Novel interaction of selenium-binding protein with glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase of Arabidopsis thaliana

2006 ◽  
Vol 33 (9) ◽  
pp. 847 ◽  
Author(s):  
Adamantia Agalou ◽  
Herman P. Spaink ◽  
Andreas Roussis
Keyword(s):  
Arabidopsis Thaliana ◽  
Metabolic Regulation ◽  
Binding Protein ◽  
Binding Assays ◽  
Fructose Bisphosphate ◽  
Metabolic Role ◽  
Fructose Bisphosphate Aldolase ◽  
Direct Binding ◽  
High Degree

The metabolic role and regulation of selenium, particularly in plants, is poorly understood. One of the proteins probably involved in the metabolic regulation of this element is the selenium-binding protein (SBP) with homologues present across prokaryotic and eukaryotic species. The high degree of conservation of SBP in different organisms suggests that this protein may play a role in fundamental biological processes. In order to gain insight into the biochemical function of SBP in plants we used the yeast two-hybrid system to identify proteins that potentially interact with an Arabidopsis thaliana (L.) Heynh. homologue. Among the putative binding partners of SBP, a NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a fructose-bisphosphate aldolase (FBA) were found as reliable positive candidates. The interaction of these proteins with SBP was confirmed by in vitro binding assays. Previous findings in Escherichia coli, demonstrated the direct binding of selenium to both GAPDH and aldolase. Therefore our results reveal the interaction, at least in pairs, of three proteins that are possibly linked to selenium and suggest the existence of a protein network consisting of at least SBP, GAPDH and FBA, triggered by or regulating selenium metabolism in plant cells.

scholarly journals Reinvestigation of the dysbindin subunit of BLOC-1 (biogenesis of lysosome-related organelles complex-1) as a dystrobrevin-binding protein

2006 ◽  
Vol 395 (3) ◽  
pp. 587-598 ◽  
Author(s):  
Ramin Nazarian ◽  
Marta Starcevic ◽  
Melissa J. Spencer ◽  
Esteban C. Dell'Angelica
Keyword(s):  
Recombinant Proteins ◽  
Binding Protein ◽  
Coiled Coil ◽  
Direct Interaction ◽  
Muscle Pathology ◽  
Binding Assays ◽  
Genetic Studies ◽  
Binding Interface ◽  
Lysosome Related Organelles

Dysbindin was identified as a dystrobrevin-binding protein potentially involved in the pathogenesis of muscular dystrophy. Subsequently, genetic studies have implicated variants of the human dysbindin-encoding gene, DTNBP1, in the pathogeneses of Hermansky–Pudlak syndrome and schizophrenia. The protein is a stable component of a multisubunit complex termed BLOC-1 (biogenesis of lysosome-related organelles complex-1). In the present study, the significance of the dystrobrevin–dysbindin interaction for BLOC-1 function was examined. Yeast two-hybrid analyses, and binding assays using recombinant proteins, demonstrated direct interaction involving coiled-coil-forming regions in both dysbindin and the dystrobrevins. However, recombinant proteins bearing the coiled-coil-forming regions of the dystrobrevins failed to bind endogenous BLOC-1 from HeLa cells or mouse brain or muscle, under conditions in which they bound the Dp71 isoform of dystrophin. Immunoprecipitation of endogenous dysbindin from brain or muscle resulted in robust co-immunoprecipitation of the pallidin subunit of BLOC-1 but no specific co-immunoprecipitation of dystrobrevin isoforms. Within BLOC-1, dysbindin is engaged in interactions with three other subunits, named pallidin, snapin and muted. We herein provide evidence that the same 69-residue region of dysbindin that is sufficient for dystrobrevin binding in vitro also contains the binding sites for pallidin and snapin, and at least part of the muted-binding interface. Functional, histological and immunohistochemical analyses failed to detect any sign of muscle pathology in BLOC-1-deficient, homozygous pallid mice. Taken together, these results suggest that dysbindin assembled into BLOC-1 is not a physiological binding partner of the dystrobrevins, likely due to engagement of its dystrobrevin-binding region in interactions with other subunits.

Abstract 15297: Discovery and Preclinical Optimization of Selective ABCA1 Inducers as Multifunctional Therapeutic Candidates

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Cutler Lewandowski ◽  
Md. Wasim Khan ◽  
Manel Ben Aissa ◽  
Brian Layden ◽  
Gregory Thatcher
Keyword(s):  
Liver Steatosis ◽  
Acid Synthesis ◽  
Luciferase Assay ◽  
Pharmacokinetic Parameters ◽  
Lead Compound ◽  
Binding Assays ◽  
Drug Candidates ◽  
Direct Binding ◽  
Isoform Selectivity

Introduction: Reduced expression of cholesterol transporter ABCA1 is critical in pathogenesis of type 2 diabetes (T2D) and related conditions, such as cardiovascular disease (CVD) and Alzheimer’s disease (AD). Thus, increasing ABCA1 represents a novel therapeutic strategy for these conditions. However, prior drug development efforts have achieved limited success at increasing ABCA1 (controlled by liver X receptor [LXR] β) while avoiding unwanted liver triglyceride production (through LXRα via transcription factor SREBP1c). Hypothesis: We hypothesized that phenotypic screening for selective ABCA1 inducers followed by medicinal chemistry optimization would bypass the isoform selectivity issues encountered in traditional target-based drug discovery and enable development of lead therapeutic candidates with preclinical efficacy and safety. Methods/Results: We screened 20k compounds for ABCA1 and SREBP1c-linked luciferase activity, followed by qPCR to validate and prioritize selective ABCA1-inducing hits. We synthesized ~70 structural analogs of the best hit, achieving substantial EC 50 (4.5 μM to 270 nM) and E max (3.5-fold to 6.0-fold vs. vehicle) improvements in ABCA1 luciferase assay while maintaining selectivity against SREBP1c. Direct binding assays confirmed selectivity for LXRβ vs. LXRα, corroborating cell-based data. Lead compounds enhanced cellular cholesterol efflux, reduced inflammation in vitro , and attenuated high-fat diet (HFD) induced weight gain, insulin resistance, and inflammation in mice. Metabolomics analysis revealed that our lead compound corrected HFD-induced perturbations in liver glucose and fatty acid synthesis. Finally, side effects associated with published LXR agonists - liver steatosis and neutropenia - were not observed with our compound. Conclusions: We established a platform to develop selective ABCA1 inducers as drug candidates. Via this platform, we identified a safe and efficacious lead compound for T2D. Our study also represents the first report of an LXR agonist characterized by metabolomics - a powerful tool to complement biochemical readouts. Continued optimization to improve pharmacokinetic parameters, plus evaluation in CVD and AD models, is ongoing.

Transactivation activity of the human cytomegalovirus IE2 protein occurs at steps subsequent to TATA box-binding protein recruitment

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 37-46 ◽  
Author(s):  
Jong-Mook Kim ◽  
Youngtae Hong ◽  
Kuan-Teh Jeang ◽  
Sunyoung Kim
Keyword(s):  
Human Cytomegalovirus ◽  
Binding Protein ◽  
Tata Box ◽  
Transactivation Domain ◽  
Binding Assays ◽  
Accelerate Rate ◽  
Tata Box Binding Protein ◽  
Protein Recruitment ◽  
Rate Limiting

The IE2 protein of human cytomegalovirus transactivates viral and cellular promoters through a wide variety of cis-elements, but the mechanism of its action has not been well characterized. Here, IE2–Sp1 synergy and IE2–TATA box-binding protein (TBP) interaction are examined by artificial recruitment of either Sp1 or TBP to the promoter. It was found that IE2 could cooperate with DNA-bound Sp1. A 117 amino acid glutamine-rich fragment of Sp1, which can interact with Drosophila TAFII110 and human TAFII130, was sufficient for the augmentation of IE2-driven transactivation. In binding assays in vitro, IE2 interacted directly with the C-terminal region of Sp1, which contains the zinc finger DNA-binding domain, but not with its transactivation domain, suggesting that synergy between IE2 and the transactivation domain of Sp1 might be mediated by other proteins such as TAF or TBP. It was also found that TBP recruitment to the promoter markedly increased IE2-mediated transactivation. Thus, IE2 acts synergistically with DNA-bound Sp1 and DNA-bound TBP. These results suggest that, in human cytomegalovirus IE2 transactivation, Sp1 functions at an early step such as recruitment of TBP and IE2 acts to accelerate rate-limiting steps after TBP recruitment.

scholarly journals Design of Epitope-Based Peptide Vaccine against Pseudomonas aeruginosa Fructose Bisphosphate Aldolase Protein Using Immunoinformatics

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Mustafa Elhag ◽  
Ruaa Mohamed Alaagib ◽  
Nagla Mohamed Ahmed ◽  
Mustafa Abubaker ◽  
Esraa Musa Haroun ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Peptide Vaccine ◽  
Multidrug Resistant ◽  
Fructose Bisphosphate ◽  
Mhc I ◽  
Mhc Ii ◽  
Fructose Bisphosphate Aldolase ◽  
Hospital Acquired

Pseudomonas aeruginosa is a common pathogen that is responsible for serious hospital-acquired infections, ventilator-associated pneumonia, and various sepsis syndromes. Also, it is a multidrug-resistant pathogen recognized for its ubiquity and its intrinsically advanced antibiotic-resistant mechanisms. It usually affects immunocompromised individuals but can also infect immunocompetent individuals. There is no vaccine against it available till now. This study predicts an effective epitope-based vaccine against fructose bisphosphate aldolase (FBA) of Pseudomonas aeruginosa using immunoinformatics tools. The protein sequences were obtained from NCBI, and prediction tests were undertaken to analyze possible epitopes for B and T cells. Three B cell epitopes passed the antigenicity, accessibility, and hydrophilicity tests. Six MHC I epitopes were found to be promising, while four MHC II epitopes were found promising from the result set. Nineteen epitopes were shared between MHC I and II results. For the population coverage, the epitopes covered 95.62% worldwide excluding certain MHC II alleles. We recommend in vivo and in vitro studies to prove its effectiveness.

Overexpression of maize transcription factor mEmBP-1 increases photosynthesis, biomass, and yield in rice

2020 ◽  
Vol 71 (16) ◽  
pp. 4944-4957 ◽  
Author(s):  
Shahnaz Perveen ◽  
Mingnan Qu ◽  
Faming Chen ◽  
Jemaa Essemine ◽  
Naveed Khan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Yield Potential ◽  
Fructose Bisphosphate ◽  
Gene Encoding ◽  
Transport Reaction ◽  
Chl A ◽  
Genes Encoding ◽  
Fructose Bisphosphate Aldolase ◽  
Direct Binding ◽  
Photosynthesis Genes

Abstract Identifying new options to improve photosynthetic capacity is a major approach to improve crop yield potential. Here we report that overexpression of the gene encoding the transcription factor mEmBP-1 led to simultaneously increased expression of many genes in photosynthesis, including genes encoding Chl a,b-binding proteins (Lhca and Lhcb), PSII (PsbR3 and PsbW) and PSI reaction center subunits (PsaK and PsaN), chloroplast ATP synthase subunit, electron transport reaction components (Fd1 and PC), and also major genes in the Calvin–Benson–Bassham cycle, including those encoding Rubisco, glyceraldehyde phosphate dehydrogenase, fructose bisphosphate aldolase, transketolase, and phosphoribulokinase. These increased expression of photosynthesis genes resulted in increased leaf chlorophyll pigment, photosynthetic rate, biomass growth, and grain yield both in the greenhouse and in the field. Using EMSA experiments, we showed that mEmBP-1a protein can directly bind to the promoter region of photosynthesis genes, suggesting that the direct binding of mEmBP-1a to the G-box domain of photosynthetic genes up-regulates expression of these genes. Altogether, our results show that mEmBP-1a is a major regulator of photosynthesis, which can be used to increase rice photosynthesis and yield in the field.

scholarly journals Effect of immunization of hamsters against recombinant P26h on fertility rates

Reproduction ◽  
2002 ◽  
pp. 307-313 ◽  
Author(s):  
C Gaudreault ◽  
L Montfort ◽  
R Sullivan
Keyword(s):  
Zona Pellucida ◽  
Binding Protein ◽  
Maltose Binding Protein ◽  
Fertilization Rate ◽  
Active Immunization ◽  
Antigenic Determinants ◽  
Binding Assays ◽  
Contraceptive Vaccine

Despite the various contraceptive methods available, an effective and inexpensive method remains to be established. Immunocontraception may help to achieve this goal. P26h has been proposed as a candidate for the development of a male contraceptive vaccine. P26h, a hamster sperm protein, interacts with the zona pellucida. Furthermore, in vivo fertilization can be blocked completely by active immunization of male hamsters against P26h. Maltose binding protein (MBP)-P26 shares antigenic determinants with the native P26h present on cauda epididymal spermatozoa. The aim of the present study was to reproduce the immunocontraceptive properties of native P26h by immunizing male hamsters against a recombinant P26h fused with a maltose binding protein (MBP). Active immunization of male hamsters with the MBP-P26h showed that specific anti-P26h circulating IgGs could be generated. Mating of immunized male hamsters with superovulated females resulted in a significant decrease, 20-25%, in the fertilization rate. This result is in agreement with results from in vitro sperm-zona pellucida binding assays. Indeed, the anti-recombinant P26h IgGs showed lower inhibitory properties when compared with anti-native P26h IgG. Despite the high anti-P26h IgG titres generated in hamsters, histological studies showed that active immunization has no pathological sequelae to the reproductive tissues. The potential of P26h as a candidate for a contraceptive vaccine is discussed.

scholarly journals Import of fructose bisphosphate aldolase into the glycosomes of Trypanosoma brucei.

1987 ◽  
Vol 105 (6) ◽  
pp. 2649-2654 ◽  
Author(s):  
C E Clayton
Keyword(s):  
Trypanosoma Brucei ◽  
Peptide Mapping ◽  
Tsetse Fly ◽  
In Vitro Translation ◽  
Fructose Bisphosphate ◽  
Structural Differences ◽  
Fructose Bisphosphate Aldolase ◽  
Made In

The glycolytic enzymes of Trypanosomatids are compartmentalized within peroxisome-like microbodies called glycosomes. Fructose bisphosphate aldolase is synthesized on free polysomes and imported into glycosomes within 5 min. Peptide mapping reveals no primary structural differences between the in vivo-synthesized protein and that made in vitro from a synthetic template. However, native aldolase from glycosomes is partially protease resistant, whereas the in vitro translation product is not. Pulse-chase results indicate that aldolase in bloodstream trypanosomes has a much longer half-life than in the procyclic tsetse fly form.

scholarly journals The Borrelia burgdorferi Glycosaminoglycan Binding Protein Bgp in the B31 Strain Is Not Essential for Infectivity despite Facilitating Adherence and Tissue Colonization

2017 ◽  
Vol 86 (2) ◽  
Author(s):  
Samantha Schlachter ◽  
Janakiram Seshu ◽  
Tao Lin ◽  
Steven Norris ◽  
Nikhat Parveen
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Binding Protein ◽  
Mammalian Host ◽  
Infection Model ◽  
Binding Assays ◽  
Content Type ◽  
Mouse Infection Model ◽  
Successful Infection

ABSTRACTThe Lyme disease-causing organismBorrelia burgdorferiis transmitted into the mammalian host by an infected-tick bite. Successful infection relies on the ability of this extracellular pathogen to persist and colonize different tissues.B. burgdorferiencodes a large number of adhesins that are able to interact with host ligands to facilitate adherence and tissue colonization. Multiple glycosaminoglycan binding proteins present inB. burgdorferioffer a degree of redundancy of function during infection, and this highlights the importance of glycosaminoglycans as host cell receptors for spirochete adherence. Of particular interest in this study isBorreliaglycosaminoglycan binding protein (Bgp), which binds to heparin-related glycosaminoglycans. The properties of abgptransposon mutant and atrans-complemented derivative were compared to those of the wild-typeB. burgdorferiin thein vitrobinding assays and in infection studies using a C3H/HeJ mouse infection model. We determined that the loss of Bgp impairs spirochete adherence, infectivity, and tissue colonization, resulting in a reduction of inflammatory manifestations of Lyme disease. Although Bgp is not essential for infectivity, it is an important virulence factor ofB. burgdorferithat allows adherence and tissue colonization and contributes to disease severity.

scholarly journals The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase

2019 ◽  
Vol 295 (7) ◽  
pp. 1867-1878 ◽  
Author(s):  
Jeroen G. Koendjbiharie ◽  
Shuen Hon ◽  
Martin Pabst ◽  
Robert Hooftman ◽  
David M. Stevenson ◽  
...  
Keyword(s):  
Pentose Phosphate ◽  
Dihydroxyacetone Phosphate ◽  
Cooperative Binding ◽  
Fructose Bisphosphate ◽  
Pentose Metabolism ◽  
Fructose Bisphosphate Aldolase ◽  
Clostridium Thermosuccinogenes ◽  
High Degree ◽  
Pentose Sugars ◽  
Substrate Concentrations

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C5 precursors (such as ribose) during growth on other (non-C5) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PPi-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PPi-PFKs of Clostridium thermosuccinogenes and Clostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli, which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PPi-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of C. thermosuccinogenes that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its K½ value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.

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