Lipid metabolism in Trypanosoma brucei, the causative agent of African sleeping sickness, differs from its human host in several fundamental ways. This has lead to the validation of a plethora of novel drug targets, giving hope of novel chemical intervention against this neglected disease. Cytidine diphosphate diacylglycerol (CDP-DAG) is a central lipid intermediate for several pathways in both prokaryotes and eukaryotes, being produced by CDP-DAG synthase (CDS). However, nothing is known about the single T. brucei CDS gene (Tb927.7.220/EC 2.7.7.41) or its activity. In this study we show TbCDS is functional by complementation of a non-viable yeast CDS null strain and that it is essential in the bloodstream form of the parasite via a conditional knockout. The TbCDS conditional knockout showed morphological changes including a cell-cycle arrest due in part to kinetoplast segregation defects. Biochemical phenotyping of TbCDS conditional knockout showed drastically altered lipid metabolism where reducing levels of phosphatidylinositol detrimentally impacted on glycoylphosphatidylinositol biosynthesis. These studies also suggest that phosphatidylglycerol synthesized via the phosphatidylglycerol-phosphate synthase is not synthesized from CDP-DAG, as was previously thought. TbCDS was shown to localized the ER and Golgi, probably to provide CDP-DAG for the phosphatidylinositol synthases.

The type 1 ribosome inactivating protein from Momordica balsamina (MbRIP1) has been shown to interact with purine bases, adenine and guanine of RNA/DNA. We report here the binding and structural studies of MbRIP1 with a pyrimidine base, cytosine; cytosine containing nucleoside, cytidine; and cytosine containing nucleotide, cytidine diphosphate. All three compounds bound to MbRIP1 at the active site with dissociation constants of 10-4 M-10-7 M. As reported earlier, in the structure of native MbRIP1, there are 10 water molecules in the substrate binding site. Upon binding of cytosine to MbRIP1, four water molecules were dislodged from the substrate binding site while five water molecules were dislodged when cytidine bound to MbRIP1. Seven water molecules were dislocated when cytidine diphosphate bound to MbRIP1. This showed that cytidine diphosphate occupied a larger space in the substrate binding site enhancing the buried surface area thus making it a relatively better inhibitor of MbRIP1 as compared to cytosine and cytidine. The key residues involved in the recognition of cytosine, cytidine and cytidine diphosphate were Ile71, Glu85, Tyr111 and Arg163. The orientation of cytosine in the cleft is different from that of adenine or guanine indicating a notable difference in the modes of binding of purine and pyrimidine bases. Since adenine containing nucleosides/nucleotides are suitable substrates, the cytosine containing nucleosides/nucleotides may act as inhibitors.

Dopamine receptor-coupled stimulation of inositol phosphate formation has been characterized extensively, but little is known about the diacylglycerol arm of this dual-signaling pathway. This study examined several parameters of cytidine diphosphate-diacylglycerol (CDP-DG) accumulation as an index of agonist-stimulated DG formation. Rat brain slices pre-labeled with 5-[3H]cytidine were incubated with various test agents in the presence of LiCl and accumulated CDP-DG analyzed. Dopamine and SKF38393 significantly and dose-dependently stimulated CDP-DG accumulation. SKF38393 responses were inhibited by neomycin and reversed by myo-inositol or by exclusion of LiCl. Compared to inositol phosphate formation in 2-[3H]inositol-prelabeled slices, the CDP-DG responses were proportionately greater, while the agonist EC50 values were similar between the two assays. The D1-receptor antagonist SCH23390 inhibited SKF38393-mediated responses at 0.1-10 microM concentrations, whereas greater concentrations reversed the inhibition. SKF38393 effects were completely blocked by the DG kinase inhibitor R59022, thus precluding any role for phospholipase-D or de novo phosphatidate synthesis in the dopaminergic response. D609 which inhibits phosphatidylcholine-specific phospholipase-C (PLC), potently inhibited both CDP-DG accumulation and inositol phosphate formation. These findings demonstrate that the selective D1-receptor antagonist SCH23390 is a partial agonist at the D1-like dopamine receptor that couples to phosphoinositide signaling, that dopaminergic facilitation of phosphoinositide signaling is independent of de novo phosphatidate synthesis, and that the widely used enzyme inhibitor, D-609, is probably not selective for phosphatidylcholine-specific PLC in brain slice preparations. The greater sensitivity of the CDP-DG measurement presents this assay as a reliable and possibly superior index of dopamine receptor-coupled PLC activation in intact tissues.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all living organisms. The catalytic cycle of E. coli RNR involves a long-range proton-coupled electron transfer (PCET) from a tyrosyl radical (Y122˙) in subunit β2 to a cysteine (C439) in the active site of subunit α2, which subsequently initiates nucleotide reduction. This oxidation occurs over 35 Å and involves a specific pathway of redox active amino acids (Y122 ↔ [W48?] ↔ Y356 in β2 to Y731 ↔ Y730 ↔ C439 in α2). The mechanisms of the PCET steps at the interface of the α2β2 complex remain puzzling due to a lack of structural information for this region. Recently, DFT calculations on the 3-aminotyrosyl radical (NH2Y731˙)-α2 trapped by incubation of NH2Y731-α2/β2/CDP(substrate)/ATP(allosteric effector) suggested that R411-α2, a residue close to the α2β2 interface, interacts with NH2Y731˙ and accounts in part for its perturbed EPR parameters. To examine its role, we further modified NH2Y731-α2 with a R411A substitution. NH2Y731˙/R411A generated upon incubation of NH2Y731/R411A-α2/β2/CDP/ATP was investigated using multi-frequency (34, 94 and 263 GHz) EPR, 34 GHz pulsed electron-electron double resonance (PELDOR) and electron-nuclear double resonance (ENDOR) spectroscopies. The data indicate a large conformational change in NH2Y731˙/R411A relative to the NH2Y731˙ single mutant. Particularly, the inter-spin distance from NH2Y731˙/R411A in one αβ pair to Y122˙ in a second αβ pair decreases by 3 Å in the presence of the R411A mutation. This is the first experimental evidence for the flexibility of pathway residue Y731-α2 in an α2β2 complex and suggests a role for R411 in the stacked Y731/Y730 conformation involved in collinear PCET. Furthermore, NH2Y731˙/R411A serves as a probe of the PCET process across the subunit interface.

In mycobacteria, phosphatidylinositol (PI) acts as a common lipid anchor for key components of the cell wall, including the glycolipids phosphatidylinositol mannoside, lipomannan, and lipoarabinomannan. Glycolipids in Mycobacterium tuberculosis, the causative agent of tuberculosis, are important virulence factors that modulate the host immune response. The identity-defining step in PI biosynthesis in prokaryotes, unique to mycobacteria and few other bacterial species, is the reaction between cytidine diphosphate-diacylglycerol and inositol-phosphate to yield phosphatidylinositol-phosphate, the immediate precursor to PI. This reaction is catalyzed by the cytidine diphosphate-alcohol phosphotransferase phosphatidylinositol-phosphate synthase (PIPS), an essential enzyme for mycobacterial viability. Here we present structures of PIPS from Mycobacterium kansasii with and without evidence of donor and acceptor substrate binding obtained using a crystal engineering approach. PIPS from Mycobacterium kansasii is 86% identical to the ortholog from M. tuberculosis and catalytically active. Functional experiments guided by our structural results allowed us to further characterize the molecular determinants of substrate specificity and catalysis in a new mycobacterial species. This work provides a framework to strengthen our understanding of phosphatidylinositol-phosphate biosynthesis in the context of mycobacterial pathogens.

Herein, we described a washing- and label-free clustered regularly interspaced short palindromic repeats (CRISPR)/LwaCas13a-based RNA detection method utilizing a personal glucose meter (PGM), which relies on the trans-cleavage activity of CRISPR/Cas13a and kinase reactions. In principle, the presence of target RNA activates the trans-cleavage of CRISPR/Cas13a, generating 2',3'-cyclic phosphate adenosine, which is converted to adenosine monophosphate (AMP) by the T4 polynucleotide kinase. Subsequently, the AMP is converted to adenosine diphosphate (ADP) through phosphorylation by a myokinase; ADP is then used as a substrate in the cascade enzymatic reaction promoted by pyruvate kinase and hexokinase. The overall reaction leads to the continuous conversion of glucose to glucose-6-phosphate, resulting in a reduction of glucose concentration proportional to the level of target RNA, which can therefore be indirectly measured with a PGM. By employing this novel strategy, severe acute respiratory syndrome coronavirus-2 RNA can be successfully detected with excellent specificity. In addition, we were able to overcome non-specific responses of CRISPR/Cas13a and distinguish single nucleotide polymorphisms by introducing a single-base mismatch in the complementary RNA. Our study provides an alternative coronavirus disease 2019 detection technology that is affordable, accessible, and portable with a fast turnaround time and excellent selectivity.

Microinjection, but not extracellular application, of cytidine-5'-diphosphate-D-glucose (CDPG) has been shown to elicit Ca(2+)-dependent currents in Xenopus laevis oocytes. These responses were comparable to those of inositol-1,4,5-trisphosphate (InsP3) in being both rapid and dose dependent. For example, maximal amplitudes of CDPG-induced current were similar (approximately 365 +/- 75 nA at 1 microM CDPG) to those of InsP3. The CDPG currents were insensitive to removal of extracellular Ca2+, indicating the dependence on Ca2+ release from intracellular Ca2+ stores but not on Ca2+ entry through plasma membrane. CDPG-induced currents were reduced or abolished by pretreatment with thapsigargin, by injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or by extracellular perfusion of the Cl- channel blocker niflumic acid but were insensitive to injection of the InsP3 antagonist heparin. These results suggest that CDPG induces Ca2+ discharge from intracellular Ca2+ stores via a mechanism distinct from that of InsP3 in Xenopus oocytes. Another pyrimidine nucleotide-glucose derivative, uridine-5'-diphosphate-alpha-D-glucose, also induced Ca(2+)-dependent currents, but the activity was lower than that of CDPG (maximal amplitude, 272 +/- 62 nA). Other nucleotide-glucose compounds (adenosine-5'-diphosphate-D-glucose, guanosine-5'-diphosphate-D-glucose, and thymidine-5'-diphosphate-D-glucose) had no current responses when injected into oocytes. After injection of CDPG, CDPG-induced Ca2+ release appeared to couple to a Ca2+ entry pathway similar to that coupled to InsP3. These results indicate that pyrimidine nucleotide-glucose conjugates may provide novel pharmacological tools for the study of Ca2+ signaling in oocytes.

Phospholipids are elemental building-block molecules for biological membranes. Biosynthesis of phosphatidylinositol, phosphatidylglycerol and phosphatidylserine requires a central liponucleotide intermediate named cytidine-diphosphate diacylglycerol (CDP-DAG). The CDP-DAG synthetase (Cds) is an integral membrane enzyme catalysing the formation of CDP-DAG, an essential step for phosphoinositide recycling during signal transduction. Here we report the structure of the Cds from Thermotoga maritima (TmCdsA) at 3.4 Å resolution. TmCdsA forms a homodimer and each monomer contains nine transmembrane helices arranged into a novel fold with three domains. An unusual funnel-shaped cavity penetrates half way into the membrane, allowing the enzyme to simultaneously accept hydrophilic substrate (cytidine 5'-triphosphate (CTP)/deoxy-CTP) from cytoplasm and hydrophobic substrate (phosphatidic acid) from membrane. Located at the bottom of the cavity, a Mg(2+)-K(+) hetero-di-metal centre coordinated by an Asp-Asp dyad serves as the cofactor of TmCdsA. The results suggest a two-metal-ion catalytic mechanism for the Cds-mediated synthesis of CDP-DAG at the membrane-cytoplasm interface.

FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/traJ) for the plasmid-encoded FinO protein, to transcriptome-wide activity as documented for chromosomally encoded ProQ proteins. Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level.

Saccharomyces cerevisiae, commonly known as baker's yeast, is one of the most comprehensively studied model organisms in science. Yeast has been used to study a wide variety of human diseases, and the yeast model system has proved to be an especially amenable tool for the study of lipids and lipid-related pathophysiologies, a topic that has gained considerable attention in recent years. This review focuses on how yeast has contributed to our understanding of the mitochondrial phospholipid cardiolipin (CL) and its role in Barth syndrome (BTHS), a genetic disorder characterized by partial or complete loss of function of the CL remodeling enzyme tafazzin. Defective tafazzin causes perturbation of CL metabolism, resulting in many downstream cellular consequences and clinical pathologies that are discussed herein. The influence of yeast research in the lipid-related pathophysiologies of Alzheimer's and Parkinson's diseases is also summarized.

ATP is universally conserved as the principal energy currency in cells, driving metabolism through phosphorylation and condensation reactions. Such deep conservation suggests that ATP arose at an early stage of biochemical evolution. Yet purine synthesis requires 6 phosphorylation steps linked to ATP hydrolysis. This autocatalytic requirement for ATP to synthesize ATP implies the need for an earlier prebiotic ATP equivalent, which could drive protometabolism before purine synthesis. Why this early phosphorylating agent was replaced, and specifically with ATP rather than other nucleoside triphosphates, remains a mystery. Here, we show that the deep conservation of ATP might reflect its prebiotic chemistry in relation to another universally conserved intermediate, acetyl phosphate (AcP), which bridges between thioester and phosphate metabolism by linking acetyl CoA to the substrate-level phosphorylation of ADP. We confirm earlier results showing that AcP can phosphorylate ADP to ATP at nearly 20% yield in water in the presence of Fe3+ ions. We then show that Fe3+ and AcP are surprisingly favoured. A wide range of prebiotically relevant ions and minerals failed to catalyse ADP phosphorylation. From a panel of prebiotic phosphorylating agents, only AcP, and to a lesser extent carbamoyl phosphate, showed any significant phosphorylating potential. Critically, AcP did not phosphorylate any other nucleoside diphosphate. We use these data, reaction kinetics, and molecular dynamic simulations to infer a possible mechanism. Our findings might suggest that the reason ATP is universally conserved across life is that its formation is chemically favoured in aqueous solution under mild prebiotic conditions.

Adhesion G Protein-Coupled Receptor L4 (ADGRL4/ELTD1) is an endothelial cell adhesion G protein-coupled receptor (aGPCR) which regulates physiological and tumour angiogenesis, providing an attractive target for anti-cancer therapeutics. To date, ADGRL4/ELTD1's full role and mechanism of function within endothelial biology remains unknown, as do its ligand(s). In this study, ADGRL4/ELTD1 silencing, using two independent small interfering RNAs (siRNAs), was performed in human umbilical vein endothelial cells (HUVECS) followed by transcriptional profiling, target gene validation, and metabolomics using liquid chromatography-mass spectrometry in order to better characterise ADGRL4/ELTD1's role in endothelial cell biology. We show that ADGRL4/ELTD1 silencing induced expression of the cytoplasmic metabolic regulator ATP Citrate Lyase (ACLY) and the mitochondria-to-cytoplasm citrate transporter Solute Carrier Family 25 Member 1 (SLC25A1) but had no apparent effect on pathways downstream of ACLY (fatty acid and cholesterol synthesis or acetylation). Silencing induced KIT expression and affected the Notch signalling pathway, upregulating Delta Like Canonical Notch Ligand 4 (DLL4) and suppressing Jagged Canonical Notch Ligand 1 (JAG1) and Hes Family BHLH Transcription Factor 2 (HES2). The effect of ADGRL4/ELTD1 silencing on the cellular metabolic profile was modest but several metabolites were significantly affected. Cis-aconitic acid, uridine diphosphate (UDP)-glucoronate, fructose 2,6-diphosphate, uridine 5-diphosphate, and aspartic acid were all elevated as a result of silencing and phosphocreatine, N-acetylglutamic acid, taurine, deoxyadenosine triphosphate, and cytidine monophosphate were depleted. Metabolic pathway analysis implicated ADGRL4/ELTD1 in pyrimidine, amino acid, and sugar metabolism. In summary, this study shows that ADGRL4/ELTD1 impacts core components of endothelial metabolism and regulates genes involved in endothelial differentiation/homeostasis and Notch signalling.

Candida albicans is an opportunistic human fungal pathogen that causes life-threatening systemic infections, as well as oral mucosal infections. Phospholipids are crucial for pathogenesis in C. albicans, as disruption of phosphatidylserine (PS) and phosphatidylethanolamine (PE) biosynthesis within the cytidine diphosphate diacylglycerol (CDP-DAG) pathway causes avirulence in a mouse model of systemic infection. The synthesis of PE by this pathway plays a crucial role in virulence, but it was unknown if downstream conversion of PE to phosphatidylcholine (PC) is required for pathogenicity. Therefore, the enzymes responsible for methylating PE to PC, Pem1 and Pem2, were disrupted. The resulting pem1Δ/Δ pem2Δ/Δ mutant was not less virulent in mice, but rather hypervirulent. Since the pem1Δ/Δ pem2Δ/Δ mutant accumulated PE, this led to the hypothesis that increased PE synthesis increases virulence. To test this, the alternative Kennedy pathway for PE/PC synthesis was exploited. This pathway makes PE and PC from exogenous ethanolamine and choline, respectively, using three enzymatic steps. In contrast to Saccharomyces cerevisiae, C. albicans was found to use one enzyme, Ept1, for the final enzymatic step (ethanolamine/cholinephosphotransferase) that generates both PE and PC. EPT1 was overexpressed, which resulted in increases in both PE and PC synthesis. Moreover, the EPT1 overexpression strain is hypervirulent in mice and causes them to succumb to system infection more rapidly than wild-type. In contrast, disruption of EPT1 causes loss of PE and PC synthesis by the Kennedy pathway, and decreased kidney fungal burden during the mouse systemic infection model, indicating a mild loss of virulence. In addition, the ept1Δ/Δ mutant exhibits decreased cytotoxicity against oral epithelial cells in vitro, whereas the EPT1 overexpression strain exhibits increased cytotoxicity. Taken altogether, our data indicate that mutations that result in increased PE synthesis cause greater virulence and mutations that decrease PE synthesis attenuate virulence.

As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

The causative agent of Legionnaires disease, Legionella pneumophila, translocates the phosphocholine transferase AnkX during infection and thereby posttranslationally modifies the small guanosine triphosphatase (GTPase) Rab1 with a phosphocholine moiety at S76 using cytidine diphosphate (CDP)-choline as a cosubstrate. The molecular basis for Rab1 binding and enzymatic modification have remained elusive because of lack of structural information of the low-affinity complex with AnkX. We combined thiol-reactive CDP-choline derivatives with recombinantly introduced cysteines in the AnkX active site to covalently capture the heterocomplex. The resulting crystal structure revealed that AnkX induces displacement of important regulatory elements of Rab1 by placing a β sheet into a conserved hydrophobic pocket, thereby permitting phosphocholine transfer to the active and inactive states of the GTPase. Together, the combination of chemical biology and structural analysis reveals the enzymatic mechanism of AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins.

Candida albicans is a commensal fungus, opportunistic pathogen, and the most common cause of fungal infection in humans. The biosynthesis of phosphatidylcholine (PC), a major eukaryotic glycerophospholipid, occurs through two primary pathways. In Saccharomyces cerevisiae and some plants, a third PC synthesis pathway, the PC deacylation/reacylation pathway (PC-DRP), has been characterized. PC-DRP begins with the acylation of the lipid turnover product, glycerophosphocholine (GPC), by the GPC acyltransferase, Gpc1, to form Lyso-PC. Lyso-PC is then acylated by lysolipid acyltransferase, Lpt1, to produce PC. Importantly, GPC, the substrate for Gpc1, is a ubiquitous metabolite available within the host. GPC is imported by C. albicans, and deletion of the major GPC transporter, Git3, leads to decreased virulence in a murine model. Here we report that GPC can be directly acylated in C. albicans by the protein product of orf19.988, a homolog of ScGpc1. Through lipidomic studies, we show loss of Gpc1 leads to a decrease in PC levels. This decrease occurs in the absence of exogenous GPC, indicating that the impact on PC levels may be greater in the human host where GPC is available. A gpc1Δ/Δ strain exhibits several sensitivities to antifungals that target lipid metabolism. Furthermore, loss of Gpc1 results in both a hyphal growth defect in embedded conditions and a decrease in long-term cell viability. These results demonstrate for the first time the importance of Gpc1 and this alternative PC biosynthesis route (PC-DRP) to the physiology of a pathogenic fungus.

The cytidine diphosphate diacylglycerol synthases (CDSs) gene encodes the cytidine diphosphate-diacylglycerol (CDP-DAG) synthase enzyme that catalyzes the formation of CDP-diacylglycerol from phosphatidic acid. At present, there are no reports of CDS2 in birds. Here, we identified chicken CDS2 transcripts by combining conventional RT-PCR amplification, 5' rapid amplification of cDNA ends (RACE), and 3' RACE, explored the spatio-temporal expression profiles of total CDS2 and the longest transcript variant CDS2-4, and investigated the effect of exogenous insulin on the mRNA level of total CDS2 via quantitative RT-PCR. Four transcripts of chicken CDS2 (CDS2-1, -2, -3, and -4) were identified, which were alternatively spliced at the 3'-untranslated region (UTR). Both total CDS2 and CDS2-4 were prominently expressed in adipose tissue, and exhibited low expression in liver and pectoralis of 49-day-old chickens. Regarding the spatio-temporal expression patterns of CDS2 in chicken, total CDS2 exhibited a similar temporal expression tendency with a high level in the later period of incubation (embryonic day 19 [E19] or 1-day-old) in the brain, liver, and pectoralis. While CDS2-4 presented a distinct temporal expression pattern in these tissues, CDS2-4 levels peaked at 21 d in the brain and pectoralis, while liver CDS2-4 mRNA levels were highest at the early stage of hatching (E10). Total CDS2 (P < 0.001) and CDS2-4 (P = 0.0090) mRNA levels in the liver were differentially regulated throughout the development of the chicken. Total CDS2 levels in the liver of Silky chickens were higher than that of the broiler in the basal state and after insulin stimulation. Exogenous insulin significantly down-regulated the level of total CDS2 at 240 min in the pectoralis of Silky chickens (P < 0.01). In conclusion, chicken CDS2 isoforms with variation at the 3'-UTR were identified, which was prominently expressed in adipose tissue. Total CDS2 and CDS2-4 presented distinct spatio-temporal expression patterns, that is they were differentially regulated with age in brain, liver, and pectoralis. Insulin could regulate chicken CDS2 levels in a breed- and tissue-specific manner.

Schwann cells are integral cellular components of the dense cholinergic presynaptic plexus (nerve plate) which innervates each electrocyte in skate electric organ. Using the Ca2+-sensitive dye fura-2, we have followed the response in these cells to various chemical challenges. In K+ depolarized nerve plates nerve terminals consistently responded with a rapid and sustained Ca2+ signal. Schwann cell responses to depolarization were rarely seen but, when observed, were always delayed in onset when compared to nerve terminal response (6-10 s later). The possibility that these responses were triggered by mediators released from nerve terminals was tested by direct application of candidate substances. Schwann cells were found to respond to adenosine triphosphate and adenosine diphosphate with a biphasic increase of intracellular Ca2+ concentration, a rapid peak response being followed in the majority of cells by a sustained plateau phase. In the absence of external Ca2+ only the transient peak response was observed. Depletion of internal Ca2+ stores with thapsigargin completely inhibited the adenosine triphosphate-stimulated rise in Schwann cell Ca2+. The response to adenosine triphosphate was concentration-dependent (EC50 2.8 microM) and was reversibly blocked by two antagonists of P2 purinoceptors: suramin and reactive blue 2. Adenosine diphosphate and 2-methylthio-adenosine triphosphate were equipotent with adenosine triphosphate and at high concentrations (100 microM) diadenosine tetraphosphate produced responses comparable to low concentrations of adenosine triphosphate. Adenosine, adenosine monophosphate, the alpha beta-methylene analogues of adenosine triphosphate and adenosine diphosphate, uridine triphosphate, cytidine triphosphate and guanosine triphosphate were without significant effect. These results show that, in skate electric organ Schwann cells, the release of Ca2+ from intracellular stores is triggered by adenosine triphosphate acting on P(2gamma) receptors and suggest that Schwann cells may be targets for synaptically-released adenosine triphosphate in the electric organ model of the neuromuscular junction.

Glycosylation is an essential post-translational modification that underlies many biological processes and diseases. α-dystroglycan (α-DG) is a receptor for matrix and synaptic proteins that causes muscular dystrophy and lissencephaly upon its abnormal glycosylation (α-dystroglycanopathies). Here we identify the glycan unit ribitol 5-phosphate (Rbo5P), a phosphoric ester of pentose alcohol, in α-DG. Rbo5P forms a tandem repeat and functions as a scaffold for the formation of the ligand-binding moiety. We show that enzyme activities of three major α-dystroglycanopathy-causing proteins are involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain-containing (ISPD) is cytidine diphosphate ribitol (CDP-Rbo) synthase. Fukutin and fukutin-related protein are sequentially acting Rbo5P transferases that use CDP-Rbo. Consequently, Rbo5P glycosylation is defective in α-dystroglycanopathy models. Supplementation of CDP-Rbo to ISPD-deficient cells restored α-DG glycosylation. These findings establish the molecular basis of mammalian Rbo5P glycosylation and provide insight into pathogenesis and therapeutic strategies in α-DG-associated diseases.

Proteostasis declines with age, characterized by the accumulation of unfolded or damaged proteins. Recent studies suggest that proteins constituting pathological inclusions in neurodegenerative diseases also enter and accumulate in mitochondria. How unfolded proteins are managed within mitochondria remains unclear. Here, we found that excessive unfolded proteins in the mitochondrial matrix of yeast cells are consolidated into solid-phase inclusions, which we term deposits of unfolded mitochondrial proteins (DUMP). Formation of DUMP occurs in mitochondria near endoplasmic reticulum-mitochondria contact sites and is regulated by mitochondrial proteins controlling the production of cytidine 5'-diphosphate-diacylglycerol. DUMP formation is age dependent but accelerated by exogenous unfolded proteins. Many enzymes of the tricarboxylic acid cycle were enriched in DUMP. During yeast cell division, DUMP formation is necessary for asymmetric inheritance of damaged mitochondrial proteins between mother and daughter cells. We provide evidence that DUMP-like structures may be induced by excessive unfolded proteins in human cells.