Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline

Background

An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N-acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one.

Methods

We carried out the transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk.

Results

Endo-M-N175Q showed efficient transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield.

Conclusions

Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed transglycosylation.

General significance

Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.     

O-GlcNAc modification affects the ATM-mediated DNA damage response

Background

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. The role of O-GlcNAcylation in the ataxia-telangiectasia mutated (ATM)-mediated DNA damage response is unknown. It is unclear whether ATM, which is an early acting and central component of the signal transduction system activated by DNA double strand breaks, is an O-GlcNAc-modified protein.

Methods

The effect of O-GlcNAc modification on ATM activation was examined using two inhibitors, PUGNAc and DON that increase and decrease, respectively, levels of protein O-GlcNAcylation. To assess O-GlcNAcylation of ATM, immunoprecipitation and immunoblot analyses using anti-ATM or anti-O-GlcNAc antibody were performed in HeLa cells and primary cultured neurons. Interaction of ATM with O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc to target proteins, was examined by immunoprecipitation and immunoblot analyses using anti-ATM.

Results

Enhancement of protein O-GlcNAcylation increased levels of X-irradiation-induced ATM activation. However, decreases in protein O-GlcNAcylation did not affect levels of ATM activation, but these decreases did delay ATM activation and ATM recovery processes based on assessment of de-phosphorylation of phospho-ATM. Thus, activation and recovery of ATM were affected by O-GlcNAcylation. ATM was subjected to O-GlcNAcylation, and ATM interacted with OGT. The steady-state O-GlcNAc level of ATM was not significantly responsive to X-irradiation or oxidative stress.

General significance

ATM is an O-GlcNAc modified protein, and dynamic O-GlcNAc modification affects the ATM-mediated DNA damage response.     

Addressing the use of PDIF-CN2 molecules in the development of n-type organic field-effect transistors for biosensing applications

Background

There is no doubt that future discoveries in the field of biochemistry will depend on the implementation of novel biosensing techniques, able to record biophysiological events with minimal biological interference. In this respect, organic electronics may represent an important new tool for the analysis of structures ranging from single molecules up to cellular events. Specifically, organic field-effect transistors (OFET) are potentially powerful devices for the real-time detection/transduction of bio-signals. Despite this interest, up to date, the experimental data useful to support the development of OFET-based biosensors are still few and, in particular, n-type (electron-transporting) devices, being fundamental to develop highly-performing circuits, have been scarcely investigated.

Methods

Here, films of N,N′-1H,1H-perfluorobutyldicyanoperylene-carboxydi-imide (PDIF-CN₂) molecules, a recently-introduced and very promising n-type semiconductor, have been evaporated on glass and silicon dioxide substrates to test the biocompatibility of this compound and its capability to stay electrically-active even in liquid environments.

Results

We found that PDIF-CN₂ transistors can work steadily in water for several hours. Biocompatibility tests, based on in-vitro cell cultivation, remark the need to functionalize the PDIF-CN₂ hydrophobic surface by extra-coating layers (i.e. poly-l-lysine) to favor the growth of confluent cellular populations.

Conclusions

Our experimental data demonstrate that PDIF-CN₂ compound is an interesting organic semiconductor to develop electronic devices to be used in the biological field.

General significance

This work contributes to define a possible strategy for the fabrication of low-cost and flexible biosensors, based on complex organic complementary metal-oxide-semiconductor (CMOS) circuitry including both p- (hole-transporting) and n-type transistors. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

VP2118 has major roles in Vibrio parahaemolyticus response to oxidative stress

Background

Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus, a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus. This study aims to clarify the role of V. parahaemolyticus SODs against ROS.

Methods

V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured.

Results

Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance.

Conclusion

VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses.

General significance

The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.     

Purification,characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells

Background

Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.

Methods

Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.

Results

A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates, and GalNAcβ1–4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells.

Conclusions

The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.

General significance

CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.     

A pentapeptide signature motif plays a pivotal role in Leishmania DNA topoisomerase IB activity and camptothecin sensitivity

Background

Leishmania donovani – the causative agent of visceral leishmaniasis – has several evolutionary characteristics that make the disease difficult to combat. Among these differences, a rare heterodimeric DNA topoisomerase IB has been reported thus opening a new promising field in the therapy of leishmaniasis. Several studies of the human enzyme have pointed to the importance of the linker domain in respect to camptothecin sensitivity. At present, it has been impossible to pinpoint the regions that make up the linker domain in Leishmania.

Methods

Several site-directed mutations as well as internal and linear truncations involving both subunits were assayed on both, relaxation activity and sensitivity to camptothecin.

Results

Truncations performed on the trypanosomatids conserved motif (RPPVVRS) of the small subunit of leishmanial DNA topoisomerase IB demonstrated that elimination of pentapeptide RPPVV produced a nonfunctional enzyme. However, the removal of the dipeptide RS led to an enzyme with reduced relaxation activity and less sensitivity to camptothecin. The basic structure, both sensitive to camptothecin and able to fully relax DNA, composed of amino acids 1–592 and 175–262 in the large and small subunits, respectively.

Conclusion

It has been established that the region between amino acids 175 and 180 (RPPVV) of the small subunit plays a pivotal role in both interaction with the large subunit and sensitivity to camptothecin in Leishmania.

General significance

The present report describes a functional analysis of the leishmanial DNA topoisomerase IB regions directly involved both in sensitivity to poisons and in the conformation of the linker domain.     

A plant peptide: N-glycanase orthologue facilitates glycoprotein ER-associated degradation in yeast

Background

The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme involved in the ER-associated degradation (ERAD) process, while ERAD-independent activities are also reported. Previous biochemical analyses indicated that the cytoplasmic PNGase orthologue in Arabidopsis thaliana (AtPNG1) can function as not only PNGase but also transglutaminase, while its in vivo function remained unclarified.

Methods

AtPNG1 was expressed in Saccharomyces cerevisiae and its in vivo role on PNGase-dependent ERAD pathway was examined.

Results

AtPNG1 could facilitate the ERAD through its deglycosylation activity. Moreover, a catalytic mutant of AtPNG1 (AtPNG1(C251A)) was found to significantly impair the ERAD process. This result was found to be N-glycan-dependent, as the AtPNG(C251A) did not affect the stability of the non-glycosylated RTA? (ricin A chain non-toxic mutant). Tight interaction between AtPNG1(C251A) and the RTA? was confirmed by co-immunoprecipitation analysis.

Conclusion

The plant PNGase facilitates ERAD through its deglycosylation activity, while the catalytic mutant of AtPNG1 impair glycoprotein ERAD by binding to N-glycans on the ERAD substrates.

General significance

Our studies underscore the functional importance of a plant PNGase orthologue as a deglycosylating enzyme involved in the ERAD.     

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

Background

F₁F_O ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.

Methods

We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.

Results

We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F₁F_O ATP synthases.

Conclusions

Our system now allows performing previously impossible structural and functional studies on A. aeolicus F₁F_O ATP synthase.

General significance

More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.     

Oxidation of melatonin by AAPH-derived peroxyl radicals: Evidence of a pro-oxidant effect of melatonin

Background

Melatonin is well-established as a powerful reducing agent of oxidant generated in the cell medium. We aimed to investigate how readily melatonin is oxidized by peroxyl radicals ROO⋅ generated by the thermolysis of 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and the role of glutathione (GSH) during the reaction course.

Methods

Chromatographic, mass spectroscopy, and UV–visible spectrometric techniques were used to study the oxidation of melatonin by ROO⋅ or horseradish peroxidase (HRP)/H₂O₂. Our focus was the characterization of products and the study of features of the reaction.

Results

We found that N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK) and a monohydroxylated derivative of melatonin were the main products of the reaction between melatonin and ROO⋅. Higher pH or saturation of the medium with molecular oxygen increased the yield of AFMK but did not affect the reaction rate. Melatonin increased the depletion of intracellular GSH mediated by AAPH. Using the HRP/H₂O₂ as the oxidant system, the addition of melatonin promoted the oxidation of GSH to GSSG.

Conclusions

These results show, for the first time, that melatonin radical is able to oxidize GSH.

General significance

We propose that this new property of melatonin could explain or be related to the recently reported pro-oxidant activities of melatonin.     

Different endocytic functions of AGEF-1 in C. elegans coelomocytes

Background

ADP-ribosylation factors (ARFs) are a family of small GTP-binding proteins that play roles in membrane dynamics and vesicle trafficking. AGEF-1, which is thought to act as a guanine nucleotide exchange factor of class I ARFs, is required for caveolin-1 body formation and receptor-mediated endocytosis in oocytes of Caenorhabditis elegans. This study explores additional roles of AGEF-1 in endocytic transport.

Methods

agef-1 expression was knocked down by using RNAi in C. elegans. Markers that allow analysis of endocytic transport in scavenger cells were investigated for studying the effect of AGEF-1 on different steps of membrane transport.

Results

Knockdown of AGEF-1 levels results in two apparent trafficking defects in coelomocytes of C. elegans. First, there is a delay in the uptake of solutes from the extracellular medium. Second, there is a dramatic enlargement of the sizes of lysosomes, even though lysosomal acidification is normal and degradation still occurs.

Conclusion

Our results suggest that AGEF-1 regulates endosome/lysosome fusion or fission events, in addition to earlier steps in endocytic transport.

General significance

AGEF-1 is the first identified GTPase regulator that functions at the lysosome fusion or fission stage of the endocytic pathway. Our study provides insight into lysosome dynamics in C. elegans.     

LRRK2, but not pathogenic mutants,protects against H2O2 stress depending on mitochondrial function and endocytosis in a yeast model

Background

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). Studies in the yeast Saccharomyces cerevisiae have provided valuable insights into the mechanisms of cellular dysfunction associated with the expression of faulty PD genes.

Methods

We developed a yeast model for full-length LRRK2 studies. We expressed wild-type (wt) LRRK2 and mutations and evaluated their role during oxidative stress conditions. The involvement of mitochondria was assessed by using rho-zero mutants and by evaluating reactive oxygen species (ROS) production and mitochondrial membrane potential by flow cytometry. The involvement of endocytosis was also studied by testing several endocytic mutants and by following the vacuolar delivery of the probe FM4-64.

Results

Expression of LRRK2 in yeast was associated to increased hydrogen peroxide resistance. This phenotype, which was dependent on mitochondrial function, was not observed for PD-mutants G2019S and R1441C or in the absence of the kinase activity and the WD40 repeat domain. Expression of the pathogenic mutants stimulated ROS production and increased mitochondrial membrane potential. For the PD-mutants, but not for wild-type LRRK2, endocytic defects were also observed. Additionally, several endocytic proteins were required for LRRK2-mediated protection against hydrogen peroxide.

Conclusions

Our results indicate that LRRK2 confers cellular protection during oxidative stress depending on mitochondrial function and endocytosis.

General significance

Both the loss of capacity of LRRK2 pathogenic mutants to protect against oxidative stress and their enhancement of dysfunction may be important for the development of PD during the aging process.     

Effect of the urease-derived peptide Jaburetox on the central nervous system of Triatoma infestans (Insecta: Heteroptera)

Background

Triatoma infestans is the main vector of Chagas'disease in Southern Cone countries. In triatomines, symptoms suggesting neurotoxicity were observed after treatment with Jaburetox (Jbtx), the entomotoxic peptide obtained from jackbean urease. Here, we study its effect in the central nervous system (CNS) of this species.

Methods

Immunohistochemistry, Western blots, immunoprecipitation, two-dimensional electrophoresis, tandem mass spectrometry and enzymatic assays were performed.

Results

Anti-Jbtx antibody labeled somata of the antennal lobe only in Jbtx-treated insects. Western blot assays of nervous tissue using the same antibody reacted with a 61 kDa protein band only in peptide-injected insects. Combination of immunoprecipitation, two-dimensional electrophoresis and tandem mass spectrometry identified UDP-N-acetylglucosamine pyrophosphorylase (UDP-GlcNAcP) as a molecular target for Jbtx. The activity of UDP-GlcNAcP increased significantly in the CNS of Jbtx-treated insects. The effect of Jbtx on the activity of nitric oxide synthase (NOS) and NO production was investigated as NO is a recognized messenger molecule in the CNS of T. infestans. NOS activity and NO levels decreased significantly in CNS homogenates of Jbtx-treated insects.

Conclusions

UDP-GlcNAcP is a molecular target of Jbtx. Jbtx impaired the activity of T. infestans nitrergic system, which may be related with early behavioral effects.

General Significance

We report that the CNS of Triatoma infestans is a target for the entomotoxic peptide and propose that a specific area of the brain is involved. Besides potentially providing tools for control strategies of Chagas' disease vectors our data may be relevant in various fields of research as insect physiology, neurobiology and protein function.     

An ectotherm homologue of human predicted gene NAT16 encodes histidine N-acetyltransferase responsible for Nα-acetylhistidine synthesis

Background

Nα-Acetylhistidine (NAH) is present in very high concentrations exclusively in the brain and lens of ectothermic vertebrates, including ray-finned fishes, amphibians and reptiles, and not in those of endothermic birds and mammals. Although NAH is known to be synthesized from l-His and acetyl-CoA by histidine N-acetyltransferase (HISAT; EC 2.3.1.33), the gene encoding HISAT has remained unknown for any organism.

Methods

HISAT was purified from the blue mackerel brain, and its partial amino acid sequences were analyzed using mass spectrometry and Edman degradation. Using the sequence information, the corresponding gene was cloned and sequenced. Recombinant proteins encoded by the fish gene and its human homologue were expressed in a cell-free translation system.

Results

HISAT was identified to be a protein encoded by a fish homologue of the human predicted gene NAT16 (N-acetyltransferase 16). HISAT is an unstable enzyme that is rapidly and irreversibly inactivated during preincubation at 37 °C in the absence of acetyl-CoA. In fish brain, the HISAT gene is expressed as two splice variants containing an identical ORF but differing lengths of 5′-UTR. Both variants are expressed exclusively in the fish brain and lens. Interestingly, the recombinant human NAT16 protein, unlike the recombinant fish HISAT, has only trace enzyme activity for NAH synthesis.

Conclusions

These results propose that the function of mammalian NAT16 has been altered from l-His acetylation (NAH synthesis) to another different biological role.

General significance

The molecular identification of HISAT will allow progress in the understanding of the physiological function of NAH in ectothermic vertebrates.     

Heme-iron utilization by Leptospira interrogans requires a heme oxygenase and a plastidic-type ferredoxin-NADP reductase

Background

Heme oxygenase catalyzes the conversion of heme to iron, carbon monoxide and biliverdin employing oxygen and reducing equivalents. This enzyme is essential for heme-iron utilization and contributes to virulence in Leptospira interrogans.

Methods

A phylogenetic analysis was performed using heme oxygenases sequences from different organisms including saprophytic and pathogenic Leptospira species. L. interrogans heme oxygenase (LepHO) was cloned, overexpressed and purified. The structural and enzymatic properties of LepHO were analyzed by UV–vis spectrophotometry and ¹H NMR. Heme-degrading activity, ferrous iron release and biliverdin production were studied with different redox partners.

Results

A plastidic type, high efficiently ferredoxin-NADP⁺ reductase (LepFNR) provides the electrons for heme turnover by heme oxygenase in L. interrogans. This catalytic reaction does not require a ferredoxin. Moreover, LepFNR drives the heme degradation to completeness producing free iron and α-biliverdin as the final products. The phylogenetic divergence between heme oxygenases from saprophytic and pathogenic species supports the functional role of this enzyme in L. interrogans pathogenesis.

Conclusions

Heme-iron scavenging by LepHO in L. interrogans requires only LepFNR as redox partner. Thus, we report a new substrate of ferredoxin-NADP⁺ reductases different to ferredoxin and flavodoxin, the only recognized protein substrates of this flavoenzyme to date. The results presented here uncover a fundamental step of heme degradation in L. interrogans.

General significance

Our findings contribute to understand the heme-iron utilization pathway in Leptospira. Since iron is required for pathogen survival and infectivity, heme degradation pathway may be relevant for therapeutic applications.     

Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition

Background

DNA replication represents a critical step of the cell cycle which requires highly controlled and ordered regulatory mechanisms to ensure the integrity of genome duplication. Among a plethora of elements, post-translational modifications (PTMs) ensure the spatiotemporal regulation of pivotal proteins orchestrating cell division. Despite increasing evidences showing that O-GlcNAcylation regulates mitotic events, the impact of this PTM in the early steps of the cell cycle remains poorly understood.

Methods and results

Quiescent MCF7 cells were stimulated by serum mitogens and cell cycle progression was determined by flow cytometry. The levels of O-GlcNAc modified proteins, O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) were examined by Western blotting and OGA activity was measured during the progression of cells towards S phase. A global decrease in O-GlcNAcylation was observed at S phase entry, concomitantly to an increase in the activity of OGA. A combination of two-dimensional electrophoresis, Western blotting and mass spectrometry was then used to detect and identify cell cycle-dependent putative O-GlcNAcylated proteins. 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated through G1/S transition were identified and the O-GlcNAc variations of Cytokeratin 8, hnRNP K, Caprin-1, Minichromosome Maintenance proteins MCM3, MCM6 and MCM7 were validated by immunoprecipitation.

Conclusions

The dynamics of O-GlcNAc is regulated during G1/S transition and observed on key proteins involved in the cytoskeleton networks, mRNA processing, translation, protein folding and DNA replication.

General significance

Our results led us to propose that O-GlcNAcylation joins the PTMs that take part in the regulation of DNA replication initiation.     

Entamoeba histolytica thioredoxin reductase: Molecular and functional characterization of its atypical properties

Background

Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. Thioredoxin reductase catalyzes the reversible transfer of reducing equivalents between NADPH and thioredoxin, a small protein that plays key metabolic functions in maintaining the intracellular redox balance.

Methods

The present work deals with in vitro steady state kinetic studies aimed to reach a better understanding of the kinetic and structural properties of thioredoxin reductase from E. histolytica (EhTRXR).

Results

Our results support that native EhTRXR is a homodimeric covalent protein that is able to catalyze the NAD(P)H-dependent reduction of amoebic thioredoxins and S‐nitrosothiols. In addition, the enzyme exhibited NAD(P)H dependent oxidase activity, which generates hydrogen peroxide from molecular oxygen. The enzyme can reduce compounds like methylene blue, quinones, ferricyanide or nitro-derivatives; all alternative substrates displaying a relative high capacity to inhibit disulfide reductase activity of EhTRXR.

Conclusions and general significance

Interestingly, EhTRXR exhibited kinetic and structural properties that differ from other low molecular weight TRXR. The TRX system could play an important role in the parasite defense against reactive species. The latter should be critical during the extra intestinal phase of the amoebic infection. So far we know, this is the first in depth characterization of EhTRXR activity and functionality.     

Verdoheme formation in Proteus mirabilis catalase

Background

Heme oxidative degradation has been extensively investigated in peroxidases but not in catalases. The verdoheme formation, a product of heme oxidation which inactivates the enzyme, was studied in Proteus mirabilis catalase.

Methods

The verdoheme was generated by adding peracetic acid and analyzed by mass spectrometry and spectrophotometry.

Results

Kinetics follow-up of different catalase reactional intermediates shows that i) the formation of compound I always precedes that of verdoheme, ii) compound III is never observed, iii) the rate of compound II decomposition is not compatible with that of verdoheme formation, and iv) dithiothreitol prevents the verdoheme formation but not that of compound II, whereas NADPH prevents both of them. The formation of verdoheme is strongly inhibited by EDTA but not increased by Fe³⁺ or Cu²⁺ salts. The generation of verdoheme is facilitated by the presence of protein radicals as observed in the F194Y mutated catalase. The inability of the inactive variant (H54F) to form verdoheme, indicates that the heme oxidation is fully associated to the enzyme catalysis.

Conclusion

These data, taken together, strongly suggest that the verdoheme formation pathway originates from compound I rather than from compound II.

General significance

The autocatalytic verdoheme formation is likely to occur in vivo.     

Methyl and isopropyl N-methylanthranilates attenuate diclofenac- and ethanol-induced gastric lesions in rats

Aims

Two natural alkaloids, methyl (M) and isopropyl (I) N-methylanthranilates, with recently demonstrated significant pharmacological activities, were assayed for their possible overall effect on intact gastric mucosa and their protective properties towards the onset of gastric lesions induced by diclofenac (a non-steroidal anti-inflammatory drug, NSAID) or ethanol.

Main methods

The influence of I and M on gastric mucosa integrity was assessed by oral administration in doses of 200 mg/kg. The gastroprotective action of I and M in doses of 50, 100 and 200 mg/kg was analyzed in the diclofenac and ethanol-induced gastric lesion models in rats. After the treatment, the stomachs of the animals were analyzed (captured by a digital camera). Ulcer scoring, morphometric and histopathological analyses of the stomachs were done.

Key findings

The oral application of these compounds on their own, even in quite high doses (200 mg/kg) did not induce gastric lesions. Both alkaloids exerted a very strong antiulcer activity, even in low doses (50 mg/kg), by decreasing the number of lesions caused by the application of either diclofenac or ethanol, eliminating them completely or reducing them to a form of mucosal hyperemia.

Significance

Their possible mechanism of action was discussed and due to their many positive properties including anxiolytic, antidepressant, antinociceptive, anti-inflammatory and gastroprotective activities, as well as a cheap and simple synthetic route for their preparation, methyl and isopropyl N-methylanthranilates, both alike, might represent a cost effective alternative sought for in the treatment of peptic ulcers and/or new safer NSAIDs for pain management.     

In vitro characterization of ligand-induced oligomerization of the S. cerevisiae G-protein coupled receptor,Ste2p

Background

The S. cerevisiae α-factor receptor, Ste2p, is a G-protein coupled receptor that plays key roles in yeast signaling and mating. Oligomerization of Ste2p has previously been shown to be important for intracellular trafficking, receptor processing and endocytosis. However the role of ligand in receptor oligomerization remains enigmatic.

Methods

Using functional recombinant forms of purified Ste2p, atomic force microscopy, dynamic light scattering and chemical crosslinking are applied to investigate the role of ligand in Ste2p oligomerization.

Results

Atomic force microscopy images indicate a molecular height for recombinant Ste2p in the presence of α-factor nearly double that of Ste2p alone. This observation is supported by complementary dynamic light scattering measurements which indicate a ligand-induced increase in the polydispersity of the Ste2p hydrodynamic radius. Finally, chemical cross-linking of HEK293 plasma membranes presenting recombinant Ste2p indicates α-factor induced stabilization of the dimeric form and higher order oligomeric forms of the receptor upon SDS-PAGE analysis.

Conclusions

α-factor induces oligomerization of Ste2p in vitro and in membrane.

General significance

These results provide additional evidence of a possible role for ligand in mediation of Ste2p oligomerization in vivo.