- Adjunctive S-adenosylmethionine (SAMe) in treating non-remittent major depressive disorder: An 8-week double-blind, randomized, controlled trial. [Journal Article]
- ENEur Neuropsychopharmacol 2018 Aug 14
- There has been increasing interest in nutraceutical augmentation strategies to boost the efficacy of antidepressants. This study assessed whether S-adenosylmethionine (SAMe), a methyl donor that occu...
There has been increasing interest in nutraceutical augmentation strategies to boost the efficacy of antidepressants. This study assessed whether S-adenosylmethionine (SAMe), a methyl donor that occurs naturally in the body, may be of such benefit. We conducted an 8-week, double-blind RCT in which 107 treatment non-remittent outpatients with DSM-5 diagnosed Major Depressive Disorder (MDD) were randomized to either SAMe or placebo adjunctively to antidepressants. One-carbon cycle nutrients, pertinent single nucleotide polymorphisms (SNPs), and BDNF were also analysed as potential moderators of response. A linear mixed-effects model revealed a significant overall reduction in Montgomery-Asberg Depression Rating Scale (MADRS) score across time, however there was no significant between-group difference observed (p = 0.51). Response rates at Week 8 were 54.3% in the SAMe group and 50.0% in the placebo group, with remission rates 43.5% for SAMe and 38.3% for placebo (all results NS). No effect of SAMe was found on any secondary outcome. Differential response to SAMe was not modified by a range of key genotypes (e.g. COMT), nor reflected in a change of homocysteine, red cell folate, or BDNF. Use of SAMe elicited no significant adverse effects beyond placebo, however it was implicated in one case of serotonin syndrome-like symptoms. This study concludes that 800 mg/day of SAMe is not an effective adjunctive treatment in MDD, and no obvious biomarker reflected any differential response to treatment. Due to such a distinctly high placebo-response (despite rigorous screening), future studies should employ a placebo run-in period and other strategies to minimize placebo response.
- Investigating the Mechanism of Action of Diketopiperazines Inhibitors of the Burkholderia cenocepacia Quorum Sensing Synthase CepI: A Site-Directed Mutagenesis Study. [Journal Article]
- FPFront Pharmacol 2018; 9:836
- Quorum sensing (QS) is a bacterial intercellular communication process which controls the production of major virulence factors, such as proteases, siderophores, and toxins, as well as biofilm format...
Quorum sensing (QS) is a bacterial intercellular communication process which controls the production of major virulence factors, such as proteases, siderophores, and toxins, as well as biofilm formation. Since the inhibition of this pathway reduces bacterial virulence, QS is considered a valuable candidate drug target, particularly for the treatment of opportunistic infections, such as those caused by Burkholderia cenocepacia in cystic fibrosis patients. Diketopiperazine inhibitors of the acyl homoserine lactone synthase CepI have been recently described. These compounds are able to impair the ability of B. cenocepacia to produce proteases, siderophores, and to form biofilm, being also active in a Caenorhabditis elegans infection model. However, the precise mechanism of action of the compounds, as well as their effect on the cell metabolism, fundamental for candidate drug optimization, are still not completely defined. Here, we performed a proteomic analysis of B. cenocepacia cells treated with one of these inhibitors, and compared it with a cepI deleted strain. Our results demonstrate that the effects of the compound are similar to the deletion of cepI, clearly confirming that these molecules function as inhibitors of the acyl homoserine lactone synthase. Moreover, to deepen our knowledge about the binding mechanisms of the compound to CepI, we exploited previously published in silico structural insights about this enzyme structure and validated different candidate binding pockets on the enzyme surface using site-directed mutagenesis and biochemical analyses. Our experiments identified a region near the predicted S-adenosylmethionine binding site critically involved in interactions with the inhibitor. These results could be useful for future structure-based optimization of these CepI inhibitors.
- Folate deficiency prevents neural crest fate by disturbing the epigenetic Sox2 repression on the dorsal neural tube. [Journal Article]
- DBDev Biol 2018 Aug 09
- Folate deficiency has been known to contribute to neural tube and neural crest defects, but why these tissues are particularly affected, and which are the molecular mechanisms involved in those abnor...
Folate deficiency has been known to contribute to neural tube and neural crest defects, but why these tissues are particularly affected, and which are the molecular mechanisms involved in those abnormalities are important human health questions that remain unanswered. Here we study the function of two of the main folate transporters, FolR1 and Rfc1, which are robustly expressed in these tissues. Folate is the precursor of S-adenosylmethionine, which is the main donor for DNA, protein and RNA methylation. Our results show that knockdown of FolR1 and/or Rfc1 reduced the abundance of histone H3 lysine and DNA methylation, two epigenetic modifications that play an important role during neural and neural crest development. Additionally, by knocking down folate transporter or pharmacologically inhibiting folate transport and metabolism, we observed ectopic Sox2 expression at the expense of neural crest markers in the dorsal neural tube. This is correlated with neural crest associated defects, with particular impact on orofacial formation. By using bisulfite sequencing, we show that this phenotype is consequence of reduced DNA methylation on the Sox2 locus at the dorsal neural tube, which can be rescued by the addition of folinic acid. Taken together, our in vivo results reveal the importance of folate as a source of the methyl groups necessary for the establishment of the correct epigenetic marks during neural and neural crest fate-restriction.
- Guidelines for Determining the Structures of Radical SAM Enzyme-Catalyzed Modifications in the Biosynthesis of RiPP Natural Products. [Journal Article]
- MEMethods Enzymol 2018; 606:439-460
- Radical S-adenosylmethionine (RaS) enzymes catalyze some of the most fascinating transformations in Nature. With only ~100 of the >300,000 members studied to date, it is safe to assume that a plethor...
Radical S-adenosylmethionine (RaS) enzymes catalyze some of the most fascinating transformations in Nature. With only ~100 of the >300,000 members studied to date, it is safe to assume that a plethora of new reactions and reaction mechanisms remain to be elucidated. It is by now relatively easy to spot RaS enzymes in microbial genomes. However, to determine the reactions that they carry out, detailed structural characterization of the product(s) is necessary, a process that still represents a significant roadblock in the study of RaS enzymes. We have recently combined natural products structural elucidation along with RaS enzymology to provide a proof of concept for how the confluence of these approaches can lead to the discovery of new natural products and RaS enzyme-mediated transformations. Herein, we provide guidelines for expressing, purifying, and reconstituting a subclass of RaS enzymes that contain a so-called SPASM domain, as well as characterizing the reactions that they catalyze using a combination of HR/MSn and NMR investigations. Application of these approaches will aid in expanding the chemical and biosynthetic repertoire of RaS enzymes in the future.
- Methods for Studying the Radical SAM Enzymes in Diphthamide Biosynthesis. [Journal Article]
- MEMethods Enzymol 2018; 606:421-438
- Diphthamide is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. Biosynthesis of diphthamide was proposed to involve four steps. The first st...
Diphthamide is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. Biosynthesis of diphthamide was proposed to involve four steps. The first step is a CC bond forming reaction catalyzed by unique radical S-adenosylmethionine (SAM) enzymes. Classical radical SAM enzymes use SAM and [4Fe-4S] clusters to generate a 5'-deoxyadenynal radical and catalyze numerous reactions. Radical SAM enzymes in diphthamide biosynthesis cleave a different CS bond in SAM to generate a 3-amino-3-carboxypropyl radical and modify a histidine residue of substrate protein EF2. Here, we describe our investigations on these unique radical SAM enzymes, including the preparation, characterization, and activity assays we have developed.
- Purification, Characterization, and Biochemical Assays of Biotin Synthase From Escherichia coli. [Journal Article]
- MEMethods Enzymol 2018; 606:363-388
- Biotin synthase (BioB) catalyzes the oxidative insertion of a sulfur atom between the C6 methylene and the C9 methyl positions in dethiobiotin. The enzyme couples oxidation of each carbon position to...
Biotin synthase (BioB) catalyzes the oxidative insertion of a sulfur atom between the C6 methylene and the C9 methyl positions in dethiobiotin. The enzyme couples oxidation of each carbon position to reduction of the S-adenosyl-l-methionine (SAM) sulfonium center, generating 5'-deoxyadenosine and l-methionine, products that are characteristic of enzymes from the radical SAM superfamily. In bacteria, biotin biosynthesis is tightly regulated by the dual-function BirA repressor/holocarboxylase synthetase, resulting in very low levels of all biotin biosynthetic enzymes such that activity-based purification of BioB from the native organism is virtually impossible. However, overexpression and purification of recombinant BioB from E. coli are straight forward and, in contrast with many radical SAM enzymes, can be carried out under aerobic conditions. The active enzyme contains two iron-sulfur clusters, and the characterization and manipulation of these clusters are essential for a thorough understanding of enzyme catalysis and stability. An optimized in vitro assay for BioB is described herein that requires use of an auxiliary protein reducing system and must be carried out under anaerobic conditions to prevent oxidative damage to the reduced iron-sulfur clusters. Three methods for detection of biotin are described, with discussion of the advantages and limitations of each method. Challenges that may be encountered in adapting these assays to other organisms are also discussed.
- Mechanistic Studies of Radical SAM Enzymes: Pyruvate Formate-Lyase Activating Enzyme and Lysine 2,3-Aminomutase Case Studies. [Journal Article]
- MEMethods Enzymol 2018; 606:269-318
- The radical SAM enzyme superfamily is large and diverse, with ever-increasing numbers of examples of characterized reactions. This chapter focuses on the methodology we have developed over the last 2...
The radical SAM enzyme superfamily is large and diverse, with ever-increasing numbers of examples of characterized reactions. This chapter focuses on the methodology we have developed over the last 25 years for working with these enzymes, with the specific examples discussed being the pyruvate formate-lyase activating enzyme (PFL-AE) and lysine 2,3-aminomutase (LAM). Both enzymes are purified from overexpressing Escherichia coli, but differ in that PFL-AE is expressed without an affinity tag and does not require iron-sulfur cluster reconstitution, while LAM purification is carried out through use of a His6 affinity tag and the enzyme benefits from cluster reconstitution. Because of radical SAM enzymes' catalytic need for a [4Fe-4S] cluster, we present methods for characterization and incorporation of a full [4Fe-4S] cluster in addition to enzyme activity assay protocols. Synthesis of SAM (S-adenosyl-l-methionine) and its analogs have played an important role in our mechanistic studies of radical SAM enzymes, and their synthetic methods are also presented in detail.
- Using Peptide Mimics to Study the Biosynthesis of the Side-Ring System of Nosiheptide. [Journal Article]
- MEMethods Enzymol 2018; 606:241-268
- Thiopeptide natural products have gained interest recently for their diverse pharmacological properties, including antibacterial, antifungal, anticancer, and antimalarial activities. Due to their inh...
Thiopeptide natural products have gained interest recently for their diverse pharmacological properties, including antibacterial, antifungal, anticancer, and antimalarial activities. Due to their inherent poor solubility and uptake, there is interest in developing new thiopeptides that mimic these unique structures, but which exhibit better pharmacokinetic properties. One strategy is to exploit the biosynthetic pathways using a chemoenzymatic approach to make analogs. However, a complete understanding of thiopeptide biosynthesis is not available, especially for those molecules that contain a large number of modifications to the thiopeptide core. This gap in knowledge and the lack of a facile method for generating a variety of thiopeptide intermediates makes studying particular enzymatic steps difficult. We developed a method to produce thiopeptide mimics based on established synthetic procedures to study the reaction catalyzed by NosN, the class C radical S-adenosylmethionine methylase involved in carbon transfer to C4 of 3-methylindolic acid and completion of the side-ring system in nosiheptide. Herein, we detail strategies for overproducing and isolating NosN, as well as procedures for synthesizing substrate mimics to study the formation of the side-ring system of nosiheptide.
- Biochemical Approaches for Understanding Iron-Sulfur Cluster Regeneration in Escherichia coli Lipoyl Synthase During Catalysis. [Journal Article]
- MEMethods Enzymol 2018; 606:217-239
- Lipoyl synthase (LipA in bacteria) is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the second step of the de novo biosynthesis of the lipoyl cofactor: the insertion of sulfur at C6 and ...
Lipoyl synthase (LipA in bacteria) is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the second step of the de novo biosynthesis of the lipoyl cofactor: the insertion of sulfur at C6 and C8 of a pendant octanoyl chain. In addition to the [4Fe4S] cluster that is characteristic of the radical SAM (RS) enzymes, LipA contains a second [4Fe4S] cluster that, though controversial, has been proposed to be degraded during turnover to supply the inserted sulfur atoms. A consequence of this proposed role is that the destruction of its iron-sulfur cluster renders the enzyme in an inactive state. Recently, it was shown that Escherichia coli proteins NfuA or IscU can confer catalytic properties to E. coli LipA in vitro. In this chapter, we present methods for characterizing LipA and analyzing its activity in vitro, and provide strategies to monitor the pathway for the regeneration of LipA's auxiliary cluster by E. coli iron-sulfur carrier protein NfuA.
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- The high degree of cystathionine β-synthase (CBS) activation by S-adenosylmethionine (SAM) may explain naked mole-rat's distinct methionine metabolite profile compared to mouse. [Letter]
- GGeroscience 2018 Aug 07