were discussed in relation to the possible role of conjugated flavonoids in plant growth.

Partial Purification and Some Properties of Flavonol 7-Sulfotransferase from Flaveria bidentis.

A novel flavonol-specific sulfotransferase was partially purified from the shoot tips of Flaveria bidentis var. Angustifolia O.K. (Asteraceae) by chromatography on 3'-phosphoadenosine 5'-phosphate-agarose affinity column and chromatofocusing on Mono P. The latter step resulted in the separation of two isoforms, both of which exhibited expressed specificity for position 7 of quercetin 3,3'- and quercetin 3,4'-disulfate. The 7-sulfotransferase isoforms I and II had a pH optimum of 7.5 in phosphate buffer, apparent pl values of 6.5 and 6.3, and an M(r) of 35,000. They had no requirement for divalent cations and were not inhibited by EDTA or SH group reagents. Their K(m) values for both the sulfate donor and flavonol acceptor were of the same order of magnitude (0.20-0.46 micromolar). This enzyme, together with the recently reported flavonol 3-, 3'-, and 4'-sulfotransferases from F. chloraefolia (L Varin, RK Ibrahim [1989] Plant Physiol 90: 977-981) form the complement involved in the biosynthesis of polysulfated flavonols in this genus. A proposed sequential order for the enzymatic sulfation in both species is described.

Partial Purification and Characterization of Three Flavonol-Specific Sulfotransferases from Flaveria chloraefolia.

Three distinct flavonol-specific sulfotransferases were partially purified from the shoot tips of Flaveria chloraefolia A. Gray by fractional precipitation with ammonium sulfate, followed by gel filtration on Sephacryl S-200, 3'-phosphoadenosine 5-phosphate-Agarose affinity chromatography and chromatofocusing on Mono P. These enzymes exhibited expressed specificity for positions 3 of various flavonol acceptors and of 3' and 4' of flavonol 3-sulfate. The three sulfotransferases had similar molecular weights (35,000), exhibited no requirement for divalent cations and were not inhibited by SH group reagents. Their K(m) values for both the sulfate donor and the flavonol acceptors were of the same order of magnitude (ca. 0.2-0.4 micromolar). Except for the 3-sulfotransferase, which exhibited two optima at pH 6.5 and 8.5, the 3' and the 4'-sulfotransferases had a pH optimum of 7.5. The three enzymes could be resolved only by chromatofocusing and were eluted at pH 5.4, pH 6.0, and pH 5.1 for the 3-, 3'- and 4'-sulfotransferases, respectively. The substrate specificity of these three enzymes is discussed in relation to the biosynthesis of polysulfated flavonols in F. chloraefolia.

Enzymatic assay for flavonoid sulfotransferase.

A novel enzyme assay for flavonoid sulfotransferase is described. It makes use of tetrabutylammonium dihydrogen phosphate which forms a pair of ions with the flavonoid sulfate esters formed. This renders the sulfate ester soluble in organic solvents such as ethyl acetate, whereas the sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate, remains in the aqueous reaction mixture. The procedure is simple, rapid, and reproducible. It eliminates the need for chromatographic separation of the reaction products, except when their identification is required, and is suitable for use in the purification and kinetic studies of sulfotransferases.

Effect of pirenzepine and PGE2 on taurocholic acid-induced gastric lesions.

Cytoprotective activity of Pirenzepine (PZ) and Prostaglandin E2 (PGE2) was investigated in the gastric damage induced by taurocholic acid (TA) in the rat. Gastric mucosal potential difference (PD) and gross mucosal erosions were measured. Intravenous PZ (18 mg/kg) and PGE2 (5 micrograms/kg) prevented both the sharp decrease of PD and the gastric lesions caused by intragastric TA (40 mM). When TA administration preceeded drug treatment, both compounds reversed the PD fall but only PZ was able to restore gross mucosal integrity. It is thought that TA causes gastric damage by producing back-diffusion of H+ ions across the mucosal layer. It is speculated that antimuscarinic, such as PZ, might reduce gastric damage either by preventing, at the submucosal level, the spreading of cytolesive process triggered by H+ retro-diffusion and/or by inhibition of acid secretion in the depth of the gastric glands.

Protection of gastric mucosa in rats. Differences between vagotomy, atropine, and PGE2.

We have studied the protective effects of truncal vagotomy, atropine, and PGE2 against gastric mucosal injury produced by necrotizing agents (0.2 N NaOH, 0.6 N HCl, absolute ethanol), acetylsalicylic acid (ASA) and HCl, or serotonin (5HT). Vagotomy, atropine, and PGE2 prevent the effects of different noxious agents. Vagotomy is protective only against 5HT and against ASA + 0.15 N or 0.35 N HCl, whereas atropine and PGE2 are also protective against the necrotizing agents. The effectiveness of vagotomy against ASA + 0.35 N HCl does not depend on the inhibition of acid secretion and supports the hypothesis that removal of the vagal drive counteracts the effect of H+ back-diffusion.

Indomethacin-induced intestinal ulcers in rats: effects of salicylazosulfapyridine and dexamethasone.

Characteristics of inflammatory bowel diseases have been hypothesized to resemble those of the syndrome of intestinal ulceration induced in the rat by non-steroidal anti-inflammatory compounds. However, no systematic studies have been undertaken to examine this possibility. Therefore, we have studied the influence of some pharmacological agents, such as steroids and salicylazosulfapyridine (SAS), which are clinically useful in the treatment of inflammatory bowel diseases, and to review published data on other pharmacological approaches commonly used for the therapy of inflammatory bowel diseases that have been shown to counteract indomethacin-induced intestinal toxicity. Orally administered SAS 100 to 800 mg/kg or dexamethasone 0.05 to 0.1 mg/kg exerted dose-related, anti-ulcer activity, with ED50 values and 95% confidence limits of 145 (95-222) mg/kg SAS and 0.184 (0.152-0.224) mg/kg dexamethasone. Other treatments, including cholestyramine, low-residue diets and antibiotics have also been reported to ameliorate clinical and experimental intestinal diseases. The clinical significance of present findings has been discussed.