Structural insights into the mechanism of PEPCK catalysis

Phosphoenolpyruvate carboxykinase catalyzes the reversible decarboxylation of oxaloacetic acid with the concomitant transfer of the gamma-phosphate of GTP to form PEP and GDP as the first committed step of gluconeogenesis and glyceroneogenesis. The three structures of the mitochondrial isoform of PEPCK reported are complexed with Mn2+, Mn2+-PEP, or Mn2+-malonate-Mn2+ GDP and provide the first observations of the structure of the mitochondrial isoform and insight into the mechanism of catalysis mediated by this enzyme. The structures show the involvement of the hyper-reactive cysteine (C307) in the coordination of the active site Mn2+. Upon formation of the PEPCK-Mn2+-PEP or PEPCK-Mn2+-malonate-Mn2+ GDP complexes, C307 coordination is lost as the P-loop in which it resides adopts a different conformation. The structures suggest that stabilization of the cysteine-coordinated metal geometry holds the enzyme as a catalytically incompetent metal complex and may represent a previously unappreciated mechanism of regulation. A third conformation of the mobile P-loop in the PEPCK-Mn2+-malonate-Mn2+ GDP complex demonstrates the participation of a previously unrecognized, conserved serine residue (S305) in mediating phosphoryl transfer. The ordering of the mobile active site lid in the PEPCK-Mn2+-malonate-Mn2+ GDP complex yields the first observation of this structural feature and provides additional insight into the mechanism of phosphoryl transfer.     

Turbidity Structures the Controls of Ecosystem Metabolism and Associated Metabolic Process Domains Along a 75-km Segment of a Semiarid Stream

Ecosystems - Stream ecosystem metabolism contributes to global carbon cycling, yet predicting metabolism across ecosystems remains elusive. Even within stream segments, spatial variation in...     

Ligneous membranitis in Scottish Terriers is associated with a single nucleotide polymorphism in the plasminogen (PLG) gene

Ligneous membranitis (LM) is a rare chronic inflammatory condition of the mucous membranes associated with plasminogen (encoded by PLG) deficiency in affected humans and dogs. In human, the condition is genetic in nature with numerous mutations and polymorphisms in PLG identified in affected individuals and related family members. The condition is uncommonly reported in dogs and, to date, no genetic studies have been performed. We identified related Scottish Terriers (littermates) with severe LM and unaffected relatives (sire, dam and a sibling from a previous litter). Plasma plasminogen activity was below normal in one affected dog but within normal reference intervals for the other. Sequencing of PLG from the affected dogs revealed a homozygous A>T single nucleotide polymorphism in an intron donor site (c.1256+2T>A). The related, unaffected dogs displayed heterozygous alleles at this position (c.1256+2T/A), whereas no mutation was detected in unaffected, non‐related control dogs. This is the first report to identify gene polymorphisms associated with LM in dogs.     

Characterizing and modeling hydrogen sulfide production in anaerobic digestion of livestock manure,agro-industrial wastes,and wastewater sludge

Hydrogen sulfide (H₂S) is the most undesirable inorganic gas in biogas from anaerobic digestion (AD). However, H₂S production in AD is complex and understanding of its processes is still limited. This study performed six controlled batch anaerobic co-digestion experiments to investigate H₂S production. Materials were obtained from four field anaerobic digester systems and co-digestion feedstocks from agroindustry. An additional precipitation experiment was conducted to further examine H₂S production dynamics. Digesters containing highly soluble, carbohydrate-based wastes had a high H₂S final specific production (FSP) value. Additionally, the FSP values were negatively correlated with the initial Fe(II):S ratios in the digester liquid of the batch tests. The precipitation experiment indicated that iron sulfide precipitation was preferred in the presence of an anaerobic community. The H₂S production as a time series was successfully modeled using a generalized additive model (R² > 0.82). This study revealed that sulfate, phosphorus, and iron concentrations are important predictors and potential inhibitors of H₂S production in AD. Further examination of real-time H₂S modeling in AD is warranted.     

Biogenesis of functional antigenic peptide transporter TAP requires assembly of pre-existing TAP1 with newly synthesized TAP2

The transporter associated with antigen processing (TAP) is essential for the delivery of antigenic peptides from the cytosol into the endoplasmic reticulum (ER), where they are loaded onto major histocompatibility complex class I molecules. TAP is a heterodimeric transmembrane protein that comprises the homologous subunits TAP1 and TAP2. As for many other oligomeric protein complexes, which are synthesized in the ER, the process of subunit assembly is essential for TAP to attain a native functional state. Here, we have analyzed the individual requirements of TAP1 and TAP2 for the formation of a functional TAP complex. Unlike TAP1, TAP2 is very unstable when expressed in isolation. We show that heterodimerization of TAP subunits is required for maintaining a stable level of TAP2. By using an in vitro expression system we demonstrate that the biogenesis of functional TAP depends on the assembly of preexisting TAP1 with newly synthesized TAP2, but not vice versa. The pore forming core transmembrane domain (core TMD) of in vitro expressed TAP2 is necessary and sufficient to allow functional complex formation with pre-existing TAP1. We propose that the observed assembly mechanism of TAP protects newly synthesized TAP2 from rapid degradation and controls the number of transport active transporter molecules. Our findings open up new possibilities to investigate functional and structural properties of TAP and provide a powerful model system to address the biosynthetic assembly of oligomeric transmembrane proteins in the ER.     

Flexibility of nutritional strategies within a mutualism: food availability affects algal symbiont productivity in two congeneric sea anemone species

Mutualistic symbioses are common, especially in nutrient-poor environments where an association between hosts and symbionts can allow the symbiotic partners to persist and collectively out-compete non-symbiotic species. Usually these mutualisms are built on an intimate transfer of energy and nutrients (e.g. carbon and nitrogen) between host and symbiont. However, resource availability is not consistent, and the benefit of the symbiotic association can depend on the availability of resources to mutualists. We manipulated the diets of two temperate sea anemone species in the genus Anthopleura in the field and recorded the responses of sea anemones and algal symbionts in the family Symbiodiniaceae to our treatments. Algal symbiont density, symbiont volume and photosynthetic efficiency of symbionts responded to changes in sea anemone diet, but the responses depended on the species of sea anemone. We suggest that temperate sea anemones and their symbionts can respond to changes in anemone diet, modifying the balance between heterotrophy and autotrophy in the symbiosis. Our data support the hypothesis that symbionts are upregulated or downregulated based on food availability, allowing for a flexible nutritional strategy based on external resources.     

Novel Use of Proton Magnetic Resonance Spectroscopy (1HMRS) to Non-Invasively Assess Placental Metabolism

Background

Placental insufficiency is a major cause of antepartum stillbirth and fetal growth restriction (FGR). In affected pregnancies, delivery is expedited when the risks of ongoing pregnancy outweigh those of prematurity. Current tests are unable to assess placental function and determine optimal timing for delivery. An accurate, non-invasive test that clearly defines the failing placenta would address a major unmet clinical need. Proton magnetic resonance spectroscopy (¹H MRS) can be used to assess the metabolic profile of tissue in-vivo. In FGR pregnancies, a reduction in N-acetylaspartate (NAA)/choline ratio and detection of lactate methyl are emerging as biomarkers of impaired neuronal metabolism and fetal hypoxia, respectively. However, fetal brain hypoxia is a late and sometimes fatal event in placental compromise, limiting clinical utility of brain ¹H MRS to prevent stillbirth. We hypothesised that abnormal placental ¹H MRS may be an earlier biomarker of intrauterine hypoxia, affording the opportunity to optimise timing of delivery in at-risk fetuses.

Methods and Findings

We recruited three women with severe placental insufficiency/FGR and three matched controls. Using a 3T MR system and a combination of phased-array coils, a 20×20×40 mm¹H MRS voxel was selected along the ‘long-axis’ of the placenta with saturation bands placed around the voxel to prevent contaminant signals. A significant choline peak (choline/lipid ratio 1.35–1.79) was detected in all healthy placentae. In contrast, in pregnancies complicated by FGR, the choline/lipid ratio was ≤0.02 in all placentae, despite preservation of the lipid peak (p<0.001).

Conclusions

This novel proof-of-concept study suggests that in severe placental insufficiency/FGR, the observed 60-fold reduction in the choline/lipid ratio by ¹H MRS may represent an early biomarker of critical placental insufficiency. Further studies will determine performance of this test and the potential role of 1H-MRS in the in-vivo assessment of placental function to inform timing of delivery.     

Genetic diseases of the Kennedy pathways for membrane synthesis

The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease.