Formaldehyde induces hyperphosphorylation and polymerization of Tau protein both in vitro and in vivo

Background

Chronic formaldehyde exposure leads to memory impairment and abnormal elevation of endogenous formaldehyde has been found in the brains of Alzheimer's disease (AD) patients. Hyperphosphorylated Tau protein with subsequent aggregates as neurofibrillary tangles (NFTs) is one of the typical pathological characteristics in AD brains. The mechanism underlying abnormally elevated concentrations of endogenous formaldehyde that induce Tau hyperphosphorylation is unknown.

Methods

N2a cells and mice were treated with formaldehyde for different time points, then Western blotting and immunocytochemistry were utilized to determine the phosphorylation and polymerization of Tau protein. HPLC was used to detect the concentration of formaldehyde in cell media.

Results

Under formaldehyde stress, Tau became hyperphosphorylated, not only in the cytoplasm, but also in the nucleus of neuroblastoma (N2a) cells, and mouse brains. Polymers of cellular phospho-Tau were also detected. Significant accumulation of glycogen synthase kinase-3β (GSK-3β) in the nucleus of N2a and mouse brain cells, and elevation of its phosphorylation at Y216, was observed under formaldehyde stress. Formaldehyde-induced Tau hyperphosphorylation was blocked in the presence of LiCl and CT99021, inhibitors of GSK-3β, and by RNAi interference.

Conclusions

Formaldehyde, which may cause age-related memory loss, can act as a factor triggering Tau hyperphosphorylation via GSK-3β catalysis and induces polymerization of Tau.

General significance

Investigation of formaldehyde-induced Tau hyperphosphorylation may provide novel insights into mechanisms underlying tauopathies.     

3D NMR structure of a complex between the amyloid beta peptide (1–40) and the polyphenol ε-viniferin glucoside: Implications in Alzheimer's disease

Background

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. There is a consensus that Aβ is a pathologic agent and that its toxic effects, which are at present incompletely understood, may occur through several potential mechanisms. Polyphenols are known to have wide-ranging properties with regard to health and for helping to prevent various diseases like neurodegenerative disorders. Thus inhibiting the formation of toxic Aβ assemblies is a reasonable hypothesis to prevent and perhaps treat AD

Methods

Solution NMR and molecular modeling were used to obtain more information about the interaction between the Aβ_1–40 and the polyphenol ε-viniferin glucoside (EVG) and particularly the Aβ residues involved in the complex.

Results

The study demonstrates the formation of a complex between two EVG molecules and Aβ_1–40 in peptide characteristic regions that could be in agreement with the inhibition of aggregation. Indeed, in previous studies, we reported that EVG strongly inhibited in vitro the fibril formation of the full length peptides Aβ_1–40 and Aβ_1–42, and had a strong protective effect against PC12 cell death induced by these peptides.

Conclusion

For the full length peptide Aβ_1–40, the binding sites observed could explain the EVG inhibitory effect on fibrillization and thus prevent amyloidogenic neurotoxicity.

General significance

Even though this interaction might be important at the biological level to explain the protective effect of polyphenols in neurodegenerative diseases, caution is required when extrapolating this in vitro model to human physiology.     

Degradation of proteins upon storage at near-neutral pH: Indications of a proteolytic/gelatinolytic activity associated with aggregates

Background

The twin phenomena of aggregation and degradation are classically associated with protein storage. However, although aggregation has been thought to be a possible consequence of protein degradation, it has never before been proposed to be a cause of degradation.

Methods

Proteins stored under physiological conditions and electrophoresed on SDS-PAGE were examined zymographically for the presence of detergent-resistant high molecular weight (HMW) forms, and association of such HMW forms with time-correlated, seeding-dependent gelatinolytic activity, under various conditions.

Results

Eight different proteins aggregate naturally during storage at near-neutral pH, with concomitant development of ‘gelatinolytic’ activity diminished greatly by storage at low temperatures, extremes of pH, arginine, imidazole, BSA, azide, EDTA, DTT, PMSF (but not AEBSF), and diisopropyl fluorophosphate (DFP), suggesting involvement of surface serine residues in a novel aggregate-borne proteolytic activity.

Conclusions

Naturally-formed aggregates of proteins appear to use surface serines to perform peptide bond hydrolysis, explaining degradation of proteins during storage, and indicating why aggregates are cytotoxic.

General significance

The study suggests that a bi-directional cause–effect relationship operates between protein aggregation, and protein degradation, providing clues to the design of better conditions for long-term protein storage.     

Astaxanthin prevents TGFβ1-induced pro-fibrogenic gene expression by inhibiting Smad3 activation in hepatic stellate cells

Background

Non-alcoholic steatohepatitis (NASH) is a subset of non-alcoholic fatty liver disease, the most common chronic liver disease in the U.S. Fibrosis, a common feature of NASH, results from the dysregulation of fibrogenesis in hepatic stellate cells (HSCs). In this study, we investigated whether astaxanthin (ASTX), a xanthophyll carotenoid, can inhibit fibrogenic effects of transforming growth factor β1 (TGFβ1), a key fibrogenic cytokine, in HSCs.

Methods

Reactive oxygen species (ROS) accumulation was measured in LX-2, an immortalized human HSC cell line. Quantitative realtime PCR, Western blot, immunocytochemical analysis, and in-cell Western blot were performed to determine mRNA and protein of fibrogenic genes, and the activation of Smad3 in TGFβ1-activated LX-2 cells and primary mouse HSCs.

Results

In LX-2 cells, ROS accumulation induced by tert-butyl hydrogen peroxide and TGFβ1 was abolished by ASTX. ASTX significantly decreased TGFβ1-induced α-smooth muscle actin (α-SMA) and procollagen type 1, alpha 1 (Col1A1) mRNA as well as α-SMA protein levels. Knockdown of Smad3 showed the significant role of Smad3 in the expression of α-SMA and Col1A1, but not TGFβ1, in LX-2 cells. ASTX attenuated TGFβ1-induced Smad3 phosphorylation and nuclear translocation with a concomitant inhibition of Smad3, Smad7, TGFβ receptor I (TβRI), and TβRII expression. The inhibitory effect of ASTX on HSC activation was confirmed in primary mouse HSCs as evidenced by decreased mRNA and protein levels of α-SMA during activation.

Conclusion

Taken together, ASTX exerted anti-fibrogenic effects by blocking TGFβ1-signaling, consequently inhibiting the activation of Smad3 pathway in HSCs.

General significance

This study suggests that ASTX may be used as a preventive/therapeutic agent to prevent hepatic fibrosis.     

Antiadipogenic effect of carnosic acid,a natural compound present in Rosmarinus officinalis, is exerted through the C/EBPs and PPARγ pathways at the onset of the differentiation program

Background

Obesity is a serious health problem all over the world, and inhibition of adipogenesis constitutes one of the therapeutic strategies for its treatment. Carnosic acid (CA), the main bioactive compound of Rosmarinus officinalis extract, inhibits 3T3-L1 preadipocytes differentiation. However, very little is known about the molecular mechanism responsible for its antiadipogenic effect.

Methods

We evaluated the effect of CA on the differentiation of 3T3-L1 preadipocytes analyzing the process of mitotic clonal expansion, the level of adipogenic markers, and the subcellular distribution of C/EBPβ.

Results

CA treatment only during the first day of 3T3-L1 differentiation process was enough to inhibit adipogenesis. This inhibition was accompanied by a blockade of mitotic clonal expansion. CA did not interfere with C/EBPβ and C/EBPδ mRNA levels but blocked PPARγ, and FABP4 expression. C/EBPβ has different forms known as LIP and LAP. CA induced an increase in the level of LIP within 24 h of differentiation, leading to an increment in LIP/LAP ratio. Importantly, overexpression of LAP restored the capacity of 3T3-L1 preadipocytes to differentiate in the presence of CA. Finally, CA promoted subnuclear de-localization of C/EBPβ.

Conclusions

CA exerts its anti-adipogenic effect in a multifactorial manner by interfering mitotic clonal expansion, altering the ratio of the different C/EBPβ forms, inducing the loss of C/EBPβ proper subnuclear distribution, and blocking the expression of C/EBPα and PPARγ.

General significance

Understanding the molecular mechanism by which CA blocks adipogenesis is relevant because CA could be new a food additive beneficial for the prevention and/or treatment of obesity.     

Systemic treatment with liver X receptor agonists raises apolipoprotein E,cholesterol, and amyloid-β peptides in the cerebral spinal fluid of rats

Background

Apolipoprotein E (apoE) is a major cholesterol transport protein found in association with brain amyloid from Alzheimer's disease (AD) patients and the ε4 allele of apoE is a genetic risk factor for AD. Previous studies have shown that apoE forms a stable complex with amyloid β (Aβ) peptides in vitro and that the state of apoE lipidation influences the fate of brain Aβ, i.e., lipid poor apoE promotes Aβ aggregation/deposition while fully lipidated apoE favors Aβ degradation/clearance. In the brain, apoE levels and apoE lipidation are regulated by the liver X receptors (LXRs).

Results

We investigated the hypothesis that increased apoE levels and lipidation induced by LXR agonists facilitates Aβ efflux from the brain to the cerebral spinal fluid (CSF). We also examined if the brain expression of major apoE receptors potentially involved in apoE-mediated Aβ clearance was altered by LXR agonists. ApoE, cholesterol, Aβ40, and Aβ42 levels were all significantly elevated in the CSF of rats after only 3 days of treatment with LXR agonists. A significant reduction in soluble brain Aβ40 levels was also detected after 6 days of LXR agonist treatment.

Conclusions

Our novel findings suggest that central Aβ lowering caused by LXR agonists appears to involve an apoE/cholesterol-mediated transport of Aβ to the CSF and that differences between the apoE isoforms in mediating this clearance pathway may explain why individuals carrying one or two copies of APOE ε4 have increased risk for AD.

     

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.     

The Global Sequence Signature algorithm unveils a structural network surrounding heavy chain CDR3 loop in Camelidae variable domains

Background

A large fraction of camelid (camels and llamas) antibodies is composed of heavy chain-only homodimers, able to recognise antigens with their variable domain. Events in somatic assembly and maturation of antibodies such as hypermutations and rearrangement of variable loops (CDRs — complementary determining regions) and selection among a wide range of framework variants are generally considered to be random processes.

Methods

An original algorithmic approach (Global Sequence Signature—GSS) was developed, able to take into account multiple functional and/or local sequence properties to detect scattered evolutionary constraints into sequences.

Results

Using the GSS approach, we show that the length of the main hypervariable loop (CDR3) is linked to the nature of 19 surrounding residues on the scaffold. Surprisingly, the relation between CDR3 size and scaffold residues strongly depends on the considered species, illustrating either significant differences in selection mechanisms or functional constraints during antibody maturation.

Conclusions

Combined with the statistical coupling analysis (SCA) approach at the level of scaffold residues, this study has unravelled a robust interaction network on antibody structure surrounding the CDR3 loop.

General significance

In addition to the general applicability of the GSS algorithm, which can bring together functional and sequence data to locate hot spots of constrained evolution, the relationship between CDR3 and scaffold discussed here should be taken into account in protein engineering when designing antibody libraries.     

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.     

In vivo formation of Plasmodium falciparum ribosomal stalk — A unique mode of assembly without stable heterodimeric intermediates

Background

The ribosomal stalk composed of P-proteins constitutes a structure on the large ribosomal particle responsible for recruitment of translation factors and stimulation of factor-dependent GTP hydrolysis during translation. The main components of the stalk are P-proteins, which form a pentamer. Despite the conserved basic function of the stalk, the P-proteins do not form a uniform entity, displaying heterogeneity in the primary structure across the eukaryotic lineage. The P-proteins from protozoan parasites are among the most evolutionarily divergent stalk proteins.

Methods

We have assembled P-stalk complex of Plasmodium falciparum in vivo in bacterial system using tricistronic expression cassette and provided its characteristics by biochemical and biophysical methods.

Results

All three individual P-proteins, namely uL10/P0, P1 and P2, are indispensable for acquisition of a stable structure of the P stalk complex and the pentameric uL10/P0-(P1-P2)₂ form represents the most favorable architecture for parasite P-proteins.

Conclusion

The formation of P. falciparum P-stalk is driven by trilateral interaction between individual elements which represents unique mode of assembling, without stable P1–P2 heterodimeric intermediate.

General significance

On the basis of our mass-spectrometry analysis supported by the bacterial two-hybrid assay and biophysical analyses, a unique pathway of the parasite stalk assembling has been proposed. We suggest that the absence of P1/P2 heterodimer, and the formation of a stable pentamer in the presence of all three proteins, indicate a one-step formation to be the main pathway for the vital ribosomal stalk assembly, whereas the P2 homo-oligomer may represent an off-pathway product with physiologically important nonribosomal role.     

Use of Wisteria floribunda agglutinin affinity chromatography in the structural analysis of the bovine lactoferrin N-linked glycosylation

Background

Over the years, the N-glycosylation of both human and bovine lactoferrin (LF) has been studied extensively, however not all aspects have been studied in as much detail. Typically, the bovine LF complex-type N-glycans include certain epitopes, not found in human LF N-glycans, i.e. Gal(α1-3)Gal(β1-4)GlcNAc (αGal), GalNAc(β1-4)GlcNAc (LacdiNAc), and N-glycolylneuraminic acid (Neu5Gc). The combined presence of complex-type N-glycans, with αGal, LacdiNAc, LacNAc [Gal(β1-4)GlcNAc], Neu5Ac (N-acetylneuraminic acid), and Neu5Gc epitopes, and oligomannose-type N-glycans complicates the high-throughput analysis of such N-glycoprofiles highly.

Methods

For the structural analysis of enzymatically released N-glycan pools, containing both LacNAc and LacdiNAc epitopes, a prefractionation protocol based on Wisteria floribunda agglutinin affinity chromatography was developed. The sub pools were analysed by MALDI-TOF-MS and HPLC-FD profiling, including sequential exoglycosidase treatments.

Results

This protocol separates the N-glycan pool into three sub pools, with (1) free of LacdiNAc epitopes, (2) containing LacdiNAc epitopes, partially shielded by sialic acid, and (3) containing LacdiNAc epitopes, without shielding by sialic acid. Structural analysis by MALDI-TOF-MS and HPLC-FD showed a complex pattern of oligomannose-, hybrid-, and complex-type di-antennary structures, both with, and without LacdiNAc, αGal and sialic acid.

Conclusions

Applying the approach to bovine LF has led to a more detailed N-glycome pattern, including LacdiNAc, αGal, and Neu5Gc epitopes, than was shown in previous studies.

General significance

Bovine milk proteins contain glycosylation patterns that are absent in human milk proteins; particularly, the LacdiNAc epitope is abundant. Analysis of bovine milk serum proteins is therefore excessively complicated. The presented sub fractionation protocol allows a thorough analysis of the full scope of bovine milk protein glycosylation. This article is part of a Special Issue entitled Glycoproteomics.     

Regulation of brown adipogenesis by the Tgf-β family: Involvement of Srebp1c in Tgf-β- and Activin-induced inhibition of adipogenesis

Background

Brown adipocytes generate heat through the expression of mitochondrial Ucp1. Compared with the information on the regulatory differentiation of white preadipocytes, the factors affecting brown adipogenesis are not as well understood. The present study examined the roles of the Tgf-β family members Bmp, Tgf-β and Activin during differentiation of HB2 brown preadipocytes.

Methods

Endogenous Bmp activity and effects of exogenous Tgf-β family members were examined. Role of Srebp1c in brown adipogenesis was further explored.

Results

Although Bmp7 has been suggested to be a potent stimulator of brown adipogenesis, it affected neither the expression of brown adipocyte-selective genes nor Ucp1 induction in response to a β adrenergic receptor agonist. Unlike in 3T3-L1 white preadipocytes, endogenous Bmp activity was not required for brown adipogenesis; treatment with inhibitors of the Bmp pathway did not affect differentiation of preadipocytes. Administration of Tgf-β1 or Activin A efficiently decreased the insulin-induced expression of brown adipocyte-selective genes. Tgf-β1 and Activin A decreased the expression of Pparγ2 and C/ebpα, suggesting the inhibition of adipogenesis. The Tgf-β- and Activin-induced inhibition of brown adipogenesis was mediated by the repression of Srebp1c expression; Tgf-β1 and Activin A blocked Srebp1c gene induction in response to the differentiation induction, and knock-down of Srebp1 expression inhibited brown adipogenesis.

Conclusion

Endogenous Bmp is dispensable for brown adipogenesis, and Srebp1c is indispensable, which is negatively regulated by Tgf-β and Activin.

General significance

Control of activity of the Tgf-β family is potentially useful for maintenance of energy homeostasis through manipulation of brown adipogenesis.     

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.     

Endothelium-dependent epithelial–mesenchymal transition of tumor cells: Exclusive roles of transforming growth factor β1 and β2

Background

Induction of epithelial–mesenchymal transition (EMT) is essential for the metastasis of tumor cells and maintaining their stemness. This study aimed to examine whether endothelial cells, which are most closely located to tumor cells in vivo, play a role in inducing EMT in tumor cells or not.

Methods

Concentrated culture medium of bovine aortic endothelial cells (BAECs) was applied to tumor cell lines (A549 and PANC-1) and epithelial cell line (NMuMg). Cadherin conversion, expressions of α-smooth muscle actin and ZO-1, actin fiber formation and cell migration were examined as hallmarks of the induction of EMT in these cell lines. Transforming growth factor β (TGFβ) antibodies were used to neutralize TGFβ₁, TGFβ₂ and TGFβ₃. Expression and release of TGFβ proteins in BAECs as well as in porcine and human endothelial cells were assessed by Western blotting and ELISA, respectively.

Results

Conditioned medium of BAEC induced EMT in the examined cell lines. All endothelial cells from various species and locations expressed TGFβ₁ and TGFβ₂ proteins and much lower level of TGFβ₃ protein. Conditioned medium from these endothelial cells contained TGFβ₁ and TGFβ₂, but TGFβ₃ could not be detected. Neutralizing antibody against each of TGFβ₁ or TGFβ₂ did not reverse endothelium-dependent EMT, but simultaneous neutralization of both TGFβ₁ and TGFβ₂ completely abolished it.

Conclusions

Endothelial cells may play a role in the induction and maintenance of EMT in tumor cells by constitutively releasing TGFβ₁ and TGFβ₂.

General significance

The present results provide a novel strategy of the inhibition of tumor metastasis by targeting vascular endothelium.     

Receptor for advanced glycation end-product (RAGE) gene polymorphism 2245G/A is associated with pro-inflammatory,oxidative-glycation markers and sRAGE in diabetic retinopathy

Background

Receptor for advanced glycation end-product (RAGE) gene polymorphism 2245G/A is associated with diabetic retinopathy (DR). However, the mechanism on how it affects the disease development is still unclear.

Aim

This study aims to investigate the relationship between 2245G/A RAGE gene polymorphism and selected pro-inflammatory, oxidative-glycation markers in DR patients.

Methods

A total of 371 unrelated type 2 diabetic patients [200 with retinopathy, 171 without retinopathy (DNR)] and 235 healthy subjects were recruited. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism method followed by DNA sequencing. The nuclear and cytosolic extracts from peripheral blood mononuclear cells were used for nuclear factor kappa B (NF-κB) p65 and superoxide dismutase activity measurement respectively. Plasma was used for glutathione peroxidase activity, advanced oxidation protein product (AOPP), monocyte chemoattractant protein (MCP)-1, pentosidine and soluble RAGE (sRAGE) measurements.

Results

DR patients with 2245GA genotype had significantly elevated levels of activated NF-κB p65, plasma MCP-1, AOPP and pentosidine but lower level of sRAGE when compared to DR patients with wild-type 2245GG.

Conclusion

The RAGE gene polymorphism 2245G/A is associated with pro-inflammatory, oxidative-glycation markers and circulating sRAGE in DR patients. Patients with 2245GA RAGE genotype could aggravate DR possibly via NF-κB mediated inflammatory pathway.     

Identification and characterization of retinoid-active short-chain dehydrogenases/reductases in Drosophila melanogaster

Background

In chordates, retinoid metabolism is an important target of short-chain dehydrogenases/reductases (SDRs). It is not known whether SDRs play a role in retinoid metabolism of protostomes, such as Drosophila melanogaster.

Methods

Drosophila genome was searched for genes encoding proteins with ∼ 50% identity to human retinol dehydrogenase 12 (RDH12). The corresponding proteins were expressed in Sf9 cells and biochemically characterized. Their phylogenetic relationships were analyzed using PHYLIP software.

Results

A total of six Drosophila SDR genes were identified. Five of these genes are clustered on chromosome 2 and one is located on chromosome X. The deduced proteins are 300 to 406 amino acids long and are associated with microsomal membranes. They recognize all-trans-retinaldehyde and all-trans-3-hydroxyretinaldehyde as substrates and prefer NADPH as a cofactor. Phylogenetically, Drosophila SDRs belong to the same branch of the SDR superfamily as human RDH12, indicating a common ancestry early in bilaterian evolution, before a protostome–deuterostome split.

Conclusions

Similarities in the substrate and cofactor specificities of Drosophila versus human SDRs suggest conservation of their function in retinoid metabolism throughout protostome and deuterostome phyla.

General significance

The discovery of Drosophila retinaldehyde reductases sheds new light on the conversion of β-carotene and zeaxantine to visual pigment and provides a better understanding of the evolutionary roots of retinoid-active SDRs.     

Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases

Background

(5R?) and (5S?) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.

Methods

Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.

Results

In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the k_cat/K_m ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.

Conclusions

The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.

General significance

The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.