Glycine Cleavage System Research Articles

Introduction of glycine synthase enables uptake of exogenous formate and strongly impacts the metabolism in Clostridium pasteurianum.

Autotrophic or mixotrophic use of one-carbon (C1) compounds is gaining importance for sustainable bioproduction. In an effort to integrate the reductive glycine pathway (rGP) as a highly promising pathway for the assimilation of CO2 and formate, genes coding for glycine synthase system from Gottschalkia acidurici were successfully introduced into Clostridium pasteurianum, a non-model host microorganism with industrial interests. The mutant harboring glycine synthase exhibited assimilation of exogenous formate and reduced CO2 formation. Further metabolic data clearly showed large impacts of expression of glycine synthase on the product metabolism of C. pasteurianum. In particular, 2-oxobutyrate (2-OB) was observed for the first time as a metabolic intermediate of C. pasteurianum and its secretion was solely triggered by the expression of glycine synthase. The perturbation of C1 metabolism is discussed regarding its interactions with pathways of the central metabolism, acidogenesis, solventogenesis, and amino acid metabolism. The secretion of 2-OB is considered as a consequence of metabolic and redox instabilities due to the activity of glycine synthase and may represent a common metabolic response of Clostridia in enhanced use of C1 compounds.

Structure-based dynamic analysis of the glycine cleavage system suggests key residues for control of a key reaction step

Molecular shuttles play decisive roles in many multi-enzyme systems such as the glycine cleavage system (GCS) for one-carbon (C1) metabolism. In GCS, a lipoate swinging arm containing an aminomethyl moiety is attached to protein H and serves as a molecular shuttle among different proteins. Protection of the aminomethyl moiety in a cavity of protein H and its release induced by protein T are key processes but barely understood. Here, we present a detailed structure-based dynamic analysis of the induced release of the lipoate arm of protein H. Based on molecular dynamics simulations of interactions between proteins H and T, four major steps of the release process showing significantly different energy barriers and time scales can be distinguished. Mutations of a key residue, Ser-67 in protein H, led to a bidirectional tuning of the release process. This work opens ways to target C1 metabolism in biomedicine and the utilization of formate and CO2 for biosynthesis.

Tracing Metabolic Fate of Mitochondrial Glycine Cleavage System Derived Formate In Vitro and In Vivo.

Folate-mediated one-carbon (1C) metabolism is a major target of many therapies in human diseases. Studies have focused on the metabolism of serine 3-carbon as it serves as a major source for 1C units. The serine 3-carbon enters the mitochondria transferred by folate cofactors and eventually converted to formate and serves as a major building block for cytosolic 1C metabolism. Abnormal glycine metabolism has been reported in many human pathological conditions. The mitochondrial glycine cleavage system (GCS) catalyzes glycine degradation to CO2 and ammonium, while tetrahydrofolate (THF) is converted into 5,10-methylene-THF. GCS accounts for a substantial proportion of whole-body glycine flux in humans, yet the particular metabolic route of glycine 2-carbon recycled from GCS during mitochondria glycine decarboxylation in hepatic or bone marrow 1C metabolism is not fully investigated, due to the limited accessibility of human tissues. Labeled glycine at 2-carbon was given to humans and primary cells in previous studies for investigating its incorporations into purines, its interconversion with serine, or the CO2 production in the mitochondria. Less is known on the metabolic fate of the glycine 2-carbon recycled from the GCS; hence, a model system tracing its metabolic fate would help in this regard. We took the direct approach of isotopic labeling to further explore the in vitro and in vivo metabolic fate of the 2-carbon from [2-13C]glycine and [2-13C]serine. As the 2-carbon of glycine and serine is decarboxylated and catabolized via the GCS, the original 13C-labeled 2-carbon is transferred to THF and yield methyleneTHF in the mitochondria. In human hepatoma cell-lines, 2-carbon from glycine was found to be incorporated into deoxythymidine (dTMP, dT + 1), M + 3 species of purines (deoxyadenine, dA and deoxyguanine, dG), and methionine (Met + 1). In healthy mice, incorporation of GCS-derived formate from glycine 2-carbon was found in serine (Ser + 2 via cytosolic serine hydroxy methyl transferase), methionine, dTMP, and methylcytosine (mC + 1) in bone marrow DNA. In these experiments, labeled glycine 2-carbon directly incorporates into Ser + 1, A + 2, and G + 2 (at C2 and C8 of purine) in the cytosol. It is noteworthy that since the serine 3-carbon is unlabeled in these experiments, the isotopic enrichments in dT + 1, Ser + 2, dA + 3, dG + 3, and Met + 1 solely come from the 2-carbon of glycine/serine recycled from GCS, re-enters the cytosolic 1C metabolism as formate, and then being used for cytosolic syntheses of serine, dTMP, purine (M + 3) and methionine. Taken together, we established model systems and successfully traced the metabolic fate of mitochondrial GCS-derived formate from glycine 2-carbon in vitro and in vivo. Nutritional supply significantly alters formate generation from GCS. More GCS-derived formate was used in hepatic serine and methionine syntheses, whereas more GCS-derived formate was used in dTMP synthesis in the bone marrow, indicating that the utilization and partitioning of GCS-derived 1C unit are tissue-specific. These approaches enable better understanding concerning the utilization of 1C moiety generated from mitochondrial GCS that can help to further elucidate the role of GCS in human disease development and progression in future applications. More studies on GCS using these approaches are underway.

Articles of Significant Interest in This Issue

Lipoic acid is a prosthetic group necessary for the activities of the glycine cleavage system and enzymes such as pyruvate dehydrogenase. Although there are archaea that possess a classical lipoyl synthase (LipA) gene homolog, many archaeal species, including the hyperthermophilic archaeon Thermococcus kodakarensis, do not. Jin et al. (e01359-20) identified two genes whose products in combination displayed lipoyl synthase activity. Disrupting either of these genes resulted in strains that display lipoic acid auxotrophy. Homologs of the two genes are widely distributed among archaea, suggesting that, in addition to LipA, the two proteins represent a new group of lipoyl synthases.

Obesity increases hepatic glycine dehydrogenase and aminomethyltransferase expression while dietary glycine supplementation reduces white adipose tissue in Zucker diabetic fatty rats.

Obesity is associated with altered glycine metabolism in humans. This study investigated the mechanisms regulating glycine metabolism in obese rats. Eight-week-old Zucker diabetic fatty rats (ZDF; a type-II diabetic animal model) received either 1% glycine or 1.19% L-alanine (isonitrogenous control) in drinking water for 6weeks. An additional group of lean Zucker rats also received 1.19% L-alanine as a lean control. Glycine concentrations in serum and liver were markedly lower in obese versus lean rats. Enteral glycine supplementation restored both serum and hepatic glycine levels, while reducing mesenteric and internal white fat mass compared with alanine-treated ZDF rats. Blood glucose and non-esterified fatty acid (NEFA) concentrations did not differ between the control and glycine-supplemented ZDF rats (P > 0.10). Both mRNA and protein expression of aminomethyltransferase (AMT) and glycine dehydrogenase, decarboxylating (GLDC) were increased in the livers of obese versus lean rats (P < 0.05). In contrast, glycine cleavage system H (GCSH) hepatic mRNA expression was downregulated in obese versus lean rats, although there was no change in protein expression. These findings indicate that reduced quantities of glycine observed in obese subjects likely results from an upregulation of the hepatic glycine cleavage system and that dietary glycine supplementation potentially reduces obesity in ZDF rats.

Regulation of glycine metabolism by the glycine cleavage system and conjugation pathway in mouse models of non-ketotic hyperglycinemia.

Glycine abundance is modulated in a tissue‐specific manner by use in biosynthetic reactions, catabolism by the glycine cleavage system (GCS), and excretion via glycine conjugation. Dysregulation of glycine metabolism is associated with multiple disorders including epilepsy, developmental delay, and birth defects. Mutation of the GCS component glycine decarboxylase (GLDC) in non‐ketotic hyperglycinemia (NKH) causes accumulation of glycine in body fluids, but there is a gap in our knowledge regarding the effects on glycine metabolism in tissues. Here, we analysed mice carrying mutations in Gldc that result in severe or mild elevations of plasma glycine and model NKH. Liver of Gldc‐deficient mice accumulated glycine and numerous glycine derivatives, including multiple acylglycines, indicating increased flux through reactions mediated by enzymes including glycine‐N‐acyltransferase and arginine: glycine amidinotransferase. Levels of dysregulated metabolites increased with age and were normalised by liver‐specific rescue of Gldc expression. Brain tissue exhibited increased abundance of glycine, as well as derivatives including guanidinoacetate, which may itself be epileptogenic. Elevation of brain tissue glycine occurred even in the presence of only mildly elevated plasma glycine in mice carrying a missense allele of Gldc. Treatment with benzoate enhanced hepatic glycine conjugation thereby lowering plasma and tissue glycine. Moreover, administration of a glycine conjugation pathway intermediate, cinnamate, similarly achieved normalisation of liver glycine derivatives and circulating glycine. Although exogenous benzoate and cinnamate impact glycine levels via activity of glycine‐N‐acyltransferase, that is not expressed in brain, they are sufficient to lower levels of glycine and derivatives in brain tissue of treated Gldc‐deficient mice.

Assembly of the [4Fe-4S] cluster of NFU1 requires the coordinated donation of two [2Fe-2S] clusters from the scaffold proteins, ISCU2 and ISCA1.

NFU1, a late-acting iron-sulfur (Fe-S) cluster carrier protein, has a key role in the pathogenesis of the disease, multiple mitochondrial dysfunctions syndrome. In this work, using genetic and biochemical approaches, we identified the initial scaffold protein, mitochondrial ISCU (ISCU2) and the secondary carrier, ISCA1, as the direct donors of Fe-S clusters to mitochondrial NFU1, which appears to dimerize and reductively mediate the formation of a bridging [4Fe-4S] cluster, aided by ferredoxin 2. By monitoring the abundance of target proteins that acquire their Fe-S clusters from NFU1, we characterized the effects of several novel pathogenic NFU1 mutations. We observed that NFU1 directly interacts with each of the Fe-S cluster scaffold proteins known to ligate [2Fe-2S] clusters, ISCU2 and ISCA1, and we mapped the site of interaction to a conserved hydrophobic patch of residues situated at the end of the C-terminal alpha-helix of NFU1. Furthermore, we showed that NFU1 lost its ability to acquire its Fe-S cluster when mutagenized at the identified site of interaction with ISCU2 and ISCA1, which thereby adversely affected biochemical functions of proteins that are thought to acquire their Fe-S clusters directly from NFU1, such as lipoic acid synthase, which supports the Fe-S-dependent process of lipoylation of components of multiple key enzyme complexes, including pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and the glycine cleavage complex.

P0339URINE METABOLOMIC ASSAY IN EARLY IGA NEPHROPATHY PATIENTS REVEALS URINE GLYCINE AS A PROGNOSTIC BIOMARKER

Abstract Background and Aims A urine metabolomic biomarker from early immunoglobulin A nephropathy (IgAN) can be a useful tool for early detection or prognostic prediction of the disease. Method We profiled the urine metabolomes, using nuclear magnetic resonance (NMR) spectrometry, from 197 IgAN patients with eGFR ≥ 60 mL/min/1.73 m2, and the patients with same kidney function range but have diagnosis of membranous nephropathy (n=81) and minimal change disease (n=50) were included as the disease-control group. Additional 146 healthy controls who received routine health screenings and donated urine samples were included. We calculated the creatinine-adjusted urine concentrations of 27 urine metabolites. Urine metabolites specifically increased in urines from IgAN patients were identified, and their associations with the prognosis of the disease, determined with the outcome of eGFR 30% reduction from baseline, were investigated. Additional kidney biopsy samples from independent patients and controls were stained for molecules included in related biological process for experimental validation. Results Among the 15 metabolites higher in the urines from IgAN patients than those from the healthy controls, only glycine was the metabolite that the median level was significantly higher also than that of the disease-control group. We found that the higher levels of glycine, alanine, citrate, threonine, and valine were significantly associated with higher risks of 30% eGFR reduction in IgAN patients. Among the molecules related to metabolism of glycine, expressions of T and H proteins from glycine cleavage system were significantly reduced in the kidney biopsy slides from independent IgAN patients when compared to the controls. Conclusion Urine glycine may be a prognostic and pathophysiology-related biomarker of IgAN. A further study confirming the clinical usefulness and pathophysiologic significance of urine metabolites in IgAN patients is warranted.

Formaldehyde formation in the glycine cleavage system and its use for an aldolase-based biosynthesis of 1,3-propanediol

Glycine cleavage system (GCS) occupies a key position in one-carbon (C1) metabolic pathway and receives great attention for the use of C1 carbons like formate and CO2 via synthetic biology. In this work, we demonstrate that formaldehyde exists as a substantial byproduct of the GCS reaction cycle. Three causes are identified for its formation. First, the principal one is the decomposition of N5,N10-methylene-tetrahydrofolate (5,10-CH2-THF) to form formaldehyde and THF. Increasing the rate of glycine cleavage promotes the formation of 5,10-CH2-THF, thereby increasing the formaldehyde release rate. Next, formaldehyde can be produced in the GCS even in the absence of THF. The reason is that T-protein of the GCS can degrade methylamine-loaded H-protein (Hint) to formaldehyde and ammonia, accompanied with the formation of dihydrolipoyl H-protein (Hred), but the reaction rate is less than 0.16% of that in the presence of THF. Increasing T-protein concentration can speed up the release rate of formaldehyde by Hint. Finally, a certain amount of formaldehyde can be formed in the GCS due to oxidative degradation of THF. Based on a formaldehyde-dependent aldolase, we elaborated a glycine-based one carbon metabolic pathway for the biosynthesis of 1,3-propanediol (1,3-PDO) in vitro. This work provides quantitative data and mechanistic understanding of formaldehyde formation in the GCS and a new biosynthetic pathway of 1,3-PDO.

Plasma Formate Is Greater in Fetal and Neonatal Rats Compared with Their Mothers

Formate can be incorporated into 10-formyl-tetrahydrofolate (10-formyl-THF), which is a substrate for purine synthesis, and after further reduction of the one-carbon group, may be used as a substrate for thymidylate synthesis and for homocysteine remethylation. We examined plasma formate concentrations and the expression of genes involved in the production and utilization of formate in fetal and neonatal rats and in pregnant and virgin female rats. In 1 experiment, plasma formate was measured by GC-MS in rats aged 1-56 d. In a second experiment, virgin female (adult) rats, 19-d pregnant rats (P) and their male and female fetuses (F), and 3-d-old (N) and 7-d-old (J) offspring had plasma and amniotic fluid analyzed for formate by GC-MS, mRNA abundance in liver and placenta by qPCR, and several plasma amino acids by HPLC. The plasma formate concentration was significantly higher in fetuses at embryonic day 19 than in the mothers. It was also significantly higher in neonatal rats but slowly returned to adult concentrations by ∼3 wk. The abundance of mitochondrial monofunctional 10-formyl-tetrahydrofolate synthetase (Mthfd1l) mRNA was significantly higher in placenta (PP) and F liver than in liver of N or J. Expression of mitochondrial bifunctional NAD-dependent methylene-tetrahydrofolate dehydrogenase/methenyl-tetrahydrofolate cyclohydrolase (Mthfd2) was significantly enriched in PP and liver of P, intermediate in F liver, and much lower in liver of N and J, relative to PP. Serine hydroxymethyltransferase 2 (Shmt2), methylenetetrahydrofolate dehydrogenase 1 (Mthfd1), and glycine decarboxylase protein of the glycine cleavage system (Gldc) mRNA expression was significantly lower in PP compared with other groups. Cytoplasmic NAD(P)-dependent 10-formyl-tetrahydrofolate dehydrogenase (Aldh1/1) and mitochondrial NAD(P)-dependent 10-formyl-tetrahydrofolate dehydrogenase (Aldh1/2) , genes responsible for the catabolism of 10-formylTHF, were very weakly expressed in PP, low in livers of F and N, and reached the significantly higher adult levels in J. Serine, glycine, and methionine concentrations in plasma of F were significantly higher than in plasma of P. Formate metabolism is highly active in fetuses and in placenta of pregnant rats.

Dual-Localized Enzymatic Components Constitute the Fatty Acid Synthase Systems in Mitochondria and Plastids.

Plant fatty acid biosynthesis occurs in both plastids and mitochondria. Here, we report the identification and characterization of Arabidopsis (Arabidopsis thaliana) genes encoding three enzymes shared between the mitochondria- and plastid-localized type II fatty acid synthase systems (mtFAS and ptFAS, respectively). Two of these enzymes, β-ketoacyl-acyl carrier protein (ACP) reductase and enoyl-ACP reductase, catalyze two of the reactions that constitute the core four-reaction cycle of the FAS system, which iteratively elongates the acyl chain by two carbon atoms per cycle. The third enzyme, malonyl-coenzyme A:ACP transacylase, catalyzes the reaction that loads the mtFAS system with substrate by malonylating the phosphopantetheinyl cofactor of ACP. GFP fusion experiments revealed that the these enzymes localize to both chloroplasts and mitochondria. This localization was validated by characterization of mutant alleles, which were rescued by transgenes expressing enzyme variants that were retargeted only to plastids or only to mitochondria. The singular retargeting of these proteins to plastids rescued the embryo lethality associated with disruption of the essential ptFAS system, but these rescued plants displayed phenotypes typical of the lack of mtFAS function, including reduced lipoylation of the H subunit of the glycine decarboxylase complex, hyperaccumulation of glycine, and reduced growth. However, these latter traits were reversible in an elevated-CO2 atmosphere, which suppresses mtFAS-associated photorespiration-dependent chemotypes. Sharing enzymatic components between mtFAS and ptFAS systems constrains the evolution of these nonredundant fatty acid biosynthetic machineries.

Hormonal Regulation of Glycine Decarboxylase and its Impact on Cellular Physiology

The amino acid glycine is involved in generation of multiple critical metabolites including glutathione, heme, and creatinine. Interestingly, in both humans and rodents, circulating glycine levels are significantly reduced in obesity, glucose intolerance, type II diabetes and non‐alcoholic fatty liver disease. The glycine cleavage system (GCS) is the predominant glycine degradation pathway in humans. The rate‐limiting enzyme of GCS is glycine decarboxylase (GLDC), and loss‐of‐function mutations of GLDC cause hyperglycinemia. Here, we show that GLDC gene expression is upregulated in livers of mouse models of diabetes and diet‐induced obesity as well as in the fasted state in normal animals. In exploring the hormonal signals that mediate these regulatory events we found that both glucagon and insulin stimulated GLDC gene expression. In primary rat hepatocytes, GLDC expression was strongly stimulated by glucagon and cAMP, and mildly with insulin while in a rat hepatoma cell line, insulin strongly stimulated GLDC expression as compared to cAMP. We identified the cAMP‐response element binding protein 1 (Creb1) and insulin responsive transcription factor Sterol regulatory element binding protein 1c (Srebp‐1c) as mediators of the glucagon and insulin stimulatory effect, respectively. By knocking down or overexpressing GLDC in cell lines we observed GLDC expression levels are strongly linked to intracellular glutathione and reactive oxygen species (ROS) levels. Our findings suggest that the hormonal regulation of GLDC may contribute to compensatory changes in glutathione production as a defense against metabolic disease‐associated oxidative stress.Support or Funding InformationThis work is supported by NIH R21AG050741, Grants Boost OVPR Wayne State University and the Michigan Diabetes Research and Training Center.

A Dictyostelium discoideum mitochondrial fluorescent tagging vector that does not affect respiratory function

Visualizing mitochondria in living Dictyostelium discoideum cells using fluorescent dyes is often problematic due to variability in staining, metabolism of the dyes, and unknown potential effects of the dyes on mitochondrial function. We show that fluorescent labelling of mitochondria, using an N-terminal mitochondrial localization sequence derived from the D. discoideum protein GcvH1 (glycine cleavage system H1) attached to a red fluorescent protein enables clear mitochondrial imaging. We also show that this labelling has no effect upon mitochondria load or respiratory function.

Reduction of Phakopsora pachyrhizi infection on soybean through host- and spray-induced gene silencing.

Asian soybean rust (ASR), caused by the obligate fungal pathogen Phakopsora pachyrhizi, often leads to significant yield losses and can only be managed through fungicide applications currently. In the present study, eight urediniospore germination or appressorium formation induced P. pachyrhizi genes were investigated for their feasibility to suppress ASR through a bean pod mottle virus (BPMV)‐based host‐induced gene silencing (HIGS) strategy. Soybean plants expressing three of these modified BPMV vectors suppressed the expression of their corresponding target gene by 45%–80%, fungal biomass accumulation by 58%–80%, and significantly reduced ASR symptom development in soybean leaves after the plants were inoculated with P. pachyrhizi, demonstrating that HIGS can be used to manage ASR. In addition, when the in vitro synthesized double‐stranded RNAs (dsRNAs) for three of the genes encoding an acetyl‐CoA acyltransferase, a 40S ribosomal protein S16, and glycine cleavage system H protein were sprayed directly onto detached soybean leaves prior to P. pachyrhizi inoculation, they also resulted in an average of over 73% reduction of pustule numbers and 75% reduction in P. pachyrhizi biomass accumulation on the detached leaves compared to the controls. To the best of our knowledge, this is the first report of suppressing P. pachyrhizi infection in soybean through both HIGS and spray‐induced gene silencing. It was demonstrated that either HIGS constructs targeting P. pachyrhizi genes or direct dsRNA spray application could be an effective strategy for reducing ASR development on soybean.

Functional cooperation of the glycine synthase-reductase and Wood-Ljungdahl pathways for autotrophic growth of Clostridium drakei.

Among CO2-fixing metabolic pathways in nature, the linear Wood-Ljungdahl pathway (WLP) in phylogenetically diverse acetate-forming acetogens comprises the most energetically efficient pathway, requires the least number of reactions, and converts CO2 to formate and then into acetyl-CoA. Despite two genes encoding glycine synthase being well-conserved in WLP gene clusters, the functional role of glycine synthase under autotrophic growth conditions has remained uncertain. Here, using the reconstructed genome-scale metabolic model iSL771 based on the completed genome sequence, transcriptomics, 13C isotope-based metabolite-tracing experiments, biochemical assays, and heterologous expression of the pathway in another acetogen, we discovered that the WLP and the glycine synthase pathway are functionally interconnected to fix CO2, subsequently converting CO2 into acetyl-CoA, acetyl-phosphate, and serine. Moreover, the functional cooperation of the pathways enhances CO2 consumption and cellular growth rates via bypassing reducing power required reactions for cellular metabolism during autotrophic growth of acetogens.

Neonatal Nonketotic Hyperglycinemia: A Rare Case from Pakistan

Nonketotic hyperglycinemia (NKH) is an autosomal recessive disorder caused by a defect in glycine cleavage enzyme. It leads to the accumulation of glycine in the body tissues, blood, and cerebrospinal fluid (CSF). Most NKH cases are diagnosed during the natal period of life and are fatal if not promptly diagnosed and managed. Here we present a case of a two-day-old child who presented with reluctant feeding and lethargy. She had reduced tone in all four limbs and a Glasgow Coma Scale score of 9. Keeping an infectious etiology in mind, she was started on cefotaxime and amikacin. The patient was shifted to the neonatal intensive care unit; however, no improvement in the patient's condition was seen and antibiotics were changed to linezolid and meropenem along with initiation of acyclovir. The patient's blood and CSF cultures were negative. Serum amino acid chromatography showed elevated levels of glycine, and a diagnosis of NKH was made. The patient was managed symptomatically but expired on the 22nd day of admission. The case highlights the importance of prompt diagnosis and management of aminoacidopathies. Nearly all metabolic disorders have similar clinical presentations, and an early diagnosis can improve the outcome in patients.

Glycine Enhances Satellite Cell Proliferation, Cell Transplantation, and Oligonucleotide Efficacy in Dystrophic Muscle.

The need to distribute therapy evenly systemically throughout the large muscle volume within the body makes Duchenne muscular dystrophy (DMD) therapy a challenge. Cell and exon-skipping therapies are promising but have limited effects, and thus enhancing their therapeutic potency is of paramount importance to increase the accessibility of these therapies to DMD patients. In this study, we demonstrate that co-administered glycine improves phosphorodiamidate morpholino oligomer (PMO) potency in mdx mice with marked functional improvement and an up to 50-fold increase of dystrophin in abdominal muscles compared to PMO in saline. Glycine boosts satellite cell proliferation and muscle regeneration by increasing activation of mammalian target of rapamycin complex 1 (mTORC1) and replenishing the one-carbon unit pool. The expanded regenerating myofiber population then results in increased PMO uptake. Glycine also augments the transplantation efficiency of exogenous satellite cells and primary myoblasts in mdx mice. Our data provide evidence that glycine enhances satellite cell proliferation, cell transplantation, and oligonucleotide efficacy in mdx mice, and thus it has therapeutic utility for cell therapy and drug delivery in muscle-wasting diseases.

Metabolic dependencies govern microbial syntrophies during methanogenesis in an anaerobic digestion ecosystem

Methanogenesis, a biological process mediated by complex microbial communities, has attracted great attention due to its contribution to global warming and potential in biotechnological applications. The current study unveiled the core microbial methanogenic metabolisms in anaerobic vessel ecosystems by applying combined genome-centric metagenomics and metatranscriptomics. Here, we demonstrate that an enriched natural system, fueled only with acetate, could support a bacteria-dominated microbiota employing a multi-trophic methanogenic process. Moreover, significant changes, in terms of microbial structure and function, were recorded after the system was supplemented with additional H2. Methanosarcina thermophila, the predominant methanogen prior to H2 addition, simultaneously performed acetoclastic, hydrogenotrophic, and methylotrophic methanogenesis. The methanogenic pattern changed after the addition of H2, which immediately stimulated Methanomicrobia-activity and was followed by a slow enrichment of Methanobacteria members. Interestingly, the essential genes involved in the Wood-Ljungdahl pathway were not expressed in bacterial members. The high expression of a glycine cleavage system indicated the activation of alternative metabolic pathways for acetate metabolism, which were reconstructed in the most abundant bacterial genomes. Moreover, as evidenced by predicted auxotrophies, we propose that specific microbes of the community were forming symbiotic relationships, thus reducing the biosynthetic burden of individual members. These results provide new information that will facilitate future microbial ecology studies of interspecies competition and symbiosis in methanogenic niches.6mWxSszdRnKL2KDYxsStzzVideo abstract.

Impaired folate 1-carbon metabolism causes formate-preventable hydrocephalus in glycine decarboxylase-deficient mice.

Ventriculomegaly and hydrocephalus are associated with loss of function of glycine decarboxylase (Gldc) in mice and in humans suffering from non-ketotic hyperglycinemia (NKH), a neurometabolic disorder characterized by accumulation of excess glycine. Here, we showed that ventriculomegaly in Gldc-deficient mice is preceded by stenosis of the Sylvian aqueduct and malformation or absence of the subcommissural organ and pineal gland. Gldc functions in the glycine cleavage system, a mitochondrial component of folate metabolism, whose malfunction results in accumulation of glycine and diminished supply of glycine-derived 1-carbon units to the folate cycle. We showed that inadequate 1-carbon supply, as opposed to excess glycine, is the cause of hydrocephalus associated with loss of function of the glycine cleavage system. Maternal supplementation with formate prevented both ventriculomegaly, as assessed at prenatal stages, and postnatal development of hydrocephalus in Gldc-deficient mice. Furthermore, ventriculomegaly was rescued by genetic ablation of 5,10-methylene tetrahydrofolate reductase (Mthfr), which results in retention of 1-carbon groups in the folate cycle at the expense of transfer to the methylation cycle. In conclusion, a defect in folate metabolism can lead to prenatal aqueduct stenosis and resultant hydrocephalus. These defects are preventable by maternal supplementation with formate, which acts as a 1-carbon donor.

Target Discovery in Ralstonia solanacearum through an Activity-Based Protein Profiling Technique Based on Bioactive Oxadiazole Sulfones.

Ralstonia solanacearum is an extremely destructive and rebellious phytopathogen that can cause bacterial wilt diseases in more than 200 plant species. To explore and discover the potential targets in R. solanacearum for the purpose of developing new agrochemicals targeting this infection, here, we exploited a typical activity-based protein profiling technique for target discovery in R. solanacearum based on an activity-based probe 1 derived from bioactive oxadiazole sulfones. A total of 65 specific targets were identified with high confidence through a quantitative chemical proteomic approach. Three representative proteins (glycine cleavage system H protein, thiol peroxidase, and dihydrolipoamide S-succinyltransferase) were validated as the targets by using the immunoblotting analysis with their respective antibodies. Additionally, the in vitro interaction between the recombinant thiol peroxidase and probe 1 further confirmed that this protein was a target of oxadiazole sulfones. We anticipated that these discovered protein targets in R. solanacearum can stimulate the discovery and development of novel agrochemicals targeting bacterial infections caused by R. solanacearum.