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.     

Cell migration—The role of integrin glycosylation

Background

Cell migration is an essential process in organ homeostasis, in inflammation, and also in metastasis, the main cause of death from cancer. The extracellular matrix (ECM) serves as the molecular scaffold for cell adhesion and migration; in the first phase of migration, adhesion of cells to the ECM is critical. Engagement of integrin receptors with ECM ligands gives rise to the formation of complex multiprotein structures which link the ECM to the cytoplasmic actin skeleton. Both ECM proteins and the adhesion receptors are glycoproteins, and it is well accepted that N-glycans modulate their conformation and activity, thereby affecting cell–ECM interactions. Likely targets for glycosylation are the integrins, whose ability to form functional dimers depends upon the presence of N-linked oligosaccharides. Cell migratory behavior may depend on the level of expression of adhesion proteins, and their N-glycosylation that affect receptor-ligand binding.

Scope of review

The mechanism underlying the effect of integrin glycosylation on migration is still unknown, but results gained from integrins with artificial or mutated N-glycosylation sites provide evidence that integrin function can be regulated by changes in glycosylation.

General significance

A better understanding of the molecular mechanism of cell migration processes could lead to novel diagnostic and therapeutic approaches and applications. For this, the proteins and oligosaccharides involved in these events need to be characterized.     

Difucosylation of chitooligosaccharides by eukaryote and prokaryote α1,6-fucosyltransferases

Background

The synthesis of eukaryotic N-glycans and the rhizobia Nod factor both involve α1,6-fucosylation. These fucosylations are catalyzed by eukaryotic α1,6-fucosyltransferase, FUT8, and rhizobial enzyme, NodZ. The two enzymes have similar enzymatic properties and structures but display different acceptor specificities: FUT8 and NodZ prefer N-glycan and chitooligosaccharide, respectively. This study was conducted to examine the fucosylation of chitooligosaccharides by FUT8 and NodZ and to characterize the resulting difucosylated chitooligosaccharides in terms of their resistance to hydrolysis by glycosidases.

Methods

The issue of whether FUT8 or NodZ catalyzes the further fucosylation of chitooligosaccharides that had first been monofucosylated by the other. The oligosaccharide products from the successive reactions were analyzed by normal-phase high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. The effect of difucosylation on sensitivity to glycosidase digestion was also investigated.

Results

Both FUT8 and NodZ are able to further fucosylate the monofucosylated chitooligosaccharides. Structural analyses of the resulting oligosaccharides showed that the reducing terminal GlcNAc residue and the third GlcNAc residue from the non-reducing end are fucosylated via α1,6-linkages. The difucosylation protected the oligosaccharides from extensive degradation to GlcNAc by hexosamidase and lysozyme, and also even from defucosylation by fucosidase.

Conclusions

The sequential actions of FUT8 and NodZ on common substrates effectively produce site-specific-difucosylated chitooligosaccharides. This modification confers protection to the oligosaccharides against various glycosidases.

General significance

The action of a combination of eukaryotic and bacterial α1,6-fucosyltransferases on chitooligosaccharides results in the formation of difucosylated products, which serves to stabilize chitooligosaccharides against the action of glycosidases.     

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.     

Generating novel recombinant prokaryotic lectins with altered carbohydrate binding properties through mutagenesis of the PA-IL protein from Pseudomonas aeruginosa

Background

Prokaryotic lectins offer significant advantages over eukaryotic lectins for the development of enhanced glycoselective tools. Amenability to recombinant expression in Escherichia coli simplifies their production and presents opportunities for further genetic manipulation to create novel recombinant prokaryotic lectins (RPLs) with altered or enhanced carbohydrate binding properties. This study explored the potential of the α-galactophilic PA-IL lectin from Pseudomonas aeruginosa for use as a scaffold structure for the generation of novel RPLs.

Method

Specific amino acid residues in the carbohydrate binding site of a recombinant PA-IL protein were randomly substituted by site-directed mutagenesis. The resulting expression clones were then functionally screened to identify clones expressing rPA-IL proteins with altered carbohydrate binding properties.

Results

This study generated RPLs exhibiting diverse carbohydrate binding activities including specificity and high affinity for β-linked galactose and N-acetyl-lactosamine (LacNAc) displayed by N-linked glycans on glycoprotein targets. Key amino acid substitutions were identified and linked with specific carbohydrate binding activities. Ultimately, the utility of these novel RPLs for glycoprotein analysis and for selective fractionation and isolation of glycoproteins and their glycoforms was demonstrated.

Conclusions

The carbohydrate binding properties of the PA-IL protein can be significantly altered using site-directed mutagenesis strategies to generate novel RPLs with diverse carbohydrate binding properties.

General significance

The novel RPLs reported would find a broad range of applications in glycobiology, diagnostics and in the analysis of biotherapeutics. The ability to readily produce these RPLs in gram quantities could enable them to find larger scale applications for glycoprotein or biotherapeutic purification.     

Bioinformatics analysis reveals disturbance mechanism of MAPK signaling pathway and cell cycle in Glioblastoma multiforme

Background & objectives

To analyze the reversal gene pairs and identify featured reversal genes related to mitogen-activated protein kinases (MAPK) signaling pathway and cell cycle in Glioblastoma multiforme (GBM) to reveal its pathogenetic mechanism.

Methods

We downloaded the gene expression profile GSE4290 from the Gene Expression Omnibus database, including 81 gene chips of GBM and 23 gene chips of controls. The t test was used to analyze the DEGs (differentially expressed genes) between 23 normal and 81 GBM samples. Then some perturbing metabolic pathways, including MAPK (mitogen-activated protein kinases) and cell cycle signaling pathway, were extracted from KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database. Cancer genes were obtained from the database of Cancer Gene Census. The reversal gene pairs between DEGs and cancer genes were further analyzed in MAPK and cell cycle signaling pathway.

Results

A total 8523 DEGs were obtained including 4090 up-regulated and 4433 down-regulated genes. Among them, ras-related protein rab-13(RAB13), neuroblastoma breakpoint family member 10 (NBPF10) and disks large homologue 4 (DLG4) were found to be involved in GBM for the first time. We obtained MAPK and cell cycle signaling pathways from KEGG database. By analyzing perturbing mechanism in these two pathways, we identified several reversal gene pairs, including NRAS (neuroblastoma RAS) and CDK2 (cyclin-dependent kinase 2), CCND1 (cyclin D1) and FGFR (fibroblast growth factor receptor). Further analysis showed that NRAS and CDK2 were positively related with GBM. However, FGFR2 and CCND1 were negatively related with GBM.

Interpretation & conclusions

These findings suggest that newly identified DEGs and featured reversal gene pairs participated in MAPK and cell cycle signaling pathway may provide a new therapeutic line of approach to GBM.     

Bauhinia forficata lectin (BfL) induces cell death and inhibits integrin-mediated adhesion on MCF7 human breast cancer cells

Background

Plant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. In this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp. forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity.

Methods

MCF7 cells were treated with BfL. Viability and adhesion alterations were evaluated using flow cytometry and western blotting.

Results

BfL inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases α₁, α₆ and β₁ integrin subunit expression, and increases α₅ subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic acid (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21.

Conclusion

BfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells.

General significance

Cell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer.     

Induction of apoptosis by Polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma L929 cells

Background

The Galanthus nivalis agglutinin (GNA)-related lectins have been reported to bear antiproliferative and apoptosis-inducing activities in cancer cells; however, the precise mechanisms by which GNA-related lectins induce cell death are still only rudimentarily understood.

Methods

In the present study, Polygonatum odoratum lectin (designated POL), a mannose-binding specific GNA-related lectin, possessed a remarkable antiproliferative activity toward murine fibrosarcoma L929 cells. And, this lectin induced L929 cell apoptosis in a caspase-dependent manner. In addition, POL treatment increased the levels of FasL and Fas-Associated protein with Death Domain (FADD) proteins and resulted in caspase-8 activation. Also, POL treatment caused mitochondrial transmembrane potential collapse and cytochrome c release, leading to activations of caspase-9 and caspase-3. Moreover, POL treatment enhanced tumor necrosis factor α (TNFα)-induced L929 cell apoptosis.

Results

Our data demonstrate for the first time that this lectin induces apoptosis through both death-receptor and mitochondrial pathways, as well as amplifies TNFα-induced L929 cell apoptosis.

General significance

These inspiring findings would provide new molecular basis for further understanding cell death mechanisms of the Galanthus nivalis agglutinin (GNA)-related lectins in future cancer investigations.     

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.     

Involvement of ASK1–p38 pathway in the pathogenesis of diabetes triggered by pancreatic ß cell exhaustion

Background

Diabetes mellitus is characterized by high blood glucose levels. Pancreatic ß cell death contributes to type 1 and type 2 diabetes. Akita mice, which harbor a human permanent neonatal diabetes-linked mutation (Cys96Tyr) in the insulin gene, are well established as an animal model of diabetes caused by pancreatic ß cell exhaustion. Mutant Insulin 2 protein (Ins2^C96Y) induces endoplasmic reticulum (ER) stress and pancreatic ß cell death in Akita mice, although the molecular mechanism of Ins^C96Y-induced cell death remains unclear.

Methods

We investigate the mechanisms of Ins2^C96Y-induced pancreatic ß cell death in vitro and in vivo, using p38 inhibitor (SB203580), MIN6 cell (pancreatic ß cell line), Akita mice and apoptosis signal-regulating kinase 1 (ASK1) knockout mice.

Results

The expression of Ins^C96Y activated the ASK1–p38 pathway. Deletion of ASK1 mitigated Ins^C96Y-induced pancreatic ß cell death and delayed the onset of diabetes in Akita mice. Moreover, p38 inhibitor suppressed Ins^C96Y-induced MIN6 cell death.

Conclusions

These findings suggest that ER stress-induced ASK1–p38 activation, which is triggered by the accumulation of Ins^C96Y, plays an important role in the pathogenesis of diabetes.

General significance

Pancreatic ß cell death caused by insulin overload appears to be involved in the pathogenesis of type 1 and type 2 diabetes. Inhibition of the ASK1–p38 pathway may be an effective therapy for various types of diabetes.     

Multiple roles of glucose-6-phosphatases in pathophysiology: State of the art and future trends

Background

The endoplasmic reticulum enzyme glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate to glucose and inorganic phosphate. The enzyme is a part of a multicomponent system that includes several integral membrane proteins; the catalytic subunit (G6PC) and transporters for glucose-6-phosphate, inorganic phosphate and glucose. The G6PC gene family presently includes three members, termed as G6PC, G6PC2, and G6PC3. Although the three isoforms show a moderate amino acid sequence homology, their membrane topology and catalytic site are very similar. The isoforms are expressed differently in various tissues. Mutations in all three genes have been reported to be associated with human diseases.

Scope of review

The present review outlines the biochemical features of the G6PC gene family products, the regulation of their expression, their role in the human pathology and the possibilities for pharmacological interventions.

Major conclusions

G6PCs emerge as integrators of extra- and intracellular glucose homeostasis. Beside the well known key role in blood glucose homeostasis, the members of the G6PC family seem to play a role as sensors of intracellular glucose and of intraluminal glucose/glucose-6-phosphate in the endoplasmic reticulum.

General significance

Since mutations in the three G6PC genes can be linked to human pathophysiological conditions, the better understanding of their functioning in connection with genetic alterations, altered expression and tissue distribution has an eminent importance.     

AtoSC two-component system is involved in cPHB biosynthesis through fatty acid metabolism in E. coli

Background

We have shown previously that AtoSC two-component system regulates the biosynthesis of E. coli cPHB [complexed poly-(R)-3-hydroxybutyrate].

Methods

The AtoSC involvement on fatty acids metabolism, towards cPHB synthesis, was studied using cPHB determination, gene expression, and fatty acid metabolic pathways inhibitors.

Results

Deletion of the atoDAEB operon from the E. coli genome resulted in a consistent reduction of cPHB accumulation. When in ΔatoDAEB cells, the atoDAEB operon and the AtoSC system were introduced extrachromosomally, a significant enhancement of cPHB levels was observed. Moreover, the introduction of a plasmid with atoSC genes regulated positively cPHB biosynthesis. A lesser cPHB enhancement was triggered when plasmids carrying either atoS or atoC were introduced. The intracellular distribution of cPHB was regulated by AtoSC or AtoC according to the inducer (acetoacetate or spermidine). Blockage of β-oxidation by acrylic acid reduced cPHB levels, suggesting the involvement of this pathway in cPHB synthesis; however, the overproduction of AtoSC or its constituents separately resulted in cPHB enhancement. Inhibition of fatty acid biosynthesis by cerulenin resulted to a major cPHB reduction, indicating the contribution of this pathway in cPHB production. Inhibition of both β-oxidation and fatty acid biosynthesis reduced dramatically cPHB, suggesting the contribution of both pathways in cPHB biosynthesis.

Conclusions

Short fatty acid catabolism (atoDAEB operon) and fatty acids metabolic pathways participate in cPHB synthesis through the involvement of AtoSC system.

General significance

The involvement of the AtoSC system in the fatty acids metabolic pathways interplay towards cPHB biosynthesis provides additional perceptions of AtoSC role on E. coli regulatory biochemical processes.     

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.     

The identification and molecular characterization of the first archaeal bifunctional exo-β-glucosidase/N-acetyl-β-glucosaminidase demonstrate that family GH116 is made of three functionally distinct subfamilies

Background

β-N-acetylhexosaminidases, which are involved in a variety of biological processes including energy metabolism, cell proliferation, signal transduction and in pathogen-related inflammation and autoimmune diseases, are widely distributed in Bacteria and Eukaryotes, but only few examples have been found in Archaea so far. However, N-acetylgluco- and galactosamine are commonly found in the extracellular storage polymers and in the glycans decorating abundantly expressed glycoproteins from different Crenarchaeota Sulfolobus sp., suggesting that β-N-acetylglucosaminidase activities could be involved in the modification/recycling of these cellular components.

Methods

A thermophilic β-N-acetylglucosaminidase was purified from cellular extracts of S. solfataricus, strain P2, identified by mass spectrometry, and cloned and expressed in E. coli. Glycosidase assays on different strains of S. solfataricus, steady state kinetic constants, substrate specificity analysis, and the sensitivity to two inhibitors of the recombinant enzyme were also reported.

Results

A new β-N-acetylglucosaminidase from S. solfataricus was unequivocally identified as the product of gene sso3039. The detailed enzymatic characterization demonstrates that this enzyme is a bifunctional β-glucosidase/β-N-acetylglucosaminidase belonging to family GH116 of the carbohydrate active enzyme (CAZy) classification.

Conclusions

This study allowed us to propose that family GH116 is composed of three subfamilies, which show distinct substrate specificities and inhibitor sensitivities.

General significance

The characterization of SSO3039 allows, for the first time in Archaea, the identification of an enzyme involved in the metabolism β-N-acetylhexosaminide, an essential component of glycoproteins in this domain of life, and substantially increases our knowledge on the functional role and phylogenetic relationships amongst the GH116 CAZy family members.     

Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells

Background

Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.

Methods

The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.

Results

We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.

Conclusions

Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.

General significance

Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.     

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.     

Mobility of the conserved glycine 155 is required for formation of the active plasmodial Pdx1 dodecamer

Background

Vitamin B6 synthesis requires a functional Pdx1 assembly that is dodecameric in vivo. We have previously shown that mutation of a catalytic lysine in the plasmodial Pdx1 protein results in a protein that is both inactive and hexameric in vitro.

Methods

Static and dynamic light scattering, circular dichroism, co-purification and enzyme assays are used to investigate the role of a glycine conserved in all Pdx1 family members.

Results

Static light scattering indicates that a glycine to alanine mutant is present as a hexamer in vitro. Subsequent circular dichroism experiments demonstrate that a significant change in secondary structure content is induced by this mutation. However, this mutant is still competent to bind and support Pdx2 activity.

Conclusions

As the mutated glycine occupies an unrestricted region of the Ramachandran plot the additional stereo-chemical restrictions imposed on alanine residues strongly support our hypothesis that significant structural rearrangement of Pdx1 is required during the transition from hexamer to dodecamer.

General significance

The presented results demonstrate that reduction in the mobility of this region in Pdx1 proteins is required for formation of the in vivo dodecamer, negatively affecting the activity of Pdx1, opening the possibility of allosteric Pdx1 inhibitors.     

Methyl pyruvate rescues mitochondrial damage caused by SIGMAR1 mutation related to amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is a disease caused by motor neuron degeneration. Recently, a novel SIGMAR1 gene variant (p.E102Q) was discovered in some familial ALS patients.

Methods

We address mechanisms underlying neurodegeneration caused by the mutation using Neuro2A cells overexpressing σ₁R^E102Q, a protein of a SIGMAR1 gene variant (p.E102Q) and evaluate potential amelioration by ATP production via methyl pyruvate (MP) treatment.

Results

σ₁R^E102Q overexpression promoted dissociation of the protein from the endoplasmic reticulum (ER) membrane and cytoplasmic aggregation, which in turn impaired mitochondrial ATP production and proteasome activity. Under ER stress conditions, overexpression of wild-type σ₁R suppressed ER stress-induced mitochondrial injury, whereas σ₁R^E102Q overexpression aggravated mitochondrial damage and induced autophagic cell death. Moreover, σ₁R^E102Q-overexpressing cells showed aberrant extra-nuclear localization of the TAR DNA-binding protein (TDP-43), a condition exacerbated by ER stress. Treatment of cells with the mitochondrial Ca^2 + transporter inhibitor Ru360 mimicked the effects of σ₁R^E102Q overexpression, indicating that aberrant σ₁R-mediated mitochondrial Ca^2 + transport likely underlies TDP-43 extra-nuclear localization, segregation in inclusion bodies, and ubiquitination. Finally, enhanced ATP production promoted by methyl pyruvate (MP) treatment rescued proteasome impairment and TDP-43 extra-nuclear localization caused by σ₁R^E102Q overexpression.

Conclusions

Our observations suggest that neurodegeneration seen in some forms of ALS are due in part to aberrant mitochondrial ATP production and proteasome activity as well as TDP-43 mislocalization resulting from the SIGMAR1 mutation.

General significance

ATP supplementation by MP represents a potential therapeutic strategy to treat ALS caused by SIGMAR1 mutation.     

Estrogen receptor alpha (ERα) promoter methylation status in tumor and serum DNA in Egyptian breast cancer patients

Background

Status of DNA methylation is one of the most common molecular alterations in human neoplasia. Because it is possible to detect these epigenetic alterations in the bloodstream of patients, we investigated the aberrant DNA methylation status of estrogen receptor alpha (ERα) in patient pretherapeutic sera and tissue.

Materials and methods

In this case control study the patient series consisted of 120 sporadic primary breast cancer cases and 100 patients with benign breast lesion. ER3, ER4, and ER5 primers were used for methylation-specific polymerase chain reaction (MSP) to analyze the CpG methylation of promoter region of ERα gene. Correlation between ER3, ER4, and ER5 methylation and clinicopathological characteristics of the patients was investigated.

Result

The methylation status of ER3, ER4 and ER5 was 65%, 26.7% and 61.7% in tissue respectively and 57.5%, 21.7% and 55.8% in serum respectively. The concordance between tumor and serum DNA methylation was 80%, 72% and 92% for ER3, ER4 and ER5 respectively.

Conclusions

This study demonstrated the potential utility of serum DNA methylation of ERα gene promoter as a non-invasive diagnostic and/or prognostic marker in patients with breast cancer.     

Cooperation of binding sites at the hydrophilic domain of cell-surface sulfatase Sulf1 allows for dynamic interaction of the enzyme with its substrate heparan sulfate

Background

Sulf1 is a cell-surface sulfatase removing internal 6-O-sulfate groups from heparan sulfate (HS) chains. Thereby it modulates the activity of HS-dependent growth factors. For HS interaction Sulf1 employs a unique hydrophilic domain (HD).

Methods

Affinity-chromatography, AFM-single-molecule force spectroscopy (SMFS) and immunofluorescence on living cells were used to analyze specificity, kinetics and structural basis of this interaction.

Results

Full-length Sulf1 interacts broadly with sulfated glycosaminoglycans (GAGs) showing, however, higher affinity toward HS and heparin than toward chondroitin sulfate or dermatan sulfate. Strong interaction depends on the presence of Sulf1-substrate groups, as Sulf1 bound significantly weaker to HS after enzymatic 6-O-desulfation by Sulf1 pretreatment, hence suggesting autoregulation of Sulf1/substrate association. In contrast, HD alone exhibited outstanding specificity toward HS and did not interact with chondroitin sulfate, dermatan sulfate or 6-O-desulfated HS. Dynamic SMFS revealed an off-rate of 0.04/s, i.e., ~ 500-fold higher than determined by surface plasmon resonance. SMFS allowed resolving the dynamics of single dissociation events in each force–distance curve. HD subdomain constructs revealed heparin interaction sites in the inner and C-terminal regions of HD.

Conclusions

Specific substrate binding of Sulf1 is mediated by HD and involves at least two separate HS-binding sites. Surface plasmon resonance K_D-values reflect a high avidity resulting from multivalent HD/heparin interaction. While this ensures stable cell–surface HS association, the dynamic cooperation of binding sites at HD and also the catalytic domain enables processive action of Sulf1 along or across HS chains.

General significance

HD confers a novel and highly dynamic mode of protein interaction with HS.