Guanosine Diphosphate Research Articles

Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.

Identification of Guanosine 5\u2032-diphosphate as Potential Iron Mobilizer: Preventing the Hepcidin-Ferroportin Interaction and Modulating the Interleukin-6/Stat-3 Pathway

Hepcidin, a peptide hormone, is a key regulator in mammalian iron homeostasis. Increased level of hepcidin due to inflammatory conditions stimulates the ferroportin (FPN) transporter internalization, impairing the iron absorption; clinically manifested as anemia of inflammation (AI). Inhibiting hepcidin-mediated FPN degradation is proposed as an important strategy to combat AI. A systematic approach involving in silico, in vitro, ex vivo and in vivo studies is employed to identify hepcidin-binding agents. The virtual screening of 68,752 natural compounds via molecular docking resulted into identification of guanosine 5′-diphosphate (GDP) as a promising hepcidin-binding agent. The molecular dynamics simulations helped to identify the important hepcidin residues involved in stabilization of hepcidin-GDP complex. The results gave a preliminary indication that GDP may possibly inhibit the hepcidin-FPN interactions. The in vitro studies revealed that GDP caused FPN stabilization (FPN-GFP cell lines) and increased the FPN-mediated cellular iron efflux (HepG2 and Caco-2 cells). Interestingly, the co-administration of GDP and ferrous sulphate (FeSO4) ameliorated the turpentine-induced AI in mice (indicated by increased haemoglobin level, serum iron, FPN expression and decreased ferritin level). These results suggest that GDP a promising natural small-molecule inhibitor that targets Hepcidin-FPN complex may be incorporated with iron supplement regimens to ameliorate AI.

Roles of Rap1 signaling in tumor cell migration and invasion.

Ras-associated protein-1 (Rap1), a small GTPase in the Ras-related protein family, is an important regulator of basic cellular functions (e.g., formation and control of cell adhesions and junctions), cellular migration, and polarization. Through its interaction with other proteins, Rap1 plays many roles during cell invasion and metastasis in different cancers. The basic function of Rap1 is straightforward; it acts as a switch during cellular signaling transduction and regulated by its binding to either guanosine triphosphate (GTP) or guanosine diphosphate (GDP). However, its remarkably diverse function is rendered by its interplay with a large number of distinct Rap guanine nucleotide exchange factors and Rap GTPase activating proteins. This review summarizes the mechanisms by which Rap1 signaling can regulate cell invasion and metastasis, focusing on its roles in integrin and cadherin regulation, Rho GTPase control, and matrix metalloproteinase expression.

Elucidation of Single Hydrogen Bonds in GTPases via Experimental and Theoretical Infrared Spectroscopy

Time-resolved Fourier transform infrared (FTIR) spectroscopy is a powerful tool to elucidate label-free the reaction mechanisms of proteins. After assignment of the absorption bands to individual groups of the protein, the order of events during the reaction mechanism can be monitored and rate constants can be obtained. Additionally, structural information is encoded into infrared spectra and can be decoded by combining the experimental data with biomolecular simulations. We have determined recently the infrared vibrations of GTP and guanosine diphosphate (GDP) bound to Gαi1, a ubiquitous GTPase. These vibrations are highly sensitive for the environment of the phosphate groups and thereby for the binding mode the GTPase adopts to enable fast hydrolysis of GTP. In this study we calculated these infrared vibrations from biomolecular simulations to transfer the spectral information into a computational model that provides structural information far beyond crystal structure resolution. Conformational ensembles were generated using 15 snapshots of several 100 ns molecular-mechanics/molecular-dynamics (MM-MD) simulations, followed by quantum-mechanics/molecular-mechanics (QM/MM) minimization and normal mode analysis. In comparison with other approaches, no time-consuming QM/MM-MD simulation was necessary. We carefully benchmarked the simulation systems by deletion of single hydrogen bonds between the GTPase and GTP through several Gαi1 point mutants. The missing hydrogen bonds lead to blue-shifts of the corresponding absorption bands. These band shifts for α-GTP (Gαi1-T48A), γ-GTP (Gαi1-R178S), and for both β-GTP/γ-GTP (Gαi1-K46A, Gαi1-D200E) were found in agreement in the experimental and the theoretical spectra. We applied our approach to open questions regarding Gαi1: we show that the GDP state of Gαi1 carries a Mg2+, which is not found in x-ray structures. Further, the catalytic role of K46, a central residue of the P-loop, and the protonation state of the GTP are elucidated.

Modelling and receptor-based virtual screening studies of GPR139

GPR139 belongs to Class A of GPCRs family and plays key role in molecular signalling through activation of receptors and promotes exchange of Guanosine Di-phosphate (GDP) to Guanosine Tri-phosphate (GTP). GPR139 is specifically involved in neuropeptide signalling pathway, phospholipase C-activating G-protein-coupled receptor signalling pathway and is coupled to IP3 second messenger (phospholipase C activating). In this article, we report 3D structure prediction of GPR139 using threading and ab initio methods. The models were validated and optimised. Molecular dynamics simulation of GPR139 was performed for 300 ns to investigate the dynamic perturbations in predicted model, particularly variations in seven transmembrane domains and active site residues. The active site residues were identified and the 3D model was screened against large databases of chemical libraries for identification of potential lead compounds which could bind to GPR139 receptor and activate signal transduction. The screened compounds were further refined through free binding energy calculations (ΔGbind) using generalised born and surface area continuum solvation for estimating ligand-binding affinities to GPR139.

Exome sequencing identifies a novel mutation of the GDI1 gene in a Chinese non-syndromic X-linked intellectual disability family.

X-linked intellectual disability (XLID) has been associated with various genes. Diagnosis of XLID, especially for non-syndromic ones (NS-XLID), is often hampered by the heterogeneity of this disease. Here we report the case of a Chinese family in which three males suffer from intellectual disability (ID). The three patients shared the same phenotype: no typical clinical manifestation other than IQ score ≤ 70. For a genetic diagnosis for this family we carried out whole exome sequencing on the proband, and validated 16 variants of interest in the genomic DNA of all the family members. A missense mutation (c.710G > T), which mapped to exon 6 of the Rab GDP-Dissociation Inhibitor 1 (GDI1) gene, was found segregating with the ID phenotype, and this mutation changes the 237th position in the guanosine diphosphate dissociation inhibitor (GDI) protein from glycine to valine (p. Gly237Val). Through molecular dynamics simulations we found that this substitution results in a conformational change of GDI, possibly affecting the Rab-binding capacity of this protein. In conclusion, our study identified a novel GDI1 mutation that is possibly NS-XLID causative, and showed that whole exome sequencing provides advantages for detecting novel ID-associated variants and can greatly facilitate the genetic diagnosis of the disease.

Mutations in GMPPB Presenting with Pseudometabolic Myopathy.

Mutations in the guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B (GMPPB) gene encoding a key enzyme of the glycosylation pathway have been described in families with congenital (CMD) and limb girdle (LGMD) muscular dystrophy with reduced alpha-dystroglycan (α-DG) at muscle biopsy.Patients typically display a combined phenotype of muscular dystrophy, brain malformations, and generalized epilepsy. However, a wide spectrum of clinical severity has been described ranging from classical CMD presentation to children with mild, yet progressive LGMD with or without intellectual disability. Cardiac involvement, including a long QT interval and left ventricular dilatation, has also been described in four cases.Two missense mutations in GMPPB gene, one novel and one already reported, have been identified in a 21-year-old man presenting with elevated CK (38,650UI/L; normal values <150UI/L) without overt muscle weakness. Major complaints included limb myalgia, exercise intolerance, and several episodes of myoglobinuria consistent with a form of metabolic myopathy. Muscle biopsy showed only minimal alterations, whereas a marked reduction of glycosylated α-DG was evident.This case further expands the phenotypic spectrum of GMPPB mutations and highlights the importance of exhaustive molecular characterization of patients with reduced glycosylation of α-DG at muscle biopsy.

Meaningful respirometric measurements of UCP1-mediated thermogenesis

Regarding the enormous interest in brown and brite/beige adipose tissue in the context of metabolic disease, reliable quantification of thermogenesis in these adipocytes is a central issue. This requires an assay specific for uncoupling protein 1 (UCP1) mediated thermogenesis in adherent intact cells. In a recent study we identified a major pitfall associated with established procedures generally applied for this purpose. Meaningful respirometry of UCP1-mediated thermogenesis imperatively requires activation of UCP1 and control over free fatty acid levels. By comparison of respiration profiles of wild-type (WT) and UCP1 knock-out (KO) cells we reproducibly quantified the thermogenic capacity enabled by UCP1 in both brown and brite adipocytes. Employing this protocol, we demonstrated that (1) brite adipocytes display a similar thermogenic capacity as classical brown adipocytes, (2) variations in brite adipogenesis known for inbred mouse strains are associated with differential capacities for thermogenesis in these cells, and (3) adipose triglyceride lipase (ATGL) activity is required for UCP1 activation in intact cells. We here further refined our cell-based respirometry assay by implementation of two strategies to inhibit UCP1 in WT cells. First, we employed the purine nucleotide guanosine diphosphate (GDP) to directly quantify the fraction of thermogenesis enabled by UCP1 activity. Second, applying siRNA mediated knockdown of UCP1 and ATGL we demonstrated the feasibility of this technology to study the functional relevance of candidate genes for thermogenesis in brown and brite adipocytes.

Effects of Prunus mume Siebold & Zucc. in the pacemaking activity of interstitial cells of Cajal in murine small intestine.

Interstitial cells of Cajal (ICCs) function as pacemaker cells in the gastrointestinal (GI) tract and therefore, serve an important role in regulating GI motility. The effects of a species of plum (Prunus mume Siebold & Zucc.) on cultured ICC cluster-induced pacemaker potentials in the mouse small intestine were investigated, and the effects of a methanolic extract of Prunus mume (m-PM) on ICC pacemaker activities were examined using the whole-cell patch-clamp technique. ICC pacemaker membrane potentials were depolarized by m-PM in a concentration dependent manner in current clamp mode. 4-Diphenylacetoxy-N-methyl-piperidine methiodide, which is a muscarinic 3 (M3) receptor antagonist, was able to block m-PM-induced pacemaker potential increases, whereas methoctramine, which is a muscarinic 2 (M2) receptor antagonist, was not. When 1 mM guanosine diphosphate β-5 was present in the pipette solution, m-PM induced slight pacemaker depolarization. Following pretreatment in bath solution of Ca2+-free solution or a Ca2+-ATPase inhibitor in endoplasmic reticulum, the pacemaker currents were inhibited. Furthermore, pretreatment with PD98059, SB203580 or SP600125, which is a c-jun NH2-terminal kinase inhibitor, blocked m-PM-induced ICC potential depolarization. Furthermore, m-PM inhibited transient receptor potential melastatin (TRPM) 7 channels, but did not affect Ca2+-activated Cl- channels. These results suggest that m-PM is able to modulate pacemaker potentials through the muscarinic M3 receptor, via G-protein and external and internal Ca2+, in a mitogen-activated protein kinase and TRPM7-dependent manner. Therefore, m-PM may provide a basis for the development of a novel gastroprokinetic agent.

Improved production of 2′-fucosyllactose in engineered Escherichia coli by expressing putative α-1,2-fucosyltransferase, WcfB from Bacteroides fragilis

2′-Fucosyllactose (2-FL) is one of most abundant oligosaccharides in human milk, which is involved in many biological functions for infant health. Since 2-FL has a great potential in application to functional food materials and pharmaceuticals, several microbial systems for mass production of 2-FL have been developed in recent years. Microbial production of 2-FL was suggested to be influenced by a number of factors including fucosylation activity of α-1,2-fucosyltransferase. In the present study, the wcfB gene coding for α-1,2-fucosyltransferase from Bacteroides fragilis was screened from eleven candidates of putative α-1,2-fucosyltransferase. Introduction of the wcfB gene allows the lacZ-deleted strain of E. coli expressing the genes for guanosine 5′-diphosphate (GDP)-l-fucose biosynthetic enzymes to produce 2-FL. As a result of fed-batch fermentation, 15.4g/L extracellular concentration of 2-FL with 2-FL yield of 0.858g/g lactose and productivity of 0.530g/L/h were obtained. In addition, the feasibility of industrial production of 2-FL using this microbial system was demonstrated by performing fed-batch fermentation in a 75L bioreactor.

Impact of G12 Mutations on the Structure of K-Ras Probed by Ultraviolet Photodissociation Mass Spectrometry.

Single-residue mutations at Gly12 (G12X) in the GTP-ase protein K-Ras can lead to activation of different downstream signaling pathways, depending on the identity of the mutation, through a poorly defined mechanism. Herein, native mass spectrometry combined with top-down ultraviolet photodissociation (UVPD) was employed to investigate the structural changes occurring from G12X mutations of K-Ras. Complexes between K-Ras or the G12X mutants and guanosine 5'-diphosphate (GDP) or GDPnP (a stable GTP analogue) were transferred to the gas phase by nano-electrospray ionization and characterized using UVPD. Variations in the efficiencies of backbone cleavages were observed upon substitution of GDPnP for GDP as well as for the G12X mutants relative to wild-type K-Ras. An increase in the fragmentation efficiency in the segment containing the first 50 residues was observed for the K-Ras/GDPnP complexes relative to the K-Ras/GDP complexes, whereas a decrease in fragmentation efficiency occurred in the segment containing the last 100 residues. Within these general regions, the specific residues at which changes in fragmentation efficiency occurred correspond to the phosphate and guanine binding regions, respectively, and are indicative of a change in the binding motif upon replacement of the ligand (GDP versus GDPnP). Notably, unique changes in UVPD were observed for each G12X mutant with the cysteine and serine mutations exhibiting similar UVPD changes whereas the valine mutation was significantly different. These findings suggest a mechanism that links the identity of the G12X substitution to different downstream effects through long-range conformational or dynamic effects as detected by variations in UVPD fragmentation.

Fucosylation Deficiency in Mice Leads to Colitis and Adenocarcinoma

De novo synthesis of guanosine diphosphate (GDP)-fucose, a substrate for fucosylglycans, requires sequential reactions mediated by GDP-mannose 4,6-dehydratase (GMDS) and GDP-4-keto-6-deoxymannose 3,5-epimerase-4-reductase (FX or tissue specific transplantation antigen P35B [TSTA3]). GMDS deletions and mutations are found in 6%-13% of colorectal cancers; these mostly affect the ascending and transverse colon. We investigated whether a lack of fucosylation consequent to loss of GDP-fucose synthesis contributes to colon carcinogenesis. FX deficiency and GMDS deletion produce the same biochemical phenotype of GDP-fucose deficiency. We studied a mouse model of fucosylation deficiency (Fx-/- mice) and mice with the full-length Fx gene (controls). Mice were placed on standard chow or fucose-containing diet (equivalent to a control fucosylglycan phenotype). Colon tissues were collected and analyzed histologically or by enzyme-linked immunosorbent assays to measure cytokine levels; T cells also were collected and analyzed. Fecal samples were analyzed by 16s ribosomal RNA sequencing. Mucosal barrier function was measured by uptake of fluorescent dextran. We transplanted bone marrow cells from Fx-/- or control mice (Ly5.2) into irradiated 8-week-old Fx-/- or control mice (Ly5.1). We performed immunohistochemical analyses for expression of Notch and the hes family bHLH transcription factor (HES1) in colon tissues from mice and a panel of 60 human colorectal cancer specimens (27 left-sided, 33 right-sided). Fx-/- mice developed colitis and serrated-like lesions. The intestinal pathology of Fx-/- mice was reversed by addition of fucose to the diet, which restored fucosylation via a salvage pathway. In the absence of fucosylation, dysplasia appeared and progressed to adenocarcinoma in up to 40% of mice, affecting mainly the right colon and cecum. Notch was not activated in Fx-/- mice fed standard chow, leading to decreased expression of its target Hes1. Fucosylation deficiency altered the composition of the fecal microbiota, reduced mucosal barrier function, and altered epithelial proliferation marked by Ki67. Fx-/- mice receiving control bone marrow cells had intestinal inflammation and dysplasia, and reduced expression of cytokines produced bycytotoxic T cells. Human sessile serrated adenomas and right-sided colorectal tumors with epigenetic loss of MutL homolog 1 (MLH1) had lost orhad lower levels of HES1 than other colorectal tumor types ornontumor tissues. In mice, fucosylation deficiency leads to colitis and adenocarcinoma, loss of Notch activation, and down-regulation of Hes1. HES1 loss correlates with the development of human right-sided colorectal tumors with epigenetic loss of MLH1. These findings indicate that carcinogenesis in a subset of colon cancer is consequent to a molecular mechanism driven by fucosylation deficiency and/or HES1-loss.

Structures of NS5 Methyltransferase from Zika Virus

SUMMARYThe Zika virus (ZIKV) poses a major public health emergency. To aid in the development of antivirals, we present two high-resolution crystal structures of the ZIKV NS5 methyltransferase: one bound to S-adenosylmethionine (SAM) and the other bound to SAM and 7-methyl guanosine diphosphate (7-MeGpp). We identify features of ZIKV NS5 methyltransferase that lend to structure-based antiviral drug discovery. Specifically, SAM analogs with functionalities on the Cβ atom of the methionine portion of the molecules that occupy the RNA binding tunnel may provide better specificity relative to human RNA methyltransferases.

Recent advances in congenital myasthenic syndromes

AbstractCongenital myasthenic syndromes (CMS) are heterogeneous disorders caused by germline mutations in genes expressed at the neuromuscular junction. Mutations have been identified in 24 genes encoding acetylcholine receptor subunits (CHRNA1, CHRNB1, CHRND, CHRNE and CHRNG), skeletal muscle sodium channel (SCN4A), signaling molecules driving acetylcholine receptor subunits clustering (AGRN, LRP4, MUSK and DOK7), synaptic structural proteins (COLQ, LAMB2 and COL13A1), postsynaptic structural proteins (RAPSN and PLEC), presynaptic molecules (CHAT, SYT2), glycosylation enzymes (GFPT1, DPAGT1, ALG2, ALG14 and GMPPB), and other less characterized molecules (PREPL and SCL25A1). CMS are recessive disorders, except for slow channel CMS and synaptotagmin 2 (SYT2)‐CMS. Onsets are largely less than 2 years, but adult‐onset is not rare, especially in slow‐channel CMS and limb‐girdle type CMS caused by glycosylation defects and by DOK7 mutations. Clinical features include fatigable muscle weakness, amyotrophy and minor facial anomalies. Eye, facial and bulbar muscles are frequently affected, but sparing of these muscles is observed, especially in limb‐girdle type CMS. Serum creatine kinase levels are frequently elevated in slow‐channel CMS, glutamine‐fructose‐6‐phosphate aminotransferase 1 (GFPT1)‐CMS, and guanosine diphosphate mannose pyrophosphorylase B (GMPPB)‐CMS. Electrophysiological findings supporting compromised neuromuscular signal transmission are a prerequisite for diagnosing CMS. Most CMS patients are likely to be underdiagnosed, and recognition of CMS in undiagnosed muscle weakness and/or amyotrophy is critical for diagnosing CMS.

Rhodomyrtone Target Exploration: Computer Aided Search on Staphylococcus aureus Key Proteins as a Potential Therapeutic Target.

Methicillin-resistant Staphylococcus aureus (MRSA) has been a global public health problem because MRSA infection often leads to poor clinical outcomes. Currently, the search for an effective candidate has been ongoing. Rhodomyrtone, a natural compound, has been exhibited strong anti-MRSA activity comparable to that of vancomycin, a drug of choice for MRSA treatment. An important procedure to develop the compound in clinical use is elucidation of its molecular mechanism. However, previous attempts were performed to clarify the mechanism but ambiguity still exists. With this aspect, computer aided techniques to identify drug targets is able to enhance a success rate in drug discovery. Fifty MRSA proteins, playing roles in vital processes, was screened rhodomyrtone molecular targets. The molecular docking study was operated using AutoDock4. To confirm two possible targets, checkerboard assay and cell visualization were further carried out. Rhodomyrtone exhibited an interesting efficacy towards one-fifth of the given proteins. Moreover, metaldependent phosphate binding proteins were excluded from possible targets because of electrostatic forces. Amongst chosen proteins, rhodomyrtone, both enantiomers, displayed significant potency to dihydrofolate reductase (DHFR) and filamenting temperature-sensitive Z (FtsZ) proteins, compared to their natural substrates/inhibitors. However, protein cofactors such as nicotinamide adenine dinucleotide phosphate or guanosine diphosphate decreased rhodomyrtone binding affinity. This information suggested a cofactor free DHFR and a ligand-unbound FtsZ are likely to prove to be rhodomyrtone targets for MRSA inhibition. In addition, checkerboard assay and cell visualization gave a hint on target confirmation. We have proposed potential rhodomyrtone targets, and DHFR and FtsZ caught our interest. Further studies will need to focus on profound molecular information concerning the rhodomyrtone response to these proteins, both in experimental and computational views.

High-Sensitivity and Low-Toxicity Fucose Probe for Glycan Imaging and Biomarker Discovery

Fucose, a terminal sugar in glycoconjugates, criticallyregulates various physiological and pathological phenomena, including cancer development andinflammation. However, there are currently no probes for efficient labeling and detection of this sugar. We chemically synthesized a novel series of alkynyl-fucose analogs as probe candidates and found that 7-alkynyl-fucose gave the highest labeling efficiency and low cytotoxicity. Among the fucose analogs, 7-alkynyl-fucose was the best substrate against all five fucosyltransferases examined. We confirmed its conversion to the corresponding guanosine diphosphate derivative in cells and foundthat cellular glycoproteins were labeled much moreefficiently with 7-alkynyl-fucose than with an existing probe. 7-Alkynyl-fucose was detected in the N-glycan core by mass spectrometry, and 7-alkynyl-fucose-modified proteins mostly disappeared in core-fucose-deficient mouse embryonic fibroblasts, suggesting that this analog mainly labeled core fucose in these cells. These results indicate that 7-alkynyl-fucose is a highly sensitive and powerful tool for basic glycobiology research and clinical application for biomarker discovery.

Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins.

Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV) and murine norovirus (MNV). These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5′ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP) moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

Metabolic engineering of Escherichia coli to produce 2'-fucosyllactose via salvage pathway of guanosine 5'-diphosphate (GDP)-l-fucose.

2'-Fucosyllactose (2-FL) is one of the key oligosaccharides in human milk. In the present study, the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway from fucose was employed in engineered Escherichia coli BL21star(DE3) for efficient production of 2-FL. Introduction of the fkp gene coding for fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the fucT2 gene encoding α-1,2-fucosyltransferase from Helicobacter pylori allows the engineered E. coli to produce 2-FL from fucose, lactose and glycerol. To enhance the lactose flux to 2-FL production, the attenuated, and deleted mutants of β-galactosidase were employed. Moreover, the 2-FL yield and productivity were further improved by deletion of the fucI-fucK gene cluster coding for fucose isomerase (FucI) and fuculose kinase (FucK). Finally, fed-batch fermentation of engineered E. coli BL21star(DE3) deleting lacZ and fucI-fucK, and expressing fkp and fucT2 resulted in 23.1 g/L of extracellular concentration of 2-FL and 0.39 g/L/h productivity. Biotechnol. Bioeng. 2016;113: 2443-2452. © 2016 Wiley Periodicals, Inc.

Fatty liver disease induced by perfluorooctane sulfonate: Novel insight from transcriptome analysis

Perfluorooctane sulfonate (PFOS), a hepato-toxicant and potential non-genotoxic carcinogen, was widely used in industrial and commercial products. Recent studies have revealed the ubiquitous occurrence of PFOS in the environment and in humans worldwide. The widespread contamination of PFOS in human serum raised concerns about its long-term toxic effects and its potential risks to human health. Using fatty liver mutant foie gras (fgr−/−)/transport protein particle complex 11 (trappc11−/−) and PFOS-exposed wild-type zebrafish embryos as the study model, together with RNA sequencing and comparative transcriptomic analysis, we identified 499 and 1414 differential expressed genes (DEGs) in PFOS-exposed wild-type and trappc11 mutant zebrafish, respectively. Also, the gene ontology analysis on common deregulated genes was found to be associated with different metabolic processes such as the carbohydrate metabolic process, glycerol ether metabolic process, mannose biosynthetic process, de novo’ (Guanosine diphosphate) GDP-l-fucose biosynthetic process, GDP-mannose metabolic process and galactose metabolic process. Ingenuity Pathway Analysis further highlighted that these deregulated gene clusters are closely related to hepatitis, inflammation, fibrosis and cirrhosis of liver cells, suggesting that PFOS can cause liver pathogenesis and non-alcoholic fatty liver disease in zebrafish. The transcriptomic alterations revealed may serve as biomarkers for the hepatotoxic effect of PFOS.

Interaction of Heterotrimeric G-Protein Components with Receptor-like Kinases in Plants: An Alternative to the Established Signaling Paradigm?

Heterotrimeric G proteins comprising Gα, Gβ, and Gγ subunits (hereafter termed G proteins) mediate important signaling processes in all eukaryotes. As per the established paradigm, during resting phase the Gα is guanosine diphosphate (GDP)-bound and the three subunits form an inactive trimeric complex GDP⋅Gαβγ. Signal perception occurs at the G-protein coupled receptors (GPCRs), which act as guanine nucleotide exchange factors (GEFs) and facilitate the exchange of GDP for guanosine triphosphate (GTP) on Gα.